Supplementary material

Polybrominated diphenyl ethers (PBDEs) and hydroxylated -PBDEs in human serum from Shanghai, China: a study on their presence and correlations

Bentuo Xua,MinghongWua,MingnanWangb,Chenyuan Pana,WenhuiQiuc, Liang Tanga,Gang Xua*

a School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, People’s Republic of China.

bYinyuan Hospital, Jiading District, Shanghai 201800, People’s Republic of China.

cSchool of Environmental Science and Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 518055, People’s Republic of China.

Corresponding author at: *G. Xu, Institute of Applied Radiation, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, People’s Republic of China, Phone: +86-21-66137521, E-mail: .

Six tables (Table S1–S6) and one figure (Fig. S1)

Lipid content determination.

Briefly, hydrochloric acid and 2-propanol were added to serum samples to denaturize the proteins and release the lipids and the organohalogen compounds. The analytes were extracted by hexane and MTBE (v/v, 1:1). The organic phase was washed with a solution of potassium chloride (1%), followed by evaporating to dryness for gravimetric determination of extracted lipid content.

Extraction and Analysis of PBDEs and HO-PBDEs.

Each serum sample (0.5-2 g) were placed into 15 mL polypropylene centrifuge tubes and spiked with 100 μL of the mixed internal standard working solutions. After equilibration was reached at room temperature after about an hour, 9 mL of water and 1 mL of formic acid were added to the sample. The sample was mixed thoroughly by vortexing and sonicated for 20 min in an ultrasonic bath. PBDEs and OH-PBDEs were enriched and separated from lipids by Oasis MAXSPE cartridges (6 cc, 150 mg, 40 μm) and all processing was performed on a SPE vacuum manifold. SPE cartridges were conditioned by 2 × 3 mL of 2% formic acid in acetone; 3 mL 1 M NaOH aqueous solution, and then 2 × 4 mL water. The sample solution was loaded on the SPE cartridge with flow rate of about 0.5-1 mL/min. The sample tube was rinsed with 1 mL of water and a rising solution was also loaded on the SPE cartridge. Finally, 4 mL of 0.03% NH4 aqueous solution was added to the cartridge and eluted. The cartridge was then washed with 2 mL of water and dried for 20 min by vacuum. The cartridge was then first eluted with 3 × 2 mL of acetone, and this fraction contained neutral contaminants PBDEs. The eluent was finally concentrated and solvent-exchanged to isooctane to constant volumes of 200 μL under a gentle N2 stream and transferred to GC vial for GC-MS analysis. OH-PBDEs were eluted out from the SPE cartridge with a second elution phase of 3 × 2 mL of 2% formic acid in acetone. The eluent was collected in a conical centrifuge tube and concentrated to about 200 μL by a stream of nitrogen. A 5 mL volume of hexanes was added to the tube. After vortexing and separation by centrifugation, the hexanes phase was transfer to another conical centrifuge tube. The sample (hexanes solution) was then evaporated to dryness under a stream of nitrogen. The residue was then reconstituted in 100 μL acetonitrile containing 0.1% formic acid and transferred to LC conical vial for LC-MS/MS analysis.

Instrumentation analysis of PBDEs and HO-PBDEs.

Identification and quantification of the 13 PBDE congeners (excluding BDE-209) was performed by GC-NCI/MS (Shimadzu 2010 Plus, Japan) equipped with an Rtx-5MS capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Restek, Bellefonte, PA). Aliquots of 1 μl were injected in splitless mode at 280 °C with helium as the carrier gas and methane as reagent gas at 1.2 mL min-1. The oven temperature was programmed as follows: 90 °C (2 min), then 25 °C min-1 to 210 °C, 10 °C min-1 to 275 °C (10 min), and finally being held at 330 °C for 10 min. Ion source was at 250 °C and transfer line at 280 °C. For detecting BDE-209, a DB-5HT capillary column (15 m × 0.25 mm i.d. × 0.1 μm film thickness; J&W Scientific, Folsom, CA) was used for separation. The oven temperature was programmed as follows: 110 °C was maintained for 1 min, raised at 25 °C min-1 to 300 °C and maintained for 10 min. Targeted monitored ions were as followed: 13 PBDE congeners (79, 81), BDE-209 (486.7, 488.7) and 13C12-BDE-209 (494.6, 496.6).

6 OH-PBDEs analysis was performed Agilent 1260 liquid chromatograph coupled to an Agilent 6460 triple quadrupole mass spectrometer. The chromatographic separation was carried out using a Poroshell 120 EC-C18 reversed-phase column (3 × 100 mm, 2.7 μm, Agilent). The temperature of LC column was kept at 40 °C. The mobile phases consisted of acetonitrile containing 0.1% formic acid (A) and water (B), and the LC flow rate was 0.4 mL/min. The volume for injections on the LC was 5 μL. The gradient profile (excluding the method of 4’-OH-BDE17) started with 30% B and linearly decreased to 10% B in 11 min. The column was allowed to equilibrate for 5 min for next injection. When 4’-OH-BDE17 was detected in the instrument, the elution program was keeping 100% A for 5 min.

The mass system was operated in electro-spray ionization (ESI) negative ion mode with multiple reactions monitoring (MRM). Nitrogen was used as the nebulizing and dissolvating gas. Argon was used as the collision gas. The crucial MS/MS parameters were as follows: The drying gas temperature: 350 °C, drying gas flow: 8 L min-1, nebulizer Pressure: 40 psi, sheath gas temperature: 350 °C, sheath gas flow: 11 L min-1, capillary voltage (–): 3500 V and nozzle voltage (–): 1000 V. The cone voltage (CV) and collision energy (CE) for each analyte and internal standards are summarized in the supplementary data (Table S2).

Table S1

Personal information of the 25serum samples.

Number / Age / Sex / Occupation / Health / Domicile
1 / 40 / Male / Work outside / Fine / Jiading, Shanghai
2 / 40 / Female / Office staff indoor / Fine / Jiading, Shanghai
3 / 42 / Female / Office staff indoor / Fine / Jiading, Shanghai
4 / 44 / Female / Office staff indoor / Fine / Jiading, Shanghai
5 / 44 / Female / Office staff indoor / Fine / Jiading, Shanghai
6 / 53 / Female / Work outside / Fine / Jiading, Shanghai
7 / 58 / Female / Office staff indoor / Fine / Jiading, Shanghai
8 / 58 / Female / Work outside / Fine / Jiading, Shanghai
9 / 60 / Female / Work outside / Fine / Other place
10 / 62 / Male / Not clear / Fine / Jiading, Shanghai
11 / 63 / Female / Office staff indoor / Fine / Jiading, Shanghai
12 / 64 / Male / Not clear / Fine / Jiading, Shanghai
13 / 66 / Female / Office staff indoor / Hypertension / Jiading, Shanghai
14 / 66 / Female / Not clear / Coronary disease / Other place
15 / 66 / Male / Not clear / Fine / Jiading, Shanghai
16 / 66 / Female / Work outside / Fine / Jiading, Shanghai
17 / 68 / Female / Not clear / Fine / Jiading, Shanghai
18 / 68 / Female / Office staff indoor / Fine / Jiading, Shanghai
19 / 68 / Female / Work outside / Hypertension / Other place
20 / 70 / Female / Not clear / Fine / Jiading, Shanghai
21 / 70 / Male / Electrical maintenance worker / Fine / Jiading, Shanghai
22 / 71 / Female / Work outside / Fine / Jiading, Shanghai
23 / 72 / Female / Not clear / Fine / Jiading, Shanghai
24 / 73 / Male / Work outside / Fine / Jiading, Shanghai
25 / 75 / Male / Work outside / Hypertension / Other place

Table S2

MS/MS parameters for the determination of OH-PBDEs.

Compound / CV (V) / CE (eV) / RT (min) / Quantitative ions (m/z) / Qualitative ions (m/z)
3’-OH-BDE7 / 3 / 30 / 2.97 / 342.9>79 / 342.9>81
3-OH-BDE47 / 3 / 24 / 5.02 / 500.7>418.7 / 500.7>79
6-OH-BDE47 / 3 / 30 / 5.58 / 500.7>81 / 500.7>79
2’-OH-BDE68 / 3 / 24 / 6.02 / 500.7>81 / 500.7>79
6’-OH-BDE99 / 3 / 19 / 7.32 / 578.6>79 / 578.6>81
6-OH-BDE137 / 3 / 30 / 8.01 / 658.5>79 / 658.5>81
Internal standard
4’-OH-BDE17 / 3 / 44 / 1.52 / 420.8>79 / 420.8>81

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Table S3

Linear range, correlation coefficient, spiked recoveries and limits of detection.

Compound / Linear range
(μgL-1) / Correlation coefficient
(R2) / Recoveries in the spiked matrices (%) / Recoveries in the spiked blanks
(%) / LOQ
(ng g-1) / LOD
(ng g-1)
BDE-17 / 1-20 / 0.9998 / 97% / 85% / 0.03 / 0.01
BDE-28 / 1-20 / 0.9995 / 92% / 91% / 0.04 / 0.01
BDE-47 / 1-20 / 0.9989 / 82% / 109% / 0.04 / 0.01
BDE-100 / 1-20 / 0.9988 / 82% / 114% / 0.04 / 0.01
BDE-99 / 1-20 / 0.9996 / 67% / 108% / 0.05 / 0.02
BDE-85 / 1-20 / 0.9993 / 87% / 67% / 0.10 / 0.03
BDE-154 / 1-20 / 0.9996 / 66% / 112% / 0.04 / 0.01
BDE-153 / 1-20 / 0.9990 / 81% / 94% / 0.07 / 0.02
3’-OH-BDE7 / 1-20 / 0.9988 / 107% / 80% / 0.02 / 0.01
3-OH-BDE47 / 1-20 / 0.9995 / 99% / 94% / 0.04 / 0.01
6-OH-BDE47 / 1-20 / 0.9996 / 73% / 73% / 0.02 / 0.01
2’-OH-BDE68 / 1-20 / 0.9994 / 95% / 95% / 0.04 / 0.01
6’-OH-BDE99 / 1-20 / 0.9992 / 93% / 93% / 0.02 / 0.01
6-OH-BDE137 / 1-20 / 0.9997 / 80% / 80% / 0.05 / 0.02

Table S4

Concentrations of individual PBDE (ng g-1lw) in serum samples and the internal standard recovery. (ND: not detected)

No. / age / BDE-17 / BDE-28 / BDE-47 / BDE-100 / BDE-99 / BDE-85 / BDE-154 / BDE-153 / ΣPBDEs / Recovery of 13C12-BDE209
1 / 40 / ND / 0.08 / 0.62 / 0.20 / ND / 0.14 / 0.06 / ND / 1.10 / 89%
2 / 40 / ND / ND / 0.03 / ND / 0.13 / 0.29 / ND / ND / 0.72 / 90%
3 / 42 / ND / 0.25 / 2.30 / ND / 0.55 / 0.80 / 0.69 / <LOD / 4.59 / 89%
4 / 44 / ND / ND / 0.23 / ND / ND / 0.05 / ND / ND / 0.28 / 84%
5 / 44 / ND / ND / 0.16 / 0.09 / ND / 0.11 / 0.06 / ND / 0.42 / 91%
6 / 53 / ND / ND / 0.70 / ND / 0.22 / 0.14 / ND / ND / 1.06 / 89%
7 / 58 / ND / ND / 0.68 / 0.35 / ND / 0.32 / 0.24 / <LOD / 1.58 / 71%
8 / 58 / 0.19 / 0.36 / 5.38 / ND / ND / 3.37 / 2.97 / 0.07 / 12.33 / 68%
9 / 60 / ND / ND / 2.39 / 1.25 / ND / 1.38 / 1.07 / 0.04 / 6.13 / 86%
10 / 62 / 0.03 / 0.09 / 0.66 / ND / 0.38 / 0.53 / 0.42 / <LOD / 2.12 / 90%
11 / 63 / ND / 0.07 / 0.44 / ND / ND / 0.25 / 0.18 / ND / 0.95 / 75%
12 / 64 / ND / ND / 0.64 / ND / ND / 0.27 / 0.23 / ND / 1.13 / 89%
13 / 66 / ND / ND / 1.42 / 0.71 / ND / 0.78 / 0.51 / <LOD / 3.43 / 90%
14 / 66 / ND / ND / 1.47 / 1.02 / ND / 0.98 / 0.75 / 0.04 / 4.26 / 88%
15 / 66 / ND / 0.14 / 0.85 / ND / ND / 0.66 / 0.51 / ND / 2.16 / 90%
16 / 66 / 0.16 / ND / 0.51 / ND / ND / 0.21 / ND / ND / 0.89 / 89%
17 / 68 / 0.29 / ND / 0.75 / ND / ND / 0.17 / 0.10 / ND / 1.31 / 89%
18 / 68 / 0.19 / ND / 0.75 / ND / ND / 0.19 / ND / ND / 1.13 / 74%
19 / 68 / ND / 0.09 / 0.73 / ND / 0.31 / 0.40 / 0.29 / ND / 1.82 / 90%
20 / 70 / 0.19 / ND / 0.51 / ND / ND / 0.05 / ND / ND / 0.75 / 79%
21 / 70 / 0.18 / ND / 0.53 / ND / ND / 0.17 / ND / ND / 0.88 / 83%
22 / 71 / 0.52 / ND / 1.07 / ND / 0.11 / 0.26 / 0.33 / ND / 2.28 / 89%
23 / 72 / 0.21 / ND / 0.54 / ND / ND / 0.25 / ND / ND / 1.00 / 80%
24 / 73 / 0.20 / ND / 0.49 / ND / ND / 0.05 / ND / <LOD / 0.76 / 89%
25 / 75 / 0.27 / ND / 0.59 / ND / ND / 0.11 / ND / ND / 0.98 / 88%

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Table S5

Concentrations of individual OH-PBDE (pgg-1lw) in serum samples and the internal standard recovery.

No. / age / 3’-OH-BDE7 / 3-OH-BDE47 / 6-OH-BDE47 / 2’-OH-BDE68 / 6’-OH-BDE99 / 6-OH-BDE137 / Σ6OH-PBDEs / Recovery of
4’-OH-BDE17
1 / 40 / 55 / 62 / 90 / 23 / 10 / 56 / 296 / 104%
2 / 40 / 49 / 46 / 59 / 20 / 10 / ND / 184 / 95%
3 / 42 / 20 / 47 / 74 / 20 / <LOD / 25 / 186 / 73%
4 / 44 / 58 / 126 / 73 / ND / 10 / ND / 267 / 109%
5 / 44 / 20 / 34 / 58 / <LOD / <LOD / ND / 112 / 78%
6 / 53 / 42 / 61 / 97 / 27 / <LOD / 25 / 251 / 80%
7 / 58 / 25 / 20 / 53 / 20 / <LOD / ND / 118 / 85%
8 / 58 / ND / 22 / 86 / 20 / ND / <LOD / 139 / 82%
9 / 60 / 36 / 58 / 56 / 20 / 10 / ND / 180 / 74%
10 / 62 / 37 / 20 / 34 / <LOD / <LOD / ND / 91 / 78%
11 / 63 / 25 / 20 / 37 / <LOD / <LOD / <LOD / 81 / 86%
12 / 64 / 58 / 91 / 115 / 20 / 10 / 25 / 319 / 82%
13 / 66 / ND / 20 / 25 / <LOD / ND / ND / 45 / 87%
14 / 66 / 48 / 68 / 150 / 23 / 10 / 50 / 348 / 104%
15 / 66 / 74 / 101 / 130 / 20 / 39 / ND / 363 / 104%
16 / 66 / 21 / 20 / 60 / 20 / ND / 25 / 146 / 79%
17 / 68 / 37 / <LOD / 81 / <LOD / ND / <LOD / 143 / 72%
18 / 68 / 43 / 20 / 94 / ND / ND / 25 / 182 / 73%
19 / 68 / 47 / 41 / 122 / 20 / 10 / 25 / 265 / 119%
20 / 70 / 46 / <LOD / 51 / <LOD / ND / 13 / 111 / 90%
21 / 70 / 25 / 20 / 35 / <LOD / ND / <LOD / 81 / 85%
22 / 71 / 29 / 20 / 124 / <LOD / ND / <LOD / 173 / 73%
23 / 72 / 68 / 45 / 176 / 22 / <LOD / 25 / 336 / 106%
24 / 73 / 27 / <LOD / 28 / <LOD / ND / <LOD / 55 / 74%
25 / 75 / 37 / 34 / 109 / <LOD / ND / 25 / 205 / 73%

Table S6.

Comparison of PBDEs concentrations (ng g-1lw) in human blood among different regions.

Location / PBDEs congeners / Concentration (ng g-1lw) / References
In China
(A). Dalian / BDE-30, 28, 35, 37, 75, 47, 66, 100, 99, 116, 155, 154, 153, 183, 181, 190 and 209 / 31.6 / Chen et al. 2010
(B). Tianjin / BDE-17, 28, 47, 85, 99, 100, 139 , 140, 153, 154, 183, 196, 197, 203 , 204, 205, 207, 208 and 209 / 46.3 / Zhu et al. 2009
(C). Laizhou Bay / BDE-28, 47, 99, 100, 153, 154, 183 and 209 / 240 / He et al. 2013
(D). Shanghai / BDE-17, 28, 47, 100, 99, 85 and 154 / 2.1 / In this study
(E). Taizhou / BDE-15, 28, 47, 85, 99, 100, 153, 154 and 183 / 107.5 / Xu et al. 2015
(F). Wenzhou / BDE-3, 15, 28, 47, 99, 100, 153, 154 , 183, 203, 207 and 209 / 4.3 / Wang et al. 2012a
(G). Shantou / BDE-196, 197, 203, 206, 207, 208 and209 / 115.9 / Ren et al. 2011
(H). Hong Kong / BDE-47, 28 and 99 / 9.2 / Wang et al. 2012b
In the world
Shanghai, China / BDE-17, 28, 47, 100, 99, 85 and 154 / 2.1 / In this study
Ulsan, Korea / BDE-47, 99 and 153 / 7.2 / Kim et al. 2015
Queensland, Australia / BDE-47, 99, 100 and 153 / 16.0 / Toms et al. 2009
Wellington, New Zealand / BDE-47, 99, 100, 153, 154 and 183 / 7.2 / Harrad and Porter 2007
California, USA / BDE-28, 47, 99, 100, 153 and 154 / 62.0 / Zota et al. 2008
Austin, USA / BDE-28, 47, 66, 85, 99, 100, 153, 154 and 183 / 57.6 / Schecter et al. 2010
North Carolina, USA / BDE-47, 99, 100 and 153 / 42.9 / Stapleton et al. 2012
Catalonia, Spain / BDE-17, 28, 47, 66, 71, 85, 99, 100, 153, 154, 138, 183, 190 and 209 / 15 / Garí and Grimalt 2013
Oslo, Norway / BDE-28, 47, 100, 99, 154, 153 / 3.3 / Thomsen et al. 2002
Stockholm, Sweden / BDE-47, 99, 100, 153, 154 and 209 / 3.6 / Weiss et al. 2006
Attika, Greece / BDE-28, 47, 100, 99, 154, 153, 183 and 209 / 1.1 / Kalantzi et al. 2011

Fig. S1. Total PBDEs and OH-PBDEs concentrations in the human blood of different sex and work place.

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Reference

Chen CE, Chen J, Zhao H, Xie Q, Yin Z, Ge L(2010) Levels and patterns of polybrominated diphenyl ethers in children's plasma from Dalian, China. Environ Int 36:163-167

Garí M, Grimalt JO (2013) Inverse age-dependent accumulation of decabromodiphenyl ether and other PBDEs in serum from a general adult population. Environ Int 54:119-127

Harrad S, Porter L(2007) Concentrations of polybrominated diphenyl ethers in blood serum from New Zealand. Chemosphere 66:2019-2023

He S, Li M, Jin J, Wang Y, Bu Y, Xu M, Yang X, Liu A(2013) Concentrations and trends of halogenated flame retardants in the pooled serum of residents of Laizhou Bay, China. Environ.Toxicol Chem / SETAC 32:1242-1247

Kalantzi OI, Geens T, Covaci A, Siskos PA (2011) Distribution of polybrominated diphenyl ethers (PBDEs) and other persistent organic pollutants in human serum from Greece. Environ Int 37:349-353

Kim J, Kang JH, Park H, Baek SY, Kim YH, Chang YS(2012) Assessment of polybrominated diphenyl ethers (PBDEs) in serum from the Korean general population. Environ Pollut 164:46-52

Ren G, Yu Z, Ma S, Zheng K, Wang Y, Wu M, Sheng G, Fu J(2011) Determination of polybrominated diphenyl ethers and their methoxylated and hydroxylated metabolites in human serum from electronic waste dismantling workers. Anal Methods 3:408-413

Schecter A, Colacino J, Sjödin A, Needham L, Birnbaum L(2010) Partitioning of polybrominated diphenyl ethers (PBDEs) in serum and milk from the same mothers. Chemosphere 78:1279-1284

Stapleton HM, Eagle S, Sjödin A, Webster TF(2012) Serum PBDEs in a North Carolina toddler cohort: associations with handwipes, house dust, and socioeconomic variables. EnvironHealth Persp 120104:9

Thomsen C, Lundanes E, Becher G(2002) Brominated flame retardants in archived serum samples from Norway: a study on temporal trends and the role of age. Environ Sci Technol 36:1414-1418

Toms ML, Sjödin A, Harden F, Hobson P, Jones R, Edenfield E, Mueller JF(2009) Serum polybrominated diphenyl ether (PBDE) levels are higher in children (2-5 years of age) than in infants and adults. Environ Health Persp 117:1461

Wang C, Lin Z, Dong Q, Lin Z, Lin K, Wang J, Huang J, Huang X, He Y, Huang C(2012a) Polybrominated diphenyl ethers (PBDEs) in human serum from Southeast China. Ecotox Environ Safe 78:206-211

Wang HS, Chen ZJ, Ho KL, Ge LC, Du J, Lam MH, Giesy JP, Wong MH, Wong CK(2012b) Hydroxylated and methoxylated polybrominated diphenyl ethers in blood plasma of humans in Hong Kong. Environ Int 47:66-72

Weiss J, Wallin E, Axmon A, Jönsson BA, Åkesson H, Janák K, Hagmar L, Bergman Å(2006) Hydroxy-PCBs, PBDEs, and HBCDDs in serum from an elderly population of Swedish fishermen's wives and associations with bone density. Environ Sci Technol 40:6282-6289

Xu P, Lou X, Ding G, Shen H, Wu L, Chen Z, Han J, Wang X(2015) Effects of PCBs and PBDEs on thyroid hormone, lymphocyte proliferation, hematology and kidney injury markers in residents of an e-waste dismantling area in Zhejiang, China. Sci.Total Environ536:215-222

Zhu L, Ma B, Hites RA(2009) Brominated flame retardants in serum from the general population in northern China. Environ Sci Technol 43:6963-6968

Zota AR, Rudel RA, Morello-Frosch RA, Brody JG(2008) Elevated house dust and serum concentrations of PBDEs in California: unintended consequences of furniture flammability standards? Environ Sci Technol 42:8158-8164

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