Presence of 31 mycotoxins
in pig feed in Spain

Focusing on emerging mycotoxins, researchers from the Universities of Granada, Murcia and Cordoba analyzed the presence of 31 mycotoxins in the samples of pig feed in Spain.

L. Gámiz-Gracia1, N. Arroyo-Manzanares2, V. Rodríguez-Estévez3 and A.M. García-Campaña1

1Department of Analytical Chemistry, Faculty of Sciences, University of Granada.
2Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, Spain.
3Department of Animal Production, Faculty of Veterinary Medicine, University of Cordoba.

Mycotoxins are fungal toxins produced by several hundred species of molds that can grow on crops or food under certain conditions. The most important are those produced by molds of the genera Aspergillus, Fusarium and Penicillium.

Mycotoxins have become one of the 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.

According to the Food and Agriculture Organization of the United Nations (FAO), it is estimated that 25% of cereal production and 20% of plant production worldwide are affected by mycotoxins, 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 fungi in the coming years3.

 

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, B2, G1, G2 and M1, ochratoxin A, patulin, deoxynivalenol, zearalenone, fumonisins B1 and B2, T-2 and HT-2 toxins, citrinin) as well as for ergot sclerotia, in various food intended for human consumption4,5.

In the case of animal feed, the number of mycotoxins with maximum permitted or recommended levels is 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.

Among farm animals, pigs are considered to be one of the most vulnerable species to the effects of mycotoxins.

Such effects may include changes in immune response (resulting in reduced vaccination efficacy), loss of appetite, abortions, agalactia, etc.

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.

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.

ERGOT ALKALOIDS

Ergot alkaloids, or rye ergot, characterized by the spur or “horn” that the fungus produces on the grain as it grows, are produced by different fungi from the orders Hypocreales and Eurotiales, Claviceps purpurea 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 considered an eradicated disease in humans, sporadic outbreaks of ergotism in cattle have been described in recent decades12,13.

This disease can cause different symptoms depending on the species in question. Thus, in pigs, ergot alkaloids can cause poor performance, loss of appetite, agalactia, reproductive problems, neonatal mortality, liver damage and gangrene13-16. This wide variety of symptoms makes it difficult to identify the problem.

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.

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.

 

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.

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.

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.

ENNIATINS Y BEAUVERICIN

ENNIATINS

Enniatins are produced by fungi of the genus Fusarium, such as F. avenaceum, F. oxysporum, F. poae or F. tricinctum, and present cyclic hexadepsipeptide structure alternating D-α-hydroxyisovaleric acids and N- methyl-L-amino acids.

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, especially in cereals and derived products.

BEAUVERICIN

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.

The structure of the main enniatins and beauvericin is shown in Figure 2.

Figure 2. Chemical structure of the main enniatins (ENN) (a) and beauvericin (b).

Enniatins inhibit the acyl-CoA and cholesterol acyl transferase enzymes11,23.

Like the enniatins, beauvericin facilitates the transport of mono or divalent cations across the cell membrane, altering the normal physiological concentrations of these ions.

Thus, beauvericin has insecticidal properties capable of inducing apoptosis in mammalian cells. It also has antiviral, cytotoxic and immunosuppressive activity23-25.

 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.

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:

  • Mycotoxins with permitted or recommended limits: aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, fumonisins B1 and B2, and T-2 and HT-2 toxins.
  • Enniatins (B, B1, A and A1)
  • Beauvericin
  • 12 most common ergot alkaloids
  • OOther mycotoxins considered of interest: citrinin, fusarenon X and sterigmatocystin

The results were published in two research articles33,34.

 The samples (2 grain corn samples and 226 feed samples, of which 183 were meal and 43 pellets) were obtained from Spanish farms and feedmills between February and August 2017 and included:

  • 71 feed and 2 corn samples destined to fattening pigs
  • 42 feeds for sows
  • 111 feeds for piglets

The mycotoxins were extracted from the matrix by solid-liquid extraction, while liquid chromatography with a fluorescence detector (LC-FLD) was used for the analysis of aflatoxins, and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the rest of the mycotoxins.

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.

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)
  • Beauvericin (98.2% of samples)
  • Fumonisin B1 (66.2% of samples)

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 concentrations were recorded for zearalenone (7,681 μg/kg), fumonisin B1 (3,959 μg/kg) and enniatin B (1,222 μg/kg).

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

However, some samples (3.1%) 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)
  • 1 feed for fattening pigs (290 μg/kg)

Figure 3. Incidence of mycotoxins found in 228 samples of pig feed.

Table 2. Summary of the results obtained in the determination of 31 mycotoxins in 228 samples of pig feed.

CO-OCCURRENCE OF MYCOTOXINS

As for the presence of several mycotoxins in the same sample (considering only those mycotoxins present at concentrations above the detection limit of the method), it can be summarized as follows:

  • 2-3 mycotoxins: 9.2%
  • 4 mycotoxins: 16.7%
  • 5 mycotoxins: 16.7%
  • 6 mycotoxins: 16.2%
  • 7 mycotoxins: 18.9%
  • 8 mycotoxins: 13.2%
  • 9-13 mycotoxins: 9.2%

Figure 4 shows the results obtained.

Figure 4. Presence of several mycotoxins in the same sample of pig feed.

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.

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 yfumonisinas 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 fumonisinsin 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 ondeoxinivalenol. 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 FoodChem 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 withergot 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 animalproduction. Front Vet Sci 3 (2016)

15. Doi:10.3389/fvets.2016.0001515. Dänicke S, Diers S. Effects of ergot alkaloids in feed on performance and liver function of piglets as evaluated by the 13C-methacetin breathtest. 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. PharmaceuticalChemistry 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 andvalidation 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 inducealterations 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.

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 inCHO-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 ofFood 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 thepresence 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 toproducts 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 wheatFusarium 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 andshow 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 emergingmycotoxins 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 swinefeeding 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 ergotalkaloids 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 Chem410 (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 theirco-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 mycotoxinsin 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/)



Micotoxicosis prevention
Sign up