Toms et al. the mean age of the pool by taking the mean of all ages of donors included in the pool. This study aimed to investigate PBDE concentration in infants and children, so we used small age ranges for younger age groups and larger age ranges for older age groups where changes in PBDE concentration were previously shown to be less apparent (Toms et al. 2008). The aim of the sample collection was to combine up to 30 samples per pool with rep- licate pools per strata where sufficient samples existed [see Supplemental Material, Table 1 (doi:10.1289/ehp.0900596.S1 via http:// dx.doi.org)]. Where possible, we used 1 mL of serum from each sample. Because of the small volume of blood obtained from infants and young children, we used < 1 mL in some pools, although we combined the same sample vol- ume from each donor to form a specific pool. We collected a total of 2,420 individual samples and combined them into 84 pools. We used disposable polyurethane pipettes to draw the sample from polypropylene tubes in which the serum was stored. The sample was placed into a 50-mL glass jar and 10 mL was aliquoted into an amber jar for transport to the laboratory. We used pooled samples because it is the most effective way to obtain large numbers of blood samples from the specific age groups. Difficulties in obtaining parental consent and cooperation of children for blood draws pre- cluded the use of individual samples, as did the volume required for PBDE analysis, which we would have been unlikely to obtain in one blood draw from an infant or young child. Ethics approval was granted on 28 June 2006 by the University of Queensland Medical Research Ethics Committee. The analysis of pooled samples by investigators at the U.S. Centers for Disease Control and Prevention (CDC) was determined not to constitute engagement in human subjects research. Analysis. The samples were analyzed at the CDC (Atlanta, GA, USA) with methodology that has been described previously (Sjödin et al. 2004b). Briefly, a set of samples was defined as 24 unknown samples with three analytical blanks and three quality assurance/ quality control samples and processed using a semiautomated sample preparation method. Human sera (2 g) were weighed into test tubes and fortified with internal standards (13C-labeled) using a 215 Liquid Handler (Gilson Inc., Middleton, WI, USA). Then for- mic acid and water were added to denature proteins and dilute the samples on the liquid handler. The target analytes were extracted into dichloromethane using the solid-phase extraction (SPE) workstation (Rapid Trace; Zymark, Hopkinton, MA, USA). Cleanup was performed on a two-layered column. The top layer comprised activated silica and the bottom layer comprised silica gel/sulfuric acid (2:1 by weight). The top layer retained polar 1462 lipids such as cholesterol, whereas the bottom layer degraded the remaining lipids to produce an extract free of biogenic material. This pro- cedure was automated using the modular SPE workstation. Samples were evaporated to 1 mL and transferred to the gas chromatograph vials, which were previously spiked with recovery standards. Samples were further evaporated to 10 µL and analyzed by gas chromatography/ high-resolution mass spectrometry with a MAT95XP instrument (ThermoFinnigan, Bremen, Germany). Chromatographic separa- tions were carried out on a 6890 gas chromato- graph (Agilent Technologies, Atlanta, GA, USA) fitted with a DB5HT capillary column (15 m, 0.25 mm inner diameter, and 0.10 µm thickness). The following congeners were tar- geted for analysis: BDE-17, -28, -47, -66, -85, -99, -100, -153, -154, and -183. BDE- 209 was not assessed in these samples because the analytical methodology for this congener was not yet fully established. The results are expressed as nanograms per gram of lipid and are reported to two significant figures, using a value of zero for congeners with concentrations below the limit of detection (LOD), which depended on sample size and blanks. Σ4PBDE concentrations are the sum of BDE-47, -99, -100, and -153. Quality control/quality assurance. To assess the reproducibility of the sampling procedures, we analyzed replicate samples collected in the same strata and compared them. In addition, we included two blind field blanks, which com- prised of bovine serum (Sigma Aldrich B8655; Sigma Aldrich, St. Louis, MO, USA) expected to have a PBDE concentration below the LOD with the analytical methodology used. In these blanks, only one congener was present at a con- centration slightly above the instrumental LOD, indicating low or negligible contamination during the sample collection and pooling. The congener that was detected in one sample was BDE-99, found at a concentration of 2.9 pg/g serum with an LOD of 2.6 pg/g serum. Statistical analysis. Statistical analysis was mostly descriptive, with average concentra- tions (means or medians) and SDs or ranges reported overall and within age, sex, and collec- tion date groups. We fitted analysis of variance (ANOVA) models (including age group, sex, collection date, and their interaction effects) to test the hypothesis that concentrations dif- fered by overall age group, overall sex, or over- all collection date or to test whether age group differences were sex specific. The age groups used in this analysis were as described above, (“Samples”). We used the conventional p-value of 5% to report results as statistically significant. We did not include BDE-17, -28, -66, -85, -154, or -183, which were detected in < 60% of samples, in the statistical analysis. We performed statistical analysis using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Results and Discussion We detected PBDEs in all 84 pools of human blood serum; the Σ4PBDE concentration ranged from 5.5 to 103 ng/g lipid. Overall, concentrations in children were higher than in adults and varied by age. For adults (both sexes) > 16 years of age, the mean ± SD of Σ4PBDEs was 16 ± 18 ng/g lipid. We detected BDE-47, -99, -100, and -153 in all samples, whereas we detected BDE-183, -154, and -85 in 43%, 25%, and 21% of samples, respec- tively. The concentrations of BDE-47, -99, -100, and -153 across both sexes and all age groups were 2.6–55.1, 0.9–24.2, 0.6–14.1, and 1.3–13.7 ng/g lipid, respectively [for full details, see Supplemental Material, Tables 1 and 2 (doi:10.1289/ehp.0900596.S1)]. We determined the coefficient of varia- tion (CV) using the results within each stra- tum to compare the reproducibility of sampling procedures between pools. For 70% of strata, the CV between replicates was < 35%. The mean (range) CV was 34% (0.3–103%), 30% (0–100%), 29% (1–106%), and 26% (3–83%) for BDE-47, -99, -100, and -153, respectively. Although variability between replicate pools was evident in some strata, most showed good agreement, indicating that sample collection and pooling procedures were uniform, and it is unlikely that contamination of the samples occurred during sampling. High variability between individuals has been reported previ- ously (Birnbaum and Cohen Hubal 2006; Ryan et al. 2002; Schecter et al. 2003), and variability between replicates is likely attributable to the inclusion in these pools of a few serum samples with an elevated concentration. The distribution of individual results in this study is unknown because only pools have been measured; how- ever, in a U.S. cohort of 2,060 individual serum samples, the reported median concentration of BDE-47 was 19.2 ng/g lipid, with a maximum concentration of 2,350 ng/g lipid (Sjödin et al. 2008b). These levels far exceed the median and would thus have caused the elevated level in a few of the serum pools as observed. Individual variability and a lack of exposure data are limi- tations of using pooled samples. Age. For children, mean Σ4PBDE concen- trations were highest in the 2- to 5-year age groups, with the maximum Σ4PBDE concentra- tion (103 ng/g lipid) detected in a pool of serum from females 2.6–3 years of age (Figure 1). We summarized samples into broader age brack- ets and ran an ANOVA with post hoc Tukey test. The mean Σ4PBDE concentrations from cord blood samples and samples from ages 0–2, 6–12, and 13–30 years were similar to each other but were significantly different from con- centrations detected in children in the 2- to 5-year age group (Table 1). We observed the lowest concentrations in the adults > 31 years of age, which were also significantly differ- ent from those in the 2- to 5-year age group. ovlume 117 | number 9 | September 2009 • Environmental Health Perspectives