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Page 4 of 20 Andjelkovic et al. J Environ Expo Assess 2024;3:23 https://dx.doi.org/10.20517/jeea.2024.22

national pooled sample, all POPs included in the Stockholm Convention were determined. The aims of the
present study were threefold: (1) to report and discuss the POP levels found both in individual and pooled
human milk samples; (2) to compare the POP levels in Belgian human milk samples (individual and
national) with results obtained in other countries and with the results from the previous Belgian survey; and
finally (3) to estimate the exposure of the nursing infants and perform a risk assessment for the different
POPs determinant in the national pooled sample for this age group.

EXPERIMENTAL
Sample collection
Between May and December 2014 a total of 31 hospitals across Belgian provinces (Flanders: Antwerpen,
Limburg, Oost-Vlaanderen, West-Vlaanderen, Vlaams-Brabant, and Wallonia: Brabant-Wallon, Hainaut,
Liège, Luxembourg, Namur) and the Brussels Capital Region were selected and visited to recruit
breastfeeding mothers, with one hospital in the urban and one in the rural area. The recruitment strategy,
along with informed consent documents, questionnaires, and all relevant documentation for participating
mothers and maternity units, was thoroughly reviewed and approved by several authorities. This included
the Commission for the Protection of Privacy (registration number HM002002523) and the Ethical
Commission of the Queen Fabiola Children’s University Hospital in Brussels, which served as the
coordinating ethical body for the multicenter study (registration number CEH 21/14). Additionally, the
ethical commissions from eight of the 31 participating maternity hospitals were involved in the process. The
other 23 maternity hospitals considered the review and approval from the coordinating ethical commission
to be adequate. Details regarding the recruitment and study protocol can be found in , while a summary of
[16]
the questionnaire is provided in Supplementary Material 1.

Preparation of the individual and pooled milk samples
The samples were registered under a code number to ensure the confidentiality of the data. Most samples
(more than 70%) consisted of a minimum of 50 mL, which was sufficient for chemical analyses. The
volumes used for each analysis and for preparing the mixed samples, the number of containers per sample,
and the province of origin were carefully documented. The sample flow is illustrated in Figure 1.

Before the analyses were conducted, individual samples were homogenized by heating to 38 °C and shaking
for 10 min. Necessary aliquots were then taken to create mixed samples that accurately represented each
province. The preparation of these samples adhered to a modified WHO protocol, taking into account two
key parameters: (1) ensuring adequate representation of each participant in the mixed sample and (2)
achieving a final volume of 110 mL for each provincial mixed sample to facilitate the planned analyses
[Supplementary Table 1].

One pooled Belgian sample was prepared following the WHO protocol . Briefly, 50 individual milk
[18]
samples were selected according to the geographical distribution [Supplementary Table 1]. The frozen
samples were first left to equilibrate at room temperature. Afterwards, each sample was homogenized by
shaking it for 5 min. From each sample, 25 mL was taken and transferred to a 2,000 mL flask, resulting in
1,250 mL milk (50 × 25 mL milk). This was kept at -20 °C and sent frozen to the European reference
laboratory for further analyses of the WHO-selected POPs. Analyses of chlorinated and brominated POPs
conducted between 2000 and 2019 were carried out at the State Institute for Chemical and Veterinary
Analysis of Food; Chemisches und Veterinäruntersuchungsamt (CVUA) in Freiburg, Germany. In contrast,
perfluoroalkane substances were analyzed from 2009 to 2019 at Örebro University in Sweden. A high level
of reliability in the results was attained .
[13]