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An overview of the multi-mycotoxin occurrence in South African produced maize ChromSAAMS 2016 12 SEPTEMBER 2016 The Southern African Grain Laboratory NPC Quality is our passion
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An overview of the multi-mycotoxin occurrence inSouth African produced maize
ChromSAAMS 201612 SEPTEMBER 2016
The Southern African Grain Laboratory NPCQuality is our passion
Evaluation of the status of occurrence of mycotoxins in South
African maize from all the production regions
Accumulation of reliable analytical data to establish a
database on the situation in South Africa to enable the maize
industry to comment on proposed maximum allowable levels
Supply reliable data as basis for targeted research projects
to effectively manage the mycotoxin levels in maize
MYCOTOXIN MONITORING OBJECTIVES
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
300 – 400 chemical toxins produced by fungi in food crops
Mycotoxins form through fungal growth during the pre- and
post – harvest period
Formation dependent on environmental conditions that
stress the plant:
drought, temperature, nitrogen deficiency, insect damage
Contaminate human food and animal feed
Toxicity of most mycotoxins not yet known but can be acute
or chronic: Aflatoxin B1 classified as carcinogenic
Regulations worldwide are different
Different maximum allowable levels for different mycotoxins
and commodities
MYCOTOXINS
INTRODUCTION
MULTI-MYCOTOXIN
ANALYSIS
SAMPLE
SELECTION
5 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
Most important mycotoxins regulated worldwide
INTRODUCTION
MULTI-MYCOTOXIN
ANALYSIS
SAMPLE
SELECTION
5 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
Mycotoxin Commodity occurrence SA Regulation
Aflatoxin B1 Maize, peanuts 5 µg/kg in foodstuffs, 20 µg/kg
in maize for animal feed
Deoxynivalenol Maize, wheat, barley 2016 proposal: max 2000 µg/kg
Fumonisins Maize 2016 proposal: Fumonisins
(B1+B2) = max 4000 µg/kg
Ochratoxin Wheat, barley
T2 and H-T2 toxin Wheat, barley
Zearalenone Maize, soya beans
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
1. REPRESENTATIVE SAMPLING
Mycotoxin contamination not homogeneously spread through crop
Presence not always visually confirmed
2. SAMPLE PRE-PREPARATION
Milling process: Particle sizeof milled products has aneffect on the extractionefficiency
MYCOTOXIN SAMPLING CHALLENGES
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
4 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
3. DIFFERENT CHEMICAL PROPERTIES INFLUENCE THE EXTRACTION
AND CLEAN-UP PROCESSES
Aflatoxins soluble in polar organic solvents but limited solubility in water
Deoxynivalenol soluble in water
Fumonisins hygroscopic, stable in acetonitrile: water but unstable in methanol
Ochratoxin A slightly soluble in water, soluble in polar solvents
T2-toxin, HT-2 toxin and zearalenone insoluble in water
MYCOTOXIN ANALYTICAL CHALLENGES
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
4 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
4. EXTRACTION EFFICIENCY
Aqueous acetonitrile mixture with addition of acids –shaking or blending
80% methanol in water – blending for 2 min
Two extraction steps:1 – Acetonitrile : water : formic acid
- 80 : 19.9 : 0.1 shaking for 60 min 2 – Acetonitrile:water:formic acid
- 20 : 79.9 : 0.1 shaking for 30 minWater : methanol : acetonitrile
– 50 : 25 : 25, blending 1 min, shaking 15 minutes and dilution
MYCOTOXIN ANALYTICAL CHALLENGES
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
5. QUANTITATIVE ANALYSIS
Availability and price of reference materials and isotope labelled internal standards
Tedious preparation of stock standards and dilutions to avoid contamination
Small volumes, regular preparation of stock solution mixtures
Matrix effects in LCMS different for different mycotoxins and in different commodities ( matrices)
Prepare different sets of matrix-matched standards for different commodities
Availability of “blank” matrix for matrix-matched standards
MYCOTOXIN ANALYTICAL CHALLENGES
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
4 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
UPLC-MS/MS METHOD – ISO 17025 ACCREDITED METHOD
MycotoxinAccuracy,
% Mean Recovery
Precision,
RSD, %
Concentration range,
µg/kg
Limit of quantitation,
(LOQ), µg/kg
Aflatoxin B1 92 11 1.25 – 80 2
Aflatoxin B2 91 13 1.25 - 80 2
Aflatoxin G1 94 12 1.25 - 80 2
Aflatoxin G2 103 15 1.25 - 80 5
Deoxynivalenol 87 13 50 - 8000 100
15-Acetyl-
Deoxynivalenol82 18 50 – 8000 100
Fumonisin B1 83 20 10 - 8000 20
Fumonisin B2 72 13 10 - 8000 20
Fumonisin B3 74 13 10 - 8000 20
Ochratoxin A 84 13 1.25 - 80 5
T2-Toxin 97 9 10 - 8000 20
HT-2 Toxin 94 12 10 - 8000 20
Zearalenone 76 13 10 - 8000 20
SA produced annually more than 10 million tons of maize;
approximately 48 % white maize and 52% yellow maize
SA is divided in 36 production regions in 9 provinces
The 3 main maize production areas are in 3 of the nine
provinces:
North West (15%), Free State (40%) and Mpumalanga (24%)
(including 22 regions)
MAIZE PRODUCTION IN SOUTH AFRICA
INTRODUCTION
MULTI-MYCOTOXIN
ANALYSIS
SAMPLE
SELECTION
5 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
A total of 1000 maize samples per season are collectedfrom commercial grain stores for the annual crop qualitysurvey
350 samples selected/ season for multi-mycotoxinanalyses over 5 seasons from 2010/2011 – 2014/2015
Samples selected represent all the production regionsproportionally
Both white and yellow maize samples selectedproportionally
MYCOTOXIN MONITORING
INTRODUCTION
MULTI-MYCOTOXIN
ANALYSIS
SAMPLE
SELECTION
5 YEAR
SUMMARY
FUMONISIN
PER
PROVINCE
DEOXYNIVALENOL
PER PROVINCE
No aflatoxin B1, B2 , G1 & G2 found in previous 4 seasons BUT 3samples found with Aflatoxin B1 > 5 µg/kg in the 2014 – 2015season
No Ochratoxin A and HT-2 toxin found
T-2 toxin only in 2 samples in 2012-2013 season
Mycotoxins found:
• Fumonisin B1, B2 and B3
• Deoxynivalenol, 15-ADON
• Zearalenone
MYCOTOXINS IN SA MAIZE CROP
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER PROVINCE
DEOXYNIVALENOL
PER PROVINCE
% Samples tested % Samples with FUM % Samples with DON % Samples with ZON
2010 - 2011 47 20 30 10
2011 - 2012 35 33 6 2
2012 - 2013 35 45 11 3
2013 - 2014 38 41 69 11
2014 - 2015 35 56 41 11
0
10
20
30
40
50
60
70
80
%
% White and yellow maize samples with mycotoxins over 5 seasons
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER PROVINCE
DEOXYNIVALENOL
PER PROVINCE
% Samples tested % Samples with FUM % Samples with DON % Samples with ZON
2010 - 2011 47 18 34 14
2011 - 2012 33 34 8 3
2012 - 2013 35 49 12 5
2013 - 2014 37 44 74 13
2014 - 2015 35 58 45 14
01020304050607080
5
% White maize samples with mycotoxins over 5 seasons3 white maize samples with Total Aflatoxins
% Samples tested % Samples with FUM % Samples with DON % Samples with ZON
2010 - 2011 47 23 25 5
2011 - 2012 38 33 4 1
2012 - 2013 35 41 9 2
2013 - 2014 39 38 65 10
2014 - 2015 35 55 37 7
01020304050607080
5
% Yellow maize samples with mycotoxins over 5 seasons
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER PROVINCE
DEOXYNIVALENOL
PER PROVINCE
Northern Cape(Region 10 -11)
North West(Region 12 - 20)
Free State(Region 21 - 28)
Mpumalanga(Region 29 -33)
Gauteng(Region 34)
Limpopo(Region 35)
KwaZulu Natal(Region 36)
2010 - 2011 75 62 47 30 44 0 26
2011 - 2012 75 47 39 27 29 33 27
2012 - 2013 71 71 53 33 36 60 65
2013- 2014 100 88 70 90 88 100 75
2014- 2015 80 83 89 75 70 75 88
0
20
40
60
80
100
120
% S
amp
les
wit
h m
yco
toxi
ns
Mycotoxin occurrence / Province over 5 seasons
3 white maize samples with Total Aflatoxins
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER PROVINCE
DEOXYNIVALENOL
PER PROVINCE
North West Free State Mpumalanga Gauteng Limpopo KwaZulu Natal
2010 - 2011 1112 456 431 298 0 262
2011 - 2012 614 1064 528 147 65 0
2012 - 2013 696 470 129 422 162 44
2013 - 2014 296 624 318 161 376 50
2014 - 2015 333 478 316 353 84 634
0200400600800
100012001400160018002000
µg/
kg
White maizeFUM (total) mean concentration (ug/kg) / Province over 5 seasons
Northern Cape North West Free State Mpumalanga Gauteng Limpopo KwaZulu Natal
2010 - 2011 454 672 1773 0 360 0 732
2011 - 2012 1195 436 525 303 0 0 102
2012 - 2013 1866 505 429 250 88 207 512
2013 - 2014 752 383 478 518 482 1332 95
2014 - 2015 435 210 242 346 199 0 183
0200400600800
100012001400160018002000
µg/
kg
Yellow maizeFUM (total) mean concentration (µg/kg) / Province over 5 seasons
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
5 YEAR
SUMMARY
FUMONISIN
PER PROVINCE
DEOXYNIVALENOL
PER PROVINCE
North West Free State Mpumalanga Gauteng Limpopo KwaZulu Natal
2010 - 2011 378 209 317 249 0 123
2011 - 2012 435 143 212 171 129 232
2012 - 2013 633 216 150 177 0 251
2013 - 2014 435 259 596 964 444 488
2014 - 2015 415 1383 504 404 0 722
0200400600800
1000120014001600
µg/
kg
White maizeDON mean concentration (ug/kg) / Province over 5 seasons
Northern Cape North West Free State Mpumalanga Gauteng Limpopo KwaZulu Natal
2010 - 2011 288 187 252 153 159 0 0
2011 - 2012 0 163 278 131 0 0 0
2012 - 2013 290 206 230 370 154 0 116
2013 - 2014 273 375 255 396 292 252 180
2014 - 2015 435 210 242 346 199 0 183
0
200
400
600
800
1000
1200
µg/
kg
Yellow maizeDON mean concentration (µg/kg) / Province over 5 seasons
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
4 YEAR
SUMMARY
FUMONISIN
PER PROVINCE
DEOXYNIVALENOL
PER PROVINCECONCLUSIONS
Fumonisins and deoxynivalenol found in samples fromall the maize producing regions
Different patterns of occurrence observed in differentseasons
Different mean concentration levels found in differentseasons
FUM - higher mean concentrations on yellow maizethan white maize from the same region
DON – higher mean concentration levels on whitemaize than yellow maize from the same region
INTRODUCTIONSAMPLE
SELECTION
MULTI-MYCOTOXIN
ANALYSIS
4 YEAR
SUMMARY
FUMONISIN
PER PROVINCE
DEOXYNIVALENOL
PER PROVINCECONCLUSIONS
This mycotoxin survey gives for the first time a representative South Africanperspective on the occurrence and concentration levels of South Africa’s annualcommercial maize crop.
The 2013/2014 average concentration levels of FUM (451 µg/kg) and DON(372 µg/kg) in SA commercial maize are 6 fold lower than the averageconcentrations reported in maize in the 2014 Biomin Mycotoxin Survey
The FUM and DON levels pose a relatively low risk when compared to themaximum allowable levels for DON and FUM in feeding stuff for animal feedand the proposed levels for human consumption but the allowable levels forfumonisin may change in future as more scientific research results becomeavailable on the fumonisin risk to animal health.
ACKNOWLEDGMENTS
CO- WORKERS: AMELIA SAVILLE, MAMSY MOTLANTHE AND ZANELE
SKHOSANA
CO-AUTHORS: WIANA LOUW AND JOLANDA NORTJE
THE SA MAIZE TRUST FOR FUNDING FROM 2010 - 2016
AFMA FOR CO-FUNDING IN 2011 AND 2012
AGBIZ GRAIN FOR COLLECTION OF THE SAMPLES EVERY SEASON
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