Validation of the micro-EROD assay with H4IIE cells for assessing sediment contamination with dioxin-like chemicals.

In vitro bioassays have been used as a bioanalytical means of detecting dioxin-like compounds (DLCs) in environmental matrices and have been suggested as a tool for quantifying DLCs in sediments. The present study evaluated the relationship between bioanalytical results from the micro-7-ethoxyresorufin-O-deethylase (EROD) bioassay and chemical analytical results in 25 sediment samples collected from rivers across Germany. Sediments were collected, polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) were extracted from the sediments, biological toxicity equivalent quotients (BEQs) were determined by micro-EROD assay and toxicity equivalent quotients (TEQs) were calculated from chemical analysis. Correlations between BEQs and TEQs were evaluated, and linear regression modeling was performed, excluding 6 samples as validation data, to derive equations for predicting TEQs from BEQs. Validation data was tested to evaluate predictive capabilities of the models. Correlations were observed between BEQ and TEQ for PCDD/Fs (r=0.987), PCBs (r=0.623), measured sum of PCDD/F and PCBs (r = 0.975) and calculated sum of PCDD/F and PCBs (r = 0.971). The modeling equations provided low variances as evaluated by mean absolute error (MAE) (≤10.3 pg/g) and root mean square error (RMSE) (≤15.8 pg/g) indicating that expected TEQs could be reasonably well calculated from BEQs. Predicted TEQs from validation data fell within the 95% probability intervals of the test data and had low variances (MAE≤6.5 pg/g) and (RMSE≤10.7 pg/g). Our results indicate that the micro-EROD bioassay can be used as a screening tool for DLCs in sediment and has the capability to be used as an alternate method to chemical analysis for quantifying dioxin-like potential of sediments.

In vitro tools for the toxicological evaluation of sediments and dredged materials: intra- and inter-laboratory comparisons of chemical and bioanalytical methods.

The implementation of in vitro bioassays for the screening of dioxin-like compounds (DLCs) into management guidelines of dredged material is of increasing interest to regulators and risk assessors. This study reports on an intra- and inter-laboratory comparison study between four independent laboratories. A bioassay battery consisting of RTL-W1 (7-ethoxy-resorufin-O-deethylase; EROD), H4IIE (micro-EROD), and H4IIE-luc cells was used to assess aryl hydrocarbon receptor-mediated effects of sediments from two major European rivers, differently contaminated with DLCs. Each assay was validated by characterization of its limit of detection (LOD) and quantification (LOQ), z-factor, reproducibility, and repeatability. DLC concentrations were measured using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) and compared to bioassay-specific responses via toxicity equivalents (TEQs) on intra- and inter-laboratory levels. The micro-EROD assay exhibited the best overall performance among the bioassays. It was ranked excellent (z-factor = 0.54), reached a repeatability > 75%, was highly comparable (r 2 = 0.87) and reproducible (83%) between two laboratories, and was well correlated (r 2 = 0.803) with TEQs. Its LOD and LOQ of 0.5 and 0.7 pM 2,3,7,8-TCDD, respectively, approached LOQs of HRGC/HRMS measurements. In contrast, cell lines RTL-W1 and H4IIE-luc produced LODs > 0.7 pM 2,3,7,8-TCDD, LOQs > 1.7 pM 2,3,7,8-TCDD, and repeatability < 70%. Based on the data obtained, the micro-EROD assay is the most favorable bioanalytical tool, and via a micro-EROD-based limit value, it would allow for the assessment of sediment DLC concentrations; thus, it could be considered for the implementation into testing and management guidelines for dredged materials.

Toward Streamlined Identification of Dioxin-like Compounds in Environmental Samples through Integration of Suspension Bioassay.

Effect-directed analysis (EDA) is a powerful strategy to identify biologically active compounds in environmental samples. However, in current EDA studies, fractionation and handling procedures are laborious, consist of multiple evaporation steps, and thus bear the risk of contamination and decreased recoveries of the target compounds. The low resulting throughput has been one of the major bottlenecks of EDA. Here, we propose a high-throughput EDA (HT-EDA) work-flow combining reversed phase high-performance liquid chromatography fractionation of samples into 96-well microplates, followed by toxicity assessment in the micro-EROD bioassay with the wild-type rat hepatoma H4IIE cells, and chemical analysis of bioactive fractions. The approach was evaluated using single substances, binary mixtures, and extracts of sediment samples collected at the Three Gorges Reservoir, Yangtze River, China, as well as the rivers Rhine and Elbe, Germany. Selected bioactive fractions were analyzed by highly sensitive gas chromatography-atmospheric pressure laser ionization-time-of-flight-mass spectrometry. In addition, we optimized the work-flow by seeding previously adapted suspension-cultured H4IIE cells directly into the microplate used for fractionation, which makes any transfers of fractionated samples unnecessary. The proposed HT-EDA work-flow simplifies the procedure for wider application in ecotoxicology and environmental routine programs.