Hyogo Pref. Inst. of Pub. Health & Env. Sci. 8th October...

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


eurofins Global Business

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GfA - History


PCDD/F PBDD/FPBCDD/FPCBPAHPCNPCBzPCPPCPh

PBDEPBBHBCDTBBPA PCTchloroparaffinesorganotinsalkylphenolsheavy metals

dioxins & more

Contaminant analysis performed by GfA – eurofins POPs competence centre –


Dioxin and dioxin-like PCBs guidelines in Japan and Germany

Japan Germany

Emission: 0,1 ng TEQ/m3 SameFly ash: 150 ng TEQ/g no law

Water: 1 pg TEQ/l no lawWaste water: 10 pg TEQ/l Groundwater 5 pg TEQ/l

Sediment: 150 pg TEQ/g no lawSoil : 1000 pg TEQ/g 100/1000/10000 pg TEQ/g Sewage sludge: no law 100 ng TEQ/kg

Chemicals: no law 1/5/100 µg/kgSubstances, Mixtures: no law 2 µg 2378 TCDD/kg


Dioxin and dioxin-like PCBs recommendations

Japan Germany

Atmosphere air: 0,6 pg TE/m3 0,016 pg TE/m3

Indoor air (after remediation): 0,5- 2 pg TE m3

recommendationLandfills: 500-1000 ng TE/kgDioxins in workplace: 2,5 pg TEQ/m3 50 pg TE/m3 TRK

Feed/Food: no law EU guidelines

TDI: 4 pg TEQ/kg/day Same

Dioxin-like PCBs: in evaluation for feed/food and emission


How are dioxins regulated? EU guidelines for feeding stuff; some examples

0

1

2

3

4

5

6

7

ng WHO-PCDD/F-TEQ/kg feeding stuff with a moisture content of 12 %

Fish oilFeed for

fishesAnimal

fatAquaticanimalsMineralsFeeding-

stuffMilk, Eggs

Binders Plants


How are they regulated? EU maximum limits for food

0

1

2

3

4

5

6

7

pg WHO-PCDD/F-TEQ/g fat basis resp. pg WHO-PCDD/F-TEQ/g fresh weight in case of muscle meat of fish

LiverVeg. oil

Pork meat

Poultry meat

Fish oil Sheep meat

Cow meat

Milk Hen eggs

Muscle meat of fish


Pizza

Tea

Orange juice

Cornflakes

Lollys

etc.Mashed potatoes

etc.Plant oilChocolate

Mineral concentratesSilageSoja souce

VitaminesPed foodetc.Sausage

etc.GlycerineAnimal fatPlant fatCroissantsetc.

Cilicic acidsAnimal mealBleaching earthNutsWhey products

authoritiesetc.TalcumMilk replacerBentoniteHoneyYoghurt

supervising Whale fatIron powderMolassed sugarProteinsOlive oilCottage cheese

VeterinaryCod liverCarbonatesCoca ShellsRape oilEggsCheese

Marine oilKaoliniteRape mealPalm oilCerealsButter

authoritiesFish oil capsulesMagnesium sulfateSugar beetsCastor oilSaladeCream powder

supervising FishZinc SulfateGrasSoja oilVegetablesCream

FoodstuffFish mealZinc OxideCitrus pelletsCocos oilFishMilk powder

Fish oilAmmonium Choridefeed of plant origeneVitaminesMeat (all kinds)Milk

(Monitoring)Hygiene products

EnvironmentFish productsFeed additivesFeedPharmaceutical / FoodDiary products

Feed/Food Matrices analysed for dioxins


EC action levels and maximum levels in food and comparison to literature data/values

vege

tabl

es

vege

tabl

e oi

l

pig

fish

oil

poul

try

milk

egg

cow

shee

p

fish

liver

9 54 14,8 8

0

1

2

3

4

5

6

7

8

pg W

HO

-PC

DD

/F-T

EQ/g

fat b

asis

resp

. ng

WH

O-P

CD

D/F

-TEQ

/kg

fres

h w

eigh

t (ve

g, fr

uit,

fish) action level

limit value

lit. cited mean values

GfA lit. cited max. values


Alerts Notifications from the EU for Food – rise is also reflected in public perception

Source: EU, Rapid Alert System for Food and Feed (http://europa.eu.int/comm/food/food/rapidalert/report2003_en.pdf)

0

100

200

300

400

500

1997 1998 1999 2000 2001 2002 2003

EU Alert Notifications


Global dioxin crisis situations (a few examples)

USA, 1958: Chicken oedema factorJapan, 1968; Taiwan, 1979: Contaminated rice oilSpain, 1982: Contaminated olive oil

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


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.


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


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


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


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.


How does the CALUX reportergene assay works?

Mouse/Rat liver cells:Stabile transfected with an AhR controlled

luciferase gene construct

　　　　　　　　　　　　　　　　　　　　　　　　　

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　

2

Spread cells in 96 well plates

Add sample and TCDD

Luciferase enzyme reaction

Luminometer: Luciferase + Luciferin ⇒Light + Oxyluciferin

Culture for 24 hrs

Culture for 24 hrs


EU-Guidelines: CALUX Applications (literature review)

Laier et al. 2001

1.5; Max. 2 - foldMother milk

Overmeire et al. 2003

1.6Max. 5 - fold

0.73.0Cow milk

Jeong et al. 2001

0.7520.10.75Feed

Cederberg et al. 2002

0.92Max. 3 - fold0.1 – 0.70.75 –4.0

Food

Jeong et al. 2001

0.851.50.73.0Pork

Yabushita et al. 2002

0.891.70.14.0Fish

ReferenceCorrelation CALUX to GC/MS: R2

CALUX-TEQ/ WHO-TEQMedian

CALUXLOQ

EU-LIMIT

Matrix


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


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


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


ELISA technologies: e.g. Ah-Immunoassay


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


Polymerase chain reaction (PCR) analysis: AhPCR kit

Receptor-Probe Complex Trapped

Activated ReceptorCaptures Probe

Probe is PCRAmplified

And Measured

Dioxin ActivatesReceptor

TM


Proteomic analysis for dioxins

Proteomic dioxin biomarker analysis:

Measurement of

Enzymes (e.g. Superoxide dismutase)

Stress proteins (e.g. hsp60),

Receptors or

Cytosketal proteins (e.g. Myosin).


What‘s possible as fastest turnaround time (TAT) for feed/food? Sample flowchart; rough estimate

ASE Rotavaporor Polyvap

Mini clean-up/ PowerPrep

N2 + DMSO

Turbovap Total time incl. detectionSteps

Time per step: 1 sample

CALUX: 8 h ; ELISA: 7-8 hHRMS: 5-6 h

Sample preparation:depending on matrix: e.g. fish: freeze drying 24 h,

feedstuff about 30 min


CALUX: 8 h ; ELISA: 7-8 hHRMS: 12-14 hrs


CALUX: 3-6 days, 10 man daysELISA: 4-6 days; 10 man days

HRMS: 9-14 days, 30 man days

30 min (R) 240 min 15 min

2.5 days 3.5 days 1.5 days

90 min

6 days

360 min60 min (P) 90 min 45 min

30 min

120 min

4 days


Time and Cost of analysis

35,000-60,000

3-5Reporter Gene Assay (Method 1-4)

AhR –basedBioassay

200,00010-20The GC/MS Method

36,000-50,000

1-3DXNs-Immunoassay(Method 7-11)

40,0001AhR PCR Assay(Method 6)

45,0003Ah-Immunoassay(Method 5)

Costs(yen/sample)

Time(day/sample)

Category


International validation studies

Sediment International calibration round robin:

Besselink et al. OC, 58, 417 (2002): 6 CALUX labs standard deviation 28%

Dioxinlike Compounds in Food using Bioassays, MTE, Örebro, University, Sweden: Several screening methods; Cod liver, fly ash, fly ash extract, salmon tissue

Validation study EU-Joint Research Center (10 labs):

CALUX-TEQ 50% (Fish oil) and 60% (Feed) lower than WHO-TEQ

National comparison study of bioassays in Japan: see presentation Ota et al.

Several screening technologies have been tested for standards, fly ashes and flue gas samples


Current research projects in the EU: DIFFERENCE

WWW.Dioxins.nl: Project Screening methods for dioxins in feed/food

Round 1: Spiked vegetable oil

-3

-2

-1

0

1

2

3

CALUX

CALUX

ASE/GC-HRM

S

GC-HRMS

GC-LRMS

GC-HRMS

ASE/CALUX

GCxGC

Analytical method

Z-sc

ore

Total TEQDioxin-TEQPCB-TEQ


Salmon muscle, Baltic Sea

2 4 5 9 15 23 24 25 28 6 8 16 20 19 13 7 10 14 17 270

5

10

15

20

25

30PCDD/Fnon-ortho PCBsmono-ortho PCBs

pg T

EQs/

g fre

sh w

eigh

t

DR-CALUXCALUX

P450

RGS

H4IIE

-luc

ERO

D im

mun

oass

ays

WHO

-TEQ

s

DR C

ALUX

TEQ

s

CALU

X TE

Qs

(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


Comparison with the GC/MS measurements Comparison with the GC/MS measurements ( Standard Samples )( Standard Samples )

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Rati

o B

ioass

ay/G

C-M

S

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11

Sample1E Sample1F Sample1G

AhR-based Bioassays

M1 – M6

DXNs-Immunoassays

M7-M11


0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Rati

o B

ioas

say/G

C-M

S

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11

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


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


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

action limits required: false positives rates increase


Future Outlook

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!


GfA staff


Bioassays for dioxins – let’s test the whole dioxin life cycle!

Hyogo Pref. Inst. of Pub. Health & Env. Sci. 8th October...

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