Endocrine activity in an urban river system and the biodegradation of estrogen-like endocrine disrupting chemicals through a bio-analytical approach using DRE- and ERE-CALUX bioassays.

The Zenne River, crossing the Brussels region (Belgium) is an extremely urbanized river impacted by both domestic and industrial effluents. The objective of this study was to monitor the occurrence and activity of Endocrine Active Substances (EAS) in river water and sediments in the framework of the Environmental Quality Standards Directive (2008/105/EC and 2013/39/EU). Activities were determined using Estrogen and Dioxin Responsive Elements (ERE and DRE) Chemical Activated Luciferase Gene Expression (CALUX) bioassays. A potential contamination source of estrogen active compounds was identified in the river at an industrial area downstream from Brussels with a peak value of 938 pg E2 eq./L water (above the EQS of 0.4 ng/L) and 195 pg E2 eq./g sediment. Estrogens are more abundantly present in the sediments than in the dissolved phase. Principal Component Analysis (PCA) showed high correlations between Suspended Particulate Matter (SPM), Particulate (POC) and Dissolved Organic Carbon (DOC) and estrogenic EAS. The dioxin fractions comply with previous data and all were above the United States Environmental Protection Agency (US EPA) low-level risk, with one (42 pg TCDD eq./g sediment) exceeding the high-level risk value for mammals. The self-purifying ability of the Zenne River regarding estrogens was examined with an in vitro biodegradation experiment using the bacterial community naturally present in the river. Hill coefficient and EC50 values (Effective Concentration at 50%) revealed a process of biodegradation in particulate and dissolved phase. The estrogenic activity was decreased by 80%, demonstrating the ability of self-purification of estrogenic compounds in the Zenne River.

Characterisation and implementation of the ERE-CALUX bioassay on indoor dust samples of kindergartens to assess estrogenic potencies.

Estrogen-like endocrine disrupting chemicals (EEDCs) can be found abundantly in the environment. Due to their low-dose effects and the large amount of unknown EEDCs, it is difficult to assess and manage possible human health risks. For young children, who are particularly vulnerable to endocrine disruption due to their development rate, indoor dust is one of the main routes of exposure. In this study, an estrogen responsive elements chemically activated luciferase gene expression (ERE-CALUX) bioassay was characterized and implemented for the analysis of 12 dust samples from kindergartens in Flanders and Brussels (Belgium). The human ovarian carcinoma BG 1CALUX cell line showed reproducible results and a low limit of detection (LOD). The effective concentration at 50% of the maximum response (EC50) yielded 497 fg/well, while the LOD was 16 fg/well. For all dust samples, full dose-response curves and their corresponding EC50 values could be calculated. All samples yielded bio-analytical equivalent concentrations (BEQs) that were significantly higher than the procedural blank level and ranged from 426 to 8710 pg E2 equivalents/g dust. A clear relationship was observed between a semi-quantitative interior score and the ERE-CALUX response of the samples. In addition, the concentration of phthalates, a major group of EEDCs used as plasticizers in plastics, was determined in the samples by GC-MS. Diisoheptyl phthalate (DiHP) and di(2-ethylhexyl) phthalate (DEHP) were present in every dust sample. A good correlation was found between ERE-CALUX activities and phthalate concentrations, when all phthalates except diisononyl phthalate (DiNP) and diisodecyl phthalate (DiDP), which do not bind to the estrogen receptor, were taken into account. This shows that the ERE-CALUX can provide relevant results concerning exposure to EEDCs from indoor dust. This article is part of a Special Issue entitled 'Endocrine disruptors & steroids'.

Dioxin levels in fertilizers from Belgium: determination and evaluation of the potential impact on soil contamination.

Dioxins are harmful persistent organic pollutants (POPs) to which humans are exposed mostly via the consumption of animal products. They can enter the food chain at any stage, including crop fertilization. Fertilizers belong to several categories: synthetic chemicals providing the essential elements (mostly N, P and K) that are required by the crops but also organic fertilizers or amendments, liming materials, etc. Ninety-seven samples of fertilizers were taken in Belgium during the year 2011 and analyzed after a soft extraction procedure for polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (DL-PCBs) using GC-IDHRMS. Only small qualitative differences could be observed between the main fertilizer categories since the PCDD:PCDF:DL-PCB average ratio obtained with the results expressed in TEQ was often close to 30:30:40 (typically for sewage sludge) or 40:30:30 (typically for compost). The median dioxin levels determined were generally lower than recorded previously and were the highest for sewage sludge and compost (5.6 and 5.5 ng TEQ/kg dry weight (dw), respectively). The levels in other fertilizers were lower including manure for which the median value was only 0.2 ng TEQ/kg dw. Several fertilization scenarios relying on the use of those fertilizers were assessed taking into consideration the application conditions prevailing in Belgium. From this assessment it could be concluded that the contribution of fertilizers to the overall soil contamination will be low by comparison of other sources of contamination such as atmospheric depositions. At the field scale, intensive use of compost and sewage sludge will increase dramatically the dioxin inputs compared with other fertilization practices but this kind of emission to the soil will still be relatively low compared to the dioxin atmospheric depositions.

Potential impact of fertilization practices on human dietary intake of dioxins in Belgium.

Dioxins can enter the food chain at any stage, including crop fertilization. Therefore, we developed a simple method for estimating the introduction of dioxins in the food chain according to various fertilization practices. Using dioxin's contamination data taken from the literature, we estimated that fertilization accounts for approximately 20% of the dioxin inputs on agricultural soils at country scale. For the estimations at the field scale, 6 fertilization scenarios were considered: sludge, compost, digestate, manure, mineral fertilizers, and a common fertilization scenario that corresponds to an average situation in Belgium and combines mineral and organic fertilizers. According to our first estimations, mineral fertilizers, common fertilization practices or manure bring less than 1 ng TEQ/m² while atmospheric deposition or digestate bring between 1 and 3 ng TEQ/m² and sludge or compost bring more than 3 ng TEQ/m². The use of solid fertilizers could potentially increase the dioxin levels in the 30 cm agricultural soil layer by 0 to ~1.5% per year (up to ~9% for the 5 cm thick surface layer). For animals, the increase in dioxin ingestion linked to the fertilization practices is lower than 1% for most scenarios with the exception of the compost scenario. Increases in human dietary intake of dioxin are estimated to be lower than 1% for conventional rearing methods (i.e. grazing animals are reared outdoor while pigs and poultry are reared indoor). Spraying liquid fertilizers on meadows and fodder crops, even if very limited in practice, deserves much more attention because this application method could theoretically lead to higher dioxin's intake by livestock (from 6 to ~300%). Considering an average half-life of dioxins in soils of 13 years, it appears that the risks of accumulation in soils and in the food chain are negligible for the various fertilization scenarios.

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.

Average daily nitrate and nitrite intake in the Belgian population older than 15 years.

The aim of this study was to assess the dietary intake of nitrate and nitrite in Belgium. The nitrate content of processed vegetables, cheeses and meat products was analysed. These data were completed by data from non-targeted official control and from the literature. In addition, the nitrite content of meat products was measured. Concentration data for nitrate and nitrite were linked to food consumption data of the Belgian Food Consumption Survey. This study included 3245 respondents, aged 15 years and older. Food intakes were estimated by a repeated 24-h recall using EPIC-SOFT. Only respondents with two completed 24-h recalls (n=3083) were included in the analysis. For the intake assessment, average concentration data and individual consumption data were combined. Usual intake of nitrate/nitrite was calculated using the Nusser method. The mean usual daily intake of nitrate was 1.38 mg kg(-1) bodyweight (bw) day(-1) and the usual daily intake at the 97.5 percentile was 2.76 mg kg(-1) bw day(-1). Exposure of the Belgian population to nitrate at a mean intake corresponded to 38% of the ADI (while 76% at the 97.5 percentile). For the average consumer, half of the intake was derived from vegetables (especially lettuce) and 20% from water and water-based drinks. The average daily intake of nitrate and nitrite from cheese and meat products was low (0.2% and 6% of the ADI at average intake, respectively). Scenario analyses with a higher consumption of vegetables or a higher nitrate concentration in tap water showed a significant higher intake of nitrate. Whether this is beneficial or harmful must be further assessed.

Estimate of intake of sulfites in the Belgian adult population.

An exposure assessment was performed to estimate the usual daily intake of sulfites in the Belgian adult population. Food consumption data were retrieved from the national food consumption survey. In a first step, individual food consumption data were multiplied with the maximum permitted use levels for sulfites, expressed as sulphur dioxide, per food group (Tier 2). In a second step, on the basis of a literature review of the occurrence of sulfites in different foods, the results of the Tier 2 exposure assessment and available occurrence data from the control programme of the competent authority, a refined list of foods was drafted for the quantification of sulphite. Quantification of sulphite was performed by a high-performance ion chromatography method with eluent conductivity detector in beers and potato products. Individual food consumption data were then multiplied with the actual average concentrations of sulfite per food group, or the maximum permitted levels in case actual levels were not available (partial Tier 3). Usual intakes were calculated using the Nusser method. The mean intake of sulfites was 0.34 mg kg(-1) bw day(-1) (Tier 2), corresponding to 49% of the acceptable daily intake (ADI) and 0.19 mg kg(-1) bw day(-1), corresponding to 27% of the ADI (partial Tier 3). The food group contributing most to the intake of sulfites was wines. The results showed that the intake of sulfites is likely to be below the ADI in Belgium. However, there are indications that high consumers of wine have an intake around the ADI.

Estimate of intake of benzoic acid in the Belgian adult population.

An exposure assessment was performed to estimate average daily benzoic acid intake for Belgian adults. Food consumption data were retrieved from the national food-consumption survey. As a first step, individual food-consumption data were multiplied with the maximum permitted use levels for benzoic acid per food group (Tier 2). As a second step, a label survey to identify the foods where benzoic acid is effectively used as an additive and a literature review of the possible occurrence of benzoic acid as a natural substance were performed. With this information, a refined list of foods was drafted for the quantification of benzoic acid, which was performed by a high-performance liquid chromatography (HPLC) method, optimized and validated for this purpose. Individual food-consumption data were then multiplied with the actual average concentrations of benzoic acid per food group (Tier 3). Usual intakes were calculated using the Nusser method. The mean benzoic acid intake was 1.58 mg kg(-1) body weight day(-1) (Tier 2) and 1.25 mg kg(-1) body weight day(-1) (Tier 3). In Tier 2, men exceeded the acceptable daily intake (ADI) of 5 mg kg(-1) body weight day(-1) at the 99th percentile. The greatest contributors to the benzoic acid intake were soft drinks. Benzoic acid as a natural substance represents only a small percentage of the total intake. The results show that actual benzoic acid intake is very likely to be below the ADI. However, there is a need to collect national food-consumption data for children as they might be more vulnerable to an excessive intake.

Optimization and validation of a liquid chromatography tandem mass spectrometry (LC/MSn) method for analysis of corticosteroids in bovine liver: evaluation of Keyhole Limpet beta-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.

PCDD/Fs and dioxin-like PCBs in home-produced eggs from Belgium: levels, contamination sources and health risks.

This paper discusses the dioxin TEQ levels as determined by the chemically activated luciferase gene expression assay (CALUX) and by HRGC-HRMS in eggs, soils, faeces and kitchen waste samples obtained in the CONTEGG study. The samples were collected in each Belgian province at private homes and in small gardens where chickens are held. The CALUX levels for eggs sampled in autumn were higher than the levels in eggs obtained at the same locations in spring (median values of 5.86 and 4.08 pg CALUX TEQ/g fat, respectively). The total WHO-TEQ levels in eggs, determined by HRGC-HRMS, ranged from 3.29 to 95.35 pg TEQ/g fat in autumn and from 1.50 to 64.79 pg TEQ/g fat in spring. In the soils on which the chickens forage, levels of 2.51-11.35 pg I-TEQ/g in autumn and 2.00-7.86 pg I-TEQ/g in spring were found. The congener pattern of PCDD/Fs in the eggs, soils and faeces was dominated by OCDD, in addition to 1,2,3,4,6,7,8-HeptaCDD, OCDF and 1,2,3,4,6,7,8-HeptaCDF. The predominant dioxin-like PCBs were PCB118, PCB 105 and PCB 156. The dioxin-like PCBs contributed on average 47%, 14% and 20% to the total WHO-TEQ in eggs, soils and faeces, respectively. Kitchen waste samples were very low-contaminated with dioxin-like compounds. The present results showed a good agreement between egg and soil TEQ levels for PCDD/Fs but not for dioxin-like PCBs. This study showed that current soil levels found in some private gardens do not lead to egg levels below the current EU maximal level of 6 pg total TEQ/g fat for dioxins and dioxin-like PCBs. The consumers of the analysed eggs attained 5-79% of the tolerable weekly intake (TWI) of 14 pg TEQ/kg bw for dioxins and dioxin-like PCBs by exposure to their home-produced eggs only.

PCB and organochlorine pesticides in home-produced eggs in Belgium.

The level of polychlorinated biphenyls (PCB) and persistent organochlorinated pesticides (OC) in home-produced eggs was investigated in Belgium. The concentration of dichlorodiphenytrichloroethane (DDT) is above the norm for 17% of the eggs collected during the spring on 58 different locations. For PCB, aldrin, dieldrin, and chlordane, 3-5% of the samples are above the norm too. These levels are surprisingly high for compounds banned for about 30 years. Higher concentrations in home-produced eggs are expected compared to battery eggs because of contact with the environment and especially the soil. For ten selected locations, the concentration in soils, excreta and feed was measured, but no simple correlation between egg and feed or soil level could be established. Hexachlorohexane, endosulfan, endrin, methoxychlor and nitrofen were not detected in any sample.

Assessment of the chemical contamination in home-produced eggs in Belgium: general overview of the CONTEGG study.

This overview paper describes a study conducted for the Belgian Federal Public Service of Health, Food Chain Safety and Environment during 2006-2007. Home-produced eggs from Belgian private owners of hens were included in a large study aiming to determine concentration levels of various environmental contaminants. By means of the analyses of soil samples and of kitchen waste samples, obtained from the same locations, an investigation towards the possible sources of contaminants was possible. Eggs, soils, faeces and kitchen waste samples were checked for the presence of dioxins, PCBs (including dioxin-like PCBs), organochlorine pesticides, trace elements, PAHs, brominated flame retardants and mycotoxins. The study design, sampling methodology and primary conclusions of the study are given. It was found that in some cases dioxin-like compounds were present at levels that are of concern for the health of the egg consumers. Therefore, measures to limit their contamination in eggs, produced by hens of private owners, were proposed and deserve further attention.

Mycotoxin analyses in some home produced eggs in Belgium reveal small contribution to the total daily intake.

Low levels of deoxynivalenol (DON, range: 2.6-17.9 ng/g) and its metabolite de-epoxy-DON (DOM-1, range: 2.4-23.7 ng/g) were found in 20 home-produced egg samples collected in Belgium during autumn 2006 (from 10 breeders) and spring 2007 (same breeders). DON intake assessment showed that the consumption of these eggs may contribute to less than 1% of the provisional maximum tolerable daily intake of 1 microg/kg body weight established by FAO/WHO. None of the egg samples analyzed had quantifiable levels of zearalenone (ZEA), alpha-zearalenol, beta-zearalenol, ochratoxin A (OTA) and citrinin (CIT). Intake of DON, ZEA, OTA and CIT via the consumption of home produced eggs seems not to be a matter of concern. Despite this, we recommend to continue in screening other eggs allowing to increase the sample size and the subsequent conclusion for mycotoxin contamination in eggs. As home produced food is generally not submitted to any compliance control and may be consumed in large quantities by their producers and other household members, it is worthwhile to further pay attention to the quality of feed as well as the environment in which the hens live.

Analysis of fluoride in toothpastes on the Belgian market.

We developed a method for the quantitative determination of sodium fluoride (NaF), sodium monofluorophosphate (SMFP) and amine fluoride (AmF) in toothpastes on the Belgian market. Samples were suspended in water and the determination was made using anion chromatography with conductivity detection after chemical suppression. The described ion chromatographic method is an easy and reliable isocratic high-performance liquid chromatography method for the determination of total soluble fluoride content in toothpastes. The analytical repeatability and reproducibility, the matrix effects and the method's decision limit of three different toothpastes containing NaF, SMFP and AmF at a concentration close to the permitted one of 0.15 g % total F are determined. All the samples analysed are in conformity with the Cosmetics Directive 76/768/EC; none exceeds the limit of 0.15 g %.

PCBs and PCDD/FS in fish and fish products and their impact on the human body burden in Belgium.

The concentrations of marker PCBs (28, 52, 101, 118, 138, 153, 180) in fish have been assessed with GC-MS: an average concentration of 540 ng-PCB g(-1) fat (5.02 ng-PCB g(-1) wet weight) was observed. The average concentration of PCDD/Fs, assessed with the CALUX bioassay, amounted to 64 pg-CALUX-TEQ g(-1) fat (0.58 pg-CALUX-TEQ g(-1) wet weight) and that of PCDD/Fs + dioxin-like PCBs amounted to 131 pg-CALUX-TEQ g(-1) fat (1.18 pg-CALUX-TEQ g(-1) wet weight). Results of the PCB congeners analyses show that PCB-153 is the most abundant congener in almost all samples, with also main contributions of the 138- and 180-congeners. For some species such as the sand sole and lemon sole, a fairly constant PCB content, independent of the fat percentage, was observed. For a second group of species such as whelks, cod, and whiting, a positive correlation was observed between their PCB concentration (ng g(-1) fat) and their % of fat. The relationship between marker PCBs and PCDD/Fs concentrations, when plotted on a log scale, fits a straight line (correlation coefficient r = 0.83). With our results on fish and literature data for other food products, intake of marker PCBs and PCDD/Fs could be calculated for the adult population in Belgium (19-60 years old). The Total Daily Intake (TDI) of marker PCBs (ng-PCB day(-1)) ranges between 1690 and 2210. The TDI of PCDD/Fs (pg-CALUX day(-1)) ranges between 80.5 and 122, that of PCDD/Fs + dioxin-like PCBs amounts to 151. When PCDD/Fs in fish are assessed with GC-HRMS, the TDI can be lower. The relative importance of fish regarding marker PCB intake amounts to 15-19%, while regarding PCDD/Fs intake it amounts to 34-51%. Using TDI, the body burden evolution of marker PCBs and PCDD/Fs, with age has been calculated.

Chemical contamination of free-range eggs from Belgium.

The elements manganese, cobalt, nickel, copper, zinc, arsenic, selenium, molybdenum, cadmium, antimony, thallium, lead and mercury, and selected persistent organochlorine compounds (dioxins, marker and dioxin-like polychlorinated biphenyls, dichlorodiphenyltricholroethane (DDT) and metabolites as well as other chlorinated pesticides) were analysed in Belgian free-range eggs obtained from hens of private owners and of commercial farms. It was found that eggs from private owners were more contaminated than eggs from commercial farms. The ratios of levels in eggs from private owners to the levels in eggs from commercial farms ranged from 2 to 8 for the toxic contaminants lead, mercury, thallium, dioxins, polychlorinated biphenyls and the group of DDT. DDT contamination was marked by the substantial presence of p,p'-DDT in eggs from private owners in addition to dichlorodiphenyldichloroethylene (p,p-DDE) and dichlorodiphenyl-dichloroethane (p,p'-DDD). It is postulated that environmental pollution is at the origin of the higher contamination of eggs from private owners. Extensive consumption of eggs from private owners is likely to result in toxic equivalent quantity intake levels exceeding the tolerable weekly intake.

Open digestion under reflux for the determination of total arsenic in seafood by inductively coupled plasma atomic emission spectrometry with hydride generation.

A method for the determination of arsenic (As) in seafood by inductively coupled plasma atomic emission spectrometry with continuous hydride generation is described. Several analytical parameters have been investigated and optimised. The analytical features of the method (recovery, precision, accuracy and limit of detection) were calculated. Practical detection limit of 3.6mug/kg fresh weight for As has been reached. The precision of the method expressed as relative standard deviation (R.S.D.) was in the range of 2.7-3.7% and the recovery percentage ranged from 98.4 to 101.8%. The reliability of the developed method was checked by analysing several certified reference materials. A complete mineralization was obtained for arsenobetaine (AsB) containing reference material with a mixture of nitric and sulphuric acids followed by adding hydrogen peroxide in an open digestion system. This method can be applied to routine analysis without any risks of interferences.

Validation of the CALUX bioassay for PCDD/F analyses in human blood plasma and comparison with GC-HRMS.

Following the dioxin crisis of 1999, several studies were conducted to assess the impact of this crisis on the dioxin body burden in the Belgian population. The Scientific Institute of Public Health identified a population from whom plasma samples were available and from whom, during the follow up survey, plasma samples were obtained in 2000. In total, 496 samples were collected for GC-HRMS and CALUX analyses to verify statistical assessment conclusions. This study was seen as an opportunity to validate the CALUX bioassay for biological sample analysis and to compare toxic equivalency (TEQ) values obtained by the reference GC-HRMS technique and by the screening method. This article focuses on the validation results of the CALUX bioassay for the analyses of the dioxin fractions of blood plasma. The sample preparation is based on a liquid-liquid extraction, followed by an acid silica in series with an activated carbon clean-up. A good recovery (82%) and reproducibility (coefficient of variation less than 25%) were found for this method. Based on 341 plasma samples, a significant correlation was established between the bioassay and chemical method (R = 0.64). However, a proportional systematic error was observed when the results obtained with the CALUX bioassay were regressed with the results from the GC-HRMS analyses. The limit of quantification (LOQ) used to calculate TEQ values from the GC-HRMS determinations, the use of the relative potency values instead of the toxic equivalent factor and the potential of CALUX bioassay to measure all compounds with affinity for the AhR may partly explain this proportional systematic error. Nevertheless, the present results suggest that the CALUX bioassay could be a promising valid screening method for human blood plasma analyses.

The international validation of bio- and chemical-analytical screening methods for dioxins and dioxin-like PCBs: the DIFFERENCE project rounds 1 and 2.

The European research project DIFFERENCE is focussed on the development, optimisation and validation of screening methods for dioxin analysis, including bio-analytical and chemical techniques (CALUX, GC-LRMS/MS, GCxGC-ECD) and on the optimisation and validation of new extraction and clean-up procedures. The performance of these techniques is assessed in an international validation study and the results are compared with the reference technique GC-HRMS. This study is set up in three rounds and is in accordance with the International Harmonized Protocol for Proficiency Studies and the ISO 5725 standard. The results of the first two rounds are very promising in particular for GC-LRMS/MS. The results obtained with this technique were as accurate as the results reported by the labs using the GC-HRMS. The initial results reported for GCxGC-ECD overestimate the dioxin concentration in the samples. The results reported by the labs using the CALUX technique underestimate the total TEQ concentrations in the samples, compared to the GC-HRMS reference method. The repeatability of the CALUX is significantly higher than the other screening techniques. It was shown that accelerated solvent extraction (ASE) is a valid alternative extraction and clean-up procedure for fish oil and vegetable oil. The results obtained with CALUX and GC-HRMS after ASE are equivalent to the results obtained with the classical extraction and purification procedures.

Importance of clean-up for comparison of TEQ-values obtained by CALUX and chemo-analysis.

This paper presents Chemically Activated LUciferine gene eXpression (CALUX) TEQ-values obtained for nine plasma samples following two different purification procedures, one of them involving fractionation. CALUX results obtained for the dioxin (DX) and dioxin + PCB (DX + PCB) fractions were then compared to the GC-HRMS TEQ-values calculated for the 17 polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (17 PCDD/F) and 17 PCDD/F + 4 cPCB congeners, respectively. The overestimation of the CALUX (DX fraction) TEQ-values in comparison with the chemo-analyses of the 17 PCDD/F is mainly explained by the presence of other AhR agonists, like brominated compounds. Otherwise, the constancy of the CALUX (DX + PCB fraction) TEQ-value which compares to increasing the GC-HRMS (17 PCDD/F + 4 cPCB) TEQ results raises questions concerning (1) the significance of CALUX results obtained without fractionation as well as (2) the toxicological effect of a cocktail of contaminants on the human health.