Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics of pregnant women

Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics of pregnant women

STOTEN-135671; No of Pages 9 Science of the Total Environment xxx (xxxx) xxx Contents lists available at ScienceDirect Science of the Total Environm...

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STOTEN-135671; No of Pages 9 Science of the Total Environment xxx (xxxx) xxx

Contents lists available at ScienceDirect

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

Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics of pregnant women Jing Dong a,1, Yanan Ma b,1, Kunkun Leng a, Lingling Wei a, Ying Wang c, Chang Su d, Ming Liu e, Jie Chen a,⁎ a

Department of Occupational and Environmental Health, School of Public Health, China Medical University, No. 77 Puhe Road, Shenyang 110122, PR China Department of Biostatistics and Epidemiology, School of Public Health, China Medical University, No. 77 Puhe Road, Shenyang 110122, PR China Department of Gynaecology and Obstetrics, Shengjing Hospital of China Medical University, Shenyang, Liaoning, PR China d Yale School of Medicine, New Haven, CT, USA e Bioinformatics and Computational Biology Program, Worcester Polytechnic Institute, Worcester, MA, USA 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

• Living near the motor vehicle traffic may increase urinary concentrations of MEHP and MEHHP. • Residential renovation may increase urinary concentrations of MEHP and MEHHP. • Using air freshener may increase urinary concentrations of MEHP. • Moldy walls may increase urinary concentrations of MEHP and MEHHP. • Using household humidifier may decrease urinary concentrations of MEHP.

a r t i c l e

i n f o

Article history: Received 18 September 2019 Received in revised form 15 November 2019 Accepted 19 November 2019 Available online xxxx Editor: Lotfi Aleya Keywords: DEHP exposure Residential characteristics Pregnant women Humidifier

a b s t r a c t Epidemiological evidence on the associations between urinary di-(2-ethylhexyl) phthalate (DEHP) metabolites and residential characteristics is limited. Therefore, we investigated the associations of urinary DEHP metabolites with the residential characteristics of pregnant women. We collected completed questionnaires and maternal spot urine samples from 616 random pregnant women in Shengjing Hospital of China Medical University in Shenyang. Urinary DEHP metabolites concentrations, including mono-(2-ethylhexyl) phthalate (MEHP) and mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), were measured and analyzed by Gas ChromatographyMass Spectrometry (GC–MS). Multivariable linear regression models were performed to obtain regression estimates (β) and 95% confidence intervals (CIs) after adjustment for sociodemographic characteristics. In all participants, the geometric mean of MEHP and MEHHP concentrations were 4.25 ± 4.34 and 5.72 ± 2.65 μg/L, respectively. In multivariable analyses after adjusting for sociodemographic characteristics, distance from residence to motor vehicle traffic (≥150 m versus b20 m) was negatively associated with MEHP (β = −0.241, 95% CI: −0.448, −0.033) and MEHHP (β = −0.279, 95% CI: −0.418, −0.140) concentrations. Compared with the one that had not recently been renovated, a renovated home was associated with higher MEHP (β = 0.194, 95% CI: 0.064, 0.324) and MEHHP (β = 0.111, 95% CI: 0.024, 0.197) concentrations. Air freshener use was associated with higher MEHP (β = 0.322, 95% CI: 0.007, 0.636) concentrations. Moldy walls were positively associated with MEHP (β = 0.299, 95% CI: 0.115, 0.482) and MEHHP (β = 0.172, 95% CI: 0.050, 0.294)

⁎ Corresponding author at: Department of Occupational and Environmental Health, School of Public Health, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang 110122, PR China. E-mail address: [email protected] (J. Chen). 1 These authors contributed equally as first co-authors.

https://doi.org/10.1016/j.scitotenv.2019.135671 0048-9697/© 2018 Published by Elsevier B.V.

Please cite this article as: J. Dong, Y. Ma, K. Leng, et al., Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135671

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concentrations. In contrast, humidifier use was associated with a lower MEHP concentration (β = −0.167, 95% CI: −0.302, −0.032). Residential characteristics were probably associated with the DEHP exposure of pregnant women in Shenyang. Living near the motor vehicle traffic, residential renovation, air freshener use, and moldy walls are likely risk factors for increased DEHP exposure, whereas using household humidifier could be considered a protective measure to reduce DEHP exposure. © 2018 Published by Elsevier B.V.

1. Introduction Di-2-ethylhexyl phthalate (DEHP) is considered as a ubiquitous environmental endocrine disruptor (Erythropel et al., 2014; Rowdhwal and Chen, 2018). As part of a group of synthetic organic chemicals, DEHP is extensively used as a plasticizer to boost the plasticity, durability, and elasticity of polymeric products, such as rubber toys, medical plastic infusion bag and tubes, food packaging, household furniture, and polyvinylchloride (PVC) flooring (Calafat and McKee, 2006). Due to the massive production of DEHP about N2 million tons each year (Chou et al., 2018), human daily exposure to DEHP through the living environment has been an increasing concern as demonstrated by the findings of in vitro and in vivo environmental and toxicological studies. In general, people spend more time indoors than outdoors, thus the indoor environment accounts for a considerable proportion of our daily exposure to DEHP (Zhu et al., 2019). In previous literatures, the presence of DEHP and its metabolites in various environmental matrices, including indoor air (Song et al., 2015), dust samples (Bergh et al., 2011), and polluted air near traffic (Lu et al., 2019), have been investigated. The DEHP concentration in the indoor environment can vary by several orders of magnitude due to different residential characteristics, such as the inner moisture content (Hsu et al., 2017), building materials of the house (Takeuchi et al., 2014), and indoor temperature (Bi et al., 2015). In addition, residential environmental factors, including recent home renovation, pet ownership and indoor dampness, were associated with an increased risk of respiratory diseases and allergic dermatitis (Dong et al., 2013; Li and Kendrick, 1995; Mendell et al., 2018). To our knowledge, only a few epidemiological studies have examined the associations between DEHP exposure and residential characteristic. Liao et al. reported that there was no significant association between residential location, window frame material and urinary DEHP metabolites (Liao et al., 2018). Xu et al. found that DEHP sources characteristics as well as ventilation rate may influence the steady-state DEHP concentration and the resulting internal exposure (Xu et al., 2010). Therefore, it is convincible that the potential associations between DEHP exposure and residential characteristics need further investigation. In the human body, DEHP is rapidly metabolized through a two-step kinetic phase: DEHP is first hydrolyzed to a monoester (mono-(2-ethylhexyl) phthalate [MEHP]) by relevant enzymes, namely, lipases and esterase; MEHP is further oxidized to its secondary metabolites, including mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) and 2-carboxymethylhexyl phthalate (MCMHP) (Koch et al., 2006). In general, the monoester or secondary oxidative metabolites of DEHP are the ultimate development toxicants (Koch et al., 2005). Zhao et al. indicated that the transformation rate of MEHP to MEHHP was more efficient in late pregnancy (Zhao et al., 2018). It was also a major metabolic pathway in humans for the transformation of MEHP to MEHHP (Ito et al., 2014). In particular, urinary MEHHP is a more sensitive and quantifiable biomarker of DEHP exposure compared with other DEHP metabolites (Barr et al., 2003; Silva et al., 2006; Tranfo et al., 2018). Increasing numbers of studies have reported urinary DEHP metabolites concentrations in general population (Huang et al., 2015), as well as specifically in pregnant women (Ferguson et al., 2017), and in children (Fromme et al., 2013; Langer et al., 2014). In addition, a single sample can provide a reasonably good estimate of individual exposure and reflect the long-term

exposure to phthalates, even back to the exposure of 3 years ago (Frederiksen et al., 2013; Johns et al., 2015). Therefore, individuals' chronic exposure to DEHP from the environment can be estimated from urinary concentrations of DEHP metabolites. Recently, the adverse effects of ubiquitous DEHP exposure on human health, particularly that of pregnant women, have attracted worldwide attention because epidemiologic studies revealed that positive associations of higher levels of DEHP metabolites with adverse reproductive health and birth outcomes, such as miscarriage, preterm birth, low birth weight, reduced anogenital distance, and behavioral syndromes in offspring development (Huang et al., 2019; Messerlian et al., 2016; Swan et al., 2015; Zhang et al., 2009; Zhao et al., 2017). Toxicological studies in laboratory animals also found that exposure to DEHP during the perinatal period may cause adverse impacts on the development of the neurological (Fu et al., 2019; You et al., 2018), immune (Kitaoka et al., 2013), reproductive system (Wang et al., 2016), even the transgenerational effects (Rattan et al., 2018). Sources of DEHP exposure are extensive, including diet, plastics, personal care products, and medical devices. Measuring related metabolites in urine is a reliable and valid method to assess the DEHP exposure of pregnant women (Grindler et al., 2018; Tsai et al., 2018). In addition, the Korean National Environmental Health Survey suggested that DEHP metabolites concentrations in urine of Korean female adults were generally higher than those reported from national biomonitoring programs in US and Canada populations (Park et al., 2019). Accordingly, urinary concentrations of the DEHP metabolites in pregnant women might provide a direct comparison of the DEHP exposure of various populations. Thus, the aims of the present study were using urinary concentrations of the DEHP metabolites (MEHP and MEHHP) to evaluate DEHP exposure in pregnant women and investigating the associations of urinary DEHP metabolites with outdoor and indoor residential characteristics. 2. Methods 2.1. Chemicals and reagents DEHP metabolite standards, namely MEHP (≥99.9%) and MEHHP (≥99.9%), and two isotopically labeled DEHP metabolites, namely 13C4MEHP (99.9%) and 13C4-MEHHP (99.9%), as well as N,O-Bis (trimethylsilyl) trifluoroacetamide with trimethylchlorosilane (BSTFATMCS), β-glucuronidase and Select HLB tube were purchased from Sigma-Aldrich (Sigma Aldrich Lab., Inc., St. Louis, MO, USA). Other reagents, including hexane, ethyl acetate, and acetonitrile, were obtained from Dikma Technologies (Beijing, China). Pyridine was ordered from Shanghai Macklin Biochemical Co. 2.2. Study population and sample collection From June 2016 to July 2017, pregnant women were invited to participate in our study from the obstetrics and gynecology department at Shengjing Hospital of China Medical University in Shenyang. The inclusion criteria to participant in our study were: 1) pregnant women in the third trimester of pregnancy, 2) age ≥ 18 years, 3) lived in northeast China, 4) signature of the written informed consent form. The exclusion criteria:1) under 18 years old, 2) not in the third trimester of pregnancy, 3) pulmonary diseases, 4) heart and kidney diseases, 5) other serious organic diseases. The flow chart of study design is shown in Fig. 1. Each

Please cite this article as: J. Dong, Y. Ma, K. Leng, et al., Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135671

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participant was required to complete a questionnaire concerning sociodemographic and residential characteristics. Maternal spot urine samples were collected and then stored at −80 °C until analysis. All of the investigated participants received an informed consent form and signed it voluntarily. A total of 706 questionnaires and maternal urine samples were collected by our professionally trained investigators. After integrating the questionnaires with samples information and excluding individuals without essential information or urine samples, the final study population included 616 valid participants. The total effective rate of urine samples and information collection was 87.25%. 2.3. Measurement of urinary DEHP metabolites The Gas Chromatography–Mass Spectrometry (GC–MS) method was used and modified to identify and quantify urinary DEHP metabolites (Bustamante-Montes et al., 2013). The linearity, limits of detection (LODs), limits of quantification (LOQs) and standard curves, were applied to determine the validity of the analytical method. The measurement of urinary DEHP metabolites were made into two steps: the extraction and derivatization of metabolites from urine samples, and the instrumental analysis of metabolites. Firstly, Urine samples were thawed in a glass tube at room temperature. Isotope internal standard and β-glucuronidase were added to the tube for enzymatic hydrolysis. The mixture was sealed and incubated at 37 °C for 120 min. Then, a Select HLB tube was activated by acetonitrile and ultrapure water, which was used for liquid phase extraction (LPE). After enzymatic hydrolysis, urine samples were filtered in an activated HLB tube. Finally, the organic phase was slowly dried using N2 gas. The derived reagents, including BSTFA, TMCS, and pyridine, were added to blow-dried glass tubes and stored at 30 °C for 30 min until analysis. Two microliters of the extract were injected in splitless mode by using an auto-sampler and HP-5 ms capillary column. High-purity helium was used as the carrier gas at a flow rate of 15 mL/min. The initial oven temperature was set at 100 °C with a holding time of 1 min, then increased by 10 °C/min to 200 °C, and further rose by 5 °C/min to 250 °C and held for 12 min. The mass spectrometer was operated in electron impact mode (70 eV) and the ion source temperature was set at 250 °C. Multi-reaction monitoring was performed by monitoring several mass ions of DEHP metabolites for quantitative and qualitative analysis (MEHP: 239, 73, and 221; MEHHP: 295, 147, and 221). The retention times (Rt) of MEHP and MEHHP were 15.7 and 19.2 min, respectively.

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2.4. Quality assurance and quality control Glass containers were used instead of plastic vessels to reduce background contamination and interference. The LODs and LOQs of MEHP and MEHHP were calculated to be 0.1 and 0.33 ng/mL using signal-tonoise ratios of 3/1 and 10/1, respectively. The standard curves of MEHP and MEHHP were obtained using GC–MS with standard concentrations in the range of 5 to 1000 μg/L and the regression coefficients of the calibration standards were above 0.99 (R2MEHP = 0.998; R2MEHHP = 0.999). The isotope internal standard method was applied to each batch of urine samples to improve detection stability and accuracy. In addition, the investigators were provided professional training programs to ensure the authenticity of the questionnaires and the principle of double parallel entry was applied to ensure the accuracy of the data. 2.5. Statistical analysis The sociodemographic characteristics of participants were presented as percentages or mean ± SD. The DEHP metabolites concentrations were shown as the geometric mean (geometric standard deviation) and percentiles. Due to left-censored data of DEHP metabolites concentrations, levels below the LODs were replaced by the detection limit divided by the square root of 2 in order to fit linear regression models (Bustamante-Montes et al., 2013). Natural log-transformation was performed for metabolites to meet the normal distribution for statistical analysis. Specific gravity (SG)-corrected concentrations were used to adjust for urine dilution. The SG formula is as follows: Pc = P [(M − 1)/(SG − 1)], where Pc is the SG-corrected concentration, P is the measured concentration, M is the median SG for all of the samples, and SG is the specific gravity for the sample (Meeker et al., 2009; Pearson et al., 2009). A molar summed measure of DEHP metabolites (∑DEHP) was calculated by converting metabolite concentrations from micrograms per liter to micromoles per liter and adding MEHP and MEHHP together: ∑DEHP (μmol/L) = [(PC(MEHP)/278) + (PC (MEHHP)/294)], where PC is the SG-corrected concentration and 278 and 294 are the molar masses of MEHP and MEHHP, respectively (Kobrosly et al., 2012). Multivariable linear regression models were used to obtain regression estimates (β) and 95% confidence intervals (CIs) for the associations between DEHP metabolites and residential characteristics. An age-adjusted model examined the individual effects of residential characteristics after adjustment for age (years) alone. A multivariable-

Fig. 1. Flow chart of study design.

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adjusted model was used to investigate the associations between DEHP metabolites and residential characteristics in an adjusted model after further adjustment for age (years), BMI (b28 versus ≥28), education (high school or below versus college or above), family income (b70,000 versus ≥70,000 ¥/year), and number of pregnancies (one versus two or more). In addition, stratified analyses were applied to test for effect modification by factors potentially influencing DEHP metabolites, including age, BMI, education, family income, and number of pregnancies. The data of questionnaire was input by Epidata 3.1. All statistical analyses were analyzed using IBM SPSS Statistics software, version 21.0 (SPSS China Corp, Shanghai, China). A two-tailed test with a p value of b0.05 was considered statistically significant. 3. Results The general characteristics of all participants are shown in Table 1. The weighted median age of the study population was 30 years and the median BMI was 27.53. Eighty-four percent of pregnant women had a college-level education or higher. Families with an annual income of N70,000 (¥/year) accounted for almost half of all participants. Fortythree percent of the pregnant women had been pregnant at least twice. The detectable rate of MEHP and MEHHP in all urine samples were 99.63% and 100%, respectively. The geometric means and percentiles for urinary DEHP metabolite concentrations are presented in Table 2. The geometric means of MEHP and MEHHP were 4.25 ± 4.34 μg/L and 5.72 ± 2.65 μg/L, respectively. Associations between residential characteristics and DEHP metabolites are shown in Fig. 2. In multivariable analyses, after adjustment for

age, BMI, education, family income and number of pregnancies, a greater distance between residence and motor vehicle traffic (≥150 m versus b20 m) was negatively associated with the urinary concentrations of MEHP (β = −0.241, 95% CI: −0.448, −0.033, p = 0.05), MEHHP (β = −0.279, 95% CI: −0.418, −0.140, p b 0.001), and ∑DEHP (β = −0.258, 95% CI: −0.410, −0.105, p = 0.001). Participants who had renovated their homes in the previous 3 years had higher MEHP (β = 0.194, 95% CI: 0.064, 0.324, p = 0.003), MEHHP (β = 0.111, 95% CI: 0.024, 0.197, p = 0.012), and ∑DEHP (β = 0.151, 95% CI: 0.057, 0.246, p = 0.002) concentrations than those who had not. Moldy walls were positively associated with the concentrations of MEHP (β = 0.299, 95% CI: 0.115, 0.482, p = 0.002), MEHHP (β = 0.172, 95% CI: 0.050, 0.294, p = 0.006), and ∑DEHP (β = 0.222, 95% CI: 0.088, 0.356, p = 0.001). Air freshener use was significantly associated with MEHP (β = 0.322, 95% CI: 0.007, 0.636, p = 0.045) and ∑DEHP (β = 0.237, 95% CI: 0.008, 0.466, p = 0.042) concentrations. In addition, humidifier use was associated with a lower MEHP concentration (β = −0.167, 95% CI: −0.302, −0.032, p = 0.016). In subgroup analyses of residential characteristics (Tables 3 and 4), significant associations of MEHP with distance between residence and motor vehicle traffic, air freshener and humidifier use were partly concealed by several strata. However, these associations still remained statistically significant across all stratified variables. We did not observe any other inconsistent associations of MEHP, MEHHP, and ∑DEHP concentrations with residential characteristics compared with the above results. Therefore, stratified analyses indicated that significant associations between these residential characteristics and DEHP metabolites were basically consistent across all pre-specified strata. 4. Discussion

t1:1 t1:2 t1:3

Table 1 Distribution of demographic, socioeconomic, and residential characteristics in all pregnant participants.

t1:4

Characteristic

Means ± SD or N (%)

t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44

Age (years) Body mass index (kg/m2)

29.92 ± 3.88 27.53 ± 3.51

Education High school or below College or above

100 (16.23%) 516 (83.77%)

Family income (¥/year) b70,000 ≥70,000

304 (49.35%) 312 (50.65%)

Number of pregnancies One Two or more

353 (57.31%) 263 (42.69%)

Distance between residence and motor vehicle traffic b20 m 20–150 m ≥150 m

71 (11.53%) 217 (35.23%) 328 (53.24%)

Distance between residence and smokestack b20 m 20–150 m ≥150 m

43 (6.99%) 139 (22.56%) 434 (70.45%)

Renovation in previous 3 years No Yes

342 (55.52%) 274 (44.48%)

Air freshener use No Yes

592 (96.10%) 24 (3.90%)

Moldy walls No Yes

534 (86.69%) 82 (13.31%)

Humidifier use No Yes

408 (66.23%) 208 (33.77%)

In this representative population sample of pregnant women of northeast China, we investigated the associations of urinary DEHP metabolites with the residential characteristics. Our results showed that several residential characteristics, including living near the motor vehicle traffic, household renovation, air fresheners, and moldy walls, were significantly associated with higher DEHP metabolites concentrations. We also found inverse association of using household humidifier and MEHP concentration. These findings indicate that the presence of these residential characteristics may have positive or negative effects on individual DEHP exposure. We measured the urine DEHP metabolites concentrations across the third trimester of pregnancy in 616 pregnant women. The geometric means of MEHP (4.25 ± 4.34 μg/L) and MEHHP (5.72 ± 2.65 μg/L) were lower than those of pregnant women in US (2006–2008) (Ferguson et al., 2015) with 10.6 ± 3.49 μg/L (MEHP) and 34.2 ± 3.41 μg/L(MEHHP), respectively. Compared to another cohort study in US population (2010−2012) (Swan et al., 2015), the MEHP concentration of our study was 2.2 fold higher (4.25 vs 1.93 μg/L), the MEHHP concentration was slightly lower (5.72 vs 6.04 μg/L). In addition, the level of MEHP in our study was slightly higher (4.25 vs 3.27 μg/L), while the MEHHP level was 0.4 fold lower (5.72 vs 14.42 μg/L) when compared to a Sweden study on prenatal phthalate exposures (2007–2010) (Bornehag et al., 2015). Several literatures reported the endocrine disruption of DEHP metabolites in humans, especially Table 2 t2:1 Distribution of DEHP metabolites measured in urine samples collected from pregnant t2:2 women. t2:3 Compound

LOD (μg/L)

%b LOD

Geometric mean (geometric SD)

MEHP (μg/L) MEHHP (μg/L) ∑DEHP (μmol/L)

0.10 0.10 /

0.37 0.00 /

4.25 (4.34) 5.72 (2.65) 0.04 (2.92)

t2:4

Percentile 25th

50th

75th

t2:5

1.53 3.16 0.02

4.02 5.77 0.04

10.25 10.80 0.07

t2:6 t2:7 t2:8

Abbreviation: LOD, limit of detection.

Please cite this article as: J. Dong, Y. Ma, K. Leng, et al., Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135671

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Fig. 2. Associations of residential characteristics with MEHP, MEHHP, and ∑DEHP concentrations. (Panel (A) indicated age-adjusted model, adjusted for age (years) alone. Panel (B) indicated multivariable-adjusted model, adjusted for age (years), BMI (b28 versus ≥28), education (high school or below versus college or above), family income (b70,000 versus ≥70,000 ¥/year), and number of pregnancies (one versus two or more). Regression coefficient (β) and 95% confidence intervals (95% CI) were obtained from linear regression to estimate the association between residential characteristics and DEHP metabolites.)

MEHP and MEHHP (Casals-Casas et al., 2008; Frederiksen et al., 2007). However, urinary concentrations of DEHP metabolites in pregnant women varied considerably among population. It may be due to regional and sociodemographic differences. Moreover, Yaghyjan et al. found that metabolic conversion rate of MEHP may be influenced by individual characteristics and lifestyle factors, including age, race, BMI, and alcohol drinking (Yaghjyan et al., 2016). In the present study, distance of residence from motor vehicle traffic was negatively associated with DEHP metabolite concentrations, indicating that traffic-related air pollution may be a potential source of DEHP exposure for pregnant women living near the motor vehicle traffic. Previous studies have reported that traffic-related air pollution is a potential risk factor for pregnancy complications and adverse outcomes, such as pre-eclampsia, hypertensive disorders, and spontaneous abortion (Green et al., 2009; Olsson et al., 2015; Pedersen et al., 2017). Further research confirmed that higher traffic-related air pollution exposure was associated with maternal oxidative stress and an inflammatory response during pregnancy (Yan et al., 2019). However, the hazardous components in traffic-polluted air have not been identified. A recent study observed the associations between traffic-polluted air phthalate concentrations and individual blood components, including DEHP concentrations and sex hormone levels, which suggested that traffic-related DEHP exposure may be a risk factor for reproductive health of people living near traffic (Lu et al., 2019). In view of the hazardous effects of traffic-related air pollution on human health, we suggest that some protective practices, such as wearing a particulate respirator or closing the doors and windows, could be adopted by

pregnant women living near the motor vehicle traffic to reduce direct air inhalation. In our findings, several indoor residential characteristics were associated with higher urinary concentrations of one or two of the DEHP metabolites. Specifically, we observed a significant positive association between DEHP metabolites and renovated home in the previous 3 years. The main refurbishment or decorative materials, including floor leather, rubbers, vinyl flooring, varnishes, and other plastic materials, can release a large amount of phthalates into the indoor air environment, especially DEHP (Blanchard et al., 2014; Xu et al., 2010). Several studies indicated that plasticizers and flame retardants in new building could cause ocular- and nasal mucosal-irritation symptoms, which may have an endocrine-disrupting effect (Legler, 2008; Legler and Brouwer, 2003; Sahlberg et al., 2013; Sheikh et al., 2016). Therefore, the relationship between maternal health and interior renovation should be comprehensively demonstrated and recognized by the general population. Our results showed that participants who used air freshener had a higher urinary MEHP concentration. This may be because air fresheners continuously release some chemical components, including benzene, formaldehyde, terpenes, toluene, and xylene, which probably can react with air to generate secondary organic aerosol and ultrafine particles (Kim et al., 2015). These particles might conglutinate DEHP in the indoor air and enter the body through the respiratory system or skin. Therefore, we suppose that air fresheners likely accelerate the accumulation of chemical components in the air and dust and thus increase DEHP exposure to human in the residential environment.

Please cite this article as: J. Dong, Y. Ma, K. Leng, et al., Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135671

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Table 3 Stratified analyses of the distance between residence and traffic and associations of residential characteristics with DEHP metabolites (regression estimates and 95% confidence intervals). Characteristic

t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23 t3:24 t3:25 t3:26 t3:27 t3:28 t3:29 t3:30 t3:31 t3:32 t3:33 t3:34 t3:35 t3:36 t3:37 t3:38 t3:39 t3:40 t3:41

Distance between residence and traffic (20–150 Age, year b30 ≥30 BMI, kg/m2 b28 ≥28 Education High school or below College or above Family income (¥/year) b70,000 ≥70,000 Number of pregnancies One Two or more

MEHP

MEHHP

ΣDEHP

β (95% CI)a

β (95% CI)

β (95% CI)

0.467(0.742, 0.193) −0.008(−0.362, 0.346)

0.332(0.518, 0.147) −0.140(−0.373, 0.092)

0.426(0.624, 0.229) −0.027(−0.291, 0.236)

−0.283(−0.573, 0.007) −0.288(−0.627, 0.052)

0.281(0.479, 0.082) −0.212(−0.432, 0.008)

0.251(0.471, 0.032) 0.279(0.520, 0.038)

−0.317(−0.809, 0.175) 0.278(0.523, 0.032)

0.424(0.769, 0.079) 0.230(0.394, 0.067)

−0.333(−0.693, 0.027) 0.258(0.440, 0.077)

−0.237(−0.562, 0.089) −0.317(−0.611, −0.023)

0.258(0.467, 0.049) 0.267(0.475, 0.060)

0.237(0.470, 0.003) 0.301(0.459, 0.027)

−0.107(−0.391, 0.178) 0.470(0.806, 0.133)

0.254(0.466, 0.041) 0.263(0.164, 0.062)

−0.187(−1.662, 0.098) 0.352(0.587, 0.117)

0.341(0.520, 0.163) −0.177(−0.397, 0.043)

0.413(0.604, 0.223) −0.006(−0.256, 0.244)

0.238(0.431, 0.045) 0.336(0.542, 0.130)

0.234(0.448, 0.020) 0.275(0.501, 0.049)

−0.201(−0.539, 0.136) 0.280(0.438, 0.123)

−0.237(−0.589, 0.114) 0.256(0.431, 0.082)

0.319(0.516, 0.123) 0.240(0.442, 0.038)

0.257(0.478, 0.036) 0.243(0.459, 0.027)

0.285(0.487, 0.084) 0.271(0.466, 0.075)

−0.162(−0.373, 0.049) 0.364(0.592, 0.135)

m)b

Distance between residence and traffic (≥150 m) Age, year b30 0.430(0.694, 0.165) ≥30 0.069(−0.267, 0.405) BMI, kg/m2 b28 −0.246(−0.528, 0.037) ≥28 −0.215(−0.533, 0.103) Education High school or below −0.264(−0.745, 0.218) College or above −0.228(−0.464, 0.008) Family income(¥/year) b70,000 −0.217(−0.524, 0.090) ≥70,000 −0.226(−0.512, 0.060) Number of pregnancies One −0.024(−0.294, 0.247) Two or more 0.475(0.803, 0.148)

Bold font indicates statistical significance at the 0.05 level. a Adjusted for age (years), BMI (b 28 versus ≥ 28), education (high school or below versus college or above), family income (b 70,000 versus ≥ 70,000 ¥/year), and number of pregnancies (one versus two or more). Variables examined in this table were not adjusted. b Distance from residence to motor vehicle traffic.

We also observed a significant association of higher concentrations of DEHP metabolites with moldy walls. This may be due to the fact that DEHP can be degraded into diesters or monoesters by fungi in damp and moldy dwellings, which is supported by the findings of previous studies (Gonzalez-Marquez et al., 2019; Lee et al., 2012). Similarly, MEHP can be produced from DEHP by microorganisms and/or fungi living in culture medium and inside houses (Nakamiya et al., 2005). Indeed, moldy walls are related to high humidity conditions and thus can cause a rapid outbreak of microbial growth (Pessi et al., 2002). Furthermore, higher DEHP concentrations were found to be emitted into the air and adsorbed onto dust from PVC-coated walls with a higher moisture content (Bornehag et al., 2005). Thus, moldy walls might increase DEHP exposure risk through accelerated DEHP emission and degradation by microbes. Interestingly, the use of a household humidifier was associated with lower urinary MEHP concentrations. In addition, humidifier use also had inverse associations with MEHHP and ∑DEHP, although regression analyses suggested no statistically significant association. We speculate that, on the one hand, water vapor generated by the humidifier can settle suspended particulate matter in the air, thus reducing human exposure through absorption. On the other hand, families who use household humidifiers may pay more attention to indoor hygiene and air quality. To our knowledge, no studies have reported an inverse association between household humidifiers and DEHP metabolites. Our study indicated that use of household humidifier might have a protective effect on DEHP exposure of indoor environments. However, in recent years, some studies have reported the relationship between indoor environments altered by humidifiers and adverse human health (Dong et al., 2013; Kim et al., 2014; Quinn and Shaman, 2017). In

addition, humidifiers are commonly used to increase the humidity in the residential environment, but higher humidity was found to be associated with more DEHP emissions in interior redecorating materials (Hsu et al., 2017). Furthermore, an exploratory study confirmed that frequent humidifier use increased microbial growth, including fungal and bacteria, in indoor environments (Lee et al., 2012), which is consistent with moldy walls found to be associated with higher DEHP by our study. Based on this evidence and our results, our findings suggest that further studies on DEHP and frequency of humidifier use from indoor environment should be conducted to determine its effect on human health. This is an original article to analyze residential factors related to DEHP exposure. We measured DEHP metabolites in the urine rather than in the air or dust of the residence, which more accurately reflects the level of DEHP exposure in the body of pregnant women. To our knowledge, this is the first study to explore the relationship between DEHP exposure of pregnant women and the use of household humidifier. Our results suggest a protective effect of humidifiers against DEHP exposure, although they do not indicate the frequency-effect relationship between humidifier and DEHP metabolites. Nonetheless, our study has some limitations. First, due to the cross-sectional analysis, our findings and the collected data cannot reveal any dynamic temporal relationships. Second, the sample size was limited and our results may not represent the general population. Third, although we included various variables related to sociodemographic, environmental, and residential factors to interpret the factors influencing DEHP exposure, some factors were still not accounted for, such as indoor ventilation, room cleaning frequency, and dietary status, and these characteristics are potentially taken into consideration in the future studies. Last, all

Please cite this article as: J. Dong, Y. Ma, K. Leng, et al., Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135671

J. Dong et al. / Science of the Total Environment xxx (xxxx) xxx t4:1 t4:2 t4:3

Table 4 Stratified analyses of indoor residential characteristics and associations of residential characteristics with DEHP metabolites (regression estimates and 95% confidence intervals). Characteristic

t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19 t4:20 t4:21 t4:22 t4:23 t4:24 t4:25 t4:26 t4:27 t4:28 t4:29 t4:30 t4:31 t4:32 t4:33 t4:34 t4:35 t4:36 t4:37 t4:38 t4:39 t4:40 t4:41 t4:42 t4:43 t4:44 t4:45 t4:46 t4:47 t4:48 t4:49 t4:50 t4:51 t4:52 t4:53 t4:54 t4:55 t4:56 t4:57 t4:58 t4:59 t4:60 t4:61 t4:62 t4:63 t4:64 t4:65 t4:66 t4:67 t4:68 t4:69 t4:70 t4:71 t4:72 t4:73 t4:74

7

Renovation in previous 3 years Age, year b30 ≥30 BMI, kg/m2 b28 ≥28 Education High school or below College or above Family income (¥/year) b70,000 ≥70,000 Number of pregnancies One Two or more Air freshener use Age, year b30 ≥30 BMI, kg/m2 b28 ≥28 Education High school or below College or above Family income (¥/year) b70,000 ≥70,000 Number of pregnancies One Two or more Moldy walls Age, year b30 ≥30 BMI, kg/m2 b28 ≥28 Education High school or below College or above Family income (¥/year) b70,000 ≥70,000 Number of pregnancies One Two or more Humidifier use Age, year b30 ≥30 BMI, kg/m2 b28 ≥28 Education High school or below College or above Family income (¥/year) b70,000 ≥70,000 Number of pregnancies One Two or more

MEHP

MEHHP

ΣDEHP

β (95% CI)a

β (95% CI)

β (95% CI)

0.186(0.022, 0.351) 0.218(−0.007, 0.443)

0.127(0.017, 0.237) 0.084(−0.061, 0.229)

0.138(0.018, 0.258) 0.159(−0.004, 0.321)

0.174(0.009, 0.339) 0.223(0.010, 0.436)

0.083(−0.028, 0.194) 0.150(0.011, 0.290)

0.140(0.017, 0.262) 0.168(0.017, 0.320)

−0.027(−0.431, 0.377) 0.220(0.082, 0.358)

0.019(−0.271, 0.309) 0.123(0.032, 0.215)

0.076(−0.216, 0.368) 0.159(0.058, 0.260)

0.232(0.039, 0.426) 0.149(−0.031, 0.330)

0.059(−0.065, 0.183) 0.155(0.029, 0.281)

0.149(0.011, 0.286) 0.142(0.006, 0.278)

0.142(−0.018, 0.302) 0.266(0.046, 0.487)

0.137(0.020, 0.255) 0.074(−0.056, 0.204)

0.119(−0.003, 0.242) 0.194(0.043, 0.346)

0.135(−0.264, 0.535) 0.616(0.094, 1.139)

0.105(−0.162, 0.371) 0.173(−0.168, 0.514)

0.152(−0.139, 0.442) 0.388(0.010, 0.765)

0.144(−0.321, 0.609) 0.454(0.010, 0.898)

−0.080(−0.393, 0.232) 0.271(−0.018, 0.560)

0.063(−0.281, 0.408) 0.368(0.054, 0.682)

0.533(−0.269, 1.334) 0.277(−0.071, 0.625)

0.248(−0.300, 0.796) 0.093(−0.138, 0.324)

0.548(−0.012, 1.107) 0.175(−0.080, 0.430)

0.335(−0.209, 0.880) 0.313(−0.067, 0.693)

0.148(−0.194, 0.490) 0.087(−0.182, 0.356)

0.272(−0.112, 0.655) 0.211(−0.076, 0.498)

0.101(−0.342, 0.544) 0.494(0.031, 0.956)

0.156(−0.170, 0.482) 0.081(−0.191, 0.353)

0.086(−0.253, 0.425) 0.359(0.041, 0.678)

0.166(−0.099, 0.431) 0.389(0.121, 0.657)

0.077(−0.101, 0.255) 0.230(0.059, 0.401)

0.102(−0.091, 0.295) 0.302(0.111, 0.494)

0.118(−0.124, 0.360) 0.539(0.240, 0.839)

0.085(−0.077, 0.247) 0.286(0.088, 0.485)

0.074(−0.106, 0.254) 0.415(0.202, 0.627)

0.568(0.093, 1.042) 0.233(0.030, 0.435)

0.144(−0.203, 0.491) 0.181(0.047, 0.314)

0.383(0.040, 0.726) 0.189(0.041, 0.337)

0.354(0.076, 0.632) 0.242(−0.003, 0.487)

0.153(−0.024, 0.330) 0.190(0.019, 0.361)

0.259(0.063, 0.456) 0.183(−0.002, 0.367)

0.258(0.040, 0.476) 0.359(0.028, 0.689)

0.177(0.016, 0.338) 0.157(−0.035, 0.349)

0.205(0.037, 0.372) 0.245(0.018, 0.472)

−0.135(−0.305, 0.034) −0.224(−0.452, 0.005)

−0.059(−0.174, 0.055) −0.079(−0.227, 0.068)

−0.055(−0.179, 0.069) −0.155(−0.319, 0.009)

0.179(0.356, 0.002) −0.158(−0.371, 0.054)

−0.094(−0.213, 0.025) −0.035(−0.177, 0.107)

−0.122(−0.252, 0.009) −0.060(−0.213, 0.094)

−0.250(−0.673, 0.174) 0.166(0.310, 0.021)

−0.051(−0.354, 0.252) −0.069(−0.164, 0.027)

−0.089(−0.396, 0.217) −0.099(−0.205, 0.007)

0.221(0.429, 0.013) −0.123(−0.302, 0.056)

−0.098(−0.230, 0.034) −0.039(−0.165, 0.088)

−0.124(−0.272, 0.024) −0.062(−0.197, 0.073)

−0.129(−0.296, 0.037) −0.219(−0.454, 0.015)

−0.079(−0.202, 0.043) −0.054(−0.193, 0.084)

−0.064(−0.192, 0.064) −0.138(−0.299, 0.023)

Bold font indicates statistical significance at the 0.05 level. a Adjusted for age (years), BMI (b 28 versus ≥ 28), education (high school or below versus college or above), family income (b 70,000 versus ≥ 70,000 ¥/year), and number of pregnancies (one versus two or more). Variables examined in this table were not adjusted.

Please cite this article as: J. Dong, Y. Ma, K. Leng, et al., Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135671

8

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participants in our study were pregnant women, and most participants were urban population of northeast China, which may limit the generalizability of our results. Therefore, further investigation or confirmation in other regions/populations was needed. 5. Conclusions In conclusion, we found that living near the motor vehicle traffic, household renovation, air fresheners, and moldy walls might be risk factors for DEHP exposure, whereas using household humidifier could be considered a protective measure to reduce DEHP exposure. Further studies are needed to confirm our findings in other regions and populations, to examine associations between the frequency of humidifier use and DEHP exposure, and to elucidate the underlying mechanisms. Declaration of competing interest The authors declare that they have no conflicts of interests. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (grant number 81472943) and the Natural Science Foundation of Liaoning Province, China (grant number 2019JH3/ 10300439). We thank the Obstetrics and Gynecology Department of Shengjing Hospital of China Medical University for their supports in the questionnaire survey. We also thank Ruochen Zhang, Yiting Li, Dandan Yu for assisting with the revision of the English language of this manuscript. We appreciate all of investigators, participants, master students and others who involved in the study. References Barr, D.B., Silva, M.J., Kato, K., Reidy, J.A., Malek, N.A., Hurtz, D., et al., 2003. Assessing human exposure to phthalates using monoesters and their oxidized metabolites as biomarkers. Environmental health perspectives 111, 1148–1151. https://doi.org/10.1289/ehp.6074. Bergh, C., Torgrip, R., Emenius, G., Ostman, C., 2011. Organophosphate and phthalate esters in air and settled dust - a multi-location indoor study. Indoor air 21, 67–76. https://doi.org/10.1111/j.1600-0668.2010.00684.x. Bi, C., Liang, Y., Xu, Y., 2015. Fate and transport of phthalates in indoor environments and the influence of temperature: a case study in a test house. Environmental science & technology 49, 9674–9681. https://doi.org/10.1021/acs.est.5b02787. Blanchard, O., Glorennec, P., Mercier, F., Bonvallot, N., Chevrier, C., Ramalho, O., et al., 2014. Semivolatile organic compounds in indoor air and settled dust in 30 French dwellings. Environmental science & technology 48, 3959–3969. https://doi.org/ 10.1021/es405269q. Bornehag, C.G., Lundgren, B., Weschler, C.J., Sigsgaard, T., Hagerhed-Engman, L., Sundell, J., 2005. Phthalates in indoor dust and their association with building characteristics. Environmental health perspectives 113, 1399–1404. https://doi.org/10.1289/ehp.7809. Bornehag, C.G., Carlstedt, F., Jonsson, B.A., Lindh, C.H., Jensen, T.K., Bodin, A., et al., 2015. Prenatal phthalate exposures and anogenital distance in Swedish boys. Environmental health perspectives 123, 101–107. https://doi.org/10.1289/ehp.1408163. Bustamante-Montes, L.P., Hernandez-Valero, M.A., Flores-Pimentel, D., Garcia-Fabila, M., Amaya-Chavez, A., Barr, D.B., et al., 2013. Prenatal exposure to phthalates is associated with decreased anogenital distance and penile size in male newborns. Journal of developmental origins of health and disease 4, 300–306. https://doi.org/10.1017/ s2040174413000172. Calafat, A.M., McKee, R.H., 2006. Integrating biomonitoring exposure data into the risk assessment process: phthalates [diethyl phthalate and di(2-ethylhexyl) phthalate] as a case study. Environmental health perspectives 114, 1783–1789. https://doi.org/ 10.1289/ehp.9059. Casals-Casas, C., Feige, J.N., Desvergne, B., 2008. Interference of pollutants with PPARs: endocrine disruption meets metabolism. International journal of obesity (2005) 32 (Suppl. 6), S53–S61. https://doi.org/10.1038/ijo.2008.207. Chou, C.K., Yang, Y.T., Yang, H.C., Liang, S.S., Wang, T.N., Kuo, P.L., et al., 2018. The impact of Di(2-ethylhexyl)phthalate on cancer progression. Archivum immunologiae et therapiae experimentalis 66, 183–197. https://doi.org/10.1007/s00005-017-0494-2. Dong, G.H., Qian, Z.M., Wang, J., Trevathan, E., Ma, W., Chen, W., et al., 2013. Residential characteristics and household risk factors and respiratory diseases in Chinese women: the Seven Northeast Cities (SNEC) study. The Science of the total environment 463–464, 389–394. https://doi.org/10.1016/j.scitotenv.2013.05.063. Erythropel, H.C., Maric, M., Nicell, J.A., Leask, R.L., Yargeau, V., 2014. Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure. Applied microbiology and biotechnology 98, 9967–9981. https:// doi.org/10.1007/s00253-014-6183-8.

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Please cite this article as: J. Dong, Y. Ma, K. Leng, et al., Associations of urinary di-(2-ethylhexyl) phthalate metabolites with the residential characteristics..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135671