Polycyclic aromatic hydrocarbons (PAHs) represent a class of ubiquitously occurring environmental compounds that are implicated in a wide range of toxicological effects. Routine measurement of PAH contamination generally involves chemical analytical analysis of a selected group of representatives, for example, EPA-16, which may result in underestimation of the PAH-related toxicity of a sample. Many high molecular weight PAHs are known ligands of the aryl hydrocarbon receptor (AhR), a nuclear receptor that mediates toxic effects related to these compounds. Making use of this property we developed a PAH CALUX assay, a mammalian, H4IIe- cell-based reporter assay for the hazard identification of total PAH mixtures. The PAH CALUX reporter cell line allows for specific, rapid (4 h exposure time) and reliable quantification of AhR-induced luciferase induction relative to benzo[a]pyrene (BaP), which is used as a positive reference PAH congener. Full dose response relationships with inductions over 100-fold were reached within only 2 h of exposure to BaP. The PAH CALUX is highly sensitive, that is, using a 4 h exposure time, a limit of detection (LOD) of 5.2 × 10(-11) M BaP was achieved, and highly accurate, that is, a repeatability of 5.9% and a reproducibility of 6.6% were established. Screening of a selection of PAHs that were prioritized by the European Union and/or the U.S. Environmental Protection Agency showed that the PAH CALUX bioassay has a high predictability, particularly for carcinogenic PAHs. Experiments with synthetic mixtures and reference materials containing complex PAH mixtures show the suitability of the assay for these types of applications. Moreover, the presented results suggest that application of the PAH CALUX will result in a lower risk of underestimation of the toxicity of a sample than chemical analytical approaches that focus on a limited set of prioritized compounds.

In the Fourth National Policy Document on Water Management in the Netherlands [ 1 ], it is defined that in 2003, in addition to the assessment of chemical substances, special guidelines for the assessment of dredged material should be recorded. The assessment of dredged material is based on integrated chemical and biological effect measurements. Among others, the DR CALUX® (dioxin responsive—chemically activated luciferase expression) bioassay has tentatively been recommended for inclusion in the dredged material assessment. To ensure the reliability of this bioassay, an intra‐ and interlaboratory validation study, or ring test, was performed, organized by the Dutch National Institute for Coastal and Marine Management (RIKZ) in cooperation with BioDetection Systems BV (BDS). The intralaboratory repeatability and reproducibility and the limit of detection (LOD) and quantification (LOQ) of the DR CALUX bioassay were determined by analyzing sediment extracts and dimethyl sulfoxide (DMSO) blanks. The highest observed repeatability was found to be 24.1%, whereas the highest observed reproducibility was calculated to be 19.9%. Based on the obtained results, the LOD and LOQ to be applied for the bioassay are 0.3 and 1.0 pM, respectively. The interlaboratory calibration study was divided into three phases, starting with analyzing pure chemicals. During the second phase, sediment extracts were analyzed, whereas in the third phase, whole sediments had to be extracted, cleaned, and analyzed. The average interlaboratory repeatability increased from 14.6% for the analysis of pure compound to 26.1% for the analysis of whole matrix. A similar increase in reproducibility with increasing complexity of handlings was observed with the interlaboratory reproducibility of 6.5% for pure compound and 27.9% for whole matrix. The results of this study are intended as a starting point for implementing the integrated chemical—biological assessment strategy and for systematic monitoring of dredged materials and related materials in the coming years.

In this paper, in order to provide a useful piece of information about inter-laboratory calibration study using in vitro bioassay in Japan and its evaluation method, outline and results of inter-laboratory calibration study for dioxins in food/feedstuff using DR-CALUX (Dioxin-Responsive Chemical-Activated LUciferase gene eXpression) assay with 21 participants were reported. The interlaboratory calibration study was divided into three phases. In Phase I, pure chemical mixtures consisted of PCDDs/PCDFs and Co-PCBs were analyzed. The TeCDD equivalents calculated using DR-CALUX assay (CALUX-TEQs) were equivalent to the theoretical CALUX-TEQs calculated chemically using concentrations of PCDDs/PCDFs and Co-PCBs and CALUX relative potencies to 2,3,7,8-TeCDD for these samples. Although lowest concentration sample indicated about 0.3 pM CALUX-TEQs per well, its reproducibility was comparatively good. During Phase II, cleaned sediment and food/feedstuff extracts prepared by organizer were analyzed. Decreases of interlaboratory reproducibility were observed with decrease of CALUX-TEQs in samples. In Phase II, samples indicating wide dispersion had low concentrations which were almost less than 0.3 pM CALUX-TEQs per well. In consideration of results for Phase I, although the limit of quantification for DR-CALUX assay was about 1 pM in general, 0.3 pM CALUX-TEQs per well might be the real limit of quantification. In Phase I and II, dispersion of data wasn't depending on a type of sample or dilution effect. In Phase III, whole fish oils and feedstuffs were extracted, cleaned, and analyzed by each participant according to protocol delivered by the organizer. The CALUX-TEQs were almost equivalent to the WHO-TEQs for these samples. Interlaboratory reproducibility tended to be higher than those during phase I and II. Increases of it were also observed with decrease of concentration in samples. The results demonstrated that the extraction and cleanup processes cause a major variation. From this study, it was indicated that Z-score provides useful information for identifying the cause of dispersion of data for each phase.