survey on t-2 e ht-2 toxins in samples of ...patologia vegetale, univ.di padova 2dipartimento di...
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SURVEY ON TSURVEY ON T--2 E HT2 E HT--2 TOXINS IN 2 TOXINS IN SAMPLES OF CEREALS AND SAMPLES OF CEREALS AND
PRODUCTS THEREOF FROM NORTH PRODUCTS THEREOF FROM NORTH ITALYITALY
11Dipartimento Dipartimento TeSAFTeSAF, , Sez.Sez. Patologia Vegetale, Patologia Vegetale, UnivUniv.di Padova.di Padova22Dipartimento di Scienze Animali, UniversitDipartimento di Scienze Animali, Universitàà di Padovadi Padova33OR SELLOR SELL44GLMGLM--AIRESAIRES
Causin R.Causin R.11, , MastellaMastella C.C.22, , MergoniMergoni V.V.33, , Pizzolato G.Pizzolato G.44, , BailoniBailoni L.L.22
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T2 and HT-2 TOXIN
Trichotecenes: contaminants of cereals, mainly in humid and coolclimates
Produced by different fungi belonging to genus Fusarium.
The most interesting specie is F. sporotrichioides; in ourarea it is a weak pathogen, psychrofile and hygrophilus.
> 0,9935Max
22,5 ÷ (27,5)Opt0,88- 2Min
AwTemp. °C It produces toxins between 8 and 25°C with opt. T < 15°C and Aw between 0.95 and 0.97. Itsurvives at 30°C but at this temp. it stops toxinogenicactivity.
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T2 and HT-2 TOXICITY
HUMAN
Toxic aleukia (ATA). Thousands of people poisoned in Siberia during the second world war
ANIMAL
Hemorrhagic syndrome, oral ulcer, gastrointestinal disease and neurotoxic effects (chicken)
IMMUNODEPRESSION
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T-2 and HT-2 estimated dietary intake exceed in most cases the t-TDI (Final Report SCOOP TASK 3.2.10)
T2 and HT-2 TOXICITY
temporary TDI 0.06 µg/kg bwcombinedT2+HT-2
Italy
Daily intake T2
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EU REGULATIONs no. 856/2005 and 1881/2006In most cases the estimated T-2 and HT-2 dietary intake exceedthe t-TDI. However, it has to be remarked that the majority of T-2 and HT-2 occurence data were obtained by analysis methods with high detection limit (DL). Moreover, considering that less than 20% of the samples has been found above the DL, the dietary intake was stronglyinfluenced by the detection limit of the analysis methods.
FINAL REPORT SCOOP TASK 3.2.10Few available data on food contamination.
AVAILABLE DATA ON T2 AND HT-2
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AIMS
To increase knowledge about the contamination by these toxins of wheat, barley and maize samples collected in experimental fields, drying units and mills.
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EXPERIMENTAL FIELD TRIAL (Maize - Year 2004)
68 hibryds: (6 Class 300; 5 Class 400; 17 Class 500; 29 Class 600; 11 Class 700)2 location (VENICE area and ROVIGO area)4 replications
Toxin T2 (ppb)
0,00
20,00
40,00
60,00
80,00
100,00
0 - 10 11- 40 41 - 70 71 - 100 101 - 130
ppb
sam
ples
% VERO
15,87Total13,48RO18,42VEAv. ppb T2Loc.
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EXPERIMENTAL FIELD TRIAL (Maize - Years 2000-2006)
Isolation of F. sporotrichioides
Low frequency (< 1% ; max. value 3,6%)
Sometimes no findings (sporadic presence)
Location and season influence
Most frequently isolated during the first period of vegetative stage (pollen and silk)
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ANALITYCAL ASSAYES ON CEREAL SAMPLES
Cereals: 21 wheat samples17 barley samples32 maize samples
Method of sampling : Dynamic
Origin: crop areas of North Italy(drying units, mills and feed stores)
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METHOD: ELISA
Range 0,025-0,5 mg/kg
Kit EZ Quant T-2® (Diagnostix) – Or sell -
<0,04Verrucarol<0,04T-2Tetraol
1,6T-2 Triol38HT-2
% Cross-reactivityChemical
Determination of T2
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METHOD HPLC (preparation)
• Method Visconti et al., 2005. LOD 5 µg per T2 e 3 µg per HT2
• 50 g of sample in 250 ml of water-methanol solution(90% methanol)
• estraction• clean-up on immunoaffinity column with monoclonal
antibody T-2 e HT-2 (EASY- EXTRACT T-2 & HT-2, RHONE LTD supplied by OR SELL)
• derivatisation (solution of D-MAP and of 1-AN in toluene)
• dried sample retake with 1 ml of acetonitrile/ water solution – 70/30.
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METHOD HPLC (cromatographic condition)
•Column: Gemini 5µ C6 – Phenyl 110 A (150 X 4.6 mm)•Mobile phase: binary gradient of water– acetonitrile with a starting composition of 70 % of acetonitrile•Column temperature: 40°C•Flow: 1 ml at minute•Injected volume: 75 µl•Wavelenght: Excitation: 381 nm
Emission: nm 470
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METHOD HPLC Chromatogramm of a STD
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METHOD HPLC: Chromatogramm of a real sample
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84.7067.23Barley
96.9493.58Maize
97.0567.27Wheat
H-T2T-2
METHOD HPLC (estimation of recovery %)
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Toxin T2 (ppb) Method ELISA
0
1020
30
40
5060
70
0 - 10 11- 40 41 - 70 71 - 100 101 - 130
ppb
sam
ples
% FOM
RESULTS
FREQUENCY (Method ELISA)
WBM
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RESULTS
SAMPLES FREQUENCY >LOD (Method HPLC)
19%26%Maize
47%18%Barley
15%10%Wheat
HT - 2T - 2Cereal
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RESULTS
AVERAGE VALUES, METHODS ELISA AND HPLC
0
10
20
30
40
50
Frumento Orzo Mais
Cereal
ppb
Toxi
n
ELISAHPLCScoop TaskCampo '04
T2 e HT-2* in wheat, barley, Maize
* T2 and HT2 values available only for the HPLC survey
15,8712,917,0316,59M
30,0225,37B
40,450,6846,29W
FieldSc.Tsk3.2.10
HPLCElisaCer
wheat barley maize
Field ‘04
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RESULTS
ISOLATION FREQUENCY F. sporotrichioides
------Maize
0,11%0,24%Barley
0,18%0,7%Wheat
Samples“LOW”
Samples“HIGH”
Cereal
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CONCLUSIONSDifferent contamination in different cereals (but the influence of the environmental factor must be considered)
In most cases samples have a low level of contaminationin comparison with other Fusarium-toxins (F. sporotrichioides is not very frequent)
Values pointed out are similar to that of Final reportSCOOP TASK 3.2.10; values with HPLC are a little higher.
Analitycal methods: ELISA overvalue or undervalue? HPLC results to be confirmed (few data over LOD) but it isevident the superposition with ELISA which reacts onlywith part of HT2.
The situation seems not worrying but…which will be the limits?