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Ecotoxicology and Human Environmental Health

Distribution of Phthalate Metabolites between Paired Maternal-Fetal Samples Xiaoying Li, Hongwen Sun, Yiming Yao, Zhen Zhao, Xiaolei Qin, Yishuang Duan, and Lei Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00838 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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Environmental Science & Technology

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Distribution of Phthalate Metabolites between Paired Maternal-Fetal

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Samples

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Xiaoying Li,† Hongwen Sun,*,† Yiming Yao,† Zhen Zhao,† Xiaolei Qin,† Yishuang

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Duan,† and Lei Wang†

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†

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Criteria, College of Environmental Science and Engineering, Nankai University,

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Tianjin, China

Ministry of Education Key Laboratory of Pollution Processes and Environmental

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Corresponding author: Hongwen Sun

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College of Environmental Science and Engineering, Nankai University

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38 Tongyan Road, Tianjin, 300350, China

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Tel: 86-22-23509241

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Fax: 86-22-23501117

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Email: [email protected]

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Submitted to: Environmental Science &Technology

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ACS Paragon Plus Environment


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ABSTRACT: Phthalic acid esters (PAEs) are readily metabolized to phthalate

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metabolites (mPAEs) in the human body. The occurrence of mPAEs in adult human

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samples is well documented; however, the maternal-fetal transmission of mPAEs has

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seldom been studied. In this study, 78 paired maternal-fetal samples, including

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maternal urine (MU), maternal serum (MS), cord serum (CS), and amniotic fluid (AF),

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were collected from pregnant women in Tianjin, China. Seven mPAEs were detected

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in MS, CS and AF, whereas all 11 investigated mPAEs were found in MU. The

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median concentration of mPAEs was the highest in MU (128 ng/mL, with a range of

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20.2–973 ng/mL), and proceeded in the order of CS (44.9, 13.9–315 ng/mL), MS

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(24.6, 3.75–156 ng/mL), and AF (10.4, 7.69–79.8 ng/mL). The values of mPAEs and

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several individual mPAEs were significantly correlated between MU and MS, with

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generally higher concentrations in MU, which indicated that urinary mPAEs is a good

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indicator of PAEs’ exposure in adults. Notably, the median CS:MS ratios of mPAEs

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(1.58) were higher than 1, indicating that fetuses were exposed to mPAEs before birth.

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Significant correlations were also observed between MS and CS, which suggested that

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mPAEs in MS provide an indication of the fetal exposure. This study presents the first

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systematic analysis of the distribution and transmission of various mPAEs between

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mothers and fetuses.

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■INTRODUCTION

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Phthalic acid esters (PAEs) are widely used as plasticizers in industrial and consumer

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products, including nail polish, plastic packaging, toys, personal care products, and

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pharmaceutical coatings.1,2 PAEs are readily released into packaged foods and


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environmental matrices as they are not chemically bound to plastic polymers.1,2 Hence,

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PAEs have been detected in various matrices such as air,3 surface water,4 soil,5 and

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foodstuffs;1,2 thus, there is a high risk of human exposure.2

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Once entering the human body, PAEs within a few hours or days are rapidly

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metabolized to their metabolites (mPAEs),6-8 which are more toxic than their parent

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compounds.9-11 These mPAEs were reported to be associated with many human health

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outcomes such as biochemical pregnancy loss,12 testosterone-related diseases,13

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hypertension,14 and diabetes.14 As they are more water soluble than PAEs, mPAEs are

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more easily excreted in urine. mPAEs have been extensively detected at a wide range

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of concentrations in human urine (from several to several hundred ng/mL), indicating

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the prevalence of human exposure to PAEs.1,2 Accordingly, the use of human urinary

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mPAEs as biomarkers for PAEs exposure has been suggested.1,2 However, the

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relationship between the mPAEs concentrations in urine and serum samples has not

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been extensively studied, nor have consistent results concerning correlations been

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reported.15,16

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A fetus can be exposed to chemical contaminants via maternal-fetal transmission.

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Several contaminants, such as polychlorinated biphenyls (PCBs),17 polybrominated

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diphenyl ethers (PBDEs),18,19 polycyclic aromatic hydrocarbons (PAHs),20,21 and poly-

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and perfluoroalkyl substances (PFASs),22,23 are known to be able to cross the placenta

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and enter the umbilical cord. To date, only a few studies have investigated the mPAEs

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in the umbilical cord and the total number of mPAEs studied is also rather limited.24,25

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On the other hand, both animal toxicity studies and epidemiological investigation



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showed the adverse effects of mPAEs on the development of fetuses.26-28 Thus, the

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data on prenatal exposure to mPAEs are highly needed so as to accurately assess the

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risk of maternal PAEs exposure on newborn infants.

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The CS:MS (maternal serum) concentrations ratio of a contaminant is often used

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as an important indicator to assess the transmission efficiency from the mother to the

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fetus. In previous studies, both the CS:MS ratios larger than 1 and less than 1 were

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reported,18-22,29 and the former indicates accumulation against the concentration

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gradient, for which active transport may be involved. Some studies reported the

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association between the transmission ratios and chemical properties. Usually, nonionic

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or highly lipid-soluble compounds (with molecular weight (MW) < 600 Da and 0.9 

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logKow5.0) can cross the placenta easily through passive diffusion.30-33 A plot of the

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transmission efficiencies of perfluoroalkyl carboxylic acids (PFCAs) between

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maternal-fetal samples against fluorine-carbon chain length presented as a U-shape;

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and the lowest value was found in perfluorodecanoic acid and both shorter-chain and

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longer-chain analogs had higher transfer efficiencies.23 As for ionic organic

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contaminants like mPAEs, they have great water solubility and low logKow and the

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speciation changes with circumstance parameters such as pH, and accordingly, their

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distribution within organisms may depend not only on the partition in the lipid phase

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but also on binding affinity to proteins and other macrobiomolecules and are likely to

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be influenced by circumstance parameters such as pH.34-36 In addition to chemical

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properties, the physiological status of the mother and fetus may also exert an impact,

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which may explain why the same chemicals have different transfer ratios in different


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studies.18,19 Besides, as the metabolites of PAEs, mPAEs in the fetus may come both

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from the transmission from the mother and from the metabolism of PAEs in the fetus.

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In summary, knowledge of the maternal-fetal transmission pathway of mPAEs is still

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incomplete.

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In addition to CS, amniotic fluid (AF) is also an important medium that reflects

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the chemical prenatal exposure in the fetus. AF is derived from urine, the excretion of

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gastrointestinal tract, and the mass exchange with umbilical cord of the fetus in the

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third trimester.37 Mass exchange occurs mainly between the fetus and AF and, to a

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lesser extent between AF and the mother through the fetal membrane.37,38 Hence, the

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distribution of mPAEs between AF and CS or MS (AF:CS and AF:MS) are also

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important indices for the evaluation of fetal exposure to chemicals. The occurrence

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of mPAEs in AF has been previously reported and significant positive correlations

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between creatinine-adjusted mono-n-butyl phthalate (MBP) in maternal urine (MU)

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and MBP in AF39 and between mono-(2-isobutyl) phthalate (MiBP) in MU and that in

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AF40 were found. However, the association of various mPAEs in AF with maternal

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and umbilical cord samples has not been previously determined.

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In this study, we collected 78 paired maternal-fetal samples from pregnant

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women in Tianjin, China. The aims of this study were 1) to investigate the

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concentrations and composition profiles of 11 mPAEs in these samples and explore

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their possible associations with maternal and fetal socio-demographic characteristics;

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2) to evaluate the distribution and associations of various mPAEs among the different

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matrices of MU, MS, CS, and AF; and 3) to elucidate the associations between the



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transmission efficiencies of the detected mPAEs and their chemical properties.

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■MATERIAL AND METHODS

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Chemicals

and

Reagents.

The

target

mPAEs

compounds

included

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mono-2-ethylhexyl phthalate (MEHP), MiBP, MBP, mono-(3-carboxypropyl)

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phthalate (MCPP), mono-ethyl phthalate (MEP), mono-methyl phthalate (MMP),

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mono-benzyl phthalate (MBzP), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP),

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mono-[(2-carboxymethyl) hexyl] phthalate (MCMHP), mono-(2-ethyl-5-oxohexyl)

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phthalate (MEOHP), and 5-mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP).

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Select properties and commercial sources for the 11 mPAEs and the corresponding

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PAEs are listed in Table S1 in the Supporting Information (SI). Detailed information

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for all chemicals and reagents is given in the SI.

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Sampling Campaign. All samples were collected from Tianjin Central Hospital

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of Gynecology Obstetrics, and 83 pregnant women were recruited during

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October-December, 2015. There were a total of 83 MU, 82 MS, 80 CS, and81 AF

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samples, among which 78 were composed of totally paired samples of

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MU-MS-AF-CS. This research was approved by the Ethics Committee of Tianjin

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Central Hospital of Gynecology Obstetrics, and the informed consent together with

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questionnaires was dispatched to participants and collected. MU and MS samples

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were collected two hours before delivery, while the CS and AF samples were sampled

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during delivery. All samples were stored at -80C before analysis. All participants

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were healthy and without any infectious diseases. A summary of detailed information

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of the study population is shown in Table 1.


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Sample Preparation and Instrumental Analysis. Samples were analyzed using

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a previously described method with some modifications (see Sample Preparation,

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SI).41,42 Briefly, mPAEs in 0.5 mL of the fluid samples(i.e., MU, MS, CS, and AF)

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were extracted using CNW Poly-Sery MAX cartridges (150 mg/6 mL, CNW ANPEL,

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Shanghai, China) to include free mPAEs and their conjugates after enzymolysis.

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The mPAEs in the treated samples were analyzed with high-performance liquid

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chromatography (Agilent 1200) interfaced to triple quadrupole tandem mass

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spectrometry (Agilent G6410B) (HPLC-MS/MS) and equipped with an electrospray

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ionization (ESI) interface in negative mode. The mPAEs were separated with a

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BETASIL C18 column (100 mm×2.1 mm×5 μm; Thermo Scientific, PA, U.S.) and

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monitored with multiple reaction monitoring (MRM). The detailed instrument

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parameters and mobile phase programs are given in the SI (Instrumental Analysis and

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Table S2).

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Quality Assurance and Quality Control. To ensure data quality, a procedural

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blank, an instrumental blank, and matrix-spiked samples were co-analyzed in every

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batch of 20 samples. The procedural blank was prepared by substitution of the real

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samples with 0.5 mL Milli-Q water and treated with the same procedure. The target

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mPAEs were not detected in the procedural or instrumental blanks. Twenty-five

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nanograms of three mixed isotopically labeled standards of mPAEs (i.e.,

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13

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extraction and analysis procedure. Standard samples (10 ng/mL) and pure solvent

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(acetonitrile) were injected every 20 samples to check the instrument conditions and

C4-MEP, and

13

13

C4-MBP,

C4-MECPP) was spiked into sample matrices for correction of



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the carryover of instrument contamination. A 10-point calibration curve (0.1–100

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ng/mL) was prepared at the beginning of every day, and the regression coefficient of

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calibration was above 0.99.

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Details of the recoveries and the method detection limits (MDL) of the 11

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mPAEs are provided in Table S3 for the four matrices. Values below MDL were

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recognized as 0. Because the concentrations of chemicals vary with urine volume at

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each sampling event, the creatinine level was employed for normalization of mPAEs

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concentrations in urine samples.41

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Statistical Analyses. Spearman rank correlation analyses were conducted with

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SPSS, Version 22.0 (IL, U.S.). A value of p95%), whereas MEHHP, MEOHP,

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MCPP, and MBzP were not detected; similarly, MEP was only detected in 15.0% of

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CS samples (Table 2). MMP (17.5 ng/mL), MBP (9.56ng/mL), and MiBP (5.36

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ng/mL), were the most abundant mPAEs in CS, contributing for 43.6%, 23.8%, and

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13.3% of mPAEs, respectively (Figure 1). These results indicated that low MW

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(LMW) mPAEs (i.e., MBP, MiBP, MMP, and MEP) presented the main risk to the

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fetuses compared with high MW (HMW) mPAEs (i.e., MCPP, MBzP, MEHP, MECPP,

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MEHHP, MEOHP, and MCMHP). Previous studies have found that LMW

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contaminants could easily cross the placental barrier.30-33 This study provided the first

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evidence for MEP exposure to fetuses even though it occurred at a low concentration

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and DR. Spearman correlations among 6 individual mPAEs and ∑mPAEs in CS were

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similar to those in MS (Table S6).


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Only limited studies have reported the occurrence of mPAEs in cord samples. In

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Italy, MEHP was investigated in CS of 84 newborns, and MEHP concentrations in CS

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(mean value: 520±610 ng/mL) were positively correlated with gestational age.25 In a

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nested case-control study in Shanghai, the MEHP concentrations in cord blood

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samples from 88 newborns with low birth weight and 113 healthy controls were 2500

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and 1100 ng/mL, respectively, whereas MBP was not detected.24 In France, the

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median value of MBP in 41 cord blood samples was found to be 2.90 ng/mL.53 Five

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mPAEs (MMP, MEP, MBP, MBzP, and MEHP) were investigated in 30 CS samples in

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Tianjin, with median concentrations of 26.9, 12.0, 17.0, 3.23, and 5.48 ng/mL,

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respectively.54 The results in this study were similar to those of the previous study in

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Tianjin,54 but were much lower than those found in a study conducted in Shanghai.24

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High levels of other emerging chemicals like PFCAs have been reported in human

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samples in Shanghai,55 which should be paid attention as a hot spot in future studies.

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Concentrations and Profiles of mPAEs in AF. MECPP, MCMHP, MEHP, MBP,

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MiBP, and MMP were the most frequently detected mPAEs and presented in more

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than 95.0% of the AF samples. The DR of MEP in AF samples (67.0%) was higher

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than those in MS and CS samples; furthermore, MEHHP, MEOHP, MCPP, and MBzP

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were not detected. This was the same as for MS and CS, which was indicative of

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associations between AF and MS or CS. The detection of mPAEs in AF provided

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further confirmation for the fetal exposure to mPAEs. The median mPAEs

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concentration (10.4 ng/mL) in AF was substantially lower than those in MU (128

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ng/mL), MS (24.6 ng/mL), and CS (44.9 ng/mL). This could be explained by the



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limited elimination capacity of the fetus, which indicated that high exposure of the

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fetus to mPAEs occurred mainly owing to the enclosed environment. Moreover, the

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mPAEs concentrations might be diluted by the peak AF volume at 38 weeks of

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gestation in the sampling time.37 In the four matrices, the median concentrations of

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LMW mPAEs were all higher than those of HMW mPAEs (Figure S2). Moreover,

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the proportion of the LMW mPAEs was higher in CS and AF as compared to MS

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and MU. This seems to indicate that LMW mPAEs accumulate in organs with long

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residence time. The correlations of mPAEs in AF were quite different from those in

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MS and CS (Table S6). The source of mPAEs in AF is complex since it can originate

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from both MS and CS.37 The median concentration of mPAEs in AF in this study was

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close to those observed mPAEs in AF samples in other regions/cities (Table S8).

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Factors Influencing the mPAEs Concentrations in MU, MS, CS, and AF. To

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establish the reliable factors that influence mPAEs concentrations in the four matrices,

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correlations

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socio-demographicindices were studied, including age, body mass index, gestational

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week, pregnancy, parity, sex, and fetal weight (Table S9). Pregnancy showed

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significant negative correlations with MECPP and MEOHP in MU, MECPP in MS,

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MEHP, MiBP, and MMP in CS, and MEHP in AF(all p

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