Document downloaded from: http://hdl.handle.net/10459.1/66413 The final publication is available at: https://doi.org/10.1007/s12550-019-00347-x Copyright (c) Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature, 2019 1 Frequency and levels of mycotoxins in beer from the Mexican market and exposure 2 estimate for deoxynivalenol mycotoxins 3 4 Hiram A. Wall-Martínez1, Xenia Pascari2, Antonio J. Ramos2, Sonia Marin2 and Vicente Sanchis2 5 1 Unit of Research and Food Development (UNIDA), Veracruz Institute of Technology, Veracruz, México. 6 Miguel Ángel de Quevedo 2779, 91860 Veracruz, México 7 2Applied Mycology Unit, Food Technology Department, University of Lleida, UTPV-XaRTA, Agrotecnio, 8 Av. Rovira Roure 191, 25198 Lleida, Spain 9 10 Corresponding author: Vicente Sanchis, email: [email protected], tel +34 973 702535; fax: +34 973 11 702596. 12 13 ABSTRACT 14 The aim of the present study was to evaluate the occurrence of 23 mycotoxins in beer purchased in Mexico and to assess 15 two exposure scenarios in the Mexican population through beer consumption. Multi-mycotoxin analysis of a total of 61 16 different beers (132 samples) was carried out using UHPLC-MS/MS equipment. Probability density functions were used to 17 describe mycotoxins contamination. The daily intake of mycotoxins was estimated using a semi-probabilistic approach, 18 applying the Monte Carlo method. Deoxynivalenol (DON) and its metabolites (deoxynivalenol-3-glucoside (DON3G) and 19 3-acetyl-deoxynivalenol (3ADON) were the mycotoxins found in higher proportions in contaminated samples. None of the 20 other mycotoxins overpassed the limit of quantification (LOQ) of the method. The combined intake of DON and its 21 analogues ranged from 5.24 to 86.59 ng kg-1 bw day-1, which represent from 1.20 to 19.83 % of the DON TDI. The results 22 suggest that depending on the individual consumption of beer and depending on the type of beer, the intake of DON via 23 beer could represent a significant percentage of the tolerable daily intake (TDI). 24 25 Keywords: mycotoxins, deoxynivalenol, beer, occurrence and estimated daily intake. 26 27 1 28 INTRODUCTION 29 Beer is the most consumed alcoholic beverage worldwide, with an annual per capita consumption greater 30 than 100 litres in some European countries (Euromonitor International 2014; Kirin 2016). Mexico, with a 31 production of 10.5 billion litres, is the country with the highest export of beer worldwide. In 2016, Mexico 32 beer exports reached 2.814 billion dollars, followed by Netherlands (1.905 billion), Belgium (1.438 billion) 33 and Germany (1.307 billion) (INEGI 2017). About 80% of Mexican beer is exported to the United States, the 34 rest being distributed to more than 184 countries (Kantar Worldpanel Mexico 2015; INEGI 2017). 35 Cereals used in brewing are mainly barley, wheat and corn (Shetty and Jespersen 2006). These cereals can be 36 subjected to contamination by different mycotoxins. Barley and wheat are mainly contaminated by ochratoxin 37 A (OTA), trichothecenes (deoxynivalenol (DON), nivalenol (NIV), T-2 and HT-2 toxins and zearalenone 38 (ZEN). Corn is usually infested by fungi-producing fumonisins (FBs) and aflatoxins (AFs). All these 39 mycotoxins have been associated with human and animal diseases (Zain 2011). Alternaria mycotoxins in 40 cereals have been largely ignored both in Europe and overseas (Müller and Korn 2013). Alternaria species 41 produces several mycotoxins, such as alternariol (AOH) and alternariol monomethyl ether (AME). Strong 42 evidence suggests that they are genotoxic (Pfeiffer et al. 2007) and mutagenic (Schrader et al. 2001; Brugger. 43 et al. 2006). 44 The International Agency for Research on Cancer (IARC) classified AFs as a human carcinogen (Class 1), 45 OTA and fumonisin B1 (FB1) as a possible human carcinogen (Class 2B), DON, ZEN, NIV and T-2/HT-2 46 toxins were not classifiable as to their carcinogenicity to humans (Class 3) (IARC 1993, 2002; FAO/WHO 47 2006; EFSA 2010b, 2014). The lack of regulation for Alternaria toxins worldwide is partially due to the 48 limited toxicity data available for them. As a consequence, the EFSA used the Threshold of Toxicological 49 Concern (TTC) approach to evaluate the relative level of concern of Alternaria toxins for human health. The 50 results demonstrated that dietary exposure to AOH and AME exceeded the TCC value of 2.5 ng/kg body 51 weight per day, indicating the need for additional toxicity data (Arcella et al. 2016; Tralamazza et al. 2018). 52 The accumulation of mycotoxins in cereals, or derived foods and feeds, has been sporadically documented in 53 Mexico, reaching concentrations higher than 1000 μg kg-1 for ZEN in wheat (Gonzalez-Osnaya and Farres 54 2011), 200 μg kg-1 for AFs in maize and maize products (Martínez-Flores et al. 2003; Castillo-Urueta et al. 2 −1 −1 55 2011), 5.8 μg kg for OTA (Reyes-Velázquez et al. 2008) and 5600 μg kg for FB1 (Robledo et al. 2001) in 56 maize silage. In Mexico, there is no comprehensive food mycotoxins monitoring program carried out by the 57 governmental agencies (Guzmán-de-Peña and Peña-Cabriales 2005). 58 AFs are the only mycotoxins legislated in Mexico, as described by the official Mexican norms number 59 NOM-187-SSA1-2002 NOM-247-SSA1-2008 and NOM-243-SSA1-2010. The maximum allowed limit of AFs 60 in cereals for human and animal consumption is 20 μg/kg. In the United States, AFs (20 μg/kg), DON (1000 61 μg/kg), FBs (2000-4000 μg/kg), and patulin (50 μg/kg) have been regulated (USDA 2015). European 62 regulations on mycotoxin set maximum levels in foodstuff for 14 compounds (European Commission 63 1881/2006; European Commission 2013/165/EU). Regulation 1881/2006 establishes a limit for fumonisin 64 content in maize-based foods (applicable to beer) intended for human consumption to 1000 μg/kg. However, 65 specific regulations for mycotoxins in beer do not exist in any of these countries. 66 Mycotoxin contamination can occur during cereal growth in the field, during post-harvest storage or during 67 malting (Bertuzzi et al. 2011). Considering mycotoxins thermal stability (AFs, ZEN, and DON) and solubility 68 in water (DON and FBs), they can be partially transferred from cereals to malt and then to beer (Rodríguez- 69 Carrasco et al. 2015). Several authors have studied the occurrence of mycotoxins in industrial and craft beers 70 sold in Argentina (Molto et al. 2000), Brazil (Piacentini et al. 2017), Spain (Torres et al. 1998; Rodríguez- 71 Carrasco et al. 2015; Pascari et al. 2018b), Poland (Kuzdraliński et al. 2013), Belgium (Tangni et al. 2002), 72 and other European countries (Papadopoulou-Bouraoui et al. 2004; Bertuzzi et al. 2011). There are no studies 73 on the occurrence of mycotoxins in beer consumed in Mexico or in the United States, however, some of the 74 surveys mentioned above included Mexican beers in their study detecting: OTA, AOH, DON and ZEN. 75 To estimate dietary exposure, it is necessary to combine data on food consumption and contamination levels 76 in order to allow conclusions to be drawn about the amount of a substance being consumed by the population 77 (FAO/WHO 2006). Monte Carlo simulation is a statistical method commonly used in probabilistic approach 78 assessment. Monte Carlo simulation relies on a sequence of random numbers to carry out a simulation. This 79 allows a probability distribution to be obtained and studied, instead of a single value to represent this risk 80 (Landau and Binder 2015). 81 3 82 Among the studies of exposure to mycotoxin through beer intake that have been made so far, none has been 83 conducted exclusively in Mexico. Therefore, the objective of this work was to assess two exposure scenarios 84 to mycotoxins throughout beer consumption, focusing on data for the Mexican population (daily beer 85 consumption, average body weight). 86 87 MATERIALS AND METHODS 88 Chemicals and reagents 89 The standards of Mycotoxins: aflatoxin B1 (AFB1); aflatoxin B2 (AFB2); aflatoxin G1 (AFG1); aflatoxin G2 90 (AFG2), sterigmatocystin (STE); OTA; roquefortin C (ROQ-C); AOH; AME; T-2 toxin (T-2); HT-2 toxin (HT- 91 2); neosolaniol (NEO); diacetoxyscirpenol (DAS); DON; 3-acetyl-deoxynivalenol (3ADON); 15-acetyl- 92 deoxynivalenol (15ADON);; deoxynivalenol-3-glucoside (DON3G); NIV; fusarenon-X (F-X); ZEN; fumonisin 93 B1 (FB1); fumonisin B2 (FB2) and fumonisin B3(FB3) were obtained from Sigma Aldrich (Bornem, Belgium). 94 An internal standard of deepoxy-deoxynivalenol (DOM-1) was obtained from Romer Labs (Getzersdorf, 95 Austria). All mycotoxin solid standards were dissolved in methanol (1 mg/mL) and stored at -18 °C. 96 Water was obtained from a Milli-Q® SP Reagent water system from Millipore Corp. (Brussels, Belgium). 97 Disinfectol® (denaturated ethanol with 5% ether) was supplied by Chem-Lab (Zedelgem, Belgium). 98 Methanol (LCMS grade) was purchased from BioSolve (Valkenswaard, the Netherlands), while acetonitrile 99 (Analar Normapur) was obtained from VWR International (Zaventem, Belgium). Acetic acid (glacial, 100%) 100 was supplied by Merck (Darmstadt, Germany). Magnesium sulphate (MgSO4) and sodium chloride (NaCl) 101 were purchased from Fischer Scientific (New Jersey, USA). 102 Samples 103 Various types of bottled and canned beers (n=61) were bought from supermarkets and beer stores of Veracruz 104 city (Mexico) between July and October 2017. Every product was purchased by duplicate or triplicate (2 or 3 105 different lots of each beer) according to their availability at the time of buying (total of 132 samples). Twenty- 106 five different beer producing companies, originating from eight countries, Mexico (40), Unites States (10), 107 Belgium (4), Germany (3), Spain (1), Netherlands (1), Argentina (1), and Guatemala (1) were chosen for the 108 analysis.