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Hammel et al. J Environ Expo Assess 2024;3:8 https://dx.doi.org/10.20517/jeea.2023.51 Page 3 of 17

compounds, have been associated with metabolic and endocrine disruption and activation of nuclear
receptors regulating adipogenic pathways in in vitro studies [34-36] . Given the long-term effects of PBDE
exposure and the growing prominence of NBFRs in consumer product use and environmental presence,
there is a need to elucidate dominant pathways of exposure for these brominated compounds. This is of
particular concern since children tend to have higher levels of exposure to chemicals including PBDEs,
[37]
compared to adults .
Here, we report concentrations of PBDEs, HBCDDs, as well as NBFRs and dechlorane plus in breast milk
collected from women in the Copenhagen area in 2007-2008. This was a critical point in BFR history with
the phase-out and global ban of PBDEs in 2009 changing the use pattern of BFRs. Further, the breast milk
concentrations were compared to house dust concentrations of HBCDDs and NBFRs collected at the same
time as well as and to previously published paired PBDE concentrations in placenta tissue as well as
maternal and cord blood. Exposure estimates for breast milk and dust were investigated and compared for
the infants. These data present a retrospective assessment of breast milk concentrations in Denmark
immediately following the international PBDE ban and prior to the HBCDD-ban, thus providing a
benchmark for more recent and future measurements of BFR concentrations in breast milk.

EXPERIMENTAL
Study population and sample collection
Study participants were recruited among healthy, pregnant women scheduled for caesarean section at
Copenhagen University Hospital from March to December 2007; further details are given elsewhere [15,38] .
Caesarean section might have subtle influences on nutrient composition in breast milk , but potential
[39]
influences on contaminant levels are unknown. In combination with a relatively high rate (approximately
20%) of caesarean sections in Denmark , the samples are likely to represent typical Danish FR levels at the
[40]
time. The women were asked to sample breast milk once a week for 3 months after giving birth using a
manual breast milk pump supplied by the study; after sampling, the milk was transferred to a pre-clean
amber glass. The weekly samples were combined in a composite sample stored in the participants'
household freezer. Each participant recorded the dates of sampling. Three months after birth, both milk
samples and the participants’ vacuum cleaner bags were collected. The content of the bags was sieved to
< 75 µm, as also described in Vorkamp et al. (in which the current samples correspond to the "second dust
sample") . All samples were then stored at -20 °C until analysis. The study was approved by the Regional
[41]
Ethics Committee of the Capital Region of Denmark (H-KF-327603) and the Danish Data Protection
Agency. PBDE concentrations are also available for placental tissue, maternal and umbilical cord plasma as
well as air and dust from the participants’ homes sampled before giving birth [15,41-43] ; an overview of the
biological matrices is given in Supplementary Table 1. PBDE data for placenta, fetal blood, and maternal
blood were used in this study to investigate associations with levels in breast milk and dust.

Chemical analysis
The samples were first analyzed for PBDEs and then later for NBFRs, DPs, and HBCDDs; due to a limited
sample volume, a few samples were only analyzed for PBDEs; an overview of the different analyses is given
in Supplementary Figure 1. For the analysis of PBDEs in breast milk, 20 mL of breast milk was dried with
13
diatomaceous earth, spiked with C-labelled BDE-209 and octachlorobiphenyl (PCB-198) and extracted
using Soxhlet with hexane:acetone (4:1). The milk extracts were cleaned on a multilayer column including
sulfuric acid-impregnated silica eluted with 250 mL hexane as described in Vorkamp et al. . The cleaned
[44]
extract was reduced in volume by rotary evaporator, and the final volume was approximated to 0.5 mL and
analyzed by gas chromatography with electron capture negative ionization-mass spectrometry
(GC-ECNI-MS). The analysis included 19 PBDEs (BDE-17, 28, 47, 49, 66, 71, 77, 85, 99, 100, 156, 154, 183,
197, 203, 206, 207, 208 and 209); however BDE-154 was not separated from the polybrominated biphenyl