Article pubs.acs.org/est
Temporal Levels of Urinary Neonicotinoid and Dialkylphosphate Concentrations in Japanese Women Between 1994 and 2011 Jun Ueyama,*,† Kouji H. Harada,‡ Akio Koizumi,‡ Yuka Sugiura,† Takaaki Kondo,† Isao Saito,§ and Michihiro Kamijima*,∥ †
Department of Pathophysiological Laboratory Sciences, Field of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan ‡ Department of Health and Environmental Sciences, Kyoto University Graduate School of Medicine, Yoshida, Kyoto 606-8501, Japan § Food Safety and Quality Research Center, Tokai COOP Federation, Nagakute 480-1103, Japan ∥ Department of Occupational and Environmental Health, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan ABSTRACT: Over the last two decades, usage of neonicotinoid (NEO) insecticides has increased due to their high selectivity for insects versus mammals and their effectiveness for extermination of insects resistant to conventional pesticides such as pyrethroids and organophosphates (OPs). However, historical change of the NEO exposure level in humans is poorly understood. The aim of this study is to reveal changes in the levels of NEO and OP exposure in the human body over the last two decades using biomonitoring technique. We quantified urinary concentrations of 7 NEOs (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) and 4 metabolites of OPs (dimethylphosphate, dimethylthiophosphate, diethylphosphate, and diethylthiophosphate) in 95 adult females aged 45−75 in 1994, 2000, 2003, 2009, and 2011 (n = 17−20 different individuals in each year). The results show that the detection rates of urinary NEOs in Japanese women increased significantly between 1994 and 2011, suggesting that intakes of NEOs into the human body rose during that period. In contrast, exposure to OPs having O,O-dimethyl moieties decreased steadily according to a finding that geometric means of urinary dimethylphosphate concentrations kept diminishing considerably. These changes may reflect the amounts of NEOs and OPs used as insecticides in Japan.
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INTRODUCTION Pesticides have had a broad spectrum of use in agriculture and other settings including public health and individual households throughout the world, thereby being an indispensable ubiquitous component of daily life.1,2 Of the pesticide classes, neonicotinoid (NEO) insecticides are now playing a key role in controlling agricultural pests which have developed tolerance to traditional insecticide pyrethroids and organophosphates (OPs). Seven NEOsacetamiprid (ACE), clothianidin (CLO), dinotefuran (DIN), imidacloprid (IMI), nitenpyram (NIT), thiacloprid (THD), and thiamethoxam (THM)have so far been introduced to the market and have reduced laborintensive pesticide spraying and risk of its exposure due to their low volatility, high permeability, and highly selective insecticidal properties.3 These advantages have resulted in a pronounced increase in NEO application in the world, especially in developed countries.4−7 In general, NEOs are considered good candidates for replacing OPs. Recently, some concerns about the effect of NEOs on the ecosystem and mammals have been reported. The most representative ones associate NEO use with the deterioration of bee colony growth and the decline of insectivorous birds.8,9 © XXXX American Chemical Society
There are some usage data and environmental monitoring results of NEOs, showing that aggregate NEO use and groundwater and soil contamination have increased dramatically over the past decade.7,10,11 Thus, there is an urgent need to clarify the effect of NEOs not only on the ecosystem but on human health. In this regard, in vivo high-dose administration studies conducted to identify possible hazards of NEOs have proven that THM and CLO may induce dopamine release in rat striatum, as detected by a brain microdialysis, and that THD increases thyroid hormone levels in rat plasma, which might warrant screening of neurological and endocrinological end points in exposed human populations.12,13 However, such studies have rarely been conducted except in case reports focused on suicide attempts or occupational settings.14,15 To begin with, epidemiological evidence available about the level of net NEO exposure through every route in daily life is scarce, although it is absolutely required for making a risk assessment of NEO. Received: June 25, 2015 Revised: October 19, 2015 Accepted: November 10, 2015
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DOI: 10.1021/acs.est.5b03062 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Table 1. Demographic Data of Subjects in this Study year of sample collection analytes
1994
2000
2003
2009
2011
number of subjects age (years) urinary creatinine (g/L)
20 53.3 ± 5.7 0.9 ± 0.5
20 52.6 ± 5.0 1.1 ± 0.7
20 62.6 ± 4.3 1.0 ± 0.7
17 64.0 ± 8.1 1.1 ± 0.8
18 68.1 ± 5.2 1.3 ± 0.8
Table 2. Distribution of Concentrations of Neonicotinoids and Dialkylphosphates (μg/g creatinine or nmol/g creatinine) among Samples from Japanese Women Obtained between 1994 and 2011a years of sample collection NEOs IMI
ACE
NIT
THM
THD
CLO
DIN
a
selected percentiles 25th 50th 75th 90th max. 25th 50th 75th 90th max. 25th 50th 75th 90th max. 25th 50th 75th 90th max. 25th 50th 75th 90th max. 25th 50th 75th 90th max. 25th 50th 75th 90th max.
years of sample collection
1994
2000
2003
2009
2011
DAPs
− − − 0.39 1.53 − − − − − − − − − − − − − −
− − − 0.48 0.55 − − − − 0.03 − − − − − − − 0.35 0.50 0.52 − − − − − − − − − − − − − −
− − − 0.55 0.56 − − − 0.02 0.08 − − − − 0.15 − 0.23 0.58 1.35 1.68 − − − 0.38 3.09 − − − − − − − 0.2 5.9
− 0.39 1.26 3.58 3.84 − − 0.03 0.09 0.20 − − − 0.08 0.40 0.16 0.63 1.03 2.42 2.57 − − − 0.92 2.64 − − − 2.50 12.50 − 0.5 2.7 10.2 16.0
− − 0.43 0.88 2.46 − − − − 0.03 − − 0.20 0.65 0.82 0.32 0.57 1.58 4.64 7.25 − − 0.18 0.37 1.44 − − − 0.55 1.67 − 1.8 5.7 14.0 21.4
DMP
− − − − − − − − − − − − − −
DMTP
DEP
DETP
∑DAP
selected percentiles
1994
2000
2003
2009
2011
25th 50th 75th 90th max. 25th 50th 75th 90th max. 25th 50th 75th 90th max. 25th 50th 75th 90th max. 25th 50th 75th 90th max.
22.4 34.1 67.0 108.2 149.9 4.8 9.7 36.3 47.4 156.7 6.1 8.3 14.0 26.2 71.4 0.3 1.1 2.2 6.6 11.2 294.0 483.0 855.0 1127.1 2770.5
16.6 30.2 58.0 170.5 291.1 5.9 14.4 31.7 67.8 137.5 2.4 5.3 9.8 36.8 124.3 0.1 0.4 0.8 2.0 6.8 248.9 421.9 1050.1 1697.1 2646.3
18.2 31.3 43.4 62.7 96.1 3.5 8.9 23.1 113.4 208.6 2.0 4.9 7.4 17.4 26.3 0.2 1.0 3.0 11.3 15.8 221.7 339.8 659.5 1187.3 2089.0
11.5 25.8 50.8 92.4 114.1 4.7 19.6 35.2 100.1 219.1 5.0 15.7 19.6 79.6 163.0 0.3 0.6 3.0 9.4 12.8 188.8 528.5 895.0 1935.5 2115.8
8.3 13.8 20.7 32.6 36.2 3.8 8.6 15.1 44.1 44.3 3.7 6.6 17.1 48.5 160.0 0.1 0.2 2.0 8.7 10.7 152.9 250.4 449.7 749.6 1477.1
−, less than LOD.
Japan are metabolized to the following four dialkylphosphates (DAPs), dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP),23 while about 75% of OPs registered by the U.S. Environmental Protection Agency are metabolized to the above DAPs.24 Thus, monitoring of the DAPs in urine is a reasonable approach to assess the level of net OP exposure in daily life. However, the historical comparison of the exposure levels, especially in relation to NEO exposure ones, has never been made. Therefore, the objective of the present work was to clarify changes in the exposure levels of NEOs and OPs in daily life by
One of the plausible reasons for the paucity of the data is the technical difficulty in evaluation of NEO exposure levels. Recently, we have developed the biomonitoring method of urinary NEOs,16 based on the fact that they are excreted in urine as unchanged compounds due to their high water solubility,17,18 and that the monitoring of the parent compounds better reflects individual exposure than their metabolites monitoring (unpublished data). In contrast, the exposure assessment method of OPs has been established as the metabolites monitoring, and the urinary metabolite levels have been investigated in Japan19,20 as well as in Europe21 and the U.S.22 About 70% of OP compounds registered by the Ministry of Agriculture, Forestry, and Fisheries, B
DOI: 10.1021/acs.est.5b03062 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 1. Detection rates of urinary NEOs in Japanese (A) and the amount of domestic shipments of NEOs in Japan (B) between 1994 and 2011. Inverted triangles represent years when each NEO came on the market in Japan. Panel A shows p for the Cochran-Armitage trend test, Pearson’s correlation coefficients (r) with p-values, and 95% confidence intervals of the regression lines.
University Graduate School of Medical Sciences approved the study protocol. Urinary NEOs and DAPs Analyses. Concentrations of NEOs and DAPs in urine were measured according to the methods reported previously.16,19 Briefly, one milliliter of urine was pipetted into a test tube containing 1 mL phosphate solution (2%) and 10 μL internal standard (I.S.) (5 mg/L cotinine-d3 for NIT and 10 mg/L acetamiprid-d6 for the other NEOs). The sample was applied to a solid phase extraction (SPE) procedure (Bond Elut PCX; Agilent Technologies, Santa Clara, CA). ACE, IMI, THD, THM, CLO and DIN were eluted with 0.5 mL methanol. NIT was eluted with 0.5 mL methanol and acetonitrile (1:1, v/v) containing 5% NH3. Each eluate was injected into liquid chromatography with tandem mass spectrometry (LC−MS/MS). The LC−MS/MS was composed of Agilent 1200 infinity LC coupled with an Agilent 6430 Triple Quadrupole LC/MS System (Agilent Technologies). In the measurement of four DAPs, i.e., DMP, DMTP, DEP, and DETP, 1 mL of urine was pipetted into a test tube, and 1 mL distilled water, 20 μL formic acid and 20 μL I.S. (30 mg/L DMP sodium salt-d6, 30 mg/L DMTP potassium salt-d6, 5 mg/L DEP ammonium salt-d10 and 5 mg/L DETP potassium salt-d10) were
quantification of NEOs and DAPs in urine samples collected from adult females between 1994 and 2011 in Japan.
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MATERIALS AND METHODS Study Subjects. Local residents in Kyoto and surrounding areas in Japan who attended any of the cross-sectional healthcare checkup programs conducted in 1994, 2000, 2003, 2009, and 2011 were asked to donate a spot urine specimen to the Kyoto University Human Specimen Bank.25 After being stored at −80 °C, the urine specimens analyzed in the present study (n = 17−20 different individuals in each year) were randomly sampled from the Bank using statistical software JMP Pro 11 (SAS Institute Inc., Cary, NC) on the basis that they were collected from women 45−75 (59.8 ± 8.3, mean ± SD) years old with no occupational histories related to pesticide exposure. Thus, the subjects were more likely exposed to pesticides from their diet and drinking water than directly from their surrounding environment. Specimens from males were not included because subjects of the same age range were not available during the period. The Ethics Committees of the Nagoya University Graduate School of Medicine, Kyoto University Graduate School of Medicine, and Nagoya City C
DOI: 10.1021/acs.est.5b03062 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology added. After gentle shaking, the test tube was incubated at 37 °C in a water bath for 10 min to decrease urinary turbidity. Then, the sample was loaded onto the SPE column (Oasis WAX SPE column; Waters Corporation, Milford, MA). Passed samples were collected and injected into LC−MS/MS for DMP and DEP measurement. DMTP and DETP were eluted from the SPE column with 1 mL mixture of NH3 solution (2.5%) and acetonitrile (1:1, v/v) after washing procedure with 1 mL acetonitrile. The eluate was injected into the LC−MS/MS for DMTP and DETP measurement. Two quality control urines spiked with different concentrations of the standard for each were used in the urinary NEO and DAP assays. These urine samples were collected from three healthy volunteers who had neither received medication nor had been occupationally exposed to OP or NEO beforehand, and were pooled for quality control purposes. Then, a standard NEO and DAP solution dissolved in acetonitrile was added and consequently, two final concentrations of quality control urine were prepared (1.25 and 5 μg/L for NEOs, 12.8 and 63.4 μg/L for DMP, 6.5 and 24.2 μg/L for DMTP, 1.6 and 5.7 μg/L for DEP, and 0.9 and 4.4 μg/L for DETP). Those quality control urines and blanks (distilled water) were analyzed every 20 samples. The limits of detection (LODs) were 0.03 μg/L for ACE, 0.1 μg/L for NIT and DETP, 0.2 μg/L for THM, 0.3 μg/L for IMI, DIN, THD and DEP, 0.4 μg/L for DMP and DMTP, and 1.1 μg/L for CLO. The LOD was defined as the peaks that gave a signal-to-noise ratio of 3. The limits of quantitation (LOQ) levels were defined as the lowest concentrations for which the signal-to-noise ratio was ≥10, and the precision, the percent of relative standard deviation (%RSD), was
