1

Behnisch, Peter A.; Besselink, Harrieand Brouwer, Bram

GERMAN DIOXIN CRISIS 2011 –

EXPRESS ANALYSIS OF PCB-AND PCDD/F-TEQ

IN FOOD AND FEED MATRICES

BY THE HTPS DR CALUX®METHOD

DIOXIN 2011, Brussels

2

3

Situation in January2011

•4700 farmsat firstclosed–issuetakenup by

international media

•70% of Germansdoubted, iftheirbreakfasteggsare

dioxin-free

•Ca. 70% lowerrevenuesforfarmers/industry–rough

calculationstalkedfrom20 MioEuro/weekdamage

•Most localtestingneeded> 10 days= lack of local

testingcapacity

•Screeningvia DR CALUX offerstime and costsaving

•Morethan95% of theca1800 samplesfrom

Germany testedas express analysisin 2-3 days

4

WHO-TEF/DR CALUX REP valuesof pollutedfoodfat

83

18

1800

H7D

197

121

SUM

Low riskof false

negative!

123

SUM All

18

27

270

123789-H6D

85

61

610

123678-H6D

3,6

1,2

12

123478-H6D

7,6

14

14

12378-P5D

DR CALUX-TEQ

WHO-TEQ

Conc.

5

Situation in January/February2011

•Morethan15 countriesbannedGerman meat

•German Meat Association (VDF) in cooperationwith

German Authorities(BMELV) and theRussianImport

Authoritiesacceptedmorethan40 labsforthe

German meatexports–only4 non-German

laboratories

•BDS onlyacceptednon-Germanlab usingscreening

technology DR CALUX

•Regardingacceptanceof ISO 17025 accreditedlabs

pleasenoticethataccordingEC/764/2008 all reports

of such accreditedlabshaveto beacceptedin all EC

memberstates

•BDS ISO 17025 accreditedsince2005 and new

approvedin mid2011 until2014

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BDS ISO 17025 accreditation(Dutch RvA)

Quantitative screeninganalysisaccordingto current

EC guidelines:

•BDS newlyaccreditedin mid2011 byDutch RvA

•BDS twosuccessfullPT in 2010

•BDS usesseveralrelevant referencematerialswith

precisionbelow30%

•Linearity(R

2>0.98), sensitivity(1/5th EC level) and

precision(below30%)

•All in 2011 byHRGC/HRMS cross-checkednegative

samples(2%) didnotshowanyfalsenegative results

byDR CALUX

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BDS DR CALUX vsHRGC/HRMS –

170 comparisons: <1% falsenegativ and <8% falsepositive

Matrix

N False negative

False positive

egg

18

0 3

fish

14

0 0

Poultry

meat

9 0

1

Rumina

nt me

at 6

0 0

Pig m

eat

14

0 1

fish m

eal

8 0

0

Fish o

il 15

0

1

milk

33

0 3

Feed

, plan

t orig

in 18

0

4

Feed

, veg

etable

oil

7 0

1

8

Examplesof DR CALUX usagein such

crisissituationsorforNational Monitoringprojects

•I) Examplesof such crisissituationsin whichtheDR CALUX

performedbyBDS has beeninvolved:

–Ireland(2009)

–Food/Feed

–Chile (2009)

–Food/Feed

–Italy(2008)

–Milk

–Netherlands(2005) –

Milk

–Belgium(1998)

-Eggs

•II) Examplesof governmentalmonitoringprojectsof feed/food

in whichDR CALUX byBDS has beeninvolved:

Chile (2008), Israel (2011), Morocco(2011), Mauretania(2010),

Netherlands(m

anyyears)

9

EarlierDutch reportsaboutdioxinsin eggs

analysedbyDR CALUX and HRGC/HRMS

•Controlling egg dioxin levels from laying hens with outdoor run by A. Kijlstra,

W. Traag and R. Hoogenboom (2008)

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

18,0

-4,0

-2,0

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

18,0

GC/MS (pg TEQ/g vet)

CALUX (pg TCDD eq/g vet)

•Bio-eggsin Dutch farmsbyJ. de Vries (2002)

At 2002 from 68 “biological”eggs 9 (13%) eggswere suspected in the DR-CALUX.

HRGC/MS showed, that total TEQ varied between 2.6 and 14.9 pg TEQ per gram of fat.

DR CALUX-TEQ

HRGC-HRMS TEQ

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DIOXIN 2011 e-poster: ISZ Brescia, Italy

DR CALUX vsHRGC/HRMS:

100% of milk samplesin compliantwindow–

91% of egg samplesin non-compliantwindow

APPLICATION OF DR-CALUX TO MILK AND EGG SAMPLES:

COMPARISON BETWEEN HRGC-HRMS AND SCREENING DATA

Gasparini , Bertasi and Ferretti et al

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HTPS DR CALUX®Methode

I) Rapid HTP Extraktion

30 samples/person/3 hrs

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A) Clean-up:15 Proben/hrs

Low costs: ca. <15 Euro/sample

HTPS DR CALUX®Methode

II)Clean-Up and PCB/PCDD/PCDF Separation

B) DL-PCB and PCDD/F separation

(alumina oxide method according to

DIN 2464): ca. 15 Proben/hr

Low costs: <15 Euro/sample

13

HTPS DR CALUX®Methode

III) Robot forSeeding cells

and Dosingstandards/samples

40 samples/hr

Hamilton MicrolabStarlet

14

Evaluation seeding/dosingvia robot

vs. Manual analysis

Dose-response curve DR CALUX

LOG[TCDD] (M)

Signal

-14

-13

-12

-11

-10

-9

0510

MICROLA

B STARlet

Man

ual

Samples

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HTPS DR CALUX®Methode

IV) MeasurementPCDD/F-and separate PCB-TEQ

DR CALUX®:

40 sample/hrforseparate

Dioxin and PCB-TEQ

(TristarMultimode Reader

Berthold Technologies

withstacker)

For comparison:

HRGC/HRMS:

0.5 sample/hrforseparate

Dioxin and PCB-TEQ

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German DIOXIN Crisis2011 –

ResultsbyDR CALUX testing

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German pigmeat> 97,6% negative;

lessthan2.5 % falsepositive byDR CALUX

0,1-

1,47

0,1-

0,77

0,1-

1,1

Pig

Meat;

Range

0,43

0,21

0,23

Pig

Meat;

Mean

89%

94%

97,6

99,8%

502

496

496

Pig

Meat; N

Sample

compliant

TOTAL

-50%

Sample

compliant

PCDD/F

-50%

Sample

complian

tTOTAL

-25%

Sample

compliant

PCDD/F

-25%

Total

-TEQ

PCDD/F

-TEQ

PCB

- TEQ

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DR CALUX analysisforPCB + PCDD/F-TEQ(# 502 samples):

German pigmeatDistribution

Total-PCDD/F/dl-PCB-TEQ by DR CALUX for German pig

meat in Jan/Feb 2011 in pg PCDD/F/dl-PCB-TEQ/g fat

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

1,60

0100

200

300

400

500

600

Numbers of samples

PCDD/D/dl-PCB-TEQ by

DR CALUX

EC guidedlevel1.5 ng PCDD/F/PCB-TEQ/kg fat

19

DR CALUX analysisforPCDD/Fs(# 496 samples):

German pigmeatDistribution

Distribution PCDD/F-TEQ by DR CALUX for German pig meat in Jan/Feb 2011

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

0100

200

300

400

500

600

Number of tested samples

PCDD/F-TEQ by DR CALUX

EC guidedlevel1.0 ng PCDD/F-TEQ/kg fat

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German pigliver> 97% negative;

lessthan3 % falsepositive byDR CALUX

0,1-

5,2

0,1-

3,4

0,1-

2,1

PigLiver;

Range

1,57

0,78

0,80

PigLiver;

Mean

90%

93%

97%

99%

34

37

32

PigLiver;

N

Sample

compliant

TOTAL

-50%

Sample

compliant

PCDD/F

-50%

Sample

compliant

TOTAL

-25%

Sample

compliant

PCDD/F

-25%

Total-

TEQ

PCDD/F

-TEQ

PCB-

TEQ

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German poultrymeat> 98% negative;

lessthan2 % falsepositive byDR CALUX

0,6-

3,8

0,3 –

1,7

0,3-

3,3

Chicken

Meat;

Range

1,3

0,56

0,74

Chicken

Meat;

Mean

88%

93%

98%

99,5%

207

201

201

Chicken

Meat; N

Sample

compliant

TOTAL

-50%

Sample

compliant

PCDD/F

-50%

Sample

compliant

TOTAL

-25%

Sample

compliant

PCDD/F

-25%

Total-

TEQ

PCDD/F

-TEQ

PCB

-TEQ

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DR CALUX analysisforPCDD/F/PCB-TEQ(# 207samples):

German poultrymeatDistribution

PCDD/F/dl-PCB-TEQ by DR CALUX for German

poultry meat in Jan/Feb 2011

01234

050

100

150

200

250

Number of samples

PCDD/F/dl-PCB-TEQ by

DR CALUX in ng

TEQ/kg fat

Reeks1

EC guidedlevel4 ng PCDD/F/PCB-TEQ/kg fat

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DR CALUX analysisforPCDD/F-TEQ(# 201 samples):

German poultrymeatDistribution

PCDD/F-TEQ by DR CALUX of German poultry in

Jan/Feb 2011 (cut-off 1,4 pg PCDD/F-TEQ/gr fat)

0

0,51

1,52

050

100

150

200

250

Number of samples

PCDD/F-TEQ by DR

CALUX in ng TEQ/kg

fat

EC guidedlevel2 ng PCDD/F-TEQ/kg fat

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German eggsand feedingstuff> 98% negative;

lessthan2 % falsepositive byDR CALUX

100%

100%

16

Feed;

N

0,23

Feed;

Mean

0,1-

0,65

Feed;

Range

0,1-

2,6

0,1-

2,3

0,1-

1,0

Eggs;

Range

1,48

1,21

0,67

Eggs;

Mean

89%

94%

98%

99,8%

36

99

Eggs;

N

Sample

compliant

TOTAL

-50%

Sample

compliant

PCDD/F

-50%

Sample

compliant

TOTAL

-25%

Sample

compliant

PCDD/F

-25%

Total-

TEQ

PCDD/F-

TEQ

PCB-

TEQ

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DR CALUX –higherfood/feedqualitythrougha highersafety

bycoveringalso PBDD/Fs, PBBs, PBDE, PCNs…….

0.0004

2,3,6,7-TCN

0.080

PBB-77

0.16

PBB-126

4.4E-4

Benzo[a]pyrene

0.00013

PBDE 126

0.0011

8-Iod-2,3,4-TriCDF

0.0002

8-Fluor-2,3,4-TriCDF

0.01

8-Methyl-2,3,7-Tri CDD

0.1

2,3,4,7,8-PBDF

0.60

2,3,7,8-TBDF

0.28

1-Br-2,3,7,8-TCDD

0.21

1,2,3,7,8-PBDD

0.67

2-Br-3,7,8-triCDD

0.86

2,3-diBr-7,8-diCDD

0.77

2,3,7,8-TetraBrDD

0.033

2,3,7-TriBrDD

0.0015

2,3,7-TriCDD

DR CALUX REPs

= chemical

analysis

below10%

false

negative

results

regarding

dioxin-like

toxicity

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Take homemessage

•During the German DIOXIN crisis in February-March 2011 more than 1800

samplesfrom Germany have been tested by DR CALUX in just 8 weeks time

with an average TAT of 3 days –a significant time/cost saving!

•Here we report results of ca. 500 samples of pig meat, 200 of chicken meat, 40

of pig liver, several egg and feed samples

•For more than 90%of the samples analyzed by BDS, the PCB-TEQand

PCDD/F-TEQhave been reported separately by using a modified protocol of

the DIN 2464 method.

• •In case of feeding materials and premixes tested by DR CALUX, all samples

have been complaintwith levels below 50% of the in the EC/152/2009 regulated

levels.

•In case of the analyzed pig and poultry samples, more than 97%of the samples

have been complaint

•In case of the analyzed pig and poultry samples more than 88%of the samples

have been even lower than 50%of the EC regulation levels.

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•In case of compliant DR CALUX results you are also able to exclude

relevant effects of PBDD/Fs, PBBsor PCNs= higher feed/food quality

and safety!!!

•Now please join us to break the 100 €/140 USD/11000 Yen cost barrier

of PCDD/F/PCB-TEQ analysis (under ISO 17025 mode) via Robotics!

•For the price of a typical HRGC/HRMS analysis you are now able to

analyze PXDD/PXDF/PXB as well as obesogens(PPAR CALUX),

hormones (CALUX panel) and all kinds of endocrine disrupting

chemicals (CALUX panel) –to obtain a higher quality of feed/food

safety!

•The results of this study shows again that the DR CALUX®bioassay

for screening of PCDD/Fs and dioxin-like PCBs in feed and food is an

important device to identify the few percentage of the EU limit

exceeding samples among the bulk of the compliant samples….!!!

Take homemessage

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•Thankyouverymuchforyourtime and interest…

•Herzlichen Dank für Ihre Zeit…

•Dank u wel…

•Mercibeaucouppour votretemps..

•Domo arrigato…

•Muchasgracias

………………..