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Rawn et al. J Environ Expo Assess 2024;3:16 https://dx.doi.org/10.20517/jeea.2024.04 Page 7 of 17

Although similar certified reference materials with known HBCD concentrations were not available during
the work, the HBCD concentrations in the SRMs were determined. Only α-HBCD was frequently detected
in both the unfortified and fortified milk, with β- and γ-HBCD generally present below the limits of
detection. Concentrations of α-HBCD in the SRMs were within two standard deviations of the mean
concentration, similar to the results from testing the laboratory internal QC sample.

13
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Average surrogate recoveries ranged from 35.0% ( C PBDE 15) to 93.3% ( C PBDE 100) in the human milk
samples analyzed. HBCD average surrogate recoveries ranged from 68.8% to 83.5% (β-HBCD and γ-HBCD,
respectively), while the average α-HBCD recovery was 77.1%. All concentrations in the samples were
corrected for recovery.

In addition, representative breast pumps, similar to those sent to participants were examined for
background PBDE and HBCD levels. Testing was performed by rinsing the pumps with purified water and
the rinse water was prepared for analysis as samples alongside samples of purified water. Any detectable
concentrations observed in the pump rinses were considered to be a result of the water used for this testing.

Limits of detection
Instrumental limits of detection (LOD) were determined for each PBDE based on a 3:1 signal to background
noise ratio for each sample individually and method detection limits (MDLs) were then calculated to
account for variation in instrument sensitivity and sample sizes. Average PBDE MDLs ranged from
-1
-1
-1
0.023 pg·g sample; 0.769 pg·g lipid (PBDE 15) to 0.712 pg·g sample; 23.5 pg·g lipid (PBDE 209),
-1
-1
respectively. PBDE 183 had the second highest MDL observed in the samples (0.187 pg·g sample, 6.32
-1
pg·g lipid). Limits of detection for the individual HBCD isomers were determined using low-level
standards and adjusted for sample weight and instrument sensitivity, resulting in MDLs of 0.617 pg·g
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-1
sample, 21.6 pg·g lipid (α-HBCD); 0.385 pg·g sample, 13.4 pg·g lipid (β-HBCD); and 0.693 pg·g sample,
-1
-1
-1
24.0 pg·g lipid (γ-HBCD) in the human milk samples.
-1
Statistical analysis
Statistical analyses were performed using SigmaPlot 12.5 (Systat Software Inc.). For those compounds below
the MDL in any sample, the analyte concentrations were adjusted to 1/2 MDL (i.e., MDL/2) established for
the samples prior to initiating data summary and statistical analysis. In addition to developing descriptive
statistics of these data, they were also studied to consider whether concentrations were related to some of
the personal characteristics of the participants (e.g., age, number of children the participant had prior to this
pregnancy/parity). The concentration data were not normally distributed; therefore, one-way analysis of
variance (ANOVA) tests were performed using Kruskal-Wallis ANOVA on ranks. Relationships were
considered statistically significant if the P-value was less than 0.05.

RESULTS AND DISCUSSION
The lipid content determined in the human milk samples ranged from 0.75% to 7.84%, with mean and
median lipid concentrations of 3.26% and 3.22%, respectively. PBDEs and HBCD are lipophilic compounds;
therefore, results are reported on a lipid-adjusted basis throughout this manuscript. While PBDEs were
detected in all 298 of the milk samples collected from across Canada, HBCD was detected in 94.0% (n = 280)
of the samples. PBDE 47 was the dominant contributor to ΣPBDE concentrations, followed by 153 > 99 >
[30]
100 > 28 > 209. Consistent with previous HBCD concentrations determined in Canadian serum , α-HBCD
was the predominant isomer (93.3%) in human milk samples, with β-HBCD and γ-HBCD detected in 9.7%
and 28.5% of the samples, respectively.