Exposure assessment of phthalate esters in Japanese pregnant women by using urinary metabolite analysis
Environ Health Prev Med
Exposure assessment of phthalate esters in Japanese pregnant women by using urinary metabolite analysis
Yayoi Suzuki 0 1 2 3
Mayu Niwa 0 1 2 3
Jun Yoshinaga 0 1 2 3
Chiho Watanabe 0 1 2 3
Yoshifumi Mizumoto 0 1 2 3
Shigeko Serizawa 0 1 2 3
Hiroaki Shiraishi 0 1 2 3
0 Y. Mizumoto Department of Obstetrics and Gynecology, Central Hospital of Self-Defense Force , Tokyo , Japan
1 C. Watanabe Department of Human Ecology, The University of Tokyo , Tokyo , Japan
2 Y. Suzuki (&) M. Niwa J. Yoshinaga Department of Environmental Studies, The University of Tokyo , Kashiwanoha 5-1-5, Kashiwa, Chiba 270-8563 , Japan
3 S. Serizawa H. Shiraishi National Institute for Environmental Studies , Ibaraki , Japan
Objectives Our objectives were (1) to evaluate whether single spot urine is suitable media for longer-term phthalate esters exposure assessment, and (2) to estimate intake level of phthalate esters of Japanese pregnant women using urinary metabolites as an indicator of prenatal exposure level in their offspring. Methods We analyzed nine metabolites (MMP, MEP, MnBP, MBzP, MEHP, MEOHP, MEHHP, MINP, MnOP) of seven phthalate esters in spot urine samples from 50 pregnant women by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Using four urine samples collected from each of 12 subjects from 50 pregnant women within 5-12 weeks, we compared intra- and interindividual variation in urinary metabolites by calculation of intraclass correlation coefficient (ICC). We estimated daily intakes of 50 pregnant women from their urinary metabolite concentrations.
Phthalate esters; Biomarkers; Urinary metabolites; Intraclass correlation coefficient; Daily intake
Results ICCs for seven phthalate metabolite
concentrations in single spot urine samples were: MMP (0.57), MEP
(0.47), MnBP (0.69), MBzP (0.28), MEHP (0.51), MEHHP
(0.43), and MEOHP (0.41) in 12 pregnant women.
Phthalate ester metabolites had high detection rates in 50
subjects. The mean daily intake ranged from 0.01 to 2 lg/kg
per day. The daily intake levels in all subjects were lower
than corresponding tolerable daily intake (TDI) set by the
European Food Safety Authority (EFSA), though
maximum value for DnBP of 6.91 lg/kg per day accounted for
70% of TDI value.
Conclusions Higher ICCs indicated that phthalate
metabolite levels in single spot urine could reflect longer-term
exposure to the corresponding diesters of subjects. Although
the current exposure level was less than TDIs, further studies
and exposure monitoring are needed to reveal the toxicity of
phthalate esters to sensitive subpopulation.
Phthalate esters are produced in abundance all over the
world because of their multiple uses. For instance,
di(2ethylhexyl)phthalate (DEHP) has been produced as one of
the major plasticizers for many years [
]. Other phthalate
esters, such as di-n-butyl phthalate (DnBP) and diethyl
phthalate (DEP), are used in paints and consumer products,
and as plasticizers [
]. Taking the abundance of plastics
and other phthalate-ester-containing materials in our
immediate environment into consideration, it is highly
probable that humans are exposed to various phthalate
esters in daily life.
The recognition that some phthalate esters have adverse
effects on male reproductive system in experimental
animals, especially via in utero exposure, has been increasing
]. Testosterone production of fetal male rodent is reduced
following in utero phthalate ester exposure, which results
in malformation of male reproductive tract, such as
reduced anogenital distance (AGD), retained nipples,
hypospadias, cleft phallus, undescended testis epididymal
agenesis, and testicular atrophy [
Moreover, several epidemiological studies have
suggested adverse health effects that were similar to
reproductive toxicities observed in experimental animals
]; for example, Swan et al. reported the relationship
between mother’s phthalate ester exposure levels in
gestational period and their male newborn’s anogenital
distance index (AGI). This report suggested that prenatal
phthalate ester exposure could affect human reproductive
health. They also speculated that humans might be more
sensitive than experimental animals to reproductive effect
of phthalate ester because the exposure levels of the human
subjects in that report were very low compared with those
administered to animals in the in vivo experiments [
Therefore, it has become much more important to evaluate
phthalate ester exposure levels in human population of
sensitive period (prenatal, newborn, and infant), and also to
assess human health effects caused by exposure.
Once phthalate esters are taken into human body, they
are rapidly metabolized to monoesters by hydrolysis and
oxidation and then are conjugated with glucuronide;
subsequently most of the phthalate esters are excreted in the
urine within 24 h [
]. This metabolic process can to a
certain extent differ due to the length of alkyl chain of the
phthalate esters; for example, phthalate esters with shorter
alkyl chain are mainly excreted as monoesters, while those
with longer alkyl chain are further metabolized to more
hydrophilic oxidative form .
Since Blount et al. first reported a sensitive analytical
method for phthalate ester metabolites in human urine
using high-performance liquid chromatography-tandem
mass spectrometry (HPLC-MS/MS) [
assessment of phthalate esters has become routine.
Although single spot urine sample was usually used for
exposure assessment [
], concern has been raised
over whether monoester concentration in single spot urine
represents the subject’s long-term exposure to phthalate
esters because biological half-life in human body is short.
Hoppin et al. , Hauser et al. [
], and Teitelbaum et al.
] reported utility of single spot urine for phthalate esters
exposure assessment; however, Fromme et al. [
] did not.
Hence it is still unclear whether spot urine is usable for
human exposure assessment of phthalate esters.
The main purposes of the present study are (1) to
evaluate whether single spot urine is suitable media for the
assessment of longer-term phthalate ester exposure, and (2)
to estimate intake level of phthalate esters of Japanese
pregnant women using urinary metabolite concentrations
as an indicator of prenatal exposure level of their offspring.
Materials and methods
Fifty healthy Japanese pregnant women, who were
outpatients of a department of obstetrics and gynecology of a
hospital in Tokyo, participated in this study during 2005–
2006. They were randomly chosen from outpatients who
had no pathological symptoms as diagnosed by a
gynecologist. The subjects agreed to participate after being
explained the purpose and the procedure of this study by
the gynecologist. Ethical Committee of the hospital
approved this study.
We collected single spot urine samples from the subjects at
one of their regular maternal health check-ups. Sampling
was done at 25–40 gestational weeks. The urine samples
were collected in polypropylene (PP) bottle that was
washed with ultrapure water and methanol prior to use, and
stored at -20 C until analysis. One spot urine sample was
collected from all of the 50 subjects to assess phthalate
exposure during pregnancy. Additional three urine samples
were collected with 1–6 weeks interval from 12 of the 50
subjects to evaluate intra- and interindividual variation of
urinary phthalate metabolite concentrations.
Standard solutions (100 lg/ml) of nine phthalate
monoesters (monomethyl, monoethyl, mono-n-butyl, monobenzyl,
monoisononyl, mono-n-octyl, mono-2-ethylhexyl,
mono-2ethyl-5-hydroxyhexyl, mono-2-ethyl-5-oxohexyl) ([98%
purity) were purchased from Cambridge Isotope
Laboratories, Inc. (Andover, MA). 13C4-labeled phthalate
monoesters were purchased from Cambridge Isotope
Laboratories, Inc. (Andover, MA) for an internal standard.
Acetonitrile, formic acid, and acetic acid were purchased
from Nacalai Tesque Co Ltd. (Kyoto, Japan). Ammonium
acetate and ammonia water were purchased from Wako
Pure Chemical Industries Co Ltd. (Osaka, Japan).
b-Glucuronidase (E. coli K12 origin; 200 units/ml) was
obtained from Roche Diagnostics (Mannheim, Germany).
Creatinine test Wako for Jaffe reaction was purchased
from Wako Pure Chemical Industries Co Ltd. (Osaka,
All of the glassware used in this study was washed by
sonication, rinsed with ultrapure water, dried, and heated at
400 C. Volumetric glassware was washed similarly except
for the 400 C heating, which was replaced by methanol
One milliliter of urine was taken to a glass centrifuge tube
to which was then added 300 ll mixed internal standard
solution (100 ng/ml), 250 ll 1 mol/l ammonium acetate
buffer (pH 6.5), and b-glucuronidase (5 ll). Subsequently,
urine sample was vortex-mixed, and incubated at 37 C for
60 min for the hydrolysis of glucuronide. After
incubation, 3 ml ammonium water (pH 8.0) was added to urine
samples, and then loaded onto solid-phase extraction
cartridge (OASIS-MAX 150 mg/6 cc, Nihon Waters Co
Ltd. Tokyo, Japan), which had been conditioned with
acetonitrile (10 ml) and ultrapure water (5 ml). After
washing the cartridge with ultrapure water (5 ml) and
acetonitrile (5 ml), target monoesters were eluted with
5 ml acetonitrile containing 1% formic acid to a new
glass centrifuge tube. The eluent was evaporated to
dryness with a gentle stream of nitrogen gas; the residue was
redissolved in 200 ll ultrapure water and transferred to an
autosampler vial. The nine phthalate monoesters were
determined by HPLC-MS/MS. The HPLC was from
Agilent technologies (Agilent 1100, CA, USA) and the
MS/MS was from Micromass (Micromass Quattro Ultima,
Creatinine concentrations in urine samples were
determined by Jaffe reaction.
Daily intake estimates
We used Eq. 1 for the estimation of daily intake of seven
phthalate diesters from urinary monoester concentrations,
as employed by David [
] and Koch et al. [
Daily intake ðlg=kg=day)
1; 000 ðmg=gÞ
ME is creatinine-adjusted monoester concentration, CE
is creatinine excretion rate normalized by body weight as
18 mg/kg per day for women [
]. FUE is the molar
fraction of the urinary-excreted monoester related to the
parent diesters. FUE values were as follows: for MnBP,
0.69 was used based on report of Anderson et al. [
for MMP and MEP were unknown and substituted with that
for MnBP. For MEHP, MEHHP, and MEOHP, 0.059,
0.233, and 0.154 were used, respectively [
]. FUE value of
0.0215 was used for MINP based on report of Koch et al.
]. For MnOP, 0.043 was used from the animal data of
Albro and Moore [
]. MWd and MWm are the molecular
weight of phthalate diesters and phthalate monoesters,
Phthalate metabolite concentrations were corrected for
urine volume by urinary creatinine concentration (lg/g
cre). In the following statistical analyses, we used
logtransformed phthalate metabolite concentrations adjusted
creatinine concentrations because the seven phthalate
monoester concentrations in urine samples from the
pregnant women were log-normally distributed. To calculate
inter- and intraindividual variance in urinary metabolite
concentrations through 5–12 weeks, we employed mixed
models by using SAS proc mixed version 9.1 (SAS
Institute inc., Cary, NC). Intraclass correlation coefficient (ICC)
was defined as the proportion of interindividual variance to
Analysis of urinary phthalate metabolites
Quantitative recoveries of added metabolites were
obtained: 71% (MINP) to 117% (MMP) for 3 ng/ml added
level and 96% (MEP, MINP, MnOP) to 112% (MMP) for
30 ng/ml added level. Those values were means of four
replicates. The reproducibility of analysis expressed as
relative standard deviation ranged from 3.4% for MEOHP
Intra- and interindividual variation
in urinary metabolite concentrations
Four spot urine samples were taken from each of 12
subjects within 5–12 weeks in late pregnancy.
Creatinineadjusted phthalate ester metabolite concentrations in the 48
urine samples were analyzed for inter- and intraindividual
variance. Since MINP and MnOP were hardly detected in
the urines, these metabolites were not included in the
statistical analyses. Table 1 shows the results of inter- and
intraindividual variance and ICCs in log-transformed
n = 48, four spot urine samples from 12 pregnant women, within 1–
6 weeks intervals during 5–12 sampling period
Log-transformed urinary metabolite concentrations were used in this
statistical analysis because the seven phthalate monoester
concentrations in 48 single spot urine samples from 12 pregnant women were
a ICC = Interindividual variance/(interindividual variance ?
All urinary metabolite concentrations were log-normally distributed in 50 pregnant women
to 27.8% for MEHP. These results indicated that our
monoesters analysis was accurate in terms of adequate
precision and recoveries. No phthalate metabolites were
detected in procedural or travel blanks.
creatinine-adjusted urinary metabolite concentrations. The
ICCs for seven urinary metabolites were MMP (0.57),
MEP (0.47), MnBP (0.69), MBzP (0.28), MEHP (0.51),
MEHHP (0.43), and MEOHP (0.41).
Phthalate metabolites levels in Japanese
Table 2 shows limit of detection, percentage of subjects
with detectable concentration in urine, and median
concentrations of urinary metabolites with min–max ranges for
the 50 Japanese pregnant women. Limit of detections were
0.008-0.07 ng/ml as urinary level for the nine phthalate
ester metabolites. All of the nine creatinine-unadjusted
and creatinine-adjusted urinary metabolite concentrations
showed log-normal distribution and, therefore, geometric
mean, geometric standard deviation, and median
concentration were shown in this table. MMP, MEP, MnBP,
MBzP, MEOHP, and MEHHP were detected in 100%
of the subjects. Urinary MnBP concentration (median
66.6 lg/g cre) was highest among the nine monoesters
analyzed while those of MINP and MnOP were low.
Comparison of the estimated daily intake level
of phthalate esters based on urinary metabolite levels
with other estimates
Phthalate diesters DEHP DEHP
Swan et al. 
Median (lg/kg per day)
DMP dimethyl phthalate, DEP diethyl phthalate, DnBP di-n-butyl phthalate, BBzP butylbenzyl phthalate, DEHP di(2-ethylhexyl)phthalate,
DINP di-isononyl phthalate, DnOP di-n-octyl phthalate
a The value was based on mean values of DEHP intake from the three DEHP metabolite concentrations for each subject
b These diesters were statistically significant with reduced AGI in the Swan et al. [
] study and calculated by Marsee et al. [
References: TDI for DBP [
], DEHP [
], and BBzP [
]. Maximum DEHP and DnBP intake levels of the
present subjects accounted for 50% and 70% of the TDIs,
Intra- and interindividual variations in urinary
Intra- and interindividual variations in urinary metabolite
concentrations were compared based on calculation of
ICCs. Except for MBzP, the ICCs were greater than 0.4,
which indicated that urine metabolite data were
reproducible over the sampling period (5–12 weeks) for most of the
phthalates. According to Rosner [
], ICC of 0.4–0.75 is
considered ‘‘fair to good’’ reproducibility. Based on the
statistical analysis and higher ICCs, we concluded that the
urinary metabolite levels in single spot urine could reflect
longer-term (approximately 2-month) exposure level of the
corresponding diester of the subject.
To date, a couple of studies have been carried out to
evaluate the utility of spot urine for the exposure
assessment of phthalate esters by analyzing inter- and
intraindividual variance of urinary excretion of
metabolites. Our results of ICCs, 0.28 to 0.69, in urinary
metabolite concentrations over 5–12 weeks were at the
same levels or relatively higher than those reported. Hauser
et al. [
] reported ICCs for MMP (0.27), MEP (0.43),
MnBP (0.71), MBzP (0.55), and MEHP (0.54) in 3 months.
Teitelbaum et al. [
] reported MEP (0.26), MnBP (0.35),
MBzP (0.62), MEHP (0.29), MEHHP (0.24), and MEOHP
(0.23) in 6 months. Hoppin et al. [
], Hauser et al. [
and Teitelbaum et al. [
] concluded that spot urine was
usable for longer-term (up to a couple of months) exposure
assessment. On the contrary, the conclusion of Fromme
et al. [
] was not similar to ours. They measured
concentrations of ten urinary metabolites in 50 subjects during
eight consecutive days and analyzed for within-subject
variance to find substantial day-to-day variation in urinary
monoester levels of the subjects. ICCs were moderate and
they concluded that spot urine was not suitable for
Differences in exposure patterns, including exposure
pathway, exposure sources, and exposure timing, might
result in such inconsistency among studies on
reproducibility of phthalate metabolite concentration in spot urine
Theoretically, utility of spot urine would be limited for
long-term exposure assessment of a chemical with short
biological half-life, such as phthalate esters, if exposure
level varied randomly either interindividually or
intraindividually. In spite of this limitation, higher ICCs found in
this study and other previous ones suggested that phthalate
ester exposure level of an individual was primarily
determined by a relatively steady habit (e.g., food and personal
care products) of the individual. Until the utility of single
spot urine for exposure assessment of phthalate esters is
established, however, multiple urine samples may ideally
be collected from subjects in epidemiologic studies, as
Hauser et al. [
] pointed out. This obviously requires cost
and effort of both subjects and examiners.
Urinary phthalate metabolite levels
in 50 pregnant women
MEHP, MINP, and MnOP were detected in 98%, 14%, and
28% of subjects, respectively. Frequent detection of
urinary metabolites shows that subjects were exposed to
various phthalate esters. This may allow us to assume that
the general Japanese population is also exposed to them on
a daily basis. In the case of MINP, several studies have
shown that MINP is not main metabolite of DINP, and is
further metabolized to oxidative forms [
]. This may
be related to the lower MINP concentrations in this study.
For MMP, MEP, MnBP, MBzP, and MEHP, we
compared median phthalate monoester concentrations in the
present Japanese subjects with those reported in the
previous studies, although the sample size and characteristics
of the subject population was not comparable. There were
greater differences between our results and literature values
for MnBP and MEP (Fig. 1) than those for other phthalate
metabolites. Urinary MEP concentration in this study
(9.4 lg/g cre) was lower than those in other countries/
regions by one order of magnitude (Fig. 1). This may be
related to the much lower production of DEP in Japan
(700 tons in 2002 [
]) than in the USA (11,700 tons in
]). On the contrary, urinary MnBP concentration
was higher in the present study and in European countries
] than in the USA, except for Hoppin et al.’s 
study. However, production of DBP was 4,135 tons in
] and 7,752 tons in USA [
], thus it is unlikely
that the difference in MnBP concentration between
countries was related to production amount of DBP. Rather the
This sBtuloduynt et aHl.o[p4p2i]n et al. [D18u]ty et alK.[o7c]h et al. S[4il3v]a et al. [S1w6]an etJaöln.s[s8o]n et al. [
difference in urinary MnBP concentration between the two
countries may reflect the difference in the usage of
products containing DBP in the proximate environment of
In contrast, MEHP level in this study was similar to in
other studies. Fujimaki et al. [
] measured MEHP,
MEOHP, and MEHHP concentrations in spot urine
samples from pregnant women in Tokyo and obtained median
values of 9.83, 10.4, and 10.9 lg/g cre, respectively, which
were of similar order to the present results. The level of
MBzP was slightly higher in this study than in others (data
not shown). Thus exposure levels of some but not all
phthalate esters are different among countries or regions,
probably due to differences in use and application of
phthalate esters among countries/regions.
Daily intake levels of phthalate diesters
and exposure sources
Figure 2 shows comparison of daily exposure level of
selected phthalates estimated in this study and those by the
Ministry of the Environment (MOE), Japan. The MOE
estimate was the sum of the diester intakes from food,
indoor air, and drinking water, which were based on the
reported mean concentrations in each media in Japan
]. Estimated mean daily intake of DMP, DEP, and
DnBP in this study was from two to four times greater than
the MOE values, suggesting the presence of exposure
sources of these phthalate esters other than food, air, and
drinking water for the present subjects (Fig. 2). The
difference in the estimation based on urine analysis and
environmental monitoring has also been pointed out
recently by Itoh et al. [
]. One possible exposure sources
that the MOE failed to include was personal care products
such as cosmetics. DEP, DMP, and DBP are the phthalate
esters used in personal care products as well as in plastics
and others. Considering this use, people are likely to be
exposed to DMP, DEP, and DBP, especially via dermal
absorption through the usage of cosmetics. Duty et al. [
reported significant association between urinary MEP
concentration and frequency of usage of personal care
products. The MOE estimation did not include dermal
exposure, which might have resulted in underestimation of
exposure. The contribution from personal care products has
to be quantitatively estimated for Japanese population to
fully assess exposure level of phthalate esters.
On the contrary, our estimated intake of DEHP was
lower than the MOE estimation (Fig. 2). In 2000, the
Ministry of Health and Welfare of Japan restrained DEHP
use in polyvinyl chloride (PVC) gloves because it was
suspected that handling of foods with PVC gloves
contaminated foods with DEHP. After this regulation, DEHP
concentration in food decreased and average daily intake of
DEHP decreased from 519 lg/day in 1999 to 160 lg/day
in 2001 [
]. Since the MOE used DEHP concentration in
food reported in 1998 for intake estimation, this might have
resulted in overestimation of daily DEHP intake level.
When we used newer exposure information of DEHP from
] and indoor air [
], calculated DEHP intake was
3.33 lg/kg per day, which was consistent with the present
estimations from urinary metabolite concentration (1.1–
2.2 lg/kg per day). This consistency may support our
notion that DEHP exposure from food had recently
decreased substantially in Japan.
Exposure levels to phthalate esters
in 50 pregnant women
As shown in Table 3, our estimates of daily intakes of
phthalate diesters were lower than TDIs set by the EFSA
based on reproductive and developmental effects to
offspring in animal experiment. Swan et al. [
relationships between prenatal phthalate exposure and
anogenital distance (AGD) of male infants and indicated
that humans might be more sensitive to prenatal exposure
to phthalate esters than were rodents. The phthalate
exposure levels in this study were similar to those in US
pregnant women who had male infants with reduced AGD
by prenatal phthalate exposure in the Swan et al. study [
(Table 3). Therefore the present phthalate ester exposure
levels might not completely be effect-free, although the
intake levels of phthalate esters in this study were lower
than TDI values set by the EFSA.
The present study revealed the fact that metabolites of
various phthalate esters are detected in urine samples
collected from Japanese pregnant women, showing that they
were exposed to phthalate esters on a daily basis. It is not
clear whether this exposure level of Japanese pregnant
women is safe enough for their offspring, though the level
was less than TDIs. Further studies are needed to reveal the
toxicity of phthalate esters to fetus and other sensitive
subpopulations. Exposure monitoring of sensitive
subpopulation to phthalates has to be continued as well.
Acknowledgments The authors greatly appreciate K. Omura and
H. Nitta, National Institite for Environmental Studies, for their advice
on statistical analysis. The authors sincerely appreciate the subjects of
this study for supplying urine samples and also the technical staff of
the Central Hospital of Self-Defense Force for sampling.
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