Occurrence and Profile Characteristics of the Pesticide Imidacloprid

Nov 16, 2015 - Concretely, 16 adults who never engaged in pesticide-related work (named Group UA, including 8 males and 8 females aged 23 to 60 years)...
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Occurrence and profile characteristics of the pesticide imidacloprid, the preservative parabens, and their metabolites in human urine from rural and urban China Lei Wang, Tianzhen Liu, Fang Liu, Junjie Zhang, Yinghong Wu, and Hongwen Sun Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04037 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 20, 2015

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Occurrence and profile characteristics of the pesticide imidacloprid,

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the preservative parabens, and their metabolites in human urine

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from rural and urban China

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Lei Wang†,*, Tianzhen Liu†, Fang Liu†, Junjie Zhang†, Yinghong Wu‡, Hongwen

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Sun†

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Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria/Tianjin Key Laboratory of Environmental Remediation and Pollution Control, Nankai University, Tianjin 300071, China



Tianjin Centers for Disease Control and Prevention, Tianjin 300171, China

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*Corresponding author: L. Wang Nankai University 94 Weijin Road Tianjin, China 300071 Tel: 86-22-2350-9241 Fax: 86-22-2350-8807 E-mail: [email protected]

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For submission to: Environmental Science & Technology

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Abstract

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Knowledge of human exposure to imidacloprid, the most extensively used

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insecticide, and para-hydroxybenzoic acid esters (parabens), the most extensively

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used preservative, is insufficient. In this study, 295 urine samples collected from rural

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and urban areas in China were analyzed for imidacloprid and four parabens, namely,

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methyl paraben, ethyl paraben, propyl paraben, and butyl paraben, as well as their

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major metabolites, namely, 6-chloronicotinic acid (6-ClNA) and para-hydroxybenzoic

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acid (p-HB). Imidacloprid was detected in 100% of the urine samples from rural

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Chinese and 95% of the urine samples from urban Chinese. Concentrations of urinary

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imidacloprid detected in rural Chinese (geometric mean (GM) = 0.18 ng/mL) were

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slightly higher than those detected in urban Chinese (GM = 0.15 ng/mL) when the

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effect of pesticide spraying was excluded. However, concentrations of urinary

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imidacloprid detected in rural adults increased significantly in the subsequent days of

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pesticide spraying (GM = 0.62 ng/mL), which could return to the normal levels within

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3 days. By contrast, concentrations of urinary parabens detected in rural Chinese (GM

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= 6.90 ng/mL) were lower than that in urban Chinese (GM = 30.5 ng/mL). In addition,

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the metabolism characteristics of imidacloprid to 6-ClNA and parabens to p-HB were

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discussed preliminarily.

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INTRODUCTION

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With the increasing application of artificial chemicals, human exposure to these

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compounds is of significant concern, due to the potential association of some

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compounds with some adverse healthy effects.1,2 A total of 265 chemicals have been

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included in the newest National Report on Human Exposure to Environmental

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Chemicals published by the United States Centers for Disease Control and

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Prevention3 based on the National Health and Nutrition Examination Survey of the

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US. Nevertheless, knowledge of the exposure levels and metabolism characteristics of

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chemicals is still insufficient compared with the rapid growth of the application of

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new chemicals.

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Imidacloprid [1-6(chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine]

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is a typical neonicotinoid insecticide, which was launched to the market in the early

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1990s.4 Imidacloprid is currently the most extensively used insecticide in the world,

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which represents approximately 20% of the global pesticide market,5 and is

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distributed in more than 120 countries.6 Imidacloprid exhibits a lower acute toxicity in

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mammals than insects.6 However, the affinity of imidacloprid to mammalian nicotinic

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acetylcholine receptors (nAChRs) and its toxicity to mammals have been observed.7

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Most recently, imidacloprid was reported to induce changes to the biochemical

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parameters of the kidney of male rats, at no observed adverse effect level.8 Adverse

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effects, such as thyroid disturbance, neurobehavioral impairments, and lipid

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peroxidation induced by exposure to low subchronic doses of imidacloprid, were also

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reported in birds9 and aquatic organisms.10,11

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Although the use of imidacloprid has been gaining popularity in agricultural

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settings, human exposure to imidacloprid has not been fully evaluated. 12-15 China is a

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major producer and user of imidacloprid, with an annual capacity of 25,000 tons and a

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domestic demand of 3,000 tons per year to 4,000 tons per year.16 As such, the Chinese

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population might be subject to high exposure risk.

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Parabens (para-hydroxybenzoic acid esters) are the most extensively used

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preservatives in packaged foods, cosmetics, and personal care products. 17 After the

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estrogenic activities of methyl paraben (MeP), ethyl paraben (EtP), propyl paraben

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(PrP), and butyl paraben (BuP) were reported in 1998,18 increasing evidence of

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endocrine and reproductive toxicities has been reported in numerous in vitro and in

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vivo assays.19–23 The potential bioaccumulation of parabens in humans was also

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proposed in several studies,24–26 and their exposure was associated with several

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adverse health outcomes in epidemiological studies.2,27 Widespread exposure of

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humans to parabens has been identified in certain populations of different countries,

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including China.3,28–36 However, knowledge of the exposure of the rural Chinese

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population to parabens is scarce, even if over 600 million Chinese live in rural areas.

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In this study, the concentrations of imidacloprid and its metabolite,

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6-chloronicotinic acid (6-ClNA), as well as four major paraben analytes, i.e., MeP, EtP,

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PrP, and BuP, and their metabolite, para-hydroxybenzoic acid (p-HB), were

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determined in 295 urine samples, including 120 samples collected from rural adults,

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58 samples from rural children, 57 samples from rural elders, 48 samples from urban

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adults, and 12 samples from urban children in China. The objectives of this study

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were to (i) determine the concentrations of urinary imidacloprid in the Chinese

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population and evaluate the effects of pesticide spraying; (ii) elucidate the rural–urban

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differences in human exposure to imidacloprid and parabens; and (iii) conduct a

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preliminary discussion of the metabolism characteristics of imidacloprid and parabens

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in humans.

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MATERIALS AND METHODS

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Reagents and standards. Standards of imidacloprid, p-HB, MeP, EtP, and PrP

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were purchased from Sigma-Aldrich (St. Louis, MO, USA). Standards of 6-ClNA and

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BuP were purchased from Alfa Aesar (Tianjin, China). The structures and

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physicochemical properties of the target analytes are shown in Table 1 and Figure S1

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(in the Supporting Information). The isotope-labelled standard d4-imidacloprid was

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purchased from Sigma-Aldrich (St. Louis, MO, USA), whereas

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13

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Laboratories (Andover, MA, USA). β-Glucuronidase from Helix pomatia (145,700

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units/mL β-glucuronidase and 887 units/mL sulfatase) was purchased from

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C6-BuP (99%), and

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C6-MeP (99%),

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C6-p-HB (99%) were purchased from Cambridge Isotope

Sigma-Aldrich (St. Louis, MO, USA).

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Sample collection. During April and May 2014, 10 rural families, including 20

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adults (named Group RA, including 9 males and 11 females aged 25 years to 60

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years), 10 children (named Group RC, including 8 males and 2 females aged 4 years

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to 9 years), and 11 elders (named Group RE, including 4 males and 7 females aged 65

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years to 85 years) (Table S1 in the Supporting Information), from a village in

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Shandong Province in China, which is known for fruit cultivation, were invited as

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rural volunteers. Three days before and three days after the spraying of imidacloprid

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pesticide in their orchards, the first morning urine specimens from the adult farmers

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who sprayed the pesticides and the children and elders in their family were collected

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daily. More detailed information of pesticide spraying is provided in Table S2.

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In the same period, urine specimens were also collected from healthy urban

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volunteers in a nearby town (~25 km far from the village). Concretely, 16 adults who

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never engaged in pesticide-related work (named Group UA, including 8 males and 8

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females aged 23 years to 60 years) and 4 children (named Group UC, including 4

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males aged 4 years to 9 years) were randomly selected. The first morning urine

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specimens were collected daily for three days. All urine specimens were collected in

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polypropylene (PP) tubes and stored at −80 °C prior to analysis. The urine collection

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was approved by the Institutional Review Board of Nankai University. Detailed

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information of the volunteers is shown in Table S1.

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Sample preparation. A liquid–liquid extraction method, similar to that reported

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in our previous study33 with minor modifications, was employed for the extraction of

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imidacloprid, parabens, and their metabolites. Briefly, 2 mL of urine was transferred

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into a 15 mL PP tube and spiked with 100 µL of methanol containing 10 ng

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d4-imidacloprid, 10 ng 13C6-MeP, 10 ng 13C6-EtP, and 50 ng 13C6-p-HB. Then, 300 µL

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of hydrolytic enzyme buffer solution containing 77 units of β-glucuronidase was

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transferred into the PP tube and enzymolyzed for 12 h at 37 °C. Afterward, the

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specimens were extracted two times with 8 and 5 mL of ethyl acetate. For each

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extraction, the mixture was shaken in an oscillator shaker for 60 min and centrifuged

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at 3,000 r/min for 5 min. The supernatants were combined and washed with 1 mL of

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Milli-Q water, shaken in an oscillator shaker for 10 min, and centrifuged at 3,000

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r/min for 5 min. The supernatant was transferred into a glass tube and concentrated to

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dryness under a gentle nitrogen stream. Finally, 0.5 mL of methanol was added and

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vortex mixed for analysis by ultra-performance liquid chromatography–tandem mass

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spectrometry (UPLC-MS/MS).

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Instrumental Analysis. Separation and detection of imidacloprid, parabens, and

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their metabolites were accomplished by using the Waters 7100C Series

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UPLC-MS/MS (Waters Corporation, Milford, MA, USA) interfaced with a triple

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quadrupole mass spectrometer Xevo TQ-S (Waters Corporation, Milford, MA, USA).

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Ten microliters of the extract was injected into an analytical column (Waters BEH

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Shield C18, 100 × 3.0 mm, 1.7 µm; Waters Corporation, Milford, MA, USA)

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connected serially to a Javelin guard column (Waters BEH Shield C18, 20 × 2.1 mm,

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1.7 µm; Waters Corporation, Milford, MA, USA). The mobile phase comprised 100%

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acetonitrile (A) and Milli-Q water that contained 0.01% formic acid (B). A gradient

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elution at a flow rate of 0.4 mL/min was used for the analysis, with detailed

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information shown in Table S3. The MS/MS was operated in multiple reaction

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monitoring, with the negative and positive modes used, respectively. Imidacloprid and

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d4-imidacloprid were operated in positive ionization mode, while the others were

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operated in negative ionization mode. The parameters were optimized by infusion of

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individual analytes. The results are shown in Table S4.

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Quality assurance and quality control. Procedural blanks were analyzed in

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every batch of 25 specimens. Duplicate analysis of selected specimens showed a

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coefficient of variation of