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Page 4 of 17 Rawn et al. J Environ Expo Assess 2024;3:16 https://dx.doi.org/10.20517/jeea.2024.04

study, 2,001 agreed to participate. As part of the requirement, participants were given lifestyle and
demographic questionnaires during each of the first and third trimesters for their completion and
approximately 50% (n = 1,017) of the recruited participants provided samples of their milk for analysis of
multiple classes of chemicals (e.g., persistent organic pollutants, metals, bisphenol-A).

Health Canada analytical laboratories involved in the human milk analyses did not have the capacity to
analyze all of the samples collected as part of the current study. As a result, a plan to distribute the milk
samples among analytical laboratories was established for all analytes to ensure that samples from across all
sampling sites were analyzed for each class of analyte. Data from the Canadian Community Health Survey
(CCHS) and estimates of the number of women who would continue to breastfeed for a period extending
[47]
beyond two weeks of the birth of their babies were considered as part of the framework. This information
was coupled with the number of participants from each of the research centres to enable the development of
an estimate of samples available for distribution between laboratories. Statisticians then prepared a sample
distribution framework to randomly select samples from each location to be analyzed by each of the
laboratories. Factors considered while developing the sampling plan included the number of samples able to
be handled by each lab, to reach a target of 300 total samples for legacy persistent organic pollutants (e.g.,
PBDEs and HBCD), and a representative distribution of ages and number of previous pregnancies/births by
participant [Table 1]. The plan was developed in advance of all samples being received, and given that
sample collection took place from 2008 until 2011, the plan had to allow for flexibility and to ensure that the
pan-Canadian situation would be captured by each laboratory analyzing a subset of the samples. Among the
1,017 participants providing milk, 298 were identified and analyzed for PBDEs and HBCD in the Health
Canada laboratory [Figure 1].

Consistent with previous Canadian studies, milk was collected by the study participants, who were asked to
hand express their milk (fore- and hindmilk). If participants experienced difficulty with hand expression, a
Medela® (Medela International, Zug, Switzerland) manual breast pump was provided, along with
instructions for either manually expressing or using the breast pump to collect the milk. Milk samples were
collected between two and 10 weeks following delivery of the babies, recognizing that milk was to be
analyzed for a variety of different analytes/classes of compounds, 250 mL were requested of participants
(125 mL in glass containers, 125 mL in plastic containers). In general, 250 mL were received from
participants, although occasionally, the volumes received were lower. Aliquots of the samples for PBDE/
HBCD analysis were collected into 500 mL wide-mouth amber I-CHEM® glass jars with fluoropolymer
resin-liner polypropylene closure (Thermo Fisher Scientific, Rockwood, TN, USA). Participant instructions
for collection allowed for them to express the milk over a period of time, and keep it in their refrigerator
(4 °C) if collected over a few days, but if it took a longer period of time, the milk was to be stored at -20 °C
in their freezers. Once collected and frozen, the samples were shipped to the coordination centre in Ste.
Justine, Québec and then shipped to the laboratory where aliquots of the samples were prepared and then
transferred to the laboratories for chemical analysis.

Extraction and clean-up
Sample preparation followed the protocol for analysis of PBDEs and HBCD described previously for the
other persistent organics (e.g., polychlorinated biphenyls) . Briefly, samples (~25 g) were transferred into
[48]
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Erlenmeyer flasks and surrogate standards were added to the samples ( C PBDEs 15, 28, 47, 99, 153, 154,
183 and 209; C α-, β- and γ-HBCD) (Cambridge Isotope Laboratories, Andover, MA, USA). An aliquot of
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each sample (5%) was taken for gravimetric determination of lipid content in each sample. Prior to
extraction by homogenizing in 2:1 (v/v) acetone: hexane (Residue analysis grade, EMD; Ottawa, ON,
Canada), the samples were set aside on the lab bench for approximately 30 min following the addition of
surrogate standards. Lipids present were digested by washing extracts with sequential aliquots of