Ann Colles

Persistent organic pollutants (POPs) in human milk: A biomonitoring study in rural areas of Flanders (Belgium)

To collect information on the concentrations of persistent organic pollutants (POPs) in the rural areas in Flanders (Belgium), 84 breastfeeding mothers were recruited in rural communities in East and West Flanders and Flemish Brabant in 2009-2010. Polychlorinated biphenyl (PCB) congeners, organochlorine pesticides, brominated flame retardants, perfluorinated compounds, polychlorinated dibenzodioxines and dibenzofurans, and dioxin-like PCBs were measured in individual milk samples and in a pooled milk sample, while some additional pollutants were only measured in the pooled sample. For most pollutants, the concentrations in this study were lower or comparable to the concentrations measured in the pooled Belgian sample of the WHO human milk study of 2006, except for the pesticides dichlorodiphenyltrichloroethane DDT (+25% for ΣDDT and metabolites) and trans-nonachlor (+94%), and for the brominated flame retardant hexachlorocyclododecane HBCD (+153%). Perfluorinated compounds were for the first time determined in human milk samples from Belgium and the concentrations were comparable to those from other European countries. Also, interesting associations were found between the concentrations of POPs measured in human milk and personal characteristics as well as dietary habits of the study population. PFOS en PFOA concentrations were significantly higher in milk of primiparous participants compared to mothers who gave birth to their second child. Lower brominated PBDE congeners increased with increasing BMI of the mothers (p=0.01 for BDE 47, p=0.02 for BDE 99 and p=0.02 for BDE 100). Participants consuming milk or dairy products daily had significant higher concentrations of ΣDDTs (p=0.03) and oxychlordane (p=0.047) in their human milk samples.

Concept of the Flemish human biomonitoring programme

Since 2002 a human biomonitoring network has been established in Flanders (Belgium) as part of a programme on environmental health surveillance. The human biomonitoring network should support environmental health policy by identifying priorities for further action. The first cycle of the programme (2002-2006) confirmed the hypotheses that living in areas with different environmental pressure is reflected in different loads of environmental chemicals in the residents. In the second cycle of the programme (2007-2011) the number of environmental chemicals for which human biomonitoring data were obtained was expanded substantially. The goal of the Flemish programme is to use and translate the scientific results into policy actions. Its further orientation in the second cycle to human biomonitoring in hot spots and sensitive age groups or susceptible persons with underlying complications (e.g. persons with diabetes) are linked to these goals. Interaction with stakeholders is embedded in the programme emphasizing transparency of the choices that are made and direct communication. The Flemish human biomonitoring programme is organized centrally with major involvement of research partners from different disciplines which allows engrafting environmental health research on the programme. One of the major focuses is the question whether combinations of pollutants in the general population are associated with biological effects. The objective of this paper is to review and discuss the options that were taken in the human biomonitoring programme in order to achieve its goals.

Chapter 2F:The Flemish Environment and Health Study (FLEHS) – Second Survey (2007–2011): Establishing Reference Values for Biomarkers of Exposure in the Flemish Population

The second Flemish human biomonitoring survey (2007–2011) generated information on the distribution of biomarker values for a large number of environmental pollutants in a representative sample of the Flemish population. The study was implemented by the Flemish Centre of Expertise for Environment and Health, which was funded and steered by the Flemish government. From May 2008 to July 2009, 255 newborns and their mothers, 210 adolescents (14–15 years old) and 204 adults (20–40 years old) were recruited as a representative sample of the Flemish population. In all age groups, invitation letters, information brochures, letter of informed consent and self-administered questionnaires were distributed. The collected samples consisted of cord blood from the newborns, blood from the mothers, the adolescents and the adults, urine from the adolescents and the adults, and hair from the mothers. In addition to historical compounds (i.e. heavy metals, persistent chlorinated compounds, 1-hydroxy pyrene and t,t-muconic acid), new emerging pollutants were analyzed for the first time in individual samples from the Flemish population. Perfluorinated compounds and polycyclic musk compounds could be detected in all analyzed blood samples. Levels of brominated flame retardants were under the limit of quantification in most individual blood samples. Bisphenol A, metabolites of phthalates and para-hydroxybenzoic acid, a metabolite of parabens, could be detected in 90% or more of the urine samples. For metabolites of organophosphate pesticides, the highest detection frequency was observed for DMTP, detected in 90–95% of the individual urine samples. In addition, 2,5-DCP, a metabolite of para-dichlorobenzene, could be detected in over 80% of the individual urine samples. Margins of safety (MOS) were calculated by dividing the P90 obtained in FLEHSII by the available health based biomonitoring equivalants (BEs). MOS below 10 were found for toxicologically relevant arsenic, cadmium, lead, MeHg, HCB and phthalates.

Identification of PM10 characteristics involved in cellular responses in human bronchial epithelial cells (Beas-2B).

Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM10 in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP). In 2013-2014 PM10 was sampled (24h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with different pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM10 particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days. PM10 particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples. Associations between PM10 characteristics and biological effects of PM10 were assessed by single and multiple regression analyses. The reduction in cell viability was significantly correlated with BC, Cd and Pb. The induction of IL-8 in Beas-2B cells was significantly associated with Cu, Ni and Zn and endotoxin. Endotoxin levels explained 33% of the variance in IL-8 induction. A significant interaction between ambient temperature and endotoxin on the pro-inflammatory activity was seen. No association was found between OP and the cellular responses. This study supports the hypothesis that, on an equal mass basis, PM10 induced biological effects differ due to differences in PM10 characteristics. Metals (Cd, Cu, Ni and Zn), BC, and endotoxin were among the main determinants for the observed biological responses.

Determination of PCDD/Fs, PBDD/Fs and dioxin-like PCBs in human milk from mothers residing in the rural areas in Flanders, using the CALUX bioassay and GC-HRMS.

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast and inexpensive tool for the determination of dioxin-like compounds in a large number of samples and requires only small sample volumes, the use of this technique in human biomonitoring programs provides a good alternative to GC-HRMS. In this study, a new CALUX method for the separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in small amounts of human milk samples with the new sensitive H1L7.5c1 cell line was used to analyze 84 human milk samples, collected from mothers residing in the Flemish rural communities. The geometric mean CALUX-Bioanalytical Equivalent (CALUX-BEQ) values, reported for the 84 mothers from the study area were 10.4 (95% CI: 9.4-11.4) pg CALUX-BEQ per gram lipid or 0.41 (95% CI: 0.37-0.45) pg CALUX-BEQ per gram milk for the PCDD/Fs and 1.73 (1.57-1.91) pg CALUX-BEQ per gram lipid or 0.07 (95% CI: 0.06-0.08) pg CALUX-BEQ per gram milk for the dioxin-like PCBs. Multiple regression analysis showed significant associations between PCDD/Fs and weight change after pregnancy, smoking and consumption of local eggs. One pooled human milk sample was analyzed with both CALUX and GC-HRMS. The ratio of CALUX and GC-HRMS results for this sample were respectively 1.60, 0.58 and 1.23 for the PCDD/Fs, the dl-PCBs and the sum of both fractions, when using the 2005-TEF values. Additionally, also low levels of certain brominated dioxins and furans were detected in the pooled sample with GC-HRMS.

Health effects in the Flemish population in relation to low levels of mercury exposure: From organ to transcriptome level

Due to possible health risks, quantification of mercury accumulation in humans was included in the Flemish biomonitoring programmes FLEHS I (2002-2006) and FLEHS II (2007-2011). The general objective of FLEHS I was to assess regional exposure levels in order to link possible differences in these internal exposure levels to different types of local environmental pressure. Therefore, Hg and MMHg (methylmercury) were only measured in pooled blood samples per region and per age class. In FLEHS II, mercury concentrations were measured in hair of each participant. About 200 adolescents and 250 mothers (reference group) and two times 200 adolescents (2 hotspots) were screened. The main objectives of the FLEHS II study were: (1) to determine reference levels of mercury in hair for Flanders; (2) to assess relations between mercury exposure and possible sources like fish consumption; (3) to assess dose-effect relations between mercury exposure and health effect markers. The results showed that mercury concentrations in the Flemish population were rather low compared to other studies. Mercury levels in the Flemish populations were strongly related to the age of the participants and consumption of fish. Significant negative associations were observed between mercury in hair and asthma, having received breast feeding as a newborn, age at menarche in girls, allergy for animals and free testosterone levels. Significant correlations were also observed between mercury in hair and genes JAK2, ARID4A, Hist1HA4L (boys) and HLAdrb5, PIAS2, MANN1B1, GIT and ABCA1 (girls).

Trace metal concentrations measured in blood and urine of adolescents in Flanders, Belgium: Reference population and case studies Genk-Zuid and Menen

In the Flemish human biomonitoring programme FLEHS II (2007-2011) trace metals (Cd, Pb, Cr, Ni, Cu, Mn, Tl, Sb, As and toxic relevant arsenic) were analysed in the blood and urine of adolescents (14-15 years old) in the reference population in Flanders and in areas of important industrial activities: Genk-Zuid and Menen. After adjustment of the results for confounding factors, the adolescents living in Genk-Zuid had higher levels of Cr, Cu and Tl in blood, higher levels of Cd and toxic relevant arsenic (TRA) in urine, but lower levels of Ni in blood and Sb in urine compared to the reference population. In Menen higher levels of Cd and Cu in urine, higher concentrations of Tl in blood but lower concentrations of Pb in blood and lower Ni, Sb and As in urine were found compared to the reference population. For both the reference population and the hot spots the concentrations are within the ranges found in other countries. Compared to the previous biomonitoring programme FLEHS I (2002-2006) a decrease in the concentrations of Cd and especially of Pb in blood was observed. However, it cannot be excluded that differences between the two campaigns are partially due to different sampling strategies.

Quantification of PCDD/Fs and dioxin-like PCBs in small amounts of human serum using the sensitive H1L7.5c1 mouse hepatoma cell line: optimization and analysis of human serum samples from adolescents of the Flemish human biomonitoring program FLEHS II

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast and inexpensive tool for the throughput analysis of dioxin-like compounds in a large number of samples and requires only small sample volumes, the use of this technique in human biomonitoring programs provides a good alternative to GC-HRMS. In this study, a method for the separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in human serum with the new sensitive H1L7.5c1 mouse hepatoma cell line was optimized. Sample dilution factors of 5 and 2.4 were selected for routine analysis of respectively the PCDD/Fs and dl-PCBs. The validation studies showed that repeatability and within-lab reproducibility for the quality control (QC) standard were within the in-house criteria. A long-term within-lab reproducibility of 25% for the PCDD/F fraction and 41% for the dl-PCB fraction for the analysis of pooled serum samples, expressed as pg BEQ/g fat, was determined. CALUX recoveries of the spiked procedural blanks were within the acceptable in-house limits of 80-120% for both fractions and the LOQ was 30.3 pg BEQ/g fat for the PCDD/Fs and 14.5 pg BEQ/g fat for the dl-PCBs. The GC-HRMS recovery of a C13-spiked pooled serum sample was between 60 and 90% for all PCDD/F congeners and between 67 and 82% for the non-ortho PCBs. An adequate separation between both fractions was found. The CALUX/GC-HRMS ratio for a pooled serum sample was respectively 2.0 and 1.4 for the PCDD/Fs and the dl-PCBs, indicating the presence of additional AhR active compounds. As expected, a correlation was found between human serum samples analyzed with both the new H1L7.5c1 cell line and the more established H1L6.1c3 cell line. The geometric mean CALUX-BEQ values, reported for the adolescents of the second Flemish Environment and Health Study (FLEHS II) recruited in 2009-2010, were 108 (95% CI: 101-114) pg CALUX-BEQ/g fat for the PCDD/Fs and 32.1 (30.1-34.2) pg CALUX-BEQ/g fat for the dioxin-like PCBs.

Application of non-invasive biomarkers in a birth cohort follow-up in relation to respiratory health outcome

Asthma-related symptoms can manifest in children during the early years, but only some of the children will develop the disease. This feasibility study showed that it is possible to apply non-invasive markers (in urine, exhaled nitric oxide (FENO) and exhaled breath condensate (EBC)) in 3-year-old children, and evaluated the biomarkers in relation to health outcomes and potential modifiers. FENO was correlated with respiratory allergy, and was borderline significantly correlated with wheezing, but not with the asthma predictive index (mAPI). EBC pH and urinary 8-oxo-deoxyguanosine were not significantly correlated with these clinical outcomes. An EBC proteolytic peptide pattern was developed, which could distinguish between mAPI-positive and -negative children. Non-invasive biomarkers may become a promising tool for investigating respiratory health in children but further research is needed.

A POLICY ORIENTED MODEL SYSTEM FOR THE ASSESSMENT OF LONGTERM EFFECTS OF EMISSION REDUCTIONS ON OZONE

A methodology has been presented for the assessment of long-term effects of emission reductions on ozone concentrations in Belgium at low computational costs. Results for various scenarios show some important trends. The average values (ADM) are hardly changing or show a slight decrease. The indicators with a low cut off as threshold value (AOT40) show a slight decrease. Both trends are confirmed by observations between 1989 and 1999. Indicators representing peak values (e.g. NET60 and AOT60) decrease significantly for the various policy scenarios. From the measurements these trends are not clear, since the annual variation are dominated by the varying meteorological conditions. For the scenario’s considered the regression method gives accurate results for the ADM and AOT40 indicators, but less accurate results for the AOT60 and NET60 indicators.