**1. Introduction**

Mycotoxins, secondary metabolites produced by fungi, a ffect 25% of crops worldwide [1]. Mycotoxin contamination usually takes place during crop growth due to adverse environmental conditions, inappropriate harvesting, storage, or processing procedures [2].

Ochratoxin A (OTA), the major mycotoxin of the ochratoxins' group and the one presenting grea<sup>t</sup> toxicological concern, is produced by *Penicillium verrucosum*, *Aspergillus ochraceus*, and rarely by some strains of *Aspergillus niger* [3]. OTA has been reported as nephrotoxic, carcinogenic, teratogenic, genotoxic, and immunotoxic. This mycotoxin also disturbs blood coagulation, hinders protein synthesis, endorses cell membrane peroxidation, abolishes calcium homeostasis, and constrains mitochondrial respiration [4]. In addition, it is epidemiologically associated to the human Balkan endemic nephropathy (BEN) disease and to urinary tract tumors [5,6]. Moreover, it is described as a cumulative mycotoxin since it is easily assimilated by the digestive tract and is gradually excreted by the urinary system [4]. Since 1993, OTA has been described as a possible carcinogen to humans, group 2B, by the International Agency for Research on Cancer (IARC) [7].

OTA generally occurs in numerous food products. This includes cereals, oleaginous seeds, green coffee, wine, meat, cocoa, spices, and fruit berries, which are contaminated at levels that vary according to environmental and processing conditions [8,9]. Moreover, some studies report that the prevalence and levels of OTA in organic cereals is higher when compared to non-organic cereals [10].

Due to the fact that OTA widely occurs in cereals, beer, which is a cereal product, has a potential contamination risk [11]. OTA was first reported in beer in 1983 [12]. Since then, several analytical methodologies were developed to study the natural incidence of OTA in this beverage. Most studies employed solid phase extraction through immunoaffinity columns (IAC) [12–17]. For detection and quantification, most authors employed liquid chromatography with fluorescence detection (LC-FD) [8,13,14,18,19], with limits of detection (LODs) varying between 0.002 μg/<sup>L</sup> and 1 μg/L. Liquid chromatography with tandem mass detection (LC-MS-MS) or ultra-pressure liquid chromatography with mass detection (UPLC-MS) was also applied, with LODs varying between 0.75 and 0.0003 μg/L, respectively [12,20].

Worldwide, several studies have investigated the presence of OTA in beer. Its occurrence was reported in Brazil [14], South Africa [18], Iran [21,22], Turkey [23], China [24], Japan [25], and Europe [13,20]. Namely, European studies reported it in Germany [26], Belgium [17], Spain [2], Italy [21,27], and the Czech Republic [19,28].

According to the scientific literature, the occurrence of OTA in beer samples is usually at low levels. In these studies, the minimum levels found ranged between 0.0009 and 2.7 μg/L, in Iran and Europe, respectively [20,22]. Some studies reported higher concentrations, up to 18 μg/<sup>L</sup> in Brazil [14]. However, one of the studies showed significantly higher values, up to 2340 μg/<sup>L</sup> [18].

The incidence of OTA in beer depends on the contamination of brewing materials, such as barley and barley malt, with ochratoxigenic fungi species [12]. During the production of beer, considerable OTA losses (40–89%) have been perceived in the grist during mashing, most possibly owing to proteolytic degradation [11]. Another 16% may be eliminated with spent grains [11]. With the fermentation process, OTA decreases in the range of 2% to 69% [11]. The remaining OTA passes on to the final beer product [12].

This contaminant may eventually reach consumers, and a frequent consumption of contaminated products could suppose a risk for human health [20]. There is no maximum allowable limit established by the European Commission (EC) for OTA content in beer [12]. Although there is no defined limit for beer, a maximum of 3 μg/kg for malt has been established by the European Union [29]. However, there are guideline levels established by the Netherlands (0.5 μg/L), Finland (0.3 μg/L), and Italy (0.2 μg/L) [8]. A tolerable weekly intake (TWI) of 120 ng/kg body weight (b.w.) was set for OTA in 2006 by the European Food Safety Authority (EFSA) [30].

Our main goal was to verify, for the first time, the OTA contamination of the beer marketed and consumed in Portugal, as well as to calculate the human estimated daily intake and risk assessment.

#### **2. Results and Discussion**

#### *2.1. Validation and Quality Control*

Validation and quality control fulfilled the European guidelines [31] and the results are presented in Table 1.


**Table 1.** Analytical quality control data obtained for OTA in beer spiked samples.

Linearity results, both on standard and matrix-matched assays, were suitable, with correlation coe fficients (*r*2) of 0.998 and 0.999, respectively. The obtained limit of detection (LOD) and limit of quantification (LOQ) were 0.14 μg/<sup>L</sup> and 0.43 μg/L, respectively. The value of matrix e ffect (ME) obtained was considered negligible, 109%.

Accuracy and precision were adequate. Recoveries varied from 81.0% to 86.0%. Intra-day repeatability ranged from 1.72% to 2.43% and inter-day repeatability ranged from 2.74% to 6.25%. Both accuracy and precision results were adequate according to the requirements established by the EC 401/2006 directive [31].

#### *2.2. Frequency and Occurrence of OTA in Beer Samples*

In the present study, from the 85 analyzed beers, nine samples (10.6%) were contaminated at concentrations ranging from < LOQ to 11.25 μg/L, with an average level of 3.14 ± 4.09 μg/<sup>L</sup> (median of 1.74%). One of the contaminated samples presented a concentration between the LOD and the LOQ. Therefore, for results' analysis, half of the LOQ value was considered (Table 2).


**Table 2.** OTA contamination levels (μg/L) found in the positive samples.

Among the contaminated samples, three contained maize, two contained wheat, and the other four indicated only the presence of barley and/or barley malt. The concentrations found in samples containing maize were 1.22, and 0.54, μg/L. In samples containing wheat, the contamination values were similar, 1.81 μg/<sup>L</sup> and 0.50 μg/L. In samples that indicated the presence of barley alone, the values were higher, at 1.81, 1.74, 9.21, and 11.25 μg/L, with the latter two being homemade beer samples. Samples with just barley in their composition represented 44.4% of the contaminated samples (Table 3). A similar higher percentage was found in samples with mixed cereals (55.5%). The di fference in the OTA levels was 6.00 μg/<sup>L</sup> (median 5.51 μg/L) versus 0.86 μg/<sup>L</sup> (median 0.54 μg/L), which was due to the high levels found in the two homemade samples that were analyzed. Regarding these homemade samples, a limited number was possible to achieve. Although these are preliminary results, we thought it was interesting to include them in the study.

Although there is evidence that organic foods often contain high concentrations of natural toxins produced by fungi [10], whereas conventional foods tend to contain more synthetic compounds such as pesticide residues, none of the contaminated samples were of organic origin. In addition, the use of fungicides or preservatives in insu fficient amounts can lead to a more serious situation due to stress caused in fungi, which leads to a stimulation of mycotoxin production [32].

Regarding the type of production, homemade beers presented the highest contamination levels at 10.23 ± 1.44 μg/L. Industrial and craft beers showed higher frequencies at 44.4% and 33.3%, but lower mean levels which were measured at 1.25 ± 0.74 μg/<sup>L</sup> (median 1.48 μg/L) and 0.95 ± 0.66 μg/<sup>L</sup> (median 0.54 μg/L), respectively.


**Table 3.** Frequency (%), OTA mean, and median contamination levels (μg/L) in di fferent categories of contaminated beer.

Two homemade samples showed really high levels, with 9.21 μg/<sup>L</sup> and 11.25 μg/L. Given that the maximum level of OTA is not currently established for beer but taking into account the limit established for wine (2 μg/L) (EC No. 1881/2006), these two samples exceeded at least 4.5 times and 5.5 times, respectively, this limit. Cereals used in homemade brewing are sold in plastic packaging presenting a greater risk of inadequate storage due to the moisture content, which favors their contamination. Another reason is that these types of beers are not subject to rigorous quality control and the cereals used are not as strictly controlled as those used in craft/industrial brewed beers.

Mycotoxins are highly stable and able to resist high temperatures and pH levels. The procedures involved in beer production use maximum operation temperatures lower than those able to destroy mycotoxins. However, they may impact mycotoxin levels given the physical, chemical, and biochemical alterations that take place [33]. The presence of OTA has been associated with barley malt contamination with ochratoxigenic species, namely *Penicillium verrucosum*. The OTA produced in the grains passes on to the wort and, although fermentation decreases its levels, the toxin is not completely removed [34]. Steeping, kilning, mashing, fermentation, and clarification are the most important steps of beer production presenting a negative impact on mycotoxins' levels. During these phases, mycotoxins may be removed through the drainage water, with the spent grains or with the fermentation residue. Moreover, they can also be diluted or destroyed as a result of the thermic treatment [33].

In the present study, every contaminated sample was pale beer. Drying (kilning) conditions are unfavorable for fungi (especially the first phase with a temperature of 50 ◦C and grain moisture of 45%). Knowing that the intensity of kilning and roasting (if applied) is crucial in malt flavor and color formation [33], fungi are removed during this process but thermostable mycotoxins produced before kilning persist [35]. Nowadays, the largest percentage of malt in most beers is pale malt which is only mildly dried at moderate temperature and might explain our results. Lager beers presented a higher contamination frequency (55.5%) but lower mean levels 0.86 ± 0.65 μg/<sup>L</sup> (median 0.54 μg/L) when compared to ale beers, the latter of which presented a frequency of 44.4% and 6.00 ± 4.95 μg/<sup>L</sup> (median 3 μg/L) mean contamination levels. On the contrary to Mateo et al. [34], a longer fermentation process and, consequently, a higher alcohol content, did not reduced the OTA level.

The studied samples were purchased from di fferent retail outlets located in Coimbra (Portugal) but originated from 10 di fferent countries. It was observed that from the nine contaminated samples, six (66.7%) were produced in Portugal, which provides evidence for the necessity of a greater control. Nonetheless, the mean levels found in the Portuguese samples which were 1.02 ± 0.70 μg/<sup>L</sup> (median 0.88 μg/L), were lower than in those from abroad.

The levels found in the present study are similar to those of other studies carried out in different countries. According to the scientific reported literature, the mean OTA content varies from 0.02 μg/<sup>L</sup> to 1.47 μg/L, for the samples analyzed in Turkey [23] and Germany [26], respectively. The minimum and maximum values vary between 0.012–0.045 μg/<sup>L</sup> and 1.5–2,34 μg/<sup>L</sup> in samples from Turkey [23] and South Africa [18], respectively. More recently, in 2017 Peters et al. reported the presence of mycotoxins, including OTA, in more than 1000 beers collected from 47 countries. OTA was found in five samples from Norway and England, ranging from 0.3 to 0.6 μg/<sup>L</sup> [36]. Bertuzzi et al. found OTA in the most sold beers in Italy with a mean concentration of 0.007 μg/L, with a maximum value of 0.07 μg/<sup>L</sup> [37].

With regard to the incidence of contamination, a frequency of 100% was found in samples collected in countries such as Spain [15], Hungary [2], Italy [27], Iran [22], Germany [26], and the Czech Republic [19]. The lowest OTA frequency was found in countries such as Brazil (0–5.3%) [14], China (0%) [24], Korea [16] and Turkey (14%) [23]. Recently, OTA was found in 45.8% of the most sold beers in Italy [37].

#### *2.3. Human Estimated Daily Intake and Risk Assessment*

The consumption of wine and beer make up part of the European culture. While Southern Europe is usually associated with wine consumption, Northern Europe is associated with beer. Nonetheless, since the 1960s wine consumption in Portugal has declined and beer is now the most consumed alcoholic beverage [38]. The number of brewing companies and the number of microbreweries increased, reflecting growth in the craft beer and specialty beer segmen<sup>t</sup> [39].

Three different scenarios were used to perform three EDI evaluations: the OTA contamination levels of the total analyzed samples (I); the mean OTA content regarding the nine positive samples; and the worst case scenario using the highest OTA level. In the first evaluation the EDI was 0.67 ng/kg b.w./day, in the second a value of 6.35 ng/kg b.w./day was obtained, and for the worst case scenario the value was 22.74 ng/kg b.w./day. When considering the estimated weekly intake (EWI) the obtained results for the three different scenarios were 4.71, 44.48, and 159.16 ng/kg b.w./week, respectively (Table 4).


**Table 4.** Estimated daily intake and risk assessment.

*a* n = 85 samples. *b* n = 9 samples. *c* The most contaminated sample was considered. *d* TWI of 120 ng/kg b.w./week was considered [30].

For risk assessment, the most recent tolerable weekly intake (TWI) value established by EFSA [30] was used (120 ng/kg b.w./week). In the first evaluation scenario, the percentage of estimated weekly intake (EWI) versus TWI was 3.92%. In the second, it was 37.06%, and in the third scenario it was 132.63%. In the first two situations, the ingestion of OTA through the consumption of beer presents no risk to the respective consumers. The inverse situation was observed for the worst case scenario.

This risk evaluation has its limitations since it is based on consumption and occurrence data which contained a high number of negative samples in this study. Nonetheless, it is a contribution to assess the human risk posed by the consumption of beer.

In food monitoring studies, the driving force is often enforcement of legal limits [40]. Current legislation does not include limits for the occurrence of OTA in beer, but the identified concentrations, especially in homemade beers, should be considered.
