CONCENTRATION LEVELS OF PCDD/F’S AND DIOXIN-LIKE PCB’S IN HUMANMILK SAMPLES FROM THE RURAL AREAS OF FLANDERS (BELGIUM).
Croes K1,5*, Vandermarken T1, Colles A2, Bruckers L3, Koppen G2, Govarts E2, Nelen V4, Van de Mieroop E4,Schoeters G2, Van Larebeke N5, Denison MS6, Kotz A7, Van Langenhove K1, Elskens M1 , Baeyens W1
1Vrije Universiteit Brussel, Department of Analytical and Environmental Chemistry (ANCH), Pleinlaan 2, 1050
Brussels, Belgium; 2VITO, Environmental Health and Risk, Boeretang 200, 2400 Mol, Belgium; 3Interuniversity
Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Belgium; 4Provincial Institute forHygiene, Antwerp, Belgium; 5Ghent University Hospital, Study Centre for Carcinogenesis and Primary
Prevention of Cancer, Ghent, Belgium; 6Department of Environmental Toxicology, University of California,
Davis, CA 95616, USA; 7State Institute for Chemical and Veterinary Analysis of Food, Bissierstrasse 5,Freiburg, Germany

Introduction:
In the first Flemish Environment and Health survey run by the Flemish Centre of Expertise on Environment andHealth (FLEHS I 2002-2006) increased concentrations of PCBs, dioxin-like substances and chlorinatedpesticides (a metabolite of DDT and hexachlorobenzene) were observed in cord blood of newborns and in bloodof 14-15 year-old adolescents and 50-65 year-old adults living in low populated rural communities of East andWest Flanders and Flemish Brabant compared to other Flemish regions. Due to the health concern associatedwith chlorinated compounds follow-up of pollutant levels in this area is of importance. Therefore, human breast
milk from mothers living in these regions was collected (2009-2010) for analysis of different POPs.
For the quantification of PCDD/Fs and/or dioxin-like PCBs in (human) milk samples both GC-HRMS and theCALUX bioassay are used in routine analysis. However, most CALUX methods use high amounts of milk(varying between 10 and 60 mL)1, 2. Since for the Flemish human breast milk survey in the rural areas not only
PCDD/Fs and dioxin-like PCBs were analyzed, but also other POPs like p,p’-DDE, PBDEs, marker PCBs, HCB,HCH, perfluorinated compounds, etc., the amount of milk available for the each assay was limited. Therefore,
the CALUX bioassay was used for the analysis of PCDD/Fs and dl-PCBs in the individual milk samples, while
GC-HRMS was used to define the concentration level of a pooled human milk sample.
Materials and methods:
In this study, 5 mL human breast milk samples obtained from 84 18-35 year-old breastfeeding mothers residingin the rural areas of Flanders (24 low-populated rural communities in East and West Flanders), were analyzed for
PCDD/Fs and dl-PCBs with a new sensitive CALUX mouse hepatoma cell line. Additionally, from each of the84 individual samples, 10 mL was taken to compose a pooled sample, which was analyzed with both theCALUX assay (VUB, ANCH, Brussels, Belgium) and by GC-HRMS (WHO reference laboratory, State Institutefor Chemical Analysis and Veterinary of Food, Freiburg, Germany). Details about inclusion criteria andcharacteristics of the study population are described by Colles et al., 20113.
CALUX analysis was performed at the VUB, using the methodology described by Croes et al. (2011 andsubmitted) 4,5. Methods used for analysis of the pooled milk sample by the WHO reference laboratory (StateInstitute for Chemical Analysis of Food) in Freiburg, Germany, are described elsewhere 6,7,8.
Results and discussion:
Five millilitre milk samples were analyzed with the CALUX bioassay for all 84 mothers participating in thestudy. All samples were above the LOQ, both for the PCDD/Fs and the dl-PCBs. The geometric mean PCDD/F
and dioxin-like PCB concentrations (raw data) in the total study population were respectively 10.4 (95% CI: 9.411.4) pg CALUX-BEQ per g fat or 0.41 (95% CI: 0.37-0.45) pg CALUX-BEQ per g milk for the PCDD/Fs and1.73 (1.57-1.91) pg CALUX-BEQ per g fat or 0.07 (95% CI: 0.06-0.08) pg CALUX-BEQ per g milk for thedioxin-like PCBs.
Univariate analysis of the raw data showed that participants who lost weight after the pregnancy had significant
higher values of dioxin-like PCBs (p=0.02) and PCDD/Fs (p=0.002) compared to participants who gained

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weight. The CALUX-BEQ values increased with the age of the mother (p=0.001 for PCDD/Fs and p=0.048 fordl-PCBs). PCDD/F levels were also higher for mothers consuming local eggs (p=0.023). A non-significant trend
was observed between smoking and the measured activity of the dioxin-like PCBs and PCDD/Fs. Levels ofdioxin-like activities were higher in milk samples from current smokers (active and passive) compared to nonsmokers. This can be expected since dioxins are present in cigarette smoke. For the PCDD/Fs BEQ values of10.7, 11.7 and 9.85 pg BEQ per g fat were found for respectively active, passive and non-smokers. For the dlPCBs BEQ values of 1.93, 1.98 and 1.57 pg BEQ per g fat were found for respectively active, passive and nonsmokers. The small (non-significant) differences between dioxin levels in active and passive smokers can be due
to amount of cigarettes smoked a day. When only taking into account active smoking, higher BEQs (for bothPCDD/Fs and dl-PCBs) were found for ex-smokers compared to non-smokers or current smokers. Afterexclusion of outliers, PCDD/F levels were borderline significantly higher in mothers with lower BMI (p=0.047).
No association was found between dl-PCBs and PCDD/Fs on the one hand and the education level of the mother,highest education level in the family and household income on the other hand. Sex of the baby, the number ofyears living in the study area and parity were also not significantly associated with the concentration of dl-PCBsor PCDD/Fs. It is however expected that the total concentration of POPs is lower in multiparous breastfeeding
mothers compared to primiparous breastfeeding women, since dioxin and PCBs are off-loaded to the infantduring gestation and lactation. Lorber and Philips (2002)9 estimated that maternal PCB/dioxin body burdens
decrease 20% to 70% during six months of exclusive breastfeeding. This means that a mother will transfer morePOPs to her first child compared to the later born children. In our study, also lower CALUX-BEQ values were
found in the human milk sample of mother giving birth to a second child compared to the milk samples fromprimiparous women (2.1 versus 1.8 pg BEQ per g lipid with p=0.20 for dl-PCBs and 12.2 versus 10.5 pg BEQ
per g lipid with p= 0.17 for PCDD/Fs).
In multiple regression analysis (after correction for predefined confounders and covariates that were significant
in a stepwise model) no significant relationships were found for the dl-PCBs. A borderline non-significant
relation was found between smoking before pregnancy and PCDD/Fs (p=0.05). The BEQ-levels were higher in
ex-smoker compared to current smokers or women who never smoked. For the PCDD/Fs also significant
relationships were found with weight change after pregnancy (p=0.003 for ln PCDD/Fs and p=0.0001 for the
non-transformed PCDD/Fs in the model, both expressed in pg BEQ per g lipid) and consumption of local eggs.
Participants who lost weight after pregnancy relative to their weight before pregnancy had significant higher
values of PCDD/Fs compared to participants who gained weight. Weight losses result in a decrease of adipose
tissue and thus in a release of POPs into blood and human milk. Participant who never consumed local eggs
showed significantly lower PCDD/F BEQ values (expressed per g lipid), with p-values of respectively 0.05 and0.04 for the Ln-transformed and the non-transformed exposure marker.
The CALUX-BEQ values on the pooled breast milk sample were in good agreement with the GC-HRMS data forboth the PCDD/F and the dl-PCB fractions (Table 1). The PCDD/F BEQ values were 32 % and 60 % higher
with CALUX compared to GC-HRMS, when using respectively the 1998 and 2005 TEF scheme. For the dlPCBs a better agreement was found between the two techniques with the 2005 TEF scheme (a CALUX/GCHRMS ratio of 0.58 compared to 0.37 for the 1998 TEFs). As already reported by various researchers, the lower
dl-PCB values are probably due to the difference between the WHO-TEFs and the CALUX-REPs, while thedifferences obtained for the PCDD/Fs are probably due to other AhR agonists. The concentrations levels (in pgper g lipid) for some PBDD/F congeners, analyzed in the pooled human milk sample with GC-HRMS, are shownin Table 2. Although only a few congeners were measured, it is clear that these compounds have a relatively low
contribution to the total TEQ/BEQ. When using the same TEF/REP values as for the chlorinated analogs, theTEQ would raise 0.42, 0.34, 0.41 pg TEQ per g lipid (medium bound, using the WHO-TEFs of 1998 or 2005, orthe CALUX-REPs from Brown et al. (2001)10, respectively). This would mean an increase of only 5 % to thetotal PCDD/F WHO-TEQ value and an adapted CALUX/GC-HRMS ratio of 1.26/1.53/1.24.
In a human milk study in Sweden (samples collected in 2002-2003), also low concentration levels of brominatedcompounds were found. The levels of 2,3,7,8-TBDF and 2,3,4,7,8-PeBDF were detected at concentrations ofrespectively 0.55 pg per g fat and 0.33 pg per g fat, which is comparable to the levels found in the Flemish
pooled milk sample. 1,2,3,7,8-PeBDF was not detected in the Swedish milk samples (Flemish rural sample: 0.2
pg per g lipid), while 1,2,3,4,7,8-/1,2,3,6,7,8-HxBDF was found at a concentration of 3.8 pg per g fat (FlemishOrganohalogen Compounds

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rural sample: below LOQ)11. Kotz et al. (2005)12 reported mean upper and lower bound PBDD/F TEQ levels of
1.08 and 0.32 in human milk samples from the third round of the WHO human milk survey (2001-2002).
Dominant PBDD/Fs congeners were 2,3,7,8-TBDF (average concentration 0.7 pg per g fat, range < 0.1 - 2.7 pg
per g fat) and 2,3,4,7,8-PeBDF (average 0.23 pg per g fat, range < 0.1 - 1.1 pg per g fat). Some other congeners,
e.g. 2,3,7,8-TBDD (concentrations 0.06 - 0.28 pg per g fat), 1,2,3,7,8-PeBDD (0.14 - 1.0 pg per g fat), 1,2,3,7,8PeBDF and 1,2,3,4,7,8-HxBDF could only be quantified in some of the samples. The PBDD/Fs contributed
about 12 % to the total WHO-TEQ. In human adipose tissue, PBDD/Fs were found in a Swedish study from2007. Depending on the TEFs/REPs used for calculating the upper and lower bound TEQ, the PBDD/Fs
contributed 1 - 15 % to the total PCDD/F WHO-TEQ11.
The concentrations of the PCDD/Fs and dl-PCBs measured in the pooled sample of the rural population in 20092010 by the WHO reference lab (CVUA, Freiburg, Germany) followed the ongoing declining trend that wasobserved in the WHO-coordinated human milk surveys (1989-2006).
In all Belgian campaigns, it was seen that the concentrations of dl-PCBs in human milk were much lower thanthe concentration levels of the PCDD/Fs. When comparing the Flemish PCDD/F levels, measured in the ruralarea, to the results from other European countries participating the 4th WHO human milk survey from 2006
(Figure 1), the Belgian levels are still quite high despite the declining trend 13. The dl-PCBs levels measured inthis study were lower compared to other European and non-European studies14.
Acknowledgements:
The authors acknowledge all mothers who kindly accepted to participate to this study, as well as thefieldworkers, midwifes, gynecologists, doctors and directors of the participating maternities, whose cooperation
was essential for the successful results of this study.
This study was commissioned, financed and steered by the Environment, Nature and Energy Department of theFlemish government.
The authors are grateful to M. Denison and the other members of the University of California-Davis (USA) forgenerously providing our laboratory with the H1L7.5c1 cell lines which was developed with funding from aSuperfund Research Program grant (ES04699) from the National Institute of Environmental Health Sciences.
References:
1. Leng J-H, Kayama F, Wang P-Y, Nakamura M, Nakata T, Wang Y. (2009); Chemosphere 75: 634–639
2.Van Overmeire I, Van Loco J, Roos P, Carbonnelle S, Goeyens L. (2004); Talanta 63: 1241–1247
3. Colles A, Koppen G, Van De Mieroop E, Covaci A, Croes K, Kotz A, Mampaey M, Schoeters G. (2011);
Organohalogen Compounds 73: 1555-1558.
4. Croes K, Vandermarken T, Colles A, Bruckers L, Koppen G, Govaerts E, Nelen V, Van de Mieroop E,Schoeters G, Van Larebeke N, Denison M, Kotz A, Van Langenhove K, Elskens M, Baeyens W. (2011);
Organohalogen Compounds 73:1582-1585.
5. Croes K, Colles A, Koppen G, Govarts E, Bruckers L, Van de Mieroop E, Nelen V, Covaci A, Dirtu A.C,Thomsen C, Haug L.S, Becher G, Mampaey M, Schoeters G, Van Larebeke N, Baeyens W. (2012);
Chemosphere submitted.
6. Malisch R, van Leeuwen FXR. (2002); Organohalogen Compounds 56: 317-320
7. Kotz A, Malisch R, Kypke K, Oehme M. (2005); Organohalogen Compounds 67: 1540-1544
8. Hui LL, Hedley AJ, Kypke K, Cowling BJ, Nelson EAS, Wong TW, van Leeuwen FXR, Malisch R. (2008);
Chemosphere 73: 50-55
9. Lorber M., Phillips L. (2002); Environmental health perspectives 110: A325.
10. Brown D.J, Chu M.D, Van Overmeire I, Chu A, Clark G. (2001); Organohalogen Compounds 53: 211-214.
11. Ericson Jogsten I, Hagberg J, Lindström G, Bavel B. (2010); Chemosphere 78: 113-120.
12. Kotz A, Malisch R, Kypke K, Oehme M. (2005); Organohalogen Compounds 67: 1540-1544.
13. World Health Organization (2009); Fact Sheet 4.3; European Environment and Health Information System(ENHIS)
14. Malisch R, Kypke K, Van Leeuwen R, Moy G, Park S. (2008); Organohalogen Compounds 70, 228-232.
pg WHO-TEQ/g lipid

GC-HRMS

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Mean

Ratio: mean758

and(TEF 1998)(TEF 2005)
CALUX-BEQ CALUX/GC-HRMS
pg BEQ/ g lipid(n=2)(TEF 1998/2005)
8.41
6.95
11.09
1.32/1.60
PCDD/Fs
5.80
3.77
2.17
0.37/0.58
dl-PCBs
14.21
10.72
13.26
0.93/1.24
Total dl-compounds
Table 1: The CALUX-BEQ and GC-HRMS WHO-TEQ values, expressed per g lipid, for the PCDD/F anddl-PCB fractions of a pooled human milk sample. TEF values from 1998 and 2005 were used.
Polybrominated dioxins (pg/g lipid)
2,3,7,8-TBDD
1,2,3,7,8-PeBDD

Polybrominated furans (pg/g lipid)
2,3,7,8-TBDF
0.7
1,2,3,7,8-PeBDF
0.2
2,3,4,7,8-PeBDF
0.4
1,2,3,4,7,8/1,2,3,6,7,8-HxBDD<0.07
1,2,3,4,7,8/1,2,3,6,7,8-HxBDF<0.3
1,2,3,7,8,9-HxBDD<0.04
1,2,3,7,8,9-HxBDF
Na
2,3,4,6,7,8-HxBDF
Na
1,2,3,4,6,7,8-HpBDD
Na
1,2,3,4,6,7,8-HpBDF
Na
1,2,3,4,7,8,9-HpBDFNaOBDDNa
OBDFNaTable 2: Overview of the concentration levels (in pg/g lipid) of PBDD/F congeners, measured in the pooledhuman milk sample. < are concentration levels below the LOQ. These concentrations were set at half theLOQ for TEQ determination. Na= result not available.
0.05
<0.07

8.4 pg TEQ/g fat(2010)

Figure 1: overview of the PCDD/F results of the four WHO-coordinated human milk surveys in different
European countries, and comparison with the TEQ found in this study. Measurements were done with
GC-HRMS and WHO-TEFs from 1998 were used the calculate the TEQ (World Health Organization,2009).

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