Multi-Mycotoxin Contamination in Serbian Maize During 2021-2023: Climatic Influences and Implications for Food and Feed Safety

Abstract

Mycotoxin contamination in maize poses significant food and feed safety risks, particularly in regions with variable climatic conditions like Serbia. This study investigated the occurrence of regulated mycotoxins in maize harvested across the Republic of Serbia from 2021 to 2023, emphasizing the impact of climatic factors. A total of 548 samples of unprocessed maize grains were analysed for the presence of key mycotoxins, including aflatoxins, ochratoxin A, zearalenone, deoxynivalenol, fumonisins, and trichothecenes type A (T-2 and HT-2 toxins), using validated analytical methods. The results revealed high contamination frequencies, with aflatoxins and fumonisins being the most prevalent. The results revealed substantial temporal variability and frequent co-contamination of mycotoxins. Aflatoxin B₁ (AFB₁) was the most concerning contaminant, with 73.2% of the samples in 2022 exceeding the European regulatory limit for human consumption (5 µg/kg) for un processed maize grains, reaching peak concentrations of 527 µg/kg, which is 105.4 times higher than the allowed limit. For animal feed, the limit of 20 µg/kg was exceeded in 40.5% of the samples, with the highest concentration being 26.4 times greater than the maximum allowable level. In 2021, the non-compliance rates for AFB₁ in food and feed were 8.3% and 2.3%, respectively, while in 2023, they were 23.2% and 12.2%, respectively. Fumonisins contamination was also high, particularly in 2021, with fumonisin B₁ (FB₁) detected in 87.1% of samples and average concentrations reaching 4532 µg/kg. Although levels decreased in 2023 (70.7% occurrence, average 885 µg/kg), contamination remained significant. Deoxynivalenol (DON) contamination was consistently high (>70% of samples), with peak concentrations of 606 µg/kg recorded in 2021. Zearalenone (ZEN) and ochratoxin A (OTA) occurred less frequently, but ZEN levels peaked in 2022 at 357.6 µg/kg, which is above the regulatory limit of 350 µg/kg for food. Trichothecenes (HT-2 and T-2 toxins) were detected sporadically, with concentrations well below critical thresholds. Co-occurrence of mycotoxins was frequent, with significant mixtures detected, particularly between aflatoxins and fumonisins, as well as other fusarial toxins. The analysis demonstrated that temperature, humidity, and rainfall during both the growing and harvest seasons strongly influenced mycotoxin levels, with the most severe contamination occurring under specific climatic conditions. Notably, the highest mycotoxin levels, like aflatoxins, were linked to warmer temperatures and lower rainfall. The high non-compliance rates for aflatoxins and fumonisins and co-contamination pose significant food and feed safety risks. From a public health perspective, chronic exposure to contaminated maize increases the likelihood of carcinogenesis and reproductive disorders. Reduced productivity and bioaccumulation in animal tissues/products represent serious economic and safety concerns for livestock. This study provides insights into the potential risks to food and feed safety and the need for enhanced regulatory frameworks, continuous monitoring, and mitigation strategies in Serbia as well as other geographical regions.

Keywords: Serbia; aflatoxins; climate; co-contamination; food safety; fumonisins; maize; mycotoxins.

Figure 1

Clustered columns indicating the proportion of samples with co-contamination of (A) aflatoxins, (B) Fusarium mycotoxins, and (C) regulated mycotoxins in Serbian maize samples harvested during 2021–2023 (The values are available in Supplementary Table S1).

Figure 2

Heatmap indicating the co-occurrence (%) of regulated mycotoxins (and mycotoxin groups) contaminating Serbian maize samples harvested during 2021–2023. Darker blue areas indicate high-risk combinations that warrant particular attention for food and feed safety assessments, while lighter shades suggest less frequent pairings.

Figure 3

T Heatmap illustration of the Spearman’s correlation coefficients (Rho) among levels of regulated mycotoxins contaminating Serbian corn samples harvested during 2021–2023 and the climatic conditions (humidity, rainfall, temperature, and altitude) of their respective localities of the maize origin. The color gradient from deep blue to deep red represents the strength and direction of correlations, with blue shades indicating positive correlations (Rho values from 0 to +1.0), red shades indicating negative correlations (Rho values from −1.0 to 0), and white areas indicating no correlation (Rho values close to 0). The intensity of the color reflects the magnitude of the correlation, where darker colors indicate stronger relationships. Correlation coefficients marked with an asterisk (*) are statistically significant (p-value < 0.01), emphasising robust associations that are unlikely to have occurred by chance. All the p-values are available in supplementary data Table S2).

Figure 4

Three-dimensional mesh graphs representing the relationship between the concentrations of aflatoxins, relative humidity (%), and temperature (°C) during the growing season (June to September) in Serbian corn samples harvested between 2021 and 2023. The panels illustrate the effect of climatic factors on aflatoxin concentrations, including (A) aflatoxin B₁ (A, AFB₁), (B) aflatoxin B₂ (AFB₂), (C) aflatoxin G₁ (AFG₁), and (D) ochratoxin A (OTA), as well as (E) total aflatoxins (sum of AFB₁, AFB₂, AFG₁, and AFG₂). Higher aflatoxin concentrations are observed under elevated temperature and relative humidity, with prominent peaks highlighting the climatic conditions most conducive to contamination. The color reflects the level of contamination.

Figure 5

Three-dimensional mesh graphs representing the relationship among the concentrations of various mycotoxins, monthly relative humidity (%), and temperature (°C) during the growing season (June to September) in Serbian corn samples harvested between 2021 and 2023. The graphs illustrate the influence of climatic factors on mycotoxin contamination levels in maize, with warmer and humid conditions often associated with higher concentrations. The panels represent (A) fumonisins (FB₁ + FB₂), (B) fumonisin B₁ (FB₁), (C) fumonisin B₂, (D) deoxynivalenol (DON), (E) zearalenone (ZEN), (F) T-2 toxin, (G) HT-2 toxin, (H) Fusarium mycotoxins (Sum of DON, ZEA, FB₁, and FB₂), and (I) total mycotoxins (Sum of all detected mycotoxins). The color reflects the level of contamination.

Figure 6

Three-dimensional mesh graph representing the relationship between co-contamination (number of mycotoxins per sample), relative humidity (%), and temperature (°C) during the growing season (June to September) in Serbian corn samples harvested between 2021 and 2023. The graph illustrates how variations in climatic factors influence the degree of mycotoxin co-contamination, with higher levels of co-contamination observed under increased humidity and elevated temperatures. Peaks in the graph indicate the conditions most favourable for multi-mycotoxin contamination detected in the analysed samples. The color reflects the co-contamination level.

Figure 7

Map of the Republic of Serbia illustrates the locations of the analysed maize samples.