ARTICLE pubs.acs.org/est
Occurrence of Phthalate Metabolites in Human Urine from Several Asian Countries Ying Guo,† Husam Alomirah,‡ Hyeon-Seo Cho,§ Tu Binh Minh,^ Mustafa Ali Mohd,|| Haruhiko Nakata,# and Kurunthachalam Kannan*,†,z †
)
Wadsworth Center, New York State Department of Health, Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, New York 12201-0509, United States ‡ Biotechnology Department, Kuwait Institute for Scientific Research, P.O. Box 24885, 13109 Safat, Kuwait § College of Fisheries and Ocean Sciences, Chonnam National University, Yeosu 550-749, South Korea ^ Vietnam Environment Administration, 409 Kim Ma Street, Ba Dinh, Hanoi, Vietnam Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia # Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan z International Joint Research Center for Persistent Toxic Substances, State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
bS Supporting Information ABSTRACT: The occurrence of 14 phthalate metabolites was found in human urine samples collected from seven Asian countries: China, India, Japan, Korea, Kuwait, Malaysia, and Vietnam. Phthalate metabolites were found in all samples, indicating widespread exposure of humans to phthalates in these Asian countries. The highest total (the sum of 14 phthalates) phthalate metabolite concentrations were found in samples collected from Kuwait (median: 1050 ng/mL), followed in decreasing order by samples from India (389 ng/ mL), China (234 ng/mL), Vietnam (133 ng/mL), Japan (120 ng/mL), Korea (117 ng/mL), and Malaysia (94.9 ng/mL). The creatinine-adjusted median concentrations of total phthalates for urine samples from Kuwait, India, China, Vietnam, Japan, Korea, and Malaysia were 692, 506, 289, 119, 103, 104, and 169 μg/g creatinine, respectively. Monomethyl phthalate (mMP), monoethyl phthalate (mEP), mono (2-isobutyl phthalate) (miBP), mono-n-butyl phthalate (mBP), and metabolites of di-(2-ethylhexyl) phthalate (DEHP) were the dominant compounds, collectively accounting for >95% of the total concentrations in the samples from the seven countries. The profiles of urinary phthalate metabolite concentrations varied among the samples collected from the seven countries. Urine samples from Kuwait contained the highest concentrations of mEP (median: 391 ng/mL), mBP (94.1 ng/mL), and the metabolites of DEHP (202 ng/mL), whereas samples from China and Japan contained the highest concentrations of miBP (50.8 ng/mL) and mMP (17.5 ng/mL), respectively. mEP was the predominant metabolite in urine samples from India and Kuwait (accounting for 49% of the total), mBP and miBP were the predominant compounds in samples from China (52%), and DEHP metabolites were the predominant compounds in samples from Korea (46%) and Vietnam (52%). Based on the urinary concentrations of mEP, mBP, miBP, and DEHP metabolites of the samples from the seven Asian countries, we estimated daily intake rates of diethyl phthalate (DEP), dibutyl phthalate (DBP), and DEHP. The results indicated that people in the seven Asian countries are exposed to DEP, DBP, and DEHP at levels well below the reference doses (RfD) suggested as unsafe by the U.S. Environmental Protection Agency (EPA). The estimated exposure doses to DEHP in Kuwait, however, were above the RfD recommended by the EPA.
’ INTRODUCTION Phthalates are esters of phthalic acid and are used as plasticizers and solvents in industrial, medical, and consumer products. Globally, over 470 million pounds of phthalates are produced every year.1 The major (90%) use of phthalates is to impart flexibility and r 2011 American Chemical Society
Received: November 18, 2010 Accepted: February 21, 2011 Revised: January 28, 2011 Published: March 11, 2011 3138
dx.doi.org/10.1021/es103879m | Environ. Sci. Technol. 2011, 45, 3138–3144
Environmental Science & Technology malleability to several plastic materials, including polyvinylchloride (PVC). The annual production of PVC in Asia was over 16 million tons, accounting for 46.5% of the global production in 2007. Phthalates are also present in toys and several personal care products (e.g., cosmetics, detergents, soaps), pesticides, and food wrap.2 Human exposure to phthalates is a concern because studies have shown that these compounds can affect the reproductive system in laboratory animals.3-6 Several epidemiological studies have shown an association between high phthalate exposure and effects on the human endocrine or reproductive system,7-10 and on children’s intelligence and behavior.11,12 As a result, phthalates have been added to the list of “chemicals of concern” by the U.S. EPA.1 Urinary phthalate metabolites are used as biomarkers of recent human exposure to phthalates.2,13,14 Phthalates are rapidly metabolized to their respective monoesters in the human body, and some primary metabolites then can be further metabolized.15-17 The biological half-lives of parent compounds and identified metabolites of phthalates are in the order of hours. The National Health and Nutrition Examination Survey (NHANES) of the Centers for Disease Control and Prevention (CDC) in the U.S. has determined the presence of phthalate metabolites in urine samples from a representative U.S. population since 1994,18-20 with the data indicating widespread human exposure to phthalates. Phthalate metabolites also have been measured in urine samples collected between 1988 and 2003 in Germany; the results also suggest widespread exposure of the German population to phthalates.21 Similarly, studies have reported urinary phthalate metabolites in samples from Sweden,22 Mexico,23 The Netherlands,24 Japan,25,26 and Korea.27 Despite the large production and consumption of phthalates in Asian countries, very little is known about the exposure of the general population to phthalates in these countries.25-27 A survey of urinary phthalate levels in 25 Koreans showed a 212 fold reduction in phthalate metabolite concentrations following the practice of a vegetarian diet for 5 consecutive days.27 The results of a survey in Liaoning, China, showed high concentrations of mono-n-butyl phthalate (mBP) and mono (2ethylhexyl) phthalate (mEHP) in urine samples of occupationally exposed populations.28 Phthalate concentrations also have been reported in infertile women with endometriosis in Japan.25 In the present study, we measured 14 phthalate metabolites in 262 urine samples collected from the general populations of seven Asian countries, China (n = 40), India (n = 22), Japan (n = 35), Korea (n = 60), Kuwait (n = 46), Malaysia (n = 29), and Vietnam (n = 30), with the aims of investigating the geographic distribution of concentrations and profiles of phthalate exposures in Asia and establishing baseline levels needed for determining future trends in exposure among populations. ’ MATERIAL AND METHODS Sample Collection. Urine samples (2-10 mL) were collected from seven Asian countries, China (number of samples for females/males: 21/19), India (15/7), Japan (8/27), Kuwait (22/24), Malaysia (19/10), and Vietnam (16/14), from May to July 2010. Samples from Korea (n = 60, gender not available) were collected during 2006-2007 (Table S1 in the Supporting Information). The samples from China, India, Japan, Korea, Kuwait, Malaysia, and Vietnam originated from the cities of Guangzhou/Shanghai/Qiqihaer, Mettupalayam, Ehime/Kumamoto, Seoul/Busan/Yeosu, Al-Asma/Al-Jahra governorates, Kuala Lumpur, and Hanoi, respectively. Urine samples collected from Kuwait were from fasting subjects. The age of donors for the
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seven countries ranged from 21 to 49 years. Spot urine samples were collected in 15-mL polypropylene tubes from healthy volunteers. Institutional Review Board approvals were obtained from the New York State Department of Health (NYSDOH) for the analysis of urine samples. Sample Preparation and Analysis. Phthalate metabolites in urine samples (0.5 mL) were analyzed after enzymatic deconjugation, followed by solid phase extraction, as described in Silva et al.29 A total of 14 phthalate metabolites were analyzed: mono (3-carboxypropyl) phthalate (mCPP), monomethyl phthalate (mMP), monoethyl phthalate (mEP), mono (2-isobutyl phthalate) (miBP), mono-nbutyl phthalate (mBP), mono (2-ethyl-5-carboxypentyl) phthalate (mECPP), mono-[(2-carboxymethyl) hexyl] phthalate (mCMHP), mono (2-ethyl-5-oxohexyl) phthalate (mEOHP), mono (2-ethyl-5hydroxyhexyl) phthalate (mEHHP), monocyclohexyl phthalate (mCHP), monobenzyl phthalate (mBzP), mono (2-ethylhexyl) phthalate (mEHP), monoisonoyl phthalate (mNP), and monooctyl phthalate (mOP). For instrumental analysis, seven isotopically labeled phthalate metabolites (13C4-mMP, 13C4-mEP, 13C4-mBP, 13C4mECPP, 13C4-mEHP, 13C4-mBzP, and D4-miBP) and 13C4-4-methylumbelliferone were used as internal standards. An API 2000 electrospray triple quadrupole mass spectrometer (ESI-MS/MS; Applied Biosystems, Foster City, CA), equipped with an Agilent 1100 Series HPLC system (Agilent Technologies, Santa Clara, CA), was used for the analysis of phthalate metabolites and urinary creatinine concentrations. Chromatographic separation was achieved using a Betasil C18 column (Thermo Electron, Bellefonte, PA; 100 mm 2.1 mm, 5 μm). Target compounds were determined by multiple-reaction monitoring (MRM) in the negative ionization mode. The limits of quantification (LOQ) of phthalate metabolites varied from 0.1 to 0.5 ng/ mL. Detailed information regarding sample preparation and instrumental analysis is presented in the Supporting Information. Quality Assurance/Quality (QA/QC) Control and Data Analysis. For each batch of 25 samples, a method blank, a spiked blank, and a pair of matrix-spiked sample/duplicates were processed. The QA/QC results are shown in Table S2. Mean concentrations of 13C-labeled internal standards spiked into urine samples ranged from 73% to 104% for all target analytes, except for 13C-mMP, which showed a low recovery of 44%. However, the average recovery of mMP in matrix-spiked samples was 102%, when corrected for 13C-mMP recoveries, and, therefore, the values for mMP in the present study were considered acceptable. Trace levels of mBP, miBP, and mEHP (mean values of 0.8, 2.1, and 3.1 ng/mL, respectively) were detected in procedural blanks (water passed through the entire analytical procedure), and sample concentrations for these compounds were subtracted from blank values. The blanks were prepared in polypropylene tubes to reflect contamination that could arise from the use of such tubes during sampling. The sum concentrations of all 14 metabolites, and the sum of the five metabolites of di-(2-ethylhexyl) phthalate (DEHP) (mEHP, mECPP, mCMHP, mEHHP, and mEOHP), were defined as Σ14 phthalates and Σ5 mEHP, respectively. Concentrations below the LOQ were assigned a value of zero for data analysis. The reported concentrations were not creatinine adjusted unless indicated otherwise. Data analysis was conducted using SPSS, Version 17.0. Comparisons of Σ14 phthalate concentrations among countries were conducted using ANOVA (log transformed values were used, Duncan’s multiple range test). Comparison between individual compounds was examined using nonparametric statistical tests (Kruskal-Wallis H and Mann-Whitney U). Statistical significance was set at p < 0.05. 3139
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Figure 1. Concentrations of urinary phthalate metabolites (Σ14 phthalates) in samples from seven Asian countries: (a) ng/mL; (b) μg/g creatinine. The horizontal lines represent the 10th, 50th, and 90th percentiles, and the boxes represent the 25th and 75th percentiles. Outliers are shown as individual points.
’ RESULTS AND DISCUSSION Total Phthalate Concentrations. Phthalate metabolites were found in all urine samples from seven Asian countries, and concentrations of Σ14 phthalates ranged from 2.6 (Korea) to 19 300 ng/mL (Kuwait) (median: 185 ng/mL or 207 μg/g creatinine), indicating widespread human exposure to phthalates in Asian countries. Concentrations of Σ14 phthalates varied significantly among the seven Asian countries (p < 0.0001). The highest concentration of Σ14 phthalates was found in urine samples from Kuwait (median: 1050 ng/mL, Figure 1a and Table 1), and the mean concentration was ten times higher than the concentrations found for the samples collected from Japan, Korea, and Vietnam. Urine samples from India (389 ng/ mL) and China (234 ng/mL) contained the next highest concentrations of Σ14 phthalates. The concentrations of Σ14 phthalates in samples from Japan (120 ng/mL), Korea (117 ng/mL), Malaysia (94.9 ng/mL), and Vietnam (133 ng/mL) were lower than those found for Kuwait, India, and China. No significant difference in the urinary concentrations of Σ14 phthalates was found among the samples from Japan, Korea, Malaysia, and Vietnam. When the urinary concentrations were adjusted for creatinine levels, the distribution of Σ14 phthalates concentrations among the countries was similar to that found for unadjusted concentrations (Figure 1b), and the rank order was, Kuwait (median: 692 μg/g creatinine), India (506 μg/g), China (289 μg/g), Malaysia (169 μg/g), and the other three countries (∼100 μg/g). The details (range and mean) of metabolite concentrations are shown in Table S3. The concentrations of Σ14 phthalates in urine samples from Kuwait were the highest values ever reported, on a worldwide
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basis, for the general population (Table S4). Phthalate metabolite concentrations in urine from Kuwait were approximately 2- to 7-fold higher than the concentrations reported for the general populations from the U.S. in 2003-04 (median: 458 ng/ mL or 375 μg/g creatinine),19 Germany (median: 386 ng/mL or 327 μg/g creatinine),16 and The Netherlands (median: 272 ng/ mL or 448 μg/g creatinine),24 as well as in Swedish females (median: 150 ng/mL or 179 μg/g creatinine).22 Geographical Distribution of Phthalate Metabolite Profiles. Relationships between urinary concentrations of creatinine-adjusted and unadjusted (i.e., volume based) phthalate metabolites indicated that not all the correlations were significant (Table S5). A recent study by the CDC30 showed that the differences in urinary phthalate concentrations (based on volume) between term and preterm birth groups of infants were reduced after creatinine correction. Another study, which analyzed phthalate metabolite concentrations in repeat urine samples,31 also demonstrated that creatinine adjustment actually increased within-person variability and reduced the reproducibility of urinary concentrations of phthalate metabolites. Therefore, all the following discussions on phthalate concentrations were based on unadjusted (i.e., not creatinine corrected) values. The profiles of urinary phthalate metabolite concentrations varied among the seven Asian countries (Figure 2). We investigated the relationships between the concentrations of total or individual phthalate metabolites and age or gender of donors in the seven countries. No relationships were observed between gender, except for India, for which mMP concentrations were higher in females than in males (p < 0.03), and among different age groups, except for Kuwait, for which concentrations of mEP (p < 0.0001) and Σ14 phthalates (p < 0.03) were lower in children (age e10) than in other age groups. mMP. mMP was frequently (>95%) found in urine samples from Japan and India as well as in 33% of the samples from Malaysia and in approximately 50% of the samples from other countries. Concentrations of mMP were not different among samples from China (median: 7.1 ng/mL), India (5.8 ng/mL), and Japan (17.5 ng/mL) (p > 0.09) or among samples from Malaysia (not detectable [ND]), Korea (3.4 ng/mL), Kuwait (ND), and Vietnam (ND) (p > 0.4). mEP. Only two samples in the present study did not contain mEP. As shown in Figure 2, concentrations of mEP in urine samples from Kuwait were the highest among the seven countries studied (median: 391 ng/mL), in which concentrations in 11 samples exceeded 1000 ng/mL. Concentrations of mEP in urine from Kuwait were 2-fold higher than the values reported by the U.S. 2003-04 NHANES (208 ng/mL). Except for the samples from India, which also contained high mEP levels (131 ng/mL) (p < 0.0001), no differences in mEP concentrations were found among China (19.5 ng/mL), Korea (13.2 ng/mL), Japan (14.8 ng/mL), Malaysia (30.5 ng/mL), and Vietnam (5.1 ng/mL) (p > 0.1). mBP and miBP. mBP and miBP were found in more than 97% of the samples analyzed. Similar to mEP, the highest mBP concentrations were found in samples from Kuwait (94.1 ng/mL) (p < 0.0001), followed by China (59.7 ng/mL). No significant differences in mBP concentrations were found in urine samples from the other five Asian countries (range for median values: 13.420.1 ng/mL) (p > 0.1). Concentrations of miBP in samples from Kuwait (49.4 ng/mL) and China (50.8 ng/mL) were higher than in samples from India (17.6 ng/mL), Vietnam (12.9 ng/mL), Malaysia (9.3 ng/mL), Japan (8.4 ng/mL), and Korea (5.3 ng/mL). The concentrations of mBP or miBP in urine from Korea were much lower than those reported recently by Ji et al.27 Earlier studies from the U.S., Mexico, and Korea have shown that urinary concentrations 3140
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Table 1. Concentrations (ng/mL) of Urinary Phthalate Metabolites in Seven Asian Countries
China
median
(n = 40) GM
c
medianc GM India
median
(n = 22) GM median GM Japan
median
mEP
mCPP
mBP
7.1
19.5
1.2
59.7
50.8
15.7
14.2
7.1
4.9
16.5
20.7
1.3
49.6
44.0
15.1
14.2
7.1
4.8
9.7
17.0
0.9
69.5
52.2
11.5
13.5
7.1
4.7
16.4
22.5
1.5
53.6
47.6
16.3
15.3
7.7
5.2
nd a 0.5 nd 7.0 nd
nd 3.0 nd 2.3 nd
17.6
20.6
25.9
15.9
10.7
1.4
nd
nd
22.3
26.0
16.3
9.9
0.6
1.4
3.4
nd
nd
380
5.8 8.6
120 132
0.2 0.5
14.9 13.1
19.1 18.9
23.4 23.0
26.2 28.4
16.1 17.0
11.2 11.2
nd 0.6
nd 1.4
1.6 3.4
nd nd
nd nd
506 557
nd
nd
8.4
14.7
8.6
6.9
4.0
nd
13.1
8.6
6.9
3.9
nd
median
11.1
10.0
0.5
11.5
5.9
9.8
6.1
5.3
3.0
nd
GM
12.9
12.1
0.9
13.1
5.5
9.7
6.4
5.1
2.9
nd
1.4
Korea median (n = 60) GM
3.4 10.0
13.2 13.4
nd 1.4
19.2 16.7
5.3 4.5
17.4 16.8
11.4 12.6
6.4 5.6
3.7 3.9
nd 0.1
median
2.7
12.2
nd
19.8
5.1
17.8
12.8
7.0
4.9
nd
GM
9.1
13.7
17.0
4.6
17.1
12.9
5.7
3.9
GM
276
18.3
7.5
median
3.8
255 289
14.4
15.7
(n = 29) GM
nd
234
13.0
17.7
Malaysia median
1.9
2.6
0.1
0.8
GM
nd
nd
0.5
1.3
median
3.0
1.0
131
14.8
median
1.9
nd
150
16.4
(n = 46) GM
3.0
5.8
17.5
Kuwait
2.3
8.6
18.2
(n = 35) GM
1.2
nd
391
4.2
10.1
411
4.8
nd
296
2.7
272
3.1
6.2 nd 6.3 nd 5.8
94.1
49.4
67. 7
71.6
28.0
18.2
54.1
57.5
76.4
23.5
16.1
78.7
31.2
37.5
49.2
18.1
12.5
74.1
35.5
38.8
51.2
15.9
10.9
113
30.5
0.2
13.4
9.3
6.9
8.9
6.1
4.0
18.6
0.7
10.5
10.8
7.5
8.5
4.5
3.9
38.6
0.2
15.1
15.2
12.1
13.1
10.3
5.5
38.6
1.0
14.5
16.6
13.9
12.4
9.3
6.4
0.2 nd 0.4 nd 0.3 nd 1.2 nd 1.1
389
2.0
nd
2.6
nd
1.3
nd
1.7
nd
3.2 3.7
1.9 4.7
nd 0.6
nd nd
117 107
2.4
1.2
nd
nd
104
3.9
4.0
nd
109
3.1
6.2
nd
1050
5.6
6.9
2.1
4.1
3.2
4.6
2.5 nd
1.0
2.0
1.5
3.1
2.0
2.5
2.4
4.7
0.8 nd 2.1 nd 1.2 nd 0.6 nd 1.0
1.1 nd 0.6
4.6 nd 0.2 nd 1.8 nd 1.1
120 128 103 94.1
1030 692 683 94.9 86.1 169 179
Vietnam median
nd
5.1
0.9
20.1
12.9
27.5
17.7
6.8
5.2
nd
nd
2.7
nd
nd
133
(n = 30) GM median
8.4 nd
7.2 5.8
1.6 0.5
19.1 14.1
13.6 10.3
21.0 19.1
20.0 18.3
7.6 6.7
4.8 4.1
1.9 nd
1.7 nd
3.3 1.7
0.8 nd
1.6 nd
129 119
6.2
6.5
1.2
17.0
12.2
18.8
17.8
6.7
4.3
1.4
1.2
2.4
1.0
0.7
115
GM a
miBP mECPP mCMHP mEHHP mEOHP mCHP mBzP mEHP mOP mNP Σ14phthalates
mMP
nd = Not detectable. c GM = Geometric mean. c Bold italic: median creatinine corrected concentration (μg/g).
of mBP were 1 order of magnitude higher than the concentrations of miBP.19 However, urine samples from our study showed different patterns of mBP and miBP concentrations. Ratios of miBP to mBP (ratio = 1 means equal concentrations of the two compounds) varied among the seven countries (Figure 3). Similar to the results for the U. S., most urine samples from Korea, Japan, and Kuwait contained higher concentrations of mBP than miBP, whereas most samples from India contained lower concentrations of mBP than miBP. Urine samples from China, Vietnam, and Malaysia showed mixed patterns in the concentration of mBP and miBP. The ratios of miBP to mBP indicate different exposure routes or usage patterns of dibutyl phthalate (DBP) and di-iso-butyl phthalate (DiBP) among the seven countries. DEHP Metabolites. Five urinary metabolites of DEHP, namely, mECPP, mCMHP, mEOHP, mEHHP, and mEHP, were analyzed in the present study. mEHP was found in >77% of the samples, and the other four metabolites were found in >98% of the samples. The five DEHP metabolites were highly correlated with each other (p < 0.0001, Pearson correlation test). A previous study, which
analyzed eight metabolites of DEHP in human urine, indicated that the five metabolites analyzed in the present study accounted for 94% (87%, except for mEHP) of the total DEHP metabolite concentrations.32 The concentrations of Σ5 mEHP were the highest in urine samples from Kuwait (median: 202 ng/mL), followed by India (74.7 ng/mL), Vietnam (68.3 ng/mL), China (44.8 ng/mL), Korea (42.4 ng/mL), Japan (37.4 ng/mL), and Malaysia (32.4 ng/ mL). The concentrations of Σ5 mEHP in samples from India and Vietnam were similar to the concentrations reported for the U.S. population (median: 73.0 ng/mL, sum of mECPP, mEHHP, mEHP, and mEOHP). The relative concentrations of each of the five DEHP metabolites to Σ5 mEHP concentrations varied among the countries studied. In general, mECPP and mCMHP were the most concentrated compounds (∼30% each), followed by mEHHP (∼15%), mEOHP (∼10%), and mEHP, (∼5%) (Table S6). mBzP. mBzP was found in >80% of the samples from Malaysia and Korea, 65% of the samples from Kuwait, 33% of the samples from India, China, and Japan, and 23% of the samples from Vietnam. The concentrations of mBzP (median range: ND-3.2 ng/mL) in urine 3141
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Figure 2. Geographic distribution of concentrations (geometric mean, ng/mL) of mMP, mBP, miBP, mBzP, and Σ5 mEHP in urine samples from seven Asian countries and the U.S.19
Figure 4. Compositions of phthalate metabolites (to Σ14 phthalates) in urine samples from seven Asian countries. Others = sum of mCPP, mCHP, mOP, and mNP. Figure 3. Concentration ratios of miBP to mBP in urine samples from seven Asian countries.
samples from the seven Asian countries were 1 order of magnitude lower than the concentrations reported for the U.S. (median: 17.4 ng/ mL),19 Germany (median: 21 ng/mL),16 and Sweden (13 ng/mL).22 Studies have shown that benzyl butyl phthalate (BzBP) can be metabolized to both mBzP and mBP ( 0.02). Considering the low concentrations of mBzP and the lack of relationship between mBzP and mBP, human exposure to BzBP is not high, and BzBP is not the main source of urinary mBP in Asian countries. mCPP, mCHP, mOP, and mNP. The detection frequencies of mCPP, mCHP, mOP, and mNP in urine samples were 66%, 5%,
6%, and 3%, respectively, for all seven countries studied. Urinary concentrations of these compounds were low in all seven countries studied (Table S3). Composition of Phthalate Metabolites. The distribution of urinary phthalate metabolite concentrations (to Σ14 phthalates) was different among the samples from the seven countries (Figure 4). The sum of mMP, mEP, mBP, miBP, and five metabolites of DEHP accounted for more than 95% of the total urinary phthalate metabolite concentrations. In India and Kuwait, mEP was the dominant compound (49%), followed by the metabolites of DEHP (32% and 25% in India and Kuwait, respectively), similar to the findings for the U.S.18,19 miBP and mBP were the dominant phthalate metabolites in China (49%), but these two compounds accounted for only 10% of the total concentrations in India, and 20-30% of the total for the other Asian countries. The metabolites of DEHP were dominant in 3142
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Table 2. Estimated Human Daily Exposure to Phthalates in Seven Asian Countries (μg/day) reference dose
China
India
Japan
Korea
Malaysia
Kuwait
Vietnam
DEP
800a
16000b
285
1228
181
177
693
3900 (2)c
DBP
100
2000
580 (2)
178
140
110
124
822 (5)
173
20
400
182 (2)
339 (7)
102
97.7 (1)
435 (21)
112
DEHPd
92.5
64.0
a
Reference doses of U.S. EPA (μg/kg body weight/day). b Estimated reference doses from U.S. EPA (μg/day, the body weight assumed as 20 kg). c Number of samples exceeded the estimated reference doses. d Average values calculated from urinary mEHHP and mEOHP.
urine samples from Korea (46%) and Vietnam (52%), and these metabolites accounted for 25-35% of the total concentrations in the other Asian countries. mMP accounted for < 8% of the total concentrations in all countries, except for Japan, which contained mMP at 20% of the total concentrations. The profiles of urinary phthalate metabolites indicate that the Chinese population is exposed predominantly to DBP and DiBP, whereas Indians and Kuwaitis are frequently exposed to diethyl phthalate (DEP), and Koreans and Vietnamese are more exposed to DEHP and Malaysians to DEP and DEHP. Human Exposure to Phthalates. Based on the urinary concentrations of mEP, miBP, mBP, mEHHP, and mEOHP, as well as a simple steady-state exposure model, as shown in eq 1, human exposure levels of DEP, DBP, and DEHP were estimated for the seven Asian countries. M1 1 DI ¼ CV M2 f
ð1Þ
where DI is the total daily intake of phthalate (μg/day), C is the urinary phthalate metabolite concentration (μg/L), V is human daily excretion volume of urine (L/day), M1 and M2 are the respective molecular weights of parent phthalate and its metabolite (g/mol), and f is the molar fraction of the urinary monoester metabolite excreted in relation to the ingested amount of phthalate. For V, we assumed a volume of 2.0 L; for f, the values for mEP, mBP, mEHHP, and mEOHP were reported to be 0.69, 0.70, 0.23, and 0.15, respectively.26,34 Concentrations of mBP and miBP were summed to estimate exposures to the common parent compound, DBP. The estimated daily exposures to phthalates by the populations of the seven Asian countries are shown in Table 2. The estimated daily intakes are probably overestimated due to the high volume of 2.0 V, assumed for human daily excretion of urine; studies have shown that 24 h-average urine excretion volume of 0.66 and 1.7 L for children and adults, respectively.35,36 Nevertheless, the average body weight of 20 kg, used in the estimation of reference doses (RfD) for daily intakes (μg/day), as suggested by the U.S. EPA (800, 100, and 20 μg/kg bw/day for DEP, DBP, and DEHP, respectively), was underestimated for most of the samples in the present study [the average body weight for toddlers (1-3 yrs) in the U.S. is 19 kg37,38]. The estimated mean daily exposures to DEP and DBP in the seven Asian countries were well below (100-fold for DEP and 10-fold for DBP) the RfD values of the U.S. EPA. The estimated exposure doses to DEHP for populations in India and Kuwait were close to the RfD values of the U.S. EPA. The mean daily exposure dose estimated for DEHP by Kuwaitis was 435 μg/day, and 21 of the 46 samples exceeded the RfD of 400 μg/day. Although some of these values may be overestimated, our results suggest the need for identifying sources of exposure to DEHP in India and Kuwait. Our study has several limitations, as indicated above, and it should be considered as a pilot study aimed at elucidating the occurrence, profiles, and exposure doses of phthalates in Asian
countries. This study is the first to document the elevated levels of phthalates in several Asian countries and suggests the need for further studies with large sample size along with detailed demographic information.
’ ASSOCIATED CONTENT
bS
Supporting Information. The method of sample preparation and instrumental analysis, additional tables containing detailed information about samples, QA/QC results, phthalate metabolite concentrations reported in previous studies for several countries, and relative abundances of DEHP metabolites to Σ5 mEHP. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Tel: 1-518-474-0015; fax: 1-518-473-2895; e-mail: kkannan@ wadsworth.org.
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