SUPPORTING INFORMATION FOR: CONCENTRATIONS OF POLYBROMINATED DIPHENYL ETHERS, HEXABROMOCYCLODODECANES AND TETRABROMOBISPHENOL-A IN BREAST MILK FROM

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1 SUPPORTING INFORMATION FOR: CONCENTRATIONS OF POLYBROMINATED DIPHENYL ETHERS, HEXABROMOCYCLODODECANES AND TETRABROMOBISPHENOL-A IN BREAST MILK FROM UNITED KINGDOM WOMEN DO NOT DECREASE OVER TWELVE MONTHS OF LACTATION Stuart Harrad and Mohamed Abou-Elwafa Abdallah 22 pages, 11 Tables, 2 Figures, and Method Descriptions SI for Harrad and Abdallah (2015) Page SI1 of 22

2 α-hbcd Table SI-1a: Concentrations (ng/g lipid weight) of HBCDs and PBDEs in Individual Human Milk samples Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother Mother Mother Mother Mother Mother Mother Mother γ HBCD Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother Mother Mother Mother Mother Mother Mother Mother SI for Harrad and Abdallah (2015) Page SI2 of 22

3 β-hbcd Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother Mother Mother Mother Mother Mother Mother Mother ΣHBCDs Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother Mother Mother Mother Mother Mother Mother Mother SI for Harrad and Abdallah (2015) Page SI3 of 22

4 BDE-47 Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother Mother Mother Mother Mother Mother Mother Mother BDE-99 Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother < Mother Mother Mother Mother Mother Mother Mother BDE-153 Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother Mother SI for Harrad and Abdallah (2015) Page SI4 of 22

5 Mother Mother Mother Mother Mother < < Mother SI for Harrad and Abdallah (2015) Page SI5 of 22

6 Table SI-1b: Concentrations (pg/g lipid weight) of TBCDs, BDE-209 and TBBP-A in Individual Human Milk samples ΣTBCDs Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother 2 24 < <13 < Mother Mother Mother <13 <13 14 <13 18 <13 15 < Mother Mother Mother Mother < Mother BDE-209 Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother Mother Mother Mother Mother Mother Mother Mother SI for Harrad and Abdallah (2015) Page SI6 of 22

7 TBBP-A Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother 1 <50 <50 <50 <50 54 <50 < Mother 2 < <50 <50 <50 32 <50 54 <50 55 <50 Mother 3 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 Mother <50 59 < Mother <50 56 < <50 < <50 Mother Mother 7 37 <50 <50 <50 61 <50 52 <50 <50 53 <50 57 Mother 8 <50 <50 <50 <50 53 <50 <50 <50 <50 49 <50 <50 Mother Mother <50 < < <50 <50 49 <50 SI for Harrad and Abdallah (2015) Page SI7 of 22

8 Detailed Descriptions of Analytical Methods and QA/QC Data Determination of lipid weight in milk: Lipid weight was determined gravimetrically according to the European Standard EN :1996, Part 2. Briefly, ~ 10 ml of freeze-dried human milk was weighted and transferred into 100 ml separating funnel. One millilitre saturated potassium oxalate was then added together with 10 ml of ethanol and 20 ml of hexane/diethyl ether (1:1, v/v). The mixture was shaken for about 15 min and the bottom layer was removed into another separating funnel and another 10 ml of hexane/diethyl ether (1:1, v/v) together with 5ml of ethanol was added. The lower layer was removed out and upper layer was put together with upper layer from the first funnel. Ten millilitre of water was added to this organic phase and the lower layer was again removed. The organic phase was filtered through anhydrous sodium sulphate and the lipid content gravimetrically determined. Multi-residue LC-ESI-MS/MS analysis for analysis of HBCDs and TBBP-A Separation of TBCDs, PBCDs, HBCDs and TBBP-A was achieved using a dual pump Shimadzu LC-20AB Prominence liquid chromatograph (Kyoto, Japan) equipped with SIL-20A autosampler, a DGU-20A3 vacuum degasser and a Varian Pursuit XRS3 C 18 analytical column (150 mm 2 mm I.D., 3 µm particle size). A mobile phase of (a) 1:1 methanol/water with 2 mm ammonium acetate and (b) methanol at a flow rate of 120 µl min -1 was applied for elution of the target compounds; starting at 50 % (b) then increased linearly to 100 % (b) over 3 min, held for 5 min, followed by a linear decrease to 65 % (b) over 2.5 min and held for 5.5 min. TBBP-A was eluted as a single peak at 9 min. The three main HBCD diastereomers were baseline separated on the C 18 column with retention times of 12.3, 12.9 and 13.3 min for α-, β- and γ- HBCD, respectively. Three well-resolved peaks were obtained SI for Harrad and Abdallah (2015) Page SI8 of 22

9 for PBCD isomers at retention times 11.0, 11.6 and 11.9 min (figure 2a) while 2 TBCD isomers eluted at retention times 9.2 and 9.7 min (figure 1b). Mass spectrometric analysis was performed using a Sciex API 2000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) operated in electrospray negative ionization (ESI) mode. MS/MS detection operated in the multiple reaction monitoring (MRM) mode was used for quantitative determination of HBCD isomers based on m/z , m/z and m/z for the native, 13 C-labelled and d 18 -labelled diastereomers, respectively. TBCDs and PBCDs were monitored via the transitions m/z and m/z respectively, while m/z , m/z were used to monitor native and 13 C-labelled TBBP-A. Specific instrumental calibration parameters are given in table SI-2. SI for Harrad and Abdallah (2015) Page SI9 of 22

10 Table SI-2: Optimized MS/MS parameters* for the analysis of HBCDs and TBBP-A. Parameter Curtain gas Turbo gas temperature Ion spray voltage Declustering potential Focusing potential Collision gas Collision energy Cell entrance potential Collision cell exit potential Value (units) 35 (a.u.) 500 ( C) (V) -5(V) -365(V) 5 (a.u.) 40 (ev) -6(V) -10(V) a.u. arbitrary units * These parameters were obtained using direct infusion experiments of the target compounds (TBBP-A, α-, β- and γ- HBCDs, native and 13 C- labelled isomers, 2 ng µl -1 each in MeOH) into the MS/MS system via a built-in Harvard syringe pump at a flow rate of 10 µl min -1. SI for Harrad and Abdallah (2015) Page SI10 of 22

11 Multi-residue LC-APPI-MS/MS analysis for analysis of PBDEs Target PBDEs (28, 47, 85, 99, 100, 153, 154, 183, 196, 197, 203, 206, 207, 208 and 209) were separated using a dual pump Shimadzu LC-20AB Prominence liquid chromatograph (Shimadzu, Kyoto, Japan) equipped with a SIL-20A auto sampler, a DGU-20A3 vacuum degasser and a Varian Pursuit XRS3 (Varian, Inc., Palo Alto, CA, USA) C 18 reversed phase analytical column (250 mm 4.6 mm i.d., 3 µm particle size). A mobile phase program based upon (mobile phase A) 85:10:5 methanol/toluene/water and (mobile phase B) 1:1 methanol/water at a flow rate of 500 µl min -1 was applied for elution of the target compounds; starting at 85% (mobile phase A) then increased linearly to 100% (mobile phase A) over 30 min then held for 25 min. The column was equilibrated with 85% (mobile phase A) for 5 min between runs. Mass spectrometric analysis was performed using a Sciex API 2000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with an APPI ion source operated in negative ion mode. Toluene was used as a doping agent introduced via a separate dopant port of the APPI source using a dedicated isocratic HPLC pump (Jasco PU-2800, Easton, MD, USA) at 12% of the flow rate of the mobile phase. Direct infusion of the target compounds into the MS/MS system was performed using a built-in Harvard syringe pump at a flow rate of 10 µl min -1. The infusion experiments served for tuning and adjusting the source and the compound-specific parameters (tables SI- 3, SI-4) during method development. SI for Harrad and Abdallah (2015) Page SI11 of 22

12 Table SI-3: Optimized MS/MS parameters for the analysis of PBDEs. Parameter Curtain gas Ion source temperature Ion transfer voltage Declustering potential Focusing potential Collision gas Collision energy Cell entrance potential Collision cell exit potential Value (units) 25 (a.u.) 400 ( C) (V) 8-14 (V) -365(V) 5 (a.u.) (ev) -6(V) -10(V) SI for Harrad and Abdallah (2015) Page SI12 of 22

13 Table SI-4: Optimized compound-specific parameters, linearity, LODs and LOQs of target compounds. BDE # Precursor Fragment DP * CE** LOD (pg) LOQ (pg) (m/z) (m/z) (V) (V) *Declustering potential ** Collision energy SI for Harrad and Abdallah (2015) Page SI13 of 22

14 Quality assurance/quality control Good recoveries (73-105%) of the 13 C-labelled internal standards were obtained for all the studied compounds (table SI-5). Further inspection of the method extraction/clean up performance was achieved via spiking milk samples (n=5) with d 18 -α-hbcd and 13 C-BDE-154 after freeze drying and excellent recoveries (>88%) were obtained (table SI-6). No target compounds were detected in method blanks (n=10; consisting of 2 g pre-extracted anhydrous sodium sulfate treated exactly as a sample) or field blanks (n=10; consisting of ~2 g of broken pieces of the milk containers treated exactly as a sample). Therefore, there was no need for blank correction of the results and method limits of quantification (LOQ) were estimated based on 10:1 S:N ratios and were 50, 36, and 13 pg g -1 lw for TBBP-A, HBCDs, and TBCDs, respectively. For PBDEs the LOQ values were 43, 45, 51, 55, 53, 58, 59 and 72 pg g -1 lw for BDEs # 47, 49, 85, 99, 100, 153, 154 and 209, respectively. In the absence of an appropriate standard reference material for TBBP-A and HBCDs, the accuracy and precision of the analytical method for HBCDs was assessed via replicate analysis (n=10) of NIST SRM The results obtained compared favourably with the indicative values reported elsewhere (Keller et al., 2007)(table SI-7). For TBBP-A, a standard addition or matrix spike method at 3 concentration levels (n=5 at each level) was used to assess the accuracy and precision of the method and good results were obtained (table SI-8). Finally, method accuracy for PBDEs was assessed by replicate (n=10) analysis of NIST SRM Results were satisfactory and are shown in Figure SI-2. Reference Keller, J. M.; Stapleton, H. M.; Heltsley, R.; Peck, A.; Kucklick, J. R.; Schantz, M.; Wise, S. A. SRMs Available from NIST for the Analysis of Brominated Flame Retardants; Poster presented at BFR07, Amsterdam, April, SI for Harrad and Abdallah (2015) Page SI14 of 22

15 Table SI-5: Average recoveries (expressed as percent) of the 13 C-labelled internal (surrogate) standards (added before freeze-drying). Internal standard Average recovery (%) ± SD 13 C-α-HBCD 86 ± C-β-HBCD 78 ± 7 13 C-γ-HBCD 90 ± C-TBBP-A 80 ± C-BDE ± C-BDE ± C-BDE ± C-BDE ± C-BDE ± 17 Table SI-6: Recoveries (expressed as percent) of d 18 -α-hbcd and 13 C-BDE-154 used as extraction/clean-up evaluation standard (added after-freeze drying). Sample # d 18 -α-hbcd 13 C-BDE Table SI-7: Concentrations of HBCD isomers in SRM2585 compared to the indicative values. Average concentration (ng g -1 ) ± standard deviation (n=10) Measured Indicative α-hbcd 22 ± ± 3.7 β-hbcd 4.9 ± ± 1.1 γ-hbcd 141 ± ± 22 SI for Harrad and Abdallah (2015) Page SI15 of 22

16 Table SI-8: Summary of standard addition matrix spike method results for TBBP-A. Mass added (ng) Mass recovered (ng) Recovery (%) RSD (%)(n=5) Table SI-9: Summary of standard addition matrix spike method results for HBCDs. Mass added (ng) Isomer Mass recovered (ng) Recovery (%) RSD (%)(n=5) α-hbcd β-hbcd γ-hbcd α-hbcd β-hbcd γ-hbcd α-hbcd β-hbcd γ-hbcd SI for Harrad and Abdallah (2015) Page SI16 of 22

17 Table SI-10: Summary of standard addition matrix spike method results for PBDEs. Mass added (ng) Congener Mass recovered (ng) Recovery (%) RSD (%)(n=5) BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE SI for Harrad and Abdallah (2015) Page SI17 of 22

18 Table SI-11: Age of the milk donors and lipid content of the analysed milk samples. Age Lipid content (%) Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12 Mother Mother Mother Mother Mother Mother Mother Mother Mother Mother SI for Harrad and Abdallah (2015) Page SI18 of 22

19 Figure SI-2: Comparison of certified and indicative PBDE concentrations (ng g - 1 ) in SRM 2585 with the average values (n=10) obtained in this study. Error bars represent one standard deviation. Concentration (ng/g) This Study Certified/indicative(i) values (i) 197(i) BDE congener SI for Harrad and Abdallah (2015) Page SI19 of 22

20 Figure SI-2 Concentrations of α, β, γ-hbcd, γ BDE-99, BDE-209, and TBBP-A normalized to the first sample α-hbcd % of First Sample R = ; p> Time Since First Sample (Months) β-hbcd % of First Sample R = ; p> Time Since First Sample (Months) SI for Harrad and Abdallah (2015) Page SI20 of 22

21 γ HBCD % of First Sample R = ; p> Time Since First Sample (Months) BDE R = ; p>0.1 % of First sample Time Since First Sample (Months) SI for Harrad and Abdallah (2015) Page SI21 of 22

22 BDE-209 % of First Sample R = ; p> Time Since First Sample (Months) TBBP-A % of First Sample R = ; p> Time Since First Sample (Months) SI for Harrad and Abdallah (2015) Page SI22 of 22