# Fate of Mycotoxins in Local-Race Populations of Maize Collected in the Southwest of France, from the Field to the Flour and Meal in Organic Farms

Jean-Michel Savoie, Laetitia Pinson-Gadais, Rodolphe Vidal, Camille Vindras-Fouillet

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Jean-Michel Savoie, Laetitia Pinson-Gadais, Rodolphe Vidal, Camille Vindras-Fouillet. Fate of Mycotoxins in Local-Race Populations of Maize Collected in the Southwest of France, from the Field to the Flour and Meal in Organic Farms. Agriculture, 2025, 15, 10.3390/agriculture15101064. hal-05072897

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Article

# Fate of Mycotoxins in Local-Race Populations of Maize Collected in the Southwest of France, from the Field to the Flour and Meal in Organic Farms

Jean-Michel Savoie<fnref n="1" />*, Laetitia Pinson-Gadais<fnref n="1" />, Rodolphe Vidal<fnref n="2" /> and Camille Vindras-Fouillet<fnref n="2" />

**Abstract:** Both organic and conventional farmers are confronted with the issue of mycotoxin contamination of maize, but organic farming is considered by the public to present a higher risk. There are also concerns about the sanitary quality of maize processed as a foodstuff and marketed on farms through short distribution channels, and there is a need for data on mycotoxin contamination in such a farming system. With the objective to assess the diversity of contamination levels at harvest and to track the post-harvest fate of mycotoxins, maize grain samples were collected at organic farms from southwest France after harvest, storage and milling. There was a wide range of levels of contamination by trichothecenes A and B, zearalenone, and fumonisins. The presence of ochratoxin A and aflatoxins was scarce. In some farms, but not all, the technique of drying and initial storage in cribs resulted in increased levels of contamination by *Fusarium* toxins, but not aflatoxins. The transfer of mycotoxins in milling products was higher for flour than for meal. Data are discussed in terms of mycotoxin co-occurrence, correlations between concentrations, and compliance with European Union regulations.

**Keywords:** *Fusarium*; corn; contaminant transfer

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Academic Editor: Zoltán Györi

Received: 15 April 2025

Revised: 7 May 2025

Accepted: 12 May 2025

Published: 15 May 2025

**Citation:** Savoie, J.-M.; Pinson-Gadais, L.; Vidal, R.; Vindras-Fouillet, C. Fate of Mycotoxins in Local-Race Populations of Maize Collected in the Southwest of France, from the Field to the Flour and Meal in Organic Farms. *Agriculture* **2025**, *15*, 1064. https://doi.org/10.3390/agriculture15101064

**Copyright:** © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Maize (*Zea mays* L.) is an important food security and income-generating crop in various areas of the world. Improvements in yields had been obtained by selection of hybrids, mechanisation, irrigation, use of mineral fertilisers and phytosanitary products, leading to an increase in the world production associated with global trades. Besides this agroeconomic system, farmers produce maize as a subsistence crop using local resources. Even in Europe, groups of organic farmers wishing to offer quality products, preserve cultivated biodiversity and the environment, and re-appropriate the age-old practice that is the self-production of seeds preserved and develop landrace populations. Landrace populations consist of a mixture of genotypes, all of which are reasonably adapted to the region in which they evolved, but which differ in detail, such as reaction to water stress and soil fertility or to pests and diseases [1]. Among the most important pathogens, *Fusarium* spp. pose a threat to worldwide maize production due to their ability to cause ear and kernel rots named Fusarium ear rot (FER) and to synthesise mycotoxins in non-rotted infected kernels that can be accumulated above safety levels for humans and animals.

Many comparisons of *Fusarium* contamination and amounts of mycotoxins in organic versus conventional foods have been performed without leading to definite conclusions on

<footnote n="1">¹ UR 1264 Mycology and Food Safety (MycSA), INRAE (National Research Institute for Agriculture Food and Environment), F-33882 Villenave d'Ornon, France; laetitia.pinson-gadais@inrae.fr</footnote>

<footnote n="2">² ITAB (Institut Technique de l'Agriculture Biologique), 149 Rue de Bercy, F-75595 Paris, CEDEX 12, France; rodolphe.vidal@itab.asso.fr (R.V.); camille.vindras@gmail.com (C.V.-F.)</footnote>

<footnote>* Correspondence: jean-michel.savoie@inrae.fr</footnote>
the relative contamination risks. In Brazil, working with 50 organic and 50 conventional corn cobs samples, Peres et al. (2018) [2] found significantly higher concentrations of fumonisins in conventional maize than in organic maize. In Romania, only low levels of fumonisins, aflatoxins, and DON, were measured in organic maize, and many samples contained none of these mycotoxins [3]. In Spain, the occurrence and concentration of fumonisins and DON did not differ between conventional and organic maize [4,5]. Finally, most studies published in Europe in the 2000s show insignificant differences between organic and conventional production chains, or differences that are difficult to interpret [6] because of the significant influence of uncontrollable factors such as the climate which is the prime factor of regulation of mycotoxin-producing fungi and mycotoxin contamination, and of the diversity of practices inside each system including the use of different varieties. However, it is interesting to monitor the situation in both production systems independently [7].

In contrast to the extensive research comparing organic and conventional farming, information on the fate of mycotoxins in fully integrated on-farm food chains of small-scale organic systems is scarce. Despite farmers involved in this value-added production in developed countries being motivated to contribute to new sustainable farming and food quality, they lack sufficient food safety awareness and have limited knowledge of applicable food safety regulations [8,9]. In the conventional agriculture system in Europe, stakeholders address the issue of mycotoxins by sampling, analysing and discarding contaminated batches throughout the food chain. In alternative systems, primarily organic, where farmers use self-produced seeds, store their crop at the farms before processing it themselves and sell at local markets, the opportunities for detecting contaminated kernels are non-existent. Even when the farmers are aware of the mycotoxin issues, the cost of the analysis serves as a deterrent. Consequently, there is a lack of public data on the contamination levels in such an agricultural chain. Studying small-scale organic systems is required to fill this gap. The collected information would be invaluable to raise farmers' awareness of the risks of mycotoxin contamination and to contribute to designing the technical solutions available to mitigate them throughout the value chain. This is all the more important as European legislation on mycotoxin limits in maize and maize products is evolving to include new mycotoxins and modify threshold values (Table 1).

<table><caption>**Table 1.** Evolution in the European Commission regulations on the maximal levels (µg kg⁻¹) of mycotoxins in maize and some maize products.</caption><thead><tr><th rowspan="2">Maize Products</th><th rowspan="2">Mycotoxins</th><th colspan="5">Commission Regulation (EU) Number</th></tr><tr><th>2006/1881</th><th>2007/1126</th><th>2023/915</th><th>2024/1022</th><th>2024/1038</th></tr></thead><tbody><tr><td rowspan="4">Unprocessed grains</td><td>DON</td><td>1750</td><td>1750</td><td>1750</td><td>1500</td><td></td></tr><tr><td>ZEA</td><td>200</td><td>350</td><td>350</td><td></td><td></td></tr><tr><td>FB1 + FB2</td><td>2000</td><td>4000</td><td>4000</td><td></td><td></td></tr><tr><td>T-2 + HT-2</td><td></td><td></td><td></td><td></td><td>100</td></tr><tr><td rowspan="4">Grains intended for direct human consumption</td><td>DON</td><td>750</td><td></td><td></td><td>750</td><td></td></tr><tr><td>ZEA</td><td>200</td><td></td><td>100</td><td></td><td></td></tr><tr><td>FB1 + FB2</td><td>1000</td><td></td><td>100</td><td></td><td></td></tr><tr><td>T-2 + HT-2</td><td></td><td></td><td></td><td></td><td>50</td></tr><tr><td rowspan="4">Milling fractions of maize with at least 90% of the particles in the milling product have a size ≤500 µm; flour not placed on the market for the final consumer</td><td>DON</td><td></td><td>750</td><td>750</td><td>750</td><td></td></tr><tr><td>ZEA</td><td></td><td>300</td><td>300</td><td></td><td></td></tr><tr><td>FB1 + FB2</td><td></td><td>2000</td><td>2000</td><td></td><td></td></tr><tr><td>T-2 + HT-2</td><td></td><td></td><td></td><td></td><td>20</td></tr><tr><td rowspan="4">Milling fractions of maize with less than 90% of the particles in the milling product have a size ≤500 µm; meal not placed on the market for the final consumer</td><td>DON</td><td></td><td>1250</td><td>1250</td><td>750</td><td></td></tr><tr><td>ZEA</td><td></td><td>200</td><td>200</td><td></td><td></td></tr><tr><td>FB1 + FB2</td><td></td><td>1400</td><td>1400</td><td></td><td></td></tr><tr><td>T-2 + HT-2</td><td></td><td></td><td></td><td></td><td>20</td></tr></tbody></table>
In the present work, samples were collected at farms after harvest, after storage for 7–9 months and after milling. All farmers cultivated landrace populations of maize, storing the crop at the farms and milling either at the farm or in a local mill. They were located in the southwest of France, a region characterised by a temperate oceanic climate favourable to the colonisation of ears and kernels by *Fusarium* species. The objective was to assess the diversity of contamination levels at harvest and to track the post-harvest fate of mycotoxins.

**2. Materials and Methods**

2.1. *Sample Collection*

Samples were obtained from 13 smallholders’ organic farms in the Aquitaine Region, France, in the 2020 or 2021 growing seasons (Figure 1). All were cultivated landrace populations of maize in small-scale farming for short food circuits. Maize on the cobs was collected all along the harvest of the central part of a field as 5 to 7 incremental samples of 3 to 4 kg. The aggregated sample obtained for a field was shelled to obtain a lot of 5 kg of grains.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Sampling location in the Aquitaine region (France) during the 2020 or 2021 growing seasons.</figcaption></figure>

On farm storage was either as cobs of maize in cribs (outdoor structures made of narrow wire mesh with a roof) or as grains in galvanised silos or big bags. Samples were collected in the same farms after 7 to 9 months of storage in small-scale facilities. Five incremental samples from multiple points across the cobs or grain lot were aggregated in a representative composite sample, as performed at the harvest, to obtain a lot of 5 kg of grains.

The meal and flour obtained by the farmers using dry milling processes with their own batch of grains, studied from the field, were collected in 8 out of 13 farms. For C-64-BE-1, only flour was collected. Three incremental samples of meal (particle size 300–600 µm) and of flour (particle size <212 µm) [10] were collected from different points of the batch. They were combined in an aggregate sample of meal and an aggregate sample of flour (1 kg of each).

For each origin, 1 kg of grain at harvest, 1 kg of grain after storage, 0.5 kg of meal and 0.5 kg of flour were transferred to Capinov (Landerneau, France), a private agricul-
tural analysis laboratory, for multi-mycotoxins analyses. Replicates of these samples of
1 or 0.5 kg were stored in our laboratory in a sample collection at 4 °C before *Fusarium* species detection.

## 2.2. *Mycotoxin Analyses*

The provider of the mycotoxin analysis service, Capinov, has the necessary accreditations to certify the data supplied, in accordance with COFRAC standards (COFRAC accreditation n° 1–6211) valid in Europe and worldwide. The analysed mycotoxins and the limits of quantification (LoQ) are in Table 2. The concentrations were measured by high-pressure liquid chromatography coupled to a tandem mass spectrometer for the mycotoxins produced by *Fusarium* species, or to a fluorescence detector for Aflatoxins and Ochratoxin A (internal method Capinov PR_SAT0044, 2020). The results were delivered with an uncertainty value expressing the statistical dispersion of the values attributed to a measured quantity. Two values were significantly different when there was no overlapping of the intervals of concentrations plus and minus uncertainties.

<table><caption>**Table 2.** Mycotoxins measured in maize samples.</caption><thead><tr><th>Mycotoxins</th><th>Method</th><th>LoQ <sup>(3)</sup> (µg kg<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Deoxynivalenol (DON)</td><td>HPLC/MS/MS <sup>(1)</sup></td><td>50</td></tr><tr><td>Nivalenol (Niv)</td><td>HPLC/MS/MS</td><td>50</td></tr><tr><td>3-acetyl Deoxynivalenol (3-ADON)</td><td>HPLC/MS/MS</td><td>100</td></tr><tr><td>15-acetyl Deoxynivalenol (15-ADON)</td><td>HPLC/MS/MS</td><td>100</td></tr><tr><td>Fusarenone-X (Fx)</td><td>HPLC/MS/MS</td><td>50</td></tr><tr><td>DAS</td><td>HPLC/MS/MS</td><td>10</td></tr><tr><td>T2 toxin (T-2)</td><td>HPLC/MS/MS</td><td>5</td></tr><tr><td>HT2 toxin (HT-2)</td><td>HPLC/MS/MS</td><td>5</td></tr><tr><td>Zearalenone (ZEA)</td><td>HPLC/MS/MS</td><td>10</td></tr><tr><td>Alpha-Zearalenone</td><td>HPLC/MS/MS</td><td>50</td></tr><tr><td>Beta-Zearalenone</td><td>HPLC/MS/MS</td><td>50</td></tr><tr><td>Zearalanone</td><td>HPLC/MS/MS</td><td>50</td></tr><tr><td>Fumonisin B1 (FB1)</td><td>HPLC/MS/MS</td><td>20</td></tr><tr><td>Fumonisin B2 (FB2)</td><td>HPLC/MS/MS</td><td>20</td></tr><tr><td>Fumonisin B3 (FB3)</td><td>HPLC/MS/MS</td><td>20</td></tr><tr><td>Aflatoxin B1 (Afla-B1)</td><td>HPLC/FLD <sup>(2)</sup></td><td>0.1</td></tr><tr><td>Aflatoxin B2 (Afla-B2)</td><td>HPLC/FLD</td><td>0.1</td></tr><tr><td>Aflatoxin G1 (Afla-G1)</td><td>HPLC/FLD</td><td>0.1</td></tr><tr><td>Aflatoxin G2 (Afla-G2)</td><td>HPLC/FLD</td><td>0.1</td></tr><tr><td>Ochratoxin A (OTA)</td><td>HPLC/FLD</td><td>0.2</td></tr></tbody></table>

## 2.3. Detection of *Fusarium* Species

DNA extraction was performed on grains, flours and meals using the NucleoMag® Plant kit (Macherey–Nagel, Høerdt, France) on 50 mg of ground samples at particle size <0.5 mm. The process involved homogenisation using a Precellys® Evolution (Bertin Instruments, Montigny-le-Bretonneux, France) at 6500 rpm for two 30-s cycles (with a 5 s break). Samples were then mixed with 500 µL of lysis Buffer MC1 and 10 µL RNase A, followed by incubation at 56 °C for 30 min. After centrifugation at 16,000 × g for 20 min, 100 µL of clear lysate was transferred to a separation plate containing magnetic beads in Buffer MC2. Subsequent purification steps were automated using a MagMAX™ express system (Thermo Fisher Scientific, Illkirch-Graffenstaden, France). The purification process included: incubation with magnetic beads for 5 min, washing with Buffer MC3 for 5 min, washing with Buffer MC4 for 5 min, washing with 80% ethanol for 5 min, final wash with

<footnote>(1) Liquid chromatography coupled to tandem mass spectrometry. (2) Liquid chromatography, fluorometric detection, and quantification. (3) LoQ = limit of quantification.</footnote>
Buffer MC5 for 1 min, and elution in 80 µL of Buffer MC6 for 5 min. DNA quality and quantity were assessed using 1% agarose gel electrophoresis and UV spectrophotometry, respectively.

Quantitative PCR (qPCR) analyses were conducted to assess the abundance of *Fusarium* species. Each reaction mixture contained 20 ng of template DNA in a total volume of 10 µL, utilising PowerTrack™ SYBR Green Master Mix (Thermo Fisher, Illkirch-Graffenstaden, France) and species-specific primers (Table 3). Amplification and detection were performed using a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, Illkirch-Graffenstaden, France). The thermal cycling protocol consisted of an initial denaturation step at 95 °C for 20 s, followed by 40 cycles of denaturation at 95 °C for 3 s and annealing/extension at 60 °C for 30 s. External calibration curves were generated from genomic DNA extracted from axenic cultures of *Fusarium* species (Table 3). These reference samples were also employed to validate amplification specificity through melting curve analysis. The data on DNA abundance of a *Fusarium* species are presented as classes of Cq ranges: 33 to 36 = +, 30 to 32 = ++, 26 to 29 = +++. lower than 26 = ++++.

<table><caption>**Table 3.** Primer pairs for the specific amplification of *Fusarium* species.</caption><thead><tr><th>Primer Names</th><th>Primer Sequences</th><th>Ta 1</th><th>Fusarium Species</th><th>Reference</th></tr></thead><tbody><tr><td>Fum1-656F</td><td>CGGTTGTTCATCATCTCTGA</td><td>60 °C</td><td>F. verticillioides</td><td>[11]</td></tr><tr><td>Fum1-1158R</td><td>GCTCCCGATGTAGAGCTTCTT</td><td></td><td></td><td></td></tr><tr><td>Fp82F</td><td>CAA GCA AAC AGG CTC TTC ACC</td><td>60 °C</td><td>F. poae</td><td>[12]</td></tr><tr><td>Fp82R</td><td>TGT TCC ACC TCA GTG ACA GGT T</td><td></td><td></td><td></td></tr><tr><td>Fg16N-F</td><td>ACAGATGACAAGATTCAGGCACA</td><td>60 °C</td><td>F. graminearum</td><td>[12]</td></tr><tr><td>Fg16N-R</td><td>TTCTTTGACATCTGTTCAACCCA</td><td></td><td></td><td></td></tr><tr><td>FC01-F</td><td>ATGGTGAACTCGTCGTGGC</td><td>60 °C</td><td>F. culmorum</td><td>[12]</td></tr><tr><td>FC01-R</td><td>CCCTTCTTACGCCAATCTCG</td><td></td><td></td><td></td></tr><tr><td>FspoF1</td><td>CGCACAACGCAAACTCATC</td><td></td><td></td><td></td></tr><tr><td>LanspoR1</td><td>TACAAGAAGACGTGGCGATAT</td><td>60 °C</td><td>F. sporotrichioides</td><td>[13]</td></tr></tbody></table>

## 2.4. Data Analyses

Concentrations of mycotoxins in each sample are given as measured values and uncertainty intervals given by the analysis provider. Concentrations were considered different when these intervals did not overlap. Paired samples T-test analyses were made using the non-parametric Wilcoxon signed-rank test. The relationships between the different mycotoxins and the sampling times were analysed using Kendall's Tau rank correlation coefficient. The JASP (version 0.19.3; JASP Team (2024)) statistical software was used for data analysis.

# 3. Results

## 3.1. Occurrence of Fusarium Mycotoxins in Maize Grains

Maize grains produced for use as food after processing were collected in the southwest of France (Figure 1). In the samples from 13 organic farms collected at harvest in 2020 and 2021 studied here, only one sample was not contaminated by any of the researched mycotoxins (C-64-BE-02), and another one (C-64-AR-01) was only contaminated with fumonisins at low concentrations (FB1 + FB2 = 150 +/- 82 µg kg⁻¹). In the other samples, there was a wide range of levels of contamination by DON, 15-ADON, Niv, Fx, T2, HT2, ZEA, fumonisins B1, B2, B3. The mycotoxins 3-ADON, DAS and the derivatives of ZEA were never quantifiable. At the end of storage, all the grain samples contained more than one quantifiable mycotoxin.

DON was the most prevalent mycotoxin, with 10 out of 13 farm samples (77%) having quantifiable concentrations at harvest. Half of these samples also contained quantifiable
15-ADON, but no 3-ADON was detected (Table 4). After storage, samples from two new origins were contaminated by DON, but 15-ADON became non-quantifiable in two origins previously contaminated at low concentrations (Table S1). Fumonisins were in second place in terms of prevalence, with 69% of samples containing quantifiable FB1 at harvest and 85% after storage. Most also contained FB2, one-third contained FB3 (Table 4).

<table><caption>**Table 4.** Co-occurrence of mycotoxins. Number of samples in which one or two mycotoxins were quantifiable at harvest (bold) and after storage (italics).</caption><thead><tr><th></th><th>DON</th><th>15-ADON</th><th>Niv</th><th>Fx</th><th>ZEA</th><th>T-2</th><th>HT-2</th><th>FB1</th><th>FB2</th><th>FB3</th><th>Afla</th></tr></thead><tbody><tr><td>DON</td><td>10-12</td><td>3</td><td>9</td><td>1</td><td>9</td><td>7</td><td>7</td><td>10</td><td>8</td><td>8</td><td>1</td></tr><tr><td>15-ADON</td><td>5</td><td>5-3</td><td>3</td><td>0</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>0</td></tr><tr><td>Niv</td><td>6</td><td>3</td><td>7-10</td><td>0</td><td>9</td><td>6</td><td>6</td><td>8</td><td>7</td><td>7</td><td>1</td></tr><tr><td>Fx</td><td>3</td><td>2</td><td>3</td><td>3-1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td></tr><tr><td>Zea</td><td>7</td><td>4</td><td>4</td><td>3</td><td>7-10</td><td>7</td><td>7</td><td>8</td><td>7</td><td>7</td><td>1</td></tr><tr><td>T-2</td><td>3</td><td>1</td><td>4</td><td>2</td><td>3</td><td>4-7</td><td>7</td><td>7</td><td>7</td><td>7</td><td>1</td></tr><tr><td>HT-2</td><td>7</td><td>3</td><td>5</td><td>2</td><td>6</td><td>4</td><td>7-8</td><td>9</td><td>9</td><td>9</td><td>1</td></tr><tr><td>FB1</td><td>7</td><td>2</td><td>5</td><td>2</td><td>5</td><td>4</td><td>7</td><td>9-11</td><td>9</td><td>9</td><td>1</td></tr><tr><td>FB2</td><td>6</td><td>1</td><td>4</td><td>1</td><td>4</td><td>3</td><td>6</td><td>8</td><td>8-9</td><td></td><td>1</td></tr><tr><td>FB3</td><td>5</td><td>1</td><td>4</td><td>1</td><td>4</td><td>3</td><td>5</td><td>6</td><td>6</td><td>6-9</td><td>1</td></tr><tr><td>Afla</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1-1</td></tr></tbody></table>

Niv was present in 7 samples at harvest, and 3 of them also contained Fx (Table 4). Niv co-occurred with DON in 6 samples (60%). One sample (C-64-BU-1) contained Niv but not DON. After storage, a higher number of samples contained Niv but not Fx, and the rate of co-occurrence with DON increased (83%). ZEA was present in 7/13 samples at harvest, 10/13 after storage.

A total of 7 out of 13 samples contained HT-2, and half of them also contained T-2. HT-2 co-occurred with DON or Niv at harvest (Table 4). C-64-BU-1 was the only sample positive for HT-2 but neither for DON nor ZEA. After storage, 1 new sample was contaminated with HT-2 and 3 by T-2.

## 3.2. Levels of Grain Contamination by Fusarium Mycotoxins and Fusarium Species at Harvest

At harvest, all analysed samples complied with the EU regulation 2023/915 of 25 April 2023, modified by the regulation EU 2024/1022 defining a maximal level of DON concentration at 1500 µg kg⁻¹ for unprocessed maize grains (Figure 2). C-24-VA-02, the batch having the highest concentrations (1710 µg kg⁻¹), would have been considered as conform due to the uncertainty value (504 µg kg⁻¹) (Table S1). Except C-64-IB-01 at 1370 µg kg⁻¹, the concentrations of the other samples were lower than or equal to 520 µg kg⁻¹ (Figure 2), which is the median value reported in a large world survey of contamination [14]. Despite a high level of occurrence observed, the contamination levels were low. ZEA concentrations were lower than 100 µg kg⁻¹ (Figure 2a), except in one sample (C-24-VA-02, 553 +/- 194 µg kg⁻¹), which exceeded the maximum authorised level in Europe (Table 1). ZEA concentrations were positively correlated to DON concentrations (Figure 3). The concentrations of T-2 + HT-2, between 12 and 58 µg kg⁻¹ (Figure 2a), were significantly lower than 100 µg kg⁻¹, the maximum level authorised in the new EU regulation (Table 1). There was no correlation with ZEA and DON concentrations (Figure 3), these mycotoxins being produced by different *Fusarium* species.

The concentrations of FB1 + FB2 in all the samples (Figure 2) were significantly lower than the maximal level of the previous and present EC regulation (Table 1), with 1150 +/- 360 and 350 +/- 131 µg kg⁻¹ as the highest concentration for FB1 and FB2, respectively, in C-47-BR-01. FB1 was always in significantly higher concentrations than FB2, while the
concentrations in FB3 were always the lowest (Table S1). The concentrations of FB1, FB2 and FB3 were significantly correlated. Concentration in FB1 + FB2 did not correlate with the other mycotoxins.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Box Plot of the distribution of mycotoxin concentrations in maize grains. (a): at harvest, (b): after storage, (c): after storage with a scale limited to 2000 µg kg⁻¹. Red lines are the maximal levels in the European Commission regulation.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Correlations between mycotoxin concentrations measured in maize at harvest and after storage, Kendall's tau B heatmap. * *p* < 0.05, ** *p* < 0.01.</figcaption></figure>
15-ADON, Niv and Fx are not regulated in Europe. In the five samples containing 15-ADON in addition to DON, the ratios of concentrations DON/15-ADON were from 0.8 to 2, except for C-24-VA-02, which had a ratio close to 10 (Table S1). The range of Niv + Fx concentration was 170 to 750 µg kg⁻¹ (Figure 2a). Pairwise Niv concentrations were lower than DON concentrations, except for the batch having the highest Niv concentration (731 µg kg⁻¹) and a ratio DON/Niv of 0.36 (Table S1), but the correlation between both mycotoxins was not significant (Figure 3).

Overall, presence or absence of mycotoxins and correlations of concentrations might be the results of differences in colonisation by *Fusarium* species. At harvest, *F. graminearum* was detected in all the tested samples whereas *F. culmorum* was never detected. *Fusarium verticillioides* was abundant in grains of one origin and detected in another sample, the only one where *F. sporotrichioides* was detected. *Fusarium poae* was significantly observed in grains of two origins and (Table 5).

<table><caption>**Table 5.** Incidence and relative abundance of *Fusarium* species in grains and millings of maize collected in 6 farms. See Figure 1 for localisations.</caption><thead><tr><th rowspan="2">Samples</th><th rowspan="2"></th><th colspan="5">Fusarium Species</th></tr><tr><th>F. culmorum</th><th>F. graminearum</th><th>F. poae</th><th>F. sporotrichioides</th><th>F. verticillioides</th></tr></thead><tbody><tr><td rowspan="4">C-24-VA-02</td><td>Harvest</td><td>0</td><td>++</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Storage</td><td>0</td><td>++++</td><td>++</td><td>++</td><td>++</td></tr><tr><td>Flour</td><td>0</td><td>++++</td><td>++</td><td>++</td><td>++</td></tr><tr><td>Meal</td><td>0</td><td>++++</td><td>++</td><td>++</td><td>++</td></tr><tr><td rowspan="4">C-24-VA-01</td><td>Harvest</td><td>0</td><td>++</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Storage</td><td>+</td><td>++++</td><td>++</td><td>0</td><td>++</td></tr><tr><td>Flour</td><td>0</td><td>++++</td><td>++</td><td>++</td><td>++</td></tr><tr><td>Meal</td><td>0</td><td>++++</td><td>++</td><td>++</td><td>+</td></tr><tr><td rowspan="4">C-24-VA-00</td><td>Harvest</td><td>0</td><td>++</td><td>++</td><td>++</td><td>+</td></tr><tr><td>Storage</td><td>+</td><td>++++</td><td>++++</td><td>++</td><td>+</td></tr><tr><td>Flour</td><td>+</td><td>++++</td><td>++++</td><td>+</td><td>0</td></tr><tr><td>Meal</td><td>+</td><td>++++</td><td>++++</td><td>++</td><td>+</td></tr><tr><td rowspan="4">C-24-SA-01</td><td>Harvest</td><td>0</td><td>++</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Storage</td><td>0</td><td>++</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Flour</td><td>0</td><td>++</td><td>++</td><td>++</td><td>+</td></tr><tr><td>Meal</td><td>0</td><td>++</td><td>0</td><td>++</td><td>++</td></tr><tr><td rowspan="4">C-24-SASM-01</td><td>Harvest</td><td>0</td><td>++</td><td>0</td><td>0</td><td>+++</td></tr><tr><td>Storage</td><td>0</td><td>++</td><td>++</td><td>0</td><td>+</td></tr><tr><td>Flour</td><td>0</td><td>++</td><td>0</td><td>+</td><td>++</td></tr><tr><td>Meal</td><td>0</td><td>++</td><td>0</td><td>0</td><td>++</td></tr><tr><td rowspan="4">C-47-BR-01</td><td>Harvest</td><td>+</td><td>+++</td><td>++</td><td>0</td><td>0</td></tr><tr><td>Storage</td><td>0</td><td>+++</td><td>++</td><td>+</td><td>++</td></tr><tr><td>Flour</td><td>0</td><td>+++</td><td>++</td><td>++</td><td>++++</td></tr><tr><td>Meal</td><td>0</td><td>++</td><td>++</td><td>0</td><td>++</td></tr></tbody></table>

DNA abundance of a *Fusarium* species, Cq ranges: 33 to 36 = +, 30 to 32 = ++, 26 to 29 = +++, lower than 26 = +++++, 0 = not detected.

### 3.3. Fate of *Fusarium* Mycotoxins and *Fusarium* Species During Storage

Mycotoxin contamination can continue during storage under suitable conditions for fungal activity. After harvesting, the maize cobs were either dehulled and the grains kept in galvanised steel storage bins (C-24-SA-01) or big-bags (C-24-SASM-01; C-47-BR-01), or stored whole in narrow open-air wire mesh structures known as cribs for 8 to 9 months (all the other farms). Considering all the farms, the differences in mycotoxin concentrations at harvest and after storage were not significant at *p* < 0.05 (Table 6), but significant positive correlations were observed (Figure 3).
<table><caption>**Table 6.** Overall comparisons of changes from harvest to the end of storage. Statistical analysis conducted with the Wilcoxon signed-rank test.</caption><thead><tr><th>Measure 1</th><th>Measure 2</th><th>W</th><th>z</th><th>p</th></tr></thead><tbody><tr><td>DON Harvest</td><td>DON Storage</td><td>19.000</td><td>-1.569</td><td>0.129</td></tr><tr><td>Niv Harvest</td><td>Niv Storage</td><td>17.000</td><td>-1.070</td><td>0.322</td></tr><tr><td>T2 + HT2 Harvest</td><td>T2 + HT2 Storage</td><td>11.000</td><td>-1.362</td><td>0.193</td></tr><tr><td>ZEA Harvest</td><td>ZEA Storage</td><td>9.000</td><td>-1.886</td><td>0.067</td></tr><tr><td>FB1 + FB2 Harvest</td><td>FB1 + FB2 Storage</td><td>30.000</td><td>-0.706</td><td>0.519</td></tr></tbody></table>

W = sum of rank, z represents the distance between that raw score x and the population mean in units of the standard deviation, p = probability of the z score.

Overall, there was a positive correlation between FB1 + FB2 concentrations measured at harvest and after storage. In farms where the grains were not stored in cribs, the levels of contamination did not significantly change or decrease, except for C-47-BR-01, in which the high levels of fumonisins recorded at harvest increased dramatically during storage (×4.38) (Table 7). This sample also exhibited the highest abundance of *F. verticillioides* (Table 5). In the farms with storage in cribs, quantifiable concentrations of FB1, and also other mycotoxins, appeared in C-64-BE-02, which was not contaminated at harvest. It is worth noting that no increase in fumonisins was recorded in the other farms, except for appearances of both FB1, FB2, FB 3 and *F. verticillioides* at low levels in two samples, which were highly affected by over-contamination with the other mycotoxins.

<table><caption>**Table 7.** Ratios of mycotoxin concentrations at storage to concentrations at harvest.</caption><thead><tr><th>Origin of the Samples</th><th>DON + 15-ADON</th><th>Niv + Fx</th><th>T-2 + HT-2</th><th>ZEA</th><th>FB1 + FB2 + FB3</th></tr></thead><tbody><tr><td>C-24-VA-02</td><td>3.36 *</td><td>3.45 *</td><td>14.28 *</td><td>1.54</td><td>S+</td></tr><tr><td>C-24-VA-01</td><td>2.80 *</td><td>S++</td><td>5.26 *</td><td>1.26</td><td>1.35</td></tr><tr><td>C-24-VA-00</td><td>12.32 *</td><td>S++</td><td>S+</td><td>490.20 *</td><td>S+</td></tr><tr><td>C-24-SA-01</td><td>0.34 *</td><td>0.43 *</td><td>0.42</td><td>0.67</td><td>1.52</td></tr><tr><td>C-24-SASM-01</td><td>1.44</td><td>nd</td><td>1.04</td><td>nd</td><td>2.92 *</td></tr><tr><td>C-47-BR-01</td><td>1.06</td><td>nd</td><td>S+</td><td>0.46</td><td>4.38 *</td></tr><tr><td>C-64-JA-01</td><td>0.84</td><td>2.60 *</td><td>S+</td><td>S+</td><td>0.86</td></tr><tr><td>C-64-BE-01</td><td>0.12 *</td><td>0.21 *</td><td>0</td><td>0.71</td><td>0</td></tr><tr><td>C64-AR-01</td><td>S+</td><td>nd</td><td>nd</td><td>nd</td><td>0.27</td></tr><tr><td>C-64-BE-02</td><td>S+</td><td>S+</td><td>nd</td><td>S+</td><td>nd</td></tr><tr><td>C-64-GA-01</td><td>0.34 *</td><td>0.27 *</td><td>nd</td><td>S+</td><td>nd</td></tr><tr><td>C-64-BU-1</td><td>nd</td><td>0.39 *</td><td>0</td><td>nd</td><td>0.40 *</td></tr><tr><td>C-64-IB-1</td><td>1.29</td><td>6.51 *</td><td>3.45 *</td><td>31.42 *</td><td>0.38 *</td></tr></tbody></table>

* Concentrations at storage and harvest are significantly different. S+ = no mycotoxin detected at harvest, but significant at low concentration after storage, S++ = no mycotoxin detected at harvest, but at high concentration after storage. nd = non-detected, neither at harvest nor after storage. 0 = low concentrations at harvest and non-detected after storage.

Overall, there was a positive correlation between DON concentrations measured at harvest and after storage (p < 0.01). No correlation was significant for the other trichothecenes and ZEA (Figure 3). As they were at harvest, concentrations of DON and ZEA in storage were significantly correlated positively. In addition, they were both correlated with Niv concentration. In the farms where the grains were not stored in cribs, the levels of ZEA and of trichothecenes A (TCTA) and trichothecene B (TCTB) did not significantly change during storage (Table 7). In the farms with storage in cribs, contrasting events were observed. Significant increases in the levels of mycotoxins in grains were observed after storage in cribs, but not for all the mycotoxins and sometimes significant decreases also occurred. The levels of mycotoxins decreased from the harvest to the end of storage in C-64-BE-01 and
C-64-BU-1, having low contamination levels at harvest (Table S1), while large increases in both TCTA and TCTB were observed in 3 farms from the same locality, C-24-VA-00, C-24-VA-01, C-24-VA-02 (Table 7). With a maximum level of DON at 1500 µg kg⁻¹ for unprocessed grains in EU regulation, C-24-VA-00 and C-24-VA-02 became nonmarketable to a mill after storage. In another farm at the extreme part of our study area, C-64-IB-1, the concentrations of trichothecenes and ZEA also increased while fumonisins concentrations decreased. In agreement with the changes in TCTB concentrations, an increase in abundance of *F. graminearum* and *F. poae* was noticeable for C-24-VA-00, C-24-VA-01, and C-24-VA-02. *Fusarium culmorum* became detectable in two of them (Table 5). The increase was significant for Niv but not for DON for C-64-JA-01 and C-64-IB-1. On the other hand, decreases were recorded for both DON and Niv in C-64-BE-01 and C-64-GA-01. In addition, Niv concentration decreased in C64-BU-1.

The increases in TCTA concentrations were related to increased abundances of *F. sporotrichioides* and/or *F. poae*. T2 and HT2 toxins, absent at harvest in C-47-BR-01, appeared at storage (Table 7) while *F. sporotrichioides* became detectable (Table 5).

### 3.4. Transfer of Mycotoxins into Milling Products

Comparing grain after harvest, meal and flour, from eight origins, significant positive Kendall's Tau correlations were measured ($p < 0.1$) for all the mycotoxins except for T-2 + HT-2 having a non-significant correlation between concentrations in grains and flour or meal, and FB1 + FB2, having non-significant correlation between grains and meal and between flour and meal (Table S2). Overall, highly contaminated grains will lead to highly contaminated mill products. We observed such a trend of higher concentrations in flours than in meals for all the mycotoxins measured. (Table 8).

<table><caption>**Table 8.** Ratios of mycotoxin concentrations in flour to concentrations in meal.</caption><thead><tr><th rowspan="2">Flour/Meal</th><th colspan="8">Mycotoxins</th></tr><tr><th>DON</th><th>Niv</th><th>T-2</th><th>HT-2</th><th>ZEA</th><th>FB1</th><th>FB2</th><th>FB3</th></tr></thead><tbody><tr><td>C-24-VA-02</td><td>1.35</td><td>2.06</td><td>1.63</td><td>1.87</td><td>1.56</td><td>1.82</td><td>2.31</td><td>1.59</td></tr><tr><td>C-24-VA-01</td><td>1.60</td><td>0.82</td><td>1.50</td><td>1.37</td><td>1.79</td><td>1.73</td><td>2.45</td><td>1.83</td></tr><tr><td>C-24-VA-00</td><td>1.21</td><td>0.72</td><td>1.13</td><td>0.59</td><td>1.06</td><td>1.26</td><td>1.44</td><td>1.43</td></tr><tr><td>C-24-SA-01</td><td>1.44</td><td>0.74</td><td>2.12</td><td>1.60</td><td>6.56</td><td>3.35</td><td>3.53</td><td>3.05</td></tr><tr><td>C-24-SASM-01</td><td>1.48</td><td>/</td><td>1.91</td><td>1.65</td><td>/</td><td>2.15</td><td>2.24</td><td>2.35</td></tr><tr><td>C-47-BR-01</td><td>1.22</td><td>/</td><td>2.36</td><td>1.04</td><td>1.37</td><td>1.76</td><td>2.11</td><td>2.15</td></tr><tr><td>C-64-JA-01</td><td>0.63</td><td>1.22</td><td>/</td><td>0.00</td><td>0.43</td><td>0.56</td><td>0.55</td><td>0.68</td></tr></tbody></table>

/ indicates non-quantifiable mycotoxin both in flour and meal.

Due to the uncertainty associated to the measures of TCTB, differences between grains and the milling products were significant for only two origins, and there was no general trend (Figure 4). Half of the DON measured in grain was found after milling in flour and meal for C-24-VA-02, whereas the DON concentration in milling products of C-24-VA-01 was 2 to 3 times higher than in grain and did not change in 24-VA-00. The EU regulation 2023/915 of 25 April 2023 was modified by the regulation EU 2024/1022, defining a maximal level of DON at 750 µg kg⁻¹ (Table 1). Due to the high levels in grains, the milling products from these farms exceeded the maximum. The increase was also significant for T-2 and HT-2 and dramatic for ZEA (Figure 4) in 24-VA-01, whereas for C-24-VA-00, TCTA concentrations decreased in milling products and ZEA in meal.
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Mycotoxin concentrations measured in grains after storage and milling products obtained on eight farms. (a): DON, (b): Niv, (c): ZEA, (d): T-2 and HT-2, (e): FB1, FB2 and FB3. Error bars are +/- uncertainties.</figcaption></figure>

The fumonisins concentrations in flour samples were all lower than or equal to the con-
centrations in the corresponding grains, and lower or equal concentrations were measured
in meal than in flour (Figure 4). However, the milling products for C-47-BR-01 still largely
exceeded the EU maximum level, which is 1000 µg kg⁻¹. Due to the lower maximum for
milling products than raw grains, the flours from C-24-01 and C24-SASM-01, as well as
both flour and meal for C-24-VA-01, did not comply with the EU rule, while the grain did.
Consequently, these products were not marketable as food.
### 3.5. Ochratoxin A and Aflatoxins in Maize Grains and Millings

In the samples analysed in this study, there was a wide range of levels of contamination by DON, 15-ADON, Niv, Fx, T2, HT2, ZEA, fumonisins B1, B2, B3, but the presence of OTA and Aflatoxins was scarce. The typical storage mycotoxin, OTA, was found in only one sample, C-24-VA-02, after storage at low concentrations, 0.58 (+/-0.26) µg kg⁻¹ in grains, 1.2 (+/-0.40) in flour, and 0.74 (+/-0.33). Pre-harvest contamination by aflatoxins was found in only one sample (C-64-JA-01) at a low concentration, 0.39 +/- 0.17 µg kg⁻¹ for the sum Afla-B1, Afla-B2, Afla-G1, Afla-G2. Post-harvest, we found these mycotoxins in the three farms where grains were not stored in cribs. They were at low concentrations, 0.47 (+/-0.21) and 0.87 (0.38) µg kg⁻¹ for the sum of aflatoxins in flours of C-24-SA-01 and C-24-SASM-01, respectively, but not in the meals. In both samples, aflatoxins were not quantifiable in unprocessed grains. In C-47-BR-01, grains, flour and meal were dramatically contaminated by Afla-B1 and Afla-G1, without differences between the fractions, and also contained Afla-B2 and Afla-G2 (Table 9).

<table><caption>**Table 9.** Concentrations of aflatoxins developed in storage at the farm C-47-BR-01 and were transferred into milling products. In µg kg⁻¹. Values in brackets are uncertainties.</caption><thead><tr><th></th><th>Afla-B1</th><th>Afla-B2</th><th>Afla-G1</th><th>Afla-G2</th><th>Sum</th></tr></thead><tbody><tr><td>Grain at harvest</td><td>&lt;LoQ</td><td>&lt;LoQ</td><td>&lt;LoQ</td><td>&lt;LoQ</td><td>&lt;LoQ</td></tr><tr><td>Grain after storage</td><td>99.6 (+/-43.8)</td><td>5.5 (+/-2.4)</td><td>70.0 (+/-30.8)</td><td>4.0 (+/-1.7)</td><td>179 (+/-74)</td></tr><tr><td>Flour</td><td>80.1 (+/-35.3)</td><td>4.1 (+/-1.8)</td><td>38.2 (+/-16.8)</td><td>2.5 (+/-1.1)</td><td>125 (+/-55)</td></tr><tr><td>Meal</td><td>134 (+/-58)</td><td>5.8 (+/-2.5)</td><td>54.5 (+/-24.0)</td><td>3.1 (+/-1.4)</td><td>197 (+/-81)</td></tr></tbody></table>

## 4. Discussion

### 4.1. Maize Grains Contamination by Mycotoxins and Fusarium Species

In the maize grain samples collected at harvest and after storage, we analysed in this study, there was a large diversity of occurrence and a wide range of levels of contamination by DON, 15-ADON, Niv, Fx, T2, HT2, ZEA, and fumonisins B1, B2, B3. The presence of OTA and aflatoxins was scarce.

Fumonisins have been widely reported as the prevalent mycotoxins in maize grain in southern Europe [15], and in the large international survey reported by Gruber-Dorninger et al. 2019 [14], 80% of the samples contained fumonisins. In the present work, a similar percentage of samples were contaminated with fumonisins, but the concentrations (Figure 2) were lower than those reported in Mediterranean areas. FB1 and FB2 were present in Algerian samples in a concentration range of 289–48,878 µg kg⁻¹ [16]. In Spain, the observed range of concentrations was 26.0–63,100 with a mean of 5734 µg kg⁻¹, 20% exceeding the EU limit [17].

We observed DON as the prevalent mycotoxin, with a percentage of 77%, and half of the samples were co-contaminated with 15-DON at concentrations from 50% to 125% of that of DON. These ratios were higher than those reported by Oliviera et al. (2017) in Brazil, where 15-ADON contributed, on average, to 31% of the total contamination by DON and its derivatives [18]. The present data are comparable to prior records in North America. In a 7-year survey of maize grain in the United States (711 samples), occurrences were 76% DON and 48% 15-ADON, but also 14% 3-ADON [19]. In Michigan, harvested maize grain 93 to 100% contained DON, 47 to 100% contained 15-ADON [20].

However, the observed rates of contamination by DON and its derivative (15-ADON) are higher than in most of the published works from various world regions. The frequency of DON contamination found in some Brazilian studies was lower than 9% [21,22] but reached 48% in a survey of maize samples from the south region of Brazil [18]. In South
Europe and the Mediterranean Basin, lower rates of DON occurrence were also recorded in some studies, but not in others. On a total of 20 maize samples destined for human consumption purchased from local markets in the western region of Algeria, 13 (43%) were positive for DON [16]. Studying 98 samples of maize kernels without visible signs of mould growth collected in the years 2015–2019 in 26 grain stores located in different Spanish regions, DON was quantified in 31 samples, and only 5 samples had quantifiable levels of 3-ADON + 15-ADON [17]. In northern Italy, the Emilia Romagna region, in 2014, DON was detected in 59% of the analysed samples from 51 farms, whereas it is generally rare in Italian samples. The year 2014 was reported as alarming for DON contamination in Europe [23]; 65–100% of the samples of a large survey of maize grain samples collected from 88 storage centres in Northern Italy contained DON [15]. In a global survey program to monitor mycotoxin contamination of feed raw materials collected from 2008 to 2017, 67% of 12,600 maize samples were contaminated with DON [14]. However, this large survey was based on samples voluntarily sent by farmers or the feed industry for analyses, which could be those suspected to be at risk.

Co-occurrence of mycotoxins is a common observation in maize grains, with values ranging from 60 to 90% of positive samples with more than one mycotoxin. Co-occurrence of Niv plus its derivative Fx, with DON was observed very often [24]. Here, the rates of positive samples for Niv were also high compared to published works. A Brazilian study reported only 10% and 2.5% Niv and Fx, respectively [19], while 76% of the corn samples from the south region of Brazil were contaminated with Niv [18]. In samples analysed in the United States, neither Niv nor F-x was found [20] or Niv, but no F-x was found at a rate of 7% [24]. This survey of published work shows the diversity of levels of occurrence of DON and its derivatives. The data collected from the grains of landrace maize grains from organic agriculture stored at small farms in the southwest of France tend to highlight a high level of occurrence of TCTB, but at low concentrations (<550 µg kg⁻¹) in most of the samples, and none exceeded the EU limit. However, there is a concern to consider DON derivatives and Niv plus Fx in the authorised maximal concentrations of TCTB.

As for TCTB, there was a high rate of samples contaminated with HT-2, but not T-2, which had not been reported previously, but at low concentrations. Fusilier et al. (2022) found 19 and 4% of T-2 and HT-2, respectively, in their samples [20]. In maize samples from Algerian markets, 100% of samples contained T-2 with a mean concentration of 25 µg kg⁻¹, whereas HT-2 was never found [16]. In Spain, only 5% of the samples analysed contained T-2 and HT-2 with medians lower than 12 µg kg⁻¹ [17]. Those toxins were not detected by Tonial Simões et al. (2023) in a field experiment on maize varieties [22]. The rate of positive samples recorded in the USA over 7 years was 7.5% and 5.9% with a median of 10 and 38 µg kg⁻¹ for T-2 and HT-2 toxins, respectively [14]; 12% of maize analysed worldwide showed detectable levels of T-2 and HT-2 [14].

The occurrence of ZEA (54%) and concentrations were in ranges reported in the literature. ZEA was detected in 44% of the samples in the large survey reported by Gruber-Dorninger et al. (2019) [14]; 42% were reported in the south of Brazil [18], whereas 69% of positive samples were found in Michigan [21] and 25 to 91% in Italy [15]. Values lower than 23% of positive samples were recorded in the USA [25], Algeria [16], and Spain [17]. In these studies, median values of 115 µg kg⁻¹ (mean 302) [25], median 110 µg kg⁻¹ (mean 837) [17], or mean 109 µg kg⁻¹ [16] were recorded. In Northern Italy, low concentrations with means lower than 25 µg kg⁻¹ were measured most years, but in 2014, the mean concentration was 364 µg kg⁻¹ [15]. ZEA always co-occurred with DON, with significant correlations between the concentrations. In maize intended for feed, DON and ZEA concentrations were positively correlated [14]. Both mycotoxins are known to be produced by the same species of *Fusarium*.
Most notable mycotoxigenic *Fusarium* species are members of the *Fusarium sambucinum* and *Fusarium fujikuroi* species complexes [19]. We used specific primers for assessing the presence and relative abundance of five *Fusarium* species in some maize samples being all contaminated with DON at different concentrations: *Fusarium verticillioides* as a representative member of the *Fusarium fujikuroi* Species Complex producing fumonisins and four members of the *Fusarium sambucinum* Species Complex: *F. culmorum* and *F. graminearum* known to produce DON, ZEA and Niv (some strains only), *F. poae* producing Niv and T-2/HT2, and *F. sporotrichioides* producing T-2/HT-2 and ZEA [25]. The prevalence and distribution of the *Fusarium* species depend on climatic conditions and agronomic practices. *Fusarium graminearum* tends to be the main species present in maize in cooler temperate regions, while *Fusarium fujikuroi* complex species (*F. verticillioides*, *F. subglutinans*, and *F. proliferatum*) dominate in the warmer temperate regions [26], such as Tunisia [27]. *Fusarium culmorum* is known to occur rarely in maize ears, but it is highly aggressive, and it functions better in cooler seasons [28]. Our data reflect a situation of the temperate region with a high incidence of *F. graminearum* and a low incidence of *F. verticillioides*, in contrast to data from warmer regions [27] and the absence of *F. culmorum*. In a previous study in France, the 15-ADON trichothecene chemotype predominated in 85.4% of the 185 *F. graminearum* isolates from maize, whereas the NIV chemotype was found in 14.6% [29]. According to the recorded occurrence of mycotoxins in the present study, the putative predominant chemotype was DON/15ADON but Niv/Fx was also well represented. This and co-occurrences with HT-2 indicated contaminations by different *F. graminearum* strains and contaminations by both *F. graminearum* and *F. poae*. *Fusarium sporotrichioides* was only detected in C-24-SA-01, in agreement with the contamination in T-2, but not HT-2. Actually, some *F. graminearum* strains could also produce HT-2 [30]. It is known that the conditions required for fungal growth are not necessarily the same for mycotoxin production; consequently, the presence of a mycotoxigenic *Fusarium* species does not necessarily imply mycotoxin production [31]. The levels of contamination in mycotoxins and the presence of potential producers did not match most of the time. Neither *F. culmorum* nor *F. poae* were detected in C-24-VA-02 contaminated with Niv at 175 µg kg⁻¹, whereas *F. poae* was detected in C-47-BR-01, which did not contain Niv. A strain of *F. graminearum* with the appropriate chemotype could have produced Niv, and a *Fusarium* species can be present on grains without having produced mycotoxins. Surprisingly, *F. verticillioides* was not detected in this sample, having the highest concentrations of fumonisins, whereas it was at a high level in C-24-SAMS-01, containing 7 times less FB1.

In Europe, pre-harvest aflatoxin contaminations of maize are predicted to increase in the course of climate change [32], but aflatoxins are still emerging in France. Analysing more than 500 maize samples from France taken at harvest during 2018–2020, Bailly et al. (2024) observed that only 7% were contaminated with mean levels of 3.8 µg/kg and 5.9 µg/kg for Afla-B1 and the sum of aflatoxins [33]. Actually, an aflatoxin risk index in the area covered by the present study was estimated as very low with the present climate, in agreement with the only pre-harvest contamination at a very low concentration we observed, but it is expected to increase and become high in the intermediate 2050 climate scenario [34].

## 4.2. Fate of Mycotoxin at Storage and During Dry Milling

An objective of this study was to compare the contamination in the same batch of maize before and after storage under farm conditions. Despite precautions in the sampling strategy, heterogeneity due to on-farm practices has to be considered. However, different trends in changes of mycotoxin concentrations were observed. Drying and storage in cribs as cobs is an ancient practice preserved in this part of France as part of a local traditional
way to produce the local population's maize, which was supposed to affect the levels of contamination. This hypothesis was tested.

For fumonisins and *F. verticillioides*, the only case of dramatic contamination during storage was for grains stored in big bags as a result of a drying defect. This batch was also dramatically contaminated with aflatoxins. Changes in amounts of fumonisins during storage in silos has been reported by several authors with sometime increases [26], sometime decreases [35,36]. In a Brazilian region characterised by humid tropical weather with hot and rainy summers and dry winters, the maize grains were put in 60 kg jute sacks (5 sacks per hybrid), stacked over wooden boards and stored for 12 months in a well-ventilated warehouse, located near the production area [36]. The authors related a contamination of 90% with fumonisin B1 in a range of concentrations 0.9 to 49 µg g⁻¹, and 97% with fumonisin B2 in a range of concentrations 2 to 29 µg g⁻¹. After 140 days of storage, a tendency towards decreased FB1 mean concentrations and an overall decrease in the number of CFU/g for *Fusarium* spp. was observed, although FB1 levels showed some variability [36]. In the present and prior studies, the use of cribs did not appear as a risk factor for fumonisin contamination. In most farms storing maize in cribs in Brazil studied by Quieroz et al. (2012), the total fumonisin levels did not increase throughout the storage period, except in one farm [37]. In traditional storage structures constructed using mud and wattle, commonly fitted with a grass thatched roof used in Uganda, fumonisins also decreased after 4 months of storage from an average of 5700 to 2800 µg kg⁻¹ both shelled or as cobs [38]. Maize stored in ventilated structures close to the case in cribs had significantly lower fumonisin levels than maize stored in non-ventilated structures for 6 months. In Benin, a decreasing trend in fumonisin levels detected in maize samples throughout the storage time was observed, but it was not significant in all seasons [39]. On the contrary, fumonisins in grains at less than 15% humidity increased by 3 to 45% after 6 months in different kinds of bags used for storage under local environmental conditions in Kenya, with a trend to higher rates in dry than in wet treatment [40]. Studying the evolution of mycotoxins during grain storage in bags, Gaël et al. (2020) observed that fumonisins increased slowly until month 10 and significantly from 10 to 18 months [41]. This progression was slowed down when plant biopesticides were added as dried leaves. In Mediterranean Europe, Spain, the accumulation of FB1 decreased by 63% after three months of storage [35]. In Portugal, fumonisins showed a tendency to increase (20% to 40%) during six months of storage but no correlation between the levels of fumonisins and the climatic parameters recorded in experimental silos was established, even if meanwhile temperature and CO₂ levels increased [26]. Overall, in traditional maize storage facilities, including cribs, the risk of fumonisins over-contamination appeared to be limited. This might be the result of the significant levels of air flow around the cobs in cribs that prevent high humidification of the grains and development of producers of fumonisins. The cribs are naturally ventilated storage facilities, contrary to storage in bags, corresponding to airtight storage, where humidity may accumulate due to biological activities.

Contrary to the fate of fumonisins, trichothecenes A and B and ZEA were not affected during storage in bags or silo, but concentrations changed in some farms using cribs. They tended to decrease when contaminations at harvest were low and to increase significantly for the highest contaminated batches, with associated new developments of *Fusarium* species known to produce each kind of mycotoxin. In contrast to the lack of correspondence between *Fusarium* species and mycotoxins previously observed on single samples at a given time, changes during storage in detected *Fusarium* species and their abundance were linked to increases in mycotoxin concentrations. There are not many studies on trichothecene concentration changes in maize grains during storage. In a laboratory experiment, Venslovas et al. (2022) observed decreases about two times of DON
concentration in dried maize (up to 7% moisture content) after 6 months of storage at 12 °C and 20 °C while at 4 °C after 3 months of storage it also decreased, and then after 6 months it increased to the same concentration as at the beginning of the experiment [42]. DON and T-2 absent at harvest were not found after storage [26]. Present and prior data do not allow for a conclusion on a general trend in the fate of trichothecenes in storage, but it is clear that under unfavourable conditions, dramatic over-contamination may occur, rendering batches of grain unmarketable and dangerous to human and animal health.

ZEA increased dramatically in two batches stored in cribs, overpassing the EU maximum level (350 µg kg⁻¹) and the maximum (611 µg kg⁻¹) found in a survey of the literature in Europe [43], and appeared in two other samples. During the storage period of 6 months in cribs in 10 Brazilian farms, there was no difference between zearalenone levels in samples collected at four different times from 6 farms. However, in 4 farms, there was a wide variation in these levels, with sometimes decreases. The authors attributed the wide range from one sampling period to another to the difficulty of obtaining homogeneous samples in this type of storage (ears with husk) [37]. In hermetic bags, ZEA started to increase significantly after 10 months of storage and increased by 6 times in the following months [41]. According to the present and previous data, the risk of maize contamination with ZEA during the storage period is a cause for concern. The use of cribs as drying and storage facilities tended to increase the accumulation of trichothecenes and ZEA produced by members of the *Fusarium sambucinum* Species Complex, whereas fumonisins were not affected. The difference in the optimal temperatures between the groups of species may explain this result. The middle to low air temperature during the storage period in the studied area is less favourable to the production of fumonisins.

Dry milling operations are known to cause a redistribution of mycotoxins, but do typically not affect their chemical structure [44]. The distribution of *Fusarium* mycotoxins among different fractions in the maize dry-milling fractions depends on the variety, milling method, and the specific fractions produced in different countries, and most of the published data are for industrial processing [45]. It was in most studies negatively correlated to the particle size; fractions with the largest particles generally have the lowest mycotoxin concentrations [44]. In agreement with this finding, we observed such a trend of higher concentrations in flours than in meals for all the mycotoxins measured. However, in Table 8, one can observe discrepancies with the general trend. Additionally, variations in mycotoxin contamination levels across different maize batches and unevenness in sampling can influence the final distribution of mycotoxins in various dry-milling fractions [45]. The distribution in fractions had been considered in European regulations, for which the maximum limits for DON were around 1.5 times higher in maize flour (defined as the milling product in which least 90%, measured by weight, of the particles have a size ≤ 500 µm, not placed on the market for the final consumer), than in other fractions, before the last revision (Table 1). Reduction factors from grains to milling products prevailed also in the EU regulations. Consequently, we observed here some flour and meal not complying with the EU rule, while the grains did. This highlights that when the concentrations in grains are close to the limits, it is worth checking the contamination of the milling fractions. In any case, a high level of contamination at harvest and after storage leads to highly contaminated milling products. There is a need to monitor the contamination at the production step, but also to continue monitoring the risk during storage, especially when moisture cannot be removed, and processing is not an option to count on to deliver safe products within the legal limits.

Aflatoxins are mycotoxins known to contaminate cereals after the harvest. In the worldwide survey [14], 24% of the maize samples contained Afla-B1 with a median value of concentration being 3 µg kg⁻¹. Whereas Lee and Ryu (2017) reported an average prevalence
of around 7% for aflatoxins in unprocessed maize in Europe [43], post-harvest, we found
these mycotoxins in the three farms where grains were not stored in cribs, but not in
samples of grains stored in cribs. The farm, which was dramatically contaminated at
storage, also suffered from high contamination with fumonisins, both mycotoxins being
produced in the same range of temperature. The probability of co-occurrence of Afla-B1
and FB1, FB2, FB3 was higher than what a model expected under a random association
in a 7-year survey with more than 700 grain samples in the USA [45]. Recently, in Brazil
aflatoxin-fumonisin co-exposure occurred in 8.33% of the samples from organic farming [46],
whereas previously co-occurrences between 33 and 54% had been recorded in 24 samples
of maize commercialised in São Paulo [18], and between 10 and 45% in the southern region
of Brazil [47]. Finally, the use of cribs did not always appear as a risk factor.

There are relatively few studies on the fate of aflatoxins in the dry milling of maize.
Researchers observed a reduction in Afla-B1 content in the endosperm fraction, although
this reduction was not statistically significant [45]. In the highly contaminated farm at
storage (C-47-BR-01), the contamination of aflatoxins was not reduced during processing.
The concentrations were 30 times higher than the EU maximum authorised value in the
EU (4 µg kg⁻¹) and 2.5 times the maximum previously recorded in French pre-harvest
samples [33]. In northern Italy, two out of 500 maize samples collected over 1995–1999 from
storage bins and feed mills within 3 months after harvest had Afla-B1 levels of as much as
109 to 158 µg kg⁻¹ [48]. In about 300 to 400 samples collected each year from 2011 to 2021
in 88 storage centres, the maximum of Afla-B1 concentration was 145 g kg⁻¹ [15].

**5. Conclusions**

Addressing a critical gap in data on mycotoxin contamination for a small-scale organic
farming system growing, storing and processing landrace maize grains on farm, we ob-
served a high level of occurrence of mycotoxins, but at low concentrations in most of the
samples and no one exceeded the limits of the EU regulation at harvest, except for ZEA in
one sample. Most of the samples were contaminated by two or more mycotoxins and at
least two *Fusarium* species. However, both pre-harvest and post-harvest, we have never
observed concomitant high concentrations of fumonisins, and other mycotoxins produced
by *Fusarium* species, neither pre- nor post-harvest. Experimental scale studies with natu-
rally contaminated grains under varying climatic conditions could help to decipher the
biological mechanisms leading to this mutual exclusion.

Storage was the critical stage during which additional contamination by mycotoxins produced by *Fusarium* species occurred. The use of cribs for drying and storing corn during the autumn and winter is a simple technique that saves energy compared with silos, and is in line with the needs of sustainable agriculture. However, farmers need to be cautious and monitor contamination by trichothecenes and zearalenone, whereas the risk seems less significant for fumonisins and low for aflatoxins.

Grain batches with mycotoxin concentrations higher than the EU regulation led to
milling products that are unsuitable for trade and consumption.

The data acquired in this work will be used to raise awareness of the mycotoxin issues
among farmers producing, storing and processing maize on-farm, and to assist them in
their efforts to improve the safety of their products.

**Supplementary Materials:** The following supporting information can be downloaded at
https://www.mdpi.com/article/10.3390/agriculture15101064/s1: Table S1: Mycotoxin concen-
trations in all the maize samples analysed. Table S2: Kendall's Tau correlations of mycotoxin
concentrations in grains and milling products
**Author Contributions:** Conceptualization, J.-M.S.; methodology, J.-M.S.; investigation, J.-M.S. and L.P.-G.; data curation, J.-M.S. and L.P.-G.; writing—original draft preparation, J.-M.S.; writing—reviewing and editing, J.-M.S., L.P.-G., R.V. and C.V.-F.; supervision, J.-M.S.; project administration, R.V. and C.V.-F.; funding acquisition, J.-M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the French Ministry of Agriculture, CASDAR, n° 19AIP5914 Myco3C.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data supporting the reported results can be found in the Supplementary Material of this article, and complements are available from the corresponding author upon reasonable request.

**Acknowledgments:** We are grateful to Bruno Taupier-Lettage, who initiated this project at meetings of the RMT Quasaprove. We acknowledge Nathalie Gallegos and Christine Ducos at INRAE MycSA for their work with the samples conditioning and qPCR analyses. We thank the farmers and animators at the organic farmers associations Agrobio Périgord and Biharko Lurraren Elkartea for the collection of samples.

**Conflicts of Interest:** The authors declare no conflicts of interest.

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