AFLATOXIN B1 TOXICITY IN NILE TILAPIA (Oreochromis niloticus) AND ITS CONTROL

UJJAWAL KUMAR SINGH KUSHWAHADr. S.K. SAH

Dr. D.K. SINGH

Institute of Agriculture and Animal Science (IAAS)

Tribhuvan University

Rampur, Chitwan

kush_ujjwal@yahoo.com

AbstractAflatoxin B1 is extremely toxic, mutagenic and carcinogenic compound produced by Aspergilllus flavus and Aspergillus parasiticus. It is the most common contaminant causing great economic losses in aquacultural enterprises. Improper storage of feed is one of the most important factors favoring the growth of aflatoxin producing molds and it is a major element that can be controlled by the fish producer. It has been reported that dietary aflatoxin B1 (AFB1) adversely affect the growth performance, feed conversion, apparent digestibility coefficients, physiological and biochemical parameters, non specific immunity and lesions in organs particularly in livers and pancrease leading to increase in mortality and decrease in body weight. Severity of its harmful effects correlated positively with fish age, sizes and dietary levels. An in vitro study showed that AFB1 would stimulate the release of free radicals which can cause oxidative damage to bimolecules leading eventually to many chronic diseases as well as cancer. Rasmarinus officinalis, Ocimum sanctum, parsley and β-1, 3 glucan, etc. contain a wide variety of free radical scavenging molecules such as phenolic compounds which are rich in antioxidant activity that provide protection and have a potential role in ameliorating the AFB1 induced DNA damage. Increase in awareness and constant monitoring for aflatoxin in the feed reduce the risk of aflatoxicity in fish. This review focuses on the effects of AFB1 and its toxicity control in Oreochromis niloticus. Keywords: - Aflatoxin, carcinogenic, antioxidant, hepatic, mortality

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Introduction

Aflatoxin, a mycotoxin is among the most common contaminants causing great economic losses in aquaculture enterprises (Abdelhamid et al., 1998; Jantrarotai et al., 1990). It is extremely toxic, mutagenic, and carcinogenic compounds produced by Aspergillus flavus and Aspergillus parasiticus (Kurtzman et al., 1987). Toxigenic A. flavus isolates produce aflatoxin B1 and B2 and toxigenic A. parasiticus isolates produce aflatoxin B1, B2, G1 and G2 (Kosalec and Pepeljnjak 2005, Cotty and Garcia 2007). Aflatoxin (AFB1) is classified as group 1st carcinogen by International Agency for Research on Cancer (IARC) (Anon, 1993). Ottinger and Kaattari (2000) reported that AFB1 is a polycyclic aromatic hydrocarbon that is associated with hepatic carcinogenesis and immunomodulation in a broad spectrum of vertebrates; exposure to AFB1 resulted in the reduction of cytokine, macrophase function and lymphocyte activity. Fish is an important source of protein for human beings. Regarding their importance most countries of the world care for increasing the production whether naturally or via aquaculture. A healthy natural environment is essential to maintain fish harvest levels in the face of increasing demand. Intensification can raise the risk of diseases (Delago et al., 2004). Waliyar (2005) reported that many agricultural commodities were vulnerable to attack by aflatoxin B1. Food products that could also be contaminated with this include corn, sorghum, rice, wheat, soyabean, sunflower, cotton, spices, almond, coconut and milk etc. Waliyer (2005) also reported that exposure to large doses of aflatoxin >6000 mg may cause acute toxicity with lethal effect; whereas exposure to small doses for prolonged period is carcinogenic. Interest in the toxic effect on cultured warm water fishes such as tilapia (Oreochromis sp.) and Channel catfish (Ictalurus punctatus) has increased as diets for these species are now being formulated to contain more plant and less animal ingredients. This increases the potential for development of aflatoxicosis in these species because plants ingredients have a higher potential than animal ingredients for contamination with aflatoxins (Anne et al., 2002). These subtle effects often go unnoticed and profits are lost due to decreased efficiency in production such as slow growth, reduced weight of the fish product, an increase in the amount of feed needed to reach market weight and increased medical costs (Anne et al., 2002; Abdelhamid et al., 2002 b and c). Aflatoxin B1 is a big hindarance for overall productivity of Nile tilapia. Thus to obtain maximum profit, appropriate management and its control is necessary. The main purpose of this study was to review the effects of aflatoxinB1 in Nile tilapia and recommend for control of its toxicity. Factors affecting feed contaminationFactors that increase the production of aflatoxins in feed include environmental temperature above 27oC, humidity greater than 62% and moisture levels in the feed above 14 % (Anne et al., 2002). The content of contamination varies with geographic location, feed storage practices and processing methods. Improper storage is one of the most important factors favoring the growth of aflatoxin producing molds. Poor storage of feed ingredients and nutritionally complete feed can lead to contamination with aflatoxins (Waliyar, 2005; Anne et al., 2002). In tropical and sub tropical conditions this potential is further increased due to storage under humid and hot conditions. Effect on non specific immunityEl-Boshy et al., (2008) studied the effect of aflatoxin B1 in non specific immunity and disease resistance of Oreochromis niloticus. AFB1 fed at 200µg per kg crude feed for 21 days to Nile

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tilapia. Dietary supplementation of AFB1 group showed normocytic, normochromic anaemia, lymphopenia as well as alterations related to organs damage induced by AFB1 (Abdelhamid et al., 2002 b and c; Chavez-Sanchez, 1994). The AFB1 treated group showed significant reduction in non specific immunity as reduced superoxide anion, production of blood phagocytes, serum lysozyme, serum bacterial activity, neutrophils glass adhesion and macrophage phagocytic indices. However AFB1 caused significant increase in WBC count and some enzyme activity like alkaline phosphastage, glutamic oxaloacetic tranferase and glutamic pyruvic transferase activities in aflatoxicated fish (Abdelhamid et al., 2002 b and c, 2003; 2004 a and b and El-Boshy et al., 2008). Also Abdelhamid et al., (2002 b and c) reported negative effects including reduced fish muscle area, elevated internal organ indices and causes chromosomal aberrations besides lower mitotic index of gill cells. Tuan et al., (2002) study showed that tolerance levels for tilapia can vary with the production system. El-Banna et al., (1992) concluded that AFB1 residue showed a cumulative effect related to the levels of AFB1 and feeding period. The carcinogenic effect of aflatoxin B1 has also been studied in fishes such as Salmonid, Rainbow trout, Channel catfish, tilapia, carps etc (Wu, 1998; Murjani, 2003 and Bautista et al., 1994) as cited by Cagauan et al., (2008).AFB1 proved to be immunosuppressive in Rohu even at the lowest dose(1.25mgkg-1 body weight) of toxin treatment.(Sahoo and Mukhergee,2001)

Figure1:- Histopathological changes in liver of O. niloticus injected with different levels of AFB1as compared to control (A&B). The control fish groups showing normal structure of the liver (C&D). (El-Barbery et al., 2006)

Effect on Growth and SurvivalIt has been reported that dietary aflatoxin B1 adversely affect the growth performance (Bintvihok et al., 2003; Cagauan et al., 2008; Boonyaratpalin et al., 2001; Abdelhamid et al., 2004 a and b; 2003 and 2002 b and c; Burgor and Hernadez et al, 2005; Tuan et al., 2002), caused significant increase in corrected mortality rate (Chavez-Sanchez, 1994), fat and ash contents of fish carcass (Abdelhamid et al., 2002 b and c and El-Boshy et al.,2008). Tuan et al., (2002) fed semi purified diets to Nile tilapia (2.7g) containing 0, 0.25, 2.5 and 100 mg AFB1 per kg of diets for 8 weeks. Weight gain of fish fed with 0.25 mg AFB1/kg were not significantly different from that of the control; however, diets containing higher levels of AFB1 had significantly reduced weight gain. Diets containing 100mg AFB1/kg caused weight loss and 60% of the fish in this treatment died by the end of 8 week feeding period. Mean percent survival in different treatments of Oreochromis niloticus were significantly different (Table1.). The lowest percent survival (4.17%) was obtained at 100 mg/kg AFB1 concentration while 0 mg/kg AFB1 gave 100% survival. Mortality increased as aflatoxin level increased. This suggests that the total biomass of fish was affected by the percent of survival. Thus the daily feed ration of aflatoxin treatments given to the fish was not completely consumed, an indication of the decreased appetite of fish, perhaps due to unpalatable feed. Decreased growth and appetite were observed by Royes et al., (2002) when tilapia was fed with diets containing 1800 ppb of aflatoxin for 75 days.

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Table 1:- Survival rate (SR %) of aflatoxicated fish at different concentration after 96 hr of I. P. injection.AFB1 No. of fish at different intervals h. Concentration-------------------------------------------------------------------------------------------(mg kg-1) 0 12 24 36 48 60 72 84 96 SR%

0 24 24 24 24 24 24 24 24 24 100

20 24 24 23 22 22 21 20 18 17 70.8

40 24 22 20 17 16 15 13 12 11 45.8

60 24 18 14 13 12 10 6 6 5 20.8

80 24 15 13 9 8 6 4 3 2 8.30

100 24 12 10 8 7 5 3 2 1 4.17

Chavez et al., (1994) reported that O. niloticus fingerlings were able to tolerate the immediate effect of aflatoxin but later the fish developed external and internal abnormalities as cited by Cagauan et al., (2008)Effect on liverIt had been reported that dietary aflatoxin B1 adversely cause histological changes particular on hepatopancreatic tissue ( Burgor and Hernadez et al, 2005; Tuan et al., 2002). Abdelhamid et al., (2004 a and b; 2003) showed that AFB1 led to significant decrease in crude protein and energy content of fish carcass. Similarly increases in ether extract of livers were recorded. Severe histological alterations were reported in livers of the aflatoxicated fish (Marzouk et al., 1994). Histologically livers of fish fed with diets containing 10 mg AFB1/kg contained excess lipofusin (Tuan et al., 2002). Diets containing 100mg AFB1/kg caused hepatic necrosis. The aflatoxic diets led to pathological alterations in all tested tissues of gills, intestines, livers, subcutaneous tissue and muscles, spleen, kidney and brain. Cagauan et al., (2008) reported that AFB1 ingested fishes livers were abnormal, enlarged, and yellowing. Collapsed liver were found in newly dead fishes. Similar manifestations was reported by Royes et al., (2002; Roberts, 1978; Ferguson, 1989) as cited by Cagauan et al., (2008). The livers of fishes had extensive necrosis, acute cellular swelling or ballooning necrosis, chronic granulomatus, inflammation, loss of colour where the necrotic tissues becomes paler than the surrounding normal tissue. Joner (2000) described the effect of aflatoxin in the liver as follows: first aflatoxin is absorbed from the diet in the alimentary canal and is passed to different organs. The principal target organ for aflatoxin is liver. After the invasion of aflatoxin into livers, lipid infiltrates hepatocytes and leads to necrosis. The main reason for this is that aflatoxin metabolites react negatively with different cell proteins which lead to inhibition of carbohydrates and lipid metabolism and protein synthesis, decrease in liver function, disarrangement of the blood clotting mechanism, jaundice and a decrease in essential serum proteins synthesized by the liver.

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Effect on biochemical parametersEl-Boshy et al., (2008) reported that dietary supplementation of AFB1 group showed alterations in some biochemical parameters (Abdelhamid et al., 2002 b and c and Chavez 1994) related to organ damage induced by AFB1. Abdelhamid et al., (2004 a and b; 2003) found out that AFB1 led to significant (p<0.01) decrease in haemoglobin concentration, RBC count (Abdelhamid et al., 2002 b and c; Chavez-Sanchez, 1992) and uric acid. However; it increased WBC count (Abdelhamid et al., 2002 b and c and El-Boshy et al., 2008). Alanine Aminotransferase(ALT) and Aspartate Aminotransferase (AST) activities were significantly increased in the fish groups treated with AFB1 as compared with control group (Table 2). This elevation could be attributed to hepatic injury. Several authors concluded the increase of such enzymes to the hepatotoxic effect of AFB1 (Jones et. al., 1997 and Soliman et al., 1998; Pepeljnjak et al., 2003). Hussein et al., (2000) reported elevation of liver transminase of O. niloticus that had been intoxicated with AFB1. Total protein and albumin concentrations were decreased in AFB1 treated group. This decline in serum protein concentration may be due to impaired protein synthesis or liver disorders caused by mycotoxins (Buhler et al., 2000). Several authors also reported decline in plasma protein concentration in fresh water fishes (Nile tilapia and Rainbow trout) exposed to mycotoxins (Hussein et. al.2000 and Pepeljnjak et. al., 2003). Also Sahoo and Mukherjee (2001) observed decreased in total plasma protein and albumin blood level in Labeo rohita exposed to AFB1. The plasma glucose concentrations significantly increased in fish group treated with AFB1 as compared with control group .Hypoglycemia has also been reported in O. niloticus that fed 0.5 µg or 1 µg AFB1 /kg body weight for 10 days (Hussein et al., 2000). It may be due to reducing activity of pancreatic enzymes (Jantrarotai et al., (1990) as cited by El-Boshy et al., (2008).

Table 2.Effect of dietary supplementation of AFB1 and 1, 3- β glucan on some biochemical parameters (Mean + S.E.) in Nile tilapia (Oreochromis niloticus).Group ALT

µ/mlAST µ/ml

Total protein g/dl

Albumin g/dl

Glucose mg/dl

Ureamg/dl

Creatininemg/dl

Uric acidmg/dl

Group A(control)

37.1

±3.41a

12.1

±9.4a

3.18

±0.21a

1.42

±0.11a

118

±7.2a

8.1

±0.52a

0.61

±0.10

2.01

±0.21a

Group BAFB1

65.1

±6.15b

174

±8.65b

2.65

±0.18b

1.01

±0.10b

146

±8.5b

11.8

±0.41b

0.71

±0.09

3.48

±0.37b

Group CAFB1 and Glucan

59.4

±5.6b

162

±9..12b

2.81

±0.16ab

1.09

±0.41b

131

±9.1ab

11.4

±0.68a

0.68

±0.07

2.98

±0.28b

Group DGlucan

35.1

±2.8a

124

±7.24a

3.25

±0.24a

1.36

±0.16a

121

±8.6a

7.65

±0.71a

0.58

±0.12

2.14

±1.29a

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Source:-El-Boshy et al. (2008)The elevation of urea in AFB1 treated group was reported in this study. Urea in fish is produced by liver and excreted primarily by the gills rather more the kidneys (Stoskoph, 1993). The elevation of urea in El-Boshys investigation might attribute to gill disfunction. Hussein et al., (2000) reported hyperplasia at the basal part of secondary lamellae in O. niloticus after exposure to AFB1. The creatinine value in AFB1 treated group was insignificantly different as compared with control. The insignificant increase of creatinine level in AFB1 treated group might be attributed to the renal damage that was not severe to induce renal impairments. Uric acid is formed by fish from exogenous and endogenous purines. It is converted in the liver to urea for excretion by the gills (Stoskoph, 1993). Elevation of uric acid levels in AFB1 treated groups could be attributed to liver damage induced by aflatoxin (Carlson et al., 2001; Ottinger and Kaattari, 2000).Effect on general physiologyIt has been reported that dietary aflatoxin B1 adversely affect the feed conversion, apparent digestibility coefficients and cause physiological disorders ( Burgor and Hernadez et al, 2005; Tuan et al., 2002). Residue of AFB1 (ppb) were found in the whole body of the aflatoxicated fish directly at the end of the experiment and tended to decrease after freezing periods. As well as severe histological alterations were recorded in livers, kidneys, intestine and gills of the aflatoxicated fish (Abdelhamid 2003 and 2004 a and b and Marzouk et al., 1994).Management and controlPurchase of feeds that have been recently prepared and properly stored is recommended. Control of rodents and insects are also important in maintaining nutrient quality and aflatoxin free feeds (Anne et al., 2002). A possible remedy for feeds that do contain small amount of mold is to add a toxin inactivator to the stored feeds. Increased awareness and constant monitoring for aflatoxins in the feed will reduce the risk of aflatoxicity in fish. To prevent aflatoxicosis, follow manufacturer’s recommendations regarding shelf life and try to determine feed manufacturers date. Be aware of supplier’s storage practices. Avoid using feeds that appear discoloured; lump together and smell musty. Clean feed storage bins and automatic feeders regularly (Anne et al., 2002).Abdelhamid et al. (and 2004 a and b; 2003) tested some natural materials as a feed additives (clay, egg shells, shrimp shells and betaine ) on AFB1 contaminated diets of tilapia fish. They found that the best feed additives led to significant overcoming the aflatoxic symptoms. The results also indicated that the effect of either adsorbent namely, egg shells and shrimp wastes at levels of 1 and 2% respectively were useful to reduce the toxic effects of AFB1 on Oreochromis niloticus fish via adsorbing the toxins from the fish diets. However; Hussein et al., (2000) found that dietary additions of Nigella sativa significantly ameliorated the adverse effects of dietary AFB1 on Nile tilapia. El-Boshys et al. (2008) reported that feeding of β-1, 3 glucans to healthy fish raised the non specific immunity and provided protection against bacterial infection compared with the control. Moreover the feeding of β-1, 3 glucans to AFB1 induced immunocompromised fish significantly raised the degree of resistance against S. iniae and challenge the non specific immunity level in comparison to none treated AFB1 exposed fish. The study showed that β-1, 3 glucans was able to enhance the non specific immunity and disease resistance of the Nile tilapia immuncompromised with AFB1 ( Kumari and Sahoo 2006; Selveraj et al., 2006; Guselle et al., 2007 and Sahoo and Mukhergee 2001). El- Boshys et al., (2008) observed raised serum bactericidial, lysozyme and superoxide anion activity in the beta- 1, 3 glucan treated groups in compared with AFB1 and control group. Β-1, 3 glucan stimulate

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lymphocyte activation (De Baulny et al., 1996) and acted as a stimulant for neutrophils function in Pinephales promela fish (Palic et al., 2005). It also enhanced phagocytic index that had been reported by Wang et al., (2007) and Ali et al., (2007) in Nile tilapia and Pseudosciaena crocea after treatment with β-1, 3 glucans.Supplementation of fish diet with adequate amount of activated charcoal could effectively alleviate lesions of AFB1 (Mohamed and Mokhbatly, 1987) in O. niloticus. El-Barbery (2008) concluded the damage due to AFB1 can be repaired or reduced by using Rosemary and Parsley, Particularly with the lowest levels of AFB1. Also, rosemary may be more effective in reducing the DNA damage than Parsley, especially with the highest levels of rosemary. Yet, the low level of parsley is better than the high level.

In most commercial aquaculture operations feed is one of the main production costs, thus good nutrition and feed management has a direct effect on the bottom line. Good feed production starts with raw material selection and quality control and continues through feed manufacture, storage and use on the farm.

Fish Mortality

Disturbance in metabolism and appearance of health problems Carcinogenesis and mutagenesis Formation of AFB-DNA adducts in liver cell

Release of Reactive Oxygen Species Chromosomal damages

Release of Free radicals

Formation of toxic metabolites (AFB-8, 9 epoxide) Aflatoxin B1 in livers

Antioxidants No release of Free Radicals and ROS

in livers

Different organs (Livers, kidneys, heart, pancrease etc.)

(Distribution) Good diets/Mold inhibitor

Absorption in alimentary canal

(AFB1 contaminated diets)Normal Oreochromis niloticus No Health problems

Figure2:- Frame work on mechanism of different diets in Oreochromis niloticus

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Fish that exposed to chronic or acute toxicity of aflatoxin developed various health problems including reduction of growth performance, feed utilization, increased mortality (El Barbary et al., 2006; Abdelhamid et al., 2004 a and b; 2003) and deleterious effects on reproductive traits (Mehrim et al., 2006) . Aflatoxin also causes dangerous histopathological changes in internal organs in addition to the mutagenic and carcinogenic effects (Burgor and Hernantz et al., 2005; Binthvihok et al., 2003; Boonyaratpalin et al., 2001; Murjani et al., 2003 and Thorgaard et al., 1999). The metabolic activation of AFB1 results in the formation of toxic metabolites such as AFB1 -8, 9 epoxide. The oxide subsequently covalently binds to DNA to form AFB-DNA adducts in the liver (Klaung et al., 1998). The mechanism of action of aflatoxin in the cell is mediated through the production of free radicals and reactive oxygen species (ROS) (Baynes, 1998). An in vitro study showed that AFB1 would stimulate the release of free radicals resulting in chromosomal damages (Amstad ,1984) so, ROS may in part be responsible for the carcinogenic activity of AFB1 (Shen et al., 1996)Over production of such free radicals can cause oxidative damage to bimolecules (e.g. lipids, proteins, DNA) leading eventually to many chronic diseases as well as cancer. The defence is provided by antioxidants, many herbs may contain a wide variety of free radicals scavenging molecules such as phenolic compounds which are rich in antioxidant activity (Zheng and Wang, 2001). Leaf extract of Ocimum sanctum provides protection against AFB1 carcinogenesis by acting as an antioxidant (Rastogi et al., 2007). Similarly El-Barbery reported rosemary (Rasmarinus officinalis) (Albu et al., 2007 and Troncoso et al., 2005) and parsley (Hinneburg et al., 2006; Caillet et al., 2007) repair damage due to AFB1. While Nogueria (2006) reported that Melampodium divaricatum, a medicinal plant extract had an antigenotoxic action towards the DNA damage induced by AFB1. The effect of AFB1 on O. niloticus was investigated with emphasis on the possible protective effects of Nigella sativa (Hussein et. al ., 2000) and ginger and Chamomile (Mehrim et al., 2006). Similarly El-Boshy(2008) reported β-1, 3 glucan able to reduce adverse effects of aflatoxin B1. Differences among fish species concerning the sensitivity for AFB1 were mentioned too by Ngethe et al., (1993) and Tuan et al., (2002) who concluded that there is a species differences in liver metabolism and affinity of AFB1 derived metabolites to hepatic macromolecules and that hepatoxic effects are therefore less for tilapia than for Rainbow trout. In a comparatives study on aflatoxicosis by fish, Omar et al., (1996) showed that Grey mullet is highly sensitive to AFB1 followed by Common carp, Red tilapia, and Nile tilapia respectively. Abdelhamid et al., (1997) revealed that catfish was more resistant than tilapia for aflatoxicosis though catfish contained more residual aflatoxin than tilapia. This led to a conviction that aflatoxin metabolism is different dependent on fish species

ConclusionAflatoxin contamination can cause great economic losses in aquacultural enterprises. Thus a healthy natural environment is essential to maintain fish harvest levels in the face of increasing demand. Aflatoxin B1 (AFB1) adveversly effect the growth and survival, hepatopancreatic tissues, non specific immunity, biochemical parameters and general physiology of Nile tilapia. Thus preventing fungal invasion is a must. Proper care should be given in feed processing and storage practices. Increase in awareness and constant monitoring for aflatoxin in the feed will reduce the risk of aflatoxicity in fish. Antioxidants enhance protection and ameliorate toxicity of AFB1 in Nile tilapia. Further research should be focused on search of either new or effective mold inhibitors or toxin binders in aqua feed which should be more viable and sustainable in nature.

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AcknowledgementsI express my great and deepest devotion to my parents whose inspiration and quality education that they have given, made me to write this paper. I am very thankful to my dear professors (From IAAS,Nepal) Dr.S. K. Sah and Dr. D.K. Singh whose kind help and inspiration gave me extra energy during writing this paper. I thanks my brother S.K.Kushwaha for inspiring and cooperativeness as well as my sisters Lalita and Sarita.

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