Mycotoxins in Aquaculture: Occurrence and Significance
Mycotoxins are toxic secondary metabolites produced by molds (Hussein and Brasel, 2001). They are found mainly in agricultural commodities and are produced at various stages e.g. before or after harvest, during transportation or storage. Chemically, mycotoxins display a wide range of structures, differing also in biological effects, e.g. carcinogenic, teratogenic, mutagenic, estrogenic, neurotoxic, or immunotoxic (Abd-Allah et al., 1999, El-Sayed et al., 2009, McKean et al., 2006). The effects of mycotoxins are widely studied and specific signs, symptoms and pathologies can be directly linked to mycotoxicosis. In aquaculture, mycotoxins are recognized as a threat since 1960, the first case describing negative effects in fish, where aflatoxin-contaminated cottonseed meal caused an outbreak of aflatoxicosis in hatchery-reared rainbow trout (Onchorhynchus mykiss) (Kumar et al., 2013, Wolf and Jackson, 1963). However, mycotoxicosis in aquatic species is often associated with poor growth; low feed efficiency and the lack of obvious pathological signs make it difficult to identify the source of the problem. It is therefore of utmost importance to be aware of the potential risks that mycotoxins present to farmed aquatic species, as well as a deep knowledge on the occurrence of mycotoxins in commonly used raw materials in aquatic feeds.
Mycotoxins in aquaculture
Research characterizing the adverse effects of mycotoxins on the performance and health of animals has in large part focused on terrestrial livestock species (D’Mello and Macdonald, 1997, Pestka, 2007, Rotter et al., 1996). However, in recent years, research has been carried out on the effects of mycotoxins in aquaculture species. This became even more important with the high cost of ﬁsh meal and the need to identify more economical protein sources, such as plant protein or other commercially available by-products. According to Tacon et al. (2011), plant nutrients already represent the major dietary protein source used within feeds for lower trophic level fish species like tilapia, carp or catfish, and the second major source of dietary protein and lipids after fishmeal and fish oil for high trophic level fish species. Generally, most of the mycotoxins that have the potential to reduce growth and health status of aquaculture farmed animals are produced by Aspergillus, Penicillium and Fusarium species. Toxic metabolites produced by these fungi are known to be either carcinogenic (e.g. aflatoxin B1, ochratoxin A, fumonisin B1), estrogenic (zearalenone), neurotoxic (fumonisin B1), nephrotoxic (ochratoxin), dermatotoxic (trichothecenes) or immunosuppressive (aflatoxin B1, ochratoxin A and T-2 toxin).
Mycotoxins effects in aquatic species
Rainbow trout (Oncorhynchus mykiss) are known to be sensitive to low levels of DON (Hooft et al., 2011, Woodward et al., 1983). Hooft et al. (2011) reported that low, graded levels of DON ranging from 300 to 2,600 µg kg-1 from naturally contaminated corn resulted in highly signiﬁcant decrease in growth (-40%), feed intake (-52.7%), feed efﬁciency (-76.7%) and protein and energy utilization (-74.4% and -72.1%) when compared to control. Contrarily, channel catfish (Ictalurus punctatus) fed diets containing up to 10,000 µg Kg−1 of DON from either a puriﬁed source or naturally contaminated wheat had no effects on feed consumption, growth, hematocrit values or liver weights (Manning et al., 2014). However, in Pacific white shrimp (Litopenaeus vannamei), DON levels ranging from 200 to 1,000 µg kg−1 in the diet, significantly reduced shrimp body weight and/or growth rate (Trigo-Stockli et al., 2000). Also in carp (Cyprinus carpio L.) a feeding trial using three different concentrations of DON (352, 619 and 953 µg kg−1) confirmed the immunosuppressive effects of DON even at low doses (Pietsch et al., 2014).
Scarce information is available on the effects of fumonisins on aquaculture species. It is known that rainbow trout liver is sensitive to FB, inducing changes in sphingolipid metabolism on values lower than 100 µg kg− 1 (Meredith et al., 1998) and inducing cancer in 1-month old trout (Riley et al., 2001). Tuan et al. (2003) demonstrated that feeding FB1 at levels of 10, 40, 70 and 150 mg/kg feed for 8 weeks affected growth performance of Nile tilapia fingerlings. Fumonisin B1 has not been extensively studied as a shrimp feed contaminant. However the few studies available suggest that Litopenaeus vannamei is sensitive to FB1. García-Morales et al. (2013) have shown that white shrimp fed FB1 at levels from 20 to 200 µg kg−1 had a reduction in soluble muscle protein concentration and changes in myosin thermodynamic properties were observed in shrimp after 30 days of exposure to FB. The same authors reported marked histological changes in tissue of shrimp fed a diet containing FB1 at 200 µg kg−1 and meat quality changes, after 12 days of ice storage, when fed diets containing more than 600 µg kg−1 FB.
ZEN has mostly been studied for its ability to cause disorders in the reproductive tract of farm animals (Minervini and Aquila, 2008, Zinedine et al., 2007). However, the effect of zearalenone on fish and shrimp has hardly been evaluated. The few studies existing show that ZEN can modulate ER-dependent gene expression affecting the reproduction of ﬁsh. This has been shown in zebraﬁsh (Danio rerio), where the exposure to ZEN reduced spawning frequency (Schwartz et al., 2010), or changed their relative fecundity from one generation to another (Schwartz et al., 2013). In another study, when zebrafish larvae were exposed to 500 µg L−1 or higher of ZEN, defects in heart and eye development and upward curvature of the body axis were observed (Bakos et al., 2013). Among aquaculture farmed species, when black tiger shrimp (Penaeus monodon Fabricius) were fed 500 and 1,000 µg kg−1 ZEN contaminated feed, histological changes were observed in hepatopancreatic tissue (Supamattaya et al., 2005). For carp (Cyprinus carpio L.), Pietsch et al. (2015), investigated ZEN at three different concentrations (332 µg kg−1, 621 µg kg−1 and 797 µg kg−1) for four weeks. The authors observed no effect on growth, but effects on hematological parameters were confirmed. In addition, an influence on white blood cell counts was noted, whereby granulocytes and monocytes were affected in fish fed 621 µg kg−1 and 797 µg kg−1 of ZEN in the diet. Furthermore, marginal ZEN and α-zearalenol (α-ZEL) concentrations were detected in muscle samples and the genotoxic potential of ZEN was confirmed by analyzing formation of micronuclei in erythrocytes.
Aflatoxins, mainly the toxicity of AFB1, have been considerably investigated in farmed fish and crustaceous species for aquaculture (Dirican, 2015, Santacroce et al., 2008). It is reported that seabass and rainbow trout are very sensitive to AF (LC50=180 µg kg−1 BW and 5-10 µg kg−1 BW, respectively) (El-Sayed and Khalil, 2009, Hendricks, 1994), however, the results obtained were by gavage feeding in the case of seabass and by intraperitoneal injection in trout. Also Centoducati and co-authors (2010) concluded that gilthead sea bream (Sparus aurata) hepatocytes are highly sensitive to AFB1 exposure. For tropical species, growth rate and FCR of Nile tilapia was reported to be significantly affected by AF on the feed. Values ranging from 100 to 2,500 µg kg−1 of AFB significantly affected growth performance in tilapia (Chávez-Sánchez et al., 1994, El-Banna et al., 1992, Oliveira et al., 2013, Tuan et al., 2003). It was also shown by El-Banna et al. (1992) that at lower concentrations of AF in the diets (50 µg kg−1 of AFB) vacuolization and necrosis of hepatocytes were observed. For shrimp, black tiger shrimp (Penaeus monodon Fabricius) fed AFB1 levels ranging from 5 to 20 µg kg−1 had a decrease of 46% to 59% of the body weight on the AFB1-treated groups compared to control (Bintvihok et al., 2003). On Pacific white shrimp, Ostrowski-Meissner et al. (1995), reported abnormal hepatopancreas and antennal gland tissues which were caused by 2 weeks of AFB1 at 50 µg kg−1. Feed conversion and growth were significantly affected at 400 µg kg−1 AFB1 and apparent digestibility coefficients decreased significantly at 900 µg kg−1 AFB1.
Ochratoxin A Studies on the toxicity of OTA in aquatic animals are very scarce. Severe abnormalities such as deformities of the head, tail and eyes were found in zebra fish after the developing eggs were exposed to OTA (Debeaupuis et al., 1984). In rainbow trout intoxicated with OTA (Doster et al., 1972), severe degeneration and necrosis of kidney and liver, pale kidney, light swollen livers and mortality occurred. On channel catfish (Ictalurus punctatus) fed 5, 1.0, 2.0, 4.0, or 8.0 mg OA/kg diets, the authors found significant reduction in weight gain, poorer feed conversion rate, lower survival and hematocrit. Moreover, moderate to severe histopathological lesions of liver and posterior kidney were observed (Lovell, 1992, Manning et al., 2003). For common carp (Cyprinus carpio), Agouz and Anwer (2011) showed that a natural contamination of 15 µg kg−1 of OTA in the diet resulted in decreased growth performance and feed utilization parameters. Carcass dry matter, protein and ash contents negatively correlated with OTA. In European seabass (Dicentrarchus labrax L.), El-Sayed et al. (2009) found the 96 h LC50 value at 277 µg kg-1 bw with 95% conﬁdence limits of 244 to 311 µg kg-1 bw. Nevertheless, Supamattaya et al. (2005) noticed that shrimp feeds occasionally contaminated with OTA (~1,000 µg kg−1) have no negative impact on the shrimp culture industry.
Mycotoxins occurrence in plant ingredients
From all the possible alternatives, e.g. animal by-products, fishery by-products, bacteria and algae concentrate, plant ingredients seem to be one of the most promising solution to replace fish meal. Numerous plant raw materials have been successfully tested (Gatlin et al., 2007). It is observed that an interrelationship between nutrition, immunology and disease resistance in fish and shrimp has been essential to evaluate the alternative plant ingredients. It is commonly agreed that one of the most negative aspects of plant meals are the presence of anti-nutrients that are detrimental to fish. Although there are processes to remove or inactivate many of these compounds, the same does not happen to mycotoxins that are relatively stable to processing conditions. In order to evaluate the occurrence of mycotoxins in common plant nutrients used for aquaculture feeds production, over a period of one year (January 2015 - December 2015), 2176 samples of different plant sources were analyzed within the scope of BIOMIN Mycotoxin Survey program. The plant meals selected were: Soy Bean Meal (SBM), Wheat (WH), Wheat Bran (WB), Corn (C), Corn Gluten Meal (CGM), Cottonseed Meal (CSM), Rapeseed/Canola Meal (R/CM) and Rice Bran (RB).
The occurrence and significance
All of the eight plant meals analyzed from Asia and Europe revealed the incidence of mycotoxins. Ninety-three percent of the Asian samples were found to be contaminated with mycotoxins and in European samples 78% of the samples had mycotoxins (Table 1).
Of the 67 samples of SBM analyzed, we observed that in 88% of Asian samples mycotoxins were detected, while in the European samples the contamination reached 58% of samples. Asian samples were mostly contaminated with Fusarium mycotoxins (ZEN, DON and FUM), being AF, T-2 and OTA, found in very low levels. For European samples, Fusarium metabolites were also predominant, with DON registering a maximum occurrence of 930 µg Kg-1. European samples were also marked by the presence of T-2 with a maximum value of 105 µg Kg-1. Fifty-eight percent of SBM samples from Asia had more than one mycotoxin while for European samples the co-occurrence was 32%.
Seventy-one percent of the 163 WH samples collected in Asia contained mycotoxins. These samples were characterized by the high level of DON, with an average value of 1,275 µg Kg-1 and maximum occurrence of 6,976 µg Kg-1. Of the 80% contaminated samples from Europe, DON was also prevalent with a lower average value (418 µg Kg-1), compared to Asia, to Asia, but with similar maximum occurrence of 6,219 µg Kg-1. In the case of the In the case of the European samples, FUM was also detected a relatively high, maximum occurrence of 1,628 µg Kg-1. The mycotoxin co-occurrence values for both regions were quite low, with Asia having a co-occurrence of 28% and Europe 26%.
Wheat bran samples were in lower numbers than WH, yet it was observed that all Asian samples collected were contaminated with mycotoxins and 88% of the European samples had mycotoxins present. Asian samples were contaminated by Fusarium mycotoxins, with average values of 620 µg Kg-1 for FUM, 761 µg Kg-1 for ZEN and DON with 1,660 µg Kg-1. On European samples, mycotoxins observed were mainly FUM at maximum of 5,334 µg Kg-1 and DON with average of 5,124 µg Kg-1 and maximum occurrence of 15,976 µg Kg-1. In terms of average occurrence values, we observed a much higher contamination on WB samples when compared with WH. Mankevičienė (2014) observed that the concentrations of mycotoxins in wheat bran can be several times higher than those in grain. A higher concentration of these metabolites in by-products is expected due to the high stability of mycotoxins. The mycotoxin co-occurrence values were 31% for Asia and 38% for Europe.
Corn was one of the most mycotoxin-contaminated commodities for both Europe and Asia. This commodity was mainly contaminated with FUM (average value of 2,038 µg kg−1; maximum value of 16,258 µg kg−1) in the case of Asian samples and by DON (average value of 2,469 µg kg−1; maximum value of 19,180 µg kg−1), in European samples. Beside these high value levels of DON and FUM, samples were also contaminated by other Fusarium mycotoxins and by AF.
Corn Gluten Meal
Corn Gluten Meal was the most contaminated commodity in this survey. For both regions, samples were mainly contaminated by Fusarium mycotoxins (ZEN, DON and FUM) on average values of 2,394 µg kg−1 and maximums of 8,825 µg kg−1. These values are not surprising, after observing the contamination values for corn, since CGM is a by-product, a concentration of mycotoxins in these kinds of derived products is expected. Logically the co-occurrence values were also expected to be very high. For Asian samples it was found that all samples had more than one mycotoxin and for European samples 91% also had more than one mycotoxin. On average, each sample analyzed contained 4 mycotoxins (data not shown). These co-occurrence values increase the probability of additive or synergistic effects when using this commodity.
While not having a representative number of samples for both regions (Table 1), it is important to have a preliminary idea of the occurrence of mycotoxins in this commodity. Data obtained seems to be consistent, showing that R/CM is mainly contaminated by Fusarium metabolites, in Europe by DON and FUM at low levels (average contamination below 45 µg kg−1) and in Asia by ZEN and DON. Values obtained for Asia were higher than in Europe, showing a maximum occurrence of DON of 2,431 µg kg−1.
According to Tacon (2011), CSM is mostly commercially used for channel catfish in the USA and in China for tilapia, at inclusion levels up to 25%. While not having a high number of samples, of the nine samples collected in Europe only 33% of them were contaminated by mycotoxins, whereas the contamination is very low, not representing a real threat to aquaculture species. However in Asia, the scenario is completely different and as expected, due to temperature/humidity pattern in that region, AF is the main mycotoxin present (average value of 2,038 µg kg−1; maximum value of 16,258 µg kg−1). However, Fusarium toxins (ZEN and DON) were also found in considerable amounts.
RB has been used as a supplementary feed for Pangasius farming among the traditional farmers in Vietnam and by Chinese fish farmers on intensive crucian carp rearing (Hasan, 2007). According FAO (2012), RB is mostly commercially used for tilapia, at inclusion levels that can vary from 10% to 25% depending in the region. As this commodity is mostly used in Asia, samples were not taken in Europe. In Asia was found that this raw material is mainly contaminated by ZEN (average value of 147 µg kg−1; maximum value of 545 µg kg−1) and FUM (average value of 118 µg kg−1; maximum value of 713 µg kg−1) and in lower levels by DON and AF.
With the continuous trend to replace animal-derived proteins by plant proteins sources, it is expected that the risk of mycotoxin contamination in aquaculture feeds increases in step with the more widespread use of plant materials in aquafeeds. The risk of mycotoxins in aquaculture will be directly related with the type and origin of plant material used as well to its inclusion level in aquafeeds. Generally, in Asian samples was observed that SBM, WH, WB, C, CGM, R/CM and RB were mostly contaminated with Fusarium mycotoxins (ZEN, DON and FUM). With the exception of CSM that was mainly contaminated by AF together with Fusarium toxins (ZEN and DON) in considerable amounts. European samples were contaminated mainly by Fusarium mycotoxins (Figure 1).
Mycotoxins occurrence in aquafeeds
The inclusion of plant materials contaminated with mycotoxins in compound aquafeeds will increase the risk of mycotoxin contamination in aquaculture feeds. Recently, Gonçalves et al., (2016) compared the mycotoxin occurrence levels from 41 samples of finish aquaculture feed, both shrimp and fish, in Asia and Europe, with the available literature on fish/shrimp mycotoxicoses. The authors found that levels found for the samples analyzed during 2014 were within the sensitivity level of several important species in aquaculture (Figure 2). Gonçalves et al. (2016) highlighted the fact that the mycotoxins levels found can compromise aquaculture species, even just taking into account single mycotoxins levels. According to Gonçalves et al. (2016) the number of species affected by mycotoxins would be even higher than stated in the study due to the lack of research in some important species and the existence of mycotoxins synergisms not taken into account on that study.
The occurrence and significance
From the 41 samples of finish aquaculture feed, collected in Asia and Europe, DON was the most prevalent mycotoxin, with 68% of the samples testing positive, followed closely by AF and ZEN (59% positive) and by OTA and FB, with 57%, and 51% respectively (Table 2). Concerning the contamination level, FB was the one found in higher concentration, with an average of 637 µg kg−1 on the 21 samples contaminated with this mycotoxin and showing a considerable maximum concentration of 7,534 µg kg−1. In total, 76% of the samples had more than one mycotoxin, only 17% of the samples were contaminated by one mycotoxin and just 7% of the samples did not contain detectable levels of any of the ﬁve mycotoxins (Table 2). Analyzing the mycotoxin occurrence by region, it was possible to observe a different distribution pattern when comparing the mycotoxin occurrence between Europe and Asia, likely due to the climate differences between the two. In Europe it was observed that AF and OTA, while present in 17% and 67% of the samples, respectively, had relatively low average concentrations, 0.43 and 1.53 µg kg−1, respectively. The occurrence of the same mycotoxins in Asia was relative higher in percentage (AF = 68% and OTA = 55%) and in contamination level (AF = 51.83 µg kg−1 and OTA = 2.11 µg kg−1). However, regarding ZEN, DON and FB, the higher average contamination values were found in European samples (ZEN = 118.01 µg kg−1; DON = 165.61 µg kg−1 and FB = 3,419.92 µg kg−1) (Figure 3).
Mycotoxins were found in most of the commodities and finished feeds analyzed, showing that mycotoxins might represent a risk for the development of the sector, if plant raw materials would be an option to replace less sustainable nutrient sources. While in some cases the contamination levels are rather low, in some cases the contamination levels might represent a risk for aquaculture species. Generally, in Asian samples it was observed that SBM, WH, WB, C, CGM, R/CM and RB were mostly contaminated with Fusarium mycotoxins (ZEN, DON and FUM). With the exception of CSM that was mainly contaminated by AF together with Fusarium toxins (ZEN and DON) in considerable amounts. European samples were contaminated mainly by Fusarium mycotoxins. The co-occurrence of mycotoxins in all commodities were rather high, mainly considering that to produce an finished feed several raw materials are selected, increasing the probability of co-occurrence of finished feeds. It was also observed the accumulation of mycotoxins on the processed plant nutrients, like CGM and WB when compared to the whole grain, WH and C, respectively.
Regarding the finished feeds, the values detected pose a risk for several important aquaculture species, assuming single mycotoxin contamination, i.e. excluding possible additive and synergetic effects between mycotoxins. Co-occurrence of mycotoxins in feeds may induce synergistic effects and increase the negative impact of mycotoxins in aquatic farmed species at lower levels than when present in single contamination. Where clinical signs of mycotoxins are almost unknown or not identifiable, mycotoxins could remain as a problem not correctly identified and leading to disease susceptibility and performance losses in aquaculture industry. As we see the growing importance of the plant raw materials in the future of aquaculture industry, it will be very important to increase the knowledge on the common mycotoxins found in the different raw materials in order to better avoid the risk of mycotoxicoses in aquaculture. It will be important to increase the knowledge on the effects of mycotoxins on aquaculture species in order to define acceptance levels of mycotoxins in the finished feeds, as we have it today in livestock production sector.