Screening Of Dioxins Research Articles

Improved Method for the Determination of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans (PCDD/Fs) in Sanitary Napkins

An improved method was developed for determining highly toxic 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans (PCDD/Fs) in sanitary napkins. Samples were extracted by Soxhlet (50% dichloromethane/hexane) and sonication (hexane), and a multi-layer silica column was used for clean-up after acetone precipitation to separate the superabsorbent polymers. Improved separation efficiency of PCDD/Fs from sanitary napkins with a high level of superabsorbent polymers was achieved using the acetone precipitation method. Hexane was then applied as an alternative solvent to remove residual interfering substances including sticky components contained on sanitary napkins via alumina column chromatography in the second clean-up step. Recoveries approached 100% with an average relative standard deviation of less than 15%, and recoveries of internal standards were from 70.5 to 114.8% and 0.6 to 13.6% for the two extraction methods. The mean method detection limit was 0.075–0.968 pg/g for Soxhlet extraction and 0.0032–0.091 pg/g for sonication extraction. The established method meets the quality criteria for the screening of dioxins stated in EU Regulations 589/2014 and 709/2014, and therefore provides a suitable alternative for the determination of dioxins in sanitary napkins containing superabsorbent polymers.

Development and validation of a ready to use cryo-EROD assay for the standardized screening of dioxins and dioxin-like compounds in foodstuffs

Recent European regulations have indicated the need for new bioanalytical screening methods capable of monitoring dioxin and dioxin-like compounds in foodstuffs and environmental samples, cost-effectively and with a quicker turnaround. Cryo-cells of the hepatic H4IIE line preserved in 96-well plates were exposed to sample extracts prepared from various foodstuffs and analysed for their content of dioxins and dioxin-like compounds by means of the 7-Ethoxyresorufin-O-Deethylase (EROD)-assay in two laboratories. Assay data were compared between both laboratories and results from instrumental analysis used as a confirmatory method. Additionally, cut-off values for the different studied matrices were derived. The current European regulation regarding methods of analysis for the control of foodstuffs was applied with the aim of determining the feasibility of the cryo-methodology. Results obtained in both laboratories were in congruence with the required validation parameters of the Commission Regulation (EU) No 2017/644. Cut-off values should be established matrix-dependent to reduce the rate of false compliant results and to keep the rate of false non-compliant results under control. In summary, the ready-to-use cryo-assay method for the bioanalytical screening of foodstuffs in control laboratories without cell-culture facilities has successfully proven to be accurate, far quicker and more cost effective than current methods.

Development and application of luciferase reporter gene-based bioassay system for dioxin determination

Bioassay systems have been widely applied in large-scale screening of dioxins. Among these systems, the luciferase reporter gene-based bioassay system is designed based on aryl hydrocarbon receptor signaling pathway which mediates a series of toxicological effects of dioxins. The advantages of this bioassay system include high sensitivity, wide application and strong correlation with toxicological effects. Based on recent research on several newly developed systems, the minimum detection limit of such analytical method can reach 0.1 pM. This system could be widely used in agricultural, food, environmental, government administration and the third party inspection agency. Furthermore, a broad application prospect of this bioassay system has been proposed recently in environmental health study and health risk assessment. This review describes the development, application, standardized methods and mechanism of CALUX assay, as well as the future perspective.

A modified QuEChERS approach for the screening of dioxins and furans in sediments.

A rapid extraction and cleanup method for the screening of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in sediments is described which combines a modified QuEChERS extraction with carbon reversed-phase solid phase extraction cleanup. This approach is compared to the classical Soxhlet extraction and multi-column cleanup method in terms of toxic equivalence quotients (TEQs), precision, instrumental chromatography, method detection limits (MDLs), recovery of (13)C-labelled quantitation standard, sample preparation time, workload capacity, and sustainability factors. TEQs of 32 sediment samples were found to be well correlated and differed by 16 ± 10% between the two methods. Certified and standard reference sediments differed by 4.1 and 6.7%, respectively. Precision and instrumental chromatography were comparable. While the modified QuEChERS method had higher MDLs and lower recoveries, in terms of preparation time and workload capacity, the modified QuEChERS approach can prepare approximately 30 samples per day as compared to 10-20 samples in 3 to 4days for the classic method. The modified QuEChERS method was also found to be safer and greener. The appreciable improvement in capacity makes the modified QuEChERS approach a suitable alternative to the classical method for applications where turnaround time and the number of samples that can be analyzed are more important than minimal detection limits. Graphical Abstract Created using Microsoft Paint for Windows 7 Professional A bar graph with the structures of dioxins and furans on the x axis shows agreement between two sets of data. A legend labels the first set of data as Soxhlet. The Soxhlet set is illustrated as four days crossed off of a calendar page, a Soxhlet extractor, and several packed chromatography columns. The legend identifies the second set of data as QuEChERS. The QuEChERS set is represented by a clock face marked with twenty four hours, two centrifuge tubes containing the sediment and reagents before and after salting out, and a carbon column attached to a reservoir.

An AhR-Luciferase Adenovirus Infection System for Rapid Screening of Dioxins in Soils.

Our goal was to develop a fast-screening method for measuring dioxin levels in soils. The adenovirus (Ad)-dioxin-responsive (DR) bioassay system (AdEasy-6XDRE-TATA-Luc) combined with a fast-cleanup system was examined under conventional conditions (i.e., with incubation at 37°C) and three alternative conditions [incubation at 37°C with addition of phorbol-12-myristate-13-acetate (PMA), incubation at 33°C, and incubation at 33°C with addition of PMA]. The best conditions for carrying out the Ad-DR bioassay was 33°C and no addition of PMA. The background level of soil dioxins determined by the chemical assay [6.49 ng I-TEQ/kg dry weight (dw)] was well correlated (Pearson's r = 0.873, p < 0.001) with that by the Ad-DR bioassay [expressed in ng bioanalytical equivalents (BEQ) 81.1 ng BEQ/kg dw] (n = 17). When surveyed in contaminated soil samples (n = 114) from industrial areas by the Ad-DR bioassay, dioxin levels were 117, 102, 98.5, and 112 ng BEQ/kg dw, respectively, in northern, central, southern, and eastern Taiwan.

Reliable and sensitive adenovirus-based reporter system for high-throughput screening of dioxins

Dioxin-responsive element (DRE)-driven luciferase reporter assay is a rapid and cost-effective screening method for detection and semi-quantitation of dioxins and dioxin-like chemicals in samples from a variety of matrices. However, most of such stable cell lines gradually lose their dioxin responsiveness over time, possibly due to cellular degradation and/or silence of the transfected reporter gene. The study aimed to establish a recombinant adenovirus-based DRE-driven luciferase reporter system for easy maintenance and improvement of dioxin sensitivity/responsiveness. Both the Ad-4xDR adenovirus-based and the Huh7-4xDRE-Luc cell-based reporter systems carry a luciferase reporter gene driven by the same 4xDRE-TATA cassette, i.e., 4 copies of DRE and a TATA box. Comparison of the two reporter systems indicated that the adenovirus-based system was much more sensitive and responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than the cell-based system. Among the four recombinant adenoviruses, Ad-4xDR, Ad-6xDR, Ad-9xDR, and Ad-15xDR carrying 4, 6, 9, and 15 copies of DRE, respectively, the Ad-6xDR-infected H4IIE cells showed the highest TCDD responsiveness. Following optimization of assay conditions, including temperature, dimethyl sulfoxide (DMSO) concentration, and multiplicity of infection (MOI) of adenovirus, the Ad-6xDR-based system exhibited markedly superior performance in TCDD sensitivity (limit of detection (LOD) = 0.02 pM) and responsiveness (the half maximal effective concentration (EC50)=10.41 pM and a maximum of 409.34 folds of luciferase induction at 300 pM TCDD). Analysis of soil samples showed a high correlation between the Ad-6xDR-based reporter assay and high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). The study reveals that, as compared with our previous cell-based system, the Ad-6xDR-based reporter system is more reliable and sensitive for high-throughput screening of dioxins.

The Feasibility on Application of Enzyme-Linked Immunosorbent Assay for Screening Dioxin Soil on Contaminated Site

Enzyme-Linked ImmunoSorbant Assay (ELISA) is a dioxin screening test method, which has several advantages such like low setup cost, low analysis cost, low technical barriers, simple operation, and short analysis time. The case study of this study is a dioxin contaminated site. ELISA was applied in this site due to its widely contaminated range, numerous surveys, and short remediation period. The experience earned in this case study could be the reference for similar pollution site in the future.

Analytical Method Development for the Screening and Determination of Dioxins in Clay Matrices

Dioxin‐contaminated kaolin receives great attention by workers in the fields of environmental science, soil geology, and mining, due to its impact on the areas of health and business. The concept of “natural dioxin” formation in the clay attracts researchers to look for dioxins quantities in these soils and establish the actual route of their formation, as already proved their presence in large unjustified quantities in a number of kaolinitic soil sources and in a unique profile that denies any anthropogenic sources. On the other hand, other researches revealed the absence of dioxins in other kaolinitic sources. For rapid screening of dioxins presence an analytical method was developed basically for the determination of octachlorodibenzo‐p‐dioxin (the major dioxin congener in dioxin‐contaminated kaolin) and octachlorodibenzofuran (does not exist in dioxin‐contaminated kaolin). The method was considered as a rapid, selective, accurate and sensitive method for screening octachlorodibenzo‐p‐dioxin (OCDD) and octachlorodibenzofuran (OCDF). The method achieved linearity for OCDD/F over the range 126.5–6144.0 ng/kg dry weight (dw) with limit of quantitation of 76 ng/kg dw for OCDD and 111 ng/kg dw for OCDF. The precision (RSD%) ranged between 1.87 and 8.09% for OCDD and between 3.93 and 9.48% for OCDF. Recovery was between 85.5 and 91.1% for OCDD and between 57.9 and 64.1% for OCDF, while the accuracy for OCDD ranged from 93.8 to 97.8% and from 102.9 to 125.1% for OCDF. The developed analytical method in this research is a good alternative for known methods, because it saves time, effort, supplies, and equipments for chemical analysis. It shortens the analysis time to less than a quarter (6 h) and need less chemicals. Also it is suitable for work on a normal resolution GC/MS rather than high resolution one.

Simple and rapid yeast reporter bioassay for dioxin screening: evaluation of the dioxin-like compounds in industrial and municipal waste incineration plants

The CROMIS AhR kit, a simple and rapid yeast bioassay kit, was developed and used to detect dioxins and dioxin-like compounds in 20 gas and solid samples collected from refuse incineration plants in Japan. The World Health Organization toxic equivalent (WHO-TEQ) values of the samples were also calculated using high-resolution gas chromatography/high--resolution mass spectrometry. The WHO-TEQ values of the samples varied greatly, ranging from 0.0021-6.3 ng/g to 0.00013-16 ng/m(3)N (normal cubic meter) in the solid and gas samples, respectively. 2,3,4,7,8-Pentachlorodibenzofuran (23478-PeCDF) and 1,2,3,7,8-pentachlorodibenzo-p-dioxin (12378-PeCDD) were the major contributors to the samples' WHO-TEQ values. The yeast in the bioassay responded to these congeners, and the EC50 values of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378-TeCDD), 12378-PeCDD, and 2,3,4,7,8-PeCDF were 490, 560, and 590 nM, respectively. The incinerator samples were subjected to the bioassay to obtain 2378-TeCDD equivalent values (CROMIS-TEQ values). The CROMIS-TEQ values of the solid and gas samples ranged from 0.0019 to 5.64 ng/g and from 0.14 to 20 ng/m(3)N, respectively. The CROMIS-TEQ and WHO-TEQ values displayed good correlations (r (2) = 0.94 and 0.95 in the solid and gas samples, respectively), except for those of the samples with low dioxin concentrations (below the Japanese emission standards). Therefore, the CROMIS AhR kit is a useful tool for the initial screening of samples containing dioxin-like compounds.

Dioxins in beef samples from Mexico using a low resolution GC/MS screening method

Dioxins in beef were quantified by high resolution gas chromatography coupled to low-resolution mass spectrometry (GC/LRMS). The analyses were performed according to the minimum requirements described in the USEPA 1613 method with some minor modifications. Levels found in the samples were in the range 1.02–8.04 pg WHO-TEQ PCDDs/PCDFs g−1 fat. For comparison purposes, the maximum level allowed by the European Union is 3 pg WHO-TEQ PCDDs/PCDFs g−1 fat, and some of these samples surpassed the above-mentioned limit and can be considered as contaminated food. The results confirm that a preliminary screening of dioxins in beef can be performed by GC/LRMS. As far as we know, this is the first report of dioxins in beef in Mexico. After the appropriated tests, the applied methodology could be considered as an alternative screening method for the analysis of PCDD/Fs in other food products.

Assessment of dioxin-like soil contamination in Mexico by enzyme-linked immunosorbent assay.

In this work, we describe the results of a preliminary soil assessment program for the detection of dioxins at different sites in Mexico performed by immunoassay. We studied five different sectors considered relevant sources of dioxins: Anaversa and Tekchem industrial areas where organochlorine pesticides were manufactured and released by accidental explosions, secondary smelters, brick kilns, and rural dwellings. In the context of the Agency for Toxic Substances and Disease Registry (ATSDR) guidelines, only the brick kilns sites can be considered as low-risk areas. The dioxin concentrations detected in the vicinity of the Anaversa and Tekchem chemical plants and secondary smelters exceed the screening level of 0.05 ppb set by the ATSDR, and therefore further site-specific studies are needed. The dioxin levels found in all soot samples from indigenous dwellings where wood is used for indoor cooking were above the evaluation level. Considering that the studied areas are representative examples of dioxin sources in less developed countries, our work demonstrates the useful application of dioxin immunoassays as a tool for dioxin screening for environmental assessment programs in developing countries.

High-throughput screening of dioxins in sediment and soil using selective pressurized liquid extraction with immunochemical detection

A high-throughput screening method using selective pressurized liquid extraction (SPLE) and enzyme-linked immunosorbent assay (ELISA) for monitoring dioxins in sediment and soil is described. SPLE conditions were developed by extracting sediment or soil together with alumina, 10% AgNO 3 in silica, and sulfuric acid impregnated silica (acid silica) using dichloromethane (DCM) as the solvent at 100 °C and 2000 psi. Post-extraction cleanups were not required for ELISA. Two reference sediments (National Institute of Standards and Technology SRM 1944 and Wellington Laboratories WMS01) were analyzed by the SPLE–ELISA method. The ELISA utilized a polyclonal antibody and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) as the calibrant. Recoveries of ELISA-derived TCDD equivalents (EQ) relative to the expected gas chromatography/high resolution mass spectrometry (GC/HRMS) derived dioxin toxic equivalent (TEQ) values were 116 ± 11% for SRM 1944 and 102 ± 13% for WMS01. ELISA TCDD EQs were consistent with the dioxin TEQs as measured by GC/HRMS for 25 soil/sediment samples from seven different contaminated sites. The ELISA had an approximate method detection limit of 10 pg g −1 with a precision of 2.6–29% based on the relative percentage difference (%RPD) for duplicate samples. Estimated sample throughput for the SPLE–ELISA was three times or more than that of the GC/HRMS method employing PLE with a multi-column cleanup.

Application of a Bioassay Using DR-EcoScreen Cells to the Determination of Dioxins in Ambient Air: A Comparative Study with HRGC-HRMS Analysis

There is a strong need for the development of relatively rapid and low-cost bioassays for the determination of dioxins (polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls) in environmental and food samples. In this study, we applied a bioassay system using highly sensitive DR-EcoScreen cells (DR-cell assay) to the determination of low levels of dioxins in ambient air samples. The dioxins from 80 ambient air samples were extracted, purified by cleanup procedure, and applied to both DR-cell assay and high-resolution gas chromatography/high-resolution mass spectrometry (HRGC-HRMS) analysis. Interestingly, the toxic equivalents (TEQ) values obtained from the DR-cell assay were closely correlated with those from the HRGC-HRMS analysis (r2 = 0.957). Although the cell-based TEQ values were relatively (3.66-fold) higher than the TEQ values from the HRGC-HRMS analysis, this discrepancy was thought to be due to the difference between the toxic equivalency factor (TEF) and the relative potency (DR-REP) determined for individual dioxins. Therefore, we multiplied the TEQ values from the DR-cell assay by a conversion coefficient (0.273, the reciprocal of 3.66) and could approximate the values from the HRGC-HRMS analysis. These results clearly suggest at the DR-cell assay might be a promising method for the rapid and low-cost screening of dioxins in ambient air samples.

Densitometry determination of dioxins using gold nanoparticlemodified dioxin response element probes

This study aims to develop a rapid and sensitive densitometry bioassay of dioxins. It is known that dioxin ligand can bind heterodimeric aryl hydrocarbon receptor (AhR) and trigger the formation of the complex of dioxin-AhR, AhR nuclear translocator (Arnt), and dioxin-responsive element (DRE). We have established a rapid and sensitive method using nanoparticle-modified DRE probes based on dioxin-dependent binding of DRE to AhR complex. Dioxins can trigger the binding of nanoparticle-modified DRE probe to AhR complex. The nanoparticle-modified DRE probes in the dioxin-AhR-Arnt-DRE complexes immobilized on the microplates were detected by measuring optical density after silver enhancement. Results showed that as little as 0.01 pM of TCDD could be detected. Dioxins could be quantified accurately with the linear range from 1 nM to 0.01 pM. The coefficient of variation was 5≈8% in this bioassay. The optical density of nanoparticle-based silver enhancement was produced in a dose- and time- dependent manner. The dioxin concentrations in fly ash samples determined by this bioassay were comparable with that by the standard HRGC/HRMS. Thus this bioassay is a sensitive and convenient cell-free bioassay of dioxin that is suitable for screening of dioxins in environmental or food matrices.

Dioxin screening in fish product by pattern recognition of biomarkers

Two alternative, cost- and time-effective dioxin screening methods relying on two categories of potential lipid biomarkers were investigated. A dioxin range varying from 1.1 to 47.1 pg PCDD/F TEQ-WHO/g lipid using 64 fish meal samples was used for model calibration. The methods were based on multivariate models using either (1) fatty acid composition monitored by GC-FID or (2) fluorescence landscape signals analysed using the PARAFAC model and in both cases predicting dioxin content as pgPCDD/F TEQ-WHO/g lipid. In both cases, Partial Least Squares (PLS) regression was performed for predicting the dioxin content of a sample. The GC-FID data analyses was based on automatic peak alignment and integration, enabling extraction of the area of 140 peaks from the gas chromatograms, as opposed to the 31 fatty acids usually considered for fish oil characterisation. In addition to classic PLS employing the whole dataset for calibration, a two-step local PLS modeling approach was performed based upon an initial selection of k number of calibration samples providing the best match to the prediction sample using a so-called k Nearest Neighbors (kNN) approach, then followed by PLS calibration on these kNN selected samples for dioxin prediction. Fluorescence spectroscopy offers a promising non-invasive and ultra-rapid technique, with less than two minutes analysis time. However, fluorescence spectroscopy using the pattern recognition "kNN-PLS" yielded a correlation of 0.76 (r2) and a high root mean square error of prediction of 11.4 pg PCDD/F TEQ-WHO/g lipid. The predictions were improved when the PLS calibration was performed on all the sample with a root mean square error of prediction of 7.0 pg PCDD/F TEQ-WHO/g lipid. Unfortunately, these results failed to demonstrate the potential of fluorophore monitoring as a screening method. In contrast, the overall best screening performance was obtained with the fatty acid profile, when the kNN-PLS combination employed for pattern recognition (kNN) all the areas of the 140 detected peaks and the PLS regression used the areas of 46 selected peaks. This "kNN-PLS" prediction with three latent variables and based upon the 12 nearest neighbors selected out of the 64 x 2 fatty acid profiles (duplicate analyses), yielded a correlation of 0.85 (r2) and a root mean square error of prediction of 2.1 pg PCDD/F TEQ-WHO/g lipid and resulted in a total analysis time of one and half hour per sample.

Rapid dioxin assessment in fish products by fatty acid pattern recognition.

A novel, cost- and time-effective dioxin screening method relying on fatty acid profile was developed for fish products. The method is based on multivariate covariance between fatty acid composition and dioxin. A dioxin range varying from 1.1 to 47.1 ng TEQ-WHO kg fat(-1) was investigated using 64 fish meal samples. An optimal multivariate dioxin prediction model was developed based on reduction from the original 32 to 13 fatty acids, thus increasing the parsimony and the robustness of the model. The model obtained with three partial least squares regression (PLS) components included the following 13 fatty acids: C14:1 n-5, C16:4 n-1, C18:1 n-9, C18:2 n-6, C18:3 n-6, C18:3 n-3, C20:0, C20:1 n-9, C20:4 n-6, C20:3 n-3, C22:1 n-7, C22:6 n-3, C24:1 n-9. Considering the whole investigated dioxin range, the performance of the PLS model based upon full cross-validation yielded a correlation of 0.90 (r(2)) and a prediction error of 3.31 ng PCDD/F TEQ-WHO kg fat(-1). A submodel of samples in the lower dioxin range 1 to 15 ng PCDD/F TEQ-WHO kg fat(-1) returned a r(2) of 0.88 and an error of 1.85 ng PCDD/F TEQ-WHO kg fat(-1).

Validation and use of the CALUX‐bioassay for the determination of dioxins and PCBs in bovine milk

There is a strong need for the development of relatively cheap and rapid bioassays for the determination of dioxins and related compounds in food. A newly developed CALUX (Chemical‐Activated LUciferase gene expression) bioassay was tested for its possible use to determine low levels of dioxins in bovine milk. Data show that this mammalian cell‐based test is very sensitive for 2,3,7,8‐substituted dioxins and related PCBs, thereby reflecting the relative potencies of these compounds in comparison to TCDD (TEF‐values). The limit of detection was about 50 fg of TCDD. Furthermore, the response obtained with a mixture of dioxins was additive, in accordance with the TEF‐principle. Milk fat was isolated by centrifugation followed by clean‐up of the fat with n‐pentane, removal of the fat on a 33% H2SO4 silica column, and determination of Ah receptor agonist activity with the CALUX‐bioassay. An equivalent of 67 mg fat was tested per experimental unit, resulting in a limit of quantification around 1 pg i‐TEQ/g fat. To investigate the performance of the method, butter fat was cleaned and spiked with a mixture of 17 different 2,3,7,8‐substituted PCDD and PCDF congeners at 1, 3, 6, 9, 12 and 15 pg TEQ/g fat, as confirmed by GC/MS. In this concentration range, the method showed a recovery of TEQs around 67% (58–87%). The reproducibility, determined in three independent series showed a CV varying between 4% and 54%, with the exception of the sample spiked at 1 pg i‐TEQ (CV 97%). The repeatability determined with the sample spiked at 6pg i‐TEQ/g showed a CV of 10%. Testing of 22 bovine milk samples, taken at different sites in The Netherlands, in the CALUX‐assay showed combined dioxin and dioxin‐like PCB levels equivalent to 1.6 pg TCDD/g fat (range 0.2–4.6). GC/MS analysis of these samples revealed an average level of 1.7 pg i‐TEQ/g fat, varying between 0.5 and 4.7 pg i‐TEQ/g fat. All five samples showing a GC/MS determined dioxin content of more than 2 pg i‐TEQ/g fat gave a response in the CALUX‐assay corresponding to more than 2 pg TCDD/g fat. These data clearly show that the CALUX‐bioassay is a promising method for the rapid and low cost screening of dioxins in bovine milk.

The occurrence and fate of PCDDs and PCDFs in five bleached kraft pulp and paper mills

Preliminary results from the USEPA/Paper Industry Cooperative Dioxin Screening Survey are presented. Inputs, intermediates, and exports from five bleached kraft pulp and paper mills were analyzed for 2378-TCDD and 2378-TCDF.