Subscriber access provided by UNIV OF LETHBRIDGE
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 34
Environmental Science & Technology
1
Occurrence and profile characteristics of the pesticide imidacloprid,
2
the preservative parabens, and their metabolites in human urine
3
from rural and urban China
4 5
Lei Wang†,*, Tianzhen Liu†, Fang Liu†, Junjie Zhang†, Yinghong Wu‡, Hongwen
6
Sun†
7 8 9 10 11
†
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
12 13 14 15 16 17 18 19 20 21
*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] 22
For submission to: Environmental Science & Technology
ACS Paragon Plus Environment
Environmental Science & Technology
23
Abstract
24
Knowledge of human exposure to imidacloprid, the most extensively used
25
insecticide, and para-hydroxybenzoic acid esters (parabens), the most extensively
26
used preservative, is insufficient. In this study, 295 urine samples collected from rural
27
and urban areas in China were analyzed for imidacloprid and four parabens, namely,
28
methyl paraben, ethyl paraben, propyl paraben, and butyl paraben, as well as their
29
major metabolites, namely, 6-chloronicotinic acid (6-ClNA) and para-hydroxybenzoic
30
acid (p-HB). Imidacloprid was detected in 100% of the urine samples from rural
31
Chinese and 95% of the urine samples from urban Chinese. Concentrations of urinary
32
imidacloprid detected in rural Chinese (geometric mean (GM) = 0.18 ng/mL) were
33
slightly higher than those detected in urban Chinese (GM = 0.15 ng/mL) when the
34
effect of pesticide spraying was excluded. However, concentrations of urinary
35
imidacloprid detected in rural adults increased significantly in the subsequent days of
36
pesticide spraying (GM = 0.62 ng/mL), which could return to the normal levels within
37
3 days. By contrast, concentrations of urinary parabens detected in rural Chinese (GM
38
= 6.90 ng/mL) were lower than that in urban Chinese (GM = 30.5 ng/mL). In addition,
39
the metabolism characteristics of imidacloprid to 6-ClNA and parabens to p-HB were
40
discussed preliminarily.
ACS Paragon Plus Environment
Page 2 of 34
Page 3 of 34
Environmental Science & Technology
41
INTRODUCTION
42
With the increasing application of artificial chemicals, human exposure to these
43
compounds is of significant concern, due to the potential association of some
44
compounds with some adverse healthy effects.1,2 A total of 265 chemicals have been
45
included in the newest National Report on Human Exposure to Environmental
46
Chemicals published by the United States Centers for Disease Control and
47
Prevention3 based on the National Health and Nutrition Examination Survey of the
48
US. Nevertheless, knowledge of the exposure levels and metabolism characteristics of
49
chemicals is still insufficient compared with the rapid growth of the application of
50
new chemicals.
51
Imidacloprid [1-6(chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine]
52
is a typical neonicotinoid insecticide, which was launched to the market in the early
53
1990s.4 Imidacloprid is currently the most extensively used insecticide in the world,
54
which represents approximately 20% of the global pesticide market,5 and is
55
distributed in more than 120 countries.6 Imidacloprid exhibits a lower acute toxicity in
56
mammals than insects.6 However, the affinity of imidacloprid to mammalian nicotinic
57
acetylcholine receptors (nAChRs) and its toxicity to mammals have been observed.7
58
Most recently, imidacloprid was reported to induce changes to the biochemical
59
parameters of the kidney of male rats, at no observed adverse effect level.8 Adverse
60
effects, such as thyroid disturbance, neurobehavioral impairments, and lipid
61
peroxidation induced by exposure to low subchronic doses of imidacloprid, were also
62
reported in birds9 and aquatic organisms.10,11
ACS Paragon Plus Environment
Environmental Science & Technology
63
Although the use of imidacloprid has been gaining popularity in agricultural
64
settings, human exposure to imidacloprid has not been fully evaluated. 12-15 China is a
65
major producer and user of imidacloprid, with an annual capacity of 25,000 tons and a
66
domestic demand of 3,000 tons per year to 4,000 tons per year.16 As such, the Chinese
67
population might be subject to high exposure risk.
68
Parabens (para-hydroxybenzoic acid esters) are the most extensively used
69
preservatives in packaged foods, cosmetics, and personal care products. 17 After the
70
estrogenic activities of methyl paraben (MeP), ethyl paraben (EtP), propyl paraben
71
(PrP), and butyl paraben (BuP) were reported in 1998,18 increasing evidence of
72
endocrine and reproductive toxicities has been reported in numerous in vitro and in
73
vivo assays.19–23 The potential bioaccumulation of parabens in humans was also
74
proposed in several studies,24–26 and their exposure was associated with several
75
adverse health outcomes in epidemiological studies.2,27 Widespread exposure of
76
humans to parabens has been identified in certain populations of different countries,
77
including China.3,28–36 However, knowledge of the exposure of the rural Chinese
78
population to parabens is scarce, even if over 600 million Chinese live in rural areas.
79
In this study, the concentrations of imidacloprid and its metabolite,
80
6-chloronicotinic acid (6-ClNA), as well as four major paraben analytes, i.e., MeP, EtP,
81
PrP, and BuP, and their metabolite, para-hydroxybenzoic acid (p-HB), were
82
determined in 295 urine samples, including 120 samples collected from rural adults,
83
58 samples from rural children, 57 samples from rural elders, 48 samples from urban
84
adults, and 12 samples from urban children in China. The objectives of this study
ACS Paragon Plus Environment
Page 4 of 34
Page 5 of 34
Environmental Science & Technology
85
were to (i) determine the concentrations of urinary imidacloprid in the Chinese
86
population and evaluate the effects of pesticide spraying; (ii) elucidate the rural–urban
87
differences in human exposure to imidacloprid and parabens; and (iii) conduct a
88
preliminary discussion of the metabolism characteristics of imidacloprid and parabens
89
in humans.
90
MATERIALS AND METHODS
91
Reagents and standards. Standards of imidacloprid, p-HB, MeP, EtP, and PrP
92
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Standards of 6-ClNA and
93
BuP were purchased from Alfa Aesar (Tianjin, China). The structures and
94
physicochemical properties of the target analytes are shown in Table 1 and Figure S1
95
(in the Supporting Information). The isotope-labelled standard d4-imidacloprid was
96
purchased from Sigma-Aldrich (St. Louis, MO, USA), whereas
97
13
98
Laboratories (Andover, MA, USA). β-Glucuronidase from Helix pomatia (145,700
99
units/mL β-glucuronidase and 887 units/mL sulfatase) was purchased from
100
C6-BuP (99%), and
13
C6-MeP (99%),
13
C6-p-HB (99%) were purchased from Cambridge Isotope
Sigma-Aldrich (St. Louis, MO, USA).
101
Sample collection. During April and May 2014, 10 rural families, including 20
102
adults (named Group RA, including 9 males and 11 females aged 25 years to 60
103
years), 10 children (named Group RC, including 8 males and 2 females aged 4 years
104
to 9 years), and 11 elders (named Group RE, including 4 males and 7 females aged 65
105
years to 85 years) (Table S1 in the Supporting Information), from a village in
106
Shandong Province in China, which is known for fruit cultivation, were invited as
ACS Paragon Plus Environment
Environmental Science & Technology
107
rural volunteers. Three days before and three days after the spraying of imidacloprid
108
pesticide in their orchards, the first morning urine specimens from the adult farmers
109
who sprayed the pesticides and the children and elders in their family were collected
110
daily. More detailed information of pesticide spraying is provided in Table S2.
111
In the same period, urine specimens were also collected from healthy urban
112
volunteers in a nearby town (~25 km far from the village). Concretely, 16 adults who
113
never engaged in pesticide-related work (named Group UA, including 8 males and 8
114
females aged 23 years to 60 years) and 4 children (named Group UC, including 4
115
males aged 4 years to 9 years) were randomly selected. The first morning urine
116
specimens were collected daily for three days. All urine specimens were collected in
117
polypropylene (PP) tubes and stored at −80 °C prior to analysis. The urine collection
118
was approved by the Institutional Review Board of Nankai University. Detailed
119
information of the volunteers is shown in Table S1.
120
Sample preparation. A liquid–liquid extraction method, similar to that reported
121
in our previous study33 with minor modifications, was employed for the extraction of
122
imidacloprid, parabens, and their metabolites. Briefly, 2 mL of urine was transferred
123
into a 15 mL PP tube and spiked with 100 µL of methanol containing 10 ng
124
d4-imidacloprid, 10 ng 13C6-MeP, 10 ng 13C6-EtP, and 50 ng 13C6-p-HB. Then, 300 µL
125
of hydrolytic enzyme buffer solution containing 77 units of β-glucuronidase was
126
transferred into the PP tube and enzymolyzed for 12 h at 37 °C. Afterward, the
127
specimens were extracted two times with 8 and 5 mL of ethyl acetate. For each
128
extraction, the mixture was shaken in an oscillator shaker for 60 min and centrifuged
ACS Paragon Plus Environment
Page 6 of 34
Page 7 of 34
Environmental Science & Technology
129
at 3,000 r/min for 5 min. The supernatants were combined and washed with 1 mL of
130
Milli-Q water, shaken in an oscillator shaker for 10 min, and centrifuged at 3,000
131
r/min for 5 min. The supernatant was transferred into a glass tube and concentrated to
132
dryness under a gentle nitrogen stream. Finally, 0.5 mL of methanol was added and
133
vortex mixed for analysis by ultra-performance liquid chromatography–tandem mass
134
spectrometry (UPLC-MS/MS).
135
Instrumental Analysis. Separation and detection of imidacloprid, parabens, and
136
their metabolites were accomplished by using the Waters 7100C Series
137
UPLC-MS/MS (Waters Corporation, Milford, MA, USA) interfaced with a triple
138
quadrupole mass spectrometer Xevo TQ-S (Waters Corporation, Milford, MA, USA).
139
Ten microliters of the extract was injected into an analytical column (Waters BEH
140
Shield C18, 100 × 3.0 mm, 1.7 µm; Waters Corporation, Milford, MA, USA)
141
connected serially to a Javelin guard column (Waters BEH Shield C18, 20 × 2.1 mm,
142
1.7 µm; Waters Corporation, Milford, MA, USA). The mobile phase comprised 100%
143
acetonitrile (A) and Milli-Q water that contained 0.01% formic acid (B). A gradient
144
elution at a flow rate of 0.4 mL/min was used for the analysis, with detailed
145
information shown in Table S3. The MS/MS was operated in multiple reaction
146
monitoring, with the negative and positive modes used, respectively. Imidacloprid and
147
d4-imidacloprid were operated in positive ionization mode, while the others were
148
operated in negative ionization mode. The parameters were optimized by infusion of
149
individual analytes. The results are shown in Table S4.
150
Quality assurance and quality control. Procedural blanks were analyzed in
ACS Paragon Plus Environment
Environmental Science & Technology
151
every batch of 25 specimens. Duplicate analysis of selected specimens showed a
152
coefficient of variation of
