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Urinary phthalate metabolites among elementary school children of Korea: Sources, risks, and their association with oxidative stress marker
Science of the Total Environment 472 (2014) 49–55
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Urinary phthalate metabolites among elementary school children of Korea: Sources, risks, and their association with oxidative stress marker Sunmi Kim a, Sungeun Kang a, Gowoon Lee a, Saeram Lee a, Areum Jo a, Kyunghee Kwak a, Dohyung Kim a, Dohyun Koh a, Young Lim Kho b, Sungkyoon Kim a, Kyungho Choi a,⁎ a b
Department of Environmental Health, School of Public Health, Seoul National University, Seoul, Republic of Korea School of Human and Environmental Sciences, Eulji University, Seongnam, Gyeonggi, Republic of Korea
H I G H L I G H T S • • • • •
Two first void urines were collected in 3 day interval from young teenagers. About 3–8% of the participating children showed potential risks by DEHP exposure. Phthalate metabolites showed significant positive association with urinary MDA. DEHP metabolites showed association with consumption of dairy products or meat. Use of plastic packaging and storage material was associated with DEHP or DBP.
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Article history: Received 20 July 2013 Received in revised form 30 October 2013 Accepted 30 October 2013 Available online 27 November 2013 Keywords: Phthalate Within-individual variability Daily intake DEHP Malondialdehyde
a b s t r a c t Phthalates have been used in a variety of consumer products and hence frequently been detected in humans. Children are susceptible to endocrine disrupting chemicals such as phthalates, but only limited information is available on the sources of exposure and potential adverse health effects among children. In this study, elementary school students (n = 39, aged 9–12 years) were recruited in Seoul, and first void urine samples were collected twice in three-day intervals. Then six phthalate metabolites were analyzed by high performance liquid chromatography with triple quadrupole tandem mass spectrometry. In addition, malondialdehyde (MDA) as an oxidative stress marker was measured. A questionnaire was conducted and information on food consumption and the use of plastic packaging or storage materials was gathered. The concentrations of phthalate metabolites varied substantially by sampling time even within the same subject, but all target metabolites were detected in 100% of the samples with the highest geometric mean of 107 μg/g-creatinine for mono-n-butyl phthalate (MnBP). Urinary levels of mono-isobutyl phthalate (MiBP), and MnBP among Korean children were 8 and 3 times greater than those reported for US children, but those of monoethyl phthalate (MEP) were about 5 times lower than those of US children. Estimated phthalate intakes were generally in safe range, but in 3–8% of the participating children, the hazard quotients greater than one were noted. Urinary MDA concentrations were significantly associated with several metabolite levels after adjusting covariates in regression model. Consumption of dairy products or meat, and use of a plastic material were significantly associated with the DEHP metabolites or MnBP levels in multivariate model. The results of this study provide evidence of the association between phthalate exposure and oxidative stress especially among the early teenagers, and identified major sources that can be applied to development of management plan for phthalate exposure among children. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Phthalates have been used in a variety of consumer products such as vinyl tiles, food processing materials, medical devices, plastic bags, toys, and personal care products. Because phthalates are not chemically bound to the products, they can be easily released into surrounding ⁎ Corresponding author at: School of Public Health, Seoul National University, Gwanak, Seoul 152-742, Republic of Korea. Tel.: +82 2 880 2738; fax: +82 2 745 9104. E-mail address: [email protected] (K. Choi). 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.10.118
environment such as air, water, or dust (CDC, 2009), and hence phthalates and their metabolites have been frequently detected among humans including children worldwide (Becker et al., 2009; Frederiksen et al., 2011; Hauser and Calafat, 2005; KFDA, 2007, 2011; Koch et al., 2011; Sioen et al., 2012; Song et al., 2013). Phthalate exposure has been associated with endocrine disruption and reproductive and developmental damages in many experimental studies (Andrade et al., 2006; Borch et al., 2004; Gray et al., 2000; Okubo et al., 2003; Zacharewski et al., 1998). In humans, phthalate exposure has been also related to sex hormone disturbance (Lovekamp-Swan and Davis,
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2003; Pan et al., 2006), decreased sperm quality (Duty et al., 2004; Hauser et al., 2007), and length of gestation (Adibi et al., 2009; Whyatt et al., 2009) although conflicting reports are also available (Duty et al., 2005a; Jonsson et al., 2005; Rais-Bahrami et al., 2004). The levels of exposure appeared to be generally higher among the children compared to the adult population. For example, levels of metabolites of di-n-butyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) were higher in younger population compared to the older group (Kasper-Sonnenberg et al., 2012; KFDA, 2007, 2011; Koch et al., 2004; Song et al., 2013). Because adverse effects of exposure to endocrine disrupting chemicals were considered to be also greater among the children, monitoring occurrence levels, identifying the sources, and determining potential risks of phthalate exposure among the children are important. Until now, several studies have reported the levels of phthalate exposure among the children, but only limited information is available on their sources or associated risks. Another important research gap in phthalate exposure among children is knowledge on temporal variability of exposure. Because of rapid urinary clearance, phthalate concentrations in urine are known to have significant intra-day and inter-day differences (Frederiksen et al., 2013; Fromme et al., 2007a; Preau et al., 2010). Most of the existing studies however employ spot urine samples, therefore such variation has been indicated as a cause of often conflicting epidemiological associations between phthalate exposure and health effects (Frederiksen et al., 2013). Among Korean children, only a few studies have determined the levels of several phthalate metabolites in urine (KFDA, 2007, 2011). The results of these investigations showed that the exposure pattern among Korean children is somewhat different from those reported in other countries. The concentrations of DEP metabolites in urine are generally much lower than those reported in US and other countries (KFDA, 2007), while those of DEHP and DBP metabolites are relatively higher (KFDA, 2007). In a separate study reported only DEHP metabolites among 392 children in Korea, the concentrations of DEHP metabolites were reported to be higher among children compared to those of older age group (Song et al., 2013). This study was conducted to identify the source of phthalates exposure and evaluate the association between phthalate exposure and oxidative stress among Korean children. To derive reliable exposure estimates of phthalates and oxidative stress marker, two separate first void urines were collected in three day interval from all the participating children. The results of this study will help identify phthalates of concern and their major sources among the elementary school students in Korea, which will benefit developing management options for this important group of chemicals. 2. Materials and methods 2.1. Study population, questionnaire and sample collection Children (n = 39) were recruited from three elementary schools located in Seoul, Korea in July, 2011. General characteristics of study population are summarized in Table 1. The participants were between 9 and 12 years old, and were between 4th and 6th grades of elementary school. Participating children and their parents were asked to record the type and amount of food items the children consumed, and the information regarding the use of plastic packaging and storages at Days 1 (Monday, July 4, 2011) and 4 (Thursday), in dietary questionnaire. Dietary questionnaire included pictorial guide to help participants correctly recall the type and size of the food items they consumed (for details about dietary questionnaire, see Supplementary information). In addition, demographic and socioeconomic characteristics of the family were also asked. At Days 2 and 5, first morning void urine samples were collected at home in polypropylene tube, and transferred on ice to the laboratory. The urine samples were separated into 1.7 mL microcentrifuge tubes, and stored at − 80 °C until analysis. The
institutional review board of School of Public Health, Seoul National University approved this study, and informed consent was obtained from legal guardian of the participating child.
2.2. Chemical analysis Six primary or secondary metabolites of four most frequently used phthalates, i.e., diisobutyl phthalate (DiBP), DBP, DEHP, and diethyl phthalate (DEP) were chosen (Calafat and McKee, 2006; Wittassek et al., 2007). The metabolites include mono-isobutyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), mono(2-ethyl-hexyl) phthalate (MEHP), mono(2-ethyl-5-oxo-hexyl) phthalate (5-oxo-MEHP), mono (2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), and monoethyl phthalate (MEP). Six metabolites and isotope-labeled internal standards for phthalates (13C4-monobutyl phthalate, 13C4-MEHP, 13C4-5oxo-MEHP, 13C4-5-OH-MEHP, and 13C4-MEP) were purchased from Cambridge Isotope Laboratory (Andover, MA, USA). The analyses of phthalate metabolites in urine were performed on a high performance liquid chromatography (NanospaceSI-2, Shiseido, Tokyo, Japan) with triple quadrupole tandem mass spectrometry (LC/ MS/MS; API4000, Applied Biosystems, Foster City, CA, USA) as detailed in Kho et al. (2008). After enzymatic deconjugation of urine samples by treatment with β-galactosidase (from Escherichia coli K12), on-line clean-up and separation of phthalate metabolites were accomplished using the switching-column technique with a pretreatment column (Shiseido MFC 8, 50 ∗ 4.6 mm, 5 μm), trap column for concentrate (Imtackt Cadenza CD-C18, 30 ∗ 2.0 mm, 5 μm), and analytical column for peak separation (Imtackt Cadenza CD-C18, 75 ∗ 2.0 mm, 3 μm). Limit of quantification was determined at 0.5 μg/L for MiBP, 1.4 μg/L for MnBP, 0.6 μg/L for MEHP, 0.9 μg/L for 5-oxo-MEHP, 1.2 μg/L for 5-OH-MEHP, and 1.9 μg/L for MEP. For each instrumental run, reagent blank and solvent blank were included for quality assurance. About 10% of the samples were analyzed in duplicates in order to ensure reproducibility of the measurement. Coefficients of variation of all duplicate samples were within 20%. Accuracy was considered acceptable when deviations from the spike value were within 20%. Precision was considered acceptable when intra- and inter-day coefficients of variation were within 20%. As shown in Tables S1 and S2 of Supplementary information, accuracy and precision of analysis for each compound were generally within acceptable range. Urinary creatinine concentration was analyzed using enzymatic method by Samkwang Medical Laboratories (Seoul, Korea).
2.3. Daily phthalate intake and hazard quotient Based on the urinary phthalate metabolite concentrations, daily intake (DI) of parent phthalates was estimated using the following equation (Kohn et al., 2000): DIðμg=kg bw‐dayÞ ¼ ððUE CE=1000Þ=FueÞ ðMWp=MWmÞ
where UE is the urinary concentration of a given metabolite adjusted for creatinine (μg/g creatinine), CE is daily creatinine excretion rate normalized by body weight (mg/kg-day), Fue is a fraction of urinary excretion relative to total elimination. MWp and MWm are the molecular weights of parent phthalate and metabolite, respectively. Fue for MEP was 0.69 (Calafat and McKee, 2006), for MnBP and MiBP, 0.69 (Koch et al., 2004), and for MEHP, 5-OH-MEHP, and 5-oxo-MEHP, 0.059, 0.23, and 0.15, respectively (Fromme et al., 2007b). Hazard quotient (HQ) was defined as the estimated DI divided by reference dose (RfD). RfD values were 100, 20, and 800 μg/kg bw-day for DBP (DiBP), DEHP, and DEP, respectively (US EPA). HQ greater than unity was deemed to be of potential risk.
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Table 1 Average urinary concentrations of phthalates metabolites in children of age between 9 and 12 in Seoul, Korea. Variable (unit)
N (%)
(mean ± SD) All Gender Female Male BMI (kg/m2) (17.3 ± 2.68) Underweight (b18.5) Normal (18.5–22.9) Overweight (23.0–24.9) Parent education High school diploma Bachelor's/postgraduate degree Family monthly income (USD) b1,000 1–3000 3–6000 N6000 Parents smoking status Nonsmoker Past smoker Current smoker
Median (interquartile range) (μg/g creatinine) MiBP
MnBP
MEHP
5-OH-MEHP
5-oxo-MEHP
MEP
39 (100)
55.4 (34.7–71.6)
103 (85.4–142)
13.2 (9.3–17.6)
79.1 (63.9–101.5)
46.3 (34.4–62.6)
15.6 (11.5–26.2)
17 (44) 22 (56)
57.6 (30.5–70.3) 51.9 (36.4–71.8)
124 (85.8–167) 103 (85.0–128)
13.3 (10.0–17.4) 12.8 (8.6–18.1)
87.4 (72.3–110.6) 70.1 (57.5–94.9)
48.9 (40.8–68.8) 42.5 (33.1–62.6)
17.5 (12.6–33.3) 12.8 (10.3–20.2)
27 (69) 11 (28) 1 (2)
63.6 (31.2–93.8) 51.4 (42.8–64.1) 48.4
113 (90.1–160) 91.9 (75.5–104) 100
13.3 (10.1–18.0) 12.0 (8.0–15.9) 11.6
91.1 (75.5–104) 64.5 (50.3–76.0) 79.0
49.7 (41.5–71.0) 34.5 (28.6–47.0) 36.5
17.5 (11.9–32.0) 12.6 (10.9–16.2) 7.7
14 (36) 25 (64)
64.4 (30.5–96.2) 48.4 (36.4–71.5)
136 (85.0–173) 101 (85.8–111)
12.9 (10.4–13.7) 13.2 (8.0–17.8)
85.6 (66.2–154) 79.0 (62.0–96.5)
46.0 (35.1–89.9) 46.3 (32.6–62.6)
16.2 (7.8–22.9) 15.6 (11.7–33.3)
1 (2) 15 (39) 21 (54) 2 (5)
70.5 38.8 (26.0–83.2) 55.4 (46.7–71.8) 52.2 (33.0–71.5)
135 103 (78.7–159) 105 (85.0–136) 93.1 (88.0–98.3)
12.5 13.3 (11.0–15.7) 11.8 (8.5–18.1) 10.5 (6.6–14.5)
57.5 83.8 (73.5–98.2) 79.0 (64.5–114) 55.8 (49.6–62.0)
34.5 49.7 (42.7–59.1) 44.2 (32.6–73.1) 33.1 (24.2–42.1)
15.3 17.5 (11.0–25.4) 15.0 (11.5–33.3) 14.0 (12.4–15.6)
18 (46) 10 (26) 11 (28)
44.1 (30.5–65.7) 56.9 (48.4–65.3) 84.0 (33.0–108)
95.2 (71.8–111) 109 (100–173) 131 (88.0–152)
10.9 (7.4–15.5) 15.8 (11.6–20.0) 13.4 (10.4–14.5)
72.1 (62.0–96.5) 108 (79.0–154) 79.0 (49.6–91.1)
44.2 (32.6–55.5) 67.9 (42.8–89.9) 43.4 (24.7–49.7)
16.9 (11.7–36.8) 13.8 (7.8–17.5) 15.2 (11.5–29.5)
Mean age of target population is 11.1 ± 0.88. The measurements of two urine samples collected from the same subject in 3 day interval were averaged. There were no significant differences in urinary phthalate metabolite concentrations by each of demographic factors listed above (p b 0.05 based on non-parametric tests of Kruskal–Wallis test or Wilcoxon Mann–Whitney test).
2.4. Measurement of oxidative stress Urinary MDA levels were measured as thiobarbituric acid reactive substances (TBARS) using OxiSelect™ TBARS Assay kit (Cell Biolabs, Inc., San Diego, CA, USA) following manufacturer's instructions. A spectrophotometric plate reader (TECAN infinite® M200, TECAN Group LTD., Mannedorf, Switzerland) was used for this purpose. The protein concentration was quantified using Bradford method (Bradford, 1976) and was used for normalization of MDA level. Each experiment was carried out in duplicates and average values were used. 2.5. Statistical analysis Multiple linear regression model was employed to identify potential sources of phthalate exposure. The association between phthalate exposure and oxidative stress was investigated using multiple linear regression model. Age, sex, body mass index (BMI), family income, and secondhand smoking were included as covariates in the models. For every multivariate model, phthalate metabolite concentrations and MDA levels as dependent variables were natural log-transformed. Normal distribution of residuals was confirmed by residual graphs. Because of small sample size, additional multiple regression models were conducted excluding one or two outliers to confirm the observed association. Outliers were identified by maximum value among potential outliers which showed the absolute value of modified Z-score exceeding 3.5. Statistical analyses were performed using Statistical Analysis System (SAS 9.13, Cary, NC, USA).
their short elimination times, positive relationships between two urine samples collected in 3-day interval may reflect similar exposure patterns e.g., foods, containers, and other related sources among the participating children. Hoppin et al. (2002) reported similar levels of phthalate metabolites in two consecutive morning void samples and suggested sufficiently consistent exposure pattern among the subjects (African American women, n = 46, aged 35–49 years). Teitelbaum et al. (2008) also reported that the concentrations of major phthalate metabolites detected in several spot urines of 35 healthy Hispanic and African American children over 6 months were comparable (Teitelbaum et al., 2008). Since exposure to phthalate can be influenced by many sources which can vary day-to-day, however significant variations were often reported (Preau et al., 2010). Such variation is expected to be greater among children (Teitelbaum et al., 2008). In our study, average intra-individual CVs were generally lesser than or comparable to those reported previously. The maximum CV in the present study was detected from creatinine-corrected MEP (132%), which was slightly lower than Preau et al. (2010) that reported the maximum CV of 157% over 1 week period among adults. This observation indicates that the variation in phthalate exposure among Korean children is relatively less except for DEP. Use of or exposure to personal care products can significantly influence the level of DEP exposure (Koch et al., 2013), and such use can vary especially among children, therefore multiple day sampling would be more appropriate for phthalates like DEP metabolites. For further evaluation, the concentrations of the creatininecorrected metabolites of the two first void samples were averaged for description of the level of exposure unless specifically noted as the 1st or the 2nd urine samples.
3. Results and discussion 3.2. Concentrations of phthalate metabolites in urine samples 3.1. Within-person variability of phthalate metabolites Strong positive associations were observed for the concentrations of all metabolites between two first void urine samples (Table S3 and Fig. S1 of Supplementary information). While the concentrations of three metabolites, i.e., MiBP, MnBP, and MEHP, showed significant difference between the first and second urine samples (Wilcoxon signed rank sum test, p b 0.05, Table S3), coefficients of variation (CV) determined between the levels of two urine samples are 20–24%. Considering
All phthalate metabolites were detected in 100% of the urine samples (Table 1). The geometric mean was the greatest for MnBP (107 μg/g creatinine), and the lowest for MEHP (12.6 μg/g creatinine). The concentrations of DEHP metabolites were in descending order 5-OH-MEHP N 5-oxo-MEHP N MEHP (Table 2). No significant relationships were detected between the measured metabolite concentrations and demographic characteristics of the study population, such as sex or BMI (Table 1).
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Table 2 Concentrations of major metabolites of DBP, DEHP, and DEP in urines of children: Comparison with previous results (μg/g creatinine). This studya
Study sample size (N) Phthalate
Metabolites
DiBP DBP DEHP
MiBP MnBP MEHP 5-OH-MEHP 5-oxo-MEHP MEP
DEP
Koreab
Koreac
USAd
Germanye
1st urine
2nd urine
Average
39
35
39
60
199
342
599
47.7 (48.6–103) 96.2 (95.7–184) 11.4 (11.2–33.3) 81.9 (72.0–236) 47.3 (43.2–139) 17.6 (14.3–140)
58.3 (56.3–188) 117 (109–236) 13.0 (13.9–25.6) 82.6 (80.0–155) 47.3 (45.5–102) 19.0 (15.0–151)
53.4 (55.4–135) 107 (103–208) 12.6 (13.2–24.0) 84.4 (79.1–204) 48.6 (46.3–125) 19.2 (15.6–160)
56.8 (54.7–309) 140 (95.7–5233) 17.3 (16.6–173) 80.2 (73.2–793) 81.0 (72.0–759) 26.0 (23.5–178)
49.0 (51.5–133) 61.3 (58.4–153) 7.57 (9.14–29.2) 49.9 (49.8–129.6) 29.5 (31.5–77.9) 9.74 (10.7–90.8)
6.94 (7.03–28.7) 38.4 (39.0–137) 3.00 (2.80–28.7) 39.0 (36.6–211) 26.6 (25.3–121) 101 (87.0–719)
94.3 (88.1–308) 95.6 (93.4–310) 6.4 (6.7–25.1) 47.9 (46.0–164) 37.0 (36.3–123) NA
Geometric mean and 50th–95th percentile values are presented. NA: Not available. a Age 9–12 years old. b Age 8–13 years old, values in parentheses are 50th centile-maximum values (KFDA, 2007). c Age 10–12 years old (KFDA, 2011). d Age 6–11 years old (CDC, 2009). e Age 3–14 years old (Becker et al., 2009).
The concentrations of most phthalate metabolites in urines of the Korean children are greater than those reported from USA (n = 342, 6–11 years of age; CDC, 2009) or from Germany (n = 599, 3–14 years of age; Becker et al., 2009) (Table 2). The only exception was MEP, a metabolite of DEP, which was detected on average 19.2 μg/g creatinine. MEP was detected on average at 101 μg/g creatinine among US children. The reason for much lower MEP concentrations in Korean children's urines can be found from the differences in use of cosmetics and personal care products either by age or by country. DEP has been used as a solvent in many personal care products, particularly in those containing fragrances, e.g., perfumes, colognes, deodorants, soaps, shampoos, and hand lotions. Associations between cosmetic use and urinary MEP concentrations have been reported elsewhere (Duty et al., 2005b; Janjua et al., 2008). Higher levels of MEP were reported in urine samples of female children than in those of male children in previous studies of Korea and USA (KFDA, 2007; Teitelbaum et al., 2012). US children are expected to use more cosmetics and personal care products compared to Korean children. The use of cosmetics or personal care products among elementary school students is less common in Korea. The levels of phthalate metabolites measured in the present study were in a comparable range to those reported previously for Korean children (n = 60, KFDA, 2007; n = 199, KFDA, 2011; n = 392, Song et al., 2013). Occurrence pattern of the phthalate metabolites was the same, suggesting similar source profiles of the phthalates among Korean children. Levels of phthalate exposure become generally lower as age increases. Similar decreasing trends have been reported in several other studies particularly for metabolites of DBP and DEHP (KasperSonnenberg et al., 2012; KFDA, 2007, 2011; Koch et al., 2004). Greater food consumption rates relative to their body weight, and characteristic mouthing behavior are suggested as part of the reasons for greater phthalate exposure among younger children (Wittassek et al., 2007). However, age could not explain the differences that were observed in levels of urinary phthalate metabolites among Korean children and USA or German children. Participating Korean children (9–12 years old) showed up to two fold higher levels of DEHP metabolites than those of German children (3–14 years old), and up to 7.7 fold higher levels of DiBP metabolites than those of US children (6–11 years old). This observation strongly indicates presence of additional sources or different source profiles of phthalate exposure among Korean children compared to those in Europe or North America.
bw-day for DEP. Intake estimates of DEHP ranged from 2.7 to 21.7, 3.6 to 41.0, or 2.9 to 35.8 μg/kg bw-day, based on MEHP, 5-OH-MEHP, or 5-oxo-MEHP concentrations, respectively. None of the HQs calculated for phthalates exceeded 1 except DEHP (Fig. 1). One to three participants (3–8%) were identified to show HQs greater than 1, suggesting that DEHP exposure should be considered of concern among certain Korean children. Similar observations were reported among the German children. Among German children (n = 239, 2–14 years old), up to 7.5% of children showed DEHP exposure estimates that exceeded an RfD of 20 μg/kg bw-day (Wittassek et al., 2007). 3.4. Association with oxidative stress biomarker in urine Significant positive associations between urinary MDA concentrations, which reflect lipid peroxidation levels, and several phthalate metabolites were observed after adjusting age, sex, BMI, family income, and secondhand smoking, especially in the 2nd urine samples (Table 3). The only exception was 5-oxo-MEHP, which showed marginal significance (p = 0.061 for β estimates in multiple regression analysis). Although positive trend was maintained, however, with the 1st urine samples, no metabolites showed significant associations with MDA. When urinary MDAs and phthalates levels of the 1st and 2nd samples were averaged, however, significant positive associations between
3.3. Estimated daily intake and associated risks Estimated DIs of parent phthalates were calculated following Kohn et al. (2000) using the creatinine-corrected metabolite concentrations in urine of 39 Korean children. The median DI was 2.0 (range 0.7–6.1) for DiBP, 4.2 (range 1.4–9.4) for DnBP, and 0.5 (range 0.1–13.4) μg/kg
Fig. 1. Estimated hazard quotient from measured concentration of phthalate metabolites in urine samples of 39 children in Korea. The average metabolite concentrations of two first void urine samples were used. Solid line and box denote median and 25–75th percentile values (interquartile range), respectively. Range marked indicates 10th and 90th percentiles, and black circles denote outliers.
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Table 3 Relationships between malondialdehyde (μg/g creatinine) and creatinine-adjusted phthalate metabolites (μg/g creatinine) in urine samples. Phthalate metabolites
MiBP MnBP MEHP 5-OH-MEHP 5-Oxo-MEHP MEP
1st urine (n = 39)
2nd urine (n = 35)
Average (n = 39)
β (95% Cl)
p
β (95% Cl)
p
β (95% Cl)
p
0.002 (−0.002, 0.005) 0.002 (−0.000, 0.004) 0.006 (−0.005, 0.017) 0.001 (−0.000, 0.002) 0.001 (−0.001, 0.004) 0.000 (−0.001, 0.001)
0.322 0.087 0.248 0.339 0.247 0.613
0.004 (0.001, 0.006) 0.003 (0.001, 0.004) 0.020 (0.006, 0.354) 0.003 (0.000, 0.005) 0.004 (0.000, 0.008) 0.002 (0.000, 0.004)
0.005 * 0.002 * 0.009 * 0.025 * 0.061 0.027 *
0.003 (0.000, 0.005) 0.003 (0.001, 0.004) 0.012 (0.001, 0.023) 0.001 (−0.000, 0.003) 0.002 (−0.001, 0.004) 0.000 (−0.001, 0.002)
0.022 * 0.007 * 0.028 * 0.081 0.175 0.346
Results were presented in multivariate model adjusted for age, sex, BMI of a child, family income, and smoking status of parents. Asterisk (*) denotes statistical significance (p b 0.05). Dependent variable (MDA) was used as natural-log transformed. CI: Confidence interval.
MDA and phthalates, e.g., MiBP, MnBP, or MEHP were observed. The reason for different statistical significances in the 1st urine samples is not clear, but may be found from the variability of the oxidative stress biomarkers (Arguelles et al., 2007; Browne et al., 2008). Oxidative stress markers such as MDA can be influenced by numerous factors including physical, chemical, and biological parameters, hence certain extent of within-individual variation is inevitable. For example, urinary 8-oxo7,8-dihydro-2′-deoxyguanosine (8-oxodG), another widely used biomarker of oxidative stress, showed a within-individual variation of 20% CV in the first morning void samples collected from 26 healthy nonsmoking adults (Barregard et al., 2013). While quantitative explanation is not possible, day-of-the-week variation of stress biomarkers may also contribute to the observation of the present study, e.g., the 1st set of samples were collected on Tuesday (Day 2) morning following weekend vs. the 2nd set of samples were collected on Friday (Day 5). Therefore in order to measure the status of oxidative stress, several measurements are suggested to be necessary (Arguelles et al., 2007). For this reason, it appeared to be more appropriate, in the present study, to average MDA levels of both Tuesday and Friday urines before relating them with phthalate exposure. While it is still possible that the observed associations were due to by chance, consistently positive association between phthalate metabolites and MDA levels suggests that several phthalates might be associated with oxidative stress in children. Phthalates are known as one of the important contributors for oxidative stress in both experimental and epidemiological studies (Ambruosi et al., 2011; Santhosh et al., 1998; Xu et al., 2013; Hong et al., 2009). Recent study on 1999–2006 NHANES data (N = 10,026) reported that phthalates such as DBP and DEHP were associated with oxidative stress (Ferguson et al., 2012). Among adult population who participated in 5 day Temple-stay
program in Korea, both phthalate metabolites and MDA concentrations in urines decreased after the Temple-stay, suggesting an association between phthalates and oxidative stress (Ji et al., 2010). 3.5. Potential sources of exposure Consumption of dairy products or meat and the use of plastic packaging or storing were identified to be significantly associated with urinary DEHP metabolites (Table 4). The use of plastics and bread consumption were associated with urinary MnBP concentrations but the association was not consistent. Eggs and oil product consumption showed significant negative correlation with several phthalate metabolites. However, additional regression analysis after removing one or two extreme outliers of phthalate metabolites, significance was disappeared. Based on β values derived from multiple regression analysis of DEHP metabolites, the influence of plastic packaging on DEHP exposure appeared to be greater than that of plastic storage. Plastic packaging has been suggested as source of phthalate contamination in foodstuffs (Wormuth et al., 2006), and cooked foods packed in plastic containers were expected to be highly contaminated (Cirillo et al., 2013). Several studies showed phthalates, particularly DBP and DEHP, have been detected in foodstuffs packed in plasticizer-used materials (Casajuana and Lacorte, 2004; Freire et al., 2006; Petersen and Breindahl, 2000). For general population, diets have been considered as a primary source of phthalate exposure (Schettler, 2006). One recent study also showed that DiBP, DnBP, and DEHP were detected in milk and dairy food samples from several stages in the milk chain (Fierens et al., 2013). In the present study, consumptions of dairy products or meat were significantly associated with DEHP exposure. Ji et al. (2010)
Table 4 Relationships between urinary phthalate metabolites (μg/g creatinine) and food intake or material use for food package and storage of children in Korea. Phthalate metabolites
1st urine (n = 39)
MiBP MnBP
– Plastic packaging Plastic storage Dairy Plastic packaging
MEHP
5-OH-MEHP
5-Oxo-MEHP
MEP
Food intake or material use
Dairy Meat Plastic packaging Plastic storage Dairy Meat Plastic packaging Plastic storage –
2nd urine (n = 35) β (95% Cl)
0.052 (0.009, 0095) 0.094 (0.033, 0.156) 0.001 (0.000, 0.002) 0.138 (0.012, 0.263) 0.001 (0.000, 0.001) 0.002 (0.001, 0.004) 0.182 (0.068, 0.295) 0.156 (0.036, 0.276) 0.001 (0.000, 0.001) 0.002 (0.000, 0.004) 0.162 (0.042, 0.170) 0.139 (0.013, 0.264)
Food intake or material use – Bread Oil†
Average (n = 39) β (95% Cl)
0.194 (0.051, 0.337) −1.062 (−1.702,−0.422)
Dairy Egg†
0.001 (0.000, 0.002) −0.268 (−0.485,−0.051)
Dairy Egg†
0.001 (0.000, 0.002) −0.269 (−0.491,−0.047)
Plastic storage
0.374 (0.004, 0.744)
Food intake or material use
β (95% Cl)
Oil† –
−2.016 (−3.711,−0.321)
Dairy Plastic packaging Plastic storage Dairy Plastic packaging Plastic storage Dairy Meat Plastic packaging Plastic storage Noodle†
0.001 (0.000, 0.002) 0.172 (0.027, 0.316) 0.133 (0.005, 0.262) 0.001 (0.000, 0.001) 0.199 (0.061, 0.337) 0.168 (0.047, 0.289) 0.001 (0.000, 0.001) 0.002 (0.001, 0.004) 0.203 (0.060, 0.346) 0.165 (0.039, 0.291) −1.284 (−2.471,−0.088)
Only significant results (p b 0.05) based on multivariate model adjusted for age, sex, BMI, family income, and smoking status of parents were presented. Dependent variables (phthalate metabolites) were used as natural-log transformed. Because of small sample size, additional regression model analysis was conducted after excluding extreme outliers. Without outliers, significance was disappeared. Relevant variables were marked by †. CI: Confidence interval.
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reported that urinary 5-OH-MEHP and 5-oxo-MEHP level exhibited significant positive correlations with consumption of dairy products. Meat consumption was pointed out as a significant source of DEHP exposure as well (Fromme et al., 2007b). Dietary sources are suspected for DnBP and DiBP as well. For example, bread has been suggested as one of the top contributors to DnBP and DiBP exposure among preschool children and adults in Belgium (Sioen et al., 2012). In the present study, bread consumption showed positive relationship with MnBP levels in the 2nd urine samples. Because staple foods of Korean are mainly originated from rice, bread consumption pattern among Korean however is obviously different with that of European people. Average bread including breakfast cereal consumption of Belgian population was 175–420 g/day (15–18 years: 146 ± 59 g/day) in 2004 (Vandevijvere et al., 2009), while the bread and cereal consumption rate of Korean people is on average 26 g/day for 70 kg person (0.37 ± 1.19 g/kg bw-day, (Jang et al., 2007) and 31 g/day for preschool children (Lee et al., 1998). The difference in the consumption rate may partly explain weaker influence of bread consumption on phthalate exposure among Korean children. Our finding is consistent with several previous reports that identified food as one of exposure sources of DEHP (Colacino et al., 2010; Fromme et al., 2007b; Petersen and Breindahl, 2000). The β values obtained from the regression models indicate that the contribution of plastic material was stronger than that of the food: One additional serving of dairy or meat product per month related to an average 0.002 μg 5-oxo-MEHP/g creatinine increase, while one additional use of plastic material per month related to an average increase of 0.225 μg 5-oxoMEHP/g creatinine. Our study suggests that the levels of certain phthalates especially DEHP are of potential concern among Korean children, and these exposures are potentially related to oxidative stress. Plastic food packaging and the use of plastic storage along with consumption of dairy products or meat were identified as important contributors to phthalate exposure among children. Use of plastic material as packaging or storage showed relatively greater contribution to the exposure of phthalate compared to food consumption. While these observations may require confirmation in greater sized population studies, the results of our study will help develop management plans for phthalate exposure among Korean children. Conflict of interests The authors declare no actual or potential competing interests. Acknowledgment This research was supported by the National Research Foundation of Korea (Project 2012R1A2A2A01015236). S.M. Kim is supported by BK 21 Plus program of National Research Council. This study is in part supported by School of Public Health, Seoul National University. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2013.10.118. References Adibi JJ, Hauser R, Williams PL, Whyatt RM, Calafat AM, Nelson H, et al. Maternal urinary metabolites of di-(2-ethylhexyl) phthalate in relation to the timing of labor in a US multicenter pregnancy cohort study. Am J Epidemiol 2009;169:1015–24. Ambruosi B, Filioli Uranio M, Sardanelli AM, Pocar P, Martino NA, Paternoster MS, et al. In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model. PLoS One 2011;6(11):e27452. Andrade AJ, Grande SW, Talsness CE, Gericke C, Grote K, Golombiewski A, et al. A dose response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): reproductive effects on adult male offspring rats. Toxicology 2006;228: 85–97.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Urinary phthalate metabolites among elementary school children of Korea: Sources, risks, and their association with oxidative stress marker Sunmi Kim a, Sungeun Kang a, Gowoon Lee a, Saeram Lee a, Areum Jo a, Kyunghee Kwak a, Dohyung Kim a, Dohyun Koh a, Young Lim Kho b, Sungkyoon Kim a, Kyungho Choi a,⁎ a b
Department of Environmental Health, School of Public Health, Seoul National University, Seoul, Republic of Korea School of Human and Environmental Sciences, Eulji University, Seongnam, Gyeonggi, Republic of Korea
H I G H L I G H T S • • • • •
Two first void urines were collected in 3 day interval from young teenagers. About 3–8% of the participating children showed potential risks by DEHP exposure. Phthalate metabolites showed significant positive association with urinary MDA. DEHP metabolites showed association with consumption of dairy products or meat. Use of plastic packaging and storage material was associated with DEHP or DBP.
a r t i c l e
i n f o
Article history: Received 20 July 2013 Received in revised form 30 October 2013 Accepted 30 October 2013 Available online 27 November 2013 Keywords: Phthalate Within-individual variability Daily intake DEHP Malondialdehyde
a b s t r a c t Phthalates have been used in a variety of consumer products and hence frequently been detected in humans. Children are susceptible to endocrine disrupting chemicals such as phthalates, but only limited information is available on the sources of exposure and potential adverse health effects among children. In this study, elementary school students (n = 39, aged 9–12 years) were recruited in Seoul, and first void urine samples were collected twice in three-day intervals. Then six phthalate metabolites were analyzed by high performance liquid chromatography with triple quadrupole tandem mass spectrometry. In addition, malondialdehyde (MDA) as an oxidative stress marker was measured. A questionnaire was conducted and information on food consumption and the use of plastic packaging or storage materials was gathered. The concentrations of phthalate metabolites varied substantially by sampling time even within the same subject, but all target metabolites were detected in 100% of the samples with the highest geometric mean of 107 μg/g-creatinine for mono-n-butyl phthalate (MnBP). Urinary levels of mono-isobutyl phthalate (MiBP), and MnBP among Korean children were 8 and 3 times greater than those reported for US children, but those of monoethyl phthalate (MEP) were about 5 times lower than those of US children. Estimated phthalate intakes were generally in safe range, but in 3–8% of the participating children, the hazard quotients greater than one were noted. Urinary MDA concentrations were significantly associated with several metabolite levels after adjusting covariates in regression model. Consumption of dairy products or meat, and use of a plastic material were significantly associated with the DEHP metabolites or MnBP levels in multivariate model. The results of this study provide evidence of the association between phthalate exposure and oxidative stress especially among the early teenagers, and identified major sources that can be applied to development of management plan for phthalate exposure among children. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Phthalates have been used in a variety of consumer products such as vinyl tiles, food processing materials, medical devices, plastic bags, toys, and personal care products. Because phthalates are not chemically bound to the products, they can be easily released into surrounding ⁎ Corresponding author at: School of Public Health, Seoul National University, Gwanak, Seoul 152-742, Republic of Korea. Tel.: +82 2 880 2738; fax: +82 2 745 9104. E-mail address: [email protected] (K. Choi). 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.10.118
environment such as air, water, or dust (CDC, 2009), and hence phthalates and their metabolites have been frequently detected among humans including children worldwide (Becker et al., 2009; Frederiksen et al., 2011; Hauser and Calafat, 2005; KFDA, 2007, 2011; Koch et al., 2011; Sioen et al., 2012; Song et al., 2013). Phthalate exposure has been associated with endocrine disruption and reproductive and developmental damages in many experimental studies (Andrade et al., 2006; Borch et al., 2004; Gray et al., 2000; Okubo et al., 2003; Zacharewski et al., 1998). In humans, phthalate exposure has been also related to sex hormone disturbance (Lovekamp-Swan and Davis,
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2003; Pan et al., 2006), decreased sperm quality (Duty et al., 2004; Hauser et al., 2007), and length of gestation (Adibi et al., 2009; Whyatt et al., 2009) although conflicting reports are also available (Duty et al., 2005a; Jonsson et al., 2005; Rais-Bahrami et al., 2004). The levels of exposure appeared to be generally higher among the children compared to the adult population. For example, levels of metabolites of di-n-butyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) were higher in younger population compared to the older group (Kasper-Sonnenberg et al., 2012; KFDA, 2007, 2011; Koch et al., 2004; Song et al., 2013). Because adverse effects of exposure to endocrine disrupting chemicals were considered to be also greater among the children, monitoring occurrence levels, identifying the sources, and determining potential risks of phthalate exposure among the children are important. Until now, several studies have reported the levels of phthalate exposure among the children, but only limited information is available on their sources or associated risks. Another important research gap in phthalate exposure among children is knowledge on temporal variability of exposure. Because of rapid urinary clearance, phthalate concentrations in urine are known to have significant intra-day and inter-day differences (Frederiksen et al., 2013; Fromme et al., 2007a; Preau et al., 2010). Most of the existing studies however employ spot urine samples, therefore such variation has been indicated as a cause of often conflicting epidemiological associations between phthalate exposure and health effects (Frederiksen et al., 2013). Among Korean children, only a few studies have determined the levels of several phthalate metabolites in urine (KFDA, 2007, 2011). The results of these investigations showed that the exposure pattern among Korean children is somewhat different from those reported in other countries. The concentrations of DEP metabolites in urine are generally much lower than those reported in US and other countries (KFDA, 2007), while those of DEHP and DBP metabolites are relatively higher (KFDA, 2007). In a separate study reported only DEHP metabolites among 392 children in Korea, the concentrations of DEHP metabolites were reported to be higher among children compared to those of older age group (Song et al., 2013). This study was conducted to identify the source of phthalates exposure and evaluate the association between phthalate exposure and oxidative stress among Korean children. To derive reliable exposure estimates of phthalates and oxidative stress marker, two separate first void urines were collected in three day interval from all the participating children. The results of this study will help identify phthalates of concern and their major sources among the elementary school students in Korea, which will benefit developing management options for this important group of chemicals. 2. Materials and methods 2.1. Study population, questionnaire and sample collection Children (n = 39) were recruited from three elementary schools located in Seoul, Korea in July, 2011. General characteristics of study population are summarized in Table 1. The participants were between 9 and 12 years old, and were between 4th and 6th grades of elementary school. Participating children and their parents were asked to record the type and amount of food items the children consumed, and the information regarding the use of plastic packaging and storages at Days 1 (Monday, July 4, 2011) and 4 (Thursday), in dietary questionnaire. Dietary questionnaire included pictorial guide to help participants correctly recall the type and size of the food items they consumed (for details about dietary questionnaire, see Supplementary information). In addition, demographic and socioeconomic characteristics of the family were also asked. At Days 2 and 5, first morning void urine samples were collected at home in polypropylene tube, and transferred on ice to the laboratory. The urine samples were separated into 1.7 mL microcentrifuge tubes, and stored at − 80 °C until analysis. The
institutional review board of School of Public Health, Seoul National University approved this study, and informed consent was obtained from legal guardian of the participating child.
2.2. Chemical analysis Six primary or secondary metabolites of four most frequently used phthalates, i.e., diisobutyl phthalate (DiBP), DBP, DEHP, and diethyl phthalate (DEP) were chosen (Calafat and McKee, 2006; Wittassek et al., 2007). The metabolites include mono-isobutyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), mono(2-ethyl-hexyl) phthalate (MEHP), mono(2-ethyl-5-oxo-hexyl) phthalate (5-oxo-MEHP), mono (2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), and monoethyl phthalate (MEP). Six metabolites and isotope-labeled internal standards for phthalates (13C4-monobutyl phthalate, 13C4-MEHP, 13C4-5oxo-MEHP, 13C4-5-OH-MEHP, and 13C4-MEP) were purchased from Cambridge Isotope Laboratory (Andover, MA, USA). The analyses of phthalate metabolites in urine were performed on a high performance liquid chromatography (NanospaceSI-2, Shiseido, Tokyo, Japan) with triple quadrupole tandem mass spectrometry (LC/ MS/MS; API4000, Applied Biosystems, Foster City, CA, USA) as detailed in Kho et al. (2008). After enzymatic deconjugation of urine samples by treatment with β-galactosidase (from Escherichia coli K12), on-line clean-up and separation of phthalate metabolites were accomplished using the switching-column technique with a pretreatment column (Shiseido MFC 8, 50 ∗ 4.6 mm, 5 μm), trap column for concentrate (Imtackt Cadenza CD-C18, 30 ∗ 2.0 mm, 5 μm), and analytical column for peak separation (Imtackt Cadenza CD-C18, 75 ∗ 2.0 mm, 3 μm). Limit of quantification was determined at 0.5 μg/L for MiBP, 1.4 μg/L for MnBP, 0.6 μg/L for MEHP, 0.9 μg/L for 5-oxo-MEHP, 1.2 μg/L for 5-OH-MEHP, and 1.9 μg/L for MEP. For each instrumental run, reagent blank and solvent blank were included for quality assurance. About 10% of the samples were analyzed in duplicates in order to ensure reproducibility of the measurement. Coefficients of variation of all duplicate samples were within 20%. Accuracy was considered acceptable when deviations from the spike value were within 20%. Precision was considered acceptable when intra- and inter-day coefficients of variation were within 20%. As shown in Tables S1 and S2 of Supplementary information, accuracy and precision of analysis for each compound were generally within acceptable range. Urinary creatinine concentration was analyzed using enzymatic method by Samkwang Medical Laboratories (Seoul, Korea).
2.3. Daily phthalate intake and hazard quotient Based on the urinary phthalate metabolite concentrations, daily intake (DI) of parent phthalates was estimated using the following equation (Kohn et al., 2000): DIðμg=kg bw‐dayÞ ¼ ððUE CE=1000Þ=FueÞ ðMWp=MWmÞ
where UE is the urinary concentration of a given metabolite adjusted for creatinine (μg/g creatinine), CE is daily creatinine excretion rate normalized by body weight (mg/kg-day), Fue is a fraction of urinary excretion relative to total elimination. MWp and MWm are the molecular weights of parent phthalate and metabolite, respectively. Fue for MEP was 0.69 (Calafat and McKee, 2006), for MnBP and MiBP, 0.69 (Koch et al., 2004), and for MEHP, 5-OH-MEHP, and 5-oxo-MEHP, 0.059, 0.23, and 0.15, respectively (Fromme et al., 2007b). Hazard quotient (HQ) was defined as the estimated DI divided by reference dose (RfD). RfD values were 100, 20, and 800 μg/kg bw-day for DBP (DiBP), DEHP, and DEP, respectively (US EPA). HQ greater than unity was deemed to be of potential risk.
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Table 1 Average urinary concentrations of phthalates metabolites in children of age between 9 and 12 in Seoul, Korea. Variable (unit)
N (%)
(mean ± SD) All Gender Female Male BMI (kg/m2) (17.3 ± 2.68) Underweight (b18.5) Normal (18.5–22.9) Overweight (23.0–24.9) Parent education High school diploma Bachelor's/postgraduate degree Family monthly income (USD) b1,000 1–3000 3–6000 N6000 Parents smoking status Nonsmoker Past smoker Current smoker
Median (interquartile range) (μg/g creatinine) MiBP
MnBP
MEHP
5-OH-MEHP
5-oxo-MEHP
MEP
39 (100)
55.4 (34.7–71.6)
103 (85.4–142)
13.2 (9.3–17.6)
79.1 (63.9–101.5)
46.3 (34.4–62.6)
15.6 (11.5–26.2)
17 (44) 22 (56)
57.6 (30.5–70.3) 51.9 (36.4–71.8)
124 (85.8–167) 103 (85.0–128)
13.3 (10.0–17.4) 12.8 (8.6–18.1)
87.4 (72.3–110.6) 70.1 (57.5–94.9)
48.9 (40.8–68.8) 42.5 (33.1–62.6)
17.5 (12.6–33.3) 12.8 (10.3–20.2)
27 (69) 11 (28) 1 (2)
63.6 (31.2–93.8) 51.4 (42.8–64.1) 48.4
113 (90.1–160) 91.9 (75.5–104) 100
13.3 (10.1–18.0) 12.0 (8.0–15.9) 11.6
91.1 (75.5–104) 64.5 (50.3–76.0) 79.0
49.7 (41.5–71.0) 34.5 (28.6–47.0) 36.5
17.5 (11.9–32.0) 12.6 (10.9–16.2) 7.7
14 (36) 25 (64)
64.4 (30.5–96.2) 48.4 (36.4–71.5)
136 (85.0–173) 101 (85.8–111)
12.9 (10.4–13.7) 13.2 (8.0–17.8)
85.6 (66.2–154) 79.0 (62.0–96.5)
46.0 (35.1–89.9) 46.3 (32.6–62.6)
16.2 (7.8–22.9) 15.6 (11.7–33.3)
1 (2) 15 (39) 21 (54) 2 (5)
70.5 38.8 (26.0–83.2) 55.4 (46.7–71.8) 52.2 (33.0–71.5)
135 103 (78.7–159) 105 (85.0–136) 93.1 (88.0–98.3)
12.5 13.3 (11.0–15.7) 11.8 (8.5–18.1) 10.5 (6.6–14.5)
57.5 83.8 (73.5–98.2) 79.0 (64.5–114) 55.8 (49.6–62.0)
34.5 49.7 (42.7–59.1) 44.2 (32.6–73.1) 33.1 (24.2–42.1)
15.3 17.5 (11.0–25.4) 15.0 (11.5–33.3) 14.0 (12.4–15.6)
18 (46) 10 (26) 11 (28)
44.1 (30.5–65.7) 56.9 (48.4–65.3) 84.0 (33.0–108)
95.2 (71.8–111) 109 (100–173) 131 (88.0–152)
10.9 (7.4–15.5) 15.8 (11.6–20.0) 13.4 (10.4–14.5)
72.1 (62.0–96.5) 108 (79.0–154) 79.0 (49.6–91.1)
44.2 (32.6–55.5) 67.9 (42.8–89.9) 43.4 (24.7–49.7)
16.9 (11.7–36.8) 13.8 (7.8–17.5) 15.2 (11.5–29.5)
Mean age of target population is 11.1 ± 0.88. The measurements of two urine samples collected from the same subject in 3 day interval were averaged. There were no significant differences in urinary phthalate metabolite concentrations by each of demographic factors listed above (p b 0.05 based on non-parametric tests of Kruskal–Wallis test or Wilcoxon Mann–Whitney test).
2.4. Measurement of oxidative stress Urinary MDA levels were measured as thiobarbituric acid reactive substances (TBARS) using OxiSelect™ TBARS Assay kit (Cell Biolabs, Inc., San Diego, CA, USA) following manufacturer's instructions. A spectrophotometric plate reader (TECAN infinite® M200, TECAN Group LTD., Mannedorf, Switzerland) was used for this purpose. The protein concentration was quantified using Bradford method (Bradford, 1976) and was used for normalization of MDA level. Each experiment was carried out in duplicates and average values were used. 2.5. Statistical analysis Multiple linear regression model was employed to identify potential sources of phthalate exposure. The association between phthalate exposure and oxidative stress was investigated using multiple linear regression model. Age, sex, body mass index (BMI), family income, and secondhand smoking were included as covariates in the models. For every multivariate model, phthalate metabolite concentrations and MDA levels as dependent variables were natural log-transformed. Normal distribution of residuals was confirmed by residual graphs. Because of small sample size, additional multiple regression models were conducted excluding one or two outliers to confirm the observed association. Outliers were identified by maximum value among potential outliers which showed the absolute value of modified Z-score exceeding 3.5. Statistical analyses were performed using Statistical Analysis System (SAS 9.13, Cary, NC, USA).
their short elimination times, positive relationships between two urine samples collected in 3-day interval may reflect similar exposure patterns e.g., foods, containers, and other related sources among the participating children. Hoppin et al. (2002) reported similar levels of phthalate metabolites in two consecutive morning void samples and suggested sufficiently consistent exposure pattern among the subjects (African American women, n = 46, aged 35–49 years). Teitelbaum et al. (2008) also reported that the concentrations of major phthalate metabolites detected in several spot urines of 35 healthy Hispanic and African American children over 6 months were comparable (Teitelbaum et al., 2008). Since exposure to phthalate can be influenced by many sources which can vary day-to-day, however significant variations were often reported (Preau et al., 2010). Such variation is expected to be greater among children (Teitelbaum et al., 2008). In our study, average intra-individual CVs were generally lesser than or comparable to those reported previously. The maximum CV in the present study was detected from creatinine-corrected MEP (132%), which was slightly lower than Preau et al. (2010) that reported the maximum CV of 157% over 1 week period among adults. This observation indicates that the variation in phthalate exposure among Korean children is relatively less except for DEP. Use of or exposure to personal care products can significantly influence the level of DEP exposure (Koch et al., 2013), and such use can vary especially among children, therefore multiple day sampling would be more appropriate for phthalates like DEP metabolites. For further evaluation, the concentrations of the creatininecorrected metabolites of the two first void samples were averaged for description of the level of exposure unless specifically noted as the 1st or the 2nd urine samples.
3. Results and discussion 3.2. Concentrations of phthalate metabolites in urine samples 3.1. Within-person variability of phthalate metabolites Strong positive associations were observed for the concentrations of all metabolites between two first void urine samples (Table S3 and Fig. S1 of Supplementary information). While the concentrations of three metabolites, i.e., MiBP, MnBP, and MEHP, showed significant difference between the first and second urine samples (Wilcoxon signed rank sum test, p b 0.05, Table S3), coefficients of variation (CV) determined between the levels of two urine samples are 20–24%. Considering
All phthalate metabolites were detected in 100% of the urine samples (Table 1). The geometric mean was the greatest for MnBP (107 μg/g creatinine), and the lowest for MEHP (12.6 μg/g creatinine). The concentrations of DEHP metabolites were in descending order 5-OH-MEHP N 5-oxo-MEHP N MEHP (Table 2). No significant relationships were detected between the measured metabolite concentrations and demographic characteristics of the study population, such as sex or BMI (Table 1).
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Table 2 Concentrations of major metabolites of DBP, DEHP, and DEP in urines of children: Comparison with previous results (μg/g creatinine). This studya
Study sample size (N) Phthalate
Metabolites
DiBP DBP DEHP
MiBP MnBP MEHP 5-OH-MEHP 5-oxo-MEHP MEP
DEP
Koreab
Koreac
USAd
Germanye
1st urine
2nd urine
Average
39
35
39
60
199
342
599
47.7 (48.6–103) 96.2 (95.7–184) 11.4 (11.2–33.3) 81.9 (72.0–236) 47.3 (43.2–139) 17.6 (14.3–140)
58.3 (56.3–188) 117 (109–236) 13.0 (13.9–25.6) 82.6 (80.0–155) 47.3 (45.5–102) 19.0 (15.0–151)
53.4 (55.4–135) 107 (103–208) 12.6 (13.2–24.0) 84.4 (79.1–204) 48.6 (46.3–125) 19.2 (15.6–160)
56.8 (54.7–309) 140 (95.7–5233) 17.3 (16.6–173) 80.2 (73.2–793) 81.0 (72.0–759) 26.0 (23.5–178)
49.0 (51.5–133) 61.3 (58.4–153) 7.57 (9.14–29.2) 49.9 (49.8–129.6) 29.5 (31.5–77.9) 9.74 (10.7–90.8)
6.94 (7.03–28.7) 38.4 (39.0–137) 3.00 (2.80–28.7) 39.0 (36.6–211) 26.6 (25.3–121) 101 (87.0–719)
94.3 (88.1–308) 95.6 (93.4–310) 6.4 (6.7–25.1) 47.9 (46.0–164) 37.0 (36.3–123) NA
Geometric mean and 50th–95th percentile values are presented. NA: Not available. a Age 9–12 years old. b Age 8–13 years old, values in parentheses are 50th centile-maximum values (KFDA, 2007). c Age 10–12 years old (KFDA, 2011). d Age 6–11 years old (CDC, 2009). e Age 3–14 years old (Becker et al., 2009).
The concentrations of most phthalate metabolites in urines of the Korean children are greater than those reported from USA (n = 342, 6–11 years of age; CDC, 2009) or from Germany (n = 599, 3–14 years of age; Becker et al., 2009) (Table 2). The only exception was MEP, a metabolite of DEP, which was detected on average 19.2 μg/g creatinine. MEP was detected on average at 101 μg/g creatinine among US children. The reason for much lower MEP concentrations in Korean children's urines can be found from the differences in use of cosmetics and personal care products either by age or by country. DEP has been used as a solvent in many personal care products, particularly in those containing fragrances, e.g., perfumes, colognes, deodorants, soaps, shampoos, and hand lotions. Associations between cosmetic use and urinary MEP concentrations have been reported elsewhere (Duty et al., 2005b; Janjua et al., 2008). Higher levels of MEP were reported in urine samples of female children than in those of male children in previous studies of Korea and USA (KFDA, 2007; Teitelbaum et al., 2012). US children are expected to use more cosmetics and personal care products compared to Korean children. The use of cosmetics or personal care products among elementary school students is less common in Korea. The levels of phthalate metabolites measured in the present study were in a comparable range to those reported previously for Korean children (n = 60, KFDA, 2007; n = 199, KFDA, 2011; n = 392, Song et al., 2013). Occurrence pattern of the phthalate metabolites was the same, suggesting similar source profiles of the phthalates among Korean children. Levels of phthalate exposure become generally lower as age increases. Similar decreasing trends have been reported in several other studies particularly for metabolites of DBP and DEHP (KasperSonnenberg et al., 2012; KFDA, 2007, 2011; Koch et al., 2004). Greater food consumption rates relative to their body weight, and characteristic mouthing behavior are suggested as part of the reasons for greater phthalate exposure among younger children (Wittassek et al., 2007). However, age could not explain the differences that were observed in levels of urinary phthalate metabolites among Korean children and USA or German children. Participating Korean children (9–12 years old) showed up to two fold higher levels of DEHP metabolites than those of German children (3–14 years old), and up to 7.7 fold higher levels of DiBP metabolites than those of US children (6–11 years old). This observation strongly indicates presence of additional sources or different source profiles of phthalate exposure among Korean children compared to those in Europe or North America.
bw-day for DEP. Intake estimates of DEHP ranged from 2.7 to 21.7, 3.6 to 41.0, or 2.9 to 35.8 μg/kg bw-day, based on MEHP, 5-OH-MEHP, or 5-oxo-MEHP concentrations, respectively. None of the HQs calculated for phthalates exceeded 1 except DEHP (Fig. 1). One to three participants (3–8%) were identified to show HQs greater than 1, suggesting that DEHP exposure should be considered of concern among certain Korean children. Similar observations were reported among the German children. Among German children (n = 239, 2–14 years old), up to 7.5% of children showed DEHP exposure estimates that exceeded an RfD of 20 μg/kg bw-day (Wittassek et al., 2007). 3.4. Association with oxidative stress biomarker in urine Significant positive associations between urinary MDA concentrations, which reflect lipid peroxidation levels, and several phthalate metabolites were observed after adjusting age, sex, BMI, family income, and secondhand smoking, especially in the 2nd urine samples (Table 3). The only exception was 5-oxo-MEHP, which showed marginal significance (p = 0.061 for β estimates in multiple regression analysis). Although positive trend was maintained, however, with the 1st urine samples, no metabolites showed significant associations with MDA. When urinary MDAs and phthalates levels of the 1st and 2nd samples were averaged, however, significant positive associations between
3.3. Estimated daily intake and associated risks Estimated DIs of parent phthalates were calculated following Kohn et al. (2000) using the creatinine-corrected metabolite concentrations in urine of 39 Korean children. The median DI was 2.0 (range 0.7–6.1) for DiBP, 4.2 (range 1.4–9.4) for DnBP, and 0.5 (range 0.1–13.4) μg/kg
Fig. 1. Estimated hazard quotient from measured concentration of phthalate metabolites in urine samples of 39 children in Korea. The average metabolite concentrations of two first void urine samples were used. Solid line and box denote median and 25–75th percentile values (interquartile range), respectively. Range marked indicates 10th and 90th percentiles, and black circles denote outliers.
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Table 3 Relationships between malondialdehyde (μg/g creatinine) and creatinine-adjusted phthalate metabolites (μg/g creatinine) in urine samples. Phthalate metabolites
MiBP MnBP MEHP 5-OH-MEHP 5-Oxo-MEHP MEP
1st urine (n = 39)
2nd urine (n = 35)
Average (n = 39)
β (95% Cl)
p
β (95% Cl)
p
β (95% Cl)
p
0.002 (−0.002, 0.005) 0.002 (−0.000, 0.004) 0.006 (−0.005, 0.017) 0.001 (−0.000, 0.002) 0.001 (−0.001, 0.004) 0.000 (−0.001, 0.001)
0.322 0.087 0.248 0.339 0.247 0.613
0.004 (0.001, 0.006) 0.003 (0.001, 0.004) 0.020 (0.006, 0.354) 0.003 (0.000, 0.005) 0.004 (0.000, 0.008) 0.002 (0.000, 0.004)
0.005 * 0.002 * 0.009 * 0.025 * 0.061 0.027 *
0.003 (0.000, 0.005) 0.003 (0.001, 0.004) 0.012 (0.001, 0.023) 0.001 (−0.000, 0.003) 0.002 (−0.001, 0.004) 0.000 (−0.001, 0.002)
0.022 * 0.007 * 0.028 * 0.081 0.175 0.346
Results were presented in multivariate model adjusted for age, sex, BMI of a child, family income, and smoking status of parents. Asterisk (*) denotes statistical significance (p b 0.05). Dependent variable (MDA) was used as natural-log transformed. CI: Confidence interval.
MDA and phthalates, e.g., MiBP, MnBP, or MEHP were observed. The reason for different statistical significances in the 1st urine samples is not clear, but may be found from the variability of the oxidative stress biomarkers (Arguelles et al., 2007; Browne et al., 2008). Oxidative stress markers such as MDA can be influenced by numerous factors including physical, chemical, and biological parameters, hence certain extent of within-individual variation is inevitable. For example, urinary 8-oxo7,8-dihydro-2′-deoxyguanosine (8-oxodG), another widely used biomarker of oxidative stress, showed a within-individual variation of 20% CV in the first morning void samples collected from 26 healthy nonsmoking adults (Barregard et al., 2013). While quantitative explanation is not possible, day-of-the-week variation of stress biomarkers may also contribute to the observation of the present study, e.g., the 1st set of samples were collected on Tuesday (Day 2) morning following weekend vs. the 2nd set of samples were collected on Friday (Day 5). Therefore in order to measure the status of oxidative stress, several measurements are suggested to be necessary (Arguelles et al., 2007). For this reason, it appeared to be more appropriate, in the present study, to average MDA levels of both Tuesday and Friday urines before relating them with phthalate exposure. While it is still possible that the observed associations were due to by chance, consistently positive association between phthalate metabolites and MDA levels suggests that several phthalates might be associated with oxidative stress in children. Phthalates are known as one of the important contributors for oxidative stress in both experimental and epidemiological studies (Ambruosi et al., 2011; Santhosh et al., 1998; Xu et al., 2013; Hong et al., 2009). Recent study on 1999–2006 NHANES data (N = 10,026) reported that phthalates such as DBP and DEHP were associated with oxidative stress (Ferguson et al., 2012). Among adult population who participated in 5 day Temple-stay
program in Korea, both phthalate metabolites and MDA concentrations in urines decreased after the Temple-stay, suggesting an association between phthalates and oxidative stress (Ji et al., 2010). 3.5. Potential sources of exposure Consumption of dairy products or meat and the use of plastic packaging or storing were identified to be significantly associated with urinary DEHP metabolites (Table 4). The use of plastics and bread consumption were associated with urinary MnBP concentrations but the association was not consistent. Eggs and oil product consumption showed significant negative correlation with several phthalate metabolites. However, additional regression analysis after removing one or two extreme outliers of phthalate metabolites, significance was disappeared. Based on β values derived from multiple regression analysis of DEHP metabolites, the influence of plastic packaging on DEHP exposure appeared to be greater than that of plastic storage. Plastic packaging has been suggested as source of phthalate contamination in foodstuffs (Wormuth et al., 2006), and cooked foods packed in plastic containers were expected to be highly contaminated (Cirillo et al., 2013). Several studies showed phthalates, particularly DBP and DEHP, have been detected in foodstuffs packed in plasticizer-used materials (Casajuana and Lacorte, 2004; Freire et al., 2006; Petersen and Breindahl, 2000). For general population, diets have been considered as a primary source of phthalate exposure (Schettler, 2006). One recent study also showed that DiBP, DnBP, and DEHP were detected in milk and dairy food samples from several stages in the milk chain (Fierens et al., 2013). In the present study, consumptions of dairy products or meat were significantly associated with DEHP exposure. Ji et al. (2010)
Table 4 Relationships between urinary phthalate metabolites (μg/g creatinine) and food intake or material use for food package and storage of children in Korea. Phthalate metabolites
1st urine (n = 39)
MiBP MnBP
– Plastic packaging Plastic storage Dairy Plastic packaging
MEHP
5-OH-MEHP
5-Oxo-MEHP
MEP
Food intake or material use
Dairy Meat Plastic packaging Plastic storage Dairy Meat Plastic packaging Plastic storage –
2nd urine (n = 35) β (95% Cl)
0.052 (0.009, 0095) 0.094 (0.033, 0.156) 0.001 (0.000, 0.002) 0.138 (0.012, 0.263) 0.001 (0.000, 0.001) 0.002 (0.001, 0.004) 0.182 (0.068, 0.295) 0.156 (0.036, 0.276) 0.001 (0.000, 0.001) 0.002 (0.000, 0.004) 0.162 (0.042, 0.170) 0.139 (0.013, 0.264)
Food intake or material use – Bread Oil†
Average (n = 39) β (95% Cl)
0.194 (0.051, 0.337) −1.062 (−1.702,−0.422)
Dairy Egg†
0.001 (0.000, 0.002) −0.268 (−0.485,−0.051)
Dairy Egg†
0.001 (0.000, 0.002) −0.269 (−0.491,−0.047)
Plastic storage
0.374 (0.004, 0.744)
Food intake or material use
β (95% Cl)
Oil† –
−2.016 (−3.711,−0.321)
Dairy Plastic packaging Plastic storage Dairy Plastic packaging Plastic storage Dairy Meat Plastic packaging Plastic storage Noodle†
0.001 (0.000, 0.002) 0.172 (0.027, 0.316) 0.133 (0.005, 0.262) 0.001 (0.000, 0.001) 0.199 (0.061, 0.337) 0.168 (0.047, 0.289) 0.001 (0.000, 0.001) 0.002 (0.001, 0.004) 0.203 (0.060, 0.346) 0.165 (0.039, 0.291) −1.284 (−2.471,−0.088)
Only significant results (p b 0.05) based on multivariate model adjusted for age, sex, BMI, family income, and smoking status of parents were presented. Dependent variables (phthalate metabolites) were used as natural-log transformed. Because of small sample size, additional regression model analysis was conducted after excluding extreme outliers. Without outliers, significance was disappeared. Relevant variables were marked by †. CI: Confidence interval.
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reported that urinary 5-OH-MEHP and 5-oxo-MEHP level exhibited significant positive correlations with consumption of dairy products. Meat consumption was pointed out as a significant source of DEHP exposure as well (Fromme et al., 2007b). Dietary sources are suspected for DnBP and DiBP as well. For example, bread has been suggested as one of the top contributors to DnBP and DiBP exposure among preschool children and adults in Belgium (Sioen et al., 2012). In the present study, bread consumption showed positive relationship with MnBP levels in the 2nd urine samples. Because staple foods of Korean are mainly originated from rice, bread consumption pattern among Korean however is obviously different with that of European people. Average bread including breakfast cereal consumption of Belgian population was 175–420 g/day (15–18 years: 146 ± 59 g/day) in 2004 (Vandevijvere et al., 2009), while the bread and cereal consumption rate of Korean people is on average 26 g/day for 70 kg person (0.37 ± 1.19 g/kg bw-day, (Jang et al., 2007) and 31 g/day for preschool children (Lee et al., 1998). The difference in the consumption rate may partly explain weaker influence of bread consumption on phthalate exposure among Korean children. Our finding is consistent with several previous reports that identified food as one of exposure sources of DEHP (Colacino et al., 2010; Fromme et al., 2007b; Petersen and Breindahl, 2000). The β values obtained from the regression models indicate that the contribution of plastic material was stronger than that of the food: One additional serving of dairy or meat product per month related to an average 0.002 μg 5-oxo-MEHP/g creatinine increase, while one additional use of plastic material per month related to an average increase of 0.225 μg 5-oxoMEHP/g creatinine. Our study suggests that the levels of certain phthalates especially DEHP are of potential concern among Korean children, and these exposures are potentially related to oxidative stress. Plastic food packaging and the use of plastic storage along with consumption of dairy products or meat were identified as important contributors to phthalate exposure among children. Use of plastic material as packaging or storage showed relatively greater contribution to the exposure of phthalate compared to food consumption. While these observations may require confirmation in greater sized population studies, the results of our study will help develop management plans for phthalate exposure among Korean children. Conflict of interests The authors declare no actual or potential competing interests. Acknowledgment This research was supported by the National Research Foundation of Korea (Project 2012R1A2A2A01015236). S.M. Kim is supported by BK 21 Plus program of National Research Council. This study is in part supported by School of Public Health, Seoul National University. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2013.10.118. References Adibi JJ, Hauser R, Williams PL, Whyatt RM, Calafat AM, Nelson H, et al. Maternal urinary metabolites of di-(2-ethylhexyl) phthalate in relation to the timing of labor in a US multicenter pregnancy cohort study. Am J Epidemiol 2009;169:1015–24. Ambruosi B, Filioli Uranio M, Sardanelli AM, Pocar P, Martino NA, Paternoster MS, et al. In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model. PLoS One 2011;6(11):e27452. Andrade AJ, Grande SW, Talsness CE, Gericke C, Grote K, Golombiewski A, et al. A dose response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): reproductive effects on adult male offspring rats. Toxicology 2006;228: 85–97.
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