Josefa Tolosa

A preliminary study in Wistar rats with enniatin A contaminated feed

Abstract A 28-day repeated dose preliminary assay, using enniatin A naturally contaminated feed through microbial fermentation by a Fusarium tricinctum strain, was carried out employing 2-month-old female Wistar rats as in vivo experimental model. In order to simulate a physiological test of a toxic compound naturally produced by fungi, five treated animals were fed during 28 days with fermented feed. As control group, five rats were fed with standard feed. At the 28th day, blood samples were collected for biochemical analysis and the gastrointestinal tract, liver and kidneys were removed from each rat for enniatin A detection and quantitation. Digesta were collected from stomach, duodenum, jejunum, ileum and colon. Enniatin A present in organs and in biological fluids was analyzed by liquid chromatography-diode array detector (LC-DAD) and confirmed by LC-mass spectrometry linear ion trap (MS-LIT); also several serum biochemical parameters and a histological analysis of the duodenal tract were performed. No adverse effects were found in any treated rat at the enniatin A concentration (20.91 mg/kg bw/day) tested during the 28-day experiment. Enniatin A quantitation in biological fluids ranged from 1.50 to 9.00 mg/kg, whereas in the gastrointestinal organs the enniatin A concentration ranged from 2.50 to 23.00 mg/kg. The high enniatin A concentration found in jejunum liquid and tissue points to them as an absorption area. Finally, two enniatin A degradation products were identified in duodenum, jejunum and colon content, probably produced by gut microflora.

Natural occurrence of emerging Fusarium mycotoxins in feed and fish from aquaculture.

A new analytical method for the simultaneous determination of enniatins (ENs) and beauvericin (BEA) in fish feed and fish tissues by liquid chromatography coupled to mass spectrometry with linear ion trap (LC-MS/MS-LIT) was developed. Results showed that the developed method is precise and sensitive. The presence of emerging Fusarium mycotoxins, ENs and BEA, was determined in samples of aquaculture fish and feed for farmed fish, showing that all feed samples analyzed were contaminated with mycotoxins, with 100% coexistence. In aquacultured fish samples, the highest incidence was found in edible muscle and liver. As for the exposure assessment calculated, it was found that average consumer intake was lower than tolerable daily intake (TDI) values for other Fusarium mycotoxins.

Multi‐Mycotoxin Analysis in Durum Wheat Pasta by Liquid Chromatography Coupled to Quadrupole Orbitrap Mass Spectrometry

A simple and rapid multi-mycotoxin method for the determination of 17 mycotoxins simultaneously is described in the present survey on durum and soft wheat pasta samples. Mycotoxins included in the study were those mainly reported in cereal samples: ochratoxin-A (OTA), aflatoxin B1 (AFB1), zearalenone (ZON), deoxynivalenol (DON), 3-and 15-acetyl-deoxynivalenol (3-AcDON and 15-AcDON), nivalenol (NIV), neosolaniol (NEO), fusarenon-X, (FUS-X), T-2 toxin (T-2) and HT-2 toxin (HT-2), fumonisin B1 and B2 (FB1 and FB2), and four emerging mycotoxins: three enniatins (ENA, ENA1, and ENB), and beauvericin (BEA). Twenty-nine samples were analyzed to provide an overview on mycotoxin presence: 27 samples of durum wheat pasta, and two samples of baby food. Analytical results concluded that trichothecenes showed the highest incidence, mainly DON, NIV, and HT-2 toxin, followed by ZON and ENB, while NEO, FUS-X, OTA, AFB1, and FUM were not detected in any sample. The highest contents corresponded to ENB and ranged from 91.15 µg/kg to 710.90 µg/kg.

Multimycotoxin analysis in water and fish plasma by liquid chromatography-tandem mass spectrometry.

High performance liquid chromatography-mass spectrometry was used for the determination of 15 mycotoxins in water and fish plasma samples, including aflatoxins, fumonisins, ochratoxin A, sterigmatocistin, fusarenon-X and emerging Fusarium mycotoxins. In this work, dispersive liquid-liquid microextraction (DLLME) was assessed as a sample treatment for the simultaneous extraction of mycotoxins. Results showed differences in recovery assays when different extraction solvents were employed. Ethyl acetate showed better recoveries for the major part of mycotoxins analyzed, except for aflatoxins B2, G1 and G2, which showed better recoveries when employing chloroform as extractant solvent. Fumonisins and beauvericin exhibited low recoveries in both water and plasma. This method was validated according to guidelines established by European Commission and has shown to be suitable to be applied in dietary and/or toxicokinetic studies in fish where is necessary to check mycotoxin contents in rearing water and fish plasma.

Mitigation of enniatins in edible fish tissues by thermal processes and identification of degradation products

Emerging mycotoxins, such as enniatins and beauvericin, are common contaminants in vegetal matrices, but recently, the occurrence of mycotoxins in foodstuffs from animal origin has been also reported as they can be present in edible tissues of animals fed with contaminated feedstuffs. Sea bass, sea bream, Atlantic salmon and rainbow trout from aquaculture analyzed in the present survey showed contamination by emerging Fusarium mycotoxins enniatins (ENs). ENs were extracted from raw and cooked fish with acetonitrile and analyzed by Liquid Chromatography coupled to Mass Spectrometry. In this study, the stability of ENs was evaluated during food processing by the application of different cooking methods (broiling, boiling, microwaving and baking treatments). All treated samples showed a reduction in mycotoxin levels with different percentages depending on the type of EN and the fish species. Thus, the reduction obtained ranged from 30 to 100%. The thermal treatments have shown to be a good strategy to mitigate ENs content in edible fish tissues. On the other hand, some ENs degradation products originated during the application of thermal treatments were identified.