Aflatoxin B1 toxicity and its reduction by using coumarin and vitamin E in Nile tilapia

Introduction

Egyptian aquaculture has grown rapidly, with annual production reaching 705,000 tons in 2009 – up from 57,000 tons in 1994. Per capita consumption of fish in Egypt rose from8.5 kg to 15.4 kg/ capita/ year between 1996 and 2008. Population increase and rising consumer search for lower-cost alternatives to expensive meat and poultry are driving this demand. Fish are important protein source for human beings in many countries. Most countries of the world care for increasing the fish production whether naturally or via aquaculture. Aquaculture is currently the largest single source of fish supply in Egypt accounting for almost 65% of the total fish production of the country with over 99% produced from private owned farms (GAFRD, 2009).

Mycotoxins are secondary metabolites of some filamentous fungi, mainly those belonging to Aspergillus, Penicillium and Fusarium and they are toxic. They can be found in raw materials and products of food industry as well as in feedstuffs. Mycotoxin can be present in an organism as a result of food intake producing mycotoxicosis (Mishra and Chitrangada, 2003). The contamination of animal, fish and human feeds with mycotoxins represent a worldwide problem constituting a real threat to the health of livestock for animals, aquaculture industry and human by the continuing intermittent occurrence in their feeds (Vekiru et al., 2007).

In the family of aflatoxins, aflatoxin B₁ is the most prevalent and toxic for human, animals and aquatic organisms, mostly by its strong carcinogenic, mutagenic and teratogenic effects (IARC, 1993; Santacroce et al., 2008 and Han et al., 2008). The mechanism of action of aflatoxin B₁ on the cell is mediated through the production of free radicals and reactive oxygen species (Baynes, 1991 and Van Dam et al., 1995). Amstad et al. (1984) showed that aflatoxin B₁ could stimulate the release of free radicals resulting in chromosomal damages. So, reactive oxygen species may in part be responsible for the carcinogenic activity of aflatoxin B₁ (Shen et al., 1996). A reduction in growth is one of the major negative effects reported due to aflatoxin B₁ contamination. Several studies report reduced growth rates in channel catfish (10 ppm aflatoxin B₁/kg; Janrarotai and Lovell, 1990) and Nile tilapia, (exposed 100 ppb aflatoxin B₁ - Encarnacao et al., 2009, Chavez-Sanches et al., 1994 and El-Banna et al., 1992). In addition, mortality rates of 17% were reported in Nile tilapia fed diets with 0.2 ppm AFB1 (El-Banna et al., 1992). Many countries have established very low maximum permitted levels of aflatoxin in foods, usually in the range of 1 to 25 μg/kg total aflatoxin. The Codex Committee on Food Additives and Contaminants of the Codex Alimentarius Commission has recommended to this Commission that the limit for foods in international trade be set at 15 μg/kg total aflatoxin (Van Egmond, 1989).

Coumarin can be found in many green plants. It is used mainly in the treatment of cancer and can also be used as a blood thinner (Tulayakul et al., 2007).

Vitamin E is biological antioxidants function as biological antioxidants to protect cellular macromolecules (DNA, protein, lipids) and other antioxidant molecules from uncontrolled oxidation by free radicals during normal metabolism or under the conditions of oxidative challenge such as infection, stress, and pollution (Chen et al., 2004).

This study was conducted to evaluate the ability of some treatments, namely coumarin and vitamin E to detoxify the effects of aflatoxin B₁ contaminations in feed for farmed fish.

Materials and methods

The present study was carried out at the wet Laboratory of the Animal Production Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt. The experimental period lasted 98 days from June to September, 2012.

Fish and Experimental Conditions

Healthy fingerlings ofNile tilapia (Oreochromis niloticus) provided by the Fish Hatchery of Central Laboratory for Aquaculture Research at Abbassa, Sharkia governorate, Egypt. Two hundred and seventy fingerlings (weighing approximately7.3 g after adaptation period for three weeks under normal laboratory conditions) were randomly distributed into 18 glass aquaria (35 X 40 X70 cm) containing 75L of water, representing 6 treatments (3 replicates per treatment) maintained aerated de-chlorinated tap water from a storage tank. The system installed in an environmental-controlled laboratory with a photoperiod of 12 hr., light and 12hr., darkness. Continuous aeration was provided by an air blower.

Fish of the first 3 groups were given basal diet without aflatoxin B₁, while the other 3 groups were given basal diets containing 0.25 ppm aflatoxin B₁. Within each the previous two groups they were divided into other 3 subgroups: the first group fed diet without any treatment (control), the second group was treated with coumarin (1, 2 benzopyrone) supplemented (5 g kg^-1 diet) in fish diets, the third group was treated with vitamin E supplemented (50 mg kg^-1 diet) in fish diets.

The diet remains and fish wastes of each aquarium were collected by siphoning before the second daily feeding to further analysis and minimize leaching. Every second day, each aquarium was partially cleaned including the fish feces and the water partially changed (about 50%).

Fish Diet

All fish groups were fed on basal pelleted diet consistent of fish meal 30.0%, soybean meal 20.0%, corn 20.0%, wheat bran 15.0%, alfalfa hay 10.0%, sunflower oil 2.5%, minerals mixture 0.5%, vitamin mixture 1.0% and carboxymethyl cellulose 1.0%. The chemical composition of the diet was crude protein 40.12%, ether extract 6.40%, crude fiber 5.32% and gross energy 4280.0 Kcal/Kg. The feeding rate was 5% of live body weight for the first four weeks and 3% for the remainder of the experimental period. The amount of food was adjusted bi-weekly based on the actual body weight changes. Fish were fed to apparent satiation three times per day (8.00hr., 12:00hr. and 16:00hr.) for 14 weeks.

Preparation and Administration of Aflatoxin

Aflatoxin B₁ used was produced through the fermentation of liquid media (2% yeast extract and 20% sucrose) by Aspergillus flavus MD 341. The fungus was purified by single spore isolation and culturing on Sabaruoud’s dextrose agar media at25°C for 4 days. Polished raw rice was autoclaved 3 times for 3 successive days. A block of fungus from the edge of growing colonies was inoculated into1 kg of sterilized rice and incubated at25°C for 7 days. The rice was autoclaved twice to kill the fungus. The contaminated rice was dried in an oven at56°C for 24 hr. The amount of aflatoxin B1 in the rice was measured by HPLC (AOAC, 2005).

Fish Weigh and Growth

All fish were individually weighed to the nearest 0.1 g at the beginning of the experiment and bi-weekly intervals throughout the experimental period three months. Food consumption was calculated as g/fish/day by dividing the amount of food consumed each day by the number of fish in the aquarium. Feed conversion ratio (FCR) was calculated according to Berger and Halver (1987) according to the following equation: FCR = cumulative feed delivered to aquarium/fish biomass gain.

Chemical Analysis of Fish Body and Aflatoxin Residual

The proximate composition including crude protein, crude fat, crude ash and moisture of body composition was determined using the standard procedures of AOAC (2005).

Samples from three fish of the same group were pooled and thoroughly homogenized in a mortem. Aflatoxins were extracted, filtrated and quantitatively analyzed by HPLC (AOAC, 2005) with a reverse phase column. The mobile phase consisted of 45% methanol and was pumped through the system at a flow rate of 1 ml/min. The column temperature was set to40°C, and analyses were detected using a fluorescence detector. Aflatoxin standards were purchased from Sigma-Aldrich (USA).

Physiological Parameters

Fish were anesthetized with 120 mg/l amino-benzoic acid (Sigma-Aldrich) before the drawing of blood. The body surface of the peduncle region was cleaned and dried using adsorbent paper. Blood samples were collected from the caudal blood vessels at the end of the experimental period using a disposable 1 cc tuberculin syringe. Whole blood was collected in a small sterile vial containing EDTA as an anticoagulant. Samples were used to determine the hemoglobin (Hb) content using a commercial kit (Diamond Diagnostic, Egypt), and the hematocrit (Hct) was measured according to Stoskopf (1993). The total erythrocyte (RBCs), platelets and leukocyte (WBCs) counts were determined using an Ao Bright–Line Haemocytometer (Neubauer improved, Precicolor HBG, Germany) according to the methods described by Jain (1993).

Other blood samples for serum separation were collected without the addition of anticoagulants and then centrifuged at 3000 rpm for 20 min and stored at –20°C until further biochemical analyses. Total protein, albumin (Sundeman, 1964) and plasma transaminase enzymes (AST; aspartate amino transferase and ALT; alanine amino transferase (Reitman and Frankel, 1957) were determined by using the commercial kits from Diamond Diagnostics Company, Egypt.

Water Quality

Water quality control was measured bi-weekly analysis before replacing the water in the aquarium during the experimental period. Total ammonium, nitrite and pH levels using the Hach kit model HI 83205 (Multipurameter Bench Photometer, Hanna Instruments, Romania), dissolved oxygen by HI 9146 (Oxygen and Temperature Meter, Hanna Instruments, Romania). Dissolved oxygen level was maintained above 7 mg by setting the air pump.

Economic Evaluation

Economic evaluation was calculated according to Ayyat (1991) as the following equation: profit = Income from body gain weight - feed cost. Other overhead costs were assumed constant. Price of one kg of diet was 2.159 LE (Egyptian pound = 0.185 US$) and price of selling of one kg live body weight of fish was 10.0 LE.

Statistical Analysis

The data were statistically analyzed with SAS (2002) according to the following model:

Y_ijk = µ + A_i+ T_j+ AT_ij + e_ijk

Where, µ is the overall mean, A is the fixed effect of aflatoxin (i = 1 …2), T is the fixed effect of treatments (j = 1 …3), AT is the fixed effect of the interaction between aflatoxin and treatments and e_ijkis random error. Significant differences between treatments were tested with Duncan’s multiple range test (Duncan, 1955).

Results and discussion

Water Quality

All tested water quality criteria were suitable for rearing Nile tilapia O. niloticus fingerlings. Water temperature, oxygen, pH, ammonia and nitrite (overall mean) were 27.01±0.051 °C, 8.200±0.033 mg/L, 7.895±0.080, 0.327±0.012 mg/L and 0.107±0.007 mg/L, respectively. Ranges of water quality parameters within the acceptable ranges required for normal growth of tilapia as mentioned by Boyd (1990).

Growth Performance and Feed Efficiency

Final live body weight and daily gain were affected significantly (P<0.001) with aflatoxin B₁ contamination (Table 1). Live body weight and daily gain were reduced by 9.69 and 12.42% in fish fed diets contaminated with aflatoxin B₁, when compared with those fed diets without aflatoxin B₁ (basal diet). Feed conversion was impaired significantly (P<0.01) in fish fed diets contaminated with aflatoxin B₁ by 14.91% than those fed diets without aflatoxin B₁. The mortality rate was 8.15% in fish fed diets contaminated with aflatoxin, while it was 2.96% in the fish group fed the control diet. Data in the present results confirmed that the drastic effects of dietary aflatoxin B₁ on the growth performance parameters of Nile tilapia are in agreement with those recorded by Zychowski et al. (2013); Encarnacao et al. (2009); Shehata et al. (2009) and Chavez-Sanches et al. (1994) who reported the reduction in growth rate due to aflatoxin B₁ contamination. Additional effects of aflatoxin B₁ toxicity include a reduction in the feed conversion, while the mortality rate increased. These findings were in agreement with those obtained by Zychowski et al. (2013), who reported that aflatoxin B₁ at concentrations of 1.5 and 3.0 ppm, significantly (P<0.05) decreased weight gain and feed efficiency in tilapia. The reduction on growth rate may be attributed to the reduction in protein synthesis.

Aflatoxin is known to impair protein biosynthesis by forming adducts with DNA, RNA and proteins (Busby and Wogan, 1984), inhibits RNA synthesis, DNA dependent RNA polymerase activity and causes degranulation of endoplasmic reticulum (Groopman et al., 1988 and Verma and Nair, 2004).

Experimental treatments affected significantly (P<0.01) on the final live body weight, daily gain and feed intake (Table 1). On the other hand, feed conversion insignificantly affected.

Table 1. Growth performance, feed efficiency and survival rate of Nile tilapia fish as affected by aflatoxin B₁ toxicity and some treatments to reduce its effect.

N.S. = Not significant, *** P<0.001 and ** P<0.01.
Means in the same column within each classification with different letters differ significantly (P<0.05).

The highest final live body weight and daily gain (P<0.05) were determined in the group treated with coumarin and vitamin E than the control treatment.

Moreover, coumarin treatment recorded the highest final body weight and daily gain followed by the fish group fed diet supplemented with vitamin E and the recorded values were 12.78 and 11.28%, respectively than that of the control treatment. The best fed conversion was obtained in the same order in the fish group treated with coumarin and vitamin E compared to the control group.

Coumarin can be found in many green plants. It is used mainly in the treatment of cancer and can also be used as a blood thinner. Our results showed that coumarin treatment improved growth rate and feed conversion and decreased the mortality rate in fish group fed diet contaminated with aflatoxin. The results of Tulayakul et al. (2007) showed that coumarin treatment caused the reduction of the metabolic activity of microsomes to convert aflatoxin B₁ to the aflatoxin B₁–DNA adduct, but enhanced the activity to convert aflatoxin B₁ to aflatoxicol in the liver. Coumarin enhanced the glutathione S-transferase (GST) activity to conjugate aflatoxin B₁ to glutathione S-transferase in the intestine, although no effects were noted in the liver.

In the present study, vitamin E supplementation in fish diets reduced the toxicity effect of aflatoxin B₁. High level of vitamin E raised the specific immunity, nonspecific resistance factors and disease resistance capacity when compared with aflatoxin B₁ exposed Indian major carp, Labeo rohita (Sahoo and Mukherjee, 2001). Vitamin E (2 mg/ animal/day) pre-treatment to low dose treated groups significantly ameliorated aflatoxin induced changes (Verma and Nair, 2004). Vitamin E may be involved indirectly in modulating growth by the effect on immunological parameters (Pearce et al., 2003 and Chen et al., 2004).

The interaction between aflatoxin and treatments insignificantly affected final body weight, daily gain and feed conversion (Table 1). Within each aflatoxin toxicity treated fish group with coumarin and vitamin E increased the final body weight, growth rate and improved feed conversion. The higher final body weight and daily gain and the best feed conversion were obtained in the fish group treated with coumarin.

Blood Hematological Parameters

Data of hematological parameters are illustrated in Table 2 which showed insignificant differences as affected by aflatoxin contamination in fish diet. Also the main effect of binder aflatoxin material or the interaction between aflatoxin and treatments were insignificantly affected the studied hematological parameters (Table 2).

Table 2. Hematological parameters of Nile tilapia fish as affected by aflatoxin B₁ toxicity and some treatments to reduce its effect.

Aflatoxin B1 toxicity and its reduction by using coumarin and vitamin E in Nile tilapia - Image 2

Hb= Hemoglobin, RBCs = Red blood cells (Erythrocytes), Hct= Hematocrite, WBCs= White blood cells and Platelets = Blood platelets (Thrombocytes). N.S. = Not significant.

Serum Biochemical Parameters

Serum biochemical parameters were significantly affected (P<0.001, 0.01 or 0.05) with aflatoxin B₁ contamination, except serum createnine was insignificantly affected (Table 3). Serum total protein and albumin in fish group fed control diet (without aflatoxin contamination), was decreased while serum ALT, AST, urea-N and Createnine increased in fish groups fed diets contaminated with aflatoxin B₁. These findings agreed with those found by Zaki et al. (2010) who found that the concentration of AST, ALT, urea and creatinine (which are indicative of abnormal liver and kidney functions) significant increased in fish fed diet contaminated with aflatoxin, while there was significant decrease in total protein level.

Cagauan et al. (2004) cited that the effect of aflatoxin in the liver as follows; aflatoxin is absorbed from the diet in the alimentary canal and is passed to different organs. The principal target organ for aflatoxins is the liver. After the invasion of aflatoxins into the liver, lipids infiltrate hepatocytes and leads to necrosis or liver cell death. The main reason for this is that aflatoxin metabolites react negatively with different cell proteins, which leads to inhibition of carbohydrate and lipid metabolism and protein synthesis. In relation with the decrease in liver function, there is a derangement of the blood clotting mechanism, jaundice, and a decrease in essential serum proteins synthesized by the liver.

Serum biochemical parameters were affected insignificantly with experimental treatments, excepted serum total protein which was affected significantly (P<0.01). Serum total protein increased more in fish groups fed diets supplemented with coumarin and vitamin E than the control group (Table 3). Results of Tulayakul et al. (2007) suggest that feeding with a diet containing coumarin affects aflatoxin B₁ metabolism to enhance aflatoxin B₁ detoxification through the suppression of enzyme activity in the liver and the enhancement of glutathione S-transferase activity in the intestine.

The interaction between aflatoxin and treatments insignificantly affected all serum biochemical studied parameters (Table 3).

Table 3. Blood components of Nile tilapia fish as affected by aflatoxin B₁ toxicity and some treatments to reduce its effect.

Aflatoxin B1 toxicity and its reduction by using coumarin and vitamin E in Nile tilapia - Image 3

N.S. = Not significant, *** P<0.001, ** P<0.01 and * P<0.05.
Means in the same column within each classification with different letters differ significantly (P<0.05).

Fish Body Composition

No statistical differences were observed in fish body dry matter, ether extract, crude protein and ash as affected by aflatoxin B₁ contaminated, experimental treatments or interaction between aflatoxin and experimental treatments (Table 4).

Aflatoxin B₁ Residual in Fish Body

The bioaccumulation of aflatoxin B₁ in the whole body of fish was measured at the end of the experimental period. Residual of aflatoxin was increased significantly (P<0.001) in fish group received aflatoxin B₁ contaminated diet (Table 4). Residual of aflatoxin reduced by 95.88% in fish body fed the control diet compared with those fed diet contaminated with aflatoxin B₁.

Residual of aflatoxin was affected significantly (P<0.001) with the experimental treatments. The residual of aflatoxin in treated groups with coumarin and vitamin E showed significant decrease (P<0.05) when compared with those fed the control diet (Table 4). Fish group treated with coumarin and vitamin E decreased the aflatoxin by 91.56 and 90.48%, respectively when compared with the control group.

The residual of aflatoxin significantly (P<0.001) affected with the interaction between aflatoxin and experimental treatments (Table 4). In fish groups fed diets without aflatoxin B₁ and treated with coumarin and vitamin E showed insignificant effect on aflatoxin residual. On the other hand, in fish groups fed diets with aflatoxin B₁ contamination and treated with coumarin and vitamin E showed significant effect on aflatoxin residual when compared with fish group without any treatments.

The lowest aflatoxin residual was obtained in fish group fed diets supplemented with coumarin and those supplemented with vitamin E.

Table 4. Body composition of Nile tilapia fish as affected by aflatoxin toxicity and some treatments to reduce its effect.

Aflatoxin B1 toxicity and its reduction by using coumarin and vitamin E in Nile tilapia - Image 4

N.S. = Not significant and *** P<0.001.
Means in the same column within each classification with different letters differ significantly (P<0.05).

Profit Analysis

Return from body gain and final profit increased in fish group fed diets without aflatoxin (control diet) than those fed the contamination diet with aflatoxin B₁ (Table 5). Fish groups fed diets contaminated with aflatoxin recorded lower return from body gain and final margin by 12.38 and 30.00%, respectively, when compared with the control groups (basal diet).

Table 5. Economic visibility of Nile tilapia fish as affected by aflatoxin B₁ toxicity and some treatments to reduce its effect.

Aflatoxin B1 toxicity and its reduction by using coumarin and vitamin E in Nile tilapia - Image 5

Feed cost, return from body gain and final profit increased in fish groups treated with coumarin and vitamin E. The return from body gain increased by 11.88 and 11.23%, respectively, in fish fed diets supplemented with coumarin and vitamin E when compared with fish group fed diet without feed additives. Also, the same trend for the final margin were 25.93 and 16.30%, respectively.

Within each aflatoxin contamination, feed cost, return from body gain and final profit increased in fish groups treated with coumarin and vitamin E. Higher return from body gain and final profit were obtained in fish group fed diet supplemented with coumarin than the other experimental groups.

It could be concluded from this experiment that aflatoxin contamination of fish diets caused many drastic effects in all tested parameters and it is very dangerous from the point of view of fish production and public health. Coumarin and vitamin E treatment as an oxidizing agent were the best treatments to reduce the harmful effect of aflatoxin. It could be recommended for the use vitamin E or coumarin treatments to alleviate the toxic effects of aflatoxin B₁ contaminated fish diets.

This article was originally published in Zagazig Journal of Agricultural Research, Vol. 41 No. (1) 2014.

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