CNR, Institute of Sciences of Food Production (ISPA) - Bari, Italy

Occurrence of T-2/HT-2 toxins in durum wheat and pasta

Fate of T-2/HT-2 toxins during durum wheat processing

SECTION B: Presence of T-2 and HT-2 toxin in cereals and cereal products

Seventh Fusarium Forum 2010 ● 1st-2nd February 2010

The European semolina industry in a few figuresThe European semolina industry in a few figuresThe European semolina industry implies:

231 production units:162 production units in Italy;16 production units in Greece;8 production units in Spain;7 production units in France;5 production units in Germany.

7,300,000 tons of durum wheat transformed:5,100,000 t in Italy;675,000 t in France;550,000 t in Spain.

4,850,000 tons of durum wheat semolina produced.

Union of Associations of Semolina Producersof the EU Countries

The Union of Associations of Semolina Producers of the EU countries has the purpose of ensuring the representation and promotion of the interests of the semolina industry of the European Union at European and International levels.

With an annual production, calculated on the basis of the last ten years, of approximately 35 Mt, durum wheat represents, at world level, only a «secondary» cereal, as regards quantity.

In the EU, and limited to the years subsequent to the Reform of the CAP Agenda 2000, this production has amounted to between 7.1 and 11.1 Mt, for an average annual production of 8.4 Mt.

- An extremely localised world productionAn extremely localised world productionThe Mediterranean basin and the North American continent alone represent almost 80% of the world production of durum wheat;

-- A quantitatively fluctuating world productionA quantitatively fluctuating world productionThe agronomic situation and the particular climatic conditions of the areas producing durum wheat lead to an extreme variability in production which may take the form, year by year, of quantitative fluctuations in the order of 35%.

Durum Wheat: a rare and precious cerealDurum Wheat: a rare and precious cereal

Production (Mt) Imports (Mt) import/production (%)

2000 8.8 0.9 10.2%2001 7.3 1.5 20.6%2002 9.2 0.6 6.5%2003 7.9 2.0 25.3%2004 11.1 1.6 14.4%2005 7.1 1.6 22.5%2006 7.8 1.6 20.5%2007 7.1 1.9 26.7%2008 9.9 1.4 14.1%2009 (estimates) 7.9 1.7 21.5.%

Quantity of durum wheat transformed 7,300,000 tProduction of semolina 4,850,000 t

The production of Durum Wheat in the EUThe production of Durum Wheat in the EUThe EU production of durum wheat is not able to cover the quantitative requirements, but neither the qualitative requirements, of the semolina industry of the EU.

Intake estimates indicate that the presence of T-2 and HT-2 toxin can be of concern for public health. Therefore, the development of a reliable and sensitive method, collection of more occurrence data and more investigations/research in the factors involved in the presence of T-2 and HT-2 toxin in cereals and cereal products, in particular in oats and oat products, is necessary and of high priority.

COMMISSION REGULATION (EC) No 1881/2006COMMISSION REGULATION (EC) No 1881/2006setting maximum levels for certain contaminants in foodstuffssetting maximum levels for certain contaminants in foodstuffs

TT--2 and HT2 and HT--2 toxins2 toxinsProvisional Maximum Tolerable Daily IntakeProvisional Maximum Tolerable Daily Intake

PMTDI = 60 PMTDI = 60 ngng / kg body weight / day/ kg body weight / dayLOEL (lowestLOEL (lowest--observed effect level) = 29 observed effect level) = 29 µµg/kg of body weight per day (pigs) g/kg of body weight per day (pigs) -- Safety factor = 500Safety factor = 500

Fate of T2/HT2 toxins during Fate of T2/HT2 toxins during durum wheat processingdurum wheat processing

Determination of T-2 and HT-2 toxins in durumwheat and milling fractions

LC-MS/MS determinationMultiple Reaction Monitoring (MRM) mode (5 transitions monitored per toxin, internal standard used)

Immunoaffinity columnclean-up

(T-2 Test™ HPLC)

ImmunoaffinityImmunoaffinity columncolumncleanclean--upup

(T(T--2 2 TestTest™™ HPLC)HPLC)

Extraction(with methanol:water + NaCl)

Filtration (Whatman Nº 4)Dilution with water (1:5)Filtration (Whatman GF/A)

13C24T2 toxin and 13C24HT2 toxin (Biopure, Austria)

RecoveriesRecoveries andand LimitLimit of of detectionsdetections (LOD)(LOD)

0.70.5101 (5)102 (3)semolina

0.20.1102 (2)101 (10)pasta

0.70.5107 (2)104 (3) wheat kernels

1.40.991 (7)103 (14)red dog

0.90.692 (9)90 (5)bran

1.71.585 (6)98 (3)screenings

1.50.985 (5)92 (5)fine middlings

HT-2T-2HT-2T-2

Millingfraction

LOD(ng/g)

Recovery (CV)*(%)

*spiking levels: 100 ng/g T-2 and 100 ng/g HT-2 (n= 3)

Field trials:Field trials: 2007-2008 and 2008-2009 growing seasons (Northern and Southern Italy).

Fungal Fungal inoculuminoculum:: Fusarium sporotrichioides (ITEM 707)(ITEM 707) from the collection of the CNR-ISPA (www.ispa.cnr.it/Collection).

Wheat variety:Wheat variety: Levante (Triticum durum Desf.).

Experimental design:Experimental design: rrandomised blocks with 4 replicate plots per trial (plot area 20 m2). Treatments with three different fungicides (prochloraz, tebuconazole, prothioconazole), one Fusarium inoculated control and one non-inoculated control.

Fungicide treatments:Fungicide treatments: wwithin 24 hours before fungal inoculation(beginning of anthesis, BBCH 61).

Durum wheat samples originDurum wheat samples originInoculated test plotsInoculated test plots

100 100 -- 235 235 -- 450 450 -- 590590

Harvest 2008 Harvest 2008 –– 4 level of contamination: 4 level of contamination:

Harvest 2009Harvest 2009 –– 7 level of contamination: 7 level of contamination:

TT--2+HT2+HT--2 2 ((ng/gng/g))

TT--2+HT2+HT--2 2 ((ng/gng/g)) 221 221 -- 507 507 -- 640 640 -- 720 720 -- 1160 1160 1440 1440 -- 59545954

ProcessedProcessed samplessamplesUncleanedUncleaned durumdurum wheatwheat

Pilot Pilot processprocess flowflow

Uncleaned wheat

Screenings

Cleaned Wheat

Bran Semolina

Pasta

MillingBühler Mill Model MLU 202

Tempering(final moisture 17%, 14 h + 3 h)

CleaningRationel Kornservice Mod. M220V(5 mm × 15 mm e 2 mm × 19 mm)

Red dog Fine middlings

Pasta productionSercom extruder + Braibanti dryer

LevelsLevels of Tof T--2 + HT2 + HT--2 in 2 in durumdurum wheatwheat millingmilling fractions*fractions*

* mean ± SD (n = 4)

0

50

100

150

200

250

300

350

400

Uncleanedwheat

Cleanedwheat

Bran Red dog Finemiddlings

Semolina Pasta

T-2

+ H

T-2

(n

g/

g)

HT-2

T-2

TT--2 + HT2 + HT--2 = 221 2 = 221 ±± 10 ng/g10 ng/g

221

23

46

245

68

317

Screenings834 ± 80 ng/gScreenings

834 ± 80 ng/g

2

LevelsLevels of Tof T--2 + HT2 + HT--2 in 2 in durumdurum wheatwheat millingmilling fractions*fractions*

0

200

400

600

800

1000

1200

Uncleanedwheat

Cleanedwheat

Bran Red dog Finemiddlings

Semolina Pasta

T-2

+ H

T-2

(n

g/

g)

HT-2

T-2

TT--2 + HT2 + HT--2 = 507 2 = 507 ±± 29 ng/g29 ng/g

Screenings921 ± 67 ng/gScreenings

921 ± 67 ng/g

from inoculated test plots* mean ± SD (n = 4)

507

58124

703

153

909

5

100

1

295

48

134

2212

0

50

100

150

200

250

300

350

400

450

Uncleanedwheat

Cleanedwheat

Bran Red dog Finemiddlings

Semolina Pasta

T-2

+ H

T-2

(%

) Screenings = 597% Screenings = 597%

DistributionDistribution of Tof T--2 + HT2 + HT--2 (%) in 2 (%) in millingmilling fractions*fractions*

* mean ± SD (n = 11) from processing of durum wheat contaminated with T-2 + HT2 in the range 97-5954 ng/g (four replicated experiments)

DistributionDistribution of Tof T--2 (%) in 2 (%) in millingmilling fractions*fractions*

100

1

41

160

138

24

12

0

50

100

150

200

250

Uncleanedwheat

Cleanedwheat

Bran Red dog Finemiddlings

Semolina Pasta

T-2

(%

)Screenings = 868% Screenings = 868%

* mean ± SD (n = 11) from processing of durum wheat contaminated with T-2 in therange 35-785 ng/g (four replicated experiments)

100

1

345

49

126

2212

0

100

200

300

400

500

600

Uncleanedwheat

Cleanedwheat

Bran Red dog Finemiddlings

Semolina Pasta

HT

-2 (

%)

Screenings = 560% Screenings = 560%

DistributionDistribution of HTof HT--2 (%) in 2 (%) in millingmilling fractions*fractions*

* mean ± SD (n = 11) from processing of durum wheat contaminated with HT-2 in therange 62-5169 ng/g (four replicated experiments)

An important level of T-2 + HT-2 reductionreduction occurs during the different steps of durum wheat processing relative to the uncleaned wheat:

52% in cleaned wheat88% in semolina99% in pasta

T-2 and HT-2 increaseincrease significantly in the screening and bran fractions relatively to uncleaned wheat:

~ 600% in screenings~ 300% in bran

CONCLUSIONSCONCLUSIONS

OccurrenceOccurrence of Tof T--2/HT2/HT--2 2 toxinstoxins in in durumdurum wheatwheat and pastaand pasta

DeterminationDetermination ofof TT--2 2 andand HTHT--2 2 toxinstoxins in in wheatwheat andand pastapasta

Pre-column derivatization with 1-ANHPLC/FD HPLC/FD determinationdetermination

Immunoaffinity columnclean-up

(T-2 Test™ HPLC)

Immunoaffinity columnclean-up

(T-2 Test™ HPLC)

Extraction(with methanol:water + NaCl)

Filtration (Whatman Nº 4)Dilution with water (1:5)Filtration (Whatman GF/A)

Visconti et al. (2005), J. Chromatography A, 1075, 151-158

λex. = 381 nmλem. = 470 nm

Performances of the analytical method for the determination of T-2 and HT-2 toxins in cereals

Detection limit (signal/noise = 3:1)

Range of applicability

Recovery (spiking range 25-500 ng/g)

Relative Standard Deviation

Antibody cross-reactivity

Applicability

5 ng/g (T-2)3 ng/g (HT-2)

0.01-1.4 µg/g

>80%

< 8%

100% (T-2 and HT-2)

wheat (pasta), corn, barley

Visconti et al., 2005

53Min (ng/g)

54 (59%)81 (60%)Number of positive samples

108122Max (ng/g)

24,827,3Media (ng/g)

of which:

92136Number of analyzed samples

38 (41%)55 (40%)Number of samples < LOD (<3 ng/g)

20092008T-2 + HT-2*

2008 = North (51), Center (46), South (39)2009 = North (34); Center (39), South (19)

* HPLC/FD determination

OccurrenceOccurrence of Tof T--2 + HT2 + HT--2 2 toxinstoxins in in durumdurum wheatwheat in Italyin ItalySamples collected at storage facilities through official sampling procedure (Reg. EC. 401/2006)

27,343%

118%

6649%

5540%1362008

2009

Crop

24,811%

33%

5055%

3841%92

Mean of positives

(ng/g)> 100ng/g

51-100ng/g

3-50ng/g

< 3*ng/g

Samples distribution (n)Analyzedsamples

(n)

* LOD: T-2 = 5 ng/g; HT-2 = 3 ng/g

OccurrenceOccurrence of Tof T--2 + HT2 + HT--2 2 toxinstoxins in in durumdurum wheatwheat in Italyin Italy

** LOD (LC/FD): T-2 3 ng/g; HT-2 5 ng/g* LOD (LC/MS-MS): T-2 0.5 ng/g; HT-2 0.7 ng/g

32

16

8

13

30

50

56

15 EP

5S

GR 412

45 EP

GR 415

13 EP

16 EP

22

12 EP

Sample

24341012

961062048

< LOD1< LOD< LOD

< LOD5< LOD2

50581317

801241641

77108610

58561516

5344< LOD4

4331< LOD4

< LOD6< LOD1

2515< LOD1

104871821

97801419

951361636

483498

HPLC/FD**LC/MS-MS*HPLC/FD**LC/MS-MS*

HT-2 (ng/g)T-2 (ng/g)

Confirmation by LC-MS/MS(20% analyzed durum wheat samples)

2008 2009Analyzed samples : 26- 26 “semolina” pasta

OccurrenceOccurrence of Tof T--2+HT2+HT--2 2 toxinstoxins in pastain pasta

- 5 “mix cereals” pasta- 13 “whole wheat” pasta- 88 “semolina” pasta

Analyzed samples: 106

All samples <LOD(<3 ng/g T-2; <5 ng/g HT-2)

CONCLUSIONSCONCLUSIONS

• T-2 + HT-2 presence was found in Italian durum wheat production;

• T-2 + HT-2 content in durum wheat is quite low: more than 90% of positive samples are not detectable or lower than 50 ng/gr (mean about 25 ng/gr)

• No presence of T-2 + HT-2 has been found in commercial pasta: T-2 + HT-2 <LOD for all the 132 samples analyzed.

RecommendationsRecommendations

• Due to the low presence of T-2 + HT-2 mycotoxins in commercial durum wheat and very high reduction rate occurring during cleaning and milling process, maximum limits for these mycotoxins on durum wheat does not appear necessary;

• Moreover the further reduction recorded during pasta process and the absence of T-2+ HT-2 in commercial pasta samples suggest that this product do not contribute to consumer exposure and therefore no limits are need on this product.

AcknowledgmentsAcknowledgments

RanieriRanieri R. R. SilvestriSilvestri M.M.Arlotti G.Arlotti G.Atallah M.Atallah M.

Della Ghezza I. Della Ghezza I.

Pascale M. Pascale M. Haidukowski M. Haidukowski M.

LattanzioLattanzio VVSchenaSchena R. R.

PanzariniPanzarini G.G.LippolisLippolis V.V.Visconti A. Visconti A.

CNR, Institute of Sciences of Food Production (ISPA)

Bari, ItalyBarilla G.&R. F.lli S.p.A.

Parma Italy

SECTION B: Presence of T-2 and HT-2 toxin in cereals and cereal products

Seventh Fusarium Forum 2010 ● 1st-2nd February 2010