Aquatic products represent an important food source for animal and human consumption, the rising demand of which had led to a fast development of aquaculture (Myhr and Dalmo 2005). The primary objective in fish nutrition is to provide a nutritionally balanced mixture of ingredients as finished feed to support the maintenance, growth, reproductive performance, flesh quality, and health of the animals at an acceptable cost (NRC 1993). The quality of the products used in feeds for farmed fish has become a limiting factor for activity because these feeds are ideal substrates for the growth of fungi, which, under favorable conditions, may favor the synthesis of mycotoxins. Production of these toxic metabolites can occur during the growth of the crop, during post-harvest storage, or during the storage of the compounded feed (CAST Council for Agricultural Science and Technology 2003). The use of plant-based ingredients, such as corn, in aquafeeds enhances both the risk of introducing mycotoxins into the feed at the point of feed manufacturing and mycotoxin production during storage of compounded feed (Spring and Fegan 2005).
Fumonisin is of concern to the aquaculture industry because it commonly contaminates corn and its by-products. There are several publications on the toxic effects of fumonisin B1 (FB1) in different fish species (Yildirim et al.2000; Nguyen et al. 2003; Wang et al. 1991). Aflatoxin was the first of the mycotoxins to be investigated in aquaculture. As in other animal species, aflatoxin exerts carcinogenic effects in fish (Spring and Fegan 2005). Different researchers demonstrated the presence of aflatoxin B1 (AFB1) in shrimp and fish feed (Bautista et al. 1994; Abdelhamid et al. 1998). Although ochratoxin A (OTA) has not been studied to the same extent as AFB1 in aquaculture, there are several studies demonstrating the toxic effects of this toxin in different fish species (Manning et al. 2003; Shalaby 2004; Moussa and Khattab 2003).
The number of studies addressing the effects of aflatoxins in aquatic species is very limited. The difficulty in accurately diagnosing aflatoxicosis in fish may in part explain the lack of information regarding the incidence of aflatoxicosis in farmed aquatic species. Initial findings associated with aflatoxicosis in fish include pale gills, liver damage, poor growth rates, and immune suppression. In fish, the role of fumonisins as toxic agents remains unclear. On one hand, minimal adverse effects have been reported in channel catfish fed withFusarium verticillioides culture material containing 313 ppm of FB1 for 5 weeks (Brown et al. 1994). On the other hand, for the same fish species, dietary levels of FB1 of 20 ppm or above have been shown to result in lower weight gain and significant decrease in hematocrit and red and white blood cells than those at lower doses (Lumlertdacha et al. 1995). In rainbow trout, pathological signs of ochratoxicosis included liver necrosis, pale and swollen kidneys, and high mortality (Hendricks 1994).
Continuous studies regarding the monitoring of these toxins in finished feed to be used as animal feed are being performed worldwide (Magnoli et al. 2002; Akande et al. 2006; Keller et al. 2007; Glenn 2007; Martins et al. 2008; Cavaglieri et al. 2009; Pereyra et al. 2011). However, there is little information on feed contaminated with FB1, AFB1, and OTA intended for fish feed. The aim of the present study was to determine species of the fungal genera Aspergillus,Penicillium, and Fusarium and FB1, AFB1, and OTA contamination from finished feed intended for fish farms.
Methods
Sample source
Finished feed samples were collected from two tilapia farms. The samples came from the most important region of the Rio de Janeiro State where aquaculture practice is developed. The farms were representative as much in size and composition as in the kind of storage method applied. A total of 60 pelleted samples were sampled between September 2009 and August 2010. 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 kg) from the upper layer, central layer, and lower layer were collected. Samples were properly packed in bags and immediately sent to the laboratory. Samples were immediately processed for physical and mycological analyses and kept at −4°C until FB1, AFB1, and OTA analyses.
Physical properties of samples
Water activity (aW) determinations were carried out using AQUALAB CX2 (Decagon, Devices, Inc., Pullman, WA, USA). The device was calibrated using appropriate saline solutions in the work interval - according to procedures manual - before sample measurements.
Mycological analysis
The quantitative enumeration of fungi as colony-forming units per gram of feed (CFU/g) was performed using the surface spread method described by Pitt and Hocking (1997). Ten grams of each sample was homogenized in 90 mL of distilled water solution for 30 min in an orbital shaker. Serial dilutions (10−2 to 10−4) were made, and 0.1-mL aliquots were inoculated in triplicate onto the media dichloran rose bengal chloramphenicol agar (DRBC) for estimating total culturable fungi (Abarca et al. 1994) and dichloran 18% glycerol agar (DG18) that favors xerophilic fungus development. The plates were incubated at 25°C for 5 to 7 days. All samples were also inoculated onto Nash and Snyder agar to enumerate Fusarium species (Nelson et al. 1983). Nash-Snyder plates were incubated at 24°C for 7 days under a 12-h cold white/12-h black fluorescent light photoperiod. Only plates containing 10 to 100 CFU were used for counting, with results expressed as CFU/g of sample. On the last day of incubation, individual CFU/g counts for each colony type considered to be different were recorded. Colonies representative ofAspergillus and Penicillium were transferred for sub-culturing to tubes containing malt extract agar, whereas Fusarium spp. were transferred for sub-culturing to plates containing carnation leaf agar.
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 (percentage of samples in which each genus was present) and relative density (percentage of isolation of each species among the same genus).
Fumonisin B1 detection and quantification
A commercially available enzyme-linked immunosorbent assay plate kit, AgraQuant® Total Fumonisin Assay (Romer Labs®, Campinas, Brazil), was applied for the extraction and quantification of FB1. Mycotoxin extraction and testing were performed according to the manufacturer's instructions. A 20-g portion of each sample was extracted with 100 mL of methanol for 3 min into a blend jar. The mixture was diluted in water (1:20 v/v) and an aliquot taken and placed into a culture plate. Detection limit of the technique was 0.2 μg/g.
Aflatoxin B1 and ochratoxin A determination
The extraction of AFB1 and OTA was determined according to Soares and Rodriguez-Amaya (1989). Detection and quantification evaluation was made by thin-layer chromatography. Detection limits of the used techniques were 0.003 and 0.005 μg/g for AFB1 and OTA, respectively.
Statistical analyses
Data analyses were performed by analysis of variance. Data on total fungal counts were transformed using the logarithmical function log10 (x + 1) before applying the analysis of variance. The LSD test was used to determine the significant differences between means. The analysis was conducted using the BioEstat 5.0 program.
Results
Water activity content
The fish feed samples tested had aW mean values of 0.598 ± 0.053.
Mycological isolation and identification
Fungal total counts on the DRBC and DG18 media ranged from <1 × 102 to 4.7 × 104 and from < 1 × 102 to 2.7 × 104 CFU/g, respectively. The mean fungal total counts were 2.9 × 103 ± 0.6 × 103 (medium ± standard deviation (SD)) in DRBC and 2.2 × 103 ± 3.8 ×103 (medium ± SD) in DG18 medium. Fusariumspp. were not present on the SNA medium.
Based on the frequency of fungal genera (%), eight filamentous genera were obtained. Cladosporium (85%) was predominant. Among the toxigenic genera, the genus Aspergillus (68%) was the most prevalent, followed byPenicillium species (60%). Other genera were isolated at lower frequencies such as Wallemia, Eurotium, Aureobasidium, Mucor, and Nigrospora species (Figure 1).
Figure 1 Frequency (%) of fungal genera isolated from feeds intended for fish farm.
Figure 2 shows the relative density (%) of Aspergillus spp.; Aspergillus nigeraggregate and Aspergillus flavus were the most prevalent, followed byAspergillus versicolor. Aspergillus fumigatus, Aspergillus candidus, andAspergillus oryzae were found at low relative densities.
Figure 2 Relative density (%) ofAspergillusspecies in feeds intended for fish farm.
Figure 3 shows the relative density (%) of Penicillium spp. A high diversity ofPenicillium species was found. Penicillium citrinum was the most prevalent, followed by Penicillium glabrum.
Figure 3 Relative density (%) ofPenicilliumspecies in feeds intended for fish farm.
Fumonisin B1 determination
Samples contaminated with FB1 levels (90%) ranged from 0.3 to 4.94 μg/g and 2.6 μg/g as medium level.
Aflatoxin B1 and ochratoxin A
Fifty-five percent and 3.3% of samples were individually contaminated with AFB1 and OTA, respectively, in levels detectable but not quantifiable for the technique used.
Co-occurrence
Of total samples proved to have AFB1 (55%) and FB1 (98%), 50% showed co-occurrence of these mycotoxins. The 3.3% of the samples presented co-occurrence of the three mycotoxins analyzed.
Discussion
The mycobiota and mycotoxin contamination present in finished feeds intended for fish was studied. In this study, moderate levels of colony counts were found. These results were similar to those obtained in feed from marine shrimp studied in Piaui State (Santos 2006). However, in our study, 10% of samples analyzed exceeded the levels proposed as hygienic feed quality limits in DRBC (1 × 104CFU/g; Good Manufacture Practice 2008). The DRBC medium, used to estimate total culturable fungi (Abarca et al. 1994), and DG18, which favors xerophilic fungus development, revealed high counts of mold, and this corresponds to dry feed as is being studied here.
Fungal growth leads to the reduction of the nutritional quality of fish feed samples that could affect the palatability of feed and reduce the animal's nutrient absorption, determining a low-quality substrate and an improper storage. Little published information about fungal contamination in feeds for aquaculture in Brazil has been found. Cardoso (2011) obtained counts over the proposed limits in fish feed samples in the north of Brazil, reporting high counts in 56% and 11% of samples from juvenile and growing stages, respectively. Nunes (2009), who also studied fish feed, showed that 67% feed samples had counts above the proposed limit by Good Manufacture Practice (2008).
Many studies have shown that most feeds have species of the generaAspergillus and Penicillium as predominant in pelleted feed (Keller et al.2007, 2008; Pereyra et al. 2009; Fernández Juri et al. 2009). Our results showed that Aspergillus and Penicillium species had the highest isolation frequencies. These results are similar to those obtained by Santos (2006) and Cardoso (2011) in marine shrimp and fish feed samples. However, these authors also reported Fusarium species in fish feed samples.
In this study, A. niger aggregate and A. flavus were the most prevalent species isolated from these types of samples, followed by A. versicolor. Santos (2006), Nunes (2009), and Cardoso (2011) reported high percentages of A. flavus in feed samples from aquaculture in the north of Brazil. Other studies foundAspergillus section Flavi species followed by A. niger aggregate from equine, poultry, and pet pelleted feed samples as prevalent (Keller et al. 2007; Pereyra et al. 2009; Fernández Juri et al. 2009).
Many of the Penicillium species found (P. citrinum, P. glabrum, P. minioluteum, P. citreonigrum, P. corylophilum, P. italicum, and P. variabile) can produce a very wide range of toxic compounds such as citrinin and citreoviridin (Pitt 2004). So far, there is no information about the toxicological effects of these mycotoxins in fish.
Some researchers have stressed the importance of fungal determination in order to provide information about which mycotoxin could be present (Dalcero et al. 1997, 1998; Magnoli et al. 2002). In this work, the frequency ofA. flavus and AFB1 contamination showed a positive relation, whereas both A. niger aggregate with OTA and F. verticillioides with FB1 contamination showed a negative relation. In this work, F. verticillioides strains were not isolated; however, levels of FB1 in 98% of feed samples were found. Fusariumis a genus of field fungi, of which species do not have the ability to grow in dry feed, but it is probable that FB1 has been produced in the field and comes with the raw material that composes the finished feed.
The pelleted ones are produced in a humid process with heat, where the product reaches a temperature between 85°C and 90°C, with 15.5% to 17% of humidity, for 30 to 45 s. Considering the process of feed elaboration, where the feed is subjected to high temperatures, the inactivation of fungal propagules could be expected and not FB1, which is inactivated at 150°C (WHO World Health Organization 2001).
Fusarium toxins are important not so much for their acute effects as for the chronic syndromes reported worldwide. Dietary fumonisin at 20 ppm has been shown to reduce growth rate in catfish with an average weight of 1.5 g (Yildirim et al. 2000). Two-year-old catfish only had reduced weight gain when exposed to 80 ppm of fumonisin. Toxic effects of fumonisin have been reported in the Nile tilapia at concentrations similar to those in catfish (Nguyen et al. 2003). A survey of catfish feed ingredients in Alabama and Mississippi in the USA revealed that 80% of the corn samples contained detectable levels of fumonisin in concentrations that ranged from 1.3 to 10 ppm (Lumlertdacha and Lovell 1995). In this work, the principal ingredient in the fish feed also was the corn. In our study, FB1 levels in the fish feed samples did not exceed the permissible limit from animal feed (5 ppm; Good Manufacture Practice 2008). However, chronic levels could cause adverse effects in fish. According to the European Union recommendations (2003 and 2006), the tolerable limits for fumonisins (FB1 + FB2), AFB1, and OTA are 10, 0.02, and 0.25 ppm, respectively, in animal feed (except dairy cows for AFB1).
The presence of AFB1 in this work shows a frequency higher than that found by Hashimoto et al. (2003) in feed used for aquaculture in the region of Londrina, Paraná, which was 28.5% of total aflatoxin-contaminated samples, AFB1 being the principal. In another study of commercial feeds for fish in the north and west of the state of Paraná, Buck (2005) detected total aflatoxin contamination in 17% of samples. The frequency contamination of OTA is similar to that found by Buck (2005), where 4% of feed samples analyzed showed only OTA.
Exposure to mycotoxins often occurs simultaneously. The present study has shown the simultaneous occurrence of three carcinogenic mycotoxins, FB1, AFB1, and OTA, in feed intended for fish farms. There are no studies published on feed contamination simultaneous with these three toxins intended for fish farms in Brazil.
The biological effects of mycotoxins depend on the ingested amounts, number of occurring mycotoxins, time of exposure, and animal sensitivity. Moreover, the mycotoxins' effects are not only amplified by stress production but also increased in intensively reared animals (Yiannikouris and Jouany 2002; Binder 2007).
Conclusions
The simultaneous occurrence of FB1, AFB1, and OTA emphasizes the need for further research in the area to better assess the risk to the health of fish farms and their implications for the health of consumers of this meat. The first important step in controlling the fungal and mycotoxin contamination in finished feed is to control them in the raw materials from which the feed is prepared in order to prevent the occurrence of mycotoxicosis in aquaculture, to reduce economic losses, and to minimize hazards to human health.
