Supplemental Material to Manuscript titled “Exposure to Bisphenols,

Benzophenones, Parabens and Triclosan among Pregnant Women in

different trimesters”

Hongzhi Zhao1#, Wenqian Huo2#, Jiufeng Li1, Xinli Ma 1, Wei Xia2, Zhengji Pang1,

Mingyi Xie1, Shunqing Xu2*, Zongwei Cai1*

1State Key Laboratory of Environmental and Biological Analysis, Department of

Chemistry, Hong Kong Baptist University, Hong Kong SAR, China.

2Key Laboratory of Environment and Health, Ministry of Education & Ministry of

Environmental Protection, and State Key Laboratory of Environmental Health, School

of Public Health, Tongji Medical College, Huazhong University of Science and

Technology, Wuhan, China

*Corresponding author. E-mail: zwcai@hkbu.edu.hk (Z. Cai), Tel: +852-34117070;

xust@hust.edu.cn (S. Xu), Tel: +86-27-83657705

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Table of Contents Page

Tables

Table S1 Gradient elution processes used in UHPLC S3

Table S2 Mass transitions for target analytes S4

Table S3Descriptive Statistics of total urinary concentrations (ng/mL) (free form plus conjugate species) of environmental pollutants detected in pregnant women (n = 209) of different trimesters

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Table S4 Correlations between SG-adjusted levels of each compound. S8

Table S5 Total variance explained of Principal Component Analysis S10

Table S6 The result of Mann-Whitney U test of different trimesters S11

Table S7 The ICC of different trimesters S11

Figures

Figure S1 The detection rate of each analyte in different trimesters S12

Figure S2 Metabolic pathways of BP-3 in the phase I reactions S13

Figure S3 Correlation analysis of target analyte (based on SG-adjusted urinary concentrations)

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Figure S4 Correlation analysis of SG-adjusted levels of different chemical classes

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Table S1 Gradient elution processes used in UHPLC

Retention Flow %A (water) %B (acetonitrile)0 0.2 75 25

1.5 0.2 25 2510 0.2 20 80

10.2 0.2 0 10012 0.2 0 100

12.2 0.2 75 2515 0.2 75 25

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Table S2 Mass transitions for target analytes

Analyte Abbreviation Parent ion (m/z) Product Ion (m/z) CE (eV) Internal standard LOD (ng/mL)

2,4-dihydroxybenzophenone BP-1 213.0 91.1 30 13C6-BP-3 0.10

2,2’,4,4’- tetrahydroxy benzophenone

BP-2 245.1 135.0 20 13C6-BP-3 0.01

2-hydroxy-4-methoxybenzophenone

BP-3 227.1 211.1 25 13C6-BP-3 0.20

2,2’-dihydroxy-4-methoxybenzo-bpenone

BP-8 243.1 123.1 20 13C6-BP-3 0.10

4-hydroxybenzophenone 4-OH-BP 197.1 92.1 34 13C6-BP-3 0.10/ 13C6-BP-3 233.1 217.1 25 13C6-BP-3 /

methylparaben MeP 151.0 92.1 30 13C6-MeP 0.05/ 13C6-MeP 157.0 98.1 30 / /

ethylparaben EtP 165.0 92.1 30 13C6-EtP 0.01/ 13C6-EtP 171.0 98.1 30 / /

propylparaben PrP 179.0 92.1 30 13C6-PrP 0.10/ 13C6-PrP 185 98.1 30 / /

butylparaben BuP 193 92.1 30 13C6-BuP 0.10/ 13C6-BuP 199 98.1 30 / /

benzylparaben BzP 227.1 92.1 25 13C6-BuP 0.10Triclosan TCS 287 35.2 15 13C12-TCS 0.20

/ 13C12-TCS 299 35.2 15 / /

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Abbreviations: CE, collision energy; LOD, limit of detection; MRM, multiple reaction monitoring; RT, retention time.

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Table S3 Descriptive Statistics of total urinary concentrations (ng/mL) (free form plus conjugate species) of environmental pollutants detected in pregnant women (n = 209) of different trimesters

Analyte Trimester DF (%) / GM (95%CI)Median(50th)

Selected percentilesRange (ng/mL)

25th 75th 95th

BP-1

T1 79.9Unadjusted 0.17 (0.13, 0.21) 0.19 0.04 0.53 2.62 <LOD~28.24SG-adjusted 0.20 (0.16, 0.25) 0.21 0.08 0.61 2.39 <LOD~40.79

T2 78.0Unadjusted 0.10 (0.08, 0.13) 0.10 0.07 0.26 1.06 <LOD~125.56SG-adjusted 0.11 (0.09, 0.14) 0.14 0.08 0.28 0.92 <LOD~114.14

T3 75.1Unadjusted 0.11 (0.09, 0.14) 0.12 <LOD 0.30 1.35 <LOD~378.69SG-adjusted 0.12 (0.10, 0.15) 0.12 <LOD 0.32 1.42 <LOD~236.68

BP-3

T1 85.6Unadjusted 0.76 (0.59, 0.96) 0.95 0.32 2.26 14.83 <LOD~59.41SG-adjusted 0.90 (0.71, 1.14) 1.17 0.40 2.63 12.90 <LOD~117.32

T2 73.2Unadjusted 0.34 (0.27, 0.43) 0.50 <LOD 1.19 5.09 <LOD~448.22SG-adjusted 0.37 (0.28, 0.47) 0.55 <LOD 1.38 4.87 <LOD~407.47

T3 72.7Unadjusted 0.43 (0.32, 0.57) 0.64 <LOD 1.54 9.81 <LOD~1362.73SG-adjusted 0.46 (0.35, 0.60) 0.65 <LOD 1.48 8.38 <LOD~851.71

4-OH-BP

T1 95.2Unadjusted 0.17 (0.14, 0.20) 0.19 0.08 0.44 1.45 <LOD~7.08SG-adjusted 0.20 (0.16, 0.24) 0.22 0.08 0.57 1.75 <LOD~4.00

T2 94.3Unadjusted 0.17 (0.14, 0.21) 0.20 0.07 0.46 1.41 <LOD~3.33SG-adjusted 0.18 (0.15, 0.22) 0.20 0.09 0.47 1.58 <LOD~3.01

T3 91.9Unadjusted 0.19 (0.15, 0.23) 0.22 0.07 0.46 1.39 <LOD~6.68SG-adjusted 0.20 (0.16, 0.25) 0.25 0.08 0.46 1.63 <LOD~8.35

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∑benzophen

ones (μmol/L)

T1 /Unadjusted 0.008 (0.006, 0.009) 0.007 0.004 0.016 0.083 <LOD~0.373SG-adjusted 0.009 (0.008, 0.011) 0.009 0.005 0.017 0.075 <LOD~0.594

T2 /Unadjusted 0.005 (0.004, 0.005) 0.005 0.002 0.009 0.029 <LOD~2.551SG-adjusted 0.004 (0.004, 0.006) 0.005 0.003 0.011 0.027 <LOD~2.319

T3 /Unadjusted 0.005 (0.004, 0.007) 0.005 0.002 0.012 0.059 <LOD~7.747SG-adjusted 0.006 (0.005, 0.007) 0.006 0.003 0.012 0.048 <LOD~4.842

MeP

T1 100Unadjusted 19.45 (15.06, 25.11) 17.66 4.22 77.49 422.08 0.15~1178.98SG-adjusted 23.12 (18.08, 29.55) 22.96 4.96 102.97 432.13 0.15~1256.16

T2 100Unadjusted 17.52 (13.89, 22.10) 19.37 4.44 75.94 244.39 0.32~1223.93SG-adjusted 18.87 (15.06, 23.63) 21.88 4.08 77.68 227.93 0.14~679.96

T3 99.0Unadjusted 13.58 (10.63, 17.35) 13.36 3.83 49.78 234.00 <LOD~382.68SG-adjusted 14.59 (11.47, 18.58) 13.93 3.95 47.05 223.51 <LOD~541.34

EtP

T1 95.2Unadjusted 0.81 (0.59, 1.12) 0.56 0.29 1.83 113.49 <LOD~2929.44SG-adjusted 0.96 (0.71, 1.32) 0.69 0.35 2.24 70.58 <LOD~2994.38

T2 91.4Unadjusted 0.45 (0.33, 0.62) 0.51 0.23 1.34 23.05 <LOD~302.94SG-adjusted 0.48 (0.35, 0.67) 0.56 0.21 1.44 23.90 <LOD~319.09

T3 92.3Unadjusted 0.54 (0.39, 0.76) 0.52 0.23 1.27 22.55 <LOD~2706.56SG-adjusted 0.58 (0.42, 0.80) 0.65 0.25 1.65 25.34 <LOD~1176.77

PrPT1 100

Unadjusted 3.16 (2.33, 4.29) 1.83 0.53 18.22 160.46 0.06~783.66SG-adjusted 3.75 (2.81, 5.02) 2.14 0.63 23.34 137.86 0.10~ 604.36

T2 99.5Unadjusted 2.85 (2.09, 3.89) 2.32 0.46 19.29 87.63 <LOD ~645.39SG-adjusted 3.07 (2.26, 4.17) 2.38 0.48 24.54 82.80 <LOD~430.26

T3 98.1 Unadjusted 2.35 (1.73, 3.19) 1.65 0.40 15.45 90.92 <LOD~757.48

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SG-adjusted 2.52 (1.87, 3.40) 1.99 0.45 17.07 79.11 <LOD~329.34

BuP

T1 69.4Unadjusted 0.05 (0.04, 0.07) 0.03 <LOD 0.27 2.27 <LOD~145.36SG-adjusted 0.06 (0.04, 0.08) 0.03 <LOD 0.24 3.32 <LOD~94.48

T2 66.5Unadjusted 0.04 (0.02, 0.03) 0.02 <LOD 0.22 0.75 <LOD~38.45

SG-adjusted 0.06 (0.05, 0.08) 0.02 <LOD 0.22 1.15 <LOD~17.48

T3 66.5Unadjusted 0.04 (0.03, 0.06) 0.02 <LOD 0.27 0.75 <LOD~24.77

SG-adjusted 0.05 (0.04, 0.06) 0.03 <LOD 0.24 0.99 <LOD~16.51

∑parabens (μmol/L)

T1 /Unadjusted 0.205 (0.192, 0.310) 0.218 0.041 0.770 4.335 <LOD~18.552SG-adjusted 0.244 (0.194, 0.313) 0.289 0.057 1.048 4.077 <LOD~20.098

T2 /Unadjusted 0.168 (0.170, 0.252) 0.212 0.042 0.698 2.038 <LOD~11.558SG-adjusted 0.181 (0.146, 0.228) 0.196 0.046 0.704 1.886 <LOD~6.421

T3 /Unadjusted 0.151 (0.109, 0.170) 0.178 0.042 0.681 2.810 <LOD~20.524SG-adjusted 0.162 (0.131, 0.204) 0.161 0.055 0.548 1.823 <LOD~8.923

TCS

T1 52.2Unadjusted 0.27 (0.19, 0.38) 0.16 <LOD 1.62 40.97 <LOD~179.47SG-adjusted 0.32 (0.23, 0.45) 0.18 <LOD 1.64 33.21 <LOD~234.22

T2 49.3Unadjusted / <LOD <LOD 1.22 13.72 <LOD~159.53SG-adjusted / <LOD <LOD 1.43 12.09 <LOD~106.36

T3 47.8Unadjusted / <LOD <LOD 1.23 12.40 <LOD~72.37SG-adjusted / <LOD <LOD 1.13 11.19 <LOD~723.65

Abbreviations: DF, Detection Frequency; GM, geometric mean; CI, confidence interval; SG, specific gravity; LOD, limit of detection; BP-1, 2,4-dihydroxybenzophenone; BP-3, 2-hydroxy-4-methoxybenzophenone; BP-8, 2,2’-dihydroxy-4-methoxybenzo-bpenone; 4-OH-BP, 4-hydroxybenzophenone; MeP, methylparaben; EtP, ethylparaben; PrP, propylparaben; BuP, butylparaben; BzP, benzylparaben; TCS, Triclosan.

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Data of other analytes were not reported due to the low frequency of detection in the samples (< 30 % in human urine samples). GM was reported only if the proportion of detectable more than 50 %.Undetectable concentration was accounted as a value equal to the LOD divided by the square root of 2. ∑benzophenones: the molar sum of concentrations of BP-1, BP-3, BP-2, BP-8, 4-OH-BP; ∑parabens: the molar sum of concentrations of MeP, EtP, PrP, BuP, BzP.

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Table S4 Correlations between SG-adjusted levels of each compound (n=209×3).

BPA BPS BPAF BP-1 BP-3 4-OH-BP MeP EtP PrP BuP TCS

BPA

correlation coefficient

1.00 -0.07 0.26 -0.16 0.02 0.13 0.05 0.08 0.03 -0.04 -0.03

p value(two tails)

/ 0.09 0.00 0.00 0.68 0.00 0.23 0.04 0.53 0.28 0.46

BPS

correlation coefficient

-0.07 1.00 -0.12 0.41 0.09 -0.16 0.06 0.07 0.03 0.28 0.14

p value(two tails)

0.09 / 0.00 0.00 0.03 0.00 0.12 0.06 0.45 0.00 0.00

BPAF

correlation coefficient

0.26 -0.12 1.00 -0.21 0.24 0.27 0.03 -0.06 0.06 -0.08 0.16

p value(two tails)

0.00 0.00 / 0.00 0.00 0.00 0.44 0.11 0.16 0.05 0.00

BP-1

correlation coefficient

-0.16 0.41 -0.21 1.00 0.57 0.00 0.04 0.05 0.00 0.14 0.29

p value(two tails)

0.00 0.00 0.00 / 0.00 0.93 0.30 0.19 0.96 0.00 0.00

BP-3

correlation coefficient

0.02 0.09 0.24 0.57 1.00 0.08 0.04 0.03 0.04 -0.05 0.30

p value(two tails)

0.68 0.03 0.00 0.00 / 0.03 0.30 0.50 0.33 0.23 0.00

4-OH-BP correlation 0.13 -0.16 0.27 0.00 0.08 1.00 0.04 0.07 0.04 0.19 -0.10

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coefficientp value

(two tails)0.00 0.00 0.00 0.93 0.03 / 0.27 0.06 0.32 0.00 0.01

MeP

correlation coefficient

0.05 0.06 0.03 0.04 0.04 0.04 1.00 0.24 0.68 0.26 0.00

p value(two tails)

0.23 0.12 0.44 0.30 0.30 0.27 / 0.00 0.00 0.00 0.99

EtP

correlation coefficient

0.08 0.07 -0.06 0.05 0.03 0.07 0.24 1.00 0.28 0.42 0.03

p value(two tails)

0.04 0.06 0.11 0.19 0.50 0.06 0.00 / 0.00 0.00 0.43

PrP

correlation coefficient

0.03 0.03 0.06 0.00 0.04 0.04 0.68 0.28 1.00 0.21 0.01

p value(two tails)

0.53 0.45 0.16 0.96 0.33 0.32 0.00 0.00 / 0.00 0.89

BuP

correlation coefficient

-0.04 0.28 -0.08 0.14 -0.05 0.19 0.26 0.42 0.21 1.00 -0.08

p value(two tails)

0.28 0.00 0.05 0.00 0.23 0.00 0.00 0.00 0.00 / 0.05

TCS

correlation coefficient

-0.03 0.14 0.16 0.29 0.30 -0.10 0.00 0.03 0.01 -0.08 1.00

p value(two tails)

0.46 0.00 0.00 0.00 0.00 0.01 0.99 0.43 0.89 0.05 /

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Table S5 Total variance explained of Principal Component Analysis

ComponentInitial Eigenvalue Extraction Sums of Squared Loadings

Total Variance (%) Cumulative (%) Total Variance (%) Cumulative (%)1 2.05 25.69 25.69 2.05 25.69 25.692 1.86 23.19 48.88 1.86 23.19 48.883 1.02 12.74 61.61 1.02 12.74 61.614 1.00 12.48 74.105 0.97 12.18 86.286 0.74 9.19 95.477 0.34 4.23 99.708 0.02 0.30 100.00

Extraction Method: Principal Component Analysis (based on SG-adjusted concentration)

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Table S6 The result of Mann-Whitney U test of SG-adjusted concentrations in different trimesters (n = 209×3)

Name MeP EtP4-OH-

BPPrP BP1 BuP BPAF BP3 TCS

p (two tails)

UnadjustedT1VsT2 0.60 0.06 0.86 0.64 0.001* 0.20 0.58 <0.001* 0.31T1VsT3 0.08 0.19 0.37 0.23 0.004* 0.58 0.81 <0.001* 0.20T2VsT3 0.19 0.55 0.49 0.46 0.68 0.47 0.67 0.21 0.82

SG-adjustedT1VsT2 0.26 0.01 0.55 0.33 <0.001* 0.06 0.19 <0.001* 0.11T1VsT3 0.02 0.07 0.84 0.10 <0.001* 0.20 0.40 <0.001* 0.05T2VsT3 0.13 0.49 0.24 0.46 0.81 0.63 0.74 0.35 0.99

p < 0.05(*) represent significant differences

Table S7 The ICC of different trimesters (n = 209×3)

MeP EtP PrP 4-OH-BP BP-1 BuP BP-3 TCSUnadjusted 0.39 0.01 0.23 0.53 0.07 0.21 0.14 0.33SG-adjusted 0.44 0.03 0.42 0.63 0.20 0.17 0.33 0.11

ICC: intraclass correlation coefficients

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Figure S1 the detection rate of each analyte in different trimesters.

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Figure S2 Metabolic pathways of BP-3 in the phase I reactions

Figure S3 Correlation analysis of target analyte (based on SG-adjusted urinary concentrations). (a) Correlation between EtP and BuP (b) Correlation between EtP and PrP

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Figure S4 Correlation analysis of SG-adjusted levels of different chemical classes (∑benzophenones, ∑parabens and TCS)

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