CALUX-test

Phase 2. In the seeond phase of the study, the partieipants were asked to analyze three extraeted and cleaned sediment samples using the DR CALUX bioassay. Sediments used for extraetion and cleanup were freshwater sediments from the Western Seheldt, The Netherlands. The sediment extracts were prepared by the Royal Institute for Fishery Research (RIVODLO), IJmuiden, The Netherlands, aeeording to the protoeol given here. Dilutions of the supplied sediment extracts were prepared by the partieipants in DMSO and tested for dioxin and/or dioxinlike content. On each 96-well mierotiter plate, a 2,3,7,8-TCDD standard ealibration curve was analyzed. Raw data as well as eonverted data were used for statistieal evaluation. 

On each 96-well microtiter plate, a eomplete 2,3,7,8-TCDD standard concentration range was incubated and analyzed in triplieate. A eurve fit of the 2,3,7,8-TCDD standard range was produced for the calculation of DR CALUX TEQ content in the samples tested. The analyzed relative light units (RLU) from the samples were interpolated on the 2,3,7,8-TCDD standard curve, and the DR CALUX TEQ content was quantified between the limit of quantitation (LOQ) and the concentration of 2,3,7,8-TCDD at whieh 50% of the maximum response is observed (EC50). 

The in vitro bioassay for dioxins with cleaned sediment extracts (DR-CALUX) proved to comply with the QA/QC criteria needed to guarantee the reliability of data in an inter- and intralaboratory study (Besselink et al., 2004). The chemical stability of dioxins makes it possible to apply destructive clean-up procedures which remove all matrix factors. Sample extraction and cleanup for other in vitro bioassays for specific mechanisms of toxicity require further development to make sure that the chemicals of interest are not lost or unwanted chemicals included in the sediment extract to be tested. Table 4 summarizes possible bioassays that could be performed in addition to chemical analyses with the dredged sediment in a licensing system. 

Table 5.1 Reproducibility of the CALUX assay with milk fat samples. Spiked samples were tested singly in three independent test series (adapted from 12)...

An additional screening test for TCDD-like (aryl hydrocarbon receptor, AhR, active) chemicals has been developed (Garrison et al. 1996) and is available commercially (Anonymous 1997). Dubbed the CALUX (for chemically activated luciferase gene expression) system, the assay is based on recombinant cell lines into which researchers have inserted a firefly luciferase gene. When exposed to dioxin-like compounds, the recombinant cells luminesce. The method is sensitive to ppt levels of 2,3,7,8-TCDD equivalents in blood, serum, and milk (Anonymous 1997). Samples testing positive can be subjected to more definitive and specific analytical testing. 

The CALUX assay described in Section 6.1 has been applied to Ah receptor-active compounds (not limited to dioxins) in sediments and pore waters (Murk et al. 1996) and to blood with mixed results. Sensitivities as low as 0.5 fmol of 2,3,7,8-TCDD were reported. Two polychlorinated terphenyl mixtures, the PCB-substituted Ugilec 141, polybrominated diphenyl ethers, and the PCB mixture Clophen 150 were tested in the CALUX assay and had induction potencies that were 10"4 to 10"7 compared to TCDD. Thus, this assay is more selective than earlier, induction-based assays, although clearly not as selective as GC/MS. 

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