Article pubs.acs.org/JAFC
Natural Occurrence of Emerging Fusarium Mycotoxins in Feed and Fish from Aquaculture Josefa Tolosa,* Guillermina Font, Jordi Mañes, and Emilia Ferrer Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Avenue Vicent Andrés Estellés s/n, 46100 Burjassot, Spain ABSTRACT: 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. KEYWORDS: mycotoxins, feed, farmed fish, beauvericin, enniatins
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INTRODUCTION Mycotoxins are toxic compounds produced by filamentous fungi, such as Fusarium spp., Aspergillus spp., and Penicillium spp. Trichothecenes (TC), zearalenone (ZEN), and fumonisins (FUM) are the major Fusarium mycotoxins occurring on a worldwide basis in cereal grains, pasta, nuts, and animal feeds and forages.1−3 However, this genus also produces other mycotoxins called emerging Fusarium mycotoxins because they were discovered after other Fusarium mycotoxins. The most studied emerging Fusarium mycotoxins in foods (mainly cereal-based foods) are enniatins (ENs) and beauvericin (BEA)4 (Figure 1). In vitro assays have demonstrated that ENs produce reactive oxygen species (ROS) that induce lipid peroxidation oxidative (LPO) damage, apoptosis, and necrosis via the mitochondrial pathway.5 Further studies demonstrated that ENs and BEA induced cell death by apoptosis and DNA damage.6 However, in the scientific literature there are few in vivo studies related to the biological activity of ENs and BEA.7 Recently, two studies carried out in our laboratory collected data on tissue distribution of ENA and its immunologic effects in Wistar rats.8,9 Furthermore, Devreese et al.10 and Ivanova et al.11 reported the presence of ENs in pig and poultry plasma, respectively. Research concerning the adverse effects produced by mycotoxins on animal performance and health has been focused on terrestrial livestock species.12,13 Direct consequences of mycotoxin feed intake for terrestrial animals include reduced feed intake, feed refusal, poor feed conversion, diminished body weight gain, increased disease incidence, and reduced reproductive capacities, which lead to economic losses.14,15 Moreover, mycotoxin contamination of animal feeds represents a hazard to human and animal health due to potential transmission to meat, milk, and byproducts.16−20 In this way, the occurrence of Fusarium toxins in aquaculture remains mostly unknown, and research is needed.21 In the fish organism, mycotoxins induce several disorders,22 such as changes in nutrient resorption, induce cell and organ alterations, and produce functional and morphological effects, which, in more © XXXX American Chemical Society
severe cases, lead to mortality. Thereby, losses in aquaculture caused by mycotoxins in feed can be significant. Direct loss is a consequence of increased mortality, and indirect loss is a result of production decrease and the occurrence of secondary diseases.23 Today, aquaculture accounts for more than a fourth of total world fisheries.24 In 2010, world production of cultivated edible aquatic species was 60 million tonnes, which represented an increase of 7.5% over 2009. The average annual per capita consumption (in the world) of cultivated species increased, reaching 8.7 kg in 2010. Moreover, sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) have a high production, >100 million tons in 2010.25 Cereals as energy and leguminose as protein feedstuffs are the main part of feed (up to 90%) for all kinds and categories of fish.23 Especially wheat is often used for fish feed production due to its high protein content and its benefits in preserving the pellet shape during feed processing. Thus, the amount of wheat in fish feed varies considerably, ranging from approximately 15 to 27% for carnivorous fish, whereas feed for cyprinids usually contains 20−70%.21,25 In addition, in feeds for farmed fish, fish oil and fish meal are being replaced by oilseeds and vegetable oils, including palm oils,26 which are described by some authors as raw materials contaminated with mycotoxins such as aflatoxins (AFs) and FUM.27 In this way, the use of such ingredients inevitably leads to the contamination of the final mixed feed with molds.28 Furthermore, in feed manufacturing processes, different raw materials from different origins are mixed together, producing a totally new matrix with a new risk profile.29 Thus, fish in aquaculture are commonly exposed to feedborne mycotoxins, such as AFs, ochratoxin A (OTA), ZEN, and TC, the most important mycotoxins described in fish.23,30 Received: August 6, 2014 Revised: November 27, 2014 Accepted: November 28, 2014
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dx.doi.org/10.1021/jf5036838 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Figure 1. Structure of BEA and ENs. ENA, enniatin A; ENA1, enniatin A1; ENB, enniatin B; ENB1, enniatin B1; BEA, beauvericin. The supernatant was purified using C18 cartridges (Waters, Milford, MA, USA) by applying a slight vacuum. Cartridges were previously conditioned with 5 mL of MeOH and 5 mL of deonized water, washed with 5 mL of water, and then vacuum-dried for 5 min. Finally, mycotoxins were eluted with 5 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM ammonium formate). The extract was evaporated to dryness at 30 °C using a Büchi Rotavapor R-200 (Flawil, Switzerland). Then, the solution was reconstituted with 10 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM of ammonium formate) and evaporated to dryness at 30 °C using a multisample Turbovap LV Evaporator (Zymark, Hopkinton, MA, USA). The solution was reconstituted in 1 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM of ammonium formate) and filtered through a 13 mm/0.22 μm nylon filter prior injection into the LC-MS/MS-LIT system. The samples were extracted in triplicate. Extraction with Ultrasonic Bath. For fish samples, a 10 g aliquot was homogenized with 50 mL of acetonitrile for 30 min at 30 °C using a Branson 5200 ultrasonic bath (Branson Ultrasonic Corp., Danbury, CT, USA). The extract was centrifuged at 3540g for 15 min at 5 °C. The supernatant was purified using C18 cartridges (Waters) by applying a slight vacuum. Cartridges were previously conditioned with 5 mL of MeOH and 5 mL of deonized water, washed with 5 mL of water, and then vacuum-dried for 5 min. Finally, mycotoxins were eluted with 5 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM ammonium formate). The extract was evaporated to dryness at 30 °C using a Büchi Rotavapor R-200. The solution was reconstituted in 10 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM of ammonium formate) and transferred to a 15 mL conical tube to be evaporated to dryness at 30 °C using a multisample Turbovap LV Evaporator (Zymark). After solvent evaporation, the solution was reconstituted with 1 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM of ammonium formate) and placed again in the ultrasonic bath (30 min, 30 °C). Then, the solution was washed with hexane to eliminate fat particles in liver and viscera samples and filtered through a 13 mm/0.22 μm nylon filter (Membrane Solutions) prior to injection into the LC-MS/MS-LIT system. The samples were extracted in triplicate. Extraction with Microwave. Fish samples (5 g) were extracted with 50 mL of acetonitrile during 3 min at 250 W using a microwave Saivod WP700P17-2 (Manisa, Turkey). The extract was centrifuged at 3540g for 15 min at 5 °C. The supernatant was purified using C18 cartridges (Waters) by applying a slight vacuum. Cartridges were previously conditioned with 5 mL of MeOH and 5 mL of deonized water, washed with 5 mL of water, and then vacuum-dried for 5 min. Finally, mycotoxins were eluted with 5 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM ammonium formate). The extract was evaporated to dryness at 30 °C using a Büchi Rotavapor R-200. Then, the solution was reconstituted with 10 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM of ammonium formate) and evaporated to dryness at 30 °C using a multisample Turbovap LV evaporator (Zymark). The solution was reconstituted in 1 mL of AcN/MeOH 50:50 v/v (MeOH with 20 mM ammonium formate) and filtered through a 13 mm/0.22 μm nylon filter (Membrane Solutions) prior to injection into the LC-MS/ MS-LIT system. The samples were extracted in triplicate. Instrumental and Chromatographic Conditions. LC-MS/ MS-LIT analyses were conducted on a system consisting of an Agilent
Because of the susceptibility to mycotoxin contamination in mixed feeds and the presence of these mycotoxins in animal tissues, the aim of this work was to develop a new rapid, sensitive, and reproducible analytical strategy for the determination of ENs and BEA in feeds for farmed fish and fish tissues by LC-MS/ MS-LIT.
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MATERIALS AND METHODS
Chemical and Reagents. All solvents (acetonitrile and methanol) were purchased from Merck (Darmstadt, Germany). Deionized water (
