1 September, 16 th 2004 www.dioxins.de Hyogo Pref. Inst. of Pub. Health & Env. Sci. 8th October 2004, Kobe Dr. Peter A. Behnisch (eurofins / GfA, Münster) eurofins / GfA – competence centre for dioxin analysis Current regulatory situation in the European Union
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1 September, 16th 2004www.dioxins.de
Hyogo Pref. Inst. of Pub. Health & Env. Sci.
8th October 2004, Kobe
Dr. Peter A. Behnisch(eurofins / GfA, Münster)
eurofins / GfA – competence centre for dioxin analysis
Current regulatory situation in the European Union
2 September, 16th 2004www.dioxins.de
eurofins Global Business
North North AmericaAmerica
USA
Brazil
China
EuropeEuropeEuropeEurope
Belgium
Netherlands
Denmark
Norway
Germany
Czech Republic
Switzerland
France
Competence Centres
Service laboratories
Sales offices
United Kingdom
3 September, 16th 2004www.dioxins.de
eurofins - a unique portfolio of laboratories
Food
Envi
ronm
ent
Phar
ma
4 September, 16th 2004www.dioxins.de
eurofins services
Pesticides
MycotoxinsVeterinary ResiduesAllergens
Dioxins
GMO
Comprehensive Range of chemical, microbiological and molecular biology methods
USA, 1996: Feed additives ⇒ contaminated ball clayBrazil, 1998: Improper drying of feed ⇒ Citrus pelletsBelgium, 1999: Illegal disposal of capacitor fluids Germany, 1999: Improper drying of feed ⇒ Green garbageFrance, 1999: Sewage sludge in feed premixes Spain, 2000: cholin chloride: PCP contaminated saw dust Japan, 2001: Incinerator dust on spinachFrance, USA 2002: Carbosan copperThe Netherlands 2003: Bakery wasteGlobal: Large fire accidents; metal oxide in feedAt the moment: Farm-raised fish; Eels in the Rhine
Eggs from free-ranging chicken
16 September, 16th 2004www.dioxins.de
European Dioxin Crisis situation continues–some examples of the last 12 months
Belgian Governmental Veterinary Services, Lille, 29.01.2004: Polluted eggs due to incomplete incineration nearbyFrench Agency of Medical Safety of Food (AFSSA), 03.03.2004:In fish production higher PCBs levels occurredDenmark Fish Prohibition of salmon, 01.04.2004: Contaminated salmon reportedSwedish Food Agency BLV, 26.04.2004:Bio-eggs with high dioxin levels = Feed producer reduce fish amount in chicken feedFrench Government, 10.06.2004: Tested eggs are fineGerman Agrarian Ministry, 17.06.2004;Dresden (strip packing). Scarcely 1300 tons with PCB contaminated feed from Saxonia were fed in the past months in four countries of Germany and had gone to 58 agrarian enterprises.
17 September, 16th 2004www.dioxins.de
Need for rapid, high-throughput screening methods!!!
What is currently available ?
Which applications have been already tested?
Which quality of performance?
Which validation studies have been performed?
Open Questions
18 September, 16th 2004www.dioxins.de
Milestones
1975: EROD-Bioassay
1987: Dioxin-Antibodies
1993: CALUX-Bioassay
1996: Ah-Immunoassay
1998: EGFP-bioassay or CAFLUX
2002: PCR technologies
other proteomics testing
DIOXIN 2004 News:
Several comparison studies between different technologies
19 September, 16th 2004www.dioxins.de
Which applications have already been tested with bioassays and which guidelines exist?
Notes: For all of these matrices results from bioassays exist. So far, only few governmental guidelines accept bioassays results for these matrices (sediment/soil: Japan; feed/food: Belgium/The Netherlands)
Deposition
Bio-Accumulation
Bio-AccumulationBio-Accumulation
Emission
20 September, 16th 2004www.dioxins.de
European guidelines for feed/food control for dioxins and dioxin-like compounds (I)
Commission Directive 2002/69 and 70/EG from 26.07.2002:
„Monitoring..of dioxins in foodstuff or feedstuff …..by a strategy involving a screening method in order to select those samples …..less than 30-40% below or exceed the level of interest.
They are specially designed to avoid false negatives (below 1%).“
Results:
So far, several in-house studies with bioassays have confirmed that they are able to fulfill the performance requirements (e. g. CALUX).
Depending on the matrix the false negatives are sometimes higherthan 1% : lower action limits required.
21 September, 16th 2004www.dioxins.de
How does the CALUX reportergene assay works?
Mouse/Rat liver cells:Stabile transfected with an AhR controlled
How were these LOQs determined ?• Based on sample volume and calibration curve TCDD ?• Based on set of samples around LOQ ? • Which Blanks to be used ?
What would be the CALUX-TEQ/WHO-TEQ if only dioxins and PCBs would be present ?
Was contamination with other agonists excluded ?
Open Questions
24 September, 16th 2004www.dioxins.de
Advantages and drawbacks of the CALUX technology
Currently best evaluated and most applied bioassay for dioxin screening purposes in feed/food:Advantage:
Use for high-throughput screening as red-green light decision: cheaper and faster yes/no decision possibleCovers all possible dioxin-like componds (also brominated dioxins)Degradation of unstable agonists (requires 24 h incubation)
Disadvantage: Several non-dioxin-like effects may lead to false positive results or usual higher values than the confirmation method.Recovery correction, cannot be based on internal standards
25 September, 16th 2004www.dioxins.de
Kit based bioasays: e.g. Ah-Immunoassay
•Cell-free system
•Reflects TEQ-values
•ELISA-type kit
•Simple to use
•405 nm plate reader
•5 hour assay time
•Cell-free system
•Reflects TEQ-values
•ELISA-type kit
•Simple to use
•405 nm plate reader
•5 hour assay time
26 September, 16th 2004www.dioxins.de
ELISA technologies: e.g. Ah-Immunoassay
27 September, 16th 2004www.dioxins.de
Advantages and drawbacks of dioxin ELISA kits
Advantages:Possibly cheaper and faster than CALUX, because shorter incubation time and no time consuming cell culture.ELISA technologies are widespread and standardised
Drawbacks:Not sensitive enough Clean-up systems not sufficient applied on all kind of matricesConsistent overestimation (~10-fold) depending on matrix No further degradation of unstable agonists
(Engwall et al, Örebro university, Sweden, 2004, email communication)
THE SECOND INTERNATIONAL INTERCALIBRATION OF THE SECOND INTERNATIONAL INTERCALIBRATION OF DIOXINDIOXIN--LIKE COMPOUNDS USING BIOASSAYS, 2004LIKE COMPOUNDS USING BIOASSAYS, 2004
35 September, 16th 2004www.dioxins.de
Comparison with the GC/MS measurements Comparison with the GC/MS measurements ( Standard Samples )( Standard Samples )
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M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11
Sample1E Sample1F Sample1G
AhR-based Bioassays
M1 – M6
DXNs-Immunoassays
M7-M11
36 September, 16th 2004www.dioxins.de
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Sample1A Sample2A Sample2B Sample2C
Comparison with the GC/MS measurements Comparison with the GC/MS measurements ( Flue gas samples )( Flue gas samples )
AhR-based Bioassays
M1 – M6
DXNs-Immunoassays
M7-M11
37 September, 16th 2004www.dioxins.de
Conclusion Round Robin Studies
► All bioassays detected dioxin-like effects
► Generally good repeatability in the bioassays
► Variable reproducibility between labs using the same bioassay
► Cases of underestimation compared to WHO-TEQs were seen for all types of bioassays
► No clear advantage in performance for any bioassay type
38 September, 16th 2004www.dioxins.de
Open Questions
Which matrices show high gaps between PCDD/F-TEQ and bioassay-TEQ (e. g. sewage sludge, compost, orange juice) and why?
POP convention: search for new dioxin-like POP candidates ongoing?Needs for more International round robin studies for feed/food
comparing HRMS data with bioassay dataSensitivity: Can bioassays measure dioxin-TEQ using only 1 ml of
blood as HRMS/HRGC methods are able?Can cheaper methods like PCR or ELISA technologies fulfill EU
requirements?Impact of antagonistic/agonistic PCBs to the total TEQ?False negative rates: 1% accepted according to EU guidelines? Low
Biological screening tests for dioxinlike compounds can be used according to new EU guidelines for feed/food, Samples negative when the tested levels are lower than 30-40% of the level of interest (limit).
Further research is neccesary to evaluate and compare the different screening technologies for dioxin-like compounds. (e.g. EU reference materials, international round robin studies).
Improvement and standardization of clean-up required for further reduction of variation: problem related to both bio- and immunoassays.
Combination of Bioassay screening tool and Mass-spectrometry confirmation method have been already successfully used as crisis management tools in the food/feed field!
40 September, 16th 2004www.dioxins.de
GfA staff
41 September, 16th 2004www.dioxins.de
Bioassays for dioxins – let’s test the whole dioxin life cycle!