Mycotoxicoses In Fish

This article included the story of fish mycotoxicoses under Egyptian conditions. The story started by the definition of a mycotoxin, followed by the chemical structures of different known mycotoxins and the origins of their names. After that, the occurrence of fungi and their toxins in the Egyptian aquafeeds was presented. Thereafter, the article illustrated different factors affecting the production of a mycotoxin. Also, the  mycotoxins-contaminated aquafeeds and the detection methods of mycotoxins were mentioned too. Effects of mycotoxins (mycotoxicoses) and the factors affecting their severity were discussed. Mycotoxicoses of fish, including aflatoxicosis, sterigmatocystin toxicosis, ochratoxicosis, citrinin toxicity, and toxicity of cyclopiazonic acid, t-2 toxin, zearalinone and fumonisin as well as the multi mycotoxicoses were illustrated. The article was tailed by the half lethal dose of mycotoxins besides the  possible means of prophylaxis and treatments of mycotoxicoses.


INTRODUCTION

Global consumption of fish has doubled since 1973, and the developing world has been responsible for nearly all of this growth. More and more fish production will come from aquaculture, whose share in worldwide fish production is projected to increase from 31 to 41 percent in 2020 in the baseline scenario. A healthy natural environment is essential to maintaining fish harvest levels in the face of increasing demand. Intensification can raise the risk of disease (Delago et al., 2004). Fungi (Myces) are plant-similar micro organisms, some of them are large sized (as mushrooms) and the others are microscopic, therefore they are poly-or-mono-cellular.  Some of the fungi are useful for man, since they could be eaten or used in producing drugs, dairy products, bread…. etc., and used in fungal biocontrol.   Yet, the others are harmful for man, animals and plants, since they cause diseases (mycoses) and / or intoxications (mycotoxicoses).  Therefore, fungi are responsible for crops damage (25% of the yearly production), whether in the field, during transportation, and / or during storage.  Toxic fungi can also invade various feed - and foodstuffs and hence affect agricultural animals (Abdelhamid and Saleh, 1996) and humans (Abdelhamid et al., 1999).  Moreover, these toxigenic fungi occur also in and / or on moist houses, libraries, air conditioners, feed mills, dust, air, insects, temples, banknotes, and computer disks and compact disks (Abdelhamid, 1998, 1999-b, and 2000-b).


WHAT IS MYCOTOXIN?

It is a fungal toxin, i.e. it is a secondary toxic - metabolite which produced from a toxigenic fungus.  Any mycotoxin could be produced from many fungal species, and any fungal strain can produce many mycotoxins.  Therefore, any moldy sample may contain numerous fungal genera and species (multi-infection), hence and consequently it may be contaminated with different mycotoxins.  For instance, zearalenone (F-2) is produced by Fusarium roseum, F. tricinctum, F. oxysporum, and F. moniliforme.  Also, diacetoxyscirpenol (DAS) producing fungi are F. equiseti, F. sambucinum, F. tricinctum, F. scirpi, F. solani, F. rigidiusculum, F. culmorum and F. avenaceum.   On the other hand, A. ochracious produces aflatoxins, ochratoxins, penicillic acid, cycalonic acid, viomllin….. etc., and A. flavus produces, aflatrime, aflatoxins, aspergillic acid, aspertoxin, cyclopiazonic acid, kojic acid, penetrimes, rubratoxins, sterigmatocystin, tremorgns….. etc.   So, when one mycotoxin is detected, man should suspect that others are also present in a contaminated feed (Abdelhamid, 2000-b). However, the story of mycotoxins is very new comparing with the old known story of fungi.  It began with the detection of ergot, trichothecines, aflatoxins…. and recently fumonisins. Nowadays, more than one thousand different chemically identified mycotoxins are isolated.  They are of low molecular weights. Some of them acts with each other synergistically as fumonisin-B1 and aflatoxin-B1, ochratoxin-A and aflatoxin, T2 toxin and aflatoxin. Mycotoxins cause a wide variety of adverse clinical signs depending on the nature and concentration of mycotoxins present, duration ofexposure, the animal species, its age and nutritional and health status at the time of exposure to contaminated feed (Horvath, 1998).


CHEMICAL STRUCTURES OF THE MYCOTOXINS

They are peptide derivatives (Cyclochoritme, Ergot, Gliotoxin, Sporidismine), quinone derivatives (Lotuskirin, Rogulosin), peron derivatives (Aflatoxin, Citrinin, Kojic acid, Sterigmatocystin), terpine derivatives (Fusarinone, Satratoxin, Trichothecines, Vomitoxin), nonadrid (Rubratoxin), alkaloid (Lesergic acid, Slaframin), xanthine (Sterigmatocystin), lacton (Patulin, Penicillic acid, Rubratoxin, Zearalenone), botnolid (Patulin), phynol (Zearalenone), glucose (Kojic acid), qumarin (Aflatoxin, Ochratoxin, Sterigmatocystin) as citd by Abdelhamid (2000-b).


NAMES OF MYCOTOXINS

They could be originated from the first known producing fungus, e.g. Aflatoxin (from A. flavous toxin), Ochratoxin (from A. ochracius toxin) and Citrinin (from P. citrinum), or the effect of mycotoxin, e.g. Vomitoxin (from vomiting toxin), or the chemical structure, e.g. Trichothecines (from 12, 13, epoxy- trichothecine unit), epoxy (epoxidation), P (Phenolic form), or its fluorescence color, blue (B), green (G), or the presence or not of double bond (B₁, B₂, G₁ & G₂), and excretion route, milk (M) (Abdelhamid, 2000-b).


OCCURRENCE OF FUNGI IN FISH FEEDS

Survey for microorganisms associated with the Nile tilapia fish revealed that more than 20 fungal isolates belonging to different genera and species including Saprolegnia, Trichoderma, Alternaria spp., Penicillium, Fusarium sp., Fusarium semitectum (= F. incarnatum), Cladosporium, Phoma, Nigrospora, Aspergillus niger and Aspergillus flavus were isolated from naturally diseased fish.  In the winter season of 2002, an attempt was made to isolate Saprolegnia sp. from moderately infected fish, water, and feed sampled from the saprolegniasis-affected pond. Saprolegnia sp. was successfully recovered from the infected fish and pond water samples but was not found in the aquafeed samples.  However, many saprophytic fungi were found contaminating the aquafeed samples.  In the mid summer of 2002,  Aspergillus ochraceus was found associated with a syndrome of eye dropping on Nile tilapia appeared in a commercial intensive fish culture ponds located in the area close to Kafr El-Sheikh.  This is the first report of using the fungus Trichoderma spp. for biological control of fungal diseases of fish.  Out of the present study, it can be concluded that Trichoderma viride is a promising biocontrol agent for the fish pathogens, Saprolegnia sp. and Aspergillus ochraceus.  It can be easily cultured and its inoculum can survive in water and significantly reduce saprolegniasis severity on the Nile tilapia fish. The fungus T. viride is safe and is also used for biological control purposes against plant pathogens. It can also be concluded that the fungus Alternaria eichhorniae 5 (Ae5) which did not cause any mortality to the treated fish can be safely used for the biocontrol of waterhyacinth with no negative effects on fish (Gomaa, 2004).


OCCURRENCE OF MYCOTOXINS IN FISH FEEDS

The most widely found in nature and grow and produce toxins under the proper conditions are fungal genera Aspergillus, Penicillium and Fusarium.  The latest genus requires high moisture content, so outspreads in fields and attacks vegetative substances and known as “field fungi”.  Whereas, both former genera require low humidity, so are outspreading in store houses and known as “storage fungi”.  However, moisture content greater than 14% and relative humidity greater than 70% are required for fungal growth and toxin production.  Fungal invasion negatively affects physical (texture, color, odor, flavor) and chemical (mineralization) properties as well as feeding value of the infected feed (Abdelhamid, 1993b; 1995b&c; 1999a; 2000a and 2001 and Abdelhamid et al., 1985). So, it is economically important to avoid buying damaged (mechanically or moldy) feed stuffs, maintain good conditions in store houses and do not store aquafeeds for long periods (Abdelhamid, 1985; 1989 & 1990 and Noonpugdee et al., 1986). Toxigenic fungi and their toxins are found often in various feeds of plant and animal origins including Aspergillus flavus, A. niger, Mucor, and Pencillium . The following Table illustrates some Egyptian aquafeeds and their mycotoxins content (Abdelhamid, 1980, 1983a - e, 1985, 1990, 2000b & 2005 and Abdelhamid et al., 1996):     

A. flavus producing for aflatoxins was found in dried shrimp and shrimp paste.  Also, A. ochracious, A. flavus, A. tamari and A. niger were found in smoked fish, so smoked fish contain aflatoxins and ochratoxin-A.  Fish meal contained aflatoxin-B1 and ochratoxin-A; hence, sea foods were contaminated with aflatoxin-B1 residues, therefore caused human mycotoxic food poisoning.   However, Egyptian feedstuff samples were tested for the presence of some mycotoxins and found to be contaminated, particularly with vomitoxin, aflatoxin, citrinin, zearalenone and ochratoxin, in descending order concerning the percentage of rejected (highly contaminated than the tolerable level) samples.  Aquafeeds of fish were heavily contaminated with aflatoxin up to 3388 ppb (Abdlhamid et al., 1997). However, co-occurrence of cyclopiazonic acid  was found in the aflatoxin-contaminating feed samples (Balachandran and Parthasarathy, 1996). Generally, mold toxins are more toxic to the juveniles of any species (Lim and Webster, 2001).


FACTORS AFFECTING MYCOTOXINS PRODUCTION

Each fungus requires special conditions (substrate, moisture, temperature….) for its growth and other conditions for its toxin(s) production which are different than those of the other fungi and toxins.  However, the main affecting factors on toxin production are genetic factors (related to the fungus, its strain and its genetic capability) and environmental factors  including:

1. The substrate (on which the fungus will grow) and its nutritious content.
   
   
2. Water content {water activity (aw)} of the substrate and ambient relative humidity.
   
   
3. Ambient temperature (dry growing season).
   
   
4. Ambient oxygen content (is required for fungal growth).
   
   
5. Ambient carbon dioxide (not required for fungal growth).
   
   
6. Mechanical damage (enable fungal invasion and mycotoxin production).
   
   
7. Insects invasion (enable fungal invasion and mycotoxin production).
   
   
8. Increased count of fungal spores accumulates the produced mycotoxin.
   
   
9. The growth of non-toxic fungal strains inhibits the production from the toxigenic fungi.
   
   
10. Presence of specific biota inhibit growth of fungi and mycotoxin production.
    
    
11. Time of fungal growth (after the plateau , the capability of producing  toxins decreases).
    
    
12. Cultivation operations [plants density/area unit (micro clime), agricultural rotation, fertilization, wet harvest, mechanization, storage period….. etc.].
    
    
13. Low layer thickness of a crop (< 50 cm) during drying strongly    decreases mycotoxin production  (Abdelhamid, 2000-b).

MYCOTOXINS DETECTION

The method of mycotoxin analysis depends mainly on the mycotoxin it self (or its metabolites) and the contaminated tissue or substance will be tested.  Therefore, there are many detection methods for each mycotoxin, and there are screening methods for detecting more than one mycotoxin simultaneously in the same sample.  However, each method has specific accuracy, sensitivity, recovery and reproducibility within a specific range of the mycotoxin levels (Abdelhamid, 1981, 1995a and 1996).  The principles of analysis consist of precise sampling, sample preparation and toxin extraction,  purification, derivation, elution, concentration, qualitative detection, confirmation, and quantitative detection. Methods of mycotoxins examination include biological methods (e.g. cells, tissues, eggs, shrimp, fish, chicks…etc), physical methods (e.g. UV-light), physico-chemical methods {e.g. spectrophotometer and chromatography (Paper, Column, TLC, HPTLC, LC, HPLC, GLC – MS)} and immuno-enzyme methods, e.g. ELISA (Schweighardt et al., 1980-a & b and Abdelhamid, 1985, 1996 & 2000-b).


EFFECTS OF MYCOTOXINS

These include inhibition of DNA, RNA and protein synthesis (Aflatoxin, DAS, Ochratoxin-A), genotoxic (Aflatoxin, DAS, Zearalenone), proteolytic (Oxalic acid), chromatide plaster (Destruxin-A), depression of ATPase (Oosporein), hormonotoxic (Fusaric acid), estrogenic, sexual (PR-toxin, T2, Zearalenone), cellulartoxic – free-radical and active oxygen producing (Aflatoxin), carcinogenic (Aflatoxin, Citrinin, Cyclosporins-A, Ferrocarin E, Ochratoxin-A; Oxalic acid, Patulin, Penicillic acid, Sterigmatocystine, Trichothecines), immunotoxic (Gliotoxin, Fumonisin, Vomitoxin), neurotoxic (Aspergillic acid, Fumonisin, Gibberllic acid, Kojic acid, Tremorgens, Vomitoxin), circulatory system (Aflatoxin, Citriovuridine, Cyclopiazonic acid, Cyclosorine, Ergot, T2), hepatotoxic (Aflatoxin, Fumonisin, Sterigmatocystin), nephrotoxic (Aflatoxin, Citrinin, Fumonisin, Ochratoxin, Oosporein, Oxalic acid), digestive system toxins (Lotuskerine, Rgiolocin, Rubratoxin, Sterigmatocystin), respirotoxic (Gliotoxin), and dermal toxic (DAS, Ergot, Palutin, Trichothecines, T₂), (Armbrecht, 1972; Lee et al., 1978 and Mahmoud et al.,1994).


FACTORS AFFECTING SEVERITY OF A MYCOTOXIN

It may be affected by many factors including the mycotoxin it self,  level of contamination (chronic, sub acute, acute), time of exposure, route of application, presence of other mycotoxins, the organism exposes to a mycotoxin (genetic effect on the enzyme system), sex and age of the exposed organism (hormonal effect), and clinical status of the exposed organism (hepatic enzymes status) (Abdelhamid, 2000-b).


MYCOTOXICOSES OF FISH

The outspreading of aquaculture and intensive use of artificial feeds led to fish mycotoxicoses.  So, special or specific scientific conferences are held to discuss this problem, e.g. Trout Hepatoma Research Conference, since more than four decades.  The etiology and epidemiology of trout hepatoma were described since 1967 by Ashley, Dollar et al. and Scarpelli.  An updated review of the trout hepatoma problem has appeared thereafter (Halver, 1969).  At the same period, Halver et al. (1969) reported that the resistance of Coho salmo appears to be 10 – 30 times the value found for rainbow trout, so tumors were not developed in fish fed a diet containing 20 ppb (mg/Kg) aflatoxin daily for 20 months.


1- Aflatoxicosis

The first known and very popular is the toxicity of aflatoxin.  Aflatoxin in cottonseed meal was responsible for serious economic losses (because of liver cancer, hepatoma) in hatchery-raised trout in the USA in April of 1960 (Goldblatt, 1976) until research revealed the intense sensitivity of rainbow trout to the toxin. Common effects of aflatoxicosis in finfish include poor growth, pale gills, reduced RBCs, anemia, impaired blood clotting, damage to liver, decreased immune responsiveness and increased mortality.  In rainbow trout, prolonged feeding of a low concentration of aflatoxin B₁ (AFB₁) causes liver tumors.  Rainbow trout is one of the most sensitive animals to AFB₁, so LD₅₀ for 50 g rainbow trout is 500 – 1000 ppb.  However, warm water fish, such as catfish, are less sensitive to AFB₁ (it has IP-LD₅₀ of 11.5 mg/Kg body weight).  Feeding catfish on at least 10 ppm AFB₁ – contaminated feed for 10 weeks had adverse effects on the fish.  Growth rate, PCV%, Hb concentration and erythrocyte count were lower than those from the other treatments (0, 100, 500, 2000 ppb).  At the highest level, AFB₁ caused necrosis and basophilia of hepatocytes, enlargement of blood sinusoids in the head kidney, accumulation of iron pigments in the intestinal mucosa epithelium, and necrosis of gastric glands.  However, the sub chronic toxic level of AFB₁ for catfish is approximately 6000 ppb of diet (Lovell, 1992).

Mean leukocyte count was significantly higher in the fish fed the highest concentration of AFB₁ (Jantrarotai and Lovell, 1990a).  Moreover, AFB₁ administration (12 mg/kg body weight) caused regurgitation of stomach contents by channels catfish (Jantrarotai et al., 1990). Rainbow trout fry exposed to aqueous solutions of 0.05 – 0.5 mg  AFB₁ /l for 30 min. reflected cytotoxicity with carcinogenicity which dependet on metabolism of  AFB₁  to the electrophilic 8,9-epoxide that can react covalently with cellular macromolecules and that cytotoxicity contributes to, but is not required for, hepatocarcinogenesis. It is suggested that presunptive oval cells are responsible for liver regeneration (Nunez et al., 1990).

Transmission of aflatoxin through toxic residues in fish may prove to by a potential public health hazard (Prasad et al., 1987). However, Plakas et al. (1991) found that AFB₁-residues in catfish muscles were rapidly depleted.  So, it is concluded that catfish has a very low potential for the accumulation of AFB₁ and its metabolites in the edible flesh through the consumption of AFB₁-contaminated feed. El-Banna et al. (1992) fed tilapia gingerlings AFB₁-cntaminated diets. They showed no effect on 50 ppb concerning fish performance and body composition; yet,  AFB₁-residue showed a cumulative effect related to the level  of  AFB₁ and feeding period. Walleye fish fed for 30 days on 50 or 100 ppb aflatoxin reflected 8% mortality rate, pale livers, and degenerative changes.  Residues of aflatoxins B₁, G₁ and G₂ were detected in fish muscles at up to 20 ppb.  After 2 weeks withdrawal period, no aflatoxin residues were detected but marked histo-pathological lesions were still seen (Hussain et al., 1993).

Variation between species was found also (Nakatsuru et al., 1990), since a high rate of DNA binding was observed in rainbow trout, whereas significantly lower values were evident in Coho salmon, indicating a direct relationship between binding levels and susceptibility to mycotoxin carcinogenicity.  Differences among fish species concerning their sensitivity for AFB₁ were mentioned too by Ngethe et al. (1993) who concluded that there is a species difference in liver metabolism and/or affinity of AFB₁-derived metabolites to hepatic macromolecules and that hepato-toxic effects are therefore less for tilapia than for rainbow trout.  In a comparative 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.  Dietary aflatoxin treatment decreased feed consumption, growth performance and feed and nutrient utilization. Additionally, Abdelhamid et al. (1997) revealed that catfish was more resistant than tilapia for aflatoxicosis.  Yet, catfish contained more residual aflatoxin than tilapia.  This led to a conviction that aflatoxin metabolism is different, dependent on fish species. Moreover, Troxel et al.(1997) verified that zebrafish can bioactivate AFB₁ and the resulting DNA adducts suggest sensitivity to this carcinogen.

However, dietary 17-estradiol promotes AFB₁ carcinogenesis in trout (Nunez et al., 1989). Also, Zhang et al. (1992) proved that aflatoxin-induced tumors in fish increased with environmental temperature. Since, acute shift of trout to lower temperature reduced  AFB –DNA adduct formation (Carpenter et al., 1995 and Curtis et al., 1995). In addition, dietary indole-3-carbinol promotes  AFB₁-induced hepatocarcinogensis in the rainbow trout (Oganesian et al., 1999).

Moreover, Bailey et al. (1994) found that AFB₁  is three times more carcinogenic than aflatoxicol (AFL); whereas, Bailey et al. (1998) found that relative tumorigenic potencies ofaflatoxins were  AFB₁ 1.0, AFL 0.936, aflatoxin M₁ 0.086, and AFL M₁ 0.041. 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; so, exposure to AFB₁  resulted in the reduction of cytokine, macrophage function and lymphocyte activity, i.e. trout exposed to very low concentrations of AFB₁ in feed or exposed as embryos have a very high incidence of carcinogenesis.

Anyhow, Sarcione and Black (1994) suggested that serum alpha foetoprotein measurements may be useful to confirm the appearance of hepatocellular carcinoma in experimental fish carcinogen-assay system and to detect hepatocellular neoplasia in high-risk wild fish populations exposed to carcinogenic pollutants. Also, Abd-Allah et al.(1999) suggested that the Comet assay is a useful tool for monitoring the genotoxicity of mycotoxins such as AFB₁  and for evaluating organ specific effects of these agents in different species.

Chavez – Sanchez et al. (1994) reported that Nile tilapia reflected decreases in growth and feed intake in direct relation to AFB₁ intake (0 – 3 ppm).  The liver was severely affected, it showed fatty liver, nuclear and cellular hypertrophy, nuclear atrophy, increase in number of nucleoli, cellular infiltration, hyperemia and necrosis.  In kidneys congestion, shrinking of glomeruli and melanosis were observed. Additionally, Nile tilapia fed a diet contaminated with crude aflatoxins for 22 successive weeks showed a significant decrease in growth rate, PCV, Hb conc., erythrocyte count, total leukocyte count and lymphocytes.  The mortality rate was 60% and aflatoxin residues were detected in fish at the end of week 16 (Marzouk et al., 1994). Also, Indian major carb (Labeo rohita) reflected immunosuppressive effect at a very low dose of  AFB₁ (1.25 mg/kg body weight), since they revealed a reduction of total protein,globulin levels, bacterial agglutination titre, nitroblue tetrazolim assy and serum bactericidal activities, as well as an enhanced albumin-globulin ratio without change in albumin concentration (Sahoo and Mukherjee, 2001b).

Recent studies (Abdelhamid et al., 2002b&c) on Nile tilapia aflatoxicosis confirmed the previous results. They reported negative effects including reduces in body weight, growth rates, feed conversion and survival rate.  Also, protein content of the fish as well as its utilization from the contaminated diets reduced, whereas fish fat and ash contents as well as muscular RNA increased.  Blood profile was negatively also affected, since AFB₁ reduced PCV, Hb, RBCs and protein contents, but increased some enzyme activity and WBCs.  The aflatoxic diets led to pathological alterations in all tested tissues of gills, intestine, liver, subcutaneous tissue and muscle, spleen, kidneys, and brain.  The AFB₁ – contaminated diets led to gross clinical symptoms and mortality.  It reduced fish muscles area, elevated internal organs indices, and caused chromosomal aberrations besides lower mitotic index of gill cells.  Severity of its harmful effects correlated positively with its dietary levels.  Its effects varied between fish sizes, so its dietary LC₅₀ was calculated as 1006 and 1318 ppb for Nile tilapia weighting 2 and 30 g, respectively.Hence, AFB₁-contamination of fish diets is a danger threats the fish wealth, and perhaps also the consumer.  Therefore, it is emphasized to hygienic control of aqua feeds and their ingredients (whether in their choice or storage) to avoid their fungal invasion by toxigenic fungi.  Since prophylaxis is more useful than medication (Abdelhamid et al., 2002-b & c and 2003).

Moreover, Tuan et al.(2002) fed Nile tilapia (2.7g) semipurified diets containing 0, 0.25, 2.5, 10 or 100 mg AFB/kg of diet for 8 weeks. Weight gain and hematocrit of fish fed with 0.25 mg AFB/kg were not significantly different from that of the control; however, diets containing higher levels of AFB had significantly reduced weight gain and hematocrit. Histologically, livers of fish fed with diets containing 10 mg AFB/kg conained excess lipofuscin and irregularly sized hepatocellular nuclei. Diets containing 100 mg AFB/kg caused weight loss and severe hepatic necrosis; 60% of the fish in this treatment died by the end of the 8-week feeding period. No lesions were observed in the spleen, stomach, pyloric intestine, head kidney or heart of fish in all treatments. These results indicate that acute and subchronic effects of AFB to Nile tilapia are unlike if dietary concentrations are 0.25 mg/kg or less. 


2- Sterigmatocystin

Sterigmatocystin (STC) is closely related to aflatoxin as a precursor in aflatoxin biosynthesis and classified as an IARC Group-2B carcinogen. Abdelhamid (1988) described the toxicity symptoms of dietary contamination with STC on catfish and common carp.,  since STC naturally contaminates grains and feeds.  It is a hepato- carcinogenic mycotoxin produced by aspergilli and penicillia species.  Polluted diets caused gradual decrease in growth rate as well as in muscular protein content and gradual increase in mortality, serum transaminases activity and muscular dry matter and ether extract contents in addition to some pathological findings in carp in proportion to the dietary levels of STC.  The LD₅₀ was estimated to be as 211 ppb STC in carp diet.  Three months feeding of catfish on STC (250 ppb) led to loss of body weight, increased mortality rate and muscular contents of ether extract, decrease of muscular content of protein as well as to some pathological findings in addition to the presence of residual STC in the fish muscles.


3- Ochratoxicosis

Ochratoxin-A (OCTA) is the most toxic ochratoxin.  Exposure of the developing eggs of the zebra fish resulted in a variety of severe abnormalities such as deformities of the head, tail and eyes.  These embryos hatched but did not reach the larval stages.  After the first feeding, no further malformation were observed (Debeaupuis et al., 1984).  In the same direction, Abdelhamid et al. (1997) did not find ochratoxin-A residues in toxicated Nile tilapia and cat fish.  Also, very little toxin reached muscle tissue of rainbow trout (Fuchs et al. 1986).  Lovell (1992) reported that the oral LD₅₀ for ochratoxin-A in six-month-old rainbow trout is  4.7 mg/Kg.  Pathological signs are severe necrosis of liver and kidney tissues, pale kidney, light swollen livers and death. In a recent  study, Manning et al.(2003b) fed channel catfish (6.1g) diets containing 0, 0.5, 1.0, 2.0, 4.0, or 8.0 mg OCTA/kg diet. Significant reductions in body weight gain were observed after only 2 weeks and at each successive 2-week weighing interval for catfish fed diets containing 2.0 mg OCTA/kg diet or above. At week 8, weight gain was significantly reduced in catfish fed diets containing 1.0 mg OCTA/kg or above. Feed conversion ratio was significantly poorer for catfish fed diets containing 4.0 or 8.0 mg OCTA/kg diet. Hematocrit was significantly lower for catfish fed diets containing 8.0 mg OCTA/kg, but no significant  differences in white blood cells count were observed for catfish at any levels of OCTA. Fish fed diet containing 8.0 mg OCTA/kg had significantly lower survival compared with those of the other treatments. Histopathological examination at 8 weeks revealed that there was increased incidence and severity of melanomacrophage centers in hepatopancreatic tissue and posterior kidney for catfish fed dietary concentrations of 2.0 mg OCTA/kg or above. Exocrine pancreatic cells that normally surround the hepatic portal veins of channel catfish were reduced in number or absent in livers of fish fed 1.0  mg OCTA/kg diet or greater. On Nile tilapia ochratoxicosis-A, Srour (2004) showed that increasing OCTA levels in the diet resulted in decreasing growth performance and feed utilization parameters.  Carcass dry matter, protein and ash contents were negatively correlated with OCTA levels but carcass lipids had positively correlated with OCTA levels.


4- Citrinin

Citrinin-inoculated rohu (Labeo rohita) at 12.5 or 25.0 mg/kg body weight showed damage to the kidney, liver and   intestine with depigmentation and congestion of caudal fins and death. Death was due to acute nephrotoxic and hepatotoxic effects of citrinin in fish (Sahoo et al., 1999).


5- Cyclopiazonic acid

Although Lovell (1992) mentioned that signs of fusarium toxins have not been demonstrated in fish, he gave signs of cyclopiazonic acid (CPA) toxication in channel catfish fed on 100 ppb CPA as reduced growth rate.  The highest concentration (10 ppm) caused necrosis of the gastric glands.  The IP LD₅₀ for CPA was 2.82 mg/Kg. The effects of CPA were characteristic of a neurotoxin.  Fish showed severe convulsions.  So, CPA is more toxic to catfish than AFB₁.  The fact that CPA and AFB₁ are found under similar conditions, often in combination with AFB₁ and often more frequently, indicates that CPA may be a serious contaminant in fish feeds. Cyclopiazonic acid fed for 10 weeks at a concentration of 100 µg/kg of diet had significantly growth-suppressing effect on catfish and a concentration of 10 mg/kg caused accumulations of proteinaceous granules in renal tubular epithelium and necrosis of gastric glands (Jantrarotai and Lovell, 1990b).


6- T-2 toxin

The earliest references found in literature on effects of fusarial toxins on fish are those carried out on rainbow trout (Salmo gairdnerii) as mentioned by Palti (1978). Where, Marasas et al. (1969) administered T-2 toxin derived from F. tricinctum to the trout feed pellets. Mature fish survived acute doses of T-2 higher than the single LC for fingerlings (6.1 mg/kg), although doses of 8 mg/kg severely damged the intestinal tracts og the fish. Additionally, Smalley (1973) mentioned that at LD of T-2 for trout, severe oedema and fluid accumulation in the body cavity and behind the eyes are produced in addition to the loss of the intestinal mucosa. Kravchenko et al. (1989) tested T-2 toxin on the activity of enzymes of xenobiotic metabolism in carp. Glutathione transferase activity increased moderatly, whereas the activity of lysosomal enzymes increased drastically (2-11 fold) and alkaline phosphatase activity increased 2-fold. The T-2 toxin,  a trichothecene mycotoxin, is responsible for significant reduction in growth, significantlly poor feed conversion, adversely affected hematocrit value,  low survivability and histopathological anomalies of stomach and kidneys in juvenile channel catfish (Manning et al., 2003a).


7- Zearalenone

Effects of zearalenone (ZEA) on carp have been studied by Vanyi et al. (1974). Carp was fed with maize groats containing 1,000 ppm ZEA and consumed 2-3 % of their body weight. In the testicles of treated fish severe degeneration of the caniculi was found. The alterations due to the toxin was reversible. Additionally, Arukwe et al. (1999) using a dose-dependent induction of vitellogenin (vitellogenesis and eggshell zona radiate ptoteins (zonagenesis) in rainbow trout which were observed 7 days after exposure, found that - zearalenol and ZEA possess estrogenic potencies that are approximately 50% to that of estradiol-17ß. They concluded that blood analysis of vitellogenin and eggshell zona radiate (ZR) – proteins levels provides a suitable in vivo fish model for assessing the estrogenic potencies of  ZEA and its metabolites. Celius et al. (2000) also came to the same conclusion that trout ZR – gene and proteins provide a sensitive biomarker for assessing oestrogenic activity of ZEA .


8- Fumonisin

Adult channel catfish can tolerate feed contaminated with fumonisin B₁ (FB₁ ) at concentration up to 313 mg/kg for up to 5 weeks (Brown et al., 1994). Yet, Li et al.(1994) found that FB₁ levels below 20mg/kg diet are not a problem in commercial catfish feed. Since, higher levels of FB₁ depressed growth,lowered hematocrit, increased liver glycogen, increased vacuolation in nerve fibers, and perivascular lymphohistiocytic investment in the brain of catfish.    Additionally, Lumlertdacha and Lovell (1995) reported that 2-year old channel catfish (Ictalurus punctatus) fed on 320 or 720 ppm FB₁ lost weight during the 14 weeks feeding period and experienced high mortality caused by Flexibacter columnaris . Fish fed 80 ppm FB₁ showed significantly less weight gain. When challenged by immersion in an aqueous cell suspension of a virulent strain of Edwardsiella ictaluri, fish that had been fed 80 ppm FB₁ – diet for 14 weeks had a significantly lower survival rate than the fish fed 0.3 or 20 ppm FB₁ – diets. Antibody production by fish fed 20 or 80 ppm FB₁ – diets and inoculated with killed E. ictaluri cells was significantly lower after 14 days than antibody production by inoculated control fish. These results indicate that feeding channel catfish corn material contaminated with Fusarium moniliforme and containing fumonisins can reduce the fishes' growth and resistance to E. ictaluri  infection. Also, Lumlertdacha et al. (1995) fed the same fish spices of 1- year old and 2- year old on variable levels (0.3,20,80,320,, or 720 ppm) of FB₁ for 10 and 14 weeks, respectively. They concluded that dietary contaminations with 20 ppm FB₁ or above were toxic to year-1 and year-2 channel catfish.This toxin reduced the body gain, hematocrits and red and white blood cell counts. Additionally, Petrinec et al. (2004) fed one-year-old carp  rations containing 100 or 10 mg/kg of FB₁ . The histology of fish showed that blood vessels, liver, exocrine and endocrine pancreas, excretory and hematopoietic kidney, heart and brain were sensetive to both levels of FB₁ and the rodlet cell frequency was considerably increased in and around damaged tissues.


9- Multi mycotoxins

Residues of  AFB₁ ,  OCTA , and FB₁ appear in flesh and other tissues  of channel catfish soon after the fish consume these mycotoxins in their diets. The rate at which they are retained in the tissues varies among mycotoxins and tissues. Net absorption coefficients were relatively high, being 83.5, 83.7, and 87.8%, respectively (Wu, 1999). Combinations of moniliformin (MON) and FB₁ at levels of 20:40 and 40:40 mg/kg diet did not significantly change the effeect of FB₁ on the ratio of sphingolipids. The only tissue lesions observed in liver and heart were smaller nuclei of cells in livers of fish fed diets containing the two highest levels of MON (60 & 120 mg/kg) and the combinations of the two toxins (Yildirim et al., 2000). However, Carlson et al.(2001) found that FB₁ promoted AFB₁ initiated liver tumors in rainbow trout fish fed 23 ppm FB₁ for 42 weeks; although they suggested  that short – term exposure to FB₁  will not alter phase I or phase II metabolism of AFB₁. Hashimoto et al. (2003) reported the presence of both aflatoxins and fumonisins in 23.8% of the 42 fish feed samples  indicating a risk of toxic synergism, which emphasizes the importance of monitoring mycotoxin levels in fish feeds.  However, Tuan et al. (2003) fed Nile tilapia fingerlings (2.7g) on diets containing 0,10,40,70,or 150 mg/kg of either FB₁ or  MON for 8 weeks. Fish fed MON (either at 70 or 150 mg/kg) or FB₁  (at 40 mg/kg or higher) had significantly lower weight gain than the control. Results of hematocrit, serum pyruvate,  and sphinganine/sphingosne ratio in liver  demonstrate that both mycotoxins are toxic to tilapia and could reduce the productivity of this fish.


LD₅₀ OF MYCOTOXINS

According to the sever toxicity of mycotoxins, importance of the crop and its consumer, if for import or export, economic level as well as precise determination of these toxins; different countries lay out recommendations for maximum tolerance limits of some unavoidable mycotoxins in foods, i.e. aflatoxin B₁ 0 – 1000 ppb, ochratoxin A 25 ppb (Abdelhamid, 2000b), zearalenone 0.5 – 5.0 ppm , vomitoxin 1 – 10 ppm (Ziggers, 2002) and fumonisin B₁  9 – 150 ppm (SCF, 2000). However, the organism used for estimating LD₅₀ in the following Table (Abdelhamid, 2000b) was zebra fish larva for 24 hours (otherwise it will be mentioned):

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