Monitoring of Animal Feed Contamination by Mycotoxins: Results of Five Years of Official Control by an Accredited Italian Laboratory.

Mycotoxin contamination of animal feed is a complex issue in both animal wellness and food safety. The most diffused mycotoxins subject to the official control of animal feed are Aflatoxin B1 (AF), Zearalenone (ZEA), Deoxynivalenol (DON), Ochratoxin A (OCRA), Fumonisins (FUMO), and T-2/HT-2 toxins. This work describes the results of five years of monitoring focused on the evaluation of mycotoxin contamination of animal feed. Analytical determinations were carried out by means of accredited ELISA. The obtained results showed a non-alarming scenario, with several samples resulting as "non-compliant" according to the Maximum Residue Limits (MRLs) set in European Regulation No. 574/2011. Out of 722 analyzed samples coming from 2 Italian regions, Apulia and Basilicata, 14 samples were characterized by mycotoxin concentrations higher than related MRL; in particular, 5, 4, and 5 non-compliant samples for DON, AF, and ZEA, respectively. This study also evaluated the possible correlations between mycotoxin type and feed use with a special focus on animal sensitivity to mycotoxins.

Monitoring of polyphosphate levels in animal source products collected in Italy by means of ion chromatography with suppressed conductivity detection.

Polyphosphates (PPs) constitute a class of food additives widely used due to their ability to exercise different useful activities. The food safety concern about the use of PPs in food is both the possible non-declared addition and some health effects, such as bile duct stones, decrease in oligo-element absorption, and allergic reactions in susceptible people. In this study, an analytical method based on ion chromatography with conductivity detection was applied for the detection and quantification of PPs in 238 samples of animal-derived products such as meat, dairy, and fish products. A contribution to risk assessment was also included. The monitoring confirmed the absence of non-compliant results. All concentrations of PPs were indeed lower than the legal limits set in European Regulation No. 1333/2008. Moreover, no residue of PPs was detected (> limit of quantification: 0.09 g kg-1) in samples where it was not reported on the product label. No PPs were detected in mollusks, meat-based preparations, semi-ripened, unripened, and spun paste cheese, while they are widely used in surimi, with concentrations in the range of 0.1-0.5 g kg-1. The highest concentrations were quantified in a würstel sample (4.7±0.3 g kg-1) and a spreadable cheese sample (8.9±0.7 g kg-1). Considering that the high exposure scenario together with a very susceptible population group (toddlers) were taken into account for this risk exposure study and that the highest admissible daily intake obtained was equal to 10.4%, the assessment demonstrated that the actual use of PPs in food does not pose a risk for food safety.

Determination of Nitrate and Nitrite in Swiss Chard (Beta vulgaris L. subsp. vulgaris) and Wild Rocket (Diplotaxis tenuifolia (L.) DC.) and Food Safety Evaluations.

Nitrite and nitrate levels in vegetables are a matter of concern due to their toxicity at high levels and nitrate high accumulation. Moreover, there is a lack of knowledge about their levels in some types of widely consumed vegetables such as chard and rocket. In this study, 124 Swiss chard and wild rocket samples were analyzed for determining nitrite and nitrate using validated and accredited analytical methods by ion chromatography with conductivity detection. High nitrite concentrations, up to 219.5 mg kg−1 f.w., were detected in one Swiss chard and three wild rocket samples. One Margin of Safety (MoS) value was <1. Regarding nitrate, in Swiss chard samples the mean concentration (2522.6 mg kg−1) was slightly higher than those reported in the literature for spinach and lettuce. No MoS was <1, but 83% of values were <100. Nitrate concentrations higher than the legal limit were quantified in 11 rucola samples. The verification of 25% of wild rocket samples with nitrate concentration higher than the legal limit confirmed the need for official control. This study also suggests the introduction of legal limits for nitrite/nitrate in Swiss chard and nitrite in wild rocket.

Effect of different cooking treatments on the residual level of sulphites in shrimps.

Sulphiting agents (or sulphites) are a class of food additives identified in Europe by codes E220-E228. Their addition in crustaceans is permitted with specific legal limits for avoiding the so-called "blackspot" that is a defect that compromises the marketability of these products. High levels of ingested sulphites may cause pseudoallergenic reactions in susceptible people. Moreover, they can exercise mutagenic and citotoxic effects other that destroy some vitamins such as thiamine, folic acid, nicotinamide and pyridoxal. The residual level of sulphites in crustaceans can be considerably affected by the specific method of cooking. In this study, 5 traditional procedures of cooking - grilling, oven, frying, steaming and stewed cooking - were compared to verify their effect on the residual concentration of sulphites in shrimp samples. The analytical determination was carried out using a fully validated and accredited analytical method by ion chromatography with conductivity detection. The results demonstrated that cooking leads to the decrease of sulphites levels in the products, with the highest percentage of reduction (55.3%) obtained by steaming and the lowest using oven (13.9%). The results of this study confirm that the specific method of cooking should be taken into account during "total diet studies" and risk assessment for appraising the effective number of sulphites ingested from crustaceans consumption.

Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish.

Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3−8 µg kg−1) were fulfilled. The matrix effect, ranging from −9 to 19%, allowed the use of a calibration curve in solvent (3−320 µg kg−1 in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories.

Sulfites in meat: Occurrence, activity, toxicity, regulation, and detection. A comprehensive review.

Sulfites are a class of chemical compounds, SO2 releasers, widely used as additives in food industry, due to their antimicrobial, color stabilizing, antibrowning, and antioxidant properties. As the results of these pleiotropic functions they can be added to a broad range of products including dried fruits and vegetables, seafood, juices, alcoholic and nonalcoholic beverage, and in few meat products. Sulfites ingestion has been correlated with several adverse and toxic reactions, such as hypersensitivity, allergic diseases, vitamin deficiency, and may lead to dysbiotic events of gut and oral microbiota. In many countries, these additives are closely regulated and in meat products the legislation restricts their usage. Several studies have been conducted to investigate the sulfites contents in meat and meat products, and many of them have revealed that some meat preparations represent one of the main sources of SO2 exposure, especially in adults and young people. This review discusses properties, technological functions, regulation, and health implications of sulfites in meat-based foods, and lays a special emphasis on the chemical mechanisms involved in their interactions with organic and inorganic meat components.

Quantitative determination by screening ELISA and HPLC-MS/MS of microcystins LR, LY, LA, YR, RR, LF, LW, and nodularin in the water of Occhito lake and crops.

The occurrence of harmful cyanobacterial blooms in surface waters is often accompanied by the production of a variety of cyanotoxins, and these toxins are designed to target in humans specific organs on which they act. When introduced into the soil ecosystem by spray irrigation of crops, they may affect the same molecular pathways in plants having identical or similar target organs, tissues, cells, or biomolecules. There are also several indications that terrestrial plants, including crops, can bioaccumulate cyanotoxins and present, therefore, potential health hazards for humans. During this project, for monitoring purposes, water samples were collected from lake Occhito, in which there was an algal bloom (Planktothrix rubescens) in 2009, and from three tanks which acted as hydraulic junctions. In addition, crop samples irrigated with water from the three tanks mentioned above were also picked. Finally, the characterization of principal cyanobacteria was performed, to determine the presence of cyanotoxins such as microcystins and validate a method of screening ELISA for the determination of microcystins in vegetable samples and a confirmatory method by HPLC-ESI-MS/MS. Graphical abstract Occhito lake (left), microcystin LR (center), Tomato field in Foggia (right); figures below: ELISA (left), HPLC-MS/MS (right).