Science of the Total Environment 543 (2016) 1927

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Science of the Total Environment

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Phthalate metabolites in urine of Chinese young adults: Concentration,

prole, exposure and cumulative risk assessment
Chong-Jing Gao a, Li-Yan Liu a, Wan-Li Ma a, Nan-Qi Ren a, Ying Guo b, Ning-Zheng Zhu a, Ling Jiang c,
Yi-Fan Li a,, Kurunthachalam Kannan b,
a
International Joint Research Center for Persistent Toxic Substances, State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin
Institute of Technology, Harbin 150090, China
b
Wadsworth Center, New York State Department of Health, Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O.
Box 509, Albany, NY 12201-0509, United States
c
The Second Campus Hospital, Harbin Institute of Technology, Harbin 150090, China

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

14 phthalate metabolites in urine were

analyzed for Chinese young adults.
Unique prole of urinary phthalate me-
tabolites was found.
Principal component analysis (PCA) sug-
gested two major sources of phthalates.
Half of Chinese young adults had hazard
index (HI) values exceeding the
threshold.

a r t i c l e i n f o a b s t r a c t

Article history: Phthalates are widely used in consumer products. People are frequently exposed to phthalates due to their appli-
Received 15 July 2015 cations in daily life. In this study, 14 phthalate metabolites were analyzed in 108 urine samples collected from
Received in revised form 2 November 2015 Chinese young adults using high-performance liquid chromatographytandem mass spectrometry. The total
Accepted 2 November 2015
concentrations of 14 phthalate metabolites ranged from 71.3 to 2670 ng/mL, with the geometric mean concen-
Available online 12 November 2015
tration of 306 ng/mL. mBP and miBP were the two most abundant compounds, accounting for 48% of the total
Editor: Adrian Covaci concentrations. Principal component analysis suggested two major sources of phthalates: one dominated by
the DEHP metabolites and one by the group of mCPP, mBP and miBP metabolites. The estimated daily intakes
Keywords: of DMP, DEP, DBP, DiBP and DEHP were 1.68, 2.14, 4.12, 3.52 and 1.262.98 g/kg-bw/day, respectively. In a sen-
Phthalates sitivity analysis, urinary concentration and body weight were the most inuential variables for human exposure
Urine estimation. Furthermore, cumulative risk for hazard quotient (HQ) and hazard index (HI) were evaluated. Nearly
Human exposure half of Chinese young adults had high HI values exceeding the safe threshold. This is the rst study on the occur-
Cumulative risk assessment rence and human exposure to urinary phthalate metabolites with Chinese young adults.
China
2015 Elsevier B.V. All rights reserved.

Corresponding authors.
E-mail addresses: ijrc_pts_paper@yahoo.com (Y.-F. Li), kkannan@wadsworth.org (K. Kannan).

http://dx.doi.org/10.1016/j.scitotenv.2015.11.005
0048-9697/ 2015 Elsevier B.V. All rights reserved.
20 C.-J. Gao et al. / Science of the Total Environment 543 (2016) 1927
1. Introduction
Phthalates are a group of synthetic aromatic chemicals, which
are widely used in a large range of daily life products (Aylward
et al., 2013; Hauser and Calafat, 2005; Latini, 2005; Wittassek
et al., 2011). High molecular weight phthalates including di-(2-
ethylhexyl) phthalate (DEHP), di-isononyl phthalate (DiNP), di-
isodecyl phthalate (DiDP) and di-n-octyl phthalate (DnOP) are
used as plasticizers in building materials and furnishings, whereas
low molecular weight phthalates such as diethyl phthalate (DEP),
dimethyl phthalate (DMP) and dibutyl phthalate (DBP) are used
in personal-care products (e.g., nail polishes, fragrances), lacquers,
varnishes and coatings (Schettler, 2006). People are frequently
exposed to phthalates due to their widespread applications in
consumer products. For the general population, ingestion, inhala-
tion and dermal absorption are considered as the major exposure
pathways to phthalates (Gong et al., 2014; Hauser and Calafat,
2005; Kelley et al., 2008; Schettler, 2006; Wormuth et al., 2006).
After entering into human, phthalates can be metabolized to
their hydrolytic monoesters and then some monoesters are further
transformed into oxidative metabolites after enzymatic oxidation
(Albro et al., 1973; Albro and Moore, 1974; Albro and Thomas,
1973; McKee et al., 2002; Silva et al., 2005). Phthalates are
listed as chemicals of concern by the U.S. EPA, because some
phthalates can induce reproductive anomalies (Boberg et al.,
2008; Gray et al., 2000, 2006). Furthermore, epidemiological stud-
ies have indicated that phthalates can affect human endocrine
and reproductive systems (Calafat et al., 2010; Duty et al., 2003;
Meeker et al., 2007).
Previous studies found that phthalates could occur in a variety
of body uids, tissues and organs, including urine, serum, breast
milk, cord blood, fetal liver, placenta and adipose tissue (Hogberg
et al., 2008; Latini, 2005; Wittassek et al., 2011). The ubiquitous
occurrences of phthalates have attracted more attention world-
wide recently. Urinary phthalate metabolites are considered useful
biomarkers for assessing human exposure to parent phthalates
(Barr et al., 2003; Blount et al., 2000; Calafat and McKee, 2006;
Dirven et al., 1993; Latini, 2005). To date, studies on urinary
concentrations of phthalate metabolites in general population
have been conducted in many countries, such as the U.S.,
Germany, Denmark, Canada and Belgium (Calafat et al., 2010;
Colacino et al., 2010; Dewalque et al., 2014b; Frederiksen et al.,
2013; Koch et al., 2003a; Saravanabhavan et al., 2013). There are
also several studies reported the occurrence of phthalate metabo-
lites in urine samples collected in China, which are focused on spe-
cial populations or general population in local areas (Guo et al.,
2011a, 2011b; Han, 2014; Pan et al., 2006; Wang, 2014). However,
information on phthalate metabolites in Chinese people from multi
districts is scarce, especially for the information on cumulative risk
evaluation.
International Joint Research Center for Persistent Toxic Substances
(IJRC-PTS) carried out the China PTSs Human Exposure Monitoring
Program (China-HEMP), including indoor dust sampling, blood and
urine sampling from Chinese young adults across China (Qi et al.,
2014a, 2014b). The occurrence of parabens, benzophenone-type ul-
traviolet (UV) lters and bisphenol A were analyzed in Chinese
young adults previously (Gao et al., 2015b, 2015c; Ma et al., 2013).
In the present study, 108 urine samples collected from most prov-
inces and municipalities of China were analyzed to study the occur-
rence of phthalate metabolites. The objectives of this study were:
a) to analyze concentrations and proles of phthalate metabolites in
urine samples from Chinese young adults; b) to study the difference
in concentrations between males and females, urban and rural popu-
lation; c) to investigate the potential sources of phthalate metabo-
lites; and d) to estimate the daily exposure doses and cumulative
risk of phthalates.
2. Materials and methods
2.1. Study population and sample collection
Urine sample was collected from 108 Chinese young adults (18
22 years old) in most of the mainland provinces and municipalities of
China in September 2010. All participants are healthy high school grad-
uates. The spatial distribution of the urine samples for each region can
be found in our previous study (Ma et al., 2013). The number of samples
for females/males is 67/41. All participants in this program were also
classied as urban (58) or rural (50) residents according to their
home sites. Each participant collected a rst-morning-void urine sam-
ple in polypropylene (PP) tubes. Urine samples were then stored at
20 C in laboratory. All samples were collected and processed in a
same procedure.
2.2. Sample preparation and instrumental analysis
Fourteen phthalate metabolites including mono(3-carboxypropyl)
phthalate (mCPP), monomethyl phthalate (mMP), monoethyl phthal-
ate (mEP), monoisobutyl phthalate) (miBP), mono-nbutyl phthalate
(mBP), mono(2-ethyl-5-carboxypentyl) phthalate (mECPP), mono-
[(2-carboxymethyl) hexyl] phthalate (mCMHP), mono(2-ethyl-5-
oxohexyl) phthalate (mEOHP), mono(2-ethyl-5-hydroxyhexyl) phthal-
ate (mEHHP), monocyclohexyl phthalate (mCHP), monobenzyl phthal-
ate (mBzP), mono(2-ethylhexyl) phthalate (mEHP), monoisonoyl
phthalate (mNP), and monooctyl phthalate (mOP) were analyzed in
the present study. Detailed information of all phthalate metabolites
and their parent phthalate compounds are presented in Table S1,
Supporting information.
The chemicals, reagents, applied methods for sample treatment and
instrument analysis were reported in our previous study (Guo et al.,
2011a). The detailed information can be found in Supporting Information.
Briey, 0.5 mL of urine sample was buffered with 200 L of ammonium
acetate (7.7 g ammonium acetate dissolved in 100 mL Milli-Q water
and 6 mL acetic acid; pH = 4.5) and spiked with 100 L mixture of labeled
internal standards of phthalate metabolites (250 ng/mL). Then, 50 L of -
glucuronidase (2 L/mL), 100 L of 4-methylumbelliferyl glucuronide
(500 ng/mL) and 0.5 mL of Milli-Q water were added. Urine samples
were incubated at 37 C overnight. Subsequently, phthalate metabo-
lites were extracted on a Rapid Trace Workstation (Caliper Life Sci-
ence, Hopkinton, MA, USA). Acetonitrile (1.5 mL) and phosphate
buffer (1.2 mL; 20 g sodium phosphate monobasic monohydrate dis-
solved in 1000 mL Milli-Q water and 10 mL phosphoric acid; pH = 2)
were added to condition the SPE cartridge (ABS ELUT-Nexus, Varian,
Walnut Creek, CA, USA; 60 mg/3 mL). Urine samples were diluted
with 1 mL of phosphate buffer and loaded onto the SPE cartridge at
a rate of 0.5 mL/min. The cartridge was rinsed with 2.0 mL of formic
acid (0.1 M) and 1.2 mL of Milli-Q water and dried under nitrogen for
5 min. Acetonitrile (1.2 mL) and ethyl acetate (1.1 mL) were added to
elute the target compounds. The extract was concentrated under a
stream of nitrogen to near dryness. The residue was resolved in
0.5 mL acetonitrile: Milli-Q water (1:9) for instrumental analysis.
The chromatographic separation was achieved using an Agilent
1100 Series HPLC system (Agilent Technologies, Santa Clara, CA)
equipped with a Betasil C18 column (Thermo Electron, Bellefonte,
PA; 100 mm 2.1 mm, 5 m). An API 2000 electrospray triple quad-
rupole mass spectrometer (ESIMS/MS; Applied Biosystems, Foster
City, CA) was used to identify and quantify phthalate metabolites
and urinary creatinine.
2.3. Quality assurance/quality control (QA/QC)
All the urine samples were divided into four batches. For each batch,
two method blanks, two spiked blanks, and two matrix spiked samples
were processed along with real samples. The detailed information of
 C.-J. Gao et al. / Science of the Total Environment 543 (2016) 1927
QA/QC results is shown in Table S2. Briey, the average recoveries of tar-
get compounds in spiked blanks and matrix spiked samples were
102 15% and 98 23%, respectively. The average recoveries of inter-
nal standards in blanks, matrix spiked samples and real samples were
86 17%, 71 12% and 72 15%, respectively. Trace concentrations
of mMP (mean: 3.77 ng/mL), mEP (0.06 ng/mL), mBP (0.23 ng/mL),
miBP (0.08 ng/mL) and mEHP (4.40 ng/mL) were found in method
blanks. The concentrations in the method blanks were subtracted
from the concentrations of real urine samples. The limits of quantica-
tion (LOQs) of phthalate metabolites varied from 0.1 to 0.5 ng/mL,
according to the signal to noise of 10. Concentrations below the
LOQs were replaced by values equal to half of the LOQs for statistic
analysis.
2.4. Daily intake estimation
The measured urinary concentrations of phthalate metabolites were
used to estimate daily intake of phthalates in the present study. Daily
exposure dose of phthalates was calculated using the following simple
steady-state exposure model:
M1 1 1
EDI CV   
M2 f W
where, EDI is the estimated daily intake of phthalate (g/kg-bw/day), C
is the urinary phthalate metabolite concentration (g/L), V is the human
daily excretion volume of urine (L/day), M1 and M2 are the respective
molecular weights of parent phthalate and its metabolite (g/mol), f is
the molar fraction of the urinary monoester metabolite excreted in rela-
tion to the ingested amount of phthalate, and W is the body weight (kg).
A volume of 1.7 L was used for 24 h urine volume for adults (Perucca
et al., 2007), the values of f for mMP, mEP, mBP, miBP, mEHHP, mEOHP,
and mEHP were 0.7, 0.69, 0.70, 0.7, 0.23, 0.15, and 0.059, respectively
(Anderson et al., 2001; Itoh et al., 2007; Koch et al., 2003a, 2003b,
2005). For W, the average body weights of 62.6 kg and 51.9 kg for
male and female adults were applied respectively, which were calculat-
ed based on the values reported by the General Administration of Sport
of China (http://www.sport.gov.cn/n16/n1077/n297454/2052709.
html).
2.5. Sensitivity analysis
The Monte Carlo Simulation was applied for the sensitivity analysis
with human exposure estimation for three parameters including uri-
nary concentration, body weight, and urine volume. For each parameter
in Eq. (1), 10,000 runs were conducted to test the convergence and the
stability. Correlation coefcients (r) between each input (parameter)
and the output (EDI) were used to determine the most inuential
parameter.
2.6. Cumulative risk assessment hazard quotient (HQ) and hazard index
(HI)
For public health safety purpose, the European Food Safety Authorities
(EFSA) established tolerable daily intake (TDI, a TDI is an estimate of the
amount of a substance in air, food or drinking water that can be taken
in daily over a lifetime without appreciable health risk). The values of
TDI for DBP and DEHP were 10 and 50 g/kg-bw/day, repsectively
(EFSA, 2005a, 2005b, 2005c). Kortenkamp and Faust also developed a
reference dose for anti-androgenicity (RfD AA, RfD is an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily oral expo-
sure to the human population (including sensitive subgroups) that is like-
ly to be without an appreciable risk of deleterious effects during a lifetime
(http://www.epa.gov/riskassessment/glossary.htm#r)). The values of
RfD AA for DBP, DiBP and DEHP were 100, 200 and 30 g/kg-bw/day,
respectively (Kortenkamp and Faust, 2010). Based on the EDI of
21
individual phthalate, HQ of DBP, DiBP and DEHP were calculated as
follows (Dewalque et al., 2014a):
EDI
HQ 2
Reference limit value
where, HQ is the hazard quotient for individual phthalate, the reference
limit value is the TDI or RfD AA. The reference limit value of DiBP was
assigned to be 10 g/kg-bw/day for TDI because there was no data
provided by the EFSA for DiBP (Koch, 2011; Seborg et al., 2012).
In order to evaluate the cumulative risk of phthalates, HI was calcu-
lated by summing the values of HQ of different phthalates as follows:
HITDI HQ DBP TDI HQ DiBP TDI HQ DEHP TDI 3
HIRfD AA HQ DBP RfD AA HQ DiBP RfD AA HQ DEHP RfD AA : 4
2.7. Statistical analysis
SPSS Software (Version 22) was used to perform the statistic
analysis. Non-parametric tests (the KruskalWallis H Test and the
MannWhitney U Test) were used to compare differences of individual
1
compounds between males and females, urban and rural adults, and
different regions. Statistical signicance was set at p b 0.05. Principal
component analysis was applied to investigate the potential sources of
phthalate metabolites.
3. Results and discussion
3.1. Concentration
One or more phthalate metabolites were detected in all urine sam-
ples, which indicated the ubiquitous exposure to these compounds by
Chinese young adults. Concentrations and detection rates of individual
phthalate metabolites are summarized in Table 1. Among the 14 phthal-
ate metabolites, mEP, mBP, miBP, mECPP, mCMHP, mEHHP, and mEOHP
were found in all samples. The detection rates of mCPP, mMP, mEHP,
and mBzP were 97.2%, 99.1%, 82.0%, and 39.9%, respectively. The re-
maining three metabolites (mCHP, mNP, and mOP) were only found
in less than 6% of samples, which were excluded for further analysis.
The total concentrations of phthalate metabolites (14phthalates) in
Chinese young adults ranged from 71.3 to 2670 ng/mL, with the geo-
metric mean (GM) concentration of 306 ng/mL. Signicant differences
were observed among different phthalate metabolites' concentrations
(p b 0.01) with a decreasing order as follows: mBP N miBP N mMP N
mEP N mECPP N mCMHP N mEHHP N mEOHP N mCPP N mEHP N mBzP
(Fig. 1(a)). When the concentrations were adjusted for creatinine, the dif-
ferences were still signicant (p b 0.01) (Table 1 and Fig. 1(b)).
mBP and miBP were the dominant compounds, with the GM concen-
trations of 67.0 and 57.2 ng/mL, respectively. The similar results were
also found in our previous studies for general population in China
(Guo et al., 2011b; Wang et al., 2013). However, the concentrations of
mBP in Chinese young adults were much higher than those for adults
from Chongqing, China (Han, 2014; Liu et al., 2011). Concentrations of
phthalate metabolites in urine samples worldwide are summarized in
Table S3 and presented in Fig. 2. The concentrations of mBP reported
in Japan (median: 84.3 ng/mL) (Itoh et al., 2009), Kuwait (median:
94.1 ng/mL) (Guo et al., 2011a) and Mexico (median: 82.1 ng/mL)
(Lpez-Carrillo et al., 2010) were higher than the values in China.
Germany population has the highest mBP concentration (median:
181 ng/mL) (Koch et al., 2003b). For other countries listed in Table S3,
the concentrations of mBP were all lower than the values in the present
study. The concentration of miBP found in this study was higher than all
the reported values in other countries except for Danish males (median:
58.0 ng/mL) (Joensen et al., 2012). This indicated high exposure to DiBP
by Chinese young adults.
22 C.-J. Gao et al. / Science of the Total Environment 543 (2016) 1927

Table 1
Urinary concentrations (ng/mL(g/g creatinine)) of phthalate metabolites in Chinese young adults.

Compounds GMa 5th 25th 50th 75th 95th DRb

mMP 31.8 (12.2) 8.51 (3.72) 18.8 (6.57) 35.0 (9.89) 59.5 (18.2) 113 (92.0) 99.1
mEP 37.5 (14.4) 10.1 (2.00) 21.0 (6.01) 31.7 (12.6) 68.8 (31.0) 200 (126) 100
mCPP 3.18 (1.22) 0.93 (0.32) 1.96 (0.62) 3.19 (1.18) 5.47 (1.86) 13.1 (5.22) 97.2
mBP 67.0 (25.7) 15.8 (6.38) 38.7 (12.4) 71.7 (21.9) 111 (40.6) 241 (125) 100
miBP 57.2 (21.9) 16.6 (6.10) 37.7 (12.1) 55.6 (18.9) 89.6 (32.1) 206 (82.2) 100
mECPP 24.3 (9.31) 7.80 (2.84) 15.3 (4.84) 24.1 (8.40) 36.2 (12.8) 74.4 (47.3) 100
mCMHP 15.2 (5.83) 5.94 (1.61) 9.10 (3.32) 14.1 (5.54) 23.3 (7.86) 45.2 (22.5) 100
mEHHP 14.5 (5.54) 3.97 (1.58) 8.74 (2.75) 14.2 (4.69) 22.3 (7.25) 58.1 (39.2) 100
mEOHP 9.73 (3.72) 2.85 (1.07) 5.82 (2.01) 9.32 (2.95) 14.3 (5.08) 35.5 (22.6) 100
mEHP 1.53 (0.41) 0.07 (0.02) 0.47 (0.13) 1.84 (0.47) 5.50 (1.27) 16.0 (11.4) 82.0
mBzP 0.14 (0.05) ndc (nd) nd (nd) nd (nd) 0.85 (0.31) 4.44 (3.22) 39.9
mCHP 0.21 (0.08) nd (nd) nd (nd) nd (nd) nd (nd) 0.20 (0.39) 0.03
mNP 0.11 (0.04) nd (nd) nd (nd) nd (nd) nd (nd) 0.11 (0.22) 5.41
mOP 0.19 (0.07) nd (nd) nd (nd) nd (nd) nd (nd) 0.19 (0.36) 0.01
5 DEHP metabolites 67.0 (25.7) 20.8 (7.40) 41.3 (13.8) 64.9 (21.1) 103 (34.7) 203 (127)
14 phthalates 306 (117) 95.1 (35.4) 200 (56.2) 329 (108) 468 (177) 849 (382)
a
GM = Geometric mean.
b
DR = detection rate.
c
nd = not detectable.

The concentrations of mMP in Chinese young adults (median:
31.8 ng/mL) was nearly 3 times higher than those in Chinese general
adults (median: 12.0 ng/mL) reported in our previous study (Guo
et al., 2011a). Chinese young adults also had higher concentrations of
mMP compared with the values from Japan, Korea, India and the U.S.
(Colacino et al., 2010; Guo et al., 2011a). The concentration of mMP in
Japanese people was 17.5 ng/mL, which was half of the value found in
this study (Guo et al., 2011a). The concentrations of mMP in people
Fig. 1. Concentrations of 14 phthalate metabolites in urine of Chinese young adults:
(a) ng/mL; (b) g/g creatinine (Note: The boxes represent 25th and 75th percentiles.
The horizontal lines represent the 10th, 50th, and 90th percentiles. Outliers are shown
as individual points.)
from Korea (median: 3.4 ng/mL) (Guo et al., 2011a), India (median:
5.8 ng/mL) (Guo et al., 2011a) and the U.S. (median: 1.4 ng/mL)
(Colacino et al., 2010) were one order of magnitude lower than that in
Chinese young adults. Higher concentrations of mMP in urine samples
indicated high exposure dose to DMP for Chinese young adults.
Urinary concentration of mEP in Chinese young adults (median:
37.5 ng/mL) was much lower than those from Chongqing, China (Liu
et al., 2011). But it was comparable with the values in Swedish adult
women (median: 35 ng/mL) (Hogberg et al., 2008), Malaysian general
population (median: 30.5 ng/mL) (Guo et al., 2011a) and Belgian adults
(median: 34.3 ng/mL) (Dewalque et al., 2014b). The concentration of
mEP in Chinese young adults was more than 2 times higher than
those in Japan and Korea (median: 14.8 ng/mL and 13.2 ng/mL) (Guo
et al., 2011a), but only one-third to one-fourth of the concentrations
in Germany (median: 90.2 ng/mL) (Koch et al., 2003b), Mexico
(median: 107 ng/mL) (Lpez-Carrillo et al., 2010), Netherlands
(median: 117 ng/mL) (Ye et al., 2008) and India (median: 131 ng/mL)
(Guo et al., 2011a). The U.S. National Health and Nutrition Examination
Survey found that the urinary concentrations of mEP was 208 ng/mL for
the general population in 20032004 (Colacino et al., 2010). It was near-
ly 5 or 6 times higher than the concentrations in this study. However,
adults in Kuwait (median: 391 ng/mL) had the highest concentration
of mEP, which was one order of magnitude higher than that found in
Chinese young adults (Guo et al., 2011a).
mECPP, mCMHP, mEHHP, mEOHP and mEHP are important metab-
olites of DEHP. In this study, the median concentration of the sum of
DEHP metabolites (5DEHP metabolites) was 67.0 ng/mL. Concentra-
tions of mECPP, mCMHP, mEHHP, mEOHP, and mEHP were 24.3, 15.2,
14.5, 9.73, and 1.53 ng/mL, respectively. The concentrations of DEHP
metabolites found in this study were lower than the values reported
in Kuwait (Guo et al., 2011a), Mexico (Lpez-Carrillo et al., 2010) and
U.S. (Colacino et al., 2010). Concentrations of oxidative DEHP metabo-
lites (mECPP, mCMHP, mEHHP, and mEOH) were one order of magni-
tude higher than that of mEHP. This nding maybe suggest that mEHP
can be broken down further to more oxidative metabolites (Hauser
and Calafat, 2005).
Concentrations of phthalate metabolites in males and females, as
well as urban and rural groups are summarized in Table S4. There was
no signicant difference between concentrations of phthalate metabo-
lite in males and females, as well as in urban and rural groups (p N
0.05). However, the concentrations of mMP, mCPP, mBP, miBP and all
DEHP metabolites in rural young adults were all slightly higher than
the values in urban young adults. In order to understand these differ-
ences, we divided all urine samples into four groups including urban
males and females, rural males and females. The GM concentrations of
 C.-J. Gao et al. / Science of the Total Environment 543 (2016) 1927 23

Fig. 2. Urinary concentrations (ng/mL) of phthalate metabolites in the world (Data sources: China (a), Japan (a), Korea (a), India, Kuwait, Malaysia and Vietnam: Guo et al., 2011a; China
(b): Guo et al., 2011b; China (c): Liu et al., 2011; China (d): Han, 2014; Japan (a): Itoh et al., 2009; Japan (b): Itoh et al., 2007; Korea (b), (c), (d): Song et al., 2013; Germany (a): Koch et al.,
2003b; Germany (b): Fromme et al., 2007; Mexico (a): Lpez-Carrillo et al., 2010; Mexico (b): Romero-Franco et al., 2011; Sweden: Hogberg et al., 2008; Denmark (a): Frederiksen et al.,
2013; Denmark (b): Frederiksen et al., 2013; Joensen et al., 2012; Denmark (c): Frederiksen et al., 2013; U.S.: Colacino et al., 2010; Canada: Saravanabhavan et al., 2013; Irisa: Berman et al.,
2013; Italy: Tranfo et al., 2013; Belgium: Dewalque et al., 2014b).

phthalate metabolites in urine of different subgroups are presented in metabolites in rural females were not high. Further research is needed
Table S5. It is interesting to note that the GM concentrations of all for full understanding of these courses.
phthalate metabolites in rural males were higher than those in urban
males. And the GM concentrations of all phthalate metabolites (except 3.2. Composition prole
for mMP) in urban females were higher than those in rural females.
Although these differences were not signicant (p N 0.05), they may re- The composition proles of urinary phthalate metabolites in Chinese
veal some information of these compounds from these four groups. young adults are summarized in Fig. 3. mBP, miBP and DEHP accounted
Among all four groups, the GM concentrations of mEP in urban females for 25.9%, 21.2% and 24.3% of the total concentrations of 14 phthalate
were the highest. The reason for this is most likely due to the higher metabolites, while mMP and mEP accounted for 12.6% and 14.3%, re-
usage of personal care products in urban females than the other three spectively. The sum of mBP, miBP, mMP, mEP, and DEHP metabolites
groups, as many studies suggested that personal care products are contributed N 98% of the total concentrations, while the other metabo-
major source of DEP for people exposure (Wormuth et al., 2006; Koch lites (mCPP, mBzP, mCHP, mNP and, mOP) only contributed 2%. In this
et al., 2013). The concentrations of mMP in both rural males and females study, the most abundant metabolite was mBP. This nding was similar
were higher than those in urban males and females. The sources of DMP to previous studies with Chinese general population (Guo et al., 2011a,
are not very clear. Previous studies suggested that diet and personal 2011b; Lin et al., 2011; Wang et al., 2013). In Japan, Germany, Vietnam,
care products are not the major sources of DMP for Chinese residents Korea and Sweden, mBP was also the dominant phthalate metabolite
(Guo et al., 2012, 2014; Bao et al., 2015). It seems from the present (Becker et al., 2009; Fromme et al., 2007; Guo et al., 2011a; Hogberg
study that DMP in air may be the major source for this chemical to
enter the bodies of Chinese young adults. DMP in the air is mainly de-
rived from the spray coating and the burning of various plastics (espe-
cially agricultural lm and living heating fuel) in China (Wang, 2010).
In rural areas, straw, coal and some other living heating fuels are exten-
sively used for cooking and heating every day. Then residents in rural
areas are more easily and frequently to be exposed to DMP. In addition,
DMP is extensively existed in air phase due to its low molecular weight
and high saturated vapor pressure. Therefore, DMP in air (air phase)
may be the major source of mMP in urine of rural male young adults.
The concentrations of DBP metabolite (mBP), DIBP metabolite (miBP),
and DEHP metabolites (mECPP, mCMHP, mEHHP, mEOHP and mEHP)
in urine of rural males were all higher than other subgroups, and the
reasons for this are still not clear to us. Though some studies showed im-
plications on the concentration levels for these phthalates in various
media, the sources of DBP, DIBP and DEHP are still not clear (Guo
et al., 2014; Itoh et al., 2007). The present study possibly indicated
that higher concentrations of the metabolites of DBP, DIBP and DEHP
in rural male young adults may suggest some special sources of these Fig. 3. Composition proles of 14 phthalate metabolites in urine samples from Chinese
phthalates to which the rural male young adults exposed to. These young adults (Note: Others in the legend represents the sum of mCPP, mBzP, mCHP,
special sources are not from air because the concentrations of these mNP and mOP).
24 C.-J. Gao et al. / Science of the Total Environment 543 (2016) 1927
et al., 2008; Koch et al., 2003b; Suzuki et al., 2010). In other countries,
however, the major metabolite in urine samples was different. In
Denmark, France and Israel, miBP was the major phthalate metabolite
(Berman et al., 2013; Frederiksen et al., 2013; Zeman et al., 2013).
Whereas, mEP was the dominant metabolite in urine from India,
Kuwait, Malaysia, Mexico, Netherlands, U.S., Spain, Belgium (adults)
and Peru, (Casas et al., 2011; Colacino et al., 2010; Dewalque et al.,
2014b; Guo et al., 2011a; Lpez-Carrillo et al., 2010; Ye et al., 2008). Dif-
ferent proles of phthalate metabolites may be caused by different ex-
posure sources. Compared with the population from other countries,
Chinese young adults had special composition prole of phthalate me-
tabolites. This result may indicate different major phthalate exposure
pathways in Chinese young adults or different proles of phthalate in
environmental medium, food and consumer products.
The composition proles of DEHP metabolites in urine from Chinese
young adults are summarized in Fig. S1. mECPP and mCMHP were the
dominant metabolites, accounting for 38.0% and 24.4% of the total con-
centrations of DEHP metabolite, followed by mEOHP (22.3%), mEHHP
(15.2%), and mEHP (0.25%). The composition proles of DEHP metabo-
lites in urine from other studies are also presented in Fig. S1. For
Americans, mECPP and mEHHP were the dominant metabolites of
DEHP (Colacino et al., 2010; Silva et al., 2006). For Kuwaitis, mCMHP
and mEHHP contributed to the major proportions. mECPP and
mCMHP were the dominant metabolites of DEHP in India, Japan,
Korea, Malaysia and Vietnam, which were consistent with the results
of China (Guo et al., 2011a). The special composition proles of DEHP
metabolites in urine from Chinese young adults may be caused by the
following two reasons. Firstly, the similar composition proles of
DEHP metabolites in urine from Asian countries (except for Kuwait)
may be caused by the similar exposure sources for these Asian countries
population. Secondly, different DEHP metabolites have different urinary
elimination half-lives, with the shortest elimination half-life of mEHP
(about 5 h) and the longest elimination half-lives of mCMHP and
mECPP (about 24 and 15 h, respectively) (Koch et al., 2005). Thus, sam-
pling and exposure time will also inuence the composition proles of
DEHP metabolites in urine. If sampling occurs shortly after exposure,
the relative pattern of DEHP metabolites will be different than if sam-
pling occurs at a longer time after exposure (Lorber et al., 2011). In
the present study, all samples were rst-morning voids while samples
in other studies used for comparison were collected during random
daytime hours. First morning voids likely represent sampling at a longer
time point from last exposure than spot urine samples collected during
random daytime hours. Therefore, the dominant metabolites such as
mECPP and mCMHP in urine of Chinese young adults different from
Americans may be partly caused by the long elimination half-lives of
these two metabolites.
3.3. PCA
Principal component analysis (PCA) was performed for 10 phthalate
metabolites (mMP, mEP, mCPP, mBP, miBP and the ve DEHP metabo-
lites). The results of PCA are summarized in Fig. 4. There are two princi-
pal components extracted, which accounted for 44.0% and 28.7% of the
variability, respectively. This indicated two major potential exposure
sources of phthalate for Chinese young adults. mECPP, mEHHP,
mEOHP and mEHP (metabolites of DEHP) were highly correlated with
PC1, while mCPP, mBP, miBP were highly correlated with PC2. These re-
sults suggested PC1 is dominated by the group of DEHP metabolites and
PC2 by the group of mCPP, mBP, miBP. Previous studies found that DEHP
is the most abundant phthalate in food (Guo et al., 2012). Therefore,
food may be a major exposure source of phthalate for Chinese young
adults. The sources of mCPP, mBP and miBP were more complicated.
Previous studies suggested that diet, dust, and personal care products
are not major exposure sources of DBP and DiBP for Chinese population
(Guo and Kannan, 2011; Guo et al., 2012, 2014). Thus, there may be
some other unknown exposure sources existing. Low molecular weight
Fig. 4. Principal component analysis of phthalate metabolites in urine samples from Chi-
nese young adults.
phthalates such as DBP and DiBP are widely used in lacquers, varnishes
and coatings (Calafat and McKee, 2006; Liu et al., 2014). Our earlier
study also found that the concentrations of mBP and miBP in urine
from painters were two times higher than those in general population
(Gao et al., 2015a). Therefore, exposure to these consumer products
in daily life may be an important way to DBP and DiBP for Chinese
population.
For other metabolites, mEP was positively correlated with PC2.
Earlier study suggested personal care products were the major sources
for DEP in environment (Bao et al., 2015; Guo et al., 2014). Therefore,
personal care products may be another exposure source for Chinese
young adults. Chinese young adults also had high concentrations of
mMP. But the sources of mMP are unclear until now. Previous study
suggested that the concentrations of DMP in milk products, instant noo-
dle, cookies and cakes, and salt eggs were higher than those in other
food (Guo et al., 2012). All participants in this study are high school
graduates. All food mentioned above are very popular to these high
school young adults in China. Therefore, we assumed that high concen-
trations of mMP in Chinese young adults may be related to the frequent
exposure to some special food containing DMP.
3.4. Daily intake estimation
The estimated daily intakes (EDIs) of phthalates with Chinese young
adults are shown in Table S6. The EDIs of DMP, DEP, DBP, DiBP, and
DEHP were 1.68, 2.14, 4.12, 3.52 and 1.262.98 g/kg-bw/day, respec-
tively. The EDIs of DMP, DEP, DBP, DiBP and DEHP for female adults
were all slightly higher than these for males, which was consistent
with the results of concentrations. For urban and rural adults, the EDIs
to DMP, DEP, DBP, DiBP and DEHP were comparable.
The EDIs of DEP, DBP and DEHP for Chinese young adults were
lower than the reference doses suggested by U.S. EPA (800, 100 and
20 g/kg-bw/day for DEP, DBP and DEHP, respectively) (Aylward et al.,
2009; Marsee et al., 2006). However, 12.0% of Chinese young adults
exceeded the TDI value of DBP suggested by EFSA (10 g/kg-bw/day)
and 5.6% exceeded the reference daily intake dose of DEHP suggested
by Kortenkamp and Faust (30 g/kg-bw/day) (EFSA, 2005a, 2005b,
2005c; Kortenkamp and Faust, 2010). This indicated that some Chinese
young adults are not in a safe level of phthalate exposure. Furthermore,
it should be kept in mind that most of the parameters for EDI estimation
in this study were obtained from other studies and government reports.
The uncertainty of the parameters should be considered for the inuence
on the variability of the estimation.
The result of the sensitivity analysis on EDI estimation is depicted in
Fig. S2. Human daily excretion volume of urine (V) varied from 0.8 L to
2.5 L. The body weight (W) varied from 51.1 kg to 65.6 kg (http://www.
 C.-J. Gao et al. / Science of the Total Environment 543 (2016) 1927
sport.gov.cn/n16/n1077/n297454/2052709.html). The distribution of
parameters of V and W were LN (1.7, 1.02) and LN (52, 1.04) (US EPA,
2011). The parameters of concentration and body weight are the two
most inuential parameters with correlation coefcients with EDI rang-
ing from 0.42 to 0.90 and from 0.24 to 0.95, respectively. Urine vol-
ume was the least important parameter with correlation coefcient
ranging from 0.05 to 0.24. As the values of body weight and urine
volume were not based on measurement with the participants, this
may be a limitation for this study. More exact specic parameters
should be considered in future.
3.5. Cumulative risk assessment HQ and HI
A HQ is the ratio of the potential exposure to a substance and the
level at which no adverse effects are expected. A HI is a cumulative
risk assessment which was carried out by calculating the sum of the dif-
ferent HQ based on similar toxicological endpoints. In this study, HQ
shows a risk for individual phthalate, while HI shows a cumulative
risk for total phthalates. HQ and HI values below 1 are considered as a
tolerant daily intake dose and safe adverse effect caused by phthalates.
A HQ value exceeding 1 indicates an intake above the dose considered
as acceptable. A HI value between 1 and 100 indicates a potential risk
for adverse effect, while the value exceeding 100 indicates the limit of
no observed adverse effect (Benson, 2009). The results of HQ and HI
for Chinese young adults are summarized in Table S7. The EDIs of
DEHP adopted for HQ and HI calculation were based on mEHHP. HQ of
DBP, DiBP and DEHP exceeding the threshold based on TDI were
found in Chinese young adults. It should be noted that 43 of 108
young adults in this study showed high HI values exceeding 1. This
result indicated that nearly half of Chinese young adults may have a
potential risk for adverse effect caused by phthalate exposure. For
subgroups, higher HI values were observed in female group than those
in male group, with 54.8% of females having HI values exceeding 1 com-
pared to 30.3% of males. Similar, higher HI values were observed in rural
group than those in urban group, with the percentage of 46.0% and
34.5% exceeding 1, respectively.
The results of cumulative exposure assessment are dependent on
the reference doses taken into account (Dewalque et al., 2014a). Though
HITDI was 34 folds higher than HIRfD AA, only 1.9% of Chinese young
adults showed a HI RID AA higher than 1. This indicated a considerable
level of cumulative exposure assessment of phthalate for Chinese
young adults. In addition, the cumulative risk of phthalates was
underestimated because only three phthalates (DBP, DiBP and DEHP)
were considered.
In order to quantify the uncertainty of the cumulative risk assess-
ment, the predicted probability density functions of EDI was calculated
for considering the uncertainty of measured urinary concentration
(Fig. S3). The estimated range of EDI was used for the calculation of
HQ and HI values based on TDI and RfD AA, and the results are shown
in Table S8. It is interesting to note that the values of cumulative risk as-
sessment (HQ and HI) calculated with measured concentration were
similar with those from probabilistic estimation. This indicated that
the cumulative risk assessment can be calculated with the monitoring
results directly.
4. Conclusions, limitations and implications
The concentrations, proles, sources, exposure doses and cumula-
tive risk assessment of phthalate metabolites with Chinese young adults
were analyzed in this study. mBP and miBP were the dominant com-
pounds in Chinese young adults. The concentrations of mBP, miBP and
DEHP metabolites in urine samples from East China were higher com-
pared with other regions. Rural young adults may be exposed to more
phthalates than urban young adults. The results of PCA suggested two
potential exposure sources according to the group of DEHP metabolites
and the group of mCPP, mBP, miBP. Cumulative risk assessment showed
25
nearly half of Chinese young adults had HI values exceeding the thresh-
old. This is the rst comprehensive study on the occurrence and human
exposure to urinary phthalate metabolites in young adults in China,
which provided new insight into the study of phthalates.
However, as we mentioned in above sections, there exist some
limitations for our study. First, the sample size is limited. Although the
urine samples were collected from most parts of China, the sample
size was still not enough, especially for each subgroup, which may
cause deviation in comparison between genders, urban and rural
groups, as well as different regions. Second, more specic parameters
for the exposure estimation are needed. The estimation of exposure to
phthalate metabolites produced some inaccuracies due to the parame-
ters adopted from other studies and government reports. Third, the
values of health risk to phthalates were underestimated. In the present
study, the results of cumulative risk assessment of phthalates were only
calculated with three major parent phthalates due to lack parameters of
other high detectable phthalates, such as DMP and DEP. In addition, for
some urine samples, other phthalates were also detected with higher
concentration, which were not considered due to their low detection
rate (b 30%). Therefore, the results of health risk can only be used to
represent the basic information for the major phthalates.
The ubiquitous occurrence of phthalate metabolites in urine of
Chinese young adults suggested further studies to distinguish and
evaluate the exposure pathways to phthalates. Furthermore, specic
sources of individual phthalate are still unclear. Some important expo-
sure sources like lacquers, varnishes and coatings need to be considered
and studied. Therefore, a better understanding of the major pathways of
human exposure to phthalates is necessary in future. The occurrence of
phthalate metabolites for all age groups on the nation scale should be
considered in future.
Conict of interest
The authors declared that they have no conicts of interest to this
work.
Acknowledgments
We thank all the participants for providing urine samples across
China. This research was supported by the National Natural Science
Foundation of China (21277038), Independent Research from State
Key Laboratory of Urban Water Resource and Environment (No.
2013DX14), and the Fundamental Research Funds for the Central
Universities (Grant No. HIT. NSRIF. 2014095) are highly appreciated.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2015.11.005.
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