Serum Concentrations of Brominated Flame Retardants in the United States General Population

1. Serum Concentrations of Brominated Flame Retardants in the United States General Population Andreas Sjödin, Lee-Yang Wong, Richard S. Jones, Emily Edenfield, Yalin Zhang, Carolyn Hodge, Emily Dipietro, Cheryl McClure, Wayman Turner Centers for Disease Control and Prevention 4770 Buford Highway, F-53, Atlanta, GA National Center for Environmental Health Division of Laboratory Sciences, Organic Analytical Toxicology Branch

2. The NHANES is administered by the Centers for Disease Control and Prevention (CDC) • NHANES = National Health and Nutrition Examination Survey • NCHS (National Center for Health Statistics) – Specimen Collection and Public Release of Analytical and Questionnaire Data • NCEH (National Center for Environmental Health) – Analytical Measurements • The NHANES performs a statistical representative sampling of the U.S. population with respect to age, sex, race/ethnicity and income above or below poverty level • The survey is conducted on a two year cycle; the most recently publicly available data are from the survey years 2003/2004

3. NHANES 2003/04 – Brominated Flame Retardants • A random 1/3rd subset of eligible participants (n=2,305) between the ages of 12 and 85 years was chosen. • Of these 2,062 serum samples were available for analysis. • The race/ethnic distribution was: • 71.5% non-Hispanic White (NHW) • 12.0% non-Hispanic Black (NHB) • 8.2% Mexican Americans (MA) • The Age Distribution was: • 12.2% were younger than age 20 • 17.0% were over the age of 60

4. PentaBDE BDE-99 BDE-47 BDE-183 OctaBDE BDE-153 octaBDE nonaBDE BDE-209 DecaBDE nonaBDE Three Commercial PBDE products PentaBDE(ton/year) North America 8.290 Europe 210 Asia -- Total 8.500 OctaBDE (ton/year) North America 1.375 Europe 450 Asia 2.000 Total 3.825 DecaBDE (ton/year) North America 24.300 Europe 7.500 Asia 23.000 Total 54.800

5. Human Exposure to BFRs Nursing Diet • Indoors • Dust • Dermal • Work Envir. • Electronics • Manufacturing

6. Analytical Methods • All sample preparation was performed in a clean room to minimize sample contamination originating from dust and/or particulate matter. • The supply air to this room is passed through ULPA (Ultra-Low Penetration Air) filters, which have a 99.999% efficiency for removal of particles down to 0.12 um diameter. • Inside the clean room biological safety cabinets are used as a second barrier against particle contamination during all phases of the sample handling.

7. How much dust can cause the blank contamination observed during routine sample preparation? Average Serum Blank (pg) 5 Average Dust Concentration (ng/g) 1052 Average Amount Dust / Blank (g) 4

8. Analytical Methods

9. Workflow of procedure Sample pretreatment Automated LLE Silica / Silica : H2SO4 cleanup HR-MS Analysis

10. Instrumental analysis • GC/ID-HRMS analysis • Thermo DFS (Double Focusing Sector) • Conditions for PBDE analysis • Splitless Injection • DB-5HT column (15m, 0.25mm ID, 0.1um phase thickness • Quantification Limit • 0.5pg/ul all BFRs • 5pg/ul for BDE-209

11. Blanks over time for PBDE47(One Year of data)

12. Blanks over time for PBDE209(One Year of data)

13. Quality Assurance/ Quality Control (QA/QC) • Confirmed for every Measurement • 13C-Label Recovery between 25-150% • Isotopic Distribution Ratio +/-20% from theoretical value • Relative retention time (RRT) deviation ≤ 0.004 • Confirmed for every Sample • HRMS resolution verification R>10,000 • Confirmed for every Batch • Spiking accuracy between 98-102% • Calibration curve has a r2>0.995 for each analyte • DLS QA/QC Program • Confirmed for every Study • BDE-99 / BDE-47 ratio does not indicate contamination during sample collection

14. Quality Assurance / Quality Control (QA/QC) • The relative standard deviation or CV of the method is in general less than 10% • Concentrations expressed as pg/g of serum

15. Analytical Methods Limit of Detection (LOD) is given as ng/g lipid Maximum LOD correspond to a sample size of 1.4 g of serum.

16. Statistical Methods • We used sampling weights to account for unequal probability of selection and non-response. • Geometric means are calculated for analytes detected in 60% of the participants and stratified by, age, sex, race/ethnicity, construction year of primary residence and natives vs. foreign born participants. • Weighted Person correlation coefficients were calculated • The LOD was substituted with the LOD/2^0.5

17. Statistical Methods • Regression models • In multiple regression analyses the variables included were age (continuous), sex, race (MA, NHB, NHW) and an age-squared term. We assessed all possible two-way interaction terms in the model. • The Least Square Geometric Mean (LSGM) concentration was calculated from the regression model. • To reach the final model, we used backward elimination with a threshold of p<0.05 for retaining the variable in the model. We evaluated for potential confounding by adding each of the excluded variables back into the model one by one and examined changes in the β-coefficient of the significant main effects, if a change of >10% was observed the variable was added back into the model. • Odds ratios for having a concentration of BDE-47 and BDE-153 over the 95th percentile were calculated for age (12-19, 20-59 and 60 years and older), sex and race/ethnicity (MA, NHW, NHB)

18. Selected percentiles BDE-47 concentration adjusted for sampling weights. The highest concentration observed in the NHANES 2003/04 was 2,350 ng/g lipid.

19. Geometric mean concentration of PBDEs and BB-153 in the NHANES 2003/04 survey. Adjusted by regression for differences in age, sex, and race ethnicity. 30 25 20 Geometric mean (ng/g lipid) 15 10 5 0 BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BB-153

22. Geometric mean concentration of BDE-47 and BDE-153 stratified by construction year of primary residence

23. Geometric mean concentration of BB-153 stratified by participants country of birth

24. Geometric mean concentration of BDE-47 and BDE-153 stratified by participants country of birth

25. Geometric mean concentration (ng/g lipid) and 95% CI stratified by age, sex and race/ethnicity

26. LSGM (ng/g lipid) by age group for BDE-47 A linear decrease (p=0.01) and a positive quadratic trend (p=0.01) with age

27. LSGM (ng/g lipid) by ethnic group for BDE-47Highest concentration in MA participants and significantly higher than in NHW (p=0.01). No significant difference between MA and NHB

28. LSGM (ng/g lipid) by age group for BDE-153Linearly decrease and a positive quadratic trend with age (p<0.05)

29. LSGM (ng/g lipid) by sex for BDE-153Males had significantly higher concentration than females (p<0.01).No significant difference between race/ethnic groups

30. Characterization of subjects having a serum concentration above the 95th percentile. Variables evaluated are age, sex and race/ethnicity. BDE-47:Senior adults were two times more likely to be above the 95th percentile than the 20-59 year olds (p=0.02). OR 2.0 (95% CI 1.1-3.6) BDE-153:Males were twice as likely than females to be above the 95th percentile (p=0.04). OR 2.1 (95% CI 1.0-4.3) MA were 62% less likely than NHW to be above the 95th percentile (p=0.03) OR 0.38 (95% CI 0.15-0.91)

31. Concluding Remarks on NHANES • The highest sum PBDE concentration observed in NHANES 2003/04 was 3,680 ng/g lipid. • Further studies will be needed to assess if current levels of PBDEs in the general population in the US are increasing or have reached a plateau or have started to decline. • Special attention needs to be given to children's exposures and workers in certain occupational settings that may lead to an increased risk of exposure.

32. Children’s levels of PBDEs from birth through age 13 Andreas Sjödin1, Arnold Schecter2, Lee-Yang Wong1, Richard S. Jones1, Justin Colacino3, Noor Malik2, Emily Edenfield1, Yalin Zhang1, Carolyn Hodge1, Emily Dipietro1, Cheryl McClure1, Wayman Turner1 1 Centers for Disease Control and Prevention, Atlanta, GA 2 University of Texas School of Public Health, Dallas, TX 3 University of Michigan School of Public Health, Ann Arbor, MI

33. Hypothesis • NHANES for PBDEs and many other environmental contaminants measured in serum, is limited to children above the age of 12 • Due to limitations in available serum • Exposure to PBDEs through the indoor environment is likely a major contributing factor to the overall exposure • Young children are more exposed to indoor dust than adults due to hand to mouth exposure. It can thus be hypothesized that children experience a larger exposure to PBDEs than adults. • Since young children are not included in the NHANES survey we decided to conduct a smaller complementary study to fill this data gap.

34. Study Design • Serum samples from Children’s Medical Center in Dallas, Texas were collected from August to November, 2009. • Samples were collected for pathological testing • It is not known to us if the samples were collected for routine study or if children were receiving treatment. This limitation of the study design could bias our data. • Only the sex and date of birth and sample collection date from which the child's age were calculated were available to us. • 300 serum samples were collected with approximately 25 samples per year of age.

35. Results • Detection frequency for the main PBDE congeners were good • BDE-47 (2,2’,4,4’-tetraBDE) 96% • BDE-99 (2,2’,4,4’,5-pentaBDE 95% • BDE-153 (2,2’,4,4’,5,5’-hexaBDE 90% • Using a median sample size of 1.0 g of serum • (Range: 0.12-2.0g; 5th percentile 0.39g)

36. BDE-47 (2,2’,4,4’-tetrabromodiphenyl ether)

37. BDE-153 (2,2’,4,4’,5,5’-pentabromodiphenyl ether)

38. BDE-47 (2,2’,4,4’-tetrabromodiphenyl ether)

39. BDE-153 (2,2’,4,4’,5,5’-pentabromodiphenyl ether)

40. Concluding Remarks • The serum concentration of PBDEs in children at age 13 in 2009 is similar to that observed in the NHANES 2003/04 survey • The level of PBDEs peaked between 4-8 years of age • 4-6 years of age for BDE-47 • 4-8 years of age for BDE-153 • Higher levels of PBDEs in children than adults could be explained by indoor dust ingestion. However, future research is needed to prove this hypothesis • Special attention needs to be given to children's exposures and studies involving a larger number of samples collected to better characterize the upper bound of PBDE body burden.

41. Thank you for your attention National Center for Environmental Health Division of Laboratory Sciences, Organic Analytical Toxicology Branch