Kim Croes

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.

Neurobehavioral function and low-level exposure to brominated flame retardants in adolescents: a cross-sectional study

BackgroundAnimal and in vitro studies demonstrated a neurotoxic potential of brominated flame retardants, a group of chemicals used in many household and commercial products to prevent fire. Although the first reports of detrimental neurobehavioral effects in rodents appeared more than ten years ago, human data are sparse.MethodsAs a part of a biomonitoring program for environmental health surveillance in Flanders, Belgium, we assessed the neurobehavioral function with the Neurobehavioral Evaluation System (NES-3), and collected blood samples in a group of high school students. Cross-sectional data on 515 adolescents (13.6-17 years of age) was available for the analysis. Multiple regression models accounting for potential confounders were used to investigate the associations between biomarkers of internal exposure to brominated flame retardants [serum levels of polybrominated diphenyl ether (PBDE) congeners 47, 99, 100, 153, 209, hexabromocyclododecane (HBCD), and tetrabromobisphenol A (TBBPA)] and cognitive performance. In addition, we investigated the association between brominated flame retardants and serum levels of FT3, FT4, and TSH.ResultsA two-fold increase of the sum of serum PBDE’s was associated with a decrease of the number of taps with the preferred-hand in the Finger Tapping test by 5.31 (95% CI: 0.56 to 10.05, p = 0.029). The effects of the individual PBDE congeners on the motor speed were consistent. Serum levels above the level of quantification were associated with an average decrease of FT3 level by 0.18 pg/mL (95% CI: 0.03 to 0.34, p = 0.020) for PBDE-99 and by 0.15 pg/mL (95% CI: 0.004 to 0.29, p = 0.045) for PBDE-100, compared with concentrations below the level of quantification. PBDE-47 level above the level of quantification was associated with an average increase of TSH levels by 10.1% (95% CI: 0.8% to 20.2%, p = 0.033), compared with concentrations below the level of quantification. We did not observe effects of PBDE’s on neurobehavioral domains other than the motor function. HBCD and TBBPA did not show consistent associations with performance in the neurobehavioral tests.ConclusionsThis study is one of few studies and so far the largest one investigating the neurobehavioral effects of brominated flame retardants in humans. Consistently with experimental animal data, PBDE exposure was associated with changes in the motor function and the serum levels of the thyroid hormones.

Internal exposure to pollutants and sex hormone levels in Flemish male adolescents in a cross-sectional study: associations and dose–response relationships

Flanders is densely populated with much industry and intensive farming. Hormonal status of 14- to 15-year-old male adolescents was studied in relation to internal exposure to pollutants. A total of 887 participants were selected as a random sample of the adolescents residing in the study areas. Confounding factors and significant covariates were taken into account. Serum levels of testosterone, free testosterone and estradiol, and the aromatase index showed significant positive associations with serum levels of marker polychlorobiphenyls (sum of PCBs 138, 153, and 180) and of hexachlorobenzene (HCB) and a negative association with urinary cadmium concentration. Serum levels of estradiol also showed a positive association with serum levels of dichlorodiphenyldichloroethylene (DDE). A doubling of serum concentrations of marker PCBs and HCB and of urinary concentration of cadmium were, respectively, associated with an increase of 16.4% (P<0.00001) and 16.6% (P<0.001) and a decrease of 9.6% (P<0.001) in serum testosterone concentration. Similar findings were made after additional adjustment for concurrent exposures. Associations between biological effects and internal exposures were, in terms of the regression coefficient, often stronger at exposures below the median. Environmental exposures to pollutants resulting in “normal” levels of internal exposure were associated with quite substantial differences in hormone concentrations.

Optimization and validation of a liquid chromatography tandem mass spectrometry (LC/MSn) method for analysis of corticosteroids in bovine liver: Evaluation of Keyhole Limpet β-glucuronidase/sulfatase enzyme extract

A liquid chromatography tandem mass spectrometry (LC/MS(n)) method for the determination of 12 corticosteroids in bovine liver has been optimized and validated in accordance with the European Commission Decision 2002/657/EC. A bovine liver sample was deconjugated with beta-glucuronidase/sulfatase enzyme, extracted with diethyl ether and further cleaned up with Solid Phase Extraction (SPE) before analysis with LC/MS(n). Two different enzyme extracts (originating from Helix Pomatia and Keyhole Limpet) and three SPE elution solvents (ethyl acetate, acetonitrile and methanol) were compared during the optimization. Helix Pomatia is generally known as the enzyme most being used for enzymatic hydrolysis purposes. Nevertheless, when detecting corticosteroids in the low microg kg(-1) concentration range, the Helix Pomatia extract may lead to interferences in the final LC/MS(n) chromatogram. When using the Keyhole Limpet enzyme extract, no interferences were observed and therefore, this extract was the best choice for enzymatic hydrolysis tested in this case. Ethyl acetate was used as elution solvent during the validation procedure since SPE elution with acetonitrile resulted in higher chromatographic backgrounds, while elution with methanol showed less reproducible results. Validation of the optimized method was carried out for 10 of the 12 corticosteroids, giving mean recoveries between 91 and 109%, and repeatability and reproducibility coefficients of respectively maximum 13.7 and 18.0%. The working ranges for the linear calibration curves were 5-20 microg kg(-1) for prednisolone, methylprednisolone and prednisone and 0.5-4 microg kg(-1) for the other compounds (coefficients of determination R(2)> or =0.97). Specificity, decision limit (CCalpha) and detection capability (CCbeta) were for all compounds within the EC specified limits.

Trace metals in blood and urine of newborn/mother pairs, adolescents and adults of the Flemish population (2007-2011)

The Flemish Centre for Environment and Health started with human biomonitoring in 2002 (FLEHS I: 2002-2006). The main goal of the second human biomonitoring cycle (FLEHS II: 2007-2011), was to determine mean values for a large number of pollutants in a representative sample of the general Flemish population. Values for Cd and Pb were updated, and a group of previously undetermined metals and metalloids (As, Mn, Cu and Tl) were included in some of the age groups. In this human biomonitoring program, three different age groups of the general Flemish population were monitored: 255 newborns and their mothers, 210 adolescents aged 14-15, and 204 adults between 20 and 40 years old. Trace elements were determined in cord blood and maternal blood of the mothers, in blood and urine of adolescents and in urine of adults. Determinants of life-style and personal factors were taken into account. The levels of trace elements in cord blood and maternal blood were for most elements at the lower end of the range found in literature. For Pb, As and Tl, a strong correlation (respectively r=0.43, 0.55 and 0.33; p<0.05) was found between levels in cord blood (respectively 8.6, 0.54 and 0.017 μg/L) and maternal blood (11.1, 0.64 and 0.028 μg/L), indicating that they are transported via the placenta from mother to fetus. The levels found in the adolescents and adults were compared with results from international biomonitoring studies, and were found to be in the same ranges. With the exception of Pb, all trace elements increased with increasing age group population. Finally, the results also showed that the levels of Cd and Pb in blood for this campaign (e.g. for Pb 8.6 and 14.8 μg/L in neonates and adolescents respectively) were lower compared to the first campaign (e.g. for Pb 14.7 and 21.7 μg/L in neonates and adolescents respectively), indicating a decrease over time. However, differences in sampling strategies might partially explain this observed trend.

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.

CALUX measurements: statistical inferences for the dose-response curve.

Chemical Activated LUciferase gene eXpression [CALUX] is a reporter gene mammalian cell bioassay used for detection and semi-quantitative analyses of dioxin-like compounds. CALUX dose-response curves for 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD] are typically smooth and sigmoidal when the dose is portrayed on a logarithmic scale. Non-linear regression models are used to calibrate the CALUX response versus TCDD standards and to convert the sample response into Bioanalytical EQuivalents (BEQs). Several complications may arise in terms of statistical inference, specifically and most important is the uncertainty assessment of the predicted BEQ. This paper presents the use of linear calibration functions based on Box-Cox transformations to overcome the issue of uncertainty assessment. Main issues being addressed are (i) confidence and prediction intervals for the CALUX response, (ii) confidence and prediction intervals for the predicted BEQ-value, and (iii) detection/estimation capabilities for the sigmoid and linearized models. Statistical comparisons between different calculation methods involving inverse prediction, effective concentration ratios (ECR(20-50-80)) and slope ratio were achieved with example datasets in order to provide guidance for optimizing BEQ determinations and expand assay performance with the recombinant mouse hepatoma CALUX cell line H1L6.1c3.

Radium analysis by sector field ICP-MS in combination with the Diffusive Gradients in Thin Films (DGT) technique

A new and simple method to assess low radium concentrations in water and sediment pore water was developed. The 226Ra ions are trapped on a laboratory prepared MnO2 resin (3 other ones have also been tested) that is inserted in a DGT probe (diffusive gradients in thin films). The probe can be deployed for days to weeks sufficient to accumulate enough 226Ra to be quantified. Because the High Resolution ICP-MS was operated in low resolution mode to get the highest sensitivity, isobaric interference from the Sr88Ba138 molecular ion could not be avoided. However, the pre-concentration on the MnO2 resin favours 226Ra accumulation versus that of Ba and Sr. The instrumental repeatability on a standard of 2 ng L−1226Ra was 1.4% (n = 10), while the relative standard deviation on natural samples was 8% (average of 2.2 pg L−1226Ra, n = 3). A detection limit as low as 0.022 pg L−1226Ra (0.80 mBq L−1) was achieved for a 24 h DGT deployment.

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.