Post on 20-Oct-2021
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Alternaria Toxins -Analysis & Occurrence
Theresa Zwickel Keith Richards Horst Klaffke
National Reference Laboratory forMycotoxins in Food und Feed
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 2
Outline
• The mould Alternaria
• The toxins
• Background
• Toxicology
• Our current Alternaria research project at BfR
• Future work at the BfR
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 3
Alternaria – an interesting mould speciesDivision: EumycotaClass: Deuteromycetes (Fungi imperfecti)Order: Moniliales (Hyphomycetes)Family: Dermatiaceae (Black mould)
(dark coloured mycelium and/or spores fromMelanine-occlusion)
Genus: Alternaria
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 4
Plant pathogenicityAlternaria infestation
Alternaria spp.A. alternata cerealsA. tenuissima vegetables, fruitsA. solani potatoe, tomato, pepperA. brassicae cabbageA. citri citrus fruit
Black mould on wheat earsPhoto: Astrid Oldenburg
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 5
9 8
712
1110
6
542
1
3
OOH
OHOH
OH O
H
Altertoxin I (ATX-I)
A B
C
D E
9 8
712
1110
6
542
1
3
OOH
OH
OH O
O
H
Altertoxin II (ATX-II)
9 8
712
1110
6
542
1
3
OHO
OH O
O
H H
Altertoxin III (ATX-III)
5
43
2
NH
CH3
CH2CH2
CH3
OH CH3
O
O
H
Tenuazonsäure (TeA)
N
N
N
NH
CH3
CH3
CH3
OO
O
OCH3
CH3
Tentoxin
12
3
4
O 568
910
7
OOH
O
CH3 OH
CH3
Alternariol-9-methylether (AME)
12
3
4
O 568
910
7
OOH
OH
CH3 OH
Alternariol (AOH)
1
23
4
O 568
910
7
OOH
OH
OHO
CH3
Altenuisol (ALS)
1
23
4
O 568
910
7
OOH
O
OH
CH3
CH3
OH(Iso)-Altenuen(ALT, Iso-ALT)
Alternaria toxins – chemical structures
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 6
„Alternaria-Toxins“: a short history
2003BfR statement on current status:
need for research into occurrance and toxicityof Alternaria toxins in food and feedstuffs
2011Statement from EFSA on Alternaria toxins
2012BfR initialise Alternaria toxins research project
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 7
EFSA-StatementScientific Opinion on the risks for animal and public health related to thepresence of Alternaria toxins in feed and food1
1. EFSA Journal 2011;9(10):2407 [97 pp.]. www.efsa.europa.eu/en/efsajournal/pub/2407.htm
Non - genotoxic• Tentoxin• Tenuazonic acid
Toxins fall into 2 (roughly categorised) groups:
Genotoxic• Alternariol• Alternariol monomethylether
• Very little toxicological data exists (more needed)• Exposure:no complete data
Toxicological potential TTC
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 8
TA in infant cereals in µg/kg (Rychlik et al. TUM; Mycotoxin Workshop 2013 )
Oats & barley & spelt 30 ± 1
Wheat & oats & rye & maize & barley 33 ± 2
Wheat & milk powder & banana 8 ± 0,4
Wheat & milk powder 3 ± 0,1
Sorghum/millet Lot 1 900 ± 40
Lot 2 130 ± 5
Sorghum/millet Lot 1 620 ± 60
Lot 2 900 ± 70
Sorghum/millet Lot 1 930 ± 20
Lot 2 1200 ± 100
Sorghum/millet Lot 1 370 ± 20
Lot 2 380 ± 25
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 9
Provisional Risk Assessment by the EFSA (Rychlik et al. TUM)
0
1
2
3
4
5
3 5 4 5 5 5 6 5 7 5 8 5 9 5 10 5 11 5
Aufnah
meTA
[µg/kg
KGproTag]
Körpergewicht [kg]
TTC [1,5 µg/kg KG pro Tag]
Hirsebrei [30 g; 900 µg/kg TA]
Hirsebrei [20 g; 900 µg/kg TA]
Hirsebrei [10 g; 900 µg/kg TA]
Babygläschen [200 g; 15 µg/kg]
Getreidebrei [30 g; 30 µg/kg]
Babytee [200 mL; 2 µg/l TA]
Fencheltee [200 mL;17,5 µg/l TA]
Daily intake of TA depending on consumption and body weight
TTC: threshold of toxicological concern: 1.5 µg/kg bw
30 g sorghum baby cereals(mean 900 µg/kg)
20 g sorghum baby cereals
10 g sorghum baby cereals
baby jar
other cereals
baby tea
fennel teaBodyweight (kg)
Daily intake
(ug/Kg
BW
) per day
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 10
Two main approaches for mycotoxin analysis!
Mycotoxinsin food and animal feed
Identification & characterisationof moulds(taxonomy)
Identification & quantificationof mycotoxins
• Mykological approaches• Molecular biological approaches
•Biochemical methods
• Analytical instrumental methods
Avoidance strategies
Exposure evaluation
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 11
Clean-Up
Silica SPE columns
Alternaria-Toxin-Multi-MethodI. Sample preparation
Extraction
Modified QuECHERS approach
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 12
XIC of -MRM (38 pairs): 195.849/138.700 Da ID: Tenuazonic Acid Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Turbo S... Max. 1.0e4 cps
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0.00
1.00e42.11TEA
XIC of -MRM (38 pairs): 256.999/214.700 Da ID: Alternariol Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Turbo Spray) Max. 1.3e5 cps
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0.0
1.0e510.28
AOH
XIC of -MRM (38 pairs): 290.963/229.200 Da ID: Altenuen Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Turbo Spray) Max. 3.4e4 cps
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0.0
3.4e4 10.36ALT
XIC of -MRM (38 pairs): 413.112/141.100 Da ID: Tentoxin Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Turbo Spray) Max. 2.7e5 cps
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0.0
2.0e510.92
TEN
XIC of -MRM (38 pairs): 271.020/256.000 Da ID: Alternariolmethylether Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Tur... Max. 2.2e4 cps
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0.0
2.0e412.38 AME
XIC of -MRM (10 pairs): 413.232/141.000 Da ID: Tentoxin 1 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turbo Spray) Max. 1.4e4 cps
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min
0.0
1.0e45.27
TEN
XIC of -MRM (10 pairs): 256.987/214.600 Da ID: Alternariol 1 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turbo Spray) Max. 1.0e4 cps
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min
0.00
1.00e4 4.64
AOH
XIC of -MRM (10 pairs): 270.942/256.100 Da ID: Alternariolmethylether 1 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turb... Max. 1.2e5 cps
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min
0.00
1.00e55.66
AME
XIC of -MRM (10 pairs): 291.081/229.400 Da ID: Altenuen 1 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turbo Spray) Max. 1.4e4 cps
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min
0.0
1.0e44.96
1.76 ALT
XIC of -MRM (10 pairs): 196.187/111.800 Da ID: Tenuazonic acid 2 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turbo Spr... Max. 2.1e5 cps
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min
0.0
2.0e54.12
TEA
Alternaria-Toxin-Multi-MethodII. Chromatography
Betterrentention timefor TEA!!!
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 13
Multi-Alternaria-Toxin-MethodIII. Matrix Effects – Signal Suppression / Enhancement
XIC of -MRM (10 pairs): 196.187/111.800 Da ID: Tenuazonic acid 2 from Sample 24 (KAL 7 W) of Test Tea_02-09-2013.wiff (Turbo Spray) Max. 5160.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e42.2e4
TEA wheat
TEA maize
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 14
Multi-Alternaria-Toxin-MethodIV: Linearity
AME
y = 398.52x + 1125.6R2 = 0.9994
0.00E+00
5.00E+03
1.00E+04
1.50E+04
2.00E+04
2.50E+04
3.00E+04
3.50E+04
4.00E+04
4.50E+04
0 20 40 60 80 100 120
Concentration [ng/ml]
Area
[cps
]
Alternariol methylether matrix matched calibration
Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 15
Future work on Alternaria toxins at BfR
• Validation of the quantitative Alternaria-toxins analytical multi-method (in-house)• Validation in inter-laboratory studies
• Additional Alternaria-toxins to be added to the method
• Survey of products on the market• to assess the extent of Alternaria-toxins exposure to consumers• risk assessment
Upcoming in November 2013• Inter-laboratory study (with EU-RL, NRLs and NGOs)
• Alternaria toxin standards• Food matrix (tomato juice sample)
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Bundesinstitut für Risikobewertung
Max-Dohrn-Str.8-10 D-10589 Berlin
Tel. 030 - 18412 - 3363 Fax 030 - 18412 - 3685
theresa.zwickel@bfr.bund.de www.bfr.bund.de