PRESENCE OF 31 MYCOTOXINS IN PIG FEED IN SPAIN
L. Gámiz-Gracia1, N. Arroyo-Manzanares2, V. Rodríguez-Estévez3 and A.M. García-Campaña1 Department of Analytical Chemistry, Faculty of Sciences, University of Granada.
1
Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, Spain.
2
Department of Animal Production, Faculty of Veterinary Medicine, University of Cordoba.
3
1
Mycotoxins are fungal toxins produced
According to the Food and Agriculture
by several hundred species of molds that
Organization of the United Nations
can grow on crops or food under certain
(FAO), it is estimated that 25% of cereal
conditions. The most important are
production and 20% of plant production
those produced by molds of the genera
worldwide are affected by mycotoxins,
Aspergillus, Fusarium and Penicillium.
although recent studies consider that this figure may be underestimated2.
In addition, the effects of climate change are expected to increase the production of toxigenic Mycotoxins have become one of the
fungi in the coming years3.
most reported contaminants worldwide, a fact that is reflected in the latest report from the EU Rapid Alert System for Food and Feed (RASFF)1.
It should be noted that, despite the current state of knowledge and improvements in production and storage practices, it has not been possible to eradicate the development of fungi and molds and, therefore, the presence of mycotoxins in a large number of foods.
2
Despite the wide variety of known mycotoxins with various toxicological effects, the EU has set maximum permitted or recommended levels for only 14 of them (aflatoxins B1,
Among farm animals, pigs are
B2, G1, G2 and M1, ochratoxin A, patulin,
considered to be one of the
deoxynivalenol, zearalenone, fumonisins
most vulnerable species to
B1 and B2, T-2 and HT-2 toxins, citrinin) as
the effects of mycotoxins.
well as for ergot sclerotia, in various food intended for human consumption4,5. Such effects may include changes In the case of animal feed, the number of mycotoxins with maximum permitted or recommended levels is
in immune response (resulting in reduced vaccination efficacy), loss of appetite, abortions, agalactia, etc.
even lower, being limited to aflatoxin B1, ochratoxin A, deoxynivalenol, zearalenone, fumonisins B1 and B2, T-2 and HT-2 toxins, and ergot sclerotia6,7.
Mycotoxins with established maximum permitted levels or recommendations in food intended for human consumption in the EU Aflatoxins B1, B2, G1, G2 and M1 Ochratoxin A Patulin Deoxynivalenol Zearalenone Fumonisins B1 and B2 T-2 and HT-2 toxins Citrinin
Mycotoxins with fixed maximum permitted levels or recommendations in animal feedstuffs in the EU
Aflatoxin B1 Ochratoxin A Deoxynivalenol Zearalenone Fumonisins B1 and B2 T-2 and HT-2 toxins
3
Table 1 shows a summary of the mycotoxins regulated in the European legislation for pig feed, their permitted or recommended limits, as well as their most common health effects.
Table 1. Maximum permitted or recommended values in pig feed. Mycotoxin
AFB1 a
Aspergillus flavus
Fusarium graminearum Fusarium culmorum
DON b
ZEN b
OTA
Main producing fungi
Fusarium graminearum
Penicillium verrucosum Aspergillus ochraceus Aspergillus carbonarius
b
FB1 + FB2
b
T-2 + HT-2 b
Ergot sclerotia a
Fusarium verticillioides Fusarium proliferatum
Product Raw materials for animal feed
0.02
Compound feed for pigs (except piglets)
0.02
Compound feed for piglets
0.005
Raw materials for animal feed: Cereals and cereal-based products, with the exception of corn by-products
Common effects described in pigs8,9,10 Low performance, liver damage, immunosuppression, neoplasia
8
Corn by-products
12
Compound feed for pigs
0.9
Raw materials for animal feed: Cereals and cereal-based products, with the exception of corn by-products
2
Corn by-products
3
Compound feed for piglets and gilts
0.1
Compound feed for sows and fattening pigs
0.25
Raw materials for animal feed: cereals and cereal-based products
0.25
Compound feed for pigs
0.05
Raw materials for animal feed: corn and corn-based products
60
Compound feed for pigs
5
Oat milling products (hulls)
2
Fusarium sporotrichioides Fusarium langsethiae Other cereal-based products Claviceps purpurea
Maximum or recommended content (mg/kg) c
Loss of appetite, vomiting, poor performance
Low performance, infertility, prolapse, abortions
Low performance, liver and kidney damage
0.5
Compound feed
0.25
Feed materials and compound feed containing unground cereals
1,000
Pulmonary edema, immunosuppression, intestinal disorders, weight loss Anorexia, weight loss, lesions in the mouth and esophagus, leukopenia, reproductive problems
Mycotoxins with maximum permissible contents 6 Mycotoxins with guideline values 7 c Maximum content in mg/kg in animal feed calculated on the basis of a moisture content of 12%
a
b
4
Ergot alkaloids, known since ancient times for their toxic effects, are not included in legislation (there is only a maximum content for ergot sclerotia, listed in Table 1), although they also pose a risk to livestock and it is expected that maximum contents for these compounds will be established in the near future.
Focusing on emerging mycotoxins In addition to the mycotoxins listed in Table 1, there are other mycotoxins that are currently under study as potentially toxic and for which maximum permitted levels have not yet been established.
These are the so-called “emerging mycotoxins”, including some produced by fungi of the Fusarium genus, such as enniatins and beauvericin, which colonize cereals in particular and can accumulate in the grain11. These groups of mycotoxins are discussed in more detail in the following section.
5
ERGOT ALKALOIDS Ergot alkaloids, or rye ergot,
This disease can cause different symptoms depending
characterized by the spur or “horn”
on the species in question. Thus, in pigs, ergot alkaloids
that the fungus produces on the grain
can cause poor performance, loss of appetite,
as it grows, are produced by different
agalactia, reproductive problems, neonatal mortality,
fungi from the orders Hypocreales
liver damage and gangrene13-16. This wide variety of
and Eurotiales, Claviceps purpurea
symptoms makes it difficult to identify the problem.
being the most widespread in Europe. This fungus infects grains of various cereals such as rye, wheat, barley, millet and oats, frequently used in animal feed. The toxicity of ergot alkaloids has been known since the Middle Ages, since it caused the epidemics known as “St. Anthony’s fire” or ergotism and, although it is
Ergot alkaloids
Performance Loss of appetite Reproductive problems Neonatal mortality Liver damage Gangrene
considered an eradicated disease in humans, sporadic outbreaks of ergotism in cattle have been described in recent decades12,13.
6
To date, more than 50 different ergot alkaloids with the ergoline ring as a common structure have been described (Figure 1).
Figure 1. Ergoline ring from the basic structure of ergot alkaloids.
Monitoring of six most common ergot alkaloids (ergometrine, ergotamine, ergosine, ergocristine, ergokryptine, ergocorninine) and their corresponding epimers (ergometrinine, ergotaminine, ergosinine, ergocristinine, ergokryptinine, ergocorninine) is recommended.
Main monitored ergot alkaloids Ergometrine Ergotamine Ergosine Ergocristine Ergokryptine Ergocornine
Epimers
Ergometrinine Ergotaminine Ergosinine Ergocristinine Ergokryptinine Ergocorninine
While the C8-(R) isomers are biologically active, the C8(S) epimers are considered to have little or no activity.
However, as the conversion between isomers is rapid, contaminated samples often present both forms, so, when determining the concentration of ergot alkaloids in a sample, they must be considered together17,18,19.
7
The maximum permitted levels for ergot alkaloids are currently under study, although the industry recommends maximum levels in swine feed between 0.2-0.5 mg/kg20.
However, some studies suggest that lower values consumed over prolonged periods of time could cause intestinal and liver damage21.
In the meantime, and based on the available data and to avoid vasoconstrictive effects of ergot alkaloids in cattle, an acute reference dose of 1 µg/kg body weight and a tolerable daily intake (TDI) of 0.6 µg/kg body weight per day have been estimated22. In addition, as a precautionary measure, the EU has established a maximum level for ergot sclerotia in cereals of 1,000 mg/kg6.
The European Food Safety Authority (EFSA) has recently pointed out the need to collect more information on the presence of these alkaloids and to develop analytical methods for their control.
Acute reference dose:
1 μg/kg body weight Tolerable daily intake (TDI):
Ergot alkaloids in pig feed:
0.6 μg/kg body weight per day
0.2-0.5 mg/kg
Rye ergot sclerotia in cereals:
1,000 mg/kg
8
ENNIATINS
ENNIATINS AND BEAUVERICIN
Enniatins are produced by fungi of the
The structure of the main enniatins and
genus Fusarium, such as F. avenaceum,
beauvericin is shown in Figure 2.
F. oxysporum, F. poae or F. tricinctum, and present cyclic hexadepsipeptide structure alternating D-α-hydroxyisovaleric acids and N- methyl-L-amino acids.
a)
b)
The amino acid residues of type A and B enniatins are aliphatic N-methyl-valine or N-methylisoleucine, or mixtures of these amino acids. Up to 29 different enniatins are known, enniatins A, A1, B and B1 being the most studied,
BEAUVERICIN
especially in cereals and derived products.
Beauvericin is produced by Fusarium proliferatum,
F. subglutinans, F. verticillioides and F. oxysporum, and has a structure related to that of the enniatins, although unlike the latter, the three amino acid residues are aromatic N-methyl-phenylalanines.
R1
R2
R3
ENNA
Sec-but
Sec-but
Sec-but
ENNA1
i-pr
Sec-but
Sec-but
ENNB
i-pr
i-pr
i-pr
ENNB1
i-pr
i-pr
Sec-but
Figure 2. Chemical structure of the main enniatins (ENN) (a) and beauvericin (b).
9
Thus, beauvericin has insecticidal
Enniatins inhibit the acyl-CoA and cholesterol acyl .
11,23
transferase enzymes
properties capable of inducing apoptosis in mammalian cells. It also has antiviral, cytotoxic and immunosuppressive activity23-25.
Like the enniatins, beauvericin facilitates the transport of mono or divalent cations across the cell membrane, altering the normal physiological concentrations of these ions.
Enniatins Inhibition of acyl-CoA and cholesterol acyl transferase
Beauvericin Insecticidal, antiviral, cytotoxic and immunosuppressive activity
Although in vitro studies have demonstrated the toxicity of these mycotoxins (showing possible genotoxic and reproductive effects), there is still insufficient evidence of their toxicity in vivo26. Therefore, EFSA issued a scientific opinion on the need to collect more toxicological data produced by chronic exposure to these compounds, concluding that it is necessary to have more data on the simultaneous presence of enniatins and beauvericin with other Fusarium toxins, as well as to study their possible combined effects27. Some recent studies have demonstrated the cytotoxicity of these compounds in in vitro studies, using the porcine intestinal epithelial cell line IPEC-J2, with beauvericin being the mycotoxin with the highest bioavailability28. The presence of enniatins and beauvericin in cereals has been widely documented through various studies that have revealed the high incidence of these compounds, sometimes at concentrations of the order of mg/kg29-32.
10
Case Study Presence of 31 mycotoxins in pig feed Our research group carried out the analysis of 228 samples of swine feed for the presence of 31 mycotoxins:
The samples (2 grain corn samples and 226 feed samples, of which 183 were
Mycotoxins with permitted
meal and 43 pellets) were obtained from
or recommended limits:
Spanish farms and feedmills between
aflatoxins B1, B2, G1 and G2,
February and August 2017 and included:
ochratoxin A, deoxynivalenol, zearalenone, fumonisins B1 and B2, and T-2 and HT-2 toxins. Enniatins (B, B1, A and A1)
71 feed and 2 corn samples destined to fattening pigs 42 feeds for sows 111 feeds for piglets
Beauvericin 12 most common ergot alkaloids
The mycotoxins were extracted from the matrix by solid-liquid extraction, while
Other mycotoxins considered
liquid chromatography with a fluorescence
of interest: citrinin, fusarenon
detector (LC-FLD) was used for the analysis
X and sterigmatocystin
of aflatoxins, and liquid chromatography coupled to tandem mass spectrometry
The results were published in
(LC-MS/MS) for the rest of the mycotoxins.
two research articles33,34.
It is worth mentioning that LC-MS/ MS allows the measurement of a large number of analytes with good sensitivity, selectivity and confirmatory power that have made it an essential tool for multi-mycotoxin analysis35.
11
A summary of the data obtained is presented in Figure 3 and Table 2. The most frequent mycotoxins were: Enniatin B (100% of the samples). Enniatin B1 (83.3% of the samples) Enniatin A1 (73.2% of the samples)
All samples met the requirements for maximum permitted or recommended levels for aflatoxin B1, ochratoxin A, fumonisins B1+B2, Toxins T-2 and HT-2, deoxynivalenol and sum of the twelve ergot alkaloids
Beauvericin (98.2% of samples) Fumonisin B1 (66.2% of samples) However, some samples (3.1%) On the other hand, aflatoxin G2 and the ergot alkaloids ergotaminine, ergocorninine, ergocristine, ergokryptine and ergokryptinine were not detected in any sample. As for the levels found, the highest
showed zearalenone concentrations above the recommended level: 1 sample of corn (7,681 µg/kg) 5 samples of piglet feed (concentrations between 125 and 956 µg/kg)
concentrations were recorded for zearalenone (7,681 µg/kg), fumonisin B1 (3,959 µg/kg) and
1 feed for fattening pigs (290 µg/kg)
enniatin B (1,222 µg/kg).
100% 90% 80% 70% 60% 50% 40% 30% 20% 20% 0% AFB1 AFB2 AFG1 AFG2 FB1 FB2 ZEN OTA CIT T-2 HT-2 DON F-X STE ENNA ENNA1 ENNB ENNB1 BEA Em Emn Et Etn Es Esn Eco Econ Ecr Ecrn Ekr Ekrn
Percentage of positive samples *
Figure 3. Incidence of mycotoxins found in 228 samples of pig feed.
*considering samples with concentrations above the detection limit of the method used AFB1: aflatoxin B1; AFB2: aflatoxin B2; AFG1: aflatoxin G1; AFG2: aflatoxin G2; FB1: fumonisin B1; FB2: fumonisin B2; ZEN: zearalenone; OTA: ochratoxin A; CIT: citrinin; T-2: T-2 toxin; HT-2: HT-2 toxin; DON: deoxynivalenol; F-X: fusarenon X; STE: sterigmatocystin; ENNA: enniatin A; ENNA1: enniatin A1; ENNB: enniatin B; ENNB1: enniatin B1; BEA: beauvericin; Em: ergometrine; Emn: ergometrinine; Et: ergotamine; Etn: ergotaminine; Es: ergosine; Esn: ergosinine; Eco: ergocornine; Econ: ergocorninine; Ecr: ergocristine; Ecrn: ergocristinine; Ekr: ergokriptine; Ekrn: ergokryptinine.
12
Table 2. Summary of the results obtained in the determination of 31 mycotoxins in 228 samples of pig feed. %CV *
Concentration range (µg/kg) *
Quartile 0.25 (µg/kg) *
Quartile 0.50 (µg/kg) *
Quartile 0.75 (µg/kg) *
0.94
89.9
0.29-2.91
0.46
0.57
0.95
1.3
0.60
55.0
0.28-1.06
0.38
0.47
0.77
2
0.9
0.33
33.3
0.22-0.44
0.28
0.33
0.39
AFG2
0
0.0
---
---
---
---
---
---
FB1
151
66.2
403
132.6
4.09-3,959
106
209
536
FB2
96
42.1
184
101.0
3.63-961
63,0
133
219
ZEN
31
13.6
741
244.0
101-7,681
127
137
463
OTA
2
0.9
---
---
---
---
---
---
CIT
43
18.9
147
80.3
10.7-512
73.0
109
196
T-2
17
7.5
31.9
12.4
28.0-35.9
29.9
31.9
33.9
HT-2
6
2.6
117
4.8
112-123
115
117
120
DON
35
15.4
237
50.6
153-555
165
182
229
F-X
21
9.2
291
82.5
65.1-821
111
212
343
STE
7
3.1
104
115.4
11.2-308
11.4
12,8
179
ENNA
87
38.2
9.82
170.3
3.29-65.0
3.61
3.93
6.46
ENNA1
167
73.2
19.0
127.5
4.54-140
6.69
10.5
20.4
No positive samples
Incidence (%)
AFB1
7
3.1
AFB2
3
AFG1
Average concentration (µg/kg) *
ENNB
228
100
118
137.2
2.01-1,222
14.9
54.3
164
ENNB1
190
83.3
34.3
141.3
2.58-247
6.55
15.1
37.2
BEA
224
98.2
20.7
270.2
1.71-747
4.94
8.73
19.2
Em
18
7.9
59.1
73.8
18.6-145
31.2
37.3
65.8
Emn
5
2.2
---
---
---
---
---
---
Et
8
3.5
9.47
37.5
6.32-16.0
6.58
7.42
12.8
Etn
0
0.0
---
---
---
---
---
---
Es
9
3.9
5.72
50.9
3.41-10.1
3.63
3.85
8.14
Esn
11
4.8
10.7
30.7
5.87-17.5
8.16
10.84
13.16
Eco
1
0.4
---
---
---
---
---
---
Econ
0
0.0
---
---
---
---
---
---
Ecr
5
2.2
12.2
14.0
10.2-14.9
10.5
12.5
12.6
Ecrn
0
0.0
---
---
---
---
---
---
Ekr
0
0.0
---
---
---
---
---
---
Ekrn
0
0.0
---
---
---
---
---
---
*considering only samples with concentrations above the limit of quantification of the method employed AFB1: aflatoxin B1; AFB2: aflatoxin B2; AFG1: aflatoxin G1; AFG2: aflatoxin G2; FB1: fumonisin B1; FB2: fumonisin B2; ZEN: zearalenone; OTA: ochratoxin A; CIT: citrinin; T-2: T-2 toxin; HT-2: HT-2 toxin; DON: deoxynivalenol; F-X: fusarenon X; STE: sterigmatocystin; ENNA: enniatin A; ENNA1: enniatin A1; ENNB: enniatin B; ENNB1: enniatin B1; BEA: beauvericin; Em: ergometrine; Emn: ergometrinine; Et: ergotamine; Etn: ergotaminine; Es: ergosine; Esn: ergosinine; Eco: ergocornine; Econ: ergocorninine; Ecr: ergocristine; Ecrn: ergocristinine; Ekr: ergokryptine; Ekrn: ergokryptinine.
13
CO-OCCURRENCE OF MYCOTOXINS As for the presence of several mycotoxins in the same sample (considering only those
Figure 4. Presence of several mycotoxins in the same sample of pig feed.
mycotoxins present at concentrations above the detection limit of the method),
12 mycotoxins 0.44%
it can be summarized as follows:
11 mycotoxins 0.44% 2-3 mycotoxins: 9.2% 4 mycotoxins: 16.7%
10 mycotoxins 3.51%
7 mycotoxins: 18.9%
2 mycotoxins 1.75%
9 mycotoxins 4.39% 3 mycotoxins 7.46%
5 mycotoxins: 16.7% 6 mycotoxins: 16.2%
13 mycotoxins 0.44%
8 mycotoxins 13.16%
4 mycotoxins 16.67%
8 mycotoxins: 13.2% 9-13 mycotoxins: 9.2%
Figure 4 shows the results obtained.
7 mycotoxins 18.86% 6 mycotoxins 16.23%
5 mycotoxins 16.67%
14
CONCLUSIONS Of the 228 samples analyzed, only 7 showed levels above the permitted levels for zearalenone. The low incidence of ergot alkaloids should also be noted, which could be explained by the fact that many of the feeds were made from corn, a cereal that is not very susceptible to contamination by Claviceps purpurea. On the other hand, the high incidence of emerging mycotoxins, particularly enniatin B (100% of the samples), enniatin B1 (83.3%), enniatin A1 (73.2%) and beauvericin (98.2%), not currently included in the legislation, but with evidence of toxicity to pigs, should also be highlighted.
An important aspect to take into account is the simultaneous presence of different mycotoxins in the same sample (up to 13 mycotoxins), given the possible interrelationship between the different mycotoxins consumed together, which may present additive, synergistic, potentiated or antagonistic effects on health.
These effects cannot be predicted from the individual mycotoxin effects. Moreover, there is dependence on the dose, species and toxin, and even on the factors linked to the experimental methodology used in their study. However, given the importance of this issue and taking into account that, in risk analysis, the maximum permitted or recommended levels and TDI are set according to the individual effects of each mycotoxin, these values should be reviewed taking into account these possible effects and the potential risk of chronic exposure to multiple mycotoxins, even if they are at lower levels than those permitted36,37,38.
15
REFERENCES 1. Rapid Alert System for Food and Feed (RASFF). https://ec.europa.eu/food/safety/rasff/portal_en 2. Eskola M, Kos G, Elliott CT, Hajšlová J, Mayar S, Krskay R. Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited ‘FAO estimate’ of 25%. Crit. Rev. Food Sci. Nutr. 60 (2020) 2773-2789. Doi:10.1080/10408398.2019.1658570 3. Moretti A, Pascale M, Logrieco AF. Mycotoxin risks under a climate change scenario in Europe. Trends Food Sci. Technol. 84 (2019) 38-40. Doi:10.1016/j.tifs.2018.03.008 4.Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off. J. Eur. Union L 364 (2006) 5-24. 5. Commission Recommendation of 27 March 2013 on the presence of T-2 and HT-2 toxin in cereals and cereal products. Off. J. Eur. Union L91 (2013) 12–15. 6. Recomendación de la Comisión de 17 de agosto de 2006 sobre la presencia de deoxinivalenol, zearalenona, ocratoxina A, toxinas T-2 y HT-2 y fumonisinas en productos destinados a la alimentación animal. DOUE nº L229 del 23/08/2006 7. Commission Recommendation of 17 August 2006 on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and fumonisins in products intended for animal feeding. Off. J. Eur. Union L229 (2006) 7–9. 8. Muratori Holanda D, Woo Kim S. Mycotoxin occurrence, toxicity, and detoxifying agents in pig production with an emphasis on deoxinivalenol. Toxins 12 (2021) 171. Doi:10.3390/toxins13020171 9. Yanga C, Songa G, Lim W. Effects of mycotoxin-contaminated feed on farm animals. Journal of Hazardous Materials 389 (2020) 122087. Doi:10.1016/j.jhazmat.2020.122087 10. Bertero A, Moretti A, Spicer LJ, Caloni F. Fusarium molds and mycotoxins: potential species-specific effects. Toxins 10 (2018) 244. Doi:10.3390/toxins10060244 11. Gruber-Dorninger C, Novak, B, Nagl V, Berthiller F. Emerging mycotoxins: Beyond traditionally determined food contaminants. J Agric Food Chem 65 (2017) 7052–7070. Doi:10.1021/acs.jafc.6b03413 12. Craig AM, Klotz JL, Duringer JM. Cases of ergotism in livestock and associated ergot alkaloid concentrations in feed. Front Chem 3 (2015) 1-6. Doi:10.3389/fchem.2015.00008 13. Waret-Szkuta A, Larraillet L, Oswald IP, Legrand X, Guerre P, Martineau GP. Unusual acute neonatal mortality and sow agalactia linked with ergot alkaloid contamination of feed. Porc Health Manag 5 (2019) 24. Doi:10.1186/s40813-019-0131-z 14. Coufal-Majewski S, Stanford K, McAllister T, Blakley B, McKinnon J, Vieira Chaves A, Wang Y. Impacts of cereal ergot in food animal production. Front Vet Sci 3 (2016) 15. Doi:10.3389/fvets.2016.00015 15. Dänicke S, Diers S. Effects of ergot alkaloids in feed on performance and liver function of piglets as evaluated by the 13C-methacetin breath test. Arch Anim Nutr 67 (2013) 15-36. Doi:10.1080/1745039X.2012.736279 16. Krska R, Crews C. Significance, chemistry and determination of ergot alkaloids: A review. Food Add Contam 25 (2008) 722-31. Doi: 10.1080/02652030701765756 17. Komarova E, Tolkachev O. The chemistry of peptide ergot alkaloids. Part 1: Classification and chemistry of ergot peptides. Pharmaceutical Chemistry J 35 (2001) 504-513. Doi:10.1023/A:1014050926916 18. Diana Di Mavungu J, Malysheva S, Sanders M, Larionova D, Robbens J, Dubruel P, Van Peteghem C, De Saeger S. Development and validation of a new LC-MS/MS method for the simultaneous determination of six major ergot alkaloids and their corresponding epimers. Application to some food and feed commodities. Food Chem 135 (2012) 292-303. Doi:10.1016/j.foodchem.2012.04.098 19. Crews C. Analysis of ergot alkaloids. Toxins 7 (2015) 2024. Doi:10.3390/toxins7062024 20. Alltech Canada (2015) Practical limits for mycotoxins in animal feeds to reduce negative effects on health and performance. Available from: https://www.knowmycotoxins.com/wp-content/uploads/2019/01/37-Practical-Limits-Flyer-April-2018-GLOBAL.pdf 21. Mayumi Maruo V, Bracarense AP, Metayer JP, Vilarino M, Oswald IP, Pinton P. Ergot alkaloids at doses close to EU regulatory limits induce alterations of the liver and intestine. Toxins 10 (2018) 183. Doi:10.3390/toxins10050183 22. EFSA Panel on Contaminants in the Food Chain (CONTAM); Scientific Opinion on ergot alkaloids in food and feed. EFSA Journal 10 (2012) 2798. Doi:10.2903/j.efsa.2012.2798.
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23. Escrivá L, Font G, Manyes L. In vivo toxicity studies of Fusarium mycotoxins in the last decade: A review. Food Chem Toxicol 78 (2015) 185-206. Doi: 10.1016/j.fct.2015.02.005 24. Ruiz MJ, Franzova P, Juan-Garcia A, Font G. Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro. Toxicon 58 (2011) 315-326. Doi: 10.1016/j.toxicon.2011.07.015 25. Stanciu O, Juan C, Miere D, Loghin F, Mañes J. Analysis of enniatins and beauvericin by LC-MS/MS in wheat- based products. CyTA-Journal of Food 15 (2017) 433-440. Doi: 10.1080/19476337.2017.1288661 26. Fraeyman S, Croubels S, Devreese M, Antonissen G. Emerging Fusarium and Alternaria mycotoxins: Occurrence, toxicity and toxicokinetics. Toxins 9 (2017) 228. Doi: 10.3390/toxins9070228 27. EFSA Panel on Contaminants in the Food Chain (CONTAM), “Scientific Opinion on the risks to human and animal health related to the presence of beauvericin and enniatins in food and feed”, EFSA Journal 12 (2014) 3802. (Available on: https://efsa.onlinelibrary.wiley.com/doi/ epdf/10.2903/j.efsa.2014.3802) 28. Novak B, Rainer V, Sulyok M, Haltrich D, Schatzmayr G, Mayer E. Twenty-eight fungal secondary metabolites detected in pig feed samples: their occurrence, relevance and cytotoxic effects in vitro. Toxins 11 (2019) 537. Doi:10.3390/toxins11090537 29. Stanciu O, Juan C, Miere D, Loghin F, Mañes J. Presence of enniatins and beauvericin in Romanian wheat samples: from raw material to products for direct human consumption. Toxins 9 (2017) 189. Doi: 10.3390/toxins9060189 30. Covarelli L, Beccari G, Prodi A, Generotti S, Etruschi F, Meca G, Juan C, Mañes J. Biosynthesis of beauvericin and enniatins in vitro by wheat Fusarium species and natural grain contamination in an area of central Italy. Food Microbiol 46 (2015) 618-626. Doi:10.1016/j.fm.2014.09.009 31. Svingen T, Lund Hansen N, Taxvig C, Vinggaard AM, Jensen U, Rasmussen PH. Enniatin B and beauvericin are common in Danish cereals and show high hepatotoxicity on a high-content imaging platform. Environ Toxicol 32 (2017) 1658-1664. Doi: 10.1002/tox.22367 32. Kovalsky P, Kos G, Nährer K, Schwab C, Jenkins T, Schatzmayr G, Sulyok M, Krska R. Co-occurrence of regulated, masked and emerging mycotoxins and secondary metabolites in finished feed and maize-An extensive survey. Toxins 8 (2016) 363. Doi: 10.3390/toxins8120363 33. Arroyo-Manzanares N, Rodríguez-Estévez V, Arenas-Fernández P, García-Campaña AM, Gámiz-Gracia L. Occurrence of mycotoxins in swine feeding from Spain. Toxins 11 (2019) 342. Doi: 10.3390/toxins11060342 34. Arroyo-Manzanares N, Rodríguez-Estévez V, García-Campaña AM, Castellón-Rendón E, Gámiz-Gracia L. Determination of principal ergot alkaloids in swine feeding. J Sci Food Agric. 101 (2021) 5214-5224. (2021) https://doi.org/10.1002/jsfa.11169 35. Malachová A, Stránská M, Václavíková M, Elliott CT, Black C, Meneely J, Hajšlová J, Ezekiel CN, Schuhmacher R, Krska R. Advanced LC– MS-based methods to study the co-occurrence and metabolization of multiple mycotoxins in cereals and cereal-based food. Anal Bioanal Chem 410 (2018) 801-825. Doi: 10.1007/s00216-017-0750-7 36. Lee HJ, Ryu D. Worldwide occurrence of mycotoxins in cereals and cereal-derived food products: Public health perspectives of their co-occurrence. J Agric Food Chem 65 (2017) 7034-7051. Doi: 10.1021/acs.jafc.6b04847 37. Miller JD. Significance of grain mycotoxins for health and nutrition. In: Champ BR, Highley E, Hocking AD, Pitt JI (eds.), Fungi and mycotoxins in stored products. ACIAR Proceedings No. 36, Canberra, Australia, 1991, pp. 126-135. 38. BIOMIN, “Encuesta mundial de micotoxinas. La amenaza mundial. Enero-junio 2019”. (Available on: https://www.biomin.net/es/articulos/ resultados-de-la-encuesta-mundial-de-micotoxinas-biomin-1er-semestre-2019/)
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