January | February 2010

Feature title: Recent updates on the effects of Mycotoxins in Aquafeeds

The International magazine for the aquaculture feed industry

International Aquafeed is published five times a year by Perendale Publishers Ltd of the United Kingdom.All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2009 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

Recent updates on the effects of

Mycotoxins in

aquafeeds

In recent years, the awareness of mycotoxin-related issues within the aqua industry has grown sup-ported by increasing scientific

evidence of the negative impact of mycotoxins in aquatic species and by frequent reports on the prevalence of mycotoxins in many raw materials.

For the most part, mycotoxin contami-nation of aquafeeds is greatly widespread, especially in countries with humid tropical climates owing to many factors, among which are permissive climatic conditions to mold growth and inappropriate methods of feed processing and storage.

However, the increasing globalization of trade and incorporation of imported raw materials in aquafeeds exposes feed manufacturers and their clients to the risk of combinations of mycotoxins either from multiple mycotoxins in the same raw material or from different mycotoxins in different ingredients in the same formulation (Fegan & Spring, 2008).

Furthermore, due to the rising prices of feedstuffs feed manufacturers are looking for more economical raw materials to avoid increasing feed prices.

However, the use of more affordable raw materials of lower quality might increase the risk of mycotoxin contamination in the feeds.

For example, DDGS is an economical source of energy and protein that can be used in animal feeds, but normally reports show that is contaminated with multiple mycotoxins (Rodrigues, 2008).

DiverseMycotoxins are structurally very

diverse, a characteristic that leads to a wide range of symptoms in mycotoxin affected animals going from decreased production effi-ciency to mortality.

General, unspecific symptoms associated with mycotoxin expo-sure make diagnosis difficult.

Further complications in the diagnosis of mycotoxicoses in farm animals can be caused by synergistic effects resulting from the presence of multiple mycotoxins in feeds and by secondary symptoms resulting from opportunistic disease related to the suppression of the immune system.

Additionally, sensitivity to mycotoxins varies greatly between species and is dependent on several factors which can modify the expression of toxicity including age, gender, nutritional and health status prior to exposure and environmental con-ditions (Whitlow and Hagler, 2002).

Among all known mycotoxins, aflatox-ins are the best characterized and most investigated due to their acute and chronic toxicity on aquatic species. Despite being the most studied mycotoxin, new informa-tion is now available regarding the effects of Aflatoxins in aquatic species not studied before.

Furthermore, there are new insights on the importance caused by the consump-tion of aflatoxin contaminated fish through the trophic chain in particular for human consumption.

The toxicity of AFB1 was recently studied for the Mediterranean cultured species sea bass (Dicentrarchus labrax) and Gilthead sea bream (Sparus aurata).

Highly responsive sea bassFor sea bass, El Sayed & Khalil (2009)

assessed the susceptibility and toxicity of AFB1 to European sea bass by behavioral and biochemical evaluations.

The results verified that the marine water-reared sea bass was a highly responsive species to acute AFB1 toxicosis with oral 96 h LC50 of 0.18mg/kg bwt when compared to the acute LD50 values that ranged from 0.3 to 9.0 mg/kg bwt in all species including fish.

In addition there were behavioral changes and clinical signs in sea bass consistent with similar studies in other fish species, which included skin lesions, yellowing of the body surface, abnormal swimming, hemorrhages in

by Pedro Encarnação, Biomin, Singapore

10 | InternatIonal AquAFeed | January-February 2011 January-February 2011 | InternatIonal AquAFeed | 11

F: Mycotoxins

Figure 1: Accumulation of lipid in liver cells (a) affected by AFB1 at 100 ppb for 12 weeks (H&E x 400), source (Tu, 2010).

the head and eye cataracts, as well as dam-age or gradual deterioration of the liver. In addition, the prolonged oral administration of 0.018mg/kg bwt AFB1 to sea bass for 42 successive days induced a significant increase in serum transaminases and alkaline phos-phatase activities, and significant decrease in plasma proteins (El Sayed & Khalil 2009).

For gilthead seabream, Centoducati et al. (2009) conducted an in vitro evaluation on the cytotoxic potential of AFB1 on hepato-cytes in order to grade the range of AFB1 toxicity, and the boundary between acute and long-term toxicity, using a wide range of con-centrations from 5×103 ng/ml to 2×10-5 ng/ml of AFB1 and different period of exposure (24, 48, 72 hours).

After each exposure, hepatocytes were examined for morphologic alterations and apoptosis induction. The study demonstrated that seabream hepatocytes are highly sensitive to AFB1 exposure, resulting in a significant reduction of cell viability in a dose- and time-dependent manner. Dose-response curves obtained after 24, 48 and 72 hours revealed that prolonged exposure times lead to a significant increase of the toxic potency of AFB1 (Centoducati et al., 2009).

The hapatoxicity of AFB1 was also con-firmed by Spahdari et al. (2010) in a study looking at the impacts of AFB1 on Beluga (Huso huso) at levels of 0, 25, 50, 75, and 100ppb AFB1/kg of diet during a three-months period.

In this case, results showed that the vari-ous levels of AFB1 did not significantly affect the specific growth ratio (SGR) (P<0.05) of fish in different treatments.

However, weight gain and food conver-sion ratio (FCR) varied significantly (P<0.05) between control and treatments with diets contaminated with 75 and 100ppb AFB1/kg after 90 days.

Histopathological studies showed that dif-ferent level of AFB1 can cause broad range of change in liver tissue, including progressive fat deposition, hepatocyte degeneration and necrosis, particularly at concentration of 75 and 100ppb AFB1/kg of diets after 60 days (Spahdari et al., 2010).

Recent studies in VietnamIn a recent study in Vietnam, Tu (2010)

conducted a comprehensive study on the sen-sitivity of Tra catfish (Pangasionadon hypoph-thalmus) to AFB1, analyzing the impacts of feeding AFB1 contaminated diets on growth performance, physiological response, histo-logical changes and disease resistance of Tra catfish.

After eight weeks feeding reduction in weight gain (P<0.05) was observed for fish fed diets contaminated with 50µg AFB1/kg and was reduced further with increasing levels of AFB1 in the diets. Fish fed diets contaminated with 500 and 1000µg AFB1/kg showed increased (P>0.05) hepatosomatic index (HIS), while an increase in adipose somatic index (ASI) was already observed in fish fed 50 µg AFB1/kg and above when compared to the control (Tu, 2010). After 12 weeks, blood serum analysis revealed higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in fish fed the 50, 100 and 250µg AFB1/kg suggesting occur-rence of liver damage, in particular for those fish fed the 250 µg AFB1/kg. (Tu, 2010).

Disease resistance of fish exposed to Edwardsiela ictaluri was also compromised by the presence of AFB1 in the feed and was directly related to the contamination level. Seven days after exposure, survival rates were 50, 41.7, 31.7 and 8.3 % for fish fed control, 50, 100 and 250 µg AFB1/kg, respectively.

This trial shows that, AFB1 contamination in tra catfish diets at a level of 50µg AFB1/kg and above can affect fish performance and dis-ease resistance which confirms previous obser-vation by (Phuoc et al., 2004) who reported that diet containing 60 – 300 ppb AFB1 can signifi-cantly reduced performance of tra catfish 16.59 ± 0.2 g after 10 or 22 weeks of feeding.

These findings seem to suggest that Pangasius catfish is more sensitive to AFB1 than other catfish species such as channel catfish (Ictalurus punctatus) and walking catfish (Clarias batra-chus). The study also evaluated the application of an Aflatoxin binder (Mycofix®Secure) to counteract

the negative effects of AFB1, and reported that application of 1.5kg Mycofix®Secure was effective in reducing the negatives effects of AFB1 in diets containing 500 µg AFB1/kg.

In tilapia, the mecha-nism of the toxic effects of AFB1 is still poorly under-stood, and some contra-dictory results exist in different studies. El-Banna et al. (1992) revealed that a diet contaminated with 100µg AFB1/kg of feed significantly reduced the growth of Nile tilapia for 10 weeks, and a 200µg/kg dose led to 16.7%mortality. Cagauan et al. (2004) showed in a 90-day trial that the survival rate of tilapia exposed to 5–38.62 µg AFB1/kg feed was 67% less than that of the control group, and yellowing of the tilapia surface was

10 | InternatIonal AquAFeed | January-February 2011 January-February 2011 | InternatIonal AquAFeed | 11

F: Mycotoxins

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containing 1.4 and 2.6 ppm DON (Hooft, 2010). Together, these results suggest that rainbow trout are extremely sensitive to low levels of DON from naturally contaminated plant ingredients.

For Atlantic salmon (Salmo salar), a study was conducted to evaluate the effects of DON, ZON and Ochratoxin (OTA) (Doll et al., 2010). Results from the study showed that the presence of 3700µg/kg DON in the feed caused a 31% decrease in specific growth rate and 18% increase in FCR (Doll et all., 2010). In addition, DON-dose dependent alteration of plasma enzymes suggested adverse effects in the liver, which need further confirmation by histological evaluation (Doll et al., 2010).

Recommendations for DONIt was concluded by the author’s that

the present EU recommendations for DON levels in feed (5000µg/kg) do not prevent adverse effects on performance and health of Atlantic salmon. Conversely, concentration of ZON (60 – 770µg/kg) and OTA (90 – 330µg/kg) in the feed did not affect feed intake, growth or health parameters (Doll et all., 2010). Another study on the effects of OTA was conducted in European sea bass (Dicentrarchus labrax).

In this study it was concluded that this species was highly sensitive to OTA with a 96 h LC50 of 9.23 mg/kg-1 diet (El Sayed et al., 2009).

In conclusion, an increasing number or experiments is reporting that mycotoxins are toxic for fish and the main target organs are liver, kidney and the immune function.

Although mycotoxin contamination of feed and feed ingredients represents an increase threat to aquaculture operations there are a number of options available to feed manufacturers and farmers to prevent or reduce the risk of mycotoxicosis associated with mycotoxin contamination.

These range from careful selection of raw materials, maintaining good storage conditions for feeds and raw materials, and using a good mycotoxin deactivator to combat the widest possible range of different mycotoxins that may be present.

The Mycofix® Product Line is the result of years of research and its efficacy was proven in scientific and field trials. It guar-antees a protection against a wide range of adsorbable and non-adsorbable mycotoxins by combining three strategies – Adsorption, Biotransformation and Bioprotection.

References:Cagauan, A.G., Tayaban, R.H., Somga, J.R., Bartolome, R.M., 2004. Effect of aflatoxin contaminated feeds in Nile tilapia (Oreochromis niloticus L.). In: Remedios,

Performance and health status

In addition to the negative effects of AFB1 in fish performance and health status, recent studies have also reported the pres-ence of AFB1 residues in the muscle of fish fed diets contaminated with low levels of AFB1.

The European Union established 2µg/kg as the maximum allowable concentra-tion of AFB1 in human food (FAO, 2004). Han et al. (2009) reported levels of ≈ 3µg/kg in Gibel carp (Carassius auratus gibelio) muscle when fed diets containing 10 µg/kg. Residual levels of ≈ 5µg/kg were detected in European seabass (Dicentrarchus labrax) fed diets contaminated with 18µg/kg bwt (El Sayed & Khalil, 2009).

This contrast with the findings of Deng et al. (2010) and Udomkusonsri et al., 2008 which found only traces of AFB1 residues in flesh of tilapia and walking catfish, respectively. This disagreement may be due to the different pathways of metabolism of AFB1 between these species as previously mentioned by Ngethe et al., (1993) who concluded that there are species differences in the metabolism of AFB1 in the liver and affinity of AFB1-derived metabolites to hepatic macromolecules.

Nevertheless, it raises the concern that for some species, AFB1 not only can impair fish performance, but consumption of fish exposed to AFB1 contaminated feeds can have a negative impact on human health.

Although AFB1 is the mycotoxin which receives most attention due to its high toxic-ity, other studies have been conducted to evaluate the effects of other mycotoxins in fish species.

Hooft (2010), studied the effect of DON contamination in trout (Oncorhynchus mykiss) feeds and reported that low, graded levels of DON ranging from 0.3 to 2.6ppm from naturally contaminated corn resulted in highly significant decreases in growth, feed intake, feed efficiency, protein and energy utilization of rainbow trout. Furthermore, significant differences in growth, feed effi-ciency and in protein and energy utilization between fish receiving a diet containing 2.6 ppm and fish pair-fed the control diet indicated that decreases in the perform-ance of rainbow trout associated with the consumption of DON-contaminated feed is related to direct or indirect deleterious effects on the nutrient metabolism of fish and not strictly the result of reductions in feed intake (Hooft, 2010).

This finding was further supported by evidence of histopathological changes, par-ticularly in the liver, of some fish fed diets

observed in the groups given more than 29µg AFB1/kg feed.

However, Tuan et al. (2002) demonstrated that there was no adverse effect when tilapia was fed a diet containing 250µg AFB1/kg. They also considered that only a high dose as 2.5mg AFB1/kg feed would affect the hematocrit and growth performance of tilapia.

The latest study on the effects of AFB1 in tilapia, Deng et al. (2010), focused on the toxic effects of AFB1 during long-term dietary exposure and came to the conclusion that under good culture conditions, tilapia is a rather tolerant species for dietary AFB1. No toxic effects of AFB1 were found during the first 10 weeks, but after 20 weeks, the diet with 245µg AFB1/kg or higher doses reduced the growth and induced hepatic disorder, resulting in decreased lipid content, hepato-somatic index, cytochrome P450 A1 activ-ity, elevated plasma alanine aminotransferase activity and abnormal hepatic morphology, but such dietary AFB1 doses did not affect the survival rate of experimental fish.

Figure 2. Final body weight (g/fish) of rainbow trout fed experimental diets containing low levels of DON from naturally contaminated corn (Hoof, 2010).

Figure 3 - Liver of a rainbow trout fed diet contaminated with 2.6 ppm DON showing multifocal areas of fatty infiltration (arrows) (H&E stain; bar = 50.71 μm), (Hooft, 2010).

12 | InternatIonal AquAFeed | January-February 2011 January-February 2011 | InternatIonal AquAFeed | 13

F: Mycotoxins

El-Sayed, Y.S., Khalil, R.H., Saad T.T., 2009. Acute toxicity of ochratoxin-A in marine water-reared sea bass (Dicentrarchus labrax L.) Chemosphere. 75:878–882.

Fegan, D., Spring, P., 2008. Recognizing the reality of the aquaculture mycotoxin problem: searching for a common and effective solution. Proceedings of Alltech’s 23st Annual Symposium, Lexington, Kentucky,USA. 343-354 pp.

Food and Agriculture Organization (FAO) (2004) Worldwide regulations for mycotoxins in food and feed in 2003. Food and Nutrition Paper No. 81. FAO, Rome, Italy.

Han, D., Xie, S., Zhu, X., Yang, Y., Guo, Z., 2009. Growth and hepatopancreas performances of gibel carp fed diets containing low levels of aflatoxin B1. Aquaculture Nutrition.

Hooft, J., 2010. The effects of feed-borne Fusarium mycotoxins on the performance and health of rainbow trout (Oncorhynchus mykiss). In submition of MSc Thesis. University of Guelph. Guelph, Canada. 108 p.

Ngethe, S., Horsberg, T.E., Mitema, E., Ingebrigtsen, K., 1993. Species differences in hepatic concentration of orally administered 3H AFB1 between rainbow trout (Oncorhynchus mykiss) and tilapia (Oreochromis niloticus). Aquaculture 114, 355–358.

R.B., Mair, G.C., Fitzsimmons, K. (Eds.), Proceedings of the Sixth International Symposium on Tilapia in Aquaculture, pp. 172–178.

Centoducati, G., Santacroce, M.P., Lestingi, A., Casalino, E., Crescenzo, G., 2009. Characterization of the cellular damage induced by Aflatoxin B1 in sea bream (Sparus aurata Linnaeus, 1758) hepatocytes. Ital.J.Anim.Sci. vol. 8 (Suppl. 2), 848-850.

Deng S.H, Tian, L.X., Liu, F.G., Jin, S.G., Liang, G.Y.,Yang, H.G., Du, Z.Y., Liu, Y., 2010. Toxic effects and residue of aflatoxin B1 in tilapia (Oreochromis niloticus×O. aureus) during long-term dietary exposure. Aquaculture 307: 233–240.

Doll, S., Baardsen, G., Muller, P., Koppe, W., Stubhaug, I., Danicke, S., 2010. Effects of increasing concentration of the mycotoxins deoxynivalenol, zearalenone or ochratoxin A in diets for Atlantic salmon (Salmo salar) on growth performance and health. Abstract, 14th International Symposium on Fish Nutrition and Feeding. May 31-June 4, Qingdao, China. p. 120.

El-Banna, R., Teleb, H.M., Hadi, M.M., Fakhry, F.M., 1992. Performance and tissue residue of tilapia fed dietary aflatoxin. Vet. Med. J. Giza 40, 17–23.

El-Sayed Y. S., Khalil, R.H., 2009. Toxicity, biochemical effects and residue of aflatoxin B1 in marine water-reared sea bass (Dicentrarchus labrax L.). Food and Chemical Toxicology. 47:1606–1609.

Rodrigues, I., 2008. Biomin Mycotoxin Survey Program 2008. In: Biomin Newsletter. Biomin Holding GmbH, Vol. 7.

Sepahdari, A., Mosavi, H. A. E., Sharifpour, I., Khosravi, A., Motallebi, A. A., Mohseni, M., Kakoolaki, S., Pourali, H. R., Hallajian, A., 2010. Effects of different dietary levels of AFB1 on survival rate and growth factors of Beluga (Huso huso). Iranian Journal of Fisheries Sciences. Vol. 9 No. 1 pp. 141-150

Tu, D. C., 2010. Aflatoxin B1 reduces growth performance, physiological response, histological changes and disease resistance in Tra catfish (Pangasianodon hypophthalmus). In Submition of MSc Thesis. Nong Lam University. Ho Chi Min City. Vietnam.

Tuan, N.A., Grizzle, J.M., Lovell, R.T., Manning, B.B., Rottinghaus, G.E., 2002. Growth and

hepatic lesions of Nile tilapia (Oreochromis niloticus) fed diets containing aflatoxin B1. Aquaculture 212, 311–319.

Udomkusonsri, P., Tangmunkhong, P., Chantakru, S., Arthitvong, S., Boonyawiwat, V., Kusucharit, N., 2008. Study of aflatoxin B-1 levels in muscle and liver and its effects on immune system of Walking catfish (Clarias batrachus) after chronic exposure. Proceedings of the 46th Kasetsart University Annual Conference, Kasetsart, 29 January - 1 February, 2008. pp. 588-597.

Whitlow, L.W., Hagler Jr., W.M., 2002. Mycotoxins in feeds. In: Feedstuffs: The Weekly Newspaper for Agribusiness. Vol. 74.

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F: Mycotoxins

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