**Background:** Aflatoxins (AFs) are toxic secondary metabolites produced by Aspergillus fungi, with aflatoxin B1 (AFB1) classified as a Group 1 human carcinogen and its metabolite aflatoxin M1 (AFM1) as a Group 2B probable human hepatocarcinogen. Milk is a nutrient-dense food consumed daily, especially by infants and children, making AFM1 contamination a significant public health concern. The European Union has set a strict maximum residue limit of 50 ng/L for AFM1 in milk. Climate change is expanding the geographic range of aflatoxin-producing fungi, putting regions like Northern Italy at increasing risk. This study reports results from an eight-year monitoring program (September 2013–July 2021) conducted by the Regional Breeders Association of Lombardy on raw cow's milk, and examines whether AFM1 levels in milk correlate with AFB1 contamination in feed ingredients from the same area.
**Methods:** A total of 95,882 whole raw milk samples were collected from dairy farms across Po Valley, Northern Italy, under an established sampling protocol. Samples were transported refrigerated and stored at +4°C until analysis. After centrifugation at 3500× g for 10 min at +4°C and removal of the cream layer, 100 μL of skimmed milk was analyzed using the ELISA Bio-Shield M1 ES kit (ProGnosis-Biotech, Larissa, Greece). The detection limit was 5 ng/L. Standard solutions of 0, 5, 10, 20, 40, and 80 ng/L were used for calibration. Absorbance was measured at 450 nm. Samples above 80 ng/L were diluted and retested; positive samples above 50 ng/L were confirmed using immunoaffinity column clean-up and HPLC-FLD. Milk samples were categorized into four risk levels: very low risk (<25 ng/L), low risk (25–40 ng/L), moderate risk (40–50 ng/L), and high risk (>50 ng/L). AFB1 data in feed ingredients (maize, cotton, high-moisture maize, silage maize) from the same farms and period were drawn from a previous publication by Ferrari et al. for comparison.
**Key Results:** Of the 95,882 milk samples analyzed, 92,823 (96.8%) fell into the very low risk category (<25 ng/L), 2,002 (2.1%) were low risk (25–40 ng/L), 390 (0.4%) were moderate risk (40–50 ng/L), and 667 (0.7%) exceeded the EU limit of 50 ng/L (high risk). Overall, 99.3% of samples complied with EU legislation. Mean AFM1 contamination peaked in 2013 (14.4 ng/L) and 2015 (15.1 ng/L), while in all other years the mean remained below 10 ng/L. The percentage of samples under alert (40–50 ng/L) and exceeding 50 ng/L was notably higher in 2013 and 2015 compared to subsequent years. From 2016 onward—after the regional authorities activated the 'Extraordinary operating procedures for the prevention and management of the risk of contamination by AFs'—the percentage of positive samples and alert-level samples remained consistent and low. Comparison with feed data showed parallel contamination patterns: the 2015 peak in AFM1 in milk coincided with peaks in AFB1 contamination in cotton (14.1 µg/kg) and maize (15.7 µg/kg). Silage maize showed lower contamination levels relative to milk AFM1 values. The carryover of AFB1 from feed to AFM1 in milk is reported to range from 0.3% to 6.3%, influenced by breed, health, diet, and milk yield, with high-yielding cows (>30 L/day) showing up to 6% carryover.
**Clinical Implications:** This large-scale surveillance demonstrates that rigorous monitoring programs effectively maintain AFM1 contamination in milk at very low levels, with mean contamination consistently below 10 ng/L after implementation of the regional surveillance plan in 2016. The action level of 40 ng/L set by Lombardy authorities allows early intervention before milk reaches the EU limit of 50 ng/L, enabling corrective farm-level measures such as improved feed sourcing, storage, and management. The study underscores the critical link between feed safety and milk safety, identifying maize and cotton as the highest-risk feed ingredients for AFB1 contamination, followed by high-moisture maize and silage maize. Climate change—particularly hot, dry summers—increases the risk of aflatoxin contamination in crops, as evidenced by the 2013 and 2015 peaks. Continuous monitoring of both feed and milk, combined with rapid corrective actions, is essential to protect consumer health, especially vulnerable populations such as infants and children who are major milk consumers. The economic impact of non-compliant milk (cannot be sold) also incentivizes producers to maintain rigorous controls.