Article
pubs.acs.org/est
Occurrence of Thyroid Hormone Activities in Drinking Water from
Eastern China: Contributions of Phthalate Esters
Wei Shi,† Xinxin Hu,† Fengxian Zhang,† Guanjiu Hu,‡ Yingqun Hao,‡ Xiaowei Zhang,*,† Hongling Liu,†
Si Wei,† Xinru Wang,§ John P. Giesy,†,⊥,∥,#,▼,¶ and Hongxia Yu*,†
†
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s
Republic of China
‡
State Environmental Protection Key Laboratory of Monitoring and Analysis for Organic Pollutants in Surface Water, Jiangsu
Provincial Environmental Monitoring Center, Nanjing, People’s Republic of China
§
Key Laboratory of Reproductive Medicine and Institute of Toxicology, Nanjing Medical University, Nanjing, People’s Republic of
China
⊥
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada
∥
Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan,
United States
#
Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
▼
School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China
¶
State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University,
Xiamen 361005, China
S Supporting Information
*
ABSTRACT: Thyroid hormone is essential for the development of humans. However, some synthetic chemicals with
thyroid disrupting potentials are detectable in drinking water.
This study investigated the presence of thyroid active
chemicals and their toxicity potential in drinking water from
five cities in eastern China by use of an in vitro CV-1 cellbased reporter gene assay. Waters were examined from several
phases of drinking water processing, including source water,
finished water from waterworks, tap water, and boiled tap
water. To identify the responsible compounds, concentrations
and toxic equivalents of a list of phthalate esters were quantitatively determined. None of the extracts exhibited thyroid
receptor (TR) agonist activity. Most of the water samples exhibited TR antagonistic activities. None of the boiled water displayed
the TR antagonistic activity. Dibutyl phthalate accounted for 84.0−98.1% of the antagonist equivalents in water sources, while
diisobutyl phthalate, di-n-octyl phthalate and di-2-ethylhexyl phthalate also contributed. Approximately 90% of phthalate esters
and TR antagonistic activities were removable by waterworks treatment processes, including filtration, coagulation, aerobic
biodegradation, chlorination, and ozonation. Boiling water effectively removed phthalate esters from tap water. Thus, this process
was recommended to local residents to reduce certain potential thyroid related risks through drinking water.
■
maturation and development of the brain.5 Disruption of TH
homeostasis during development of the central nervous system
of children might cause irreversible mental retardation and
neurological deficits and might affect behavior.4
Anti/thyroid hormone effects have been observed in
industrial effluents, sediment extracts, water sources in eastern
INTRODUCTION
Increasing attention has been given to the effects of environmental contaminants that can interfere with the endocrine
systems of humans or wildlife.1 Most of the efforts have
especially been focused on androgen and estrogen homeostasis.2 Limited information is available regarding disruption of
thyroid hormone (TH) function by environmental contaminants.3 Thyroid hormone is important for normal development
of the brain in higher vertebrates and postembryonic
development of lower vertebrates.4 Thyroid hormone regulates
genes, such as TSH-β and ChAT, that are involved in
© 2011 American Chemical Society
Received:
Revised:
Accepted:
Published:
1811
July 29, 2011
December 8, 2011
December 15, 2011
December 15, 2011
dx.doi.org/10.1021/es202625r | Environ. Sci. Technol. 2012, 46, 1811−1818
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China, and even in finished drinking water in Beijing.6,7
Thyroid-disrupting activities that pose a risk to human health
through drinking water and the responsible contaminants are of
concern.8 TH function can be disrupted by synthetic chemicals
from agriculture and industry, such as organochlorine (OC)
pesticides, 4-aminophenol, and phthalate esters.9,10 These
chemicals have been reported to interact with the thyroid
receptor (TR) by inhibiting binding to its endogenous ligands
or by providing additional ligands that can bind to the TR.11,7,12
Previously, concentrations of OC pesticides, polychlorinated
biphenyls, and some phenols have been determined to be
relatively small in drinking water in eastern China, but
concentrations of phthalate esters were 100- to 1000-fold
greater than the other chemicals.6
Phthalates are commonly used in a variety of products,
including building materials, cables, wires, food packaging, toys,
medical devices, bags, skin care products, clothing, insect
repellent, and medication coating.13 There are several different
phthalates, with di-2-ethylhexyl phthalate (DEHP), dibutyl
phthalate (DNBP), diisobutyl phthalate (DIBP), and din-octyl phthalate (DNOP) being the most commonly produced
phthalates (Table 1). DEHP and DNBP are commonly used
assay was used to determine anti/thyroid hormone effects in
source water, finished water from waterworks, tap water, and the
related boiled water in five cities in eastern China (see also
Figure S1, Supporting Information). The three objectives of the
study were to (1) detect TR agonist and antagonist activities in
water during treatment, including source water, finished water
from waterworks, tap water, and the related boiled water; (2)
identify the compounds responsible for thyroid-active potency
of extracts by use of a combination of instrumental analysis and
bioassays; and (3) examine the potential adverse effects posed
to the residents through drinking water.
■
EXPERIMENTAL SECTION
Chemicals and Materials. All phthalate esters were
purchased from Labor Dr. Ehrenstorfer-Schäfers (Augsburg,
Germany). Purity and abbreviations of individual chemicals are
listed in Table 1. L-3,5′,3′-triiodothyronine (T3) was obtained
from Sigma Chemical Co., St. Louis, MO, USA. Compounds
were dissolved in dimethylsulfoxide (DMSO, BDH Laboratory
Supplies, UK) and were diluted with appropriate culture
medium before use to give less than 0.5% (v/v) solvent.
Sampling Locations. Changzhou (CZ), Suzhou (SZ), Wuxi
(WX), Xuzhou (XZ), and Yancheng (YC) are five main cities in
the Yangtze River Delta which get source water from the
Yangtze River, Eastern Taihu Lake, Northern Taihu Lake,
groundwater, and Huaihe River, respectively. In August 2010,
20 L (10 L for bioassay and 10 L for chemical analysis) of water
was collected for each sample in the water treatment processes,
including source water, finished water from waterworks, tap
water, and the related boiled water from each of the 5 cities
(see also Figure S1, Supporting Information).
Sample Preparation and Determination. Samples and
procedure blanks (Milli-Q water) were extracted by use of a
previously published protocol with modification.6 Samples were
not filtered. Solid phase extraction (SPE) was performed using
500-mg Oasis HLB cartridges (Waters, USA). Cartridges were
activated and conditioned with 10 mL of high-purity hexane,
dichloromethane, acetone, methanol, and Milli-Q water
sequentially. Water was extracted under vacuum at a flow rate
of 5−8 mL/min.
Samples were separated into two aliquots and stored in
brown glass bottles for respective instrumental and biological
analysis. Approximately 2 L of sample was passed through each
column to avoid over filtration. Then the column was dried
completely under a gentle stream of nitrogen gas (99.999%
pure). Analytes were eluted with 10 mL of hexane, 10 mL of
hexane/dichloromethane (1:1), followed by 10 mL of acetone/
methanol (1:1). SPE extracts were filtered through anhydrous
sodium sulfate to remove water. Samples were dehydrated and
reduced to dryness under gentle nitrogen flow and
reconstituted in 0.1 mL of dichloromethane for quantification.
Samples used in bioassays were prepared by reduction to
dryness under gentle nitrogen flow and reconstituted in 0.2 mL
of dimethyl sulfoxide (DMSO). Extracts in DMSO were diluted
12.5-, 25-, 50-, 100-, and 200-fold relative to the original
concentrations in water for use in bioassays.
The laboratory blank consisted of Milli-Q water to exclude
endocrine disrupting chemicals during the working procedure.
The external standard was Milli-Q water spiked with each target
analyte, and analyzed using the complete procedure. Plasticizers
were quantified using a Thermo TSQ Quantum Discovery
triple-quadrupole mass spectrometer (San Jose, CA, USA) in
multiple-reaction monitoring (SRM) mode. The precision of
Table 1. CAS Number and Purities of Tested Chemicals
chemical
abbreviation
CAS No.
purity (%)
dibutyl phthalate
di-2-ethylhexyl phthalate
dimethyl phthalate
diethyl phthalate
benzyl butyl phthalate
diisodecyl phthalate
bis(2-ethylhexyl) adipate
di-n-octyl phthalate
diisononyl phthalate
diisobutyl phthalate
DNBP
DEHP
DMP
DEP
BBP
DIDP
DEHA
DNOP
DINP
DIBP
84-74-2
117-81-7
131-11-3
84-66-2
85-68-7
68515-48-0
103-23-1
117-84-0
28553-12-0
84-69-5
99.0
99.0
99.5
99.5
99.5
99.5
99.5
99.5
99.0
99.0
in most polyvinyl chloride (PVC) consumer products (toys,
shower curtains, etc.).14 DNBP is also used in some personal
care products.15 DIBP is a special plasticizer, used as a substitute
for DNBP.16 DNOP is commonly used in the manufacture of
flexible vinyl.17 Detectable concentrations of DNBP and DEHP
as great as 2.9 × 101 μg/L were found in finished water from
waterworks in Beijing.7 DNBP has also been found in finished
drinking water in Chongqing and Hangzhou, at concentrations
as great as 1.7 × 101 and 7.6 × 101 μg/L, respectively.7,18 DNBP
has been demonstrated to be a TR antagonist.6 Activated carbon
has been reported to be effective for removing organic
contaminants,19 but the use of activated carbon is not common
in drinking water treatment in eastern China. Thus, more
attention should be given to removal of phthalates and the
related toxicity during water treatment or locally at the tap.
Promoter−reporter gene assays that were rapid, sensitive, and
reproducible in in vitro methods were employed in this study to
assess the ligand−receptor interaction by receptor agonists and
antagonists. Previously, green monkey kidney fibroblast cells
(CV-1) have been used as the basis of a TH reporter gene assay
for the screening of chemicals with TR ant/agonistic properties.20 This system has been used to characterize both anti/
thyroid hormone effects of water extracts and some related
chemicals, including bisphenol A (BPA), tetrachlorobisphenol
A, carbaryl, 1-naphthol, 2-naphthol, DNBP, mono-n-butyl
phthalate (MBP), and DEHP.20 In this study, the CV-1 TH
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Relative potencies (REPs-DNBP) for antagonist activity of the
standard compounds were calculated by dividing the EC20 of DNBP
by the EC20 of the test compound. Activities of DNBP equivalents
(DNBP-EQs) were calculated by summing the product of
concentrations of individual congeners multiplied by their respective
relative potencies (REPs-DNBP) (see Supporting Information).
Values reported were mean ± SD (n = 3). In the bioassays
triplicate wells were done for each treatment. Data were analyzed
by use of one-way ANOVA, followed by Duncan’s multiple
comparisons test when appropriate using SPSS statistical
software (version 11, SPSS Inc., Chicago, IL, USA). Curvefitting analyses were carried out with GraphPad 5.4 (San Diego,
CA, USA). The level of significance was set at *p < 0.05 and
**p < 0.01. For agonistic activities, treatments were compared to
the vehicle control groups. For antagonistic activities, treatments
were compared to 5.0 × 10−9 mol/L T3 positive control groups.
the method quantified by relative standard deviation (RSD),
was determined by replicate extractions (n = 3) of a single
sample. A signal-to-noise ratio of 3:l was used as the criteria for
the analytical limit of detection (LOD). Limit of quantification
(LOQ) was defined as 10 times the noise level.
Plasmids and Cell Culture Condition. Plasmids pGal4L-TRβ and pUAS-tk-Luc were kindly provided by Dr. Ronald
M. Evans (Gene Expression Laboratory, Howard Hughes
Medical Institute, San Diego, CA, USA). The ligand binding
domain (LBD) of TRβ was fused to the DNA binding domain
(DBD) of Gal4 in plasmid pGal4-L-TRβ.
Green monkey kidney fibroblast (CV-1) cell line without
endogenous receptors (TR) was purchased from the Institute of
Biochemistry and Cell Biology in Shanghai, Chinese Academy of
Science. CV-1 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS; Gibco, Invitrogen Corporation, Carlsbad, CA,
USA), 100 U/mL penicillin (Sigma),and 100 μg/mL streptomycin (Sigma) at 37 °C in an atmosphere containing 5% CO2.
Cytotoxicity and Reporter Gene Assay. The cytotoxicity
induced by the tested chemicals and extracts was assessed using
the MTT assay according to the protocol described previously.20
For the reporter gene assay, cells were plated and transfected as
described previously.6 After an additional 12 h of incubation, the
cells were exposed to various concentrations of chemicals and
extracts for 24 h. DMSO concentrations in wells never exceed
0.5% (v/v) (see Supporting Information).
Data Analysis. The antagonist equivalency of the extracts
(Ant-TR-EQ) was derived by comparing the observed activity to
the concentration of a known TR antagonist DNBP (standard)
that produced an equivalent reduction in TR antagonist
activity.21−23 The assumptions of equal potency and efficacy
were addressed as proposed by Villeneuve et al.23 TR antagonist
activity was reported as the concentration of DNBP divided by the
sample concentration factor that produced an equivalent (20%)
depression in the bioassay response to 5.0 × 10−9 mol T3/L.
Thyroid receptor antagonistic activities of tested chemicals
were reported as the EC20 (20% relative inhibitory concentration),24,25
which refers to the concentration at which the tested chemicals
showing a 20% reduction in the activity of 5.0 × 10−9 mol/L
T3 via TRβ. By using these magnitudes of response it was possible
to avoid artifacts and biases associated with violation of the
assumptions necessary to determine relative potencies and
determine concentrations of equivalents in extracts of water.
■
RESULTS AND DISCUSSION
Cell Viability and Assay Validation. None of the tested
concentrations of individual chemicals or extracts affected
viability or proliferation of CV-1 cells alone or in the presence
of 5.0 × 10−9 mol T3/L. No cytotoxic effects of solvent or water
extracts were observed by microscopic examination throughout
the transfection assay.
The natural TR ligand T3 induced luciferase activity in a
concentration-dependent manner in the CV-1 reporter assay
(see also Figure S2, Supporting Information). T3 induced
luciferase activity in the range of 1.0 × 10−10 mol/L to 1.0 ×
10−6 mol/L, with maximal induction of 240-fold relative to that
of the vehicle control achieved at a concentration of 1.0 × 10−6
mol T3/L. The positive control, DNBP caused a typical dose−
response curve in 5.0 × 10−9 mol T3/L (Figure 1). No
significant induction of luciferase was observed in any of the
solvent controls (data not shown). Recoveries of plasticizers in
instrumental analysis were between 90% and 120%. RSDs
ranged from 3% to 12%.
TR Antagonistic Activity. In the presence of 5.0 × 10−9 mol
T3/L, DIDP caused no effect and DEHA caused only a slight
increase in expression of the reporter gene. However, all the other
phthalate esters exhibited greater TR antagonistic potencies that
resulted in concentration-dependent expression of the reporter
gene under control of the TR (Figure 1). The antagonistic potency
(EC20) reached a maximum from 6.9 × 10−7 to 3.6 × 10−4 mol/L
Figure 1. Concentration-dependent thyroid receptor antagonist activities of phthalates as measured by the CV-1 cell line TR reporter gene assay.
Results are expressed as mean ± SD (n = 3). Significant differences relative to control are indicated by asterisks (*p < 0.05 and **p < 0.01). The
dashed line depicts the control.
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extrapolation estimates of REP for BBP and DMP. The detected
TR antagonistic potency and the LOEC values of DIDP, DEHA,
DOP, and DINP in the present study were similar to those
reported by Ghisari et al.26 It has been previously reported that
agonistic compounds may appear as antagonistic once in presence
of a natural biological nuclear receptor depending on
concentration of ligand, ligand binding affinity, and the presence
of competing natural ligands.32,33 However the reason for the
mixed agonist/antagonist activities remains inconclusive. Mixed
ligand dimers may be required for antagonism, whereas sameligand dimers may promote gene activation, depending on the
ligand bound receptor induced conformational changes.34−36
None of the tested extracts displayed any TR agonistic
activity. However, 4 of the 5 tested source water extracts
exhibited TR antagonistic potencies in a concentration-dependent manner (Figure 2). Neither the procedure blank nor XZ city
source water caused TR antagonistic responses. We recalculated
the relative antagonistic potency to DNBP, because DNBP was
identified to have the most potent TR antagonist activity among
the commonly used synthetic chemicals.7 Moreover, DNBP has
been studied a lot for its potential toxicity induced by its thyroid
hormone disrupting effects. The Ant-TR-EQs for the water
sources ranged from 3.2 to 8.2 μg DNBP-EQs/L (Table 3).
Previous studies have studied the effect of DNBP on T3dependent activation of TRβ gene in T3-induced metamorphosing tadpoles and the TR antagonist response was detected at
1.1 × 103 μg DNBP/L.37 Although this concentration was more
than 100-fold greater than the TR antagonist equivalents
detected in this study, thyroid hormone modulating potential
was observed in source waters.
Significant TR antagonistic activities in waterworks and the
related tap water from SZ and WX were detected at the greatest
concentration tested (200 times the original concentration).
Because of the limited sample volumes, extracts could not be
tested at more dilutions. In these cases, the concentration of
DNBP which caused the same response as the greatest
concentration tested was divided by the enrichment factor 200
to estimate the single point Ant-TR-EQs. For the other samples
that exhibited significant potency but did not reach a maximal/
minimal response, extrapolation was used to generate Ant-TREQs from the dose−response curves.23 Almost 90% of TR
antagonistic activities were removable by treatment in waterworks
(Table 2). The measured TR antagonistic potencies of DNBP and
DEHP observed in the present study were similar to those reported
Table 2. TR Antagonistic Potency (EC20) and the Related
Potencies (REPs-DNBP) of Phthalate Esters
TR antagonistic
95% confidence limits (mol/L)
DMP
DEP
DIBP
DNBP
BBP
DEHA
DEHP
DNOP
DIDP
DINP
a
EC20 (mol/L)
REPs-DNBP
lower
upper
1.6 × 10−4
5.0 × 10−5
1.5 × 10−5
6.9 × 10−7
3.6 × 10−4
9.5 × 10−5b
3.3 × 10−5
9.1 × 10−6
1.4 × 10−5
6.22 × 10−3
1.73 × 10−2
4.48 × 10−2
1.00
1.71 × 10−3
1.48 × 10−2
5.42 × 10−2
3.29 × 10−2
-a
3.0 × 10−5
1.0 × 10−5
3.4 × 10−7
2.3 × 10−6b
1.6 × 10−5
9.0 × 10−6
8.5 × 10−6
9.7 × 10−5
2.2 × 10−5
1.3 × 10−6
3.4 × 10−5b
8.0 × 10−5
1.1 × 10−5
1.8 × 10−5
- No effect. bSynergetic effects.
previously.7,20,26 The least concentrations at which significant effects
(p < 0.05) were detected (LOEC) were similar to those reported by
Ghisari et al. based on GH3 cell growth assays,26 but were greater
than those based on yeast assays.7,27 The sensitivity of the yeast
system depends on the number of receptors, the response elements
and the choice of reporter gene used.28,29 The difference might be
due to various factors, such as different cell lines, protein synthesis
pathways, receptor affinities, and also differences in TR expression
level and cell uptake of the tested chemicals.30 In previous studies
DEP exhibited no TR antagonism,7 which is quite different from
the results in the present study. This difference might be due to
the impermeability of the cell wall of yeast cells to the test
substances.24,31 Yeast does not contain the complex generegulating network that is normally involved in responses of
mammalian cells to hormones, which can result in different
results.30 Yeast was reported to have some degree of metabolic
competence, and it is possible that the metabolites of some
chemicals would not produce these effects rather than the parent
compound.29 BBP and DMP exhibited weak antagonist activities
without reaching 20% potency. For further use of the determined
relative potencies, nonlinear regression methods were used for
Figure 2. Concentration-dependent TR antagonist activities in extracts of water as measured by the CV-1 cell line TR reporter gene assay. Extracts of
water in cell culture media were tested at 12.5, 25, 50, 100, and 200 times of the original concentration in water samples. Cells were exposed to
extracts in parallel with 5 nmol/L T3. The TR antagonist activity was expressed as relative expression versus the untreated cells (control) (mean ±
SD, *p < 0.05 and **p < 0.01).
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10−2 to 8.0 μg DNBP/L. When concentrations of DNBP-EQs
measured by the bioassay were compared with those calculated
from the concentrations of phthalates, 86−99% of the total
Ant-TR-EQ in water sources was contributed by phthalate
esters (Figure 3). With the greatest concentration and the
strong TR antagonist potency, DNBP accounted for 84.0−
98.1% of the Ant-TR-EQs. DIBP, DNOP, and DEHP
accounted for 0.1−1.2%, 0−0.7%, and 0.1−0.3% of the AntTR-EQs, respectively. The contribution of DEHP was in
agreement with the results of a previous study conducted in
Beijing, in Northern China.7 It could be speculated that DNBP
might be the major TR antagonist potency in water sources,
while DIBP, DNOP, and DEHP also contributed. Concentrations of DNBP-EQs in finished drinking water from waterworks and tap water ranged from less than 1.0 × 10−2 to 9.4 ×
10−1 μg DNBP/L. Phthalate esters contributed almost all of the
TR antagonist potency. Greater than 90% of the total
concentrations of Ant-TR-EQs were contributed by DNBP in
finished drinking water and tap water.
Humans and animals are exposed to phthalate esters from
surface water, soil, air and even in bottled water and water from
drinking fountains.41−44 In this study, waterworks were found
to effectively remove most of the phthalate esters, although
detectable concentrations of these chemicals still existed in the
tap water, but none of the target chemicals were detected in
boiled water. As semivolatile organic compounds (SVOC),
phthalate esters result in significant volatilization during the
boiling.45 In China, boiling or heating is the most widely used
means of treating water. Boiling is effective at removing
phthalate esters, and the risk posed by these chemicals to
residents through drinking is thus small. However, all available
data considering these phthalate esters showed that they are
rapidly metabolized and their metabolites were considered to
be more noxious than the parent products.20,46,47 Recently,
metabolites of phthalates have been detected in marine biota,
consumer milk, and even in human milk.48−50 The health risk
posed by the long-term human exposure to low dose of
phthalate ethers and their metabolites still warrants further
careful evaluation.
Comparisons of the toxicity equivalents from instrumental
analysis and bioassays suggested that DNBP could play a major
role in the TR antagonistic activities in water sources, while
diisobutyl phthalate (DIBP), di-n-octyl phthalate (DNOP), and
di-2-ethylhexyl phthalate (DEHP) also showed minor contributions. The waterworks treatment processes including filtration,
coagulation, aerobic biodegradation, chlorination, and ozonation
seems effective for the removal of phthalate esters and the
related TR suppression. The boiling process is effective enough
to remove the phthalate esters in tap water, and the risk posed
by these chemicals to residents through drinking is small.
However, the volatilized phthalate esters in indoor air might
enhance the risk through respiratory and skin exposure. More
attentions should be paid to the high concentrations of DNBP,
DIBP, DNOP, and DEHP in water sources and the related
surface water.
Table 3. Thyroid Receptor Antagonist Equivalents Derived
from Instrumental Analysis (DNBP-EQs) and Reporter
Gene Assays (Ant-TR-EQs) for the Water Samples (μg/L)
95% confidence limits
for Ant-TR-EQs
locations
CZ
SZ
WX
XZ
YC
a
source
waterworks
tap
source
waterworks
tap
source
waterworks
tap
source
waterworks
tap
source
waterworks
tap
DNBP-EQs
4.2
1.0 × 10−1
9.0 × 10−2
8.0
4.7 × 10−1
9.4 × 10−1
7.6
3.2 × 10−1
4.3 × 10−1
4.0 × 10−2
<0.01
<0.01
3.2
2.0 × 10−2
7.0 × 10−2
Ant-TR-EQs
4.8
9.7
3.1
4.7
4.9
-a
8.2
× 10−1
× 10−1
8.2
× 10−1
× 10−1
3.2
-
lower
upper
3.5
5.7
6.3
3.0
-
8.3
1.4 × 101
1.2 × 101
3.8
-
- No effect.
treatment processes including filtration, coagulation, aerobic
biodegradation, chlorination, and ozonation. Treatment at the
waterworks was also effective at removing the anti-TR potency.
The anti-TR potential posed by the finished water from
waterworks and the related tap water were similar, which
indicated that little toxicity was employed during the transportation process from the waterworks to the residents.
Moreover, none of the boiled water displayed the TR antagonistic
activity.
Concentrations of TR Antagonists. Individual concentrations of phthalate esters in source water, finished water from
waterworks, tap water, and the related boiled water are shown
in Table 4. Phthalate esters were detected in all samples
analyzed except for the boiled water. Concentrations of DNBP in
waters were less than the national standard in China, which is
8.0 μg/L (MHPRC 2006). However, the quantified phthalate
esters were detected in all sources of water, except groundwater
at XZ. Phthalate esters are ubiquitous environmental contaminants. Concentrations of phthalate esters were significantly less
in finished drinking water than in the source water at SZ, CZ,
and YC. This indicated effective removal of the target
compounds by water treatment processes in all these waterworks,
which include filtration, coagulation, aerobic biodegradation,
chlorination, and ozonation. The previous studies indicated that
treatment of water with ozone or chlorine removed DEHP,
DNBP, and DEP.38 Phthalate esters are degraded under both
anaerobic and aerobic conditions.39,40 Conventional water treatment processes in eastern China effectively remove phthalate
esters and their related toxicity potential in the source water.
For the waterworks in WX, the waterworks is under
reconstruction and extension to improve the water treatment
capability. Some of the chemicals have higher concentrations in
the tap water than in the finished water in WX. This may because
of the commonly used plastic pipe in WX for the transporting of
the finished water.
DNBP was identified to have the most potent TR antagonist
activity among the commonly used synthetic chemicals. 6,7
Relative antagonistic potency to DNBP was recalculated. Concentrations of DNBP-EQs in source water ranged from 4.0 ×
■
ASSOCIATED CONTENT
S Supporting Information
*
Additional data on sampling sites, applied bioassays, analytical
procedures, dose−response relationships, and cellular toxicity.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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a
4.6 × 101 ± 2.9
7.8 ± 6.0 × 10−1
8.1 × 10 ± 4.1
3.6 ± 5.0 × 10−1
2.5 ± 8.0 × 10−1
6.2 × 101 ± 2.6
2.5 × 101 ± 1.9
1.7 × 101 ± 1.2
8.4 ± 2.0 × 10−1
5.9 × 10 ± 2.1
8.3 ± 4.0 × 10−1
7.9 ± 7.0 × 10−1
3.0 × 101 ± 2.6
3.6 × 101 ±
9.0 × 10−1
6.3 × 101 ± 2.1
waterworks
tap
1.3
0.4
0.3
0.1
DNBP
BBP
2.0
0.6
7.0 × 101 ± 8.4
2.2 × 101 ± 1.4
3.2 × 103 ±
4.3 × 101
1.3
0.4
1.9 ± 3.0 × 10−1
1.3 ± 2.0 × 10−1
3.8 × 101 ± 1.2
N.D.
N.D.
2.9 ± 8.0 × 10−1
1.1 ± 1.0 × 10−1
1.3 ± 3.0 × 10−1
2.4 × 101 ± 1.0
2.5 × 101 ±
4.0 × 10−1
2.4 × 101 ± 2.7
8.0 × 10−1 ±
3.0 × 10−1
N.D.
2.9 × 10 ±
2.0 × 101
2
7.0 ± 1.0 × 10−1
9.6 ± 1.5
3.5 × 102 ±
1.5 × 101
3.5 × 101 ± 5.5
4.2 × 102 ±
1.4 × 101
3.2 × 102 ±
1.8 × 101
7.5 × 103 ±
5.6 × 101
9.3 × 102 ± 9.5
4.7 × 102 ± 8.7
7.9 × 10 ±
6.2 × 101
3
8.8 × 101 ± 7.0
9.4 × 101 ± 7.6
4.1 × 103 ±
2.7 × 101
DEHA
1.0
0.3
1.0 ± 4.0 × 10−1
1.5 ± 2.0 × 10−1
3.7 × 101 ±
6.0 × 10−1
N.D.
7.0 × 10−1 ±
3.0 × 10−1
3.4 ± 1.0 × 10−1
1.1 × 101 ± 1.9
2.5 × 101 ± 1.3
5.1 × 101 ± 1.2
5.0 × 10−1 ±
3.0 × 10−1
N.D.
6.9 × 10 ± 1.2
1
N.D.
3.3 ± 3.0 × 10−1
1.7 × 101 ± 1.2
DEHP
0.3
0.1
1.5 × 101 ± 1.2
8.5 ± 1.5
1.4 × 102 ± 2.3
7.3 ± 1.2
6.2 ± 7.0 × 10−1
1.1 × 101 ± 1.2
9.7 × 101 ± 2.4
6.7 × 101 ± 2.5
5.6 × 102 ±
1.4 × 101
2.8 × 102 ±
1.3 × 101
2.6 × 102 ±
2.3 × 101
9.8 × 10 ± 5.5
2
2.1 × 101 ± 1.9
6.4 × 101 ± 1.9
9.6 × 102 ±
1.1 × 101
DNOP
0.7
0.2
6.3 ± 7.0 × 10−1
8.6 ± 1.8
1.4 × 101 ± 1.1
N.D.
N.D.
9.0 × 10−1 ±
3.0 × 10−1
7.1 × 101 ± 7.7
2.0 × 101 ± 2.8
1.0 × 103 ±
1.1 × 101
2.0 ± 4.0 × 10−1
1.1 ± 1.0 × 10−1
9.2 × 10 ± 6.3
2
1.5 ± 0.2
1.9 × 101 ± 9.4
4.0 × 102 ±
2.3 × 101
N.D.: Not detected. LODs: Limits of detection (S/N = 3). LOQs: Limits of quantitation (S/N = 10). Concentrations are expressed as mean ± SD (n = 3).
1.3
2.1 ± 5.0 × 10−1
2.3 ± 2.0 × 10−1
0.4
2.7 × 101 ±
8.0 × 10−1
3.8 ± 1.0 × 10−1
4.4 ± 1.2
1.1 × 101 ± 1.9
1.0 × 102 ± 5.6
6.5 × 101 ± 4.9
6.4 × 101 ± 2.2
LOQs
tap
waterworks
source
N.D.
N.D.
N.D.
N.D.
4.5 × 101 ± 1.7
4.2 × 102 ±
1.0 × 101
N.D.
N.D.
waterworks
tap
N.D.
source
tap
waterworks
source
1.1 × 102 ±
1.2 × 101
1.5 × 101 ± 1.8
1.4 × 10 ±
2.0 × 101
3
1.3 × 101 ± 1.7
source
1
3.1 ± 7.0 × 10−1
2.3 ± 6.0 × 10−1
tap
1
1.9 × 101 ± 2.3
5.5 ± 3.0 × 10−1
2.7 ± 1.0
waterworks
DIBP
1.3 × 103 ±
2.2 × 101
DEP
3.3 × 101 ± 1.3
DMP
5.6 × 101 ± 5.6
source
locations
LODs
YC
XZ
WX
SZ
CZ
Table 4. Concentrations of Phthalate Esters in Groups of Source Water, Finished Water from Waterworks, and Tap Water (ng/L)a
DIDP
1.0
0.3
2.4 ± 2.0 × 10−1
2.9 ± 9.0 × 10−1
4.1 × 101 ± 1.8
1.8 ± 5.0 × 10−1
3.2 ± 6.0 × 10−1
3.4 × 101 ± 2.7
5.5 × 101 ± 1.9
3.6 × 101 ±
2.0 × 10−1
2.8 × 102 ± 8.8
9.6 × 101 ± 1.7
1.1 × 102 ±
1.2 × 101
2.6 × 10 ±
1.1 × 101
2
1.1 × 101 ± 1.2
3.2 × 101 ± 4.4
1.7 × 102 ± 2.7
DINP
1.0
0.3
7.0 × 10−1 ±
3.0 × 10−1
N.D.
1.7 × 101 ± 1.2
8.0 × 10−1 ±
1.0 × 10−1
9.0 × 10−1 ±
3.0 × 10−1
1.1 × 101 ±
7.0 × 10−1
2.9 × 101 ± 1.0
2.6 × 101 ± 2.1
1.8 × 102 ± 6.1
2.3 × 101 ± 1.2
1.9 × 101 ± 1.3
1.1 × 102 ± 8.2
7.6 ± 9.0 × 10−1
1.2 × 101 ± 1.1
1.3 × 102 ± 3.1
Environmental Science & Technology
Article
dx.doi.org/10.1021/es202625r | Environ. Sci. Technol. 2012, 46, 1811−1818
Environmental Science & Technology
Article
Figure 3. Contributions of individual phthalates to the Anti-TR-EQs in water.
■
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Author Information
Corresponding Author
*Phone: +86 25 8968 0356; fax: +86 25 8968 0356; e-mail:
zhangxw@nju.edu.cn (X.Z.). Phone: +86 25 8968 0356; fax:
+86 25 8968 0356; e-mail: yuhx@nju.edu.cn (H.Y.).
■
ACKNOWLEDGMENTS
This work was funded by Major State Basic Research
Development Program (2008CB418102), National Natural Science
Foundation of P. R. China (20737001), Science Foundation in
Jiangsu Province (BK2011032), and the Program for Postgraduates
Research and Innovation in Jiangsu Province (CX10B_022Z). W.S.
was supported by Shanghai Tongji Gao Tingyao Environmental
Science & Technology Development Foundation. J.P.G. was
supported by the Canada Research Chair program, an at large
Chair Professorship at the Department of Biology and Chemistry
and State Key Laboratory in Marine Pollution, City University of
Hong Kong, the Einstein Professor Program of the Chinese
Academy of Sciences, and the Visiting Professor Program of King
Saud University, Riyadh, Saudi Arabia.
■
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dx.doi.org/10.1021/es202625r | Environ. Sci. Technol. 2012, 46, 1811−1818
Supporting Information
Occurrence of Thyroid Hormone Activities in Drinking Water from Eastern
China: Contributions of Phthalate Esters
Wei Shi,† Xinxin Hu,† Fengxian Zhang,† Guanjiu Hu,‡ Yingqun Hao,‡ Xiaowei
Zhang,†,
*
Hongling Liu,† Si Wei,† Xinru Wang,§ John P. Giesy,†,
⊥,║, #,
∇,O
and
Hongxia Yu†, *
†
State Key Laboratory of Pollution Control and Resource Reuse, School of the
Environment, Nanjing University, Nanjing, People’s Republic of China,
‡
State Environmental Protection Key Laboratory of Monitoring and Analysis for
Organic Pollutants in Surface Water, Jiangsu Provincial Environmental Monitoring
Center, Nanjing, People’s Republic of China,
§
Key Laboratory of Reproductive Medicine & Institute of Toxicology, Nanjing
Medical University, Nanjing, People’s Republic of China,
⊥
Department of Veterinary Biomedical Sciences and Toxicology Centre, University
of Saskatchewan, Saskatoon, Saskatchewan, Canada,
║
Department of Zoology, and Center for Integrative Toxicology, Michigan State
University, East Lansing, MI, USA,
#
Zoology Department, College of Science, King Saud University, P. O. Box 2455,
Riyadh 11451, Saudi Arabia,
∇
School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China,
O
State Key Laboratory of Marine Environmental Science, College of Oceanography
and Environmental Science, Xiamen University, Xiamen 361005, China
Address correspondence to X. Zhang and H. Yu.
Prof. Xiaowei Zhang, PhD: School of the Environment, Nanjing University,
Nanjing, 210046, China. Tel.: +86 25 8968 0356, Fax: +86 25 8968 0356, E-mail:
zhangxw@nju.edu.cn.
Prof. Hongxia Yu, PhD: School of the Environment, Nanjing University, Nanjing,
210046, China. Tel.: +86 25 8968 0356, Fax: +86 25 8968 0356, E-mail:
yuhx@nju.edu.cn.
This work was funded by Major State Basic Research Development Program (No.
2008CB418102), National Natural Science Foundation of P. R. China (20737001),
Science Foundation in Jiangsu Province (BK2011032) and the Program for
Postgraduates Research and Innovation in Jiangsu Province (CX10B_022Z).
Prof.
Giesy was supported by the Canada Research Chair program, an at large Chair
Professorship at the Department of Biology and Chemistry and State Key Laboratory
in Marine Pollution, City University of Hong Kong, the Einstein Professor Program of
the Chinese Academy of Sciences and the Visiting Professor Program of King Saud
University, Riyadh, Saudi Arabia.
Supporting Information, Figure S1. Map of the chosen water sources (Changzhou
(CZ), Suzhou (SZ), Wuxi (WX), Xuzhou (XZ) and Yancheng (YC)) in Eastern China.
Supporting Information, Figure S2. Concentration-dependent luciferase activities in
CV-1 cell line TR reporter gene assay treated with T3. Results are expressed as mean
± SD (n = 3).
Relative luciferase activity
(n-fold of control)
300.00
T3
200.00
100.00
0.00
-11
-10
-9
-8
-7
log concentration (mol/L)
-6
-5
Supporting Information, Figure S3.
Concentration-dependent thyroid receptor
agonist activities of phthalates as measured by the CV-1 cell line TR reporter gene
assay. Results are expressed as mean ± SD (n = 3). Significant differences relative to
control were indicated by asterisks (* p<0.05 and ** p<0.01). The dashed line depicts
the control.
60
**
Relative luciferase activity. .
50
**
**
40
****
**
30
**
****
**
*
**
**
20
10
0
DEHA
11 nmol
nmol/L
DINP
10
nmol/L
10nmol
DEHP
BBP
10000
nmol
102 nmol/L
DIDP
DNOP
10000
nmol
103 nmol/L
DMP
DNBP
10000
nmol
104 nmol/L
DEP
DIBP
10000
nmol
105 nmol/L
Supporting Information, Figure S4. Concentration-dependent TR antagonist activities
in the water extracts with no effect measured by the CV-1 cell line TR reporter gene
assay.
Relative luciferase activity
(n-fold of 5.0×10-9 mol/L T 3)
1.2
1
0.8
0.6
0.4
0.2
0
CZ-WT P
CZ-T AP
200
XZ-SOUT H
100
XZ-WT P
50
XZ-T AP
25
YC-WT P
YC-T AP
12.5
Supporting Information,
Materials and methods
Sampling locations and collection. The Yangtze River, Huaihe River, Taihu Lake and
groundwater are the main sources of drinking water in the Yangtze River Delta, China.
Changzhou (CZ), Suzhou (SZ), Wuxi (WX), Xuzhou (XZ) and Yancheng (YC) are
five main cities in this region, which get source water from Yangtze River, Eastern
Taihu Lake, Northern Taihu Lake, groundwater and Huaihe River respectively.
In
August 2010, 20 L water were collected at each step of water processes ( including
source water, finished water from waterworks, tap water and the related boiled water)
from each of the 5 cities.
Composite water samples (20 L, 10 L for bioassay and 10 L for chemical
analysis) were collected at each location and placed into brown glass bottles.
These
bottles were pre-cleaned with nitric acid and chromic acid solution, and then rinsed
with high-purity hexane (Merck), dichloromethane (TEDIA), acetone (TEDIA) and
methanol (TEDIA).
The bottles were also washed 3 times with water samples before
sample collection. Samples were transported and stored at 4 °C and extracted within
24 h.
Cytotoxicity. The cytotoxicity induced by the tested chemicals and extracts was
assessed by use of the MTT assay according to the protocol described previously.2
Briefly, the CV-1 cells attached on culture dishes were transplanted to 96-well culture
plates at a density of 104 cells/well in DMEM with 10% charcoal-dextran-stripped
fetal bovine serum (CDS-FBS) (Sigma). After incubation for 24 hr, CV-1 cells were
treated with vehicle control or various concentrations (10−9, 10−8, 10−7, 10−6, 10−5,
10−4 mol/L) of the tested chemicals or extracts (at 12.5, 25, 50, 100 or 200 times the
original concentration in water) alone or with 5.0×10−9 mol/L T3. Then, 25 μL
3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT, 5 mg/ml in
PBS, Sigma–Aldrich, St. Louis, MO, USA) was added to each well and further
incubation for 4 hr. Formosan crystals were dissolved by adding 150 μL DMSO and
shaking for 10 min. The absorbance at 570 nm was measured with an automatic
microplate reader (EL808, Bio-Tek, Winooski, VT, USA).
Reporter gene assay. Cells were plated into 48-well culture plates at a density of
5.0×104 cells per well in the phenol red free DMEM medium containing 10%
bCDS-FBS. After 12 h, cells were transfected with 0.25 μg Gal4-responsive luciferase
reporter pUAS-tkluc, 0.1μg pGal4-L-TR which was an expression vector coding for
the ligand binding domain (LBD) of TRβ fused to the DNA binding domain of Gal4,
using 2.5 μg Sofast TM transfection reagent per well. After an additional 12 h of
incubation, the transfection medium was removed and the cells were exposed to
various concentrations of chemicals and extracts for 24 h. To determine agonistic
activity, CV-1 cells were treated with various concentrations of chemicals. For
determining antagonistic activity, CV-1 cells were exposed to various concentrations
of tested chemicals in the presence of 5×10-9 mol/L T3. DMSO concentrations in
wells never exceed 0.5% (v/v). For both agonists and antagonists, luciferase activities
of treatment groups were compared to that of the corresponding vehicle control.
Data analysis. The concentration of standard chemical which caused the same
response as the greatest concentration tested (200 times the original concentration)
was divided by the enrichment factor 200.
Results and discussion
TR agonist activity. For the agonist activities, DEHA, DINP, DEHP, BBP, DMP and
DNBP exhibited extremely week increasing potencies (Figure S3), and the maximal
response of these chemicals were far less than 5% T3 maximal response (see Figure
S2, Supporting Information).
REFERENCES
(1) Shi, W.; Wang, X.; Hu, G.; Hao, Y.; Zhang, X.; Liu, H.; Wei, S.; Wang, X.; Yu, H.
Bioanalytical and instrumental analysis of thyroid hormone disrupting compounds in water
sources along the Yangtze River. Environ. Pollut. 2011, 159, 441-448.
(2) Shen, O. X.; Du, G. Z.; Sun, H.; Wu, W.; Jiang, Y.; Song, L.; Wang, X. R. Comparison of in
vitro hormone activities of selected phthalates using reporter gene assays. Toxicol. Lett. 2009, 191
(1), 9-14.
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