Urinary phthalate metabolites among children in Saudi Arabia: Occurrences, risks, and their association with oxidative stress markers

Science of the Total Environment 654 (2019) 1350–1357

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Urinary phthalate metabolites among children in Saudi Arabia: Occurrences, risks, and their association with oxidative stress markers Inae Lee a, Raid Alakeel b, Sungmin Kim c, Yazeed A. Al-Sheikh b, Hazem Al-Mandeel d, Abdullah A. Alyousef b, Younglim Kho c,⁎, Kyungho Choi a,⁎⁎ a

School of Public Health, Seoul National University, Seoul, Republic of Korea Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University Department of Health, Environment and Safety, Eulji University, Republic of Korea d Department of Obstetrics and Gynecology, College of Medicine, King Khalid University Hospital, King Saud University b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Eighteen phthalate metabolites were analyzed in urine samples from Saudi Arabian children. • MiBP and MnBP were detected at higher levels than those reported in other countries. • Approximately 34% of the children showed potential risks (HQ N 1) from DEHP exposure. • Certain phthalate metabolites were associated with oxidative stress markers.

a r t i c l e

i n f o

Article history: Received 24 August 2018 Received in revised form 2 November 2018 Accepted 2 November 2018 Available online 5 November 2018 Editor: Adrian Covaci Keywords: Phthalates Sources Children Oxidative stress Risk

a b s t r a c t Phthalates have been used as plasticizers in numerous consumer applications and therefore, their metabolites have been detected in human urine worldwide. Despite concerns regarding their potential adverse health effects, few exposure assessments have been conducted among young populations in Middle Eastern countries. In this study, children (n = 109, aged 3–9 years) were recruited from four elementary schools in Riyadh, Saudi Arabia, in 2017, and major phthalate metabolites were measured in their urine. Their parents were asked to complete a questionnaire on their behalf to assess potential exposure sources of phthalates. In addition to 18 phthalate metabolites, malondialdehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured in urine samples by LC/MS/MS. Among the children of Saudi Arabia, urinary levels of monoisobutyl phthalate (MiBP) and monobutyl phthalate (MnBP) were higher than those reported previously in children worldwide. Monoethyl phthalate (MEP) was also detected at high levels. Several phthalate metabolites showed significant associations with the levels of MDA or 8-OHdG. Hazard quotients (HQs) derived for certain phthalates were greater than one. In particular, the HQs for di(2-ethylhexyl) phthalate (DEHP) were greater than one in 34% of the participating children. Levels of monocyclohexyl phthalate (MCHP),

Abbreviations: BBzP, butylbenzyl phthalate; BMI, body mass index; DnBP, di-n-butyl phthalate; DEHP, di(2-ethylhexyl) phthalate; DEP, diethyl phthalate; DCHP, dicyclohexyl phthalate; DI, daily intake; DiBP, diisobutyl phthalate; DiDP, diisodecyl phthalate; DiNP, diisononyl phthalate; HMW, high molecular weight; HQ, Hazard quotient; IQR, interquartile range; LMW, low molecular weight; LOD, limit of detection; LOQ, limit of quantification; MBzP, monobenzyl phthalate; MCHP, monocyclohexyl phthalate; MCMHP, mono[2(carboxymethyl)hexyl] phthalate; MCPP, mono(3-carboxypropyl) phthalate; MDA, malondialdehyde; MECPP, mono(2-ethyl-5-carboxypentyl) phthalate; MEHHP, mono(2-ethyl-5hydroxyhexyl) phthalate; MEHP, mono(2-ethylhexyl) phthalate; MEOHP, mono(2-ethyl-5-oxohexyl) phthalate; MEP, monoethyl phthalate; MHxP, monohexyl phthalate; MiBP, monoisobutyl phthalate; MiDP, monoisodecyl phthalate; MiNP, monoisononyl phthalate; MiPrP, monoisopropyl phthalate; MMP, monomethyl phthalate; MnBP, monobutyl phthalate; MOP, monooctyl phthalate; MPeP, monopentyl phthalate; RfD, reference dose; TDI, tolerable daily intake; 8-OHdG, 8-hydroxy-2′-deoxyguanosine. ⁎ Correspondence to: Y. Kho, Department of Health, Environment and Safety, Eulji University, Seongnam 13135, Republic of Korea. ⁎⁎ Correspondence to: K. Choi, Department of Environmental Health Sciences, School of Public Heath, Seoul National University, Seoul 08826, Republic of Korea. E-mail addresses: [email protected] (Y. Kho), [email protected] (K. Choi).

https://doi.org/10.1016/j.scitotenv.2018.11.025 0048-9697/© 2018 Elsevier B.V. All rights reserved.

I. Lee et al. / Science of the Total Environment 654 (2019) 1350–1357

1351

monoisodecyl phthalate (MiDP), mono(2-ethylhexyl) phthalate (MEHP), and mono[2-(carboxymethyl)hexyl] phthalate (MCMHP) in the urine samples were positively associated with the consumption frequency of certain foods. Very high levels of exposure to phthalates, along with positive associations with oxidative stress markers, outline the importance of follow-up investigations for identification of phthalate exposure sources and potential health implications among the young population of Saudi Arabia. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Phthalates have been used as plasticizers in numerous consumer products, such as food packages, medical devices, toys, building materials, personal care products, and cosmetics. Low molecular weight (LMW) phthalates, such as diethyl phthalate (DEP), have been used in cosmetics, fragrance, and, to a lesser extent, food packaging (Dodson et al., 2012; Fierens et al., 2012; Koniecki et al., 2011). High molecular weight (HMW) phthalates, such as di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DiNP), have been used in a variety of polyvinyl chloride (PVC) applications and food packaging (Fierens et al., 2012; Kawakami et al., 2011; Schecter et al., 2013). As phthalates are not chemically bound, they can leach out from these products into the surrounding environment. Humans are exposed to these chemicals either through direct intentional contacts or through indirect routes (Wormuth et al., 2006). Therefore, metabolites of these plasticizers have been widely detected in human urine around the world (Choi et al., 2017; Frederiksen et al., 2013; Guo et al., 2011; Koch et al., 2017; Smerieri et al., 2015; Zota et al., 2014). The exposure levels of phthalates can be influenced by many dietary, lifestyle, and sociodemographic factors (Larsson et al., 2014; Zota et al., 2014). Because these factors can vary by country (Guo et al., 2011), it is important to understand the current levels of occurrence of major phthalates among the general population and to identify phthalates of potential public health concern. Because of this reason, several countries such as the United States of America (USA), Germany, and Korea have conducted national human biomonitoring programs, which include metabolites of phthalates in the list of chemicals measured among the general populations (Choi et al., 2017; Koch et al., 2017; Zota et al., 2014). Young children are considered to be a population that is vulnerable to phthalates because of their adverse effects on endocrine and neurodevelopmental systems, which may lead to many chronic health consequences (Braun et al., 2013; Cho et al., 2010; Ejaredar et al., 2015; Smerieri et al., 2015). While exposure assessment of phthalates among children has been more frequently conducted worldwide, such information is mostly lacking, especially in countries of the Middle East. Only one study reported the exposure levels of phthalates in children among the general population of Saudi Arabia, but the age of the study population ranged between 1 and 87 years (Asimakopoulos et al., 2016), leaving the exposure profile of major phthalates among children largely unknown. Studies aiming at filling this knowledge gap are warranted. The present study was conducted to understand current levels of exposure to major phthalate metabolites among the children of Saudi Arabia. Their risks and association with oxidative stress markers were also assessed in order to identify phthalates of potential public health concern. In addition, the potential sources of exposure were investigated using information collected via a questionnaire survey. The results of this study will help identify phthalates of potential risk and develop appropriate mitigation measures for children in Saudi Arabia.

March and May 2017. All urine samples were collected in Falcon tubes during the early morning, between 7 and 8:30 AM, and subsequently delivered to the laboratory in Riyadh where samples were transferred into 2 mL cryovial tubes. The urine samples were then stored at −20 °C and later transported on ice to the analytical laboratory. Parents of the participating children provided the following data via questionnaire for their children: sociodemographics, food consumption, and use of personal care products (PCPs). The frequency of food consumption and use of PCPs was coded as 1: almost never, 2: 1–2 times a week, 3: 3–4 times a week, 4: 5–6 times a week, 5: once a day, and 6: more than once a day, and the frequency of eating out was coded as 1: less than once a month, 2: 1–3 times a month, 3: 1–6 times a week, 4: about once a day, and 5: more than once a day. This study was approved by the Research Ethics Committee of King Saud University (CAMS 053-37/38). 2.2. Chemical analysis Urine samples were measured for major metabolites of target phthalates using a high-performance liquid chromatography (HPLC) (Nanospace SI-2, Shiseido, Tokyo, Japan) with a triple quadrupole mass spectrometer (API 4500, AB SCIEX) following the method described in a previous study (Hong et al., 2010). Isotope-labeled internal standards were obtained from Cambridge Isotope Laboratory (Tewksbury, MA, USA). Following enzymatic hydrolysis, on-line clean-up and separation of the phthalate metabolites was performed using a column-switching technique with a pretreatment column (OASIS HLB, 20 × 2.1 mm, 5 μm, Waters, Milford, MA, USA) and an analytical column (Peakman SP C18, 150 × 2.0 mm, 3 μm, Shiseido). The measurement of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in human urine was performed using an LC-MS/MS method. The urine samples were prepared by solid-phase extraction (Oasis® HLB 96Well Plate 30 μm (30 mg)) and chromatographic separation was performed on a Kinetex column (2.6 μm HILIC 100A 150 ∗ 2.1 mm). The MDA concentration in human urine samples was determined after its conversion with 2,4-dinitrophenyl-hydrazine (DNPH) to a hydrazone (MDA-DNPH). Measurement of MDA-DNPH was performed with an LC-MS/MS method as described by Kim et al. (2017). Quality assurance and quality control information, including the limit of detection (LOD), method validation results, and LC-MS/MS parameters, are shown in Tables S1–S5 of the Supplementary information. 2.3. Estimation of daily intake amount and risks for phthalates Daily intake (DI) amounts of phthalates were estimated from concentrations of urinary phthalate metabolites following the methods outlined in Koch et al. (2007). DIðμg=kgbw =dayÞ ¼ UEðμmol=LÞ  UVðL=kgbw =dayÞ  MWpðg=molÞ=Fue

2. Materials and methods 2.1. Study population and urine sampling Children between the ages of three and nine years (n = 109) were recruited from four schools in Riyadh city, Saudi Arabia between

In the formula above, UE is the concentration of the metabolite divided by its molecular weight. UV is the daily volume of urination per body weight, and for the current population, a value of 22.5 mL/kgday was derived from a previous study where the ages of the participants ranged between 6 and 11 years (Ballauff et al., 1988). MWp is

1352

I. Lee et al. / Science of the Total Environment 654 (2019) 1350–1357

2.4. Statistical analysis

Table 1 Characteristics of the children who participated in this study (n = 109).

For phthalate metabolites with a detection frequency N75%, those that were undetected were substituted by the limit of detection (LOD) divided by the square root of 2 (Hornung and Reed, 1990). The concentrations of DEHP metabolites (MCMHP, MECPP, MEHHP, MEHP, and MEOHP) were summed (∑DEHPm) and used for the statistical analysis. Spearman correlation analysis was performed between creatininecorrected concentrations of phthalate metabolites. Associations between creatinine-corrected concentrations of phthalate metabolites, and potential exposure sources recorded as ordinal variables on the questionnaire were also investigated using Spearman correlation analysis. Linear regression model was used for associations between phthalate exposures and oxidative stress markers. Due to right-skewness, the concentrations of phthalate metabolites were natural logtransformed in regression models. In the crude model, urinary creatinine was included as a covariate (Barr et al., 2005). In adjusted models, age, sex, body mass index (BMI), and urinary creatinine were included as covariates. These covariates were selected based on previous studies that investigated associations between urinary phthalate metabolites and oxidative stress markers (Holland et al., 2016; Hong et al., 2009; Kim et al., 2014). We further performed principal component analysis (PCA) with varimax rotation on phthalate metabolites. For PCA, lntransformed molar concentrations of the target metabolites were used. All analyses were conducted by using SAS 9.3 (SAS Institute, Cary, NC, USA).

Median (IQR) or n (%) Age (years) Sex Male Female BMI (kg/m2) Parental education Degree below high school High school diploma Undergraduate and above Family monthly income (USD) b1650 1650–3300 3300–5000 N5000

5 (4–6) 60 (55%) 49 (45%) 17.2 (16.2–18.0) 10 (9.2%) 11 (10.1%) 88 (80.7%) 2 (1.8%) 73 (67.0%) 23 (21.1%) 11 (10.1%)

the molecular weight of the parent phthalate. Fue is the urinary excretion fraction, and the values 0.69, 0.84, 0.703, 0.73, 0.059, 0.233, 0.150, 0.185, and 0.042 were used for monoethyl phthalate (MEP), monobutyl phthalate (MnBP), monoisobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), mono(2-ethylhexyl) phthalate (MEHP), mono(2ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), and mono[2-(carboxymethyl)hexyl] phthalate (MCMHP), respectively (Itoh et al., 2007; Koch et al., 2005; Koch et al., 2012). The hazard quotient (HQ) was derived by the DI divided by the relevant reference dose (RfD) or tolerable daily intake (TDI). RfD or TDI values of 800, 100, 200, and 20 μg/kg bw-day were employed for diethyl phthalate (DEP), di-n-butyl phthalate (DnBP), b(2-ethylhexyl) phthalate (DEHP), respectively (U.S. Environmental Protection Agency, 1990; U.S. Environmental Protection Agency, 1993a; U.S. Environmental Protection Agency, 1993b; U.S. Environmental Protection Agency, 1993c). For diisobutyl phthalate (DiBP), both TDI and RfD are not available; therefore, the values for DnBP were used. An HQ N1 was considered as a potential risk.

3. Results and discussion 3.1. Characteristics of the study population The study population was five years of age (median) with an interquartile range (IQR) of four to six years old (Table 1), and 55% were male. The median body mass index (BMI) was 17.2 with an IQR from 16.2 to 18.0. The majority of the participants' parents possessed an undergraduate degree and above (80.7%), and 67% of the families had monthly incomes between 1650 and 3300 USD.

Table 2 Urinary levels of phthalate monoesters (unadjusted) in the population of children (n = 109). Parent phthalate

Low molecular weight phthalates

High molecular weight phthalates

DMP DEP DiBP DnBP DPeP DiPrP BBzP DCHP DHxP DOP DEHP

DiNP DiDP

Target metabolite

MMP MEP MiBP MnBP MPeP MiPrP MBzP MCHP MHxP MOP MCPP MEHP MCMHP MECPP MEHHP MEOHP ∑DEHPm MiNP MiDP

DF (%)

99.1 100 100 99.1 5.5 26.6 97.2 79.8 39.4 0 100 88.1 100 100 100 100 – 14.7 98.2

Mean ± SD (ng/mL)

17.3 ± 12.7 524.8 ± 908.6 265.2 ± 354.9 253.0 ± 268.9 – – 7.7 ± 9.1 1.2 ± 2.5 – – 37.7 ± 56.1 16.1 ± 27.8 63.1 ± 46.8 111.2 ± 78.8 99.4 ± 82.3 52.3 ± 36.8 342.1 ± 240.9 – 2.9 ± 2.3

GM (ng/mL)

12.7 277.5 146.6 155.7 – – 4.4 0.8 – – 20.9 7.8 47.2 83.6 71.9 39.4 258.3 – 2.2

GM: Geometric mean. bLOD: below limit of detection. ∑DEHPm indicates sum of DEHP metabolites including primary (MEHP) and secondary metabolites (MCMHP, MECPP, MEHHP, and MEOHP).

Percentiles (ng/mL) p25

p50

p75

p95

8.4 122 63.3 94.7 bLOD bLOD 2.3 0.4 bLOD bLOD 11.4 3.8 26.8 53.4 46.4 25.3 166.0 bLOD 1.4

14 259 155 179 bLOD bLOD 4.1 0.6 bLOD bLOD 22 8.9 52 87.4 75 41.3 272 bLOD 2.1

22.9 579 324 310 bLOD 0.4 8.5 1.2 0.3 bLOD 37.6 16.1 86.1 159 135 76.3 484 bLOD 3.6

43 1290 777 859.4 0.5 0.8 29.4 2.6 0.6 bLOD 121.4 53.5 149.8 260.4 248.6 133.4 720.6 0.71 7.9

I. Lee et al. / Science of the Total Environment 654 (2019) 1350–1357 Fig. 1. Comparisons of the concentrations of urinary phthalate metabolites, (A) MEP, (B) MiBP, (C) MnBP, and (D) MEHHP, among populations of children worldwide. Red bar indicates median concentrations of urinary phthalate metabolites of this study. More detailed information is shown in Table S6.

1353

1354

I. Lee et al. / Science of the Total Environment 654 (2019) 1350–1357 Table 3 Association between oxidative stress biomarkers and phthalate metabolites. Crude (n = 109)a

Fig. 2. (A) Hazard quotients (HQs) estimated from urinary concentrations of phthalate metabolites among the current population of children. Box plot for each phthalate shows 5th, 10th (lower whisker), 25th (lower line of the box), 75th (upper line of the box), 90th (upper whisker), and 95th percentiles of distribution. The median is shown as the line in the middle. (B) Percent (%) of HQs categorized into three groups (HQ b 0.5, 0.5 ≤ HQ b 1, and HQ ≥ 1). Number in the stacked bar indicates number of participants according to the HQ groups.

3.2. Urinary levels of phthalate metabolites All phthalate metabolites were detected in N75% of the urine samples except for monopentyl phthalate (MPeP), monoisopropyl phthalate (MiPrP), monohexyl phthalate (MHxP), monooctyl phthalate (MOP), and monoisononyl phthalate (MiNP) (Table 2). The median value of detection was greatest for MEP (259 ng/mL), and ∑DEHPm (272 ng/mL), i.e., the sum of DEHP metabolites including a primary metabolite (MEHP) and four secondary metabolites (MCMHP, MECPP, MEHHP, and MEOHP), among the measured phthalate metabolites. Among the DEHP metabolites, MECPP was detected at the highest concentrations, followed by MEHHP, MCMHP, MEOHP, and MEHP, in descending order. Similarly, in many studies, MECPP showed the highest median concentrations among the analyzed DEHP metabolites (Table S6; Casas et al., 2011; Colacino et al., 2011; Hartmann et al., 2015; Kasper-Sonnenberg et al., 2012; Koch et al., 2011; Langer et al., 2014; Liao et al., 2018; Lin et al., 2011; Teitelbaum et al., 2008; Wang et al., 2015; Wolff et al., 2007). Most of the phthalate metabolites in the urine samples of the current population of children showed relatively high levels compared to the reported values from previous studies on children worldwide (Table S6. Hartman et al., 2015; Rocha et al., 2017; Saravanabhavan et al., 2013; Wang et al., 2015; Liao et al., 2017; Frederiksen et al., 2011; Langer et al., 2014; Frederiksen et al., 2013; Colacino et al., 2011; Koch et al., 2007; Koch et al., 2011; Kasper-Sonnenberg et al., 2012; Kasper-Sonnenberg et al., 2014; Myridakis et al., 2016; KFDA, 2007; Cho et al., 2010; Casas et al., 2011; Lin et al., 2011; Hsu et al., 2012; Teitelbaum et al., 2008; Wolff et al., 2007). In particular, MiBP and MnBP were detected at the highest levels in the present population

8-OHdG MMP MEP MiBP MnBP MBzP MCHP MCPP MEHP MCMHP MECPP MEHHP MEOHP ∑DEHP MiDP MDA MMP MEP MiBP MnBP MBzP MCHP MCPP MEHP MCMHP MECPP MEHHP MEOHP ∑DEHP MiDP

Adjusted (n = 104)b

β (95% CI)

p-Value β (95% CI)

p-Value

0.339 (0.054, 0.623) 0.055 (−0.182, 0.292) 0.255 (0.035, 0.475) 0.230 (0.012, 0.447) 0.313 (0.095, 0.531) 0.099 (−0.188, 0.387) 0.293 (0.062, 0.524) 0.067 (−0.128, 0.262) 0.459 (0.153, 0.764) 0.511 (0.218, 0.805) 0.458 (0.178, 0.738) 0.427 (0.131, 0.723) 0.505 (0.197, 0.814) 0.241 (−0.073, 0.554)

0.020 0.648 0.024 0.039 0.005 0.495 0.014 0.498 0.004 0.001 0.002 0.005 0.002 0.131

0.311 (0.009, 0.612) 0.048 (−0.204, 0.300) 0.285 (0.059, 0.511) 0.269 (0.036, 0.502) 0.310 (0.080, 0.540) 0.088 (−0.218, 0.394) 0.320 (0.073, 0.567) 0.036 (−0.168, 0.239) 0.468 (0.154, 0.782) 0.493 (0.190, 0.795) 0.461 (0.176, 0.746) 0.418 (0.115, 0.721) 0.494 (0.177, 0.810) 0.214 (−0.121, 0.548)

0.044 0.705 0.014 0.024 0.009 0.570 0.012 0.730 0.004 0.002 0.002 0.007 0.003 0.208

3.173 (0.501, 5.845) 2.282 (0.098, 4.467) 0.794 (−1.322, 2.910) 2.666 (0.641, 4.690) 1.784 (−0.312, 3.880) 2.186 (−0.488, 4.860) 1.806 (−0.401, 4.013) 1.783 (−0.019, 3.586) 0.362 (−2.625, 3.349) 2.271 (−0.606, 5.148) 1.989 (−0.742, 4.719) 2.893 (0.061, 5.726) 2.164 (−0.846, 5.175) −0.782 (−3.758, 2.194)

0.020 0.041 0.458 0.010 0.094 0.108 0.108 0.053 0.811 0.121 0.152 0.045 0.157 0.604

3.766 (0.987, 6.544) 2.584 (0.288, 4.881) 0.812 (−1.359, 2.983) 2.432 (0.252, 4.612) 1.425 (−0.781, 3.631) 1.899 (−0.939, 4.736) 1.643 (−0.716, 4.003) 1.739 (−0.133, 3.611) 0.860 (−2.195, 3.914) 2.381 (−0.549, 5.310) 2.142 (−0.623, 4.908) 2.985 (0.107, 5.863) 2.370 (−0.688, 5.428) −0.854 (−3.997, 2.290)

0.008 0.028 0.460 0.029 0.203 0.187 0.170 0.068 0.578 0.110 0.128 0.042 0.127 0.591

Phthalate metabolites were ln-transformed. Boldface values indicate statistical significance (p b 0.05). a Adjusted only for urinary creatinine. b Adjusted for age, sex, BMI, and urinary creatinine.

compared to other populations of children worldwide (Fig. 1). In addition, monomethyl phthalate (MMP), MEP, and DEHP metabolites were also detected at higher levels in the current population than in the populations of children in other countries (Fig. 1, Table S6). Depending on the study, the characteristics of the study populations such as age and BMI vary, and as phthalate exposure may be associated with such characteristics (Valvi et al., 2015), direct comparison may not be possible. However, all of these studies were conducted on the children who were no older than early teens. Our observation of relatively higher urinary levels of phthalate metabolites in Saudi Arabian children is in line with two previous studies conducted in the general populations of Middle Eastern countries, even though the target populations were not limited to children (Asimakopoulos et al., 2016; Guo et al., 2011). Among general populations, compared to other Asian countries, phthalate metabolite concentrations were higher among adults in Kuwait (Guo et al., 2011). In addition, similar to our observation, MEP was detected at higher levels in urine samples from adults in Kuwait (n = 46) and in the general population of Saudi Arabia (n = 130) (Asimakopoulos et al., 2016; Guo et al., 2011). The creatinine-corrected concentrations of phthalate metabolites were correlated with each other (Table S7). In particular, concentrations of MnBP showed a relatively strong correlation (rho N0.5) with those of MBzP, mono(3-carboxypropyl) phthalate (MCPP), and secondary metabolites of DEHP. The strong correlations between metabolites of different parent phthalates suggest existence of common sources of exposure to these compounds among the children of Saudi Arabia. Similar strong correlations have been reported between several phthalate metabolites in previous studies (Hartmann et al., 2015; Larsson et al., 2014), and these correlated metabolites have been shown to have a positive correlation with the same sources of exposure (Lewis et al., 2013). For example, in a study on girls 8–13 years of age, urinary

1355

I. Lee et al. / Science of the Total Environment 654 (2019) 1350–1357 Table 4 Spearman correlations between creatinine-corrected concentrations of phthalate metabolites in urine samples and potential exposure-related sources in children. Foods or drinks

MMP MEP MnBP MiBP MBzP MCHP MCPP MEHP MCMHP MECPP MEHHP MEOHP ∑DEHP MiDP

ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value ρ p-Value

Personal care products

Eat-out (n = 106)

Hamburger (n = 107)

Ice cream (n = 105)

Take-out beverage (n = 109)

PET bottled beverage (n = 109)

Canned food (n = 108)

Jam (n = 107)

Small fish (n = 105)

Sun cream/sun spray (n = 108)

Liquid soap (n = 107)

−0.1338 0.1714 0.1721 0.0777 0.1510 0.1223 0.1323 0.1763 0.0898 0.3600 0.2210 0.0228 0.1458 0.1360 −0.0499 0.6114 0.0751 0.4440 0.0596 0.5440 0.1140 0.2445 0.0792 0.4197 0.0547 0.5777 0.0816 0.4054

0.1524 0.1172 −0.1692 0.0814 0.0265 0.7867 0.1146 0.2397 0.0115 0.9068 0.1665 0.0865 0.0618 0.5275 0.2348 0.0149 0.0617 0.5277 −0.0130 0.8940 0.0748 0.4439 0.0402 0.6810 0.0739 0.4492 0.1921 0.0474

−0.0684 0.4882 −0.0935 0.3426 −0.0091 0.9270 0.1197 0.2241 −0.0868 0.3784 0.0645 0.5135 0.1344 0.1716 0.0939 0.3406 0.2238 0.0218 0.0773 0.4333 0.1177 0.2319 0.1004 0.3081 0.1168 0.2354 −0.0382 0.6991

−0.0840 0.3853 −0.1723 0.0732 −0.1111 0.2500 −0.0630 0.5154 −0.0440 0.6494 −0.0131 0.8929 −0.1461 0.1297 −0.1967 0.0404 −0.2194 0.0219 −0.3267 0.0005 −0.2411 0.0116 −0.2664 0.0051 −0.2635 0.0056 −0.2110 0.0276

−0.1075 0.2657 −0.0955 0.3233 0.0283 0.7699 −0.0567 0.5579 0.0498 0.6074 −0.2150 0.0247 −0.0133 0.8910 −0.0537 0.5794 0.1276 0.1862 0.1669 0.0829 0.0977 0.3120 0.0893 0.3558 0.1051 0.2768 −0.0425 0.6610

0.1235 0.2030 0.0685 0.4815 −0.0586 0.5471 −0.0485 0.6181 0.0211 0.8287 −0.0316 0.7454 −0.0606 0.5330 −0.0623 0.5216 −0.0383 0.6940 −0.0023 0.9809 −0.0128 0.8957 −0.0307 0.7523 −0.0189 0.8460 0.1992 0.0387

0.0371 0.7043 −0.2744 0.0042 −0.0003 0.9972 −0.0723 0.4596 −0.0385 0.6940 −0.0790 0.4186 0.0182 0.8521 0.0940 0.3356 0.0159 0.8713 0.0055 0.9555 0.0143 0.8835 0.0096 0.9221 0.0170 0.8624 −0.0820 0.4014

−0.1545 0.1156 0.1323 0.1786 −0.1068 0.2780 −0.0259 0.7935 −0.1005 0.3077 −0.0305 0.7573 −0.1039 0.2916 −0.2283 0.0192 −0.2819 0.0036 −0.2727 0.0049 −0.2749 0.0045 −0.3042 0.0016 −0.2968 0.0021 −0.0024 0.9810

0.0282 0.7717 −0.0689 0.4784 0.0109 0.9109 −0.0437 0.6531 −0.0672 0.4893 0.0498 0.6091 0.2239 0.0198 0.0255 0.7930 −0.0061 0.9501 0.0381 0.6957 0.0139 0.8866 0.0292 0.7640 0.0451 0.6431 −0.1025 0.2910

0.0255 0.7943 −0.0630 0.5191 −0.1505 0.1219 −0.1099 0.2596 −0.2315 0.0164 −0.0191 0.8456 −0.0525 0.5912 −0.1353 0.1647 −0.1975 0.0415 −0.2236 0.0206 −0.1872 0.0535 −0.1812 0.0618 −0.2004 0.0385 0.1405 0.1490

Boldface values indicate statistical significance (p b 0.05).

concentrations of MnBP, MiBP, and DEHP metabolites were positively associated with the use of colored cosmetics and hair products (Lewis et al., 2013). Efforts to identify exposure sources of phthalates among the children of Saudi Arabia are warranted. 3.3. Estimated daily intake and associated risks Among LMW phthalates, DI estimates were highest for DEP, followed by DnBP, DiBP, and BBzP. For DEP, the median DI was estimated at 49.8 μg/kg bw-day with an IQR between 23.4 and 111.3 μg/kg bw-day. For DnBP and DiBP, the median DI was estimated at 27.0 μg/kg bw-day (IQR, 14.3–46.8), and 28.0 μg/kg bw-day (IQR, 11.4–58.4), respectively. For BBzP, however, the median DI was negligible, i.e., 0.60 μg/kg bw-day (IQR, 0.33–1.24). The median DI for DEHP metabolites was 15.3 μg/kg bw-day (IQR, 8.72–24.96). Among the participating children, approximately one-third (37 children, 33.9%) showed HQs N 1 for DEHP. For DiBP, DnBP, and DEP, 20 (18%), 8 (7.3%) and 1 (0.9%) participant(s) exceeded an HQ of 1. For BBzP, however, none exceeded an HQ of 1 (Fig. 2). Similar to the current population, DEHP has been identified as the phthalate with the greatest risk in many other countries. For example, 7.5% of the children between the ages of 2 and 14 years in Germany (n = 239), 8% in Korea (n = 39, 9–12 years old), and 17.3% in Brazil (n = 300, 6–14 years old) were estimated to have been exposed to DEHP at levels greater than the RfD suggested by the USEPA (Kim et al., 2014; Rocha et al., 2017; Wittassek et al., 2007). It is noteworthy that there were two- to fourfold more children at potential risk of DEHP exposure in the current population of Saudi Arabia compared to those in other countries. 3.4. Associations with oxidative stress markers in urine Urinary levels of phthalate metabolites were positively associated with oxidative stress markers, such as 8-hydroxy-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA), measured in the children's

urine samples (Table 3). In both crude (adjusted only for urinary creatinine) and adjusted models (adjusted for urinary creatinine, age, sex, and BMI), urinary 8-OHdG levels showed positive associations with urinary levels of MMP, MiBP, MnBP, MBzP, MCPP, secondary metabolites of DEHP, and sum of DEHP metabolites. Similar positive associations were observed between urinary MDA levels and phthalate metabolites, i.e., MMP, MEP, MnBP, and MEOHP, in both crude and adjusted models. Based on PCA with varimax rotation and regression analysis on the phthalate metabolites, several phthalate metabolites were found to be associated with the oxidative stress markers as well. Three factors (Eigenvalues N1.000) were retained; the first principal component (PC1) explained 43.5% of the total variance, and PC2 and PC3 explained 11.2% and 10.2% of the total variance, respectively. Detailed information on the factor pattern is presented in Table S8 of the Supplementary Information. In the adjusted model, both factors 1 and 2 were positively associated with 8-OHdG, and factor 2 was positively associated with MDA (Table S9). Associations between phthalates and oxidative stress have been reported in many observational studies worldwide (Asimakopoulos et al., 2016; Ferguson et al., 2017; Ferguson et al., 2011; Ferguson et al., 2012; Ferguson et al., 2015; Guo et al., 2014; Holland et al., 2016; Hong et al., 2009; Kim et al., 2014; Rocha et al., 2017; Wang et al., 2011; Wu et al., 2017). For example, in pregnant women, urinary levels of phthalate metabolites were associated with higher 8-OHdG and 8-isoprostane concentrations (Ferguson et al., 2015). Phthalate-induced oxidative stress has been demonstrated in several in vitro and in vivo studies (Aly et al., 2016; Seo et al., 2004; Shono and Taguchi, 2014; Lambrot et al., 2008). For example, in the human fetal testis in vitro, treatment with MEHP reduced the number of germ cells by causing apoptosis (Lambrot et al., 2008). The oxidative stress on the testis by phthalates was also observed in animal models (Aly et al., 2016; Shono and Taguchi, 2014). In four-week-old pubertal male rats, a 3-day exposure to MBP (chow containing 2% MBP) decreased testis weight and increased urinary 8-OHdG levels (Shono and Taguchi, 2014). In another

1356

I. Lee et al. / Science of the Total Environment 654 (2019) 1350–1357

study on adult male albino rats treated with DBP for 15 days, oxidative stress was observed in the testis (Aly et al., 2016). 3.5. Potential exposure sources Several behavioral factors were identified to be associated with higher levels of phthalate metabolites in urine samples (Table 4). Among the dietary factors, the frequency of eating out and consumption of hamburgers were identified to be positively correlated with urinary monocyclohexyl phthalate (MCHP) and MEHP concentrations, respectively. In addition, frequencies of hamburger and canned food consumption were positively correlated with urinary monoisodecyl phthalate (MiDP) levels, and frequencies of ice cream consumption were positively correlated with urinary MCMHP levels, respectively. Among personal care products (PCPs), the use of sun cream or sun spray was positively correlated with MCPP concentrations. However, consumption of takeout, PET bottled beverages, jam, and small fish, as well as the use of liquid soap, showed a negative correlation with several phthalate metabolites in the urine samples. Food has been suggested as one of the main exposure sources of HMW phthalates (Fierens et al., 2012; Koch et al., 2013; Wormuth et al., 2006). After a 48-h fast, urinary concentrations of HMW phthalate (DEHP, DiNP, diisodecyl phthalate (DiDP), and di(2-propylheptyl) phthalate (DPHP)) metabolites were reduced, whereas those for LMW phthalates did not change (Koch et al., 2013). Indeed, in a Belgian study, DEHP was detected in food products and packaging materials at the highest concentrations among eight phthalate compounds measured (Fierens et al., 2012). Dicyclohexyl phthalate (DCHP) was also detected in some food and packaging in the Belgian study. It was an interesting observation that the use of liquid soap was negatively correlated with the urinary concentrations of DEHP metabolites. A clear explanation for this negative correlation is not available, but the fact that hand-to-mouth behavior may potentially contribute to exposures to certain phthalates may be related. Inadvertent intake via the hands is one of the most well-recognized sources of exposure to chemicals, especially those in dust (Langer et al., 2010). Although relevant data are not reported for Saudi Arabia, DEHP has been detected at the highest concentrations in indoor dust in several countries (Bekö et al., 2013; Guo and Kannan, 2011; Langer et al., 2010). Therefore, the negative correlation between the use of liquid soap and urinary levels of DEHP metabolites may, in part, be explained by the reduced incidental ingestion of dust via the hands (Özkaynak et al., 2011; Watkins et al., 2011). In this study, we could not identify possible sources that may explain the urinary levels of MiBP, MnBP, and MEP. Further studies that investigate sources and pathways of exposure to their parent phthalates are warranted among the children of Saudi Arabia. 4. Conclusions Our observations clearly show that the children of Saudi Arabia are at potential risks from phthalate exposure. Several phthalate metabolites, including MiBP and MnBP, were detected at the higher levels in Saudi Arabian children than in the children populations of other countries. Many of the phthalate metabolites were positively associated with oxidative stress markers. In addition, approximately one-third of children were exposed to DEHP at levels greater than the RfD or TDI established for DEHP. While several potential sources of exposure to phthalates were suggested for this population of children, our observations are rather preliminary, and hence, more refined exposure assessment should be developed for MiBP, MnBP, and MEP. Due to the cross-sectional study design with a small number of subjects, our study has several intrinsic limitations. Therefore, causality cannot be established, and these observations should be validated in larger populations and preferably with a longitudinal study design. Our study was the first that focused on urinary concentrations of major phthalate metabolites in children from Saudi Arabia. Very high

levels of phthalate exposure along with a positive association with oxidative stress markers among the children outline the importance of follow-up efforts to identify possible health consequences of phthalate exposure. Acknowledgments This study was supported by the TRE program, King Saud University, and the Korea Ministry of Environment (MOE) (1485014553). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.11.025. References Aly, H.A., Hassan, M.H., El-Beshbishy, H.A., Alahdal, A.M., Osman, A.M., 2016. Dibutyl phthalate induces oxidative stress and impairs spermatogenesis in adult rats. Toxicol. Ind. Health 32, 1467–1477. Asimakopoulos, A.G., Xue, J., De Carvalho, B.P., Iyer, A., Abualnaja, K.O., Yaghmoor, S.S., et al., 2016. Urinary biomarkers of exposure to 57 xenobiotics and its association with oxidative stress in a population in Jeddah, Saudi Arabia. Environ. Res. 150, 573–581. Ballauff, A., Kersting, M., Manz, F., 1988. Do children have an adequate fluid intake? Water balance studies carried out at home. Ann. Nutr. Metab. 32, 332–339. Barr, D.B., Wilder, L.C., Caudill, S.P., Gonzalez, A.J., Needham, L.L., Pirkle, J.L., 2005. Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environ. Health Perspect. 113, 192–200. Bekö, G., Weschler, C.J., Langer, S., Callesen, M., Toftum, J., Clausen, G., 2013. Children's phthalate intakes and resultant cumulative exposures estimated from urine compared with estimates from dust ingestion, inhalation and dermal absorption in their homes and daycare centers. PLoS One 8, e62442. Braun, J.M., Sathyanarayana, S., Hauser, R., 2013. Phthalate exposure and children's health. Curr. Opin. Pediatr. 25, 247–254. Casas, L., Fernandez, M.F., Llop, S., Guxens, M., Ballester, F., Olea, N., et al., 2011. Urinary concentrations of phthalates and phenols in a population of Spanish pregnant women and children. Environ. Int. 37, 858–866. Cho, S.C., Bhang, S.Y., Hong, Y.C., Shin, M.S., Kim, B.N., Kim, J.W., et al., 2010. Relationship between environmental phthalate exposure and the intelligence of school-age children. Environ. Health Perspect. 118, 1027–1032. Choi, W., Kim, S., Baek, Y.W., Choi, K., Lee, K., Kim, S., et al., 2017. Exposure to environmental chemicals among Korean adults-updates from the second Korean National Environmental Health Survey (2012–2014). Int. J. Hyg. Environ. Health 220, 29–35. Colacino, J.A., Soliman, A.S., Calafat, A.M., Nahar, M.S., Zomeren-Dohm, A.V., Hablas, A., et al., 2011. Exposure to phthalates among premenstrual girls from rural and urban Gharbiah, Egypt: a pilot exposure assessment study. Environ. Health 10, 40. Dodson, R.E., Nishioka, M., Standley, L.J., Perovich, L.J., Brody, J.G., Rudel, R.A., 2012. Endocrine disruptors and asthma-associated chemicals in consumer products. Environ. Health Perspect. 120, 935–943. Ejaredar, M., Nyanza, E.C., Ten Eycke, K., Dewey, D., 2015. Phthalate exposure and childrens neurodevelopment: a systematic review. Environ. Res. 142, 51–60. Ferguson, K.K., Loch-Caruso, R., Meeker, J.D., 2011. Urinary phthalate metabolites in relation to biomarkers of inflammation and oxidative stress: NHANES 1999–2006. Environ. Res. 111, 718–726. Ferguson, K.K., Loch-Caruso, R., Meeker, J.D., 2012. Exploration of oxidative stress and inflammatory markers in relation to urinary phthalate metabolites: NHANES 1999–2006. Environ. Sci. Technol. 46, 477–485. Ferguson, K.K., McElrath, T.F., Chen, Y.H., Mukherjee, B., Meeker, J.D., 2015. Urinary phthalate metabolites and biomarkers of oxidative stress in pregnant women: a repeated measures analysis. Environ. Health Perspect. 123, 210–216. Ferguson, K.K., Chen, Y.H., VanderWeele, T.J., McElrath, T.F., Meeker, J.D., Mukherjee, B., 2017. Mediation of the relationship between maternal phthalate exposure and preterm birth by oxidative stress with repeated measurements across pregnancy. Environ. Health Perspect. 125, 488–494. Fierens, T., Servaes, K., Van Holderbeke, M., Geerts, L., De Henauw, S., Sioen, I., et al., 2012. Analysis of phthalates in food products and packaging materials sold on the Belgian market. Food Chem. Toxicol. 50, 2575–2583. Frederiksen, H., Aksglaede, L., Sorensen, K., Skakkebaek, N.E., Juul, A., Andersson, A.M., 2011. Urinary excretion of phthalate metabolites in 129 healthy Danish children and adolescents: estimation of daily phthalate intake. Environ. Res. 111, 656–663. Frederiksen, H., Nielsen, J.K., Morck, T.A., Hansen, P.W., Jensen, J.F., Nielsen, O., et al., 2013. Urinary excretion of phthalate metabolites, phenols and parabens in rural and urban Danish mother-child pairs. Int. J. Hyg. Environ. Health 216, 772–783. Guo, Y., Kannan, K., 2011. Comparative assessment of human exposure to phthalate esters from house dust in China and the United States. Environ. Sci. Technol. 45, 3788–3794. Guo, Y., Alomirah, H., Cho, H.S., Minh, T.B., Mohd, M.A., Nakata, H., et al., 2011. Occurrence of phthalate metabolites in human urine from several Asian countries. Environ. Sci. Technol. 45, 3138–3144. Guo, Y., Weck, J., Sundaram, R., Goldstone, A.E., Louis, G.B., Kannan, K., 2014. Urinary concentrations of phthalates in couples planning pregnancy and its association with 8-

I. Lee et al. / Science of the Total Environment 654 (2019) 1350–1357 hydroxy-2′-deoxyguanosine, a biomarker of oxidative stress: longitudinal investigation of fertility and the environment study. Environ. Sci. Technol. 48, 9804–9811. Hartmann, C., Uhl, M., Weiss, S., Koch, H.M., Scharf, S., Konig, J., 2015. Human biomonitoring of phthalate exposure in Austrian children and adults and cumulative risk assessment. Int. J. Hyg. Environ. Health 218, 489–499. Holland, N., Huen, K., Tran, V., Street, K., Nguyen, B., Bradman, A., et al., 2016. Urinary phthalate metabolites and biomarkers of oxidative stress in a Mexican-American cohort: variability in early and late pregnancy. Toxics 4 (1), 7. Hong, Y.C., Park, E.Y., Park, M.S., Ko, J.A., Oh, S.Y., Kim, H., et al., 2009. Community level exposure to chemicals and oxidative stress in adult population. Toxicol. Lett. 184, 139–144. Hong, S.K., Nam, H.S., Jung, K.K., Kang, I.H., Kim, T.S., Cho, S.E., et al., 2010. Development and validation of on-line column switching HPLC-MS/MS method for 10 phthalate metabolites in human urine. Kor. J. Environ. Health Sci. Eng. 36 (6), 510–517. Hornung, R.W., Reed, L.D., 1990. Estimation of average concentration in the presence of nondetectable values. Appl. Occup. Environ. Hyg. 5, 46–51. Hsu, N.Y., Lee, C.C., Wang, J.Y., Li, Y.C., Chang, H.W., Chen, C.Y., et al., 2012. Predicted risk of childhood allergy, asthma, and reported symptoms using measured phthalate exposure in dust and urine. Indoor Air 22, 186–199. Itoh, H., Yoshida, K., Masunaga, S., 2007. Quantitative identification of unknown exposure pathways of phthalates based on measuring their metabolites in human urine. Environ. Sci. Technol. 41, 4542–4547. Kasper-Sonnenberg, M., Koch, H.M., Wittsiepe, J., Wilhelm, M., 2012. Levels of phthalate metabolites in urine among mother-child-pairs - results from the Duisburg birth cohort study, Germany. Int. J. Hyg. Environ. Health 215, 373–382. Kasper-Sonnenberg, M., Koch, H.M., Wittsiepe, J., Bruning, T., Wilhelm, M., 2014. Phthalate metabolites and bisphenol A in urines from German school-aged children: results of the Duisburg birth cohort and Bochum cohort studies. Int. J. Hyg. Environ. Health 217, 830–838. Kawakami, T., Isama, K., Matsuoka, A., 2011. Analysis of phthalic acid diesters, monoester, and other plasticizers in polyvinyl chloride household products in Japan. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng. 46, 855–864. Korea Food and Drug Administration (KFDA), 2007. Exposure and Human Risk Assessment of Phthalates, Seoul. Kim, S., Kang, S., Lee, G., Lee, S., Jo, A., Kwak, K., et al., 2014. Urinary phthalate metabolites among elementary school children of Korea: sources, risks, and their association with oxidative stress marker. Sci. Total Environ. 472, 49–55. Kim, B., Jung, W., Kho, Y., 2017. Quantification of malondialdehyde in human urine by HPLC-DAD and derivatization with 2, 4-dinitrophenylhydrazine. Bull. Kor. Chem. Soc. 38 (6), 642–645. Koch, H.M., Bolt, H.M., Preuss, R., Angerer, J., 2005. New metabolites of di(2-ethylhexyl) phthalate (DEHP) in human urine and serum after single oral doses of deuteriumlabelled DEHP. Arch. Toxicol. 79, 367–376. Koch, H.M., Becker, K., Wittassek, M., Seiwert, M., Angerer, J., Kolossa-Gehring, M., 2007. Di-n-butylphthalate and butylbenzylphthalate - urinary metabolite levels and estimated daily intakes: pilot study for the German environmental survey on children. J. Expo. Sci. Environ. Epidemiol. 17, 378–387. Koch, H.M., Wittassek, M., Bruning, T., Angerer, J., Heudorf, U., 2011. Exposure to phthalates in 5–6 years old primary school starters in Germany-a human biomonitoring study and a cumulative risk assessment. Int. J. Hyg. Environ. Health 214, 188–195. Koch, H.M., Christensen, K.L., Harth, V., Lorber, M., Bruning, T., 2012. Di-n-butyl phthalate (DnBP) and diisobutyl phthalate (DiBP) metabolism in a human volunteer after single oral doses. Arch. Toxicol. 86, 1829–1839. Koch, H.M., Lorber, M., Christensen, K.L., Palmke, C., Koslitz, S., Bruning, T., 2013. Identifying sources of phthalate exposure with human biomonitoring: results of a 48 h fasting study with urine collection and personal activity patterns. Int. J. Hyg. Environ. Health 216, 672–681. Koch, H.M., Ruther, M., Schutze, A., Conrad, A., Palmke, C., Apel, P., et al., 2017. Phthalate metabolites in 24-h urine samples of the German Environmental Specimen Bank (ESB) from 1988 to 2015 and a comparison with US NHANES data from 1999 to 2012. Int. J. Hyg. Environ. Health 220, 130–141. Koniecki, D., Wang, R., Moody, R.P., Zhu, J., 2011. Phthalates in cosmetic and personal care products: concentrations and possible dermal exposure. Environ. Res. 111, 329–336. Lambrot, R., Muczynski, V., Lécureuil, C., Angenard, G., Coffigny, H., Pairault, C., et al., 2008. Phthalates impair germ cell development in the human fetal testis in vitro without change in testosterone production. Environ. Health Perspect. 117 (1), 32–37. Langer, S., Weschler, C.J., Fischer, A., Bekö, G., Toftum, J., Clausen, G., 2010. Phthalate and PAH concentrations in dust collected from Danish homes and daycare centers. Atmos. Environ. 44, 2294–2301. Langer, S., Beko, G., Weschler, C.J., Brive, L.M., Toftum, J., Callesen, M., et al., 2014. Phthalate metabolites in urine samples from Danish children and correlations with phthalates in dust samples from their homes and daycare centers. Int. J. Hyg. Environ. Health 217, 78–87. Larsson, K., Ljung Bjorklund, K., Palm, B., Wennberg, M., Kaj, L., Lindh, C.H., et al., 2014. Exposure determinants of phthalates, parabens, bisphenol A and triclosan in Swedish mothers and their children. Environ. Int. 73, 323–333.

1357

Lewis, R.C., Meeker, J.D., Peterson, K.E., Lee, J.M., Pace, G.G., Cantoral, A., et al., 2013. Predictors of urinary bisphenol a and phthalate metabolite concentrations in Mexican children. Chemosphere 93, 2390–2398. Liao, C., Liu, W., Zhang, J., Shi, W., Wang, X., Cai, J., et al., 2018. Associations of urinary phthalate metabolites with residential characteristics, lifestyles, and dietary habits among young children in Shanghai, China. Sci. Total Environ. 616–617, 1288–1297. Lin, S., Ku, H.Y., Su, P.H., Chen, J.W., Huang, P.C., Angerer, J., et al., 2011. Phthalate exposure in pregnant women and their children in Central Taiwan. Chemosphere 82, 947–955. Myridakis, A., Chalkiadaki, G., Fotou, M., Kogevinas, M., Chatzi, L., Stephanou, E.G., 2016. Exposure of preschool-age Greek children (RHEA cohort) to bisphenol A, parabens, phthalates, and organophosphates. Environ. Sci. Technol. 50, 932–941. Özkaynak, H., Xue, J., Zartarian, V.G., Glen, G., Smith, L., 2011. Modeled estimates of soil and dust ingestion rates for children. Risk Anal. 31, 592–608. Rocha, B.A., Asimakopoulos, A.G., Barbosa Jr., F., Kannan, K., 2017. Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage. Sci. Total Environ. 586, 152–162. Saravanabhavan, G., Guay, M., Langlois, E., Giroux, S., Murray, J., Haines, D., 2013. Biomonitoring of phthalate metabolites in the Canadian population through the Canadian Health Measures Survey (2007–2009). Int. J. Hyg. Environ. Health 216, 652–661. Schecter, A., Lorber, M., Guo, Y., Wu, Q., Yun, S.H., Kannan, K., et al., 2013. Phthalate concentrations and dietary exposure from food purchased in New York State. Environ. Health Perspect. 121, 473–494. Seo, K.W., Kim, K.B., Kim, Y.J., Choi, J.Y., Lee, K.T., Choi, K.S., 2004. Comparison of oxidative stress and changes of xenobiotic metabolizing enzymes induced by phthalates in rats. Food Chem. Toxicol. 42, 107–114. Shono, T., Taguchi, T., 2014. Short-time exposure to mono-n-butyl phthalate (MBP)-induced oxidative stress associated with DNA damage and the atrophy of the testis in pubertal rats. Environ. Sci. Pollut. Res. Int. 21, 3187–3190. Smerieri, A., Testa, C., Lazzeroni, P., Nuti, F., Grossi, E., Cesari, S., et al., 2015. Di-(2ethylhexyl) phthalate metabolites in urine show age-related changes and associations with adiposity and parameters of insulin sensitivity in childhood. PLoS One 10, e0117831. Teitelbaum, S.L., Britton, J.A., Calafat, A.M., Ye, X., Silva, M.J., Reidy, J.A., et al., 2008. Temporal variability in urinary concentrations of phthalate metabolites, phytoestrogens and phenols among minority children in the United States. Environ. Res. 106, 257–269. U.S. Environmental Protection Agency, 1990. Dibutyl Phthalate (CASRN 84-74-2). Integrated Risk Information System (IRIS). Available: https://www.epa.gov/iris, Accessed date: 23 August 2018. U.S. Environmental Protection Agency, 1993a. Diethyl Phthalate (CASRN 84-66-2). Integrated Risk Information System (IRIS). Available: https://www.epa.gov/iris, Accessed date: 23 August 2018. U.S. Environmental Protection Agency, 1993b. Butylbenzyl Phthalate (CASRN 85-68-7). Integrated Risk Information System (IRIS). Available: https://www.epa.gov/iris, Accessed date: 23 August 2018. U.S. Environmental Protection Agency, 1993c. Di(2-ethylhexyl)phthalate (DEHP) (CASRN 117-81-7). Integrated Risk Information System (IRIS). Available: https://www.epa. gov/iris, Accessed date: 23 August 2018. Valvi, D., Monfort, N., Ventura, R., Casas, M., Casas, L., Sunyer, J., Vrijheid, M., 2015. Variability and predictors of urinary phthalate metabolites in Spanish pregnant women. Int. J. Hyg. Environ. Health 218 (2), 220–231. Wang, Q., Wang, L., Chen, X., Rao, K.M., Lu, S.Y., Ma, S.T., et al., 2011. Increased urinary 8hydroxy-2′-deoxyguanosine levels in workers exposed to di-(2-ethylhexyl) phthalate in a waste plastic recycling site in China. Environ. Sci. Pollut. Res. Int. 18, 987–996. Wang, B., Wang, H., Zhou, W., Chen, Y., Zhou, Y., Jiang, Q., 2015. Urinary excretion of phthalate metabolites in school children of China: implication for cumulative risk assessment of phthalate exposure. Environ. Sci. Technol. 49, 1120–1129. Watkins, D.J., McClean, M.D., Fraser, A.J., Weinberg, J., Stapleton, H.M., Sjodin, A., et al., 2011. Exposure to PBDEs in the office environment: evaluating the relationships between dust, handwipes, and serum. Environ. Health Perspect. 119, 1247–1252. Wittassek, M., Heger, W., Koch, H.M., Becker, K., Angerer, J., Kolossa-Gehring, M., 2007. Daily intake of di(2-ethylhexyl)phthalate (DEHP) by German children - a comparison of two estimation models based on urinary DEHP metabolite levels. Int. J. Hyg. Environ. Health 210, 35–42. Wolff, M.S., Teitelbaum, S.L., Windham, G., Pinney, S.M., Britton, J.A., Chelimo, C., et al., 2007. Pilot study of urinary biomarkers of phytoestrogens, phthalates, and phenols in girls. Environ. Health Perspect. 115, 116–121. Wormuth, M., Scheringer, M., Vollenweider, M., Hungerbuhler, K., 2006. What are the sources of exposure to eight frequently used phthalic acid esters in Europeans? Risk Anal. 26, 803–824. Wu, H., Olmsted, A., Cantonwine, D.E., Shahsavari, S., Rahil, T., Sites, C., et al., 2017. Urinary phthalate and phthalate alternative metabolites and isoprostane among couples undergoing fertility treatment. Environ. Res. 153, 1–7. Zota, A.R., Calafat, A.M., Woodruff, T.J., 2014. Temporal trends in phthalate exposures: findings from the National Health and Nutrition Examination Survey, 2001–2010. Environ. Health Perspect. 122, 235–241.