In this study, the occurrence of
Mycotoxins are products of toxicogenic moulds that are frequently encountered as food or feed contaminants. A prominent group of mycotoxins are
Research has shown that consumption of food or animal feeding on feeds and feed mixtures contaminated with
It has been well established that factors of relevance for the nascence of
Literature data have provided evidence substantiating the need for systematic monitoring of, and control over, cereals and cereal-based products, and/or the use of decontamination techniques, so as to prevent an unfavourable impact on human and animal health as well as to cut down economic losses suffered by the food and livestock industries (
Given that data on natural occurrence of
In the 2013-2015 timeframe, a total of 257 samples of unprocessed cereals, of which 115 maize samples, 84 wheat samples, and 58 barley samples, had been harvested from fields across Bosnia and Herzegovina. For the sole purpose of this study, cereals were sampled randomly during or immediately after harvesting (in a matter of days) from agricultural fields located in different areas seated in the northern, central, eastern and western part of the county. For the purpose of this study, cereals were divided in the laboratory into several groups based on the cultivation/harvesting year (2013, 2014 and 2015), and continuously analysed during the whole sampling period.
Sampling and sample preparation was conducted in accordance with the requirements of ISO 6497:2002 and ISO 6498:1998. Prior to the determination of mycotoxin concentration that made use of the ELISA method, cereals were ground to a fine powder using an analytical mill equipped with a 1.0 mm-diameter sieve (Cylotec 1093 Tecator, Sweden), and then stored at 4 °C pending analysis.
Determination of DON, ZEA, and FUM was performed using the competitive ELISA test kits as instructed by the kit manufacturer (R-Biopharm, Darmstadt, Germany). Each kit contains a micro-titre plate with 96 wells coated with antibodies, standard solutions containing different concentrations of mycotoxins, an enzyme conjugate, an anti-antibody, a substrate, a chromogen solution (urea peroxide/tetramethylbenzidine), a stop solution, and washing and dilution buffers. Standards employed with the validation of analytical methods were provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany). All other chemicals used for analyses were of an analytical grade.
ELISA tests were performed using a ChemWell auto-analyzer (Awareness Technology Inc. 2910, USA), the absorbance thereby being measured at 450 nm. In order to determine mycotoxin concentrations in the sampled material, a standard curve was plotted for each mycotoxin analysed. When determining final mycotoxin concentrations in a given sample, the dilution factor and the mean recovery rate determined for each mycotoxin were taken into account.
Statistical analysis was performed using Statistica Ver. 10.0 Software (StatSoft Inc. 1984-2011, USA), with a statistical significance set at 95% (p= 0.05). The Shapiro Wilks test was conducted so as to determine whether the results of the analysed parameters follow the normal distribution pattern (p > 0.05). For determining the differences in concentrations of the studied mycotoxins found in various cereals during various sampling years, parametric tests like the t-test, and one-way and two-way ANOVA were used, with the statistical significance being set at p < 0.05.
The limit of detection (LOD) was calculated from the average of ten toxin-negative cereal mixture samples (containing maize, wheat and barley in equal proportions; earlier analysed for the presence of
Numerous studies have confirmed a wide representation of mycotoxins in various cereals, strongly dependent on weather conditions seen during the cultivation period and the method of storage (
In this study, the levels of
The analysis of mycotoxins was conducted using the validated quantitative ELISA method. Validation results are shown in
DON | 23.2 | 30.1 | 1478 | 50 | 92.8 | 5.2 |
100 | 94.6 | 6.1 | ||||
200 | 99.8 | 7.9 | ||||
ZEA | 3.5 | 4.2 | 367 | 50 | 87.6 | 4.3 |
100 | 94.3 | 5.8 | ||||
200 | 95.4 | 8.6 | ||||
FUM | 26.8 | 32.7 | - | 50 | 75.6 | 6.1 |
100 | 77.3 | 8.2 | ||||
200 | 80.5 | 9.4 | ||||
LOD – limit of detection; LOQ – limit of quantification |
The limit of detection (LOD) and the limit of quantification (LOQ) were the lowest for ZEA and the highest for FUM. According to the CRM-assigned values of these mycotoxins, DON and ZEA concentrations obtained with the determination of trueness were acceptable. Validation of the employed methodology resulted in the mean recovery rates of 95.7, 92.4 and 77.8% for DON, ZEA and FUM, respectively, as well as with the coefficients of variation (CV) ranging from 4.3 to 9.4%. Based on the obtained validation results and the validation criterion given under the Commission Decision 2002/657/EC, the applied quantitative ELISA method can be considered suitable for the determination of the investigated mycotoxins in cereals, as already concluded in many earlier studies (Krska et al., 2008;
The determined number (No) and the percentage of positive samples, the average (mean), the minimum (min) and the maximum (max) concentrations, together with the accompanying standard deviations (SDs), displayed for each investigated mycotoxin and each type of the studied cereal across the entire sampling period (2013-2015) are shown in
DON | Maize | 98/115 | 85 | 984 | 957 | 44 | 8,529 |
Wheat | 54/84 | 64 | 690 | 604 | 38 | 2,123 | |
Barley | 22/58 | 38 | 365 | 177 | 32 | 578 | |
ZEA | Maize | 84/115 | 73 | 326 | 314 | 10 | 2,113 |
Wheat | 49/84 | 58 | 127 | 140 | 8 | 189 | |
Barley | 20/58 | 34 | 92 | 80 | 11 | 84 | |
FUM | Maize | 77/115 | 67 | 1,259 | 1,161 | 42 | 3,275 |
Wheat | 42/84 | 50 | 414 | 316 | 35 | 328 | |
Barley | 15/58 | 26 | 145 | 105 | 38 | 232 | |
a Samples in which mycotoxin was detected (> LOD); b mean value of positives (> LOD) |
The MPLs for foodstuffs and the GVs of
DON | maize | 1,750 | 7/6.1 | 8,000 | 2/1.7 |
wheat | 1,750 | 4/4.8 | 8,000 | 0/0 | |
barley | 1,250 | 0/0 | 8,000 | 0/0 | |
ZEA | maize | 350 | 4/3.5 | 2,000 | 1/0.9 |
wheat | 100 | 5/5.9 | 2,000 | 0/0 | |
barley | 100 | 0/0 | 2,000 | 0/0 | |
FUM | maize | 4,000 | 0/0 | 60,000 | 0/0 |
aThe number/percentage of samples in which mycotoxin concentration higher than the MPL defined for foodstuffs (Commission Regulation 1881/2006) was determined; bThe number/percentage of samples in which mycotoxin concentration higher than the GVs for feedstuffs ( |
In maize, DON was detected in 85%, ZEA in 73% and FUM in 67% of samples. Their average concentrations (±SD) in maize were 984±957 μg/kg, 326±314 μg/kg, and 1,259±1,161 μg/kg, respectively. Given the MPLs of these mycotoxins in unprocessed maize (Commission Regulation 1881/2006), which equals to 1,750 μg/kg for DON, 350 μg/kg for ZEA and 4,000 μg/kg for FUM, DON levels higher than MPL were observed in 7 analyzed maize samples and those higher than ZEA MPL in 4 maize samples, while FUM concentrations higher than MPL were not determined.
Statistical analysis revealed significant (p < 0.05) mutual differences in concentrations of
In wheat, the highest mean concentration was observed for DON (690±604 μg/kg), and the lowest for ZEA (127±140 μg/kg); in barley, the highest contamination was also linked to DON (365±177 μg/kg), and the lowest to ZEA (92±80 μg/kg). Given the MPLs of these mycotoxins in unprocessed wheat and barley (Commission Regulation 1881/2006), which equals to 1,750 μg/kg and 1,250 μg/kg for DON, respectively, and 100 μg/kg for ZEA, DON levels higher than MPL were observed in 4 samples, while ZEA levels surpassing the MPL were established in 5 wheat samples. Concentrations higher than MPLs defined for these mycotoxins in barley were not determined.
In some of the samples, mycotoxin levels higher than permitted for foodstuffs and/or higher than guidance value for feedstuffs were observed, of note, all samples in which mycotoxin levels were significantly increased (non-compliant samples) were sampled in the year 2014. Increased DON and ZEA levels were found in maize and wheat. Among 257 samples taken during a three-year period, 20 samples (7.8%) were not eligible for use as raw materials intended for food production, among which three samples (1.2%) were also non-compliant as feedstuffs. Other samples non-compliant for use as foodstuffs (17 additional samples) were acceptable for use as feedstuffs.
In many European countries, data have also shown substantial variations in
In Croatia, the study by
In the study by
In this study, annual variations of DON, ZEA, and FUM concentrations during the 2013-2015 timeframe, given
Concentration of deoxynivalenol (DON) established in various types of cereals in various harvesting years
Concentration of zearalenone (ZEA) established in various types of cereals in various harvesting years
Concentration of fumonisins (FUM) established in various types of cereals in various harvesting years
The highest mean concentrations of mycotoxins were determined in all three cereals in the year 2014. The results of ANOVA revealed statistically significant differences (p <0.05) in DON, ZEA, and FUM levels between various types of cereals (i.e. maize in comparison to wheat and barley) and various cultivation/harvesting years (i.e. 2014 in comparison to 2013 and 2015). The maximum mean concentrations of mycotoxins were observed in maize sampled in 2014, with mean values of DON of 1,611±1,825 μg/kg, that of ZEA of 655±715 μg/kg, and that of FUM of 2,025±1,456 μg/kg. All samples, in which mycotoxin concentrations surpassed those stipulated for food and feed under the applicable legislation, were observed in cereals sampled during 2014.
Given the fact that high mycotoxin levels are usually associated with climate conditions, in particular humidity and temperature as the factors most critical for mould formation, and thus also mycotoxin production, in the studies of
The observed average mycotoxin concentrations revealed maize to be the most contaminated among cereals, with DON as generally the most common