Silage has been identified as a source of different microbial toxins, that may impair farm animal health and productivity as human health can also be compromised. In this sense, the aim of this study was to determine the impact of silage additives on the concentrations of deoxynivalenol (DON) and zearalenone (ZEN) mycotoxins and, eventually, to evaluate the hygienic quality of orchardgrass (Dactylis glomerata L.) silage based on the concentration of them compared to control silage. This study evaluated the influence of biological and chemical additives used in six different varieties of orchardgrass silage on DON and ZEN mycotoxin contents for the first time. The content of both fusariotoxins (DON and ZEN) in fresh matter and grass silage were below the threshold stipulated by the European Commission. The concentration of DON ranges from ~21.86 to 37.26 ng/kg, ~10.21 to 15 ng/kg, ~20.72 to 29.14 ng/kg; and ZEN range from ~3.42 to 7.87 ng/kg, ~3.85 to 8.62 ng/kg and ~2.15 to 5.08 ng/kg, in control, biological and chemical silages, respectively. In general, the biological additive was more efficient for preventing DON contamination, whereas the chemical additive was more efficient for preventing ZEN contamination in grass silage. In summary, the results obtained in this work demonstrate that biological and chemical additives can inhibit fungal growth and mycotoxin production on Dactylis glomerata L. silage and whose use could prevent animal and human diseases.

Departamento de Química Analítica Química Física e Ingeniería Química Facultad de Ciencias Universidad de Alcalá Alcalá de Henares Madrid Spain

Department of Animal Nutrition and Forage Production Faculty of AgriSciences Mendel University in Brno Brno Czech Republic

Department of Applied and Landscape Ecology Faculty of AgriSciences Mendel University in Brno Brno Czech Republic

Department of Crop Science Breeding and Plant Protection Faculty of AgriSciences Mendel University in Brno Brno Czech Republic

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Panasiuk L, Jedziniak P, Pietruszka K, Piatkowska M, Bocian L. Frequency and levels of regulated and emerging mycotoxins in silage in Poland. Mycotoxin Res. 2019;35: 17–25. doi: 10.1007/s12550-018-0327-0 PubMed DOI PMC

Vandicke J, De Visschere K, Croubels S, De Saeger S, Audenaert K, Haesaert G. Mycotoxins in Flanders’ fields: Occurrence and correlations with Fusarium species in whole-plant harvested maize. Microorganisms. 2019;7: 571. doi: 10.3390/microorganisms7110571 PubMed DOI PMC

Ogunade IM, Martinez-Tuppia C, Queiroz OCM, Jiang Y, Drouin P, Wu F, et al.. Silage review: Mycotoxins in silage: Occurrence, effects, prevention, and mitigation. J Dairy Sci. 2018;101: 4034–4059. doi: 10.3168/jds.2017-13788 PubMed DOI

Alba-Mejía JE, Dohnal V, Domínguez-Rodríguez G, Středa T, Klíma M, Mlejnková V, et al.. Ergosterol and polyphenol contents as rapid indicators of orchardgrass silage safety. Heliyon. 2023;9: e14940. doi: 10.1016/j.heliyon.2023.e14940 PubMed DOI PMC

Truong NN, Tesfamariam K, Visintin L, Goessens T, De Saeger S, Lachat C, et al.. Associating multiple mycotoxin exposure and health outcomes: Current statistical approaches and challenges. World Mycotoxin J. 2023;16: 25–32. doi: 10.3920/WMJ2022.2784 DOI

Manni K, Rämö S, Franco M, Rinne M, Huuskonen A. Occurrence of mycotoxins in grass and whole-crop cereal silages–A farm survey. Agriculture. 2022;12: 398. doi: 10.3390/agriculture12030398 DOI

Gallo A, Minuti A, Bani P, Bertuzzi T, Cappelli FP, Doupovec B, et al.. A mycotoxin-deactivating feed additive counteracts the adverse effects of regular levels of Fusarium mycotoxins in dairy cows. J Dairy Sci. 2020;103: 11314–11331. doi: 10.3168/jds.2020-18197 PubMed DOI

Silva L de A, de Mello MRB, Oliveira Pião D de, Silenciato LN, de Quadros TCO, de Souza AH, et al.. Effects of experimental exposure to zearalenone on reproductive system morphometry, plasma oestrogen levels, and oocyte quality of beef heifer. Reprod Domest Anim. 2021;56: 775–782. doi: 10.1111/rda.13917 PubMed DOI

EFSA, Knutsen H-K, Alexander J, Barregård L, Bignami M, Brüschweiler B, et al.. Risks for animal health related to the presence of zearalenone and its modified forms in feed. EFSA J. 2017;15: e04851. doi: 10.2903/j.efsa.2017.4851 PubMed DOI PMC

EFSA, Knutsen HK, Alexander J, Barregård L, Bignami M, Brüschweiler B, et al.. Risks to human and animal health related to the presence of deoxynivalenol and its acetylated and modified forms in food and feed. EFSA J. 2017;15: e04718. doi: 10.2903/j.efsa.2017.4718 PubMed DOI PMC

European Commission (EU). Regulation (576/2006) 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. 2006;229: 7–9.

Seppälä A, Heikkilä T, Mäki M, Rinne M. Effects of additives on the fermentation and aerobic stability of grass silages and total mixed rations. Grass Forage Sci. 2016;71: 458–471. doi: 10.1111/gfs.12221 DOI

Muck RE, Nadeau EMG, McAllister TA, Contreras-Govea FE, Santos MC, Kung L. Silage review: Recent advances and future uses of silage additives. J Dairy Sci. 2018;101: 3980–4000. doi: 10.3168/jds.2017-13839 PubMed DOI

Fabiszewska AU, Zielińska KJ, Wróbel B. Trends in designing microbial silage quality by biotechnological methods using lactic acid bacteria inoculants: A minireview. World J Microbiol Biotechnol. 2019;35: 76. doi: 10.1007/s11274-019-2649-2 PubMed DOI PMC

Liu Q, Lindow SE, Zhang J. Lactobacillus parafarraginis ZH1 producing anti-yeast substances to improve the aerobic stability of silage. Anim Sci J. 2018;89: 1302–1309. doi: 10.1111/asj.13063 PubMed DOI

Ma ZX, Amaro FX, Romero JJ, Pereira OG, Jeong KC, Adesogan AT. The capacity of silage inoculant bacteria to bind aflatoxin B1 in vitro and in artificially contaminated corn silage. J Dairy Sci. 2017;100: 7198–7210. doi: 10.3168/jds.2016-12370 PubMed DOI

Ferrero F, Prencipe S, Spadaro D, Gullino ML, Cavallarin L, Piano S, et al.. Increase in aflatoxins due to Aspergillus section Flavi multiplication during the aerobic deterioration of corn silage treated with different bacteria inocula. J Dairy Sci. 2019;102: 1176–1193. doi: 10.3168/jds.2018-15468 PubMed DOI

Oliveira AS, Weinberg ZG, Ogunade IM, Cervantes AAP, Arriola KG, Jiang Y, et al.. Meta-analysis of effects of inoculation with homofermentative and facultative heterofermentative lactic acid bacteria on silage fermentation, aerobic stability, and the performance of dairy cows. J Dairy Sci. 2017;100: 4587–4603. doi: 10.3168/jds.2016-11815 PubMed DOI

Wang S, Liu H, Zhao J, Dong Z, Li J, Shao T. Influences of organic acid salts and bacterial additives on fermentation profile, aerobic stability, and in vitro digestibility of total mixed ration silage prepared with wet hulless barley distillers’ grains. Agronomy. 2023;13: 672. doi: 10.3390/agronomy13030672 DOI

Kalúzová M, Kačániová M, Bíro D, Šimko M, Gálik B, Rolinec M, et al.. The change in microbial diversity and mycotoxins concentration in corn silage after addition of silage additives. Diversity. 2022;14: 592. doi: 10.3390/d14080592 DOI

Franco M, Tapio I, Pirttiniemi J, Stefański T, Jalava T, Huuskonen A, et al.. Fermentation quality and bacterial ecology of grass silage modulated by additive treatments, extent of compaction and soil contamination. Fermentation. 2022;8: 156. doi: 10.3390/fermentation8040156 DOI

Alba-Mejía JE, Skládanka J, Hilgert-Delgado A, Klíma M, Knot P, Doležal P, et al.. The effect of biological and chemical additives on the chemical composition and fermentation process of Dactylis glomerata silage. Spanish J Agric Res. 2016;14: e0604. doi: 10.5424/sjar/2016142-8040 DOI

Skládanka J, Nedělník J, Adam V, Doležal P, Moravcová H, Dohnal V. Forage as a primary source of mycotoxins in animal diets. Int J Environ Res Public Health. 2011;8: 37–50. doi: 10.3390/ijerph8010037 PubMed DOI PMC

Alshannaq A, Yu JH. Occurrence, toxicity, and analysis of major mycotoxins in food. Int J Environ Res Public Health. 2017;14: 632. doi: 10.3390/ijerph14060632 PubMed DOI PMC

Janik E, Niemcewicz M, Podogrocki M, Ceremuga M, Gorniak L, Stela M, et al.. The existing methods and novel approaches in mycotoxins’ detection. Molecules. 2021;26: 3981. doi: 10.3390/molecules26133981 PubMed DOI PMC

Nolan P, Auer S, Spehar A, Elliott CT, Campbell K. Current trends in rapid tests for mycotoxins. Food Addit Contam Part A. 2019;36: 800–814. doi: 10.1080/19440049.2019.1595171 PubMed DOI

Oplatowska-Stachowiak M, Reiring C, Sajic N, Haasnoot W, Brabet C, Campbell K, et al.. Development and in-house validation of a rapid and simple to use ELISA for the detection and measurement of the mycotoxin sterigmatocystin. Anal Bioanal Chem. 2018;410: 3017–3023. doi: 10.1007/s00216-018-0988-8 PubMed DOI

Urusov AE, Zherdev A V, Petrakova A V, Sadykhov EG, Koroleva O V, Dzantiev BB. Rapid multiple immunoenzyme assay of mycotoxins. Toxins (Basel). 2015;7: 238–254. doi: 10.3390/toxins7020238 PubMed DOI PMC

Xiong Y, Leng Y, Li X, Huang X, Xiong Y. Emerging strategies to enhance the sensitivity of competitive ELISA for detection of chemical contaminants in food samples. TrAC Trends Anal Chem. 2020;126: 115861. doi: 10.1016/j.trac.2020.115861 DOI

Rodríguez-Blanco M, Ramos AJ, Sanchis V, Marín S. Mycotoxins occurrence and fungal populations in different types of silages for dairy cows in Spain. Fungal Biol. 2021;125: 103–114. doi: 10.1016/j.funbio.2019.08.006 PubMed DOI

Santos Pereira C, Cunha SC, Fernandes JO. Prevalent mycotoxins in animal feed: Occurrence and analytical methods. Toxins (Basel). 2019;11: 290. doi: 10.3390/toxins11050290 PubMed DOI PMC

Gallo A, Fancello F, Ghilardelli F, Zara S, Froldi F, Spanghero M. Effects of several lactic acid bacteria inoculants on fermentation and mycotoxins in corn silage. Anim Feed Sci Technol. 2021;277: 114962. doi: 10.1016/j.anifeedsci.2021.114962 DOI

Queiroz OCM, Ogunade IM, Weinberg Z, Adesogan AT. Silage review: Foodborne pathogens in silage and their mitigation by silage additives. J Dairy Sci. 2018;101: 4132–4142. doi: 10.3168/jds.2017-13901 PubMed DOI

Zhang L, Zhang C, Song M, Dang D, Zhao J, Zhang L, et al.. Characterization of Lactobacillus rhamnosus GG and its effects on improving the fermentation quality of silages as a novel inoculant. Anim Feed Sci Technol. 2023;304: 115724. doi: 10.1016/j.anifeedsci.2023.115724 DOI

Skládanka J, Adam V, Doležal P, Nedělník J, Kízek R, Lindusková H, et al.. How do grass species, season and ensiling influence mycotoxin content in forage? Int J Environ Res Public Health. 2013;10: 6084–6095. doi: 10.3390/ijerph10116084 PubMed DOI PMC

Kosicki R, Błajet-Kosicka A, Grajewski J, Twarużek M. Multiannual mycotoxin survey in feed materials and feedingstuffs. Anim Feed Sci Technol. 2016;215: 165–180. doi: 10.1016/j.anifeedsci.2016.03.012 DOI

Vandicke J, De Visschere K, Ameye M, Croubels S, De Saeger S, Audenaert K, et al.. Multi-mycotoxin contamination of maize silages in Flanders, Belgium: Monitoring mycotoxin levels from seed to feed. Toxins (Basel). 2021;13: 202. doi: 10.3390/toxins13030202 PubMed DOI PMC

Cogan T, Hawkey R, Higgie E, Lee MRF, Mee E, Parfitt D, et al.. Silage and total mixed ration hygienic quality on commercial farms: Implications for animal production. Grass Forage Sci. 2017;72: 601–613. doi: 10.1111/gfs.12265 DOI

Dagnac T, Latorre A, Fernández Lorenzo B, Llompart M. Validation and application of a liquid chromatography-tandem mass spectrometry based method for the assessment of the co-occurrence of mycotoxins in maize silages from dairy farms in NW Spain. Food Addit Contam Part A. 2016;33: 1850–1863. doi: 10.1080/19440049.2016.1243806 PubMed DOI

Dänicke S, Winkler J, Meyer U, Kersten S, Wernike K, Beer M, et al.. Antibody response of growing German Holstein bulls to a vaccination against bovine viral diarrhea virus (BVDV) is influenced by Fusarium toxin exposure in a non-linear fashion. Mycotoxin Res. 2018;34: 123–139. doi: 10.1007/s12550-018-0307-4 PubMed DOI PMC

Dänicke S, Krenz J, Seyboldt C, Neubauer H, Frahm J, Kersten S, et al.. Maize and grass silage feeding to dairy cows combined with different concentrate feed proportions with a special focus on mycotoxins, Shiga Toxin (stx)-Forming Escherichia coli and Clostridium botulinum neurotoxin (BoNT) genes: Implications for animal health and food safety. Dairy. 2020;1: 91–125. doi: 10.3390/dairy1020007 DOI

Penagos-Tabares F, Khiaosa-ard R, Schmidt M, Pacífico C, Faas J, Jenkins T, et al.. Fungal species and mycotoxins in mouldy spots of grass and maize silages in Austria. Mycotoxin Res. 2022;38: 117–136. doi: 10.1007/s12550-022-00453-3 PubMed DOI PMC

Zachariasova M, Dzuman Z, Veprikova Z, Hajkova K, Jiru M, Vaclavikova M, et al.. Occurrence of multiple mycotoxins in European feedingstuffs, assessment of dietary intake by farm animals. Anim Feed Sci Technol. 2014;193: 124–140. doi: 10.1016/j.anifeedsci.2014.02.007 DOI

Thapa A, Horgan KA, White B, Walls D. Deoxynivalenol and zearalenone–Synergistic or antagonistic agri-food chain co-contaminants? Toxins (Basel). 2021;13: 561. doi: 10.3390/toxins13080561 PubMed DOI PMC

Juráček M, Felšöciová S, Bíro D, Šimko M, Gálik B, Rolinec M, et al.. The effect of lactic acid bacteria addition on microbiota and occurrence of mycotoxins in rye silages. J Cent Eur Agric. 2022;23: 342–350. doi: 10.5513/JCEA01/23.2.3540 DOI