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

Abstract

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 6 pg 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.

Dioxin-Like TEQ of women from the Seveso, Italy area by ID-HRGC/HRMS and CALUX

“…Further, high levels of some PCBs have been shown to antagonize the dioxins if not separated (Garrison et al, 1996). It has been reported that the TEQ value especially in samples containing relatively large amount of PCB 118 might be underestimated in the CALUX bioassay compared to an HRGC/HRMS determination (Bovee et al, 1998;Laier et al, 2003). Inadequate sample clean-up prior to the CALUX bioassay might result in the measurement of activity due to these interfering compounds, and an inaccurate TEQ report for the sample (Brown et al, 2002b).…”

Polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls in fish from the Netherlands: concentrations, profiles and comparison with DR CALUX® bioassay results

“…Samples of 0.25 g fish oil (kindly supplied by Nutreco, Boxmeer, The Netherlands) were purified on columns containing 10 g acid silica (33% H 2 SO 4 ), as described by Bovee et al [17]. Before total evaporation of the hexane/diethyl ether extract in a SpeedVac, 100 μl dimethyl sulfoxide (DMSO) was added as a keeper.…”

Carry-over of dioxins and PCBs from feed and soil to eggs at low contamination levels – influence of mycotoxin binders on the carry-over from feed to eggs

“…The DR CALUX Õ test was carried out as described earlier by Bovee et al (1998). An aliquot of 0.5 g fat was mixed with hexane and extracted on acid silica as described previously.…”