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Latin american journal of aquatic research

versión On-line ISSN 0718-560X

Lat. Am. J. Aquat. Res. vol.43 no.3 Valparaíso jul. 2015 

Short Communication


Screening of aflatoxin B1 and mycobiota related to raw materials and finished feed destined for fish

Monitoreo de aflatoxina B1 y micobiota relacionada a materias primas y alimentos terminados destinados a peces


Etelvina María Carvalho Gonçalves-Nunes1, Maria M. Gomes-Pereira1, Amilton P. Raposo-Costa1, Carlos A. da Rocha-Rosa2, Carina M. Pereyra3, Rodrigo M. Calvet1, Ana L. Alves-Marques1, Francisco Cardoso-Filho1 & Maria C. Sanches-Muratori1

1 Universidade Federal do Piauí, Centro de Ciências Agrárias, Teresina, Piauí 64049-550, Brazil
Departamento de Imunología e Microbiologia Veterinária, Instituto de Veterinária Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro 23890-000, Brazil
Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto Ruta 36 km 601, 5800 Río Cuarto. Córdoba, Argentina
Corresponding author: Etelvina Nunes (
Corresponding editor: Patricio Arana

ABSTRACT. The aim of the present study was to determine fungal genera, Aspergillus, Pénicillium and Fusarium species and aflatoxin B1 contamination from raw materials and finished feed intended for fish farm localized in Piaui, Brazil. Aspergillusflavus and P. citrinum were isolated with a high relative density from all samples. In general, a high percent of samples exceeded the levels proposed as feed hygienic quality limits (CFU g-1) according to Good Manufacture Practice. Aflatoxin B1 was analyzed by enzyme-linked immunosorbent assay. All raw materials and finished feed showed aflatoxin B1 levels. Although in this study AFB1 levels below recommended limits (20 μg kg-1) were found, it is important to emphasize the feed intake with toxin in low concentrations along time, since it produce chronic deleterious effects in animal production. This fact requires periodic monitoring to prevent the occurrence of chronic aflatoxicosis in aquaculture, to reduce the economic losses and to minimize hazards to animal health.

Keywords: aflatoxin B1, mycobiota, finished feed fish, raw materials, aquaculture.

RESUMEN. El objetivo de este estudio fue determinar géneros fúngicos, especies de Aspergillus, Pénicillium y Fusarium y la contaminación de aflatoxina B1 (AFB1) en materias primas y alimento terminado destinado a la cría de peces en el estado de Piauí, Brasil. Aspergillus flavus y P. citrinum fueron aisladas con alta densidad relativa. En general, un alto porcentaje de muestras excedieron los niveles fúngicos propuestos como límite de calidad higiénica (CFU g-1) según las buenas prácticas de manufactura. Aflatoxina B1 fue analizada por ensayos inmuno-enzimáticos. Todas las materias primas y el alimento terminado mostraron niveles de contaminación con AFB1. Aunque en este estudio los niveles de AFB1 estuvieron por debajo del límite recomendado (20 μg de toxina kg-1 de alimento), es importante enfatizar la ingesta de alimento con bajas concentraciones de toxina a lo largo del tiempo ya que produce efectos crónicos en animales de producción. Este factor requiere monitoreo periódico para prevenir la incidencia de aflatoxicosis crónica en acuacultura, reducir las pérdidas económicas y minimizar los riesgos de la salud animal.

Palabras clave: Aflatoxina B1, micobiota, alimento terminado, materia prima, acuicultura.


Aquaculture is currently the fastest growing animal production sector in the world. Brazil obtained the third position in production of aquaculture in America (FAO, 2011; MPA, 2011). This increase in aquaculture production must be supported by a corresponding increase in the production of formulated diets for the cultured aquatic animals. For most aquaculture systems, the cost of feed constitutes 30 to 60% of the farm operational costs (Wing-Keong, 2003). The improvement in the formulation and preparation of diets for fish requires the detection of factors that negatively affect the quality of feed, to avoid production losses (Conroy, 2000). Mycotoxins are fungal secondary metabolites associated with severe toxic effects to vertebrates and produced by important fungi including Aspergillus, Pénicillium, Fusarium, and Alternaria species (Kabak et al., 2006). Aflatoxins (AFs) are a group of naturally occurring mycotoxins produced by Aspergillus fungi, especially A. flavus and A. parasiticus. Aflatoxin B1 (AFB1), in particular, is toxic to many species of animals, as well as humans, and presents carcinogenic, teratogenic and mutagenic potential (IARC 2002, Sassahara et al., 2005). There are many reports on the contamination of mycotoxins in various raw materials and feeds for animals (Akande et al., 2006; Glenn, 2007; Keller et al., 2007; Martin et al., 2008; Cavaglieri et al., 2009; Pereyra et al., 2011). Several reports on evidence of the negative impact of mycotoxins in fish species were informed in the south of Brazil (Lopes et al., 2005, 2010; Vieira et al., 2006). However, there is no published information on feed contaminated with fungal genera and AFB1 intended for fish feed in northeast of Brazil. The aim of the present study was to determine fungal genera, Aspergillus, Penicillium and Fusarium species and AFB1 contamination from raw materials and finished feed intended for fish.

Raw materials and finished fish feed samples were collected from one industry in Piauí State, Brazil (5°5'20"S, 42°48'7"W). This industry was selected because it is the major provider of feed intended for fish farm in the northeast of Brazil. A total of 18 raw materials and 36 finished fish feed samples (pelleted) were sampled from January to March 2009. To ensure a correct sampling, each bag of 25 kg had a linear imaginary division in its length into three equal parts from which primary samples (1 k) from the upper layer, central layer and lower layer were collected. Three kinds of raw materials: soybean bran, corn bran, and other cereals (wheat meal, fishmeal and meat meal) in small amount intended for feed manufacturing were collected. The composition of the finished feed samples was composed of soybean bran (15%), corn bran (27%), other cereals (57.5%), and vitamin-mineral mix (0.5%). Samples were properly packed in bags and immediately sent to the laboratory. Samples were immediately processed for mycological analyses and kept at -4°C until AFB1 analyses.

Analysis of the mycobiota was made by the plate dilution spread method onto dichloran rose bengal chloranphenicol agar (DRBC), a general medium used for estimating total mycobiota (Abarca et al., 1994). Quantitative enumeration was done using the surface-spread method. Twenty-five grams of each sample were homogenized in 225 mL 0.1% peptone water solution for 30 min in an orbital shaker. Serial dilutions (10-2 to 10-3) were made and 0.1 mL aliquots were inoculated in duplicates onto the culture media. Plates were incubated at 25°C for 7 days in darkness. Only plates containing 10-100 colony-forming units (CFU) were used for counting. The results were expressed as CFU g-1 per sample. Representative colonies of Aspergillus and Penicillium were transferred for sub-culturing to tubes containing malt extract agar (MEA) whereas Fusarium spp. were transferred for sub-culturing to plates containing carnation leaf agar (CLA). Species of Aspergillus, Penicillium and Fusarium were identified according to Klich (2002); Samson et al. (2000); and Nelson et al. (1983), respectively. The results were expressed as isolation frequency (% of samples in which each genera was present) and relative density (% of isolation of each species among strains of the same genera).

A commercially available enzyme-linked immunosorbent assay (ELISA) plate Kit AgraQuant® Total Aflatoxin Assay (Romer Labs®) was applied for the extraction and quantification of AFB1. A 20 g portion of each sample was extracted with 100 mL methanol during 3 min into a blend jar. The mixture was diluted in water (1:20 v v-1) and an aliquot taken and placed into a culture plate. Detection limit of the technique was 1.0 μg kg-1 (ppb) for AFB1.

Data analyses were performed by analysis of variance. Total fungal counts data were transformed using a logarithmical function log10 (x+1) before applying the analysis of variance. The Student-Newman-Keuls (SNK) test was used to determine the significant differences between means. The analysis was conducted using SIGMA STAT program.

Table 1 show the fungal counts from raw materials and finished fish feed in DRBC culture media. In general, a high percent of raw materials and finished feed samples had counts higher than 1x104 CFU g-1.


Table 1. Fungal counts (CFU g-1) from raw materials and finished fish feed in DRBC culture
media. DRBC: dichloran rose bengal chloranphenicol, detection limit: 1x102 CFU g-1, maximum
recommended level: 1x104 CFU g-1 (GMP, 2008). (*) % samples exceeded the maximum
recommended level, a = the same letters represent similar results (P > 0.05%).


A mycological survey of the samples indicated the presence of nine genera of filamentous fungi (Table 2). Table 3 shows the relative density of isolated of Aspergillus spp., Penicillium spp., and Fusarium spp. isolated from raw materials and finished fish feed. Aspergillus flavus and P. citrinum were isolated with a high relative density from all raw materials and finished fish feed. Aspergillus parasiticus was isolated from all raw materials but not of finished fish feed. Aspergillus section nigri (A. carbonarius and A. niger aggregate) strains were isolated of soybean bran and other cereals with a relative density from 23.1 and 16.7%, respectively. Aspergillus versicolor, A. fumigatus and A. terreus strains were isolated at lower relative densities. Penicillium pinophylum was isolated only in corn bran with a relative density of 35% and P. funiculosum was present in the soybean bran and corn bran. Penicillium purpurogenum, P. corylophilum, P. miczynskii, P. implicatum and P. variable strains were isolated at lower relative densities. Fusarium verticillioides was only present in corn bran.


Table 2. Isolation frequency of fungal genera (%) from raw materials and finished
fish feed.


Table 3. Relative density (%) of Aspergillus spp., Pénicillium spp., and
spp. isolated from raw materials and finished fish feed.


Table 4 show the AFB1 levels in raw materials and finished fish feed. Aflatoxin B1 was detected in mean levels of 5.8 μg kg-1 (soybean bran), 1.1 μg kg-1 (corn bran), 7.4 μg kg-1 (other cereals) and 3.8 μg kg-1 (finished fish feed) with a frequency from 40, 33.3, 60 and 16.7%, respectively.


Table 4. Aflatoxin B1 levels in raw materials and finished fish feed. †Contamination
frequency (%): percentage of samples contaminated with AFB1.


Fungi and AFB1 contamination from raw materials and finished feed intended for fish farm were studied.

The quality of food is an essential prerequisite for obtaining optimal production results in fish production (Jakic-Domic et al., 2005). Fungal growth leads to reduction of the nutritional quality of the raw materials, and may contribute to the contamination of the finished fish feed by fungi (Cavaglieri et al., 2009). In this study, a high mycological contamination in raw materials and finished feed was found. The collected soybean bran (83.3%), corn bran (100%), other cereals (66.7%) and finished fish feed (66.7%) samples exceeded the limit 1x104 CFU g-1 that determines feed hygienic quality, according to good manufacturing practices (GMP, 2008). These results suggest a high fungal activity that could affect the palatability of feed and reduce the animal nutrients absorption, determining a low quality substrate (Ogundero, 1987; Martins & Martins, 2001). The screening of samples for fungal propagules is a useful exercise in itself as an indicator of contamination but also complements the analysis of mycotoxins that could be present.

High fungal diversity was found in raw materials and finished fish feed. All samples showed that Aspergillus spp. and Penicillium spp., the main toxigenic fungus, were the prevalent genera. Many studies have shown that most feed have species of Aspergillus and Penicillium genera as predominant in pelleted or extruded feed (Keller et al., 2007, 2008; Fernandez-Juri et al., 2009; Pereyra et al., 2009). Our results are similar to those obtained by Santos (2006) and Calvet (2008) in marine shrimp and Cardoso (2011) in finished fish feed samples. However, these authors did not analyze the raw materials.

In this study, a high frequency of Aspergillus species was found. Aspergillus flavus and A. parasiticus were the predominant species isolated. This species are important aflatoxins producers, mainly AFB1 (CAST, 2003). Santos (2006), Calvet (2008) and Cardoso (2011) reported high percentages of A. flavus in feed samples from aquaculture in northeast of Brazil. Other studies found Aspergillus section Flavi species from equine, poultry and pet-pelleted feed samples as prevalent (Keller et al., 2007; Fernandez-Juri et al., 2009; Pereyra et al., 2009). Many of the Penicillium species found (P. citrinum, P. purpurogenum, P. pinophylum, P. funiculosum, P. corylofhilum, P. miczynskii, P. implicatum and P. variabile), can produce a very wide range of toxic compounds such as citrinin and cetroeviridin (Pitt, 2004). So far, there is no information about the toxicological effects of these mycotoxins in fish. In this study, F. verticillioides was found only in corn bran. Other authors reported Fusarium species in marine shrimp and fish feed samples (Santos, 2006; Cardoso, 2011). Fusarium is a genus of field; these species do not have the ability to grow in dry feed such as pelleted and extruded feed.

All raw materials and finished feed showed aflatoxin B1 levels. This result agrees with those obtained by Hashimoto et al. (2003) in feed used for aquaculture in the region of Londrina, Paraná, which was 28.5% of total aflatoxins contaminated samples, being AFB1 the principal. In other study of commercial feeds for fish in the north and west of the State of Parana, Buck (2005) detected total aflatoxins contamination in 17% of samples with levels of 7.84 to 26.49 μg kg-1.

The biological effects of mycotoxins depend on the ingested amounts, number of occurring mycotoxins, and time of exposure and animal sensitivity. Moreover, the mycotoxin effects are not only amplified by stress production but also high in intensively reared animals (Yiannikouris & Jouany, 2002; Binder, 2007). Although in this study AFB1 levels below recommended limits (20 μg kg-1, GMP, 2008) were found, it is important to emphasize the feed intake with toxin in low concentrations along the time since produce chronic deleterious effects in animal production. El-Sayed & Khalil (2009) described that a prolonged feeding of European seabass with low levels of AFB1 (1.8 μg kg-1 body weight) causes not only serious health problems in exposed-fish, but also represents a high risk to consumers through AFB1 residues in fish musculature. This might also pose human health concerns as certain aflatoxin levels were recorded in the muscles of fish fed contaminated diets. Han et al. (2009) described that gibel carp, Carassius auratus gibelio (L.) fed with more than 10 μg AFB1 kg-1 diet showed accumulation of AFB1 residues in muscles and ovaries above the safety limitation of European Union (2 ppb).

It is very difficult to guarantee the absence of mycotoxins in aquaculture feeds even when appropriate measures are taken, such as good screening programs, selection of high quality raw materials and feed ingredients, and good storage conditions. It is therefore imperative to find effective ways of managing the risks posed by mycotoxin contamination.

This is the first report that provides information on the fungal and AFB1 contamination in raw materials and finished feed intended for fish farm localized in Piauí, Brazil. Future studies could be conducted to analyze other mycotoxins, as fuminisins and citrinin.


This work was carried out with grants from CAPES, Brazil. The authors thanks Alverne Barbosa for his assistance in getting the fish feed samples.



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Received: 20 May 2014;
Accepted: 26 February 2015


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