SAFEFOODERA-EMTOX project 7 FP Dr. E. Ioannou Kakouri, Chief Chemist, EFSA focal point

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1 Effect of Climate Change on Emerging Natural Toxins in Plants and Seafood Production Επιδπάζειρ ηων Κλιμαηικών Αλλαγών ζηη Δημιοςπγία Αναδςόμενων Φςζικών Τοξινών και ζηην Παπαγωγή Φςηών και Θαλαζζινών SAFEFOODERA-EMTOX project 7 FP Dr. E. Ioannou Kakouri, Chief Chemist, EFSA focal point Ministry of Health, State General Laboratory 44 Κimonos str, 1451 Nicosia, Cyprus Επγαζηήπι για ηην Πποζαπμογή ζηην Αλλαγή ηος Κλίμαηορ 2-3 Νοεμβπίος 2011 Holiday Inn, Λεςκωζία

2 State General Laboratory Ministry of Health 44 Κimonos str, 1451 Nicosia, Cyprus

3 Contents Effect of Climate Change on Emerging Natural Toxins in Plants and Seafood Production SAFEFOODERA-EMTOX project 7 FP Introduction 2. Role of SGL-WP3 3. Methodology 4. Results 5. Discussion 6. Finalization of project- Conclusions

4 Introduction Climate change has become one of the most critical issues for the future of our planet. It involves significant changes in the variability or average state of the atmosphere, such as related to temperature, precipitation and/or wind patterns, over durations ranging from decades to millions of years. The effects of climate change will have implications for food production, food security and food safety. In particular, the safety of feed and food products arising from plant and marine production systems is expected to be affected by climate change, specifically by increased occurrence of natural toxins, including mycotoxins and phycotoxins (marine biotoxins).

5 The overall objective of the current research is to project the impact of climate change on the occurrence of (possible) feed and food safety hazards in both terrestrial plant production systems and marine seafood systems in Europe. The project particularly focuses on natural toxins in cereal and shellfish production that are harmful to animal and human health, including several key mycotoxins and phycotoxins. Some of these are re-emerging. As a second objective, the project aims to quantify (as much as possible) the relationship between climate change, shifts in primary plant and marine production systems, and the occurrence of (re-)emerging natural toxins. Hereby, currently available predictive models will be used as well as reliable databases.

6 SAFEFOODERA- EMTOX project 7 FP, The project was coordinated by the RIKILT- Institute of Food Research, Netherlands, other participating countries: Cyprus (SGL) and Scandinavian countries, Sweden, Norway, Finland, Denmark. The SGL was involved in WP3. The project has 7 WP : Work Package 0 Kick off meeting - coordination Work Package 1 Climate change scenarios, DMI, DK e.g. ENSEBLES database (Global and regional climate data) Work Package 2 Cereal production Aarhus Univ, DK Work Package 3 Mycotoxins in cereals, RIKLT, NL Work Package 4 Phytoplankton production and blooms-no Work Package 5 Harmful algae and marine toxins- Del-NL Work Package 6 Integration of models RIKLT, NL

7 Role of State General Laboratory Role of SGL Participation in SAFEFOODERA- EMTOX project 7 FP, WP 0 & 3 1. Organization of Kick-off meeting 28-29/4/09 and EMTOX webpage ( (WP 0) 2. Mycotoxins in cereals (WP 3). Analysis of local cereal samples especially wheat and barley and their products for re/emerging mycotoxins Τ-2, ΗΤ- 2, DON, ZON (fusarium toxins) and OTA &AFs. 3. Availability, enrichment of the national database for the results of Τ-2, ΗΤ-2, DON, ZON, OTA and AFs. analysis in cereal samples. 4. Collection of agronomic data. Completion of questionnaire by farmers. Partners for providing samples etc: 1. S.E.DI.S LTD ( Coop. Assoc. of Cereal Farmers, samples+ quest/res ) 2. Cyprus Grain Commission (samples ) 3. Department of Agriculture ( data for mycotoxins, samples+ quest/re ) 4. Agriculture Research Institute (Samples+ Quest/re ) 5. Health Services (Samples) 6. Meteorological Service (meteorological data) 7. Farmers (Samples+ Quest/res )

8 SGL and WP 3- Mycotoxins in cereals Mycotoxins are secondary metabolites which are produced by a wide variety of fungal species in a range of crops, cereals, nuts and fruits, particularly during cultivation and/or storage stages of production. Mycotoxins are chemically stable and to a large extent - resistant against most of the feed and food processing steps, resulting in contamination of the final feed and food products. Consumption of contaminated cereal based feed and food product can cause harmful effects to human and animal health.

9 SGL- Mycotoxins control The presence of various toxic/carcinogenic Mycotoxins: Aflatoxins B1, B2, G1, G2, Ochratoxin A (OTA) and other mycotoxins in local and imported foodstuffs (cereals, nuts, dried fruits, rice etc) has been monitored and controlled systematically and effectively since 1990 in Cyprus, by the State General Lab. (SGL) and the Health Services of the Ministry of Health. The samples for official control are collected according to a specific sampling plan based on the EU Regulations, at critical control points ( HACCP approach) such as: import, primary storage, processing and market.

% SAMPLES ABOVE LIMIT 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

10 % SAMPLES ABOVE LIMIT MONITORING PROGRAMME OF AFLATOXIN (HACCP) IN CYPRUS MARKET ALL CCPs Figure 1

% POSITIVE SAMPLES 70 INCIDENCE OF AFLATOXIN M1 IN MILK IN CYPRUS 1993-2010 60 50 40 30 20 10 0 1993

11 % POSITIVE SAMPLES 70 INCIDENCE OF AFLATOXIN M1 IN MILK IN CYPRUS Fig.4 Afl M1 levels< aver 2-8 ppt

% POSITIVE SAMPLES 100 INCIDENCE OF OCHRATOXIN A IN FOODSTUFFS IN CYPRUS 1997-2010 90 80 70 60 50 RAISINS

12 % POSITIVE SAMPLES 100 INCIDENCE OF OCHRATOXIN A IN FOODSTUFFS IN CYPRUS RAISINS COFFEE WINE CEREALS OTA levels within ML Fig

13 DON levels within ML

14 Methods of Analysis of Mycotoxins Extraction With Methanol:Water or Acetonitrile:Water + NaCl or NaHCO 3 Filtration (fluted paper) Dilution with water or PBS Filtration (GF/A) Immunoaffinity column clean-up

contain specific antibodies for each mycotoxin.

15 Methodology of Analysis of Mycotoxins In immunochemical methods specific and selective immunoaffinity columns are used that contain specific antibodies for each mycotoxin. Subsequently, the process of purification based on the antibody-antigen effect takes place.

16 Methods of Analysis of Mycotoxins Aflatoxins B 1, B 2, G 1, G 2 Ochratoxin A Zearalenone (ZON) HPLC/FLD Deoxynivalenol (DON) HPLC/PDA Trichothecenes (T-2, HT-2 toxins) GC/MS Multi-Mycotoxin Analysis LC-MS/MS

17 Results Occurrence of Mycotoxins in cereals & Products Table 1. Levels of mycotoxins in cereals and their products for the years Sample Parameter Number of samples Number of local samples Number of positive samples Minimum value (ppb) Maximum value (ppb) Mean value (ppb) Median value (ppb) Number of samples over the maximum limit Wheat DON OTA ,14 0,33 0,24 0 Aflatoxins Τ ΗΤ ΖΟΝ Barley DON OTA ,26 0,26 0,26 0 Aflatoxins Τ ΗΤ ΖΟΝ Corn DON OTA Aflatoxins ,36 21,67 11,02 1 Τ ΗΤ ΖΟΝ ,7 5,4 5,1 0 Flour DON 11 8 (?) OTA ,28 1,57 0,93 0 Aflatoxins Τ ΗΤ ΖΟΝ Pasta DON 18 6 (?) 12 64, ,6 0 OTA ,06 0,54 0,25 0 Aflatoxins Τ ΗΤ

Number of Samples Number of Samples Number

Positive Samples No of Samples 20 0 DON ZON

(2008-2010) Results for ZON (2008-2010) 20

18 Number of Samples Number of Samples Number of Samples Results Occurrence of Mycotoxins in Cereals Results in cereals and their products ( ) No of Positive Samples No of Samples 20 0 DON ZON OTA AFLs T-2 HT-2 Results for DON ( ) Results for ZON ( ) No of Samples No of Positive Samples No of Samples No of Positive Samples 0 BARLEY WHEAT CORN FLOUR PASTA 0 BARLEY WHEAT CORN FLOUR

Mean value (ppb) Median value (ppb) Number of samples over ML Wheat DON 9 5 1 414 414 414 414 0 OTA 13 7 5 0,27 0,67 0,47 0,47 0 Aflatoxins 68 22 2 1,4 1,4 1,4 1,4 0 ZON 7 5 1 2,0 3,1 2,7 3,0 0

19 Number of Samples Number of Samples Number of Samples Number of Samples Results Table 2. Results of the levels of mycotoxins in wheat and barley from Sample Parameter Number of samples Number of local samples number of positive samples Minimum value (ppb) Maximum value (ppb) Mean value (ppb) Median value (ppb) Number of samples over ML Wheat DON OTA ,27 0,67 0,47 0,47 0 Aflatoxins ,4 1,4 1,4 1,4 0 ZON ,0 3,1 2,7 3,0 0 Barley DON OTA ,26 0,26 0,26 0,26 0 Aflatoxins ,8 14 9,4 8,8 0 ZON ,0 2,0 2,0 2,0 0 Results for DON ( ) Results for ηην ZON ( ) No of samples No of local samples No of positive samples 10 5 No of samples No of local samples No of positive samples 0 BARLEY WHEAT 0 BARLEY WHEAT Results for ΟΤΑ ( ) Results for AFLs ( ) No of samples No of local samples No of positive samples No of samples No of local samples No of positive samples 0 BARLEY WHEAT 0 BARLEY WHEAT

20 Results Results and Discussion 61 LOCALLY produced samples of wheat, barley & products were analyzed, 21 of which were positive to some mycotoxins: AFs, OTA. 163 analyses for 116 samples of wheat, barley, corn. & products were analyzed, 38 positive samples were found for some mycotoxins: DON, ZON, AFs, OTA. For DON 24 positive samples found, levels ranging between 64,7 and 709 ppb, within the maximum limit (ML=1250 ppb) for cereals and of 750 ppb for cereal products. For ΟΤΑ 8 positive samples were found with the levels ranging between 0,06 and1,57 ppb, within the ML of 5 ppb for cereals and of 3 ppb for cereal products. For the four Aflatoxins AFs 3 positive imported corn samples were found with the levels ranging between 0,36 and 21,67 ppb. One corn sample exceeded the ML of 10 ppb. For ΖON 3 positive samples were found with the levels ranging between 4,7 και 14 ppb within the ML of 100 ppb for cereals and of 75 ppb for cereal products.. For Τ-2 and ΗΤ-2 no positive samples were found.

No of samples No of samples Results and Discussion for local wheat + barley 2008-2010 No of samples No of

were found (1 Wheat and 1 Barley), within the maximum limit of 5 ppb for cereals.

These findings are in accordance with relevant info for DON, ZON, OTA,, Afs, Fum B1 +B2 from the Agricultural

Results for DON in Wheat and Barley Results for ΖΟΝ in Wheat and Barley 8 20 6 16 4 2 0 2008 2009 2010 No of

21 No of samples No of samples Results and Discussion for local wheat + barley No of samples No of samples 85 analyses for DON, ZON, OTA, AFs in 38 samples of local wheat+ barley Only 2 positive samples to ΟΤΑ were found (1 Wheat and 1 Barley), within the maximum limit of 5 ppb for cereals.no FUSARIUM TOXINS WERE DETECTED! These findings are in accordance with relevant info for DON, ZON, OTA,, Afs, Fum B1 +B2 from the Agricultural Services! Results for DON in Wheat and Barley Results for ΖΟΝ in Wheat and Barley No of positive samples No of local samples No of positive samples No of local samples Results for ΟΤΑ in Wheat and Barley Results for AFLs in Wheat and Barley No of positive samples No of local samples No of positive samples No of local samples

22 Finalization of the project 1. Completion of analysis of local wheat, barley etc and hay for mycotoxins, as hay is stored in the fields. 2. Completion and evaluation of questionnaires by producers/farmers. 3. Processing and utilization of the results- enrichment of the database. 4. Final meeting with the EMTOX partners of the project, final results. 5. Arrangement of Meeting for informing all stakeholders in Cyprus, may be in May Continue of monitoring of local wheat, barley etc for mycotoxins for the next years

23 Results of the completed questionnaires by farmers 8 Cypriot cereal farmers, mainly wheat 11 Questionnaires ( 1 farmer for wheat +barley, 1 farmer for wheat + barley + oat) 7 Wheat samples 3 Barley samples 1 Oat sample

24 11 questionnaires 9 wheat, 2 barley, 1 oat 1. What is your postal code? All farmers in Nicosia district area (11) 2. Optional: fill in your farm identification code 3. Do you grow wheat in 2011 in one or more plots? 5 of 8 farmers answered (8) 1 plot (1), > 1 plots (10) If so, would you please fill in the questions below for one particular random plot? The sample at harvest needs to be taken form the same plot. 4. Which name or code do you use for this plot? 7 of 8 farmers answered (10) 5. What is the soil type of this plot? Red soil (10), Different soils (1) 6. What was the main cultivation of the soil of this plot? E.g. deep tillage / surface tillage/. 7. What crop did you grow on this plot in the previous season? 8. What was the sowing date of the wheat in this plot? Surface tillage (8), Deep tillage (2), Tillage with plow (1) Wheat (2), Potatoes (5), Barley (2), Fallow (2) 12/10/ /12/2009

25 9. What wheat cultivar variety (or hybrid) did you sow and if known- what is the resistance number for Fusarium spp. for this hybrid? Cypriot wheat varieties :Ourania (6), Dimitra (1), Athieni (1), Ekavi + various genetic lines (1) 10. What is your address and phone number? All the plots are located in Nicosia district 11. What is the flowering date of the wheat in this field? The flowering date is the date at which 50% of the wheat field is flowering. 12.When do you expect to sow the wheat? Beginning of February End of April 2010 End of October 2010 January 2011 (5), After the rain (3), Fallow (1) 13. Which fungicide did you use during cultivation of the wheat and during which phenological phase did you apply this? Please fill in the scheme below Fungicide used Monival (1), Pyriphos (6), Denocamines (2), Douspan (1), Mancozeb (1), Vitavax (1), None (2) 14. What was the expected harvest date of the wheat? 15. What was the actual harvest date of the wheat? Did you postpone harvesting due to bad weather conditions? 2 If so, by how many days? Period of application (crop stage) First stage of growth (10-15 cm) (4), Beginning of March (15-20 cm) (2), cm of growth (1), Before seeding (1) Beginning of May June /6/2010 6/8/ of 8 farmers answered No (10)

26 Discussion of Questionnaires 11 Questionnaires, 8 farmers 12/10/ /12/2009, mainly wheat. Cypriot wheat varieties Ourania (6), Dimitra (1), Athieni (1), Ekavi, Most resistant for Cypriot climate is Ourania. Red soil, surface tillage more of the farmers Fungicide used: Chloropyriphos (Pyriphos+Douspan), Dithiocarbamates (Mancozeb, Vitavax). Trials are made by Agricultural Research Institute for new hybrids of wheat (wheat + rye) to improve resistance to climatic changes of Cyprus (less irrigation and more salinity). Most of the wheat consumed in Cyprus is imported and a small quantity of the locally produced is used for human consumption while the rest + barley is used as feeding stuff.

27 Conclusions No fusarium toxins (DON, ZON, HT-2, T-2) were detected in Cypriot wheat and barley samples (38) for in contrast with what is observed in other European countries. Cypriot variety of wheat Ourania is the most resistant to fusarium spp. and adapted to climate conditions Only 2 positive local samples for OTA were found. These findings are in accordance with relevant info from the Agricultural Service for DON, ZON, OTA, Afs, Fum B1 +B2! In general, all the analyzed samples (116) of wheat, barley, corn, flour and pasta were within the MLs of EU legislation. Only 38 positive samples were found for some mycotoxins: DON, ZON, AFs, OTA

28 Acknowledgements To Mrs E. Christou, Head of Food Contamination & Natural Toxins Lab of SGL To Mrs A. Anastasi, Head of IT Unit of SGL To Dr. M. Christophidou, Mrs M. Christodoulou. Mr D. Stefani, Dr D, Kafouris, K.Roti, analysts in the above Lab. of SGL To Dr H.J. (Ine) van der Fels-Klerx coordinator and other EMTOX partners To Dr Rebecca Chrysafi of Research Promotion Foundation of Cyprus

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