Mycotoxigenic Fungi and Natural Co-Occurrence of Mycotoxins in Rainbow Trout (Oncorhynchus mykiss) Feeds

 

 

Co-occurrence of at least two out of six mycotoxins was recorded in 93% (26/28) of samples analyzed. Co-occurrence of six mycotoxins was determined in 7% (2/28) of the samples.

 

 

The raw material used in animal feed production is usually the source of molds and mycotoxins [48]. In this study, it was found that 26 out of 28 samples analyzed were contaminated with fungi, whereas all of the samples with mycotoxins.

 

Moderate levels of fungal counts were determined on DRBC (<10 to 4.2 × 104 UFC/g) and DG18 (xerophilic fungi) (<10 to 3.6 × 104 UFC/g). Similar results were reported in Brazil on finished fish feeds [40,45]. However, Nunes [49] reported values lower than 1 × 107 UFC/g on ration formulated for fish feed. Regarding fungal count, 1 × 104 UFC/g on DRBC was proposed as the limit of hygienic feed quality [50]. According to our study, 10.7% of the samples were not in agreement with the limit. Barbosa et al. [45] also reported that 10% of the samples analyzed by them exceeded the limit of hygienic quality proposed. Cardoso [40] reported 56% of juvenile stage and 11% growing stages samples exceeding the limit of hygienic quality, and Nunes [49] reported 67% of samples exceeding it. High fungal counts would indicate poor quality of raw materials and/or poor manufacturing practices. Fungal growth in raw material affects the nutritional feed quality [10]. Feeds that do not meet the criteria of hygienic quality are a risk to animal health.

 

There are a few studies reporting mycobiota on finished fish feed [39,40,45] and even less on rainbow trout feeds [21]. In our study, predominant mycotoxigenic genera were Eurotium (Fr 25.00%) and Penicillium (Fr 21.43%). The most prevalent mycotoxigenic species were E. repens (21.43%) and E. rubrum (14.29%). This result differs from those obtained by Alinezhad et al. [21] on rainbow trout feed, who found the Aspergillus genus as the most prevalent (57.0%), followed by Penicillium (12.84%) and A. flavus (60.66%) as the most prevalent species. In Brazil, studies conducted on aquaculture feeds have also shown the presence of species belonging to Aspergillus and Penicillium genera. Cardoso [40] reported an occurrence of 56.16% of Aspergillus and teleomorphs in rations for fish, followed by Penicillium (19.18%), Cladosporium (16.44%) and Fusarium (8.22%), A. flavus (60%) being the most common species, followed by Eurotium sp. Barbosa et al. [45] studied the mycobiota and mycotoxin content of finished feed samples from tilapia farms and did not find species belonging to Fusarium genera. Aspergillus was the most prevalent toxicogenic genera (68%) followed by Penicillium and A. niger aggregate, A. flavus being the most prevalent species [45]. In Portugal, Almeida et al. [39] have also reported the presence of Aspergillus, Penicillium, Cladosporium and Fusarium from feed samples for farmed sea bass. Again, A. flavus was the most frequent mold found on 40.2% of the samples tested. With respect to mycotoxigenic fungi, several studies have been done regarding the toxicity of Aspergillus and Fusarium mycotoxins on fish [22,23,29–35]. Considering the frequent presence of Eurotium and Penicillium species (although found in less proportion), determined in our work and reported by other authors [40,45], further studies should be done regarding the toxicity of Eurotium and Penicillium mycotoxins in fish and its occurrence in finished fish feeds. Eurotium and Penicillium species could also be able to produce a wide range of toxic compounds. However, there is no information about the toxicity of these compounds in fish [45] or its occurrence in finished fish feeds.

 

Regarding animal feed, the EU establishes regulations/recommendations for several mycotoxins. Although is not common to observe an excess of those limits [42], a low level of contaminations and co-contamination is frequent [9,10,41–44,51]. According to this, in our work, a total of six mycotoxins were detected in the samples tested. Our values were between <1.7 to 4.19 ppb. Co-occurrence of at least two out of six mycotoxins was recorded in 93% of the samples of finished rainbow trout feeds analyzed from hatcheries in Argentina.

 

In another study on fish feed, pellets of rainbow trout feed and feed ingredients were tested for AFB1, and except gluten, all of them were contaminated with aflatoxins in the range of 0.06 to 212.18 ppb [21]. The occurrence of DON and ZEA was determined in samples of commercial fish feed collected from central Europe. A high percentage (80%) of commercial fish feed samples were found to be contaminated with DON at averages values of 289 ppb, and ZEA was found in all samples, showing an average value of 67.9 ppb [51]. Barbosa et al. [45] detected FB1 in 90% of finished fish feed samples analyzed with an average value of 2.6 ppb. Furthermore, Barbosa et al. [45] found AFB1 and OTA contaminated samples at no quantifiable levels. The intake of low levels of mycotoxins may lead to the deterioration of the immune system, causing animal health problems and economic losses due to a decrease in productivity [48].

 

Simultaneous occurrence of mycotoxins was also determined by other researchers. DON and ZEA were determined in nine out 11 samples of commercial fish feed in central Europe [51]. Furthermore, Barbosa et al. [45] determined the co-occurrence of the carcinogenic mycotoxins AFB1, OTA and FB1 in finished fish feeds from farms in Brazil. Streit et al. [44] performed a multi-mycotoxin screening on 83 samples of feed and feed ingredients from Europe, revealing the occurrence of 139 different secondary metabolites, and all of the samples analyzed contained seven to 69 metabolites, showing a high number of co-occurring metabolites. A review by Streit et al. [43] revealed that 75% to 100% of the samples of animal feeds contained more than one mycotoxin, which even at low doses are able to cause animal health problems. In South Africa, 92 samples of different animal feed types were analyzed for 11 mycotoxins (aflatoxins, ochratoxin A, fumonisins, zearalenone and trichothecenes), and except for a low number of samples, the levels of detected mycotoxins could be considered safe according to South African legislation [42]. However, mycotoxin co-occurrence was common [42]. Synergistic effects of simultaneous exposure of animals to more than one toxin could be expected as able to cause mycotoxicoses [36,42,52].

 

In our case, the presence of a particular toxin was not necessarily correlated with the presence of the possible toxin producer mold (Table 3). These results were previously observed in finished fish feeds [45]. Fungal growth and mycotoxin production on crops are influenced by several factors [52]. Particularly, Fusarium is a field fungi usually lacking the ability to grow on dry feed. However, it is possible that mycotoxins produced by the fungus in the field come with the raw material that composes the finished feed [45]. The other way around, the presence of mycotoxin-producing fungi in feedstuffs is not always conducive to contamination with mycotoxins. Besides, taking into account that the conditions of the feed manufacturing process involve feed exposure to high temperatures, fungal propagules inactivation may occur, but not for mycotoxins, since they are heat stable [45,46,53–55]. This supports the need for constant monitoring of molds and mycotoxin content in animal feed.

 

 

 

 

 

A total of 28 representative samples (1–2 kg per sample) of two different brands was collected from three hatcheries in the provinces of Río Negro and Neuquén (Argentina). Samples correspond to different fish life cycle stages: starter (13 samples), grower (13 samples: 4 pigmented and 9 unpigmented) and finisher (2 pigmented samples). 

 

Samples were processed as follows: they were homogenized to obtain 1 kg of sample that was milled afterwards. Feed samples used for mycological analysis were processed upon arrival or stored in paper bags for no longer than 3 days at room temperature (about 25 °C). Long-term storage was done at −20 °C.

 

Table 5 shows the main ingredient (I1–I8) compounds present in commercial rainbow trout feeds. All components present in the final product are listed in decreasing percentage order.

 

 

 

Enumeration and isolation of fungi were performed using the dilute plate technique [46]. This procedure consists of mixing 10 g of each sample with 90 mL 0.1% peptone solution and then shaking for 20 minutes. Then, 0.1 mL of a spore suspension dilution were inoculated onto three different culture media. For total culturable fungi enumeration, Dichloran Rose Bengal Chloramphenicol Agar (DRBC) (Dichloran: Sigma-Aldrich, Intl., Buenos Aires, Argentina; Rose Bengal: Cicarelli, San Lorenzo, Argentina; Chloramphenicol: Calbiochem, Intl., Buenos Aires, Argentina; Agar: Britania, Buenos Aires, Argentina) was used. For xerophilic fungi enumeration, Dichloran 18% Glycerol Agar (DG18) (Glycerol: Biopack, Buenos Aires, Argentina). Additionally, for the selective isolation of Alternaria and Fusarium species, Dichloran Chloramphenicol Peptone Agar (DCPA) (Peptone: Britania, Buenos Aires, Argentina) was used. Incubation time for these plates was 7 days at 25 °C. On the other hand, a 12 h of light/12 h of darkness incubation photoperiod was used for DCPA plates.

 

Plates with colonies ranging from 10 to 100 were used for counting, and the results were expressed as colony-forming units per gram of sample (CFU/g) [46]. Individual CFU/g counts for each different colony type were recorded. Mold colonies suspected to belong to the Alternaria or Fusarium genera were cultured again onto plates containing water agar (WT). Additionally, molds suspected to belong to the Aspergillus, Eurotium, Penicillium and other genera were cultured on malt extract agar (MEA). Genus level identification of filamentous fungi was performed in accordance with Samson et al. [56].

 

Fungal isolates were identified at the species level according to the leading authorities: Penicillium sp. and Aspergillus sp. according to Pitt and Hocking [46]; Fusarium sp. according to Nelson et al. [57]; Alternaria sp. according to Simmons [58]; and other fungi according to Pitt and Hocking [46]. The isolation frequency (Fr) and relative density (RD) of genus/species were calculated according to Saleemi et al. [59] as follows:

 

Fr (%) = number of samples with a genus or species/total number of samples × 100

RD (%) = number of isolates of a genus or species/total number of fungi isolated × 100

 

All of the isolates were preserved on agar slants of MEA or potato dextrose agar (PDA) for Alternaria and Fusarium at 4 °C and cryopreserved in 18% glycerol at −20 °C.

 

 

 

To evaluate mycotoxin occurrence, trout feed samples were subjected to quantitative analyses using ELISA-based analytical test kits for aflatoxin, ochratoxin A, T-2 toxin, fumonisin, deoxynivalenol and zearalenone (RIDASCREEN FAST, R-Biopharm AG, Darmstadt, Germany). The extraction procedures were according to the manufacturer’s protocol. In brief, 5 g of each ground sample were extracted with 25 mL of 70% methanol for total aflatoxins, T-2 toxin, ZEA and total fumonisins. For OTA and DON, samples were extracted with 12.5 mL of 70% methanol or 100 mL of distilled water, respectively. Afterwards, samples were shaken vigorously for 3 minutes and the extracts filtered through Whatman N°1 paper (Whatman Inc., New Jersey, NY, USA). Then, aflatoxins, OTA, T-2 toxin and ZEA filtrates were diluted with distilled water in the ratio of 1:1 and fumonisin filtrates in the ratio of 1:14. Fifty microliters of the diluted filtrate per well were used for testing [9,10]. Analytical-grade methanol (Biopack, Buenos Aires, Argentina) was used for extractions. All reagents used for mycotoxin determinations were provided with the RIDASCREEN test kit. Each kit contains a microtiter plate coated with capture antibodies, toxins standard solutions, peroxidase conjugate and an anti-mycotoxin antibody for each mycotoxin, substrate/chromogen stained red, washing buffer and stop solution 1 N sulfuric acid. Microtiter plate spectrophotometer (R-Biopharm AG, Darmstadt, Germany) was used for quantification, and absorbance was measure at 450 nm. Then, the logit/log function from the RIDA SOFT Win software (R-Biopharm AG, Darmstadt, Germany) was used for data evaluation.

 

 

For analytical purposes, mycotoxin levels below the limits of detection (LOD) were calculated with RIDA SOFT Win software. These values below LOD (often referred to as “censoring”), but above LOD/√2, were considered for statistical analysis [47,60], since they provide meaningful information at the expense of a slightly higher measurement error. Undetected levels and values below LOD/√2 were assumed to be zero. Furthermore, since there was less than 60% of censored values among the data, the described replacement method is considered suitable [47,60,61].

 

Fungal counts and toxins content were analyzed with the Kruskal-Wallis test due to the rejection of the null hypothesis on the distribution normality with the Shapiro-Wilks test. The multiple range test for variables was employed to compare means of fungal counts and toxin content of samples from different provinces and growth stages. Differences were considered significant when p < 0.05. Spearman correlation coefficients were used to evaluate correlations between mycotoxins. Statistical significance was set at p < 0.001. InfoStat Version 2013 was used for all analyses.

 

 

 

There are few studies about mycotoxigenic fungi and the natural occurrence of mycotoxins in commercial fish feed, and even less in rainbow trout finished feeds. Therefore, due to the potential risk of contamination of this kind of feed by fungi and mycotoxins, regular monitoring is highly recommended. This is the first report on mycobiota contamination and the co-occurrence of several mycotoxins in rainbow trout feed in Argentina.

 

 

 

This work was supported by Universidad Nacional de Quilmes (UNQ), Universidad Nacional de Río Negro (UNRN), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT).

 

 

Graciela Pose and Alejandro Pardo designed the study. Mariana Greco performed the experiments and analyzed the data. All authors contributed to writing the manuscript.

 

 

The authors declare no conflict of interest.

 

 

 

 

 

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