Co-Occurrence of Aflatoxin B1, Zearalenone and Ochratoxin A in Feed and Feed Materials in Central Italy from 2018 to 2022.

Mycotoxin contamination of feed and feed materials represent a serious health hazard. This study details the occurrence of aflatoxin B1 (AFB1), zearalenone (ZEN) and ochratoxin A (OTA) in 826 feed and 617 feed material samples, collected in two Italian Regions (Umbria and Marche) from 2018 to 2022 analyzed using a UPLC-FLD platform. The developed method was validated and accredited (ISO/IEC 17025) with satisfactory accuracy and precision data obtained in repeatability and intralaboratory reproducibility conditions. Feed had a higher incidence of contaminated samples (26%) with respect to feed materials (6%). AFB1 was found up to 0.1045 mg/kg in cattle feeds and 0.1234 mg/kg in maize; ZEN was detected up to 6.420 mg/kg in sheep feed while OTA was rarely reported and in lower concentrations (up to 0.085 mg/kg). Co-contamination of at least two mycotoxins was reported in 0.8% of the analyzed samples. The incidence of above maximum content/guidance level samples was 2% for feed and feed materials while almost 3-fold-higher for maize (5.8%) suggesting how mycotoxin contamination can affect some matrices more than others. Obtained data can be useful to improve official monitoring plans and therefore further raise awareness of this issue between agriculture stakeholders, healthcare entities and non-professionals.

Polar pesticides in food of animal origin: interlaboratory validation to evaluate method fitness-for-purpose of official control.

The present work reports on the design, execution and evaluation of results of an interlaboratory validation study aimed at verifying the fitness-for-purpose of a LC-MS/MS method for the detection of polar pesticides in food of animal origin in official control and monitoring programmes. To this scope, five participant laboratories, with relevant expertise, were recruited. After passing a pre-trial test, the participants were asked to analyse test samples of bovine fat, chicken eggs and cow's milk, contaminated with 11 polar pesticides (group A: Aminomethyl phosphonic acid (AMPA), cyanuric acid, ethephon, glyphosate, fosetyl aluminium, 2-hydroxyethyphosphonic acid (HEPA), maleic hydrazide, N-acetyl-glyphosate, group B: N-acetyl glufosinate (NAG), 3-methylphosphinicopropionic acid (MPP) and glufosinate ammonium) at two different levels (0.05 and 0.25 mg/kg-1 and 0.01 and 0.05 mg/kg-1 for group A and B respectively. The method was based on acidified methanol/water extraction followed by dSPE clean up with C18 sorbent. For LC-MS/MS analysis isotopically labelled standards were used for all targeted analytes. With a couple of exceptions, average recoveries ranged from 85% to 110%, with repeatability (RSDr) ranging from 3% to 25%, and reproducibility (RSDR) from 4% to 26%. The assessment by different laboratories provided also insights on key factors impacting method performance characteristics and its implementation by new users.

Preliminary Investigation about Aspergillus spp. Spread in Umbrian Avian Farms.

Among the fungi responsible for deep mycosis, the genus Aspergillus plays a predominant role both in human and veterinary medicine. From a "One Health" perspective, infections by Aspergillus spp. often represent a public health problem linked to specific occupational categories that could have a greater risk of inhaling spores and developing any respiratory disease. This preliminary investigation allowed to acquire information about the spread of Aspergillus spp. in avian livestock of the Umbria region (Central Italy), their sensitivity to antifungals, and the presence of mycotoxins in the considered farms. Environmental, feed, animal, and human samples were collected for mycological investigations; chemical analyses were also performed in feed samples. Moreover, prevalence estimated of the fungal isolates were provided for each individual farm sampled. Direct fungal identification was possible in 298 out of the 559 total samples; 162 of the samples were positive for Aspergillus spp. Mycotoxins were detected in 5 out of the 21 feed samples collected. All the aspergilli tested for antifungal susceptibility were resistant to fluconazole. The results obtained show how much the genus Aspergillus is widespread in the investigated farms; therefore, the poultry livestock represents a favorable environment for the maintenance and spread of fungal spores and their potential transmission to animals and humans.

The Current Status of Analytical Methods Applied to the Determination of Polar Pesticides in Food of Animal Origin: A Brief Review.

The use of high polar pesticides such as glyphosate and metabolites has increased due to their low cost, low persistence in the environment and high effectiveness. The use of glyphosate is currently permitted in the European Union until 15 December 2022. However, the possible toxic effects on human health and the environment are under debate. Their widespread application on various crops might lead to residues in food intended for animal consumption. For this reason, the Commission, implementing Regulation (EU) 2021/601, recommends the analyses of polar pesticides, not only in matrices of plant origin, but also in those of animal origin such as fat, liver, milk and eggs throughout the years 2022, 2023 and 2024. The determination of polar pesticides is hampered by their chemical nature, which poses challenges both in the instrumental detection (poor column retention, low molecular weight MS/MS fragments, etc.) and in the management of matrix effects, which may vary significantly from matrix to matrix within the same food commodity group. For these reasons, nowadays, there is a limited number of methods for the detection of polar pesticides in food of animal origin. This brief review discusses the different approaches for the simultaneous determination of polar pesticides in food of animal origin using both chromatographic and non-chromatographic techniques.

Monitoring Alternaria toxins in Italian food to support upcoming regulation.

The collection of occurrence data on Alternaria toxins in food and feed across the European countries is required since 2012 by the European Commission, endorsing the relevant scientific opinion by the EFSA CONTAM Panel. Within this framework, occurrence data for Alternaria toxins (Alternariol, Alternariol monomethyl ether, Tenuazonic acid, Tentoxin, and Altenuene) in 97 samples of cereal foods, tomato products, and sunflower seeds have been provided as requested by the Italian national monitoring programme (years 2017-2020). To this purpose, an LC-MS/MS method was set up and validated, obtaining fit for purpose sensitivity, recoveries (70-120%), repeatability (≤20%) and within laboratory reproducibility (≤26%). Occurrence data showed that oilseeds were the most contaminated food group with levels of Tenuazonic acid up to 16752 µg/kg and Tentoxin up to 570 µg/kg, whereas for the other mycotoxin/commodities combinations, the percentage of left censored data (below the limit of quantification) ranged from 74 to 100%.

Undertaking a New Regulatory Challenge: Monitoring of Ergot Alkaloids in Italian Food Commodities.

The present manuscript reports on monitoring data of 12 ergot alkaloids (EAs) in cereal and cereal-derived products, collected in Italy over the period 2017-2020, for official control purposes under the edge of the Commission Recommendation 2012/154/EU on the monitoring of the presence of EAs in feed and food. To these purposes, an LC-MS/MS method was set up and applied, after in-house verification of its analytical performance. Besides satisfactory recoveries and precision, the method's quantification limits proved suitable to assess the compliance of cereals and cereal-based foods with the recently issued EU maximum permitted levels (Commission Regulation 2021/1399/EU). The validity of the generated data was also evaluated through the adoption of four proficiency tests, from which acceptable z-score values (-2 ≤ z ≤ 2) were obtained. The method was then applied to analyse a total of 67 samples, collected in Italy over the period 2017-2020. The samples consisted of 18 cereal grains, 16 flours (14 of wheat and 2 of spelt) and 31 other types of cereals derivatives (including 9 for infants). Overall, the EAs analysis returned a high percentage of left-censored data (>86%). Among the positive samples, the highest contamination levels, up to 94.2 µg/kg, were found for ergocristine (12% incidence), followed by ergocristinine (7% incidence) with levels of up to 48.3 µg/kg.

A Study of the Occurrence of Aflatoxin M1 in Milk Supply Chain over a Seven-Year Period (2014-2020): Human Exposure Assessment and Risk Characterization in the Population of Central Italy.

Aflatoxin food contamination represents a rising global issue that will continue to increase due to climate change. Aflatoxin M1 (AFM1) is of high concern for the whole dairy industry. In light of AFM1's harmful potential, a human health exposure assessment and risk characterization were performed for all age populations of central Italy with regard to milk and cheese consumption by means of the margin of exposure (MOE). In total, 16,934 cow and ewe's milk samples were collected from 2014 to 2020 and analyzed by an enzyme-linked immunosorbent assay (ELISA) screening method, confirmed by high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The average concentration of AFM1 in cow's milk ranged from 0.009 to 0.015 µg/kg, while in ewe's milk, the average concentration ranged from 0.009 to 0.013 µg/kg. The average amount of AFM1 exposure ranged from 0.00005 to 0.00195 g/kg bw/day, with the main contributor represented by drinking milk, followed by the consumption of soft cheeses. A high level of public health concern related to the youngest consumers has arisen from risk characterizations highlighting the need for constant monitoring of AFM1's occurrence in milk by inspection authorities, alongside regular updates with regard to exposure assessments.