PCDD/F levels in plasma of a belgian population before and after the 1999 belgian PCB/DIOXIN incident.

We evaluated the impact of the 1999 Belgian dioxin incident on the blood plasma polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) levels among 232 Belgian blood donors (74% men, mean age 47 years). The Red Cross made plasma samples from before the incident of these donors available. A second plasma sample was collected during the second half of 2000. The sum of the 17 PCDD/F congeners was significantly lower in 2000 compared to 1998 (417 pg/g fat versus 445 pg/g fat, respectively). This could be completely attributed to the significant decrease of OctaCDD (301 pg/g fat in 2000 versus 277 pg/g fat in 1998). Moreover a slight but significant decrease was observed for 2,3,7,8-TetraCDF and for 1,2,3,4,6,7,8-HeptaCDF. 1,2,3,7,8-PentaCDF and 2,3,4,7,8-PentaCDF however showed a slight but significant increase (respective levels in 1998 were 0.004 and 14.5 pg/g fat compared to 0.006 and 17.9 pg/g fat in 2000). Given their significantly higher presence in incident related food samples this increase can be attributed to the food contamination episode. However, the total toxicity remained unchanged (22.9 in 1998 versus 23.1 pg WHO-TEQ/g fat, p>0.05). Moreover the observed congener profiles and the total PCDD/F levels were similar to those of other European non-occupationally exposed populations. In conclusion, the 1999 PCB/dioxin incident was traceable in the plasma profiles (rise of the two specific PCDF congeners), but comparison of the results for both years indicates that the changes were too small to cause an adverse public health effect.

Analysis of Ah receptor pathway activation by brominated flame retardants.

Brominated flame-retardants (BFRs) are used as additives in plastics to decrease the rate of combustion of these materials, leading to greater consumer safety. As the use of plastics has increased, the production and use of flame-retardants has also grown. Many BFRs are persistent and have been detected in environmental samples, raising concerns about the biological/toxicological risk associated with their use. Most BFRs appear to be non-toxic, however there is still some concern that these compounds, or possible contaminants in BFRs mixtures could interact with cellular receptors. In this study we have examined the interaction of decabromodiphenyl ether, Firemaster BP4A (tetrabromobisphenol A), Firemaster PHT4 (tetrabromophthalic anhydride), hexabromobenzene, pentabromotoluene, decabromobiphenyl, Firemaster BP-6 (2,2',4,4',5,5'-hexabromobiphenyl) and possible contaminants of BFR mixtures with the Ah receptor. Receptor binding and activation was examined using the Gel Retardation Assay and increased expression of dioxin responsive genes was detected using the reporter gene based CALUX assay. The results demonstrate the ability of BFRs to activate the AhR signal transduction pathway at moderate to high concentrations as assessed using both assays. AhR-dependent activation by BFRs may be due in part to contaminants present in commercial/technical mixtures. This was suggested by our comparative analysis of Firemaster BP-6 versus its primary component 2,2',4,4',5,5'-hexabromobiphenyl. Some technical mixtures of brominated flame-retardants contain brominated biphenyls, dioxins or dibenzofurans as contaminants. When tested in the CALUX assay these compounds were found to be equivalent to, or more active than their chlorinated analogues. Relative effective potency values were determined from dose response curves for these brominated HAHs.

Levels of bisphenol-A in thermal paper receipts from Belgium and estimation of human exposure.

Bisphenol-A (BPA) is a commonly used color developer in thermal paper. In this application, BPA is present in its free, unbound form and can be readily released, making thermal paper a potential source for human exposure. In this study, BPA was determined in 44 thermal paper samples collected in Belgium. BPA was detected in all the samples; 73% of the samples had concentrations between 0.9% and 2.1% (between 9 and 21 mg BPA/g paper), while the remaining 27% of the samples had concentrations below 0.01% (0.1mg BPA/g paper). The BPA concentrations measured in thermal paper were comparable with those reported in other international studies. Since thermal paper is a feedstock for paper recycling processes, contamination of other "BPA-free" papers can occur. An estimation of human exposure through thermal paper results in a median intake of 445 ng BPA/day for the general population, which corresponds to an exposure of 6.4 ng/kg bw/day for a person of 70 kg. The exposure of those people who come occupationally in contact with thermal paper can be much higher.

Multiple testing of food contact materials: a predictive algorithm for assessing the global migration from silicone moulds.

For reasons of food safety, packaging and food contact materials must be submitted to migration tests. Testing of silicone moulds is often very laborious, since three replicate tests are required to decide about their compliancy. This paper presents a general modelling framework to predict the sample's compliance or non-compliance using results of the first two migration tests. It compares the outcomes of models with multiple continuous predictors with a class of models involving latent and dummy variables. The model's prediction ability was tested using cross and external validations, i.e. model revalidation each time a new measurement set became available. At the overall migration limit of 10 mg dm(-2), the relative uncertainty on a prediction was estimated to be ~10%. Taking the default values for α and ß equal to 0.05, the maximum value that can be predicted for sample compliance was therefore 7 mg dm(-2). Beyond this limit the risk for false compliant results increases significantly, and a third migration test should be performed. The result of this latter test defines the sample's compliance or non-compliance. Propositions for compliancy control inspired by the current dioxin control strategy are discussed.

A review of dietary and non-dietary exposure to bisphenol-A.

Due to the large number of applications of bisphenol-A (BPA), the human exposure routes are multiple. We aimed to review shortly the food and non-food sources of BPA, and to evaluate their contribution to the human exposure. Food sources discussed here include epoxy resins, polycarbonate and other applications, such as paperboard and polyvinylchloride materials. Among the non-food sources, exposures through dust, thermal paper, dental materials, and medical devices were summarized. Based on the available data for these exposure sources, it was concluded that the exposure to BPA from non-food sources is generally lower than that from exposure from food by at least one order of magnitude for most studied subgroups. The use of urinary concentrations from biomonitoring studies was evaluated and the back-calculation of BPA intake seems reliable for the overall exposure assessment. In general, the total exposure to BPA is several orders of magnitude lower than the current tolerable daily intake of 50 µg/kg bw/day. Finally, the paper concludes with some critical remarks and recommendations on future human exposure studies to BPA.

Are potential sources for human exposure to bisphenol-A overlooked?

This review summarizes the numerous applications of bisphenol-A (BPA) and the potential sources for human exposure. The exposure to humans is believed to occur mainly through food contamination from polycarbonate bottles, as well as through food and beverage cans coated with epoxy resins. However, there seems to be a discrepancy between exposure assessments based on biomonitoring data and those based on food/drink concentrations. Several recent studies indicated also the importance of non-food sources. Although the main use of BPA is polymerization to polycarbonate and epoxy resins, it can also be used as an additive, from which it may be easily released. Several studies have already provided scientific evidence for the contribution of sources for dermal BPA absorption, such as thermal paper where BPA is used as an additive. Polymeric applications of BPA require further investigation regarding the amounts of BPA present, as well as the factors affecting its release and potential dermal or non-dermal exposure from these sources. It is clear that not all sources of BPA have been identified. This overview emphasizes the necessity to study also the exposure to these unexpected sources of BPA.

Intake of bisphenol A from canned beverages and foods on the Belgian market.

Bisphenol A (BPA), a contaminant which may be present in the coating of cans, was determined in 45 canned beverages and 21 canned food items from the Belgian market. Beverages had an average BPA concentration of 1.0 ng/ml, while canned foods had a higher average concentration of 40.3 ng/g. The amount of BPA present in food items was dependent on the type of can and sterilisation conditions rather than the type of food. For example, BPA was not detected in non-canned beverages (<0.02 ng/ml), while non-canned food items had a very low average concentration of 0.46 ng/g. Using detailed information from the Belgian food consumption survey, the BPA intake of adults through canned foods and beverages was estimated to be 1.05 µg/day or 0.015 µg/kg body weight/day (assuming an average adult weight of 70 kg). Intake assessments, based on urinary metabolite concentrations from the literature, resulted in slightly higher BPA intakes (range 0.028-0.059 µg/kg body weight/day). This suggests that sources other than canned foods and beverages contribute to BPA exposure in humans.

Trace elements in home-produced eggs in Belgium: levels and spatiotemporal distribution.

The purpose of this study was to evaluate the levels of arsenic, cadmium, lead, copper and zinc in home-produced eggs, soils and kitchen waste samples of private chicken owners in Belgium, and to determine spatiotemporal differences in trace element contents in eggs. Eggs were sampled in all provinces of Belgium in autumn 2006 and spring 2007. A total number of 59 private chicken owners participated in the study. Trace elements were determined by inductively coupled plasma-mass spectrometry except for mercury, which was determined by atomic absorption of mercury vapour. The mean fresh weight concentrations in eggs in autumn and spring respectively were <8.0 and <8.0 microg/kg for arsenic, 0.5 and <0.5 microg/kg for cadmium, 116 and 74 microg/kg for lead, 0.43 and 0.52 mg/kg for copper, 20.3 and 19.2 mg/kg for zinc, and 3.15 and 4.44 microg/kg for mercury. Analysis of variance determined significant differences in some trace element concentrations in eggs among seasons and regions in Belgium. Average concentrations of arsenic, cadmium and mercury corresponded well with values measured in other countries, while copper and zinc concentrations were within the same order of magnitude as in other countries. Average lead concentrations were high compared to concentrations in eggs from other countries and correlated well with lead concentrations in soil, indicating that the soil is an important source. Other sources of trace elements in eggs might be home-grown vegetables and forage (grass and herbs), and indirectly, air pollution.

Brominated flame retardants in Belgian home-produced eggs: levels and contamination sources.

The extent and the sources of contamination with brominated flame retardants (BFRs), such as polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD), in home-produced eggs from free-foraging chicken of Belgian private owners were investigated. Various factors, such as seasonal variability, exposure of chickens through diet (kitchen waste) and soil, and elimination of BFRs through eggs and faeces were assessed. PBDEs were more important than HBCD in terms of concentrations and detection frequency. Concentrations of PBDEs and HBCD in Belgian home-produced eggs were relatively low and comparable with reported levels from other European countries and the US. The concentrations of PBDEs (sum of 13 congeners, including BDE 209) ranged between not detected and 32 ng/g lipid weight (lw), with medians of 3.0 and <2.0 ng/g lw for the autumn 2006 and spring 2007 campaigns, respectively. When present, BDE 209 was the major PBDE congener (45% of sum PBDEs). When BDE 209 was not detected, the PBDE profile was composed of PentaBDE (BDE 99 and BDE 47), with, in some cases, higher contribution of OctaBDE (BDE 183 and BDE 153). HBCD was also detected (<0.4 and 2.9 ng/g lw for the autumn 2006 and spring 2007 campaigns, respectively), but at lower detection frequency. The highest HBCD value was 62 ng/g lw. The similarity between profiles and seasonal variations in the concentrations of BFRs in soil and eggs indicate that soil is an important source, but not the sole source, for eggs laid by free-foraging chicken. The contamination of eggs with PBDEs and HBCD appears to be of low concern for public health and the contribution of eggs to the total daily intake of PBDEs appears to be limited (10% for chicken owners and 5% for the average Belgian consumer).

Dioxin and dioxin-like activity in sediments of the Belgian coastal area (Southern North Sea).

Dioxin and dioxin-like activity in sediments of the North Sea, along the Belgian coast, was assessed with the bioassay CALUX (Chemically Activated LUciferase gene eXpression). Crude extracts of the samples as well as the dioxin fraction (PCDD/Fs) obtained after a thorough clean-up procedure were analyzed with the CALUX method. When analyzing the cleaned extract, a general low contamination level is observed (around 0.1 pg CALUX-TEQ/g sediment), except at the mouth of the two main rivers-the Yser and the Scheldt-where concentrations measured are about 100 times higher (10-42 pg CALUX-TEQ/g sediment). Much higher potencies are measured for the crude extracts compared to the cleaned ones. In the crude extracts, the highest dioxin-like activities were again observed at the mouth and outflow of the two rivers (600-7200 pg CALUX-TEQ/g sediment). These activities are at least two orders of magnitude higher than the ones found at the coastal and sea stations (1.3-45 pg CALUX-TEQ/g sediment). The difference in activity between cleaned and crude sediment extracts is due to the presence of dioxin-like compounds such as, for example, non-ortho and mono-ortho polychlorinated biphenyls and polybrominated dioxins, but also to PAHs. The percentage of five major PAHs in the crude samples at the river mouths, when using the average activities in those samples, varies between 25% and 50%.

Assessment of quality performance parameters for straight line calibration curves related to the spread of the abscissa values around their mean.

In validation of quantitative analysis methods, knowledge of the response function is essential as it describes, within the range of application, the existing relationship between the response (the measurement signal) and the concentration or quantity of the analyte in the sample. The most common response function used is obtained by simple linear regression, estimating the regression parameters slope and intercept by the least squares method as general fitting method. The assumption in this fitting is that the response variance is a constant, whatever the concentrations within the range examined. The straight calibration line may perform unacceptably due to the presence of outliers or unexpected curvature of the line. Checking the suitability of calibration lines might be performed by calculation of a well-defined quality coefficient based on a constant standard deviation. The concentration value for a test sample calculated by interpolation from the least squares line is of little value unless it is accompanied by an estimate of its random variation expressed by a confidence interval. This confidence interval results from the uncertainty in the measurement signal, combined with the confidence interval for the regression line at that measurement signal and is characterized by a standard deviation s(x0) calculated by an approximate equation. This approximate equation is only valid when the mathematical function, calculating a characteristic value g from specific regression line parameters as the slope, the standard error of the estimate and the spread of the abscissa values around their mean, is below a critical value as described in literature. It is mathematically demonstrated that with respect to this critical limit value for g, the proposed value for the quality coefficient applied as a suitability check for the linear regression line as calibration function, depends only on the number of calibration points and the spread of the abscissa values around their mean.

Chemically activated luciferase gene expression (CALUX) cell bioassay analysis for the estimation of dioxin-like activity: critical parameters of the CALUX procedure that impact assay results.

The chemically activated luciferase gene expression (CALUX) in vitro cell bioassay is a bioanalytical tool that is increasingly being used by research and commercial laboratories for the screening and relative quantification of dioxins and dioxin-like compounds in sample extracts. Since CALUX analyses provide a biological response to all aryl hydrocarbon receptor active compounds present in a given sample extract containing a complex mixture of chemicals, interpretation of results is significantly more complexthan of chemical analyses. Operators in the laboratory can adjust many parameters when performing CALUX analyses, and the applied procedure strongly affects the result and, hence, the interpretation of the results. This paper examines critical methodological parameters and aspects of the CALUX bioassay that can affect the quality and accuracy of the analyses. Moreover, the study aims to identify the ways that alteration of these parameters influences CALUX measurements. A greater understanding of these characteristics will lead to increased accuracy, precision, and reproducibility of the widely used CALUX bioassay within and between research laboratories.

Validation and interpretation of CALUX as a tool for the estimation of dioxin-like activity in marine biological matrixes.

Among the different analytical tools proposed as an alternative to the very expensive gas chromatography high-resolution mass spectrometry (GC-HRMS) analyses of polychlorodibenzo-p-dioxin and polychlorodibenzofurans, Chemically Activated LUciferase gene eXpression (CALUX) in vitro cell bioassay is very promising. It allows the analyses of a high number of samples since it is relatively fast, inexpensive, and sensitive. However, this technique is not yet widely applied for screening or environmental monitoring. The main reasons are probably the lack of validation and the difficulty in interpreting the global biological response of the bioassay. In this paper, the strict quality control criteria set up for the validation of CALUX are described. The validation has shown good repeatability (relative standard deviation (RSD) = 9%) and good within-lab reproducibility (RSD = 15%) of the results. The quantification limit, in the conditions applied in this paper, is 1.25 pg CALUX-TEQ/g fat. Comparison of CALUX and GC-HRMS analysis was made forvarious marine matrixes (fishes, mussels, starfishes, sea birds, and marine mammals). Good correlations are usually observed, but there are systematic differences between the results. Attempts are made to identify the origin of the discrepancy between the two methods.

Study of the interference problems of dioxin-like chemicals with the bio-analytical method CALUX.

The reporter gene expression method CALUX has proven to be a very valuable screening technique for assessing toxic equivalents of dioxins and dioxin-like compounds, because it detects all AhR ligands in a variety of sample matrices. However, the exact meaning of the CALUX response is difficult to evaluate for complex mixtures mainly since not all AhR ligands are known and since antagonistic or synergistic effects occurs. In this paper, non-additive effects on the CALUX response of dioxins were investigated for a limited number of dioxin-like compounds in concentration ranges that are 10(2)-10(8) times higher than that of 2,3,7,8-TCDD. Antagonistic effects are detected for three Aroclors (1242, 1254, 1260), Halowax 1014 (PCN), HCB and PBB 169. The ratios, Aroclor/dioxin, Halowax/dioxin and HCB/dioxin, needed to observe an antagonistic effect are 10000, 5000 and 50000, respectively. No significant deviation from additivity was observed for Aroclor 5442 (PCT) and PBB 77 in the concentration range investigated. Two clean-up procedures have also been tested: in some cases the non-additive effects disappeared or were strongly reduced. Using only an acidic silica column, the classical dioxin-like compounds investigated here (PCB, PCT, PBB, PCN, HCB) as well as the dioxins are collected and analyzed altogether in one fraction. Consequently, no major alteration of the non-additive effects is expected. An acidic silica column combined with an activated carbon column allows the separation of PCDD/F and dioxin-like PCB in two different fractions, PBB 169 is completely eluted in the dioxin fraction and PBB 77 is distributed between the PCB and dioxin fraction. HCB is completely separated from the PCDD/F fraction.

Enhanced levels of dioxins in eggs from free range hens; a fast evaluation approach.

As a consequence of an initiative of the local authorities of the city of Antwerp (Belgium), dioxin levels were determined in eggs from free range hens owned by private owners in the Northern districts of Antwerp. The reasons for this survey stem from some fears that free range eggs could be contaminated by local environmental sources (e.g. soil, grass, earthworms) as a result of the presence in this area of intensive industrial and domestic activities. The analyses revealed high levels of PCDD/F in the home-produced eggs (average = 9.9pg WHO-TEQ per g of fat; n = 15). An evaluation of the available results has been carried out by the Scientific Committee of the Belgian Federal Agency for the Safety of the Food Chain. From this evaluation, it appeared that the analysis of congener profiles was of limited use because all profiles were dominated by the OCDD congener, independently of the level of contamination. There were not enough indications allowing a causal link to be established between high dioxin levels in eggs and soil contamination and, on the other hand, it was assumed that other factors such as feeding habits, physiological state and egg laying rhythm of the hens could not be ruled out as potential causes of aggravation. A quick risk assessment led to the conclusion that the impact of this contamination is highly relevant for the consumer's health due to the important contribution of such home-produced eggs in the estimations of total body burden.

Interpretation of CALUX results in view of the EU maximal TEQ level in milk.

Analyses of dioxins in food have become increasingly important since the European Commission has enforced maximal toxic equivalent concentration (TEQ) levels in various food and feed products. Screening methodologies are usually used to exempt those samples that are below the maximum permitted limit and that can, therefore, be released to the market. In addition, one needs to select those samples that require confirmation of their dioxin TEQ level. When bioassays are used as screening tools, the interpretation of the obtained results should consider the higher variability and uncertainty associated with them. This paper explores the use of CALUX data as quantitative screening results. The validation of the method for the polychlorinated dibenzo-p-dioxins (PCDD)/F TEQ determination in milk samples is described with emphasis on the decision limit (CC(alpha)) and the precision of the method. The decision limit amounts to 4.53pg TEQ/g fat. Repeatability and within-lab reproducibility coefficients of variation are below 30%. The newly introduced parameter CC(alpha)(*) of 1.47pg TEQ/g fat delimits with CC(alpha) a range of suspicious results. These data are not significantly different from the maximum limit of 3pg TEQ/g fat and should be confirmed by a confirmatory analytical method such as HRGC-HRMS.

Importance of REP values when comparing the CALUX bioassay results with chemoanalyses results Example with spiked vegetable oils.

Differences between chemical activated luciferase gene expression (CALUX) bioassay and chemoanalyses results are observed. This paper shows that calculations of the TEQ values using REP values instead of WHO TEF values give different results. The REP values do affect the results obtained by the CALUX technique. These differences are more marked for the dioxin like PCB compounds (CALUX TEQ values are lower than WHO TEQ values) than for the dioxin compounds (CALUX TEQ values are higher than WHO TEQ values). The CALUX results were compared with the concentrations of the congeners' spiked into the oil.