1 Identification of Thyroid Receptor Antagonists in the Water from the Guanting
2 Reservoir, Beijing, China
3 Jian Li
1
*, Shujuan Ren
1
, Shaolun Han
1
,Bingli Lei
2 and Na Li
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Engineering Research Center of Groundwater Pollution Control and Remediation,
6 Ministry of Education,College of Water Sciences, Beijing Normal University, Beijing
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100875, China;
2
Institute of Environmental Pollution and Health, College of Environmental and
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Chemical Engineering, Shanghai University, Shanghai, 200444, China;
3
State Key Laboratory of Environmental Aquatic Chemistry, Research Center for
11 Eco-Environmental Sciences, Chinese Academy of Sciences, P. O. Box 2871, Beijing
12 100085, China
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* Corresponding author. Tel.: +86-010-58802738; fax:+86-010-58802738; E-mail: lijian@bnu.edu.cn

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Chemical Analysis.
Water Sample processing . The environmental water samples, procedure blanks
(Milli-Q water, conductivity of 18.2 Ω) were filtered through glass fiber filters (0.7
μm, Millipore, USA). The solid phase extraction (SPE) was performed using an HLB cartridge (500 mg, Waters, USA). The cartridges were forced under vacuum at a flow rate of approximately 6 mL/min. The cartridgeswere washed with 10 mL of dichloromethane/methanol (9/1).Then, the extracts were removed the water using anhydrous sodium and evaporated to dryness in a rotary evaporator (R-200, Buchi,
France) at 40°C to 2 mL. The dehydrated extracts were blown to dryness under a gentle nitrogen flow and reconstituted in 0.5 mL of hexane for chemical analysis.
Analysis of organicchlorine pesticides (OCPs) . The calibration mixture of 19
OCPs was purchased from Supelco, USA. Each component at the concentration of
250 µg/mL except methoxychlor at l000µg/mL in hexane: toluene (50:50, v/v). An internal standard of pentachloronitrobenzene (PCNB, Supelco Co., USA) was added to all of the samples before analysis. Analysis of OCPs was performed with an
Agilent 6890 series GC equipped with a 63Ni electron capture detector (Agilent, Palo
Alto, CA). A 30 m × 0.25 mm i.d. × 0.25 µm HP-5 column (J&W, Folsom, CA) was used for pesticide identification and quantification. High purity of nitrogen was used as carrier gas at 2.0 mL/min under the constant flow mode. Splitless injection of a 1
μL sample was performed with a 1.8 min solvent delay time. The temperature programmed was used in the oven, started at 85 °C holding for 2 min, then to 180 °C at a rate of 10 °C /min, holding for 15 min, and further to 280 °C at a rate of 20 °C
/min holding for 5 min. Injector and detector temperatures were maintained at 250 °C and 300 °C, respectively. OCPs were identified by comparing the GC peak retention times with those of authentic reference standards and quantified by peak area using an internal standard method.
Analysis of phenols . The calibration mixture of 21 phenols was purchased from
Sigma-Aldrich (USA). The mixed standard stock solution (1000 µg/mL) was prepared in isopropyl alcohol. Phenols were first derivatived by BSTFA + TMCA (99:1, v/v) which was obtained from Supelco (Supelco Park, PA). Deuterated bisphenol A (>

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99%) and deuterate dpyrene (> 99%) were purchased from Aldrich Chemistry
Corporation (Milwaukee, WI).Internal standard, deuterated pyrene or deuterated bisphenol A, was added respectively. Derivatives of phenols were dryness under a low nitrogen flow. Then capped and heated in an air bath at 65°C for 30 min. After cooling, the products of derivative were analyzed directly by GC-MS (Agilent
6890/MSD5973). The capillary columns used were HP-5 column (J&W, Folsom, CA)
(30 m × 0.25 mm i.d. × 0.25 µm film thickness). High purity of helium was used as carrier gas at 1.0 mL/min under the constant flow mode. Splitless injection of a 1 μL sample was performed with a 6 min solvent delay time. The temperature programmed was used in the oven, started at 100 °C then to 180 °C at a rate of 25 °C /min, holding for 15 min, and further to 300 °C at a rate of 20 °C /min holding for 5 min. Injector and detector temperatures were maintained at 280 °C and 300 °C, respectively. All analysis of data and control of GC and MS parameters were performed by the MSD
Productivity Chem Station for GC and GC/MSD Systems. Phenols were quantified using an internal standard method.
Analysis of phthalate esters (PAEs) . The mixture of 15 PAEs was supplied by
AccuStandard (New Haven, Michigan, USA). The samples were analyzed by GC-MS
(QP-2010, Shimadzu, Japan), operating in electron impact (EI) and selective ion monitoring modes (SIM). A DB-5ms capillary column (30 m ×250 μm, i.d.; 0.25 μm film thickness; Agilent, USA) was applied for chromatographic separation. The injector temperature and ion source temperature were maintained at 280 °C and
250 °C, respectively. The helium carrier gas (>99.999%) was controlled constant at a speed of 1 mL
/min. The GC oven temperature was initiated at 60 °C for 1.0 min, increased to 220 °C at a rate of 20 °C /min, held at 220 °C for 1.0 min, and finally ramped at 5 °C /min to 280 °C and held for 2 min. The extracts of 1 μL were injected into GC-MS in splitless mode with interface temperature of 280 °C. Quantitation was performed using the external calibration method based on five-pointed calibration curve for individual PAEs.
Quality Control.
The contamination of phthalates may occur in any step of the analytical procedure and in the bioassay procedure, resulting in overestimated results.

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Therefore all equipments were rinsed with chromic acid solution and methanol before use until no significant detection could be found in blank. In order to avoid overestimated contents, organic solvents appeared as a probably source of PAE contaminations and become important to be monitored. Meanwhile, blank samples were also tested. For further calibration purposes, all responses were corrected through the subtraction of organic solvent and blank sample contaminations (Zheng et al., 2014) .
Concentrations of the analysis were not adjusted by recoveries.
The limit of detections (LODs) listed in the Table 2 were in the condition of 20000 folds concentration of sample.
In the procedure blank, none of the OCPs, phenols or PAEs was detected.
Specificity to the TR of the water samples
To identify whether water samples affect β-galactosidase activity,
β-galactosidase, o -Nitrophenol-β-galactopyranoside (ONPG) and PBS were combined, and showed a significant yellow color. The mixture was diluted until its
β-galactosidaseacivity was nearly the same to the 10×β-galactosidaseacivity induced by T3 (2.5×10 -7 mol/L) in the TR yeast bioassays. Then water samples were incubated with the mixture (β-galactosidase + ONPG+ PBS) (9/1, v/v). The control was incubated with MilliQ water (conductivity of 18.2 Ω). Comparing with the blank
(β-galactosidase + ONPG+ PBS), it showed no inhibition activity (Figure
S1 ). Thus the water samples do not affect β-galactosidase activity.
In addition, water sample GR1, GR3 was treated with different concentrations of T3 (2.5×10 -7 , 1.0×10 -7
and 5.0×10
-8 mol/L) and the inhibition activity was detected by the modified assay. It showed that the inhibition activity of the water samples increased with decreasing doses of T3 in Figure S2 , indicating that T
3
and the thyroid disrupting chemicals (TDCs) in the water samples may be competitive bind to the TR.
These result suggested the water samples have no general suppressive effects on TR gene expression and the inhibition activities of the water samples were specific to TR.
β-galactosidase was purchased from Wako Pure Chemical Idustries, Ltd.

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(Code No. 072-04141, 10,000 units). ONPG (99%) was purchased from Sigma
Chemical (St. Louis, MO, USA). The β-galactosidase assay kit was purchased from
Tropix, Bedford (MA, USA).
120.0%
100.0%
80.0%
60.0%
40.0%
20.0%
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0.0%
GR1 GR2 GR3 GR4 GR5 GR6 GR7 GR8 control
Figure S1 Inhibition of β-galactosidase activity by the water samples. Values are presented as the average ± standard error (n=3).
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40.0%
35.0%
30.0%
2.5×10-7 -7 mol/L
1.0×10-7 -7 mol/L
5.0×10-8 -8 mol/L
25.0%
20.0%
15.0%
10.0%
5.0%
0.0%
GR1 GR3
Figure S2 Concentration-dependent relationship of thyroid receptor (TR) antagonistic activities of water samples GR1 and GR3. The antagonistic activity of the samples are

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132 represented as the percent inhibition activity relative to the β-galactosidase activity induced by 3,3’,5-triiodo-L-thyronine (T3, 2.5×10 -7 , 1×10 -7 , 5.0×10 -8 mol/L).Values are presented as the average ± standard error (n=3).
Cytotoxicity of the water samples
To ensure that increased/ reduced activities in the bioassay were caused by true agonistic/ antagonistic responses and not by cytotoxicity, viability was measured in yeast cells exposed to water samples. Yeast cells were plated, and then exposed for 2 h to water samples. And cell viability was determined spectrophotometrically as a change of cell density (OD
600 nm
) in the assay medium. The cytotoxicity of the water samples is represented as the percent inhibition activity (1-OD
600 nm-exposure medium
/
OD
600 nm-blank medium
). The results showed that there is no significant change in yeast cell viability (Fig. S3 ).
Percentage recovery of the water samples
For agonistic activity, percentage recovery in spiked samples was calculated as follows: ([T3 molar equivalent of spiked sample-T3 molar equivalent of unspiked sample]/[T3 molar equivalent of the 2.5×10 -7 mol/L T3 (the concentration of the spike, taken from the standard curve)]). For antagonistic activity, percentage recovery in spiked samples was calculated as follows: ([amiodarone hydrochloride (AH) molar equivalent of spiked sample-AH molar equivalent of unspiked sample]/[AH molar equivalent of the 10
-7 mol/L AH (the concentration of the spike, taken from the standard curve)]) (Colosi and Kney, 2011).
The average percentage recovery of the spike for all samples was 93.5%, with a standard deviation of 14.0% (Table S1). Only one of the 18 values exceeded 120% recovery, whereas no sample had less than 65% recovery.

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Figure S3 Cytotoxicity of the water samples. The cytotoxicity of the water samples is represented as the percent inhibition activity .Values are presented as the average ± standard error (n=3).
Table S1 The average percentage recovery of the spike for water samples
Samples
GR1
GR2
GR3
GR4
GR5
GR6
GR7
GR8
Blank
Percentage recovery
Agonistic activity Antagonistic activity
85.2%
90.6%
88.3%
71.1%
80.9%
90.0%
102.3%
71.2%
90.2%
105.4%
97.5%
102.8%
88.5%
96.1%
103.3%
126.9%
111.9%
81.6%

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Water quality parameters of the Guanting Reservoir
The temperature, dissolved oxygen (DO), pH, total dissolved solids (TDS) and turbidity of the water samples collected from the Guanting Reservoir were assessed with an in situ water quality analysis system as shown in the Table S2.
Table S2. Water quality parameters of the Guanting Reservoir.DO: Dissolved oxygen;
TDS: Total dissolved solids.
Temperature
(°C )
GR1 17.31
GR2 20.35
GR3 19.11
GR4 18.77
GR5 21.46
GR6 19.13
GR7 19.89
GR8 24.22
DO
(mg/L) pH
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TDS Turbidity
(g/L) (NTU)
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7.70 7.32 0.629 8.8
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11.35 8.75 0.902 0.0
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11.17 8.64 0.918 0.0
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14.25 8.49 0.925 0.6
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16.90 8.84 0.889 6.6
175
13.19 8.69 0.91 17.4
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11.70 8.86 0.903 0.0
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9.47 9.14 0.907 2.0
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Evaluation the TEQ
The concentration-dependent curve on inhibition of β-galactosidase expression by amiodarone hydrochloride (AH) in presence of submaximal T3 concentration was obtained as described previously (Li et al., 2008; Li et al., 2014). The bioassay-derived AH equivalent concentrations (TEQ bio-water sample
) were derived by the known TR antagonist AH concentration necessary to produce an equivalent reduction in TR antagonistic activity (Li et al., 2010; Li et al., 2014).
The organic extract was diluted to fit the linear part of the concentration-dependent curve for AH. The concentration-dependent response curve of the organic extract was generated to measure 20% of the maximum effect (RIC
20
) value for antagonistic assays. The bioassay-derived AH equivalent concentration

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(TEQ bio-organic extracts
) was derived by the known TR antagonist AH concentration necessary to produce an equivalent reduction (20%) in TR antagonistic activity (Li et al., 2010; Shi et al., 2011; Körner et al., 1999; Conroy et al., 2007). The TEQ bio-organic extract
was estimated as follows:
TEQ bio-organic extract
=
RIC
20-AH
RIC
20-organic extrct
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The TEQ cals was derived by the concentration of the detected chemical (C i
) and the relative potency (REP) of this chemical (Li et al., 2010). The REPs of DBP, BBP and
DEHP were 126.7, 1.9 and 1.3, respectively. The TEQ cals
was estimated as follows:
TEQ cals-i
=REP i
×C i
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