*2.1. Maize Sampling*

A total of 106 dairy farmers across Flanders were contacted to participate in this study from 2016 till 2018. The selected maize fields were scattered throughout Flanders, grown on di fferent soils and in di fferent micro climates, and the number of selected fields was proportional to the intensity of maize production in that region. Data regarding daily temperature, rainfall, relative humidity and radiation for each growing season were obtained from 17 weather stations spread across Flanders (Figure 1). A few months before harvest, each farmer received a plastic bag, a label and a manual, in which the sampling technique was explained. Sampling was done by taking at least 10 samples per trailer of harvested maize. These samples were mixed and a subsample of ca. 1 kg was put into a plastic bag. The bag was then sealed airtight and stored in a freezer until it was collected by the researchers. After sample collection, a subsample of ca. 5 g was taken for quantitative polymerase chain reaction (qPCR) analysis and stored in a freezer at –20 ◦C until further analysis; the remaining sample was dried in an airstream of 65 ◦C for four days. The dried maize sample was then milled in a 0.5 mm sieve, and stored until further mycotoxin analysis.

**Figure 1.** Location of the 106 dairy farms and 17 weather stations in Flanders, Belgium.

### *2.2. Reagents and Chemicals for LC-MS*/*MS*

Methanol (LC-MS grade), glacial acetic acid (LC-MS grade), and analytical grade acetonitrile were purchased from Biosolve B.V. (Valkenswaard, The Netherlands). Analytical grade acetic acid and ammonium acetate were obtained from Merck (Darmstadt, Germany), while analytical grade n-hexane and methanol were purchased from VWR International (Zaventem, Belgium). Water was purified using a Milli-Q Gradient System (Millipore, Brussels, Belgium).

Certified mycotoxin standard solutions in acetonitrile of OTA (10 μg/mL), aflatoxin mix (AFB1, AFB2, AFG1 and AFG2) (20 μg/mL), fumonisin mix (FB1 and FB2) (50 μg/mL), sterigmatocystin (STERIG) (50 μg/mL), DON (100 μg/mL), deepoxy-deoxynivalenol (DOM) (50 μg/mL), ZEN (100 μg/mL), NIV (100 μg/mL), neosolaniol (NEO) (100 μg/mL), T2 (100 μg/mL), 3-ADON (100 μg/mL), DAS (100 μg/mL), 15-ADON (100 μg/mL), and F-X (100 μg/mL) were obtained from Romer Labs (Tulln an der Donau, Austria). Alternariol (AOH), alternariol monomethylether (AME), zearalanone (ZAN) and enniatin B (ENN B) were purchased from Sigma-Aldrich (Bornem, Belgium), fumonisin B3 was obtained from Promec unit (Tygerberg, South Africa), and roquefortine C (ROQ-C) was purchased from Alexis Biochemicals (Enzo Life Sciences BVBA, Zandhoven, Belgium). Stock solutions were prepared in acetonitrile/water (50/50 *v*/*v*) for FB3 (1 mg/ml), methanol/dimethylformamide (60/40 *v*/*v*) for AOH and AME (1 mg/ml), and methanol for ROQ-C and ZAN (1 mg/ml). All stock solutions were stored at –20 ◦C, except for the stock solution of FB3 (4 ◦C). Working solutions were prepared by diluting the stock solutions in methanol, and were stored at –20 ◦C for three months. Three standard mixture working solutions were prepared in methanol. The first contained the mycotoxins AFB1, AFB2, AFG1 and AFG2 (2 ng/μL); OTA (5 ng/μL); ZEN and T2 (10 ng/μL); and DON, FB1 and FB2 (40 ng/μL). The second contained DAS (0.5 ng/μL); ROQ-C (1 ng/μL); 15-ADON (2.5 ng/μL); 3-ADON and STERIG (5 ng/μL); NEO and AOH (10 ng/μL); NIV, F-X and AME (20 ng/μL); and FB3 (25 ng/μL). The third contained ENN B (10 ng/μL). The standard mixtures were stored at –20 ◦C and used for a maximum of three months.
