DR-CALUX bioassay

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

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. 

For the determination of the limit of detection (LOD) and LOQ, 10 standard 2,3,7,8-TCDD calibration series were analyzed in triplicate using the DR CALUX bioassay. In Figure 1, a typical example of a standard 2,3,7,8-TCDD calibration curve is given. For each individual calibra-... 

Both the EC50 values and the 3-pM point of the 2,3,7,8TCDD ealibration curve serve as quality criteria. For each participant, the results for both data points from all 96-well plates analyzed during the presented study were collected and reeorded in Shewhart control charts. The Shewhart control chart is used to identify variations on performanee of the DR CALUX bioassay brought about by unexpected or unassigned causes. The Shewhart eontrol chart shows the mean of the EC50 and 3-pM control point and the upper and lower eontrol limits. In Figure 2, a typical Shewhart control chart is shown. Over the analysis period, none of the participants exceeded the aetion levels (AVG 3 S). 

Phase 1 Defined standard solutions. Partieipants were asked to measure the response of the two standard samples in the DR CALUX bioassay three times in triplicate. The total DR CALUX 2,3,7,8-TCDD TEQ content of both the 2,3,7,8TCDD sample as well as the mixed sample was calculated to be 7.5 uM TEQ. Since the DMSO eontent during exposure was 0.4% and the samples were diluted seven times by all partieipants, the expected DR CALUX TEQ... 

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 aim of the present study was to identify the DR CALUX bioassay performance criteria for the analysis of PHAHs in sediment samples in order to implement the bioassay in the assessment of dredged materials for systematic monitoring in the eoming years. Therefore, both an intra-and interlaboratory validation study was performed. 

Beeause the 2,3,7,8-TCDD calibration curve is used for quantification of analysis results, the stability and quality of the calibration curves is important. Furthermore, the calibration curves themselves are used as a DR CALUX bioassay quality eriterion. Aecording to the performanee eriteria set for the DR CALUX bioassay, the fitted EC50 should be within the range of 6 to 18 pM... 

The LQD and the LQQ of the DR CALUX bioassay were determined by analyzing 10 standard 2,3,7,8-TCDD ealibration series. From these analyses, it was concluded that taking into account 95% confidence, a LOD and LOQ of 0.3 and 1 pM 2,3,7,8-TCDD per well, respectively, should be applied. Hence, in case 10 g of sediment are processed and analyzed using 0.4% of DMSQ per well, the LQD and LOQ can be calculated to be 0.04 and 0.16 pg 2,3,7,8-TCDD equivalents per gram of sediment. Similar LOD and LOQ were reported by a number of authors (Bovee et al, 1998 Behnisch et al, 2001c). 

TCDD TEQ/g sediment, the 30X diluted extract gave a DR CALUX response of 34.9 6.1 pg 2,3,7,8-TCDD TEQ/g sediment. In general, an effect of dilution on the total DR CALUX TEQ content in sediment samples is observed. Although the exact nature for this observation is not known, it is hypothesized that this is due to the presence of various compounds in sediment extracts showing variable affinity toward the Ah receptor. Dose-response curves in the DR CALUX bioassay of individual compounds have been studied and showed obvious differences (Hosoe et al, 2002) both in maximum response and slope of the curve fit. 

For the determination of the intralaboratory repeatability of the DR CALUX bioassay for sediment samples, two sediment extracts were analyzed 10 times. Each analysis was performed in triplicate. As a prerequisite for a correct triplicate analysis, the percentage standard deviation in the triplicate determination should be below 15%. This is in accordance with the harmonized quality criteria for eell-based bioassay analyses of PCDDs/PCDFs in feed and food as formulated by Behnisch et al. (Behnisch et al, 2001 a) and as detailed in European Union directive 2002/69/ EC and direetive 2002/70/EC. The repeatability for the low-2,3,7,8-TCDD-content sediment... 

As with the determination of the intralaboratory repeatability, the intralaboratory reproducibility was determined by analyzing a cleaned sediment extract and a 3-pM 2,3,7,8TCDD standard on 10 separate days and by multiple persons. The reproducibility for the 3-pM 2,3,7,8-TCDD standard was found to be 13.8%, whereas the reproducibility for the cleaned sediment extraet was shown to be 19.9%. Since the observed reproducibilities are in the range of relative standard deviations for two sediment extracts analyzed in 10-fold on the same day (intralaboratory repeatability), the DR CALUX bioassay can be evaluated as a stable and robust bioanalytical tool. 

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. 

Behnisch, P.A., Hosoe, K., Brouwer, A., Sakai, S. (2001c). Cross-validation study of the DR CALUX -bioassay comparison to micro-EROD bioassay and chemical analyses. Organohalogen Corn-pounds 54 81-85. 

Schipper, C., Besselink, H., Klamer, H., Leonards, P, Verhaar, H., Felzel, E., Brouwer, B. (2001). Dioxin and/or dioxinlike analysis in sediment using the DR-CALUX bioassay an interlaboratory validation study. Society of Environmental Toxicology and Chemistry (SETAC), 22nd Annual meeting, 11-15 November 2001, USA. 

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