Assessment of estrogenic compounds in paperboard for dry food packaging with the ERE-CALUX bioassay.

Paperboard used as packaging, a non-inert material, can transfer chemicals into food. Over the years, endocrine disrupting compounds (EDCs), such as NonylPhenols (NPs), BisPhenol A (BPA) and phthalates have been shown to migrate from packaging materials into food. Due to chronic exposure and mixture effects of these EDCs, they could cause health effects even at very low doses. Many EDCs are still unknown and many more are still unregulated. The ERE-CALUX bioassay was used as a bioanalytical tool to investigate estrogenic activities of paperboard food packaging and its characteristics, including recycling rate and printing ink. A "worst case" scenario with full extraction is compared to a dry food migration experiment. By measuring an overall estrogenic activity, known and unknown estrogenic chemicals and mixture effects are taken into account and the data are compared to molecule specific analysis. Estrogenic activities ranged from 682 ±â€¯66 pg E2 eq./dm2 to 3250 ±â€¯400 pg E2 eq./dm2 for "worst case" extraction and from 347 ±â€¯30 pg E2 eq./dm2 to 1350 ±â€¯70 pg E2 eq./dm2 for migration experiments. A two-factor ANOVA revealed a relationship between estrogenic activity and the recycling rate of the paperboard, but no significant difference with printing ink was observed for these paperboard samples. Bis(2-ethylhexyl)phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) were determined in all extraction and migrations experiment samples. A Spearman rank correlation analysis showed a relationship between the estrogenic activity and the total phthalates as well as with each compound individually.

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.

Dioxins, furans and dioxin-like PCBs in sediment samples and suspended particulate matter from the Scheldt estuary and the North Sea Coast: Comparison of CALUX concentration levels in historical and recent samples.

BACKGROUND: The Scheldt estuary is historically a highly polluted river system. While several studies have focused on contamination with metals, pesticides, Polycyclic Aromatic Hydrocarbons (PAHs) and marker PolyChlorinated Biphenyls (PCBs), no data are available concerning past contamination by dioxin-like compounds. OBJECTIVES: The objective of this study is to determine spatial and time trends of PolyChlorinated Dibenzo-p-Dioxins and DibenzoFurans (PCDD/Fs) and dioxin-like PCBs (dl-PCBs) in sediment samples and Suspended Particulate Matter (SPM) from the Scheldt River basin and the North Sea Coast. METHODS: Dioxin-like compounds (PCDD/F and dl-PCB fractions) were measured with the CALUX-bioassay. Bioanalytical EQuivalent concentrations (BEQs) and Total Organic Carbon (TOC) content of historical (1982-1984) and recent (2011-2015) sediment and SPM samples from different locations in the coastal area and the estuary, were evaluated. RESULTS: A decrease in dioxin-like compound concentrations was found at all stations over time, especially for the PCDD/Fs. Dl-PCBs were relatively low in all samples. The Scheldt mouth and the Antwerp harbor yielded the highest BEQs and levels were higher in SPM than in sediment due to the higher organic carbon content in this fraction. CONCLUSIONS: Current PCDD/F and dl-PCB levels in the Belgian Coastal Zone and Scheldt estuary are much lower than their levels 30 years ago and pose a relatively low risk to the aquatic system. This is the result of a strong decrease in emissions, however, large local variabilities in sediment concentration levels can still exist because of local variability in sedimentation, erosion rates and in organic carbon content.

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'.

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.

Analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples from the Flemish measurement network: correlation between the CALUX bioassay and GC-HRMS.

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast, sensitive and inexpensive tool for the analysis of a high number of samples, the use of this technique in routine analysis of atmospheric deposition samples may be a valuable alternative for GC-HRMS. In this study, a validated CALUX method was used for the analysis of PCDD/Fs and dioxin-like PCBs in more than 90 atmospheric deposition samples for different locations in Flanders. The samples were taken in residential and agricultural areas, where a threshold limit of 21pgWHO-TEQm(-2)d(-1) for the sum of PCDD/Fs and dioxin-like PCBs was set, and in industrial zones and natural reserves, where no official threshold limit is available. The results from the Flemish measurement program showed correlation between CALUX and GC-HRMS for all the samples, originating from the different areas (R(2) of 0.81, 0.53 and 0.64 for dl-PCBs, PCDD/Fs and sum of both fractions, respectively). Median CALUX/GC-HRMS ratios of 2.0, 0.9 and 1.3 were reported for the PCDD/Fs, dioxin-like PCBs and the sum of both fractions, respectively. The results show that the CALUX bioassay is a valuable alternative tool for the classic GC-HRMS analysis of atmospheric deposition samples in the Flemish measurement network.

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.