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Page 6 of 20 Andjelkovic et al. J Environ Expo Assess 2024;3:23 https://dx.doi.org/10.20517/jeea.2024.22

California, United States) equipped with a Rxi-XLB capillary column (30 m × 0.25 mm, 0.25 µm). Injection
was done in splitless mode. Helium was used as carrier gas (1.2 mL/min). The temperature program was set
at 110 °C for 0.5 min, then increased by 25 °C/min to 200 °C, then increased by 10 °C/min to 280 °C,
followed and kept for 4 min, and finally by 25 °C/min to 300 °C and held for 3.1 min. The multiple reaction
monitoring (MRM) transitions and the MS parameters are given in Supplementary Table 2. As a quality
control, a solvent blank, a procedural blank, and a control sample were run within each batch of samples.
Average procedural blank levels were then subtracted from the sample results, and a value equal to 3 × SD of
the blank measurement was used as the limit of quantification (LOQ). For compounds absent in the blanks,
LOQs were based on a signal/noise ratio of 10 (S/N = 10). The LOQ was 2 ng/g lipid weight (lw), and the
limit of detection (LOD was 1 ng/g lw, except for oxychlordane where 5 and 2.5 ng/g lw were LOQ and
LOD, respectively).

Analysis of OCPs by GC-MS/MS in provincial samples
Composite samples were created by combining individual samples from each province, yielding a total of 11
provincial samples. Further analyses of hexachlorobutadiene, heptachlor, cis-heptachlor epoxide, trans-
heptachlor epoxide, chlordecone, and dieldrin were outsourced to an external lab. The accredited method
used for the analyses of the samples was based on GC-MS/MS detection.

Analysis of PBDEs, PeCB and BB-153 by GC-MS
The analysis of PBDEs, PeCB, and BB-153 in human milk samples was conducted in accordance with the
protocol outlined by Dimitriadou et al. (2016), incorporating minor modifications as detailed in
Supplementary Material 3 .
[20]

Analysis of HBCDs by LC-MS/MS
Composite samples of human milk were prepared according to the WHO protocol and represented the
pooled samples for each Belgian province. These samples were kept in glass containers from the first
transport until the analyses and during any further storage. The extraction was done according to the
protocol described elsewhere [20,21] . Details are in Supplementary Material 4.

Analyses of the national pooled sample by the EU reference lab
Out of 1,250 mL prepared to represent the Belgian pooled sample, 450 mL was used for analysis by the
CVUA in Freiburg, Germany, as requested by the National Coordinators of the study [22-24] . CVUA is the
WHO Reference Laboratory for the WHO-coordinated human milk study for POPs. All analytical results
were reported on a lipid basis and were related to a broad range of POPs, including PCDDs, PCDFs, and
dioxin-like PCBs. The whole list of compounds can be found in Supplementary Material 5. The results
obtained are discussed in a comparative analysis with findings from other countries.

Quality assurance/quality control
The efficiency of extraction, clean-up, and fractionation steps was evaluated by measurement of the absolute
recoveries of the internal standards [Supplementary Material 6].

Descriptive statistics
The results are outlined and summarized using descriptive statistics. Measures of central tendency (mean
and median) and variability (frequency) were calculated for the fat percentage in milk samples and the
concentrations of POPs, both overall and by compound group. Two scenarios were analyzed: lower bound
(LB) and medium bound (MB), which represent a more conservative approach to minimize the risk of
overestimation and, if necessary, to facilitate the development of realistic mitigation strategies. The upper
bound was calculated as additional information. The lower and MBs were determined by assigning a value