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Organic-diffusive gradients in thin-film samplers (o-DGT), were developed and applied for accumulation of estrogen and estrogen-like compounds on a XAD18 resin and deployed in situ in the effluents of Beijing Gaobeidian Wastewater Treatment Plant (GWWTP) and Brussels North Wastewater Treatment Plant as well as in several aquatic systems in Belgium, including the Zenne River, the Belgian Oostende Harbor and the North Sea. Estrogenic compounds accumulate on the XAD18 resin and the estrogenic activity of the resin extract was measured with the Estrogen Responsive Elements-Chemically Activated LUciferase gene eXpression (ERE-CALUX) bioassay. With this result and by applying Fick's diffusion law, it is possible to calculate the estrogenic activity in the aquatic system, if the diffusion boundary layer (DBL) is known or negligible compared to the hydrogel diffusive layer thickness. The DBL thickness in our study varied from 0.010 to 0.023 cm and ignoring the DBL thickness would for instance, underestimate the estrogenic activity by 10-20%. Estrogenic activities in the secondary effluent of GWWTP were the highest (29 ± 4 ng E2-equivalents L -1 ), while the lowest level was found at the Belgian Oostende Harbor (0.05 ± 0.01 ng E2-equivalents L -1 ). Comparable estrogenic activities in water samples measured by o-DGT and grab sampling were obtained, confirming that o-DGT can be efficiently used in various aquatic systems. The advantage of our sampling and measuring method is that very low, time averaged estrogenic activities can be determined, with a minimum of sample treatment. The risk of sample contamination is very low as well as the cost of the whole analytical procedure., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A novel type of diffusive gradients in thin-film (DGT) was combined with a chemically activated luciferase gene expression bioassay (CALUX) to measure estrogens in aquatic systems. The performance of this novel method was assessed with 17β-estradiol (E2) as the model steroid hormone, XAD 18 resin gel as the binding phase in the DGT method and VM7Luc4E2 cells (formerly BG1Luc4E2) for the Estrogen Responsive Element (ERE)-CALUX bioassay. The measured effective diffusion coefficient of E2 in agarose diffusive gel was 4.65 ± 0.37 × 10 -6 cm 2 s -1 at 25 °C. The detection limit of this combined DGT/ERE-CALUX method for 1 day of sampling (0.026 ± 0.003 ng L -1 of E2) is significantly lower than that obtained by spot sampling combined with GC-MS/MS or LC-MS/MS analysis (0.1-7.0 ng L -1 ). The method is independent of pH (5-8), ionic strength (0.001-0.5 M), and dissolved organic matter (DOM; concentrations up to 30 mg L -1 ). Field applications of this novel DGT in effluents of three sewage treatment plants in Beijing city (China) showed comparable results to conventional spot (grab) sampling. This study demonstrates that the combined DGT/ERE-CALUX approach is an effective and sensitive tool for in situ monitoring of estrogenic activity in waters and wastewaters.

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'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dioxins, furans and polychlorinated biphenyls are contaminants of high concern and as such, sensitive tools are needed to detect these persistent organic compounds in a variety of matrices. Due to the large amount of samples that need to be investigated for example for food and feed control, the CALUX bioassay (H1L6.1 clone) was developed allowing rapid and cost-efficient analysis of biological and environmental samples. Recently, a new and more sensitive clone (H1L7.5) was constructed as the third generation CALUX bioassay. This new cell line was subject of an amplification of dioxin response elements (DREs), allowing lower concentrations of target compound to be analyzed. A comparison is made between the previous, well-defined H1L6.1c3 cell line and the new H1L7.5c1 cell line: it appears that the bioassay making use of the higher number of DREs is more stable and robust, shows better repeatability and reproducibility and is; on average, 3 times more sensitive., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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, validation of new methods is urgently needed. In this study, a new method for the analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples with the CALUX bioassay was developed, optimized and validated. The method consists of 4 steps: filtration, extraction, clean up and bioassay analysis. To avoid the use of large amounts of toxic solvents, new techniques were used for filtration and extraction: a C18 filter was used instead of a liquid/liquid extraction and an Accelerated Solvent Extractor (ASE) was used instead of the traditional soxhlet extraction. After pre-oxidation of the sample extract, clean up was done using a multi-layer silica gel column coupled to a carbon column. The PCDD/F and PCB fractions were finally analyzed with the H1L7.5c1 and/or the H1L6.1c3 mouse hepatoma cell lines. The limit of quantification was 1.4pg CALUX-BEQm(-2)d(-1) for the PCBs and 5.6pgCALUX-BEQm(-2)d(-1) for the PCDD/Fs, when using the new sensitive H1L7.5c1 cell line. The GC-HRMS recovery for all PCDD/F congeners was between 55% and 112%, with a mean recovery of 90%. CALUX recoveries of spiked procedural blanks were between the accepted ranges of 80-120%. Repeatability and reproducibility were satisfactory and no interferences from metals were detected. The first results from the Flemish measurement program showed good correlation between CALUX and GC-HRMS., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 pgBEQ/gfat, 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 30.3pgBEQ/gfat for the PCDD/Fs and 14.5pgBEQ/gfat 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)pgCALUX-BEQ/gfat for the PCDD/Fs and 32.1 (30.1–34.2)pgCALUX-BEQ/gfat for the dioxin-like PCBs. [Copyright &y& Elsevier]

Abstract: The CALUX bioassay is at present the best screening method for dioxins and dioxin-like (dl) polychlorinated biphenyls (PCBs) in food and feed, and the only assay used in routine monitoring and during larger incidents. Furthermore, the use of bioassays in addition to chemical reference methods allows the discovery of novel contaminants with potentially adverse effects on human health. The CALUX bioassay shows a clear dose-related response both with dioxin standards and contaminated samples, but requires a clear strategy with respect to decision limits, necessary to deal with the many different action and tolerance limits in European Union (EU) legislation. In future, the CALUX bioassay will profit from further optimization, especially with respect to the clean-up procedure. As demonstrated in the limited number of interlaboratory studies performed so far, this should lead to an even more robust assay that can be easily introduced into less experienced laboratories. The present article discusses the different issues, based on some practical examples from the EU-DIFFERENCE project and gives recommendations for future studies. [Copyright &y& Elsevier]