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 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.

Keywords:

• dioxins
• PCBs
• polyhalogenated aromatic hydrocarbons
• bioassay
• CALUX
• validation
• milk

Notes

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