Occurrence of Thyroid Hormone Activities in Drinking Water fromEastern China: Contributions of Phthalate EstersWei 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’sRepublic of China‡State Environmental Protection Key Laboratory of Monitoring and Analysis for Organic Pollutants in Surface Water, JiangsuProvincial 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 ofChina⊥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 develop-ment of humans. However, some synthetic chemicals withthyroid disrupting potentials are detectable in drinking water.This study investigated the presence of thyroid activechemicals and their toxicity potential in drinking water fromfive cities in eastern China by use of an in vitro CV-1 cell-based reporter gene assay. Waters were examined from severalphases of drinking water processing, including source water,finished water from waterworks, tap water, and boiled tapwater. To identify the responsible compounds, concentrationsand toxic equivalents of a list of phthalate esters were quan-titatively determined. None of the extracts exhibited thyroidreceptor (TR) agonist activity. Most of the water samples exhibited TR antagonistic activities. None of the boiled water displayedthe TR antagonistic activity. Dibutyl phthalate accounted for 84.0−98.1% of the antagonist equivalents in water sources, whilediisobutyl phthalate, di-n-octyl phthalate and di-2-ethylhexyl phthalate also contributed. Approximately 90% of phthalate estersand TR antagonistic activities were removable by waterworks treatment processes, including filtration, coagulation, aerobicbiodegradation, chlorination, and ozonation. Boiling water effectively removed phthalate esters from tap water. Thus, this processwas recommended to local residents to reduce certain potential thyroid related risks through drinking water.

■ INTRODUCTIONIncreasing attention has been given to the effects of environ-mental contaminants that can interfere with the endocrinesystems of humans or wildlife.1 Most of the efforts haveespecially been focused on androgen and estrogen homeo-stasis.2 Limited information is available regarding disruption ofthyroid hormone (TH) function by environmental contami-nants.3 Thyroid hormone is important for normal developmentof the brain in higher vertebrates and postembryonicdevelopment of lower vertebrates.4 Thyroid hormone regulatesgenes, such as TSH-β and ChAT, that are involved in

maturation and development of the brain.5 Disruption of THhomeostasis during development of the central nervous systemof children might cause irreversible mental retardation andneurological deficits and might affect behavior.4

Anti/thyroid hormone effects have been observed inindustrial effluents, sediment extracts, water sources in eastern

Received: July 29, 2011Revised: December 8, 2011Accepted: December 15, 2011Published: December 15, 2011

Article

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© 2011 American Chemical Society 1811 dx.doi.org/10.1021/es202625r | Environ. Sci. Technol. 2012, 46, 1811−1818

China, and even in finished drinking water in Beijing.6,7

Thyroid-disrupting activities that pose a risk to human healththrough drinking water and the responsible contaminants are ofconcern.8 TH function can be disrupted by synthetic chemicalsfrom agriculture and industry, such as organochlorine (OC)pesticides, 4-aminophenol, and phthalate esters.9,10 Thesechemicals have been reported to interact with the thyroidreceptor (TR) by inhibiting binding to its endogenous ligandsor by providing additional ligands that can bind to the TR.11,7,12

Previously, concentrations of OC pesticides, polychlorinatedbiphenyls, and some phenols have been determined to berelatively small in drinking water in eastern China, butconcentrations of phthalate esters were 100- to 1000-foldgreater 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, insectrepellent, and medication coating.13 There are several differentphthalates, with di-2-ethylhexyl phthalate (DEHP), dibutylphthalate (DNBP), diisobutyl phthalate (DIBP), and di-n-octyl phthalate (DNOP) being the most commonly producedphthalates (Table 1). DEHP and DNBP are commonly used

in most polyvinyl chloride (PVC) consumer products (toys,shower curtains, etc.).14 DNBP is also used in some personalcare products.15 DIBP is a special plasticizer, used as a substitutefor DNBP.16 DNOP is commonly used in the manufacture offlexible vinyl.17 Detectable concentrations of DNBP and DEHPas great as 2.9 × 101 μg/L were found in finished water fromwaterworks in Beijing.7 DNBP has also been found in finisheddrinking water in Chongqing and Hangzhou, at concentrationsas great as 1.7 × 101 and 7.6 × 101 μg/L, respectively.7,18 DNBPhas been demonstrated to be a TR antagonist.6 Activated carbonhas been reported to be effective for removing organiccontaminants,19 but the use of activated carbon is not commonin drinking water treatment in eastern China. Thus, moreattention should be given to removal of phthalates and therelated 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 toassess the ligand−receptor interaction by receptor agonists andantagonists. Previously, green monkey kidney fibroblast cells(CV-1) have been used as the basis of a TH reporter gene assayfor the screening of chemicals with TR ant/agonistic proper-ties.20 This system has been used to characterize both anti/thyroid hormone effects of water extracts and some relatedchemicals, including bisphenol A (BPA), tetrachlorobisphenolA, carbaryl, 1-naphthol, 2-naphthol, DNBP, mono-n-butylphthalate (MBP), and DEHP.20 In this study, the CV-1 TH

assay was used to determine anti/thyroid hormone effects insource water, finished water from waterworks, tap water, and therelated boiled water in five cities in eastern China (see alsoFigure S1, Supporting Information). The three objectives of thestudy were to (1) detect TR agonist and antagonist activities inwater during treatment, including source water, finished waterfrom waterworks, tap water, and the related boiled water; (2)identify the compounds responsible for thyroid-active potencyof extracts by use of a combination of instrumental analysis andbioassays; and (3) examine the potential adverse effects posedto the residents through drinking water.

■ EXPERIMENTAL SECTIONChemicals and Materials. All phthalate esters were

purchased from Labor Dr. Ehrenstorfer-Schaf̈ers (Augsburg,Germany). Purity and abbreviations of individual chemicals arelisted in Table 1. L-3,5′,3′-triiodothyronine (T3) was obtainedfrom Sigma Chemical Co., St. Louis, MO, USA. Compoundswere dissolved in dimethylsulfoxide (DMSO, BDH LaboratorySupplies, UK) and were diluted with appropriate culturemedium 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 inthe Yangtze River Delta which get source water from theYangtze 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 waterwas collected for each sample in the water treatment processes,including source water, finished water from waterworks, tapwater, and the related boiled water from each of the 5 cities(see also Figure S1, Supporting Information).

Sample Preparation and Determination. Samples andprocedure blanks (Milli-Q water) were extracted by use of apreviously published protocol with modification.6 Samples werenot filtered. Solid phase extraction (SPE) was performed using500-mg Oasis HLB cartridges (Waters, USA). Cartridges wereactivated and conditioned with 10 mL of high-purity hexane,dichloromethane, acetone, methanol, and Milli-Q watersequentially. Water was extracted under vacuum at a flow rateof 5−8 mL/min.Samples were separated into two aliquots and stored in

brown glass bottles for respective instrumental and biologicalanalysis. Approximately 2 L of sample was passed through eachcolumn to avoid over filtration. Then the column was driedcompletely under a gentle stream of nitrogen gas (99.999%pure). Analytes were eluted with 10 mL of hexane, 10 mL ofhexane/dichloromethane (1:1), followed by 10 mL of acetone/methanol (1:1). SPE extracts were filtered through anhydroussodium sulfate to remove water. Samples were dehydrated andreduced to dryness under gentle nitrogen flow andreconstituted in 0.1 mL of dichloromethane for quantification.Samples used in bioassays were prepared by reduction todryness under gentle nitrogen flow and reconstituted in 0.2 mLof dimethyl sulfoxide (DMSO). Extracts in DMSO were diluted12.5-, 25-, 50-, 100-, and 200-fold relative to the originalconcentrations 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 targetanalyte, and analyzed using the complete procedure. Plasticizerswere quantified using a Thermo TSQ Quantum Discoverytriple-quadrupole mass spectrometer (San Jose, CA, USA) inmultiple-reaction monitoring (SRM) mode. The precision of

Table 1. CAS Number and Purities of Tested Chemicals

chemical abbreviation CAS No. purity (%)

dibutyl phthalate DNBP 84-74-2 99.0di-2-ethylhexyl phthalate DEHP 117-81-7 99.0dimethyl phthalate DMP 131-11-3 99.5diethyl phthalate DEP 84-66-2 99.5benzyl butyl phthalate BBP 85-68-7 99.5diisodecyl phthalate DIDP 68515-48-0 99.5bis(2-ethylhexyl) adipate DEHA 103-23-1 99.5di-n-octyl phthalate DNOP 117-84-0 99.5diisononyl phthalate DINP 28553-12-0 99.0diisobutyl phthalate DIBP 84-69-5 99.0

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the method quantified by relative standard deviation (RSD),was determined by replicate extractions (n = 3) of a singlesample. A signal-to-noise ratio of 3:l was used as the criteria forthe analytical limit of detection (LOD). Limit of quantification(LOQ) was defined as 10 times the noise level.Plasmids and Cell Culture Condition. Plasmids pGal4-

L-TRβ and pUAS-tk-Luc were kindly provided by Dr. RonaldM. Evans (Gene Expression Laboratory, Howard HughesMedical Institute, San Diego, CA, USA). The ligand bindingdomain (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 ofBiochemistry and Cell Biology in Shanghai, Chinese Academy ofScience. CV-1 cells were maintained in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% fetal bovineserum (FBS; Gibco, Invitrogen Corporation, Carlsbad, CA,USA), 100 U/mL penicillin (Sigma),and 100 μg/mL strepto-mycin (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 usingthe MTT assay according to the protocol described previously.20

For the reporter gene assay, cells were plated and transfected asdescribed previously.6 After an additional 12 h of incubation, thecells were exposed to various concentrations of chemicals andextracts for 24 h. DMSO concentrations in wells never exceed0.5% (v/v) (see Supporting Information).Data Analysis. The antagonist equivalency of the extracts

(Ant-TR-EQ) was derived by comparing the observed activity tothe concentration of a known TR antagonist DNBP (standard)that produced an equivalent reduction in TR antagonistactivity.21−23 The assumptions of equal potency and efficacywere addressed as proposed by Villeneuve et al.23 TR antagonistactivity was reported as the concentration of DNBP divided by thesample 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 chemicalsshowing a 20% reduction in the activity of 5.0 × 10−9 mol/LT3 via TRβ. By using these magnitudes of response it was possibleto avoid artifacts and biases associated with violation of theassumptions necessary to determine relative potencies anddetermine concentrations of equivalents in extracts of water.

Relative potencies (REPs-DNBP) for antagonist activity of thestandard compounds were calculated by dividing the EC20 of DNBPby the EC20 of the test compound. Activities of DNBP equivalents(DNBP-EQs) were calculated by summing the product ofconcentrations of individual congeners multiplied by their respectiverelative 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 analyzedby use of one-way ANOVA, followed by Duncan’s multiplecomparisons test when appropriate using SPSS statisticalsoftware (version 11, SPSS Inc., Chicago, IL, USA). Curve-fitting 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 tothe vehicle control groups. For antagonistic activities, treatmentswere compared to 5.0 × 10−9 mol/L T3 positive control groups.

■ RESULTS AND DISCUSSION

Cell Viability and Assay Validation. None of the testedconcentrations of individual chemicals or extracts affectedviability or proliferation of CV-1 cells alone or in the presenceof 5.0 × 10−9 mol T3/L. No cytotoxic effects of solvent or waterextracts were observed by microscopic examination throughoutthe 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 inducedluciferase 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 thatof 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). Nosignificant induction of luciferase was observed in any of thesolvent controls (data not shown). Recoveries of plasticizers ininstrumental analysis were between 90% and 120%. RSDsranged from 3% to 12%.

TR Antagonistic Activity. In the presence of 5.0 × 10−9 molT3/L, DIDP caused no effect and DEHA caused only a slightincrease in expression of the reporter gene. However, all the otherphthalate esters exhibited greater TR antagonistic potencies thatresulted in concentration-dependent expression of the reportergene 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). Thedashed line depicts the control.

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(Table 2). The measured TR antagonistic potencies of DNBP andDEHP observed in the present study were similar to those reported

previously.7,20,26 The least concentrations at which significant effects(p < 0.05) were detected (LOEC) were similar to those reported byGhisari et al. based on GH3 cell growth assays,26 but were greaterthan those based on yeast assays.7,27 The sensitivity of the yeastsystem depends on the number of receptors, the response elementsand the choice of reporter gene used.28,29 The difference might bedue to various factors, such as different cell lines, protein synthesispathways, receptor affinities, and also differences in TR expressionlevel and cell uptake of the tested chemicals.30 In previous studiesDEP exhibited no TR antagonism,7 which is quite different fromthe results in the present study. This difference might be due tothe impermeability of the cell wall of yeast cells to the testsubstances.24,31 Yeast does not contain the complex gene-regulating network that is normally involved in responses ofmammalian cells to hormones, which can result in differentresults.30 Yeast was reported to have some degree of metaboliccompetence, and it is possible that the metabolites of somechemicals would not produce these effects rather than the parentcompound.29 BBP and DMP exhibited weak antagonist activitieswithout reaching 20% potency. For further use of the determinedrelative potencies, nonlinear regression methods were used for

extrapolation estimates of REP for BBP and DMP. The detectedTR antagonistic potency and the LOEC values of DIDP, DEHA,DOP, and DINP in the present study were similar to thosereported by Ghisari et al.26 It has been previously reported thatagonistic compounds may appear as antagonistic once in presenceof a natural biological nuclear receptor depending onconcentration of ligand, ligand binding affinity, and the presenceof competing natural ligands.32,33 However the reason for themixed agonist/antagonist activities remains inconclusive. Mixedligand dimers may be required for antagonism, whereas same-ligand dimers may promote gene activation, depending on theligand bound receptor induced conformational changes.34−36

None of the tested extracts displayed any TR agonisticactivity. However, 4 of the 5 tested source water extractsexhibited TR antagonistic potencies in a concentration-depend-ent manner (Figure 2). Neither the procedure blank nor XZ citysource water caused TR antagonistic responses. We recalculatedthe relative antagonistic potency to DNBP, because DNBP wasidentified to have the most potent TR antagonist activity amongthe commonly used synthetic chemicals.7 Moreover, DNBP hasbeen studied a lot for its potential toxicity induced by its thyroidhormone disrupting effects. The Ant-TR-EQs for the watersources ranged from 3.2 to 8.2 μg DNBP-EQs/L (Table 3).Previous studies have studied the effect of DNBP on T3-dependent activation of TRβ gene in T3-induced metamorphos-ing tadpoles and the TR antagonist response was detected at1.1 × 103 μg DNBP/L.37 Although this concentration was morethan 100-fold greater than the TR antagonist equivalentsdetected in this study, thyroid hormone modulating potentialwas observed in source waters.Significant TR antagonistic activities in waterworks and the

related tap water from SZ and WX were detected at the greatestconcentration tested (200 times the original concentration).Because of the limited sample volumes, extracts could not betested at more dilutions. In these cases, the concentration ofDNBP which caused the same response as the greatestconcentration tested was divided by the enrichment factor 200to estimate the single point Ant-TR-EQs. For the other samplesthat exhibited significant potency but did not reach a maximal/minimal response, extrapolation was used to generate Ant-TR-EQs from the dose−response curves.23 Almost 90% of TRantagonistic activities were removable by treatment in waterworks

Table 2. TR Antagonistic Potency (EC20) and the RelatedPotencies (REPs-DNBP) of Phthalate Esters

TR antagonistic

95% confidence limits (mol/L)

EC20 (mol/L) REPs-DNBP lower upper

DMP 1.6 × 10−4 6.22 × 10−3 -a -DEP 5.0 × 10−5 1.73 × 10−2 3.0 × 10−5 9.7 × 10−5

DIBP 1.5 × 10−5 4.48 × 10−2 1.0 × 10−5 2.2 × 10−5

DNBP 6.9 × 10−7 1.00 3.4 × 10−7 1.3 × 10−6

BBP 3.6 × 10−4 1.71 × 10−3 - -DEHA 9.5 × 10−5b - 2.3 × 10−6b 3.4 × 10−5b

DEHP 3.3 × 10−5 1.48 × 10−2 1.6 × 10−5 8.0 × 10−5

DNOP 9.1 × 10−6 5.42 × 10−2 9.0 × 10−6 1.1 × 10−5

DIDP - - - -DINP 1.4 × 10−5 3.29 × 10−2 8.5 × 10−6 1.8 × 10−5

a- No effect. bSynergetic effects.

Figure 2. Concentration-dependent TR antagonist activities in extracts of water as measured by the CV-1 cell line TR reporter gene assay. Extracts ofwater 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 toextracts 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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treatment processes including filtration, coagulation, aerobicbiodegradation, chlorination, and ozonation. Treatment at thewaterworks was also effective at removing the anti-TR potency.The anti-TR potential posed by the finished water fromwaterworks and the related tap water were similar, whichindicated that little toxicity was employed during the trans-portation process from the waterworks to the residents.Moreover, none of the boiled water displayed the TR antagonisticactivity.Concentrations of TR Antagonists. Individual concen-

trations of phthalate esters in source water, finished water fromwaterworks, tap water, and the related boiled water are shownin Table 4. Phthalate esters were detected in all samplesanalyzed except for the boiled water. Concentrations of DNBP inwaters were less than the national standard in China, which is8.0 μg/L (MHPRC 2006). However, the quantified phthalateesters were detected in all sources of water, except groundwaterat XZ. Phthalate esters are ubiquitous environmental contami-nants. Concentrations of phthalate esters were significantly lessin finished drinking water than in the source water at SZ, CZ,and YC. This indicated effective removal of the targetcompounds by water treatment processes in all these waterworks,which include filtration, coagulation, aerobic biodegradation,chlorination, and ozonation. The previous studies indicated thattreatment of water with ozone or chlorine removed DEHP,DNBP, and DEP.38 Phthalate esters are degraded under bothanaerobic and aerobic conditions.39,40 Conventional water treat-ment processes in eastern China effectively remove phthalateesters and their related toxicity potential in the source water.For the waterworks in WX, the waterworks is underreconstruction and extension to improve the water treatmentcapability. Some of the chemicals have higher concentrations inthe tap water than in the finished water in WX. This may becauseof the commonly used plastic pipe in WX for the transporting ofthe 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. Con-centrations of DNBP-EQs in source water ranged from 4.0 ×

10−2 to 8.0 μg DNBP/L. When concentrations of DNBP-EQsmeasured by the bioassay were compared with those calculatedfrom the concentrations of phthalates, 86−99% of the totalAnt-TR-EQ in water sources was contributed by phthalateesters (Figure 3). With the greatest concentration and thestrong TR antagonist potency, DNBP accounted for 84.0−98.1% of the Ant-TR-EQs. DIBP, DNOP, and DEHPaccounted for 0.1−1.2%, 0−0.7%, and 0.1−0.3% of the Ant-TR-EQs, respectively. The contribution of DEHP was inagreement with the results of a previous study conducted inBeijing, in Northern China.7 It could be speculated that DNBPmight be the major TR antagonist potency in water sources,while DIBP, DNOP, and DEHP also contributed. Concen-trations of DNBP-EQs in finished drinking water from water-works 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 theTR antagonist potency. Greater than 90% of the totalconcentrations of Ant-TR-EQs were contributed by DNBP infinished 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 fromdrinking fountains.41−44 In this study, waterworks were foundto effectively remove most of the phthalate esters, althoughdetectable concentrations of these chemicals still existed in thetap water, but none of the target chemicals were detected inboiled water. As semivolatile organic compounds (SVOC),phthalate esters result in significant volatilization during theboiling.45 In China, boiling or heating is the most widely usedmeans of treating water. Boiling is effective at removingphthalate esters, and the risk posed by these chemicals toresidents through drinking is thus small. However, all availabledata considering these phthalate esters showed that they arerapidly metabolized and their metabolites were considered tobe 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 riskposed by the long-term human exposure to low dose ofphthalate ethers and their metabolites still warrants furthercareful evaluation.Comparisons of the toxicity equivalents from instrumental

analysis and bioassays suggested that DNBP could play a majorrole in the TR antagonistic activities in water sources, whilediisobutyl phthalate (DIBP), di-n-octyl phthalate (DNOP), anddi-2-ethylhexyl phthalate (DEHP) also showed minor contri-butions. The waterworks treatment processes including filtration,coagulation, aerobic biodegradation, chlorination, and ozonationseems effective for the removal of phthalate esters and therelated TR suppression. The boiling process is effective enoughto remove the phthalate esters in tap water, and the risk posedby these chemicals to residents through drinking is small.However, the volatilized phthalate esters in indoor air mightenhance the risk through respiratory and skin exposure. Moreattentions should be paid to the high concentrations of DNBP,DIBP, DNOP, and DEHP in water sources and the relatedsurface water.

■ ASSOCIATED CONTENT

*S Supporting InformationAdditional data on sampling sites, applied bioassays, analyticalprocedures, dose−response relationships, and cellular toxicity.This material is available free of charge via the Internet athttp://pubs.acs.org.

Table 3. Thyroid Receptor Antagonist Equivalents Derivedfrom Instrumental Analysis (DNBP-EQs) and ReporterGene Assays (Ant-TR-EQs) for the Water Samples (μg/L)

95% confidence limitsfor Ant-TR-EQs

locations DNBP-EQs Ant-TR-EQs lower upper

CZ source 4.2 4.9 3.5 8.3waterworks 1.0 × 10−1 -a - -tap 9.0 × 10−2 - - -

SZ source 8.0 8.2 5.7 1.4 × 101

waterworks 4.7 × 10−1 4.8 × 10−1 - -tap 9.4 × 10−1 9.7 × 10−1 - -

WX source 7.6 8.2 6.3 1.2 × 101

waterworks 3.2 × 10−1 3.1 × 10−1 - -tap 4.3 × 10−1 4.7 × 10−1 - -

XZ source 4.0 × 10−2 - - -waterworks <0.01 - - -tap <0.01 - - -

YC source 3.2 3.2 3.0 3.8waterworks 2.0 × 10−2 - - -tap 7.0 × 10−2 - - -

a- No effect.

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Table

4.Con

centration

sof

Phthalate

Estersin

Group

sof

Source

Water,Finished

Water

from

Waterworks,andTap

Water

(ng/L)a

locatio

nsDMP

DEP

DIBP

DNBP

BBP

DEH

ADEH

PDNOP

DID

PDIN

P

CZ

source

5.6×10

1±

5.6

3.3×10

1±

1.3

1.3×10

3±

2.2×10

14.1×10

3±

2.7×10

13.5×10

2±

1.5×10

11.7×10

1±

1.2

9.6×10

2±

1.1×10

14.0×10

2±

2.3×10

11.7×10

2±

2.7

1.3×10

2±3.1

waterworks

2.7±

1.0

5.5±

3.0×10

−1

1.9×10

1±

2.3

9.4×10

1±

7.6

9.6±

1.5

3.3±

3.0×10

−1

6.4×10

1±

1.9

1.9×10

1±

9.4

3.2×10

1±

4.4

1.2×10

1±1.1

tap

2.3±

6.0×10

−1

3.1±

7.0×10

−1

1.3×10

1±

1.7

8.8×10

1±

7.0

7.0±

1.0×10

−1

N.D.

2.1×10

1±

1.9

1.5±

0.2

1.1×10

1±

1.2

7.6±9.0×10

−1

SZsource

5.9×10

1±

2.1

8.1×10

1±

4.1

1.4×10

3±

2.0×10

17.9×10

3±

6.2×10

12.9×10

2±

2.0×10

16.9×10

1±

1.2

9.8×10

2±

5.5

9.2×10

2±

6.3

2.6×10

2±

1.1×10

11.1×10

2±8.2

waterworks

8.3±

4.0×10

−1

3.6±

5.0×10

−1

1.5×10

1±

1.8

4.7×10

2±

8.7

N.D.

N.D.

2.6×10

2±

2.3×10

11.1±

1.0×10

−1

1.1×10

2±

1.2×10

11.9×10

1±1.3

tap

7.9±

7.0×10

−1

2.5±

8.0×10

−1

1.1×10

2±

1.2×10

19.3×10

2±

9.5

8.0×10

−1±

3.0×10

−1

5.0×10

−1±

3.0×10

−1

2.8×10

2±

1.3×10

12.0±

4.0×10

−1

9.6×10

1±

1.7

2.3×10

1±1.2

WX

source

3.0×10

1±

2.6

6.2×10

1±

2.6

4.2×10

2±

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17.5×10

3±

5.6×10

12.4×10

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2.7

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8.8

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waterworks

3.6×10

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2.8

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2.9

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7.7

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source

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7.8±

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5.5

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−1

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−1

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1±

2.7

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7.0×10

−1

waterworks

N.D.

N.D.

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N.D.

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−1±

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−1

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−1±

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−1

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source

6.4×10

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1±

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waterworks

4.4±

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−1

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1±

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1.3±

2.0×10

−1

1.5±

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−1

8.5±

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8.6±

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9.0×10

−1

N.D.

tap

2.3±

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−1

2.1±

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−1

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1±

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8.4

1.9±

3.0×10

−1

1.0±

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−1

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1±

1.2

6.3±

7.0×10

−1

2.4±

2.0×10

−1

7.0×10

−1±

3.0×10

−1

LODs

0.4

0.4

0.1

0.6

0.4

0.3

0.1

0.2

0.3

0.3

LOQs

1.3

1.3

0.3

2.0

1.3

1.0

0.3

0.7

1.0

1.0

aN.D.:Not

detected.L

ODs:Limits

ofdetection(S/N

=3).L

OQs:Limits

ofquantitation(S/N

=10).Concentratio

nsareexpressedas

mean±

SD(n

=3).

Environmental Science & Technology Article

dx.doi.org/10.1021/es202625r | Environ. Sci. Technol. 2012, 46, 1811−18181816

■ 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 ResearchDevelopment Program (2008CB418102), National Natural ScienceFoundation of P. R. China (20737001), Science Foundation inJiangsu Province (BK2011032), and the Program for PostgraduatesResearch and Innovation in Jiangsu Province (CX10B_022Z). W.S.was supported by Shanghai Tongji Gao Tingyao EnvironmentalScience & Technology Development Foundation. J.P.G. wassupported by the Canada Research Chair program, an at largeChair Professorship at the Department of Biology and Chemistryand State Key Laboratory in Marine Pollution, City University ofHong Kong, the Einstein Professor Program of the ChineseAcademy of Sciences, and the Visiting Professor Program of KingSaud University, Riyadh, Saudi Arabia.

■ REFERENCES(1) Colborn, T.; Saal, F. S. V.; Soto, A. M. Developmental effects ofendocrine-disrupting chemicals in wildlife and humans. Environ. HealthPerspect. 1993, 101 (5), 378−384.(2) Kuster, M.; Diaz-Cruz, S.; Rosell, M.; de Alda, M. L.; Barcelo, D.Fate of selected pesticides, estrogens, progestogens and volatileorganic compounds during artificial aquifer recharge using surfacewaters. Chemosphere 2010, 79 (8), 880−886.(3) Jugan, M. L.; Oziol, L.; Bimbot, M.; Huteau, V.; Tamisier-Karolak, S.; Blondeau, J. P.; Levi, Y. In vitro assessment of thyroid andestrogenic endocrine disruptors in wastewater treatment plants, riversand drinking water supplies in the greater Paris area (France). Sci.Total Environ. 2009, 407 (11), 3579−3587.(4) Howdeshell, K. L. A model of the development of the brain as aconstruct of the thyroid system. Environ. Health Perspect. 2002, 110,337−348.(5) Ahmed, O. M.; El-Gareib, A. W.; El-Bakry, A. M.; Ei-Tawab, S.M. A.; Ahmed, R. G. Thyroid hormones states and brain developmentinteractions. Int. J. Dev. Neurosci. 2008, 26 (2), 147−209.(6) Shi, W.; Wang, X.; Hu, G.; Hao, Y.; Zhang, X.; Liu, H.; Wei, S.;Wang, X.; Yu, H. Bioanalytical and instrumental analysis of thyroidhormone disrupting compounds in water sources along the YangtzeRiver. Environ. Pollut. 2011, 159, 441−448.(7) Li, N.; Wang, D. H.; Zhou, Y. Q.; Ma, M.; Li, J. A.; Wang, Z. J.Dibutyl phthalate contributes to the thyroid receptor antagonisticactivity in drinking water processes. Environ. Sci. Technol. 2010, 44(17), 6863−6868.(8) Ishihara, A.; Rahman, F. B.; Leelawatwattana, L.; Prapunpoj, P.;Yamauchi, K. In vitro thyroid hormone-disrupting activity in effluents

and surface waters in Thailand. Environ. Toxicol. Chem. 2009, 28 (3),586−594.(9) Brucker-Davis, F. Effects of environmental synthetic chemicals onthyroid function. Thyroid 1998, 8 (9), 827−856.(10) Darnerud, P. O.; Lignell, S.; Glynn, A.; Aune, M.; Tornkvist, A.;Stridsberg, M. POP levels in breast milk and maternal serum andthyroid hormone levels in mother-child pairs from Uppsala, Sweden.Environ. Int. 2010, 36 (2), 180−187.(11) Zoeller, R. T. Environmental chemicals as thyroid hormoneanalogues: New studies indicate that thyroid hormone receptors are targetsof industrial chemicals? Mol. Cell. Endocrinol. 2005, 242 (1−2), 10−15.(12) Jugan, M. L.; Levy-Bimbot, M.; Pomerance, M.; Tamisier-Karolak, S.; Blondeau, J. P.; Levi, Y. A new bioluminescent cellularassay to measure the transcriptional effects of chemicals that modulatethe alpha-1 thyroid hormone receptor. Toxicol. In Vitro 2007, 21,1197−1205.(13) Cai, Y. Q.; Jiang, G. B.; Liu, J. F.; Zhou, Q. X. Multi-walledcarbon nanotubes packed cartridge for the solid-phase extraction ofseveral phthalate esters from water samples and their determination byhigh performance liquid chromatography. Anal. Chim. Acta 2003, 494(1−2), 149−156.(14) David, R. M. Exposure to phthalate esters. Environ. HealthPerspect. 2000, 108 (10), A440−A440.(15) Kohn, M. C.; Parham, F.; Masten, S. A.; Portier, C. J.; Shelby,M. D.; Brock, J. W.; Needham, L. L. Human exposure estimates forphthalates. Environ. Health Perspect. 2000, 108 (10), A440−A442.(16) Zhu, X. B.; Tay, T. W.; Andriana, B. B.; Alam, M. S.; Choi, E. K.;Tsunekawa, N.; Kanai, Y.; Kurohmaru, M. Effects of di-iso-butylphthalate on testes of prepubertal rats and mice. Okajimas Folia Anat.Jpn. 2010, 86 (4), 129−136.(17) Kruger, T.; Long, M.; Bonefeld-Jorgensen, E. C. Plasticcomponents affect the activation of the aryl hydrocarbon and theandrogen receptor. Toxicology 2008, 246 (2−3), 112−123.(18) Janjua, N. R.; Mortensen, G. K.; Andersson, A. M.; Kongshoj,B.; Skakkebaek, N. E.; Wulf, H. C. Systemic uptake of diethylphthalate, dibutyl phthalate, and butyl paraben following whole-bodytopical application and reproductive and thyroid hormone levels inhumans. Environ. Sci. Technol. 2007, 41 (15), 5564−5570.(19) Snyder, S. A.; Adham, S.; Redding, A. M.; Cannon, F. S.;DeCarolis, J.; Oppenheimer, J.; Wert, E. C.; Yoon, Y. Role ofmembranes and activated carbon in the removal of endocrine disruptorsand pharmaceuticals. Desalination 2007, 202 (1−3), 156−181.(20) Shen, O. X.; Du, G. Z.; Sun, H.; Wu, W.; Jiang, Y.; Song, L.;Wang, X. R. Comparison of in vitro hormone activities of selectedphthalates using reporter gene assays. Toxicol. Lett. 2009, 191 (1), 9−14.(21) Urbatzka, R.; van Cauwenberge, A.; Maggioni, S.; Vigano, L.;Mandich, A.; Benfenati, E.; Lutz, I.; Kloas, W. Androgenic andantiandrogenic activities in water and sediment samples from the riverLambro, Italy, detected by yeast androgen screen and chemicalanalyses. Chemosphere 2007, 67 (6), 1080−1087.(22) Conroy, O.; Saez, A. E.; Quanrud, D.; Ela, W.; Arnold, R. G.Changes in estrogen/anti-estrogen activities in ponded secondaryeffluent. Sci. Total Environ. 2007, 382 (2−3), 311−323.

Figure 3. Contributions of individual phthalates to the Anti-TR-EQs in water.

Environmental Science & Technology Article

dx.doi.org/10.1021/es202625r | Environ. Sci. Technol. 2012, 46, 1811−18181817

(23) Villeneuve, D. L.; Blankenship, A. L.; Giesy, J. P. Derivation andapplication of relative potency estimates based on in vitro bioassayresults. Environ. Toxicol. Chem. 2000, 19 (11), 2835−2843.(24) Kojima, H.; Katsura, E.; Takeuchi, S.; Niiyama, K.; Kobayashi,K. Screening for estrogen and androgen receptor activities in 200pesticides by in vitro reporter gene assays using Chinese hamster ovarycells. Environ. Health Perspect. 2004, 112 (5), 524−531.(25) Shi, W.; Zhang, F.; Hu, G.; Hao, Y.; Zhang, X.; Liu, H.; Wei, S.;Wang, X.; Giesy, J. P.; Yu, H. Thyroid hormone disrupting activitiesassociated with phthalate esters in water sources from Yangtze RiverDelta. Environ. Int. 2011, DOI: 10.1016/j.envint.2011.05.013.(26) Ghisari, M.; Bonefeld-Jorgensen, E. C. Effects of plasticizers andtheir mixtures on estrogen receptor and thyroid hormone functions.Toxicol. Lett. 2009, 189 (1), 67−77.(27) Li, J.; Ma, M.; Wang, Z. J. A two-hybrid yeast assay to quantifythe effects of xenobiotics on thyroid hormone-mediated geneexpression. Environ. Toxicol. Chem. 2008, 27 (1), 159−167.(28) Joyeux, A.; Balaguer, P.; Germain, P.; Boussioux, A. M.; Pons,M.; Nicolas, J. C. Engineered cell lines as a tool for monitoringbiological activity of hormone analogs. Anal. Biochem. 1997, 249 (2),119−130.(29) Baker, V. A. Endocrine disrupters - testing strategies to assesshuman hazard. Toxicol. In Vitro 2001, 15 (4−5), 413−419.(30) Rutishauser, B. V.; Pesonen, M.; Escher, B. I.; Ackermann, G. E.;Aerni, H. R.; Suter, M. J. F.; Eggen, R. I. L. Comparative analysis ofestrogenic activity in sewage treatment plant effluents involving threein vitro assays and chemical analysis of steroids. Environ. Toxicol. Chem.2004, 23 (4), 857−864.(31) Gray, L. E.; Kelce, W. R.; Wiese, T.; Tyl, R.; Gaido, K.; Cook, J.;Klinefelter, G.; Desaulniers, D.; Wilson, E.; Zacharewski, T.; Waller,C.; Foster, P.; Laskey, J.; Reel, J.; Giesy, J.; Laws, S.; McLachlan, J.;Breslin, W.; Cooper, R.; DiGiulio, R.; Johnson, R.; Purdy, R.; Mihaich,E.; Safe, S.; Sonnenschein, C.; Welshons, W.; Miller, R.; McMaster, S.;Colborn, T. Endocrine screening methods workshop report: Detectionof estrogenic and androgenic hormonal and antihormonal activity forchemicals that act via receptor or steroidogenic enzyme mechanisms.Reprod. Toxicol. 1997, 11 (5), 719−750.(32) Wong, C.; Kelce, W. R.; Sar, M.; Wilson, E. M. Androgenreceptor antagonist versus agonist activities of the fungicide vinclozolinrelative to hydroxyflutamide. J. Biol. Chem. 1995, 270, 19998−20003.(33) Wilson, V. S.; Bobseine, K.; Lambright, C. R.; Gray, J. L. E. Anovel cell line, MDA-kb2, that stably expresses an androgen- andglucocorticoid-responsive reporter for the detection of hormonereceptor agonists and antagonists. Toxicol. Sci. 2002, 66 (1), 69−81.(34) Allan, G. F.; Leng, X.; Tsai, S. Y.; Weigel, N. L.; Edwards, D. P.;Tsai, M. J.; O’Malley, B. W. Hormone and antihormone inducedistinct conformational changes, which are central to steroid receptoractivation. J. Biol. Chem. 1992, 267, 19513−19520.(35) Garcia, T.; Benhamou, B.; Gofflo, D.; Vergezac, A.; Philibert, D.;Chambon, P.; Gronemeyer, H. Switching agonistic, antagonistic, andmixed transcriptional responses to 11-beta-substituted progestins bymutation of the progesterone receptor. Mol. Endocrinol. 1992, 6,2071−2078.(36) Jiang, S. Y.; Langan-Fahey, S. M.; Stella, A. L.; McCague, R.;Jordan, V. C. Point mutation of estrogen receptor (ER) in the ligand-binding domain changes the pharmacology of antiestrogens in ER-negative breastcancer cells stably expressing complementary DNAs forER. Mol. Endocrinol. 1992, 6, 2167−2174.(37) Sugiyama, S.; Shimada, N.; Miyoshi, H.; Yamauchi, K. Detectionof thyroid system-disrupting chemicals using in vitro and in vivoscreening assays in Xenopus laevis. Toxicol. Sci. 2005, 88, 367−374.(38) Choi, K. J.; Kim, S. G.; Kim, C. W.; Park, J. K. Removalefficiencies of endocrine disrupting chemicals by coagulation/flocculation, ozonation, powdered/granular activated carbon adsorp-tion, and chlorination. Korean J. Chem. Eng. 2006, 23 (3), 399−408.(39) Chang, B. V.; Wang, T. H.; Yuan, S. Y. Biodegradation of fourphthalate esters in sludge. Chemosphere 2007, 69, 1116−1123.

(40) Gavala, H. N.; Alatriste-Mondragon, F.; Iranpour, R.; Ahring, B.K. Biodegradation of phthalate esters during the mesophilic anaerobicdigestion of sludge. Chemosphere 2003, 52 (4), 673−682.(41) Zeng, F.; Cui, K. Y.; Xie, Z. Y.; Liu, M.; Li, Y. J.; Lin, Y. J.; Zeng,Z. X.; Li, F. B. Occurrence of phthalate esters in water and sediment ofurban lakes in a subtropical city, Guangzhou, South China. Environ. Int.2008, 34 (3), 372−380.(42) Yamada-Okabe, T.; Aono, T.; Sakai, H.; Kashima, Y.; Yamada-Okabe, H. 2,3,7,8-tetrachlorodibenzo-p-dioxin augments the modu-lation of gene expression mediated by the thyroid hormone receptor.Toxicol. Appl. Pharmacol. 2004, 194 (3), 201−210.(43) Bergh, C.; Torgrip, R.; Emenius, G.; Ostman, C. Organo-phosphate and phthalate esters in air and settled dust - a multi-locationindoor study. Indoor Air 2011, 21 (1), 67−76.(44) Yan, H. Y.; Liu, B. M.; Du, J. J.; Row, K. H. Simultaneousdetermination of four phthalate esters in bottled water usingultrasound-assisted dispersive liquid-liquid microextraction followedby GC-FID detection. Analyst 2011, 135 (10), 2585−2590.(45) Batterman, S.; Huang, A. T.; Wang, S. G.; Zhang, L. Reductionof ingestion exposure to trihalomethanes due to volatilization. Environ.Sci. Technol. 2000, 34 (20), 4418−4424.(46) Meeker, J. D.; Calafat, A. M.; Hauser, R. Di(2-ethylhexyl)phthalate metabolites may alter thyroid hormone levels in men.Environ. Health Perspect. 2007, 115 (7), 1029−1034.(47) Hauser, R.; Meeker, J. D.; Singh, N. P.; Silva, M. J.; Ryan, L.;Duty, S.; Calafat, A. M. DNA damage in human sperm is related tourinary levels of phthalate monoester and oxidative metabolites. Hum.Reprod. 2007, 22 (3), 688−695.(48) Blair, J. D.; Ikonomou, M. G.; Kelly, B. C.; Surridge, B.; Gobas,F. Ultra-trace determination of phthalate ester metabolites in seawater,sediments, and biota from an urbanized marine inlet by LC/ESI-MS/MS. Environ. Sci. Technol. 2009, 43 (16), 6262−6268.(49) Calafat, A. M.; Slakman, A. R.; Silva, M. J.; Herbert, A. R.;Needham, L. L. Automated solid phase extraction and quantitativeanalysis of human milk for 13 phthalate metabolites. J Chromatogr. B2004, 805 (1), 49−56.(50) Mortensen, G. K.; Main, K. M.; Andersson, A. M.; Leffers, H.;Skakkebwk, N. E. Determination of phthalate monoesters in humanmilk, consumer milk, and infant formula by tandem mass spectrometry(LC-MS-MS). Anal. Bioanal. Chem. 2005, 382 (4), 1084−1092.

Environmental Science & Technology Article

dx.doi.org/10.1021/es202625r | Environ. Sci. Technol. 2012, 46, 1811−18181818

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).

-11 -10 -9 -8 -7 -6 -50.00

100.00

200.00

300.00 T3

log concentration (mol/L)

Rel

ative

luci

fera

se a

ctivi

ty(n

-fold

of c

ontro

l)

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.

***

**** ****

**

0

10

20

30

40

50

60

DEHA DINP DEHP BBP DIDP DNOP DMP DNBP DEP DIBP

Rel

ativ

e lu

cife

rase

act

ivity

. .

1 nmol 10 nmol 10000 nmol 10000 nmol 10000 nmol 10000 nmol105 nmol/L104 nmol/L103 nmol/L102 nmol/L

****

****

**

**

10 nmol/L1 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.

0

0.2

0.4

0.6

0.8

1

1.2

CZ-WTP CZ-TAP XZ-SOUTH XZ-WTP XZ-TAP YC-WTP YC-TAP

Rel

ativ

e lu

cife

rase

act

ivity

(n-f

old

of 5

.0×1

0-9 m

ol/L

T3)

200 100 50 25 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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