Dioxin Bioassays

Persistent Organic Pollutants (POPs), Aryl Hydrocarbon (Ah) Receptor, Transgenic Plants, Reporter Gene, Bioassay, Dioxins... 

Garrison, P.M., Tullis, K., and Aarts J.M.M.J.G. et al. (1996). Species specific recombinant cell lines as bioassay systems for the detection of dioxin-like chemicals. Fundamental and Applied Toxicology 30, 194-203. 

Barber, T.R., D.J. Chappie, D.J. Duda, P.C. Fuchsman, and B.L. Finley. 1998. Using a spiked sediment bioassay to establish a no-effect concentration for dioxin exposure to the amphipod Ampelisca abdita. Environ. Toxicol. Chem. 17 420-424. 

Smith, I.R., B. Marchant, M.R. van den Heuvel, J.H. Clemons, and J. Frimeth. 1994. Embryonic mortality, bioassay derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents, and organochlorine contaminants in Pacific salmon from Lake Ontario. Jour. Great Lakes Res. 20 497-509. 

Ankley, G.T., D.E. Tillitt, J.P. Giesy, P.D. Jones, and D.A. Verbrugge. 1991. Bioassay-derived 2,3,7,8-tetra-chlorodibenzo-p-dioxin equivalents in PCB-containing extracts from the flesh and eggs of Lake Michigan chinook salmon (Oncorhynchus tshawytscha) and possible implications for reproduction. Canad. Jour. Fish. Aquat. Sci. 48 1685-1690. 

Brunstrom, B., M. Engwall, K. Hjelm, L. Lindqvist, and Y. Zebuhr. 1995. EROD induction in cultured chick embryo liver a sensitive bioassay for dioxin-like environmental pollutants. Environ. Toxicol. Chem. 14 837-852. 

Whyte, J.J. Schmitt, C.J. Tillitt, D.E. 2004, The H4IIE cell bioassay as an indicator of dioxin-like chemicals in wildlife and the environment. Crit. Rev. Toxicol. 34 1-83. 

This thesis focuses on the applicability of in vitro, in vivo bioassays and bioindicators as tools for evaluating the effects of complex chemical mixtures in the process of deciding whether dredged harbour sediments can be disposed of at sea without serious adverse effects on marine ecosystem and human health. It considers the North Sea delta area in order to determine a comprehensive approach for the application of both in vitro and in vivo bioassays for hazard assessment, advanced risk assessment, and location-specific ecological impact assessment for dredged harbour sediments. To aid in the selection of appropriate, robust and reliable in vitro and in vivo bioassay and bioindication methods for these specific purposes, the uneertainty, predictability and specificity of the bioassays have been explored and the applieability in eombination with other analyses is discussed. The focus of the chosen examples is on bioassays and bioindicators for the relatively well studied dioxin-like contaminants and TBT. 

Intra- and interlaboratory calibration of the DR CALUX bioassay for the analysis of dioxins and dioxin-like chemicals in sediments... 

The infralaboratory calibration study was performed by the Institute for Environmental Studies. Sediment was extracted and cleaned up as indicated here. The determination of dioxin and/or dioxin-like content was according to the method indicated under the section DR CALUX analysis. For the intralaboratory study, the following parameters were investigated limit of detection (LOD), limit of quantitation (LOQ), and reproducibility and repeatability of the bioassay. 

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. 

Table 1 Intralaboratory repeatability and reproducibility of the dioxin response-chemically activated lucerferase (DR CALUX ) bioassay for sediment extracts 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) TEQ = toxic equivalent.

The interlaboratory results obtained from the analysis of defined standard solutions, but also from the analysis of sediment extracts prepared either by the coordinator of the study or by the participants themselves, also provide a measure of the variation between laboratories. The results show that the interlaboratory reproducibility ranges from 6.5% for the defined dioxin sample to 27.9% for the sediment sample extracted by the participants themselves. As was mentioned before, the reproducibility for this last sample is relatively high and most presumably due to the introduction of extra handlings (extraction and cleanup) to the total procedure. In addition, the fact that not all the participants had prior experience with the extraction protocol to be used could have added to the increase in variability of the process. Furthermore, the dilution factor was not dictated. This also introduces a certain degree of variation. For the reproducibility of the DR CALUX bioassay itself and not caused by differences in operating extraction conditions, the maximum variation between laboratories was observed to be 18.0%. The results for the sediment extract samples can also be used to estimate the method variability for extracts, that is, based on samples of unknown composition. Again, given the intra-as well as the interlaboratory variations observed in this study, it appears justified to conclude that the standard deviation of the means provides a reasonable estimate of the method variability, based on the overall aver-... 

Several overall conclusions can be drawn based on the statistical evaluation of the data submitted by the participants of the DR CALUX intra-and interlaboratory validation study. First, differences in expertise between the laboratories are apparent based on the results for the calibration curves (both for the curves as provided by the coordinator and for the curves that were prepared by the participants) and on the differences in individual measurement variability. Second, the average results, over all participants, are very close to the true concentration, expressed in DR CALUX 2,3,7,8-TCDD TEQs for the analytical samples. Furthermore, the interlaboratory variation for the different sample types can be regarded as estimates for the method variability. The analytical method variability is estimated to be 10.5% for analytical samples and 22.0% for sediment extracts. Finally, responses appear dependent on the dilution of the final solution to be measured. This is hypothesized to be due to differences in dose-effect curves for different dioxin responsive element-active substances. For 2,3,7,8-TCDD, this effect is not observed. Overall, based on bioassay characteristics presented here and harmonized quality criteria published elsewhere (Behnisch et al., 2001a), the DR CALUX bioassay is regarded as an accurate and reliable tool for intensive monitoring of coastal sediments. 

TBT, and the dioxin-equivalents (TEQs) based on the in vitro bioassay DR-Lue response (Table 2, Fig. 2). Altogether, the added value of the two bioassays that were ineluded in the CTT for dredged sediments and were performed with whole sediment, was not eonvineing. They apparently mainly added false positives due to sensitivity to matrix effeets (Stronkhorst et al., 2003b). 

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. 

In the next sections, the usefulness of the application of bioassays and bio-indicators for hazard, risk and impact assessment is illustrated based on experiences with dioxins and TBT. 

Example of applications of bioassay and biomarker analysis for dioxins in Boxes A, B and C... 

Table 6 Dioxin-equivalents (TEQs) determined with an in vitro bioassay (DR-Luc) in dredged harbour sediments, marine sediment, and suspended matter from the Dutch coastal zone and estuaries of the North Sea...

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