An Attept to Alleviate Aflatoxicosis on Nile Tilapia Fish by Dietary Supplementations with Chicken-Hatchery By-Products (Egg Shells) and Shrimp Processing Wastes (Shrimp Shells) - First Part

This experiment was conducted to study the drastic effects of graded levels of aflatoxin-Bon growth performance, survival rate, and feed and nutrients utilization, carcass composition, residual effects in fish, some blood constituents and histopathological changes in Nile tilapia, O. niloticus fingerlings. Also, it was conducted for experimenting the inhibiting effects of graded levels of natural, cheap and available two adsorbent agent, namely egg shells(ES) and shrimp wastes(SW) against the adverse effects of aflatoxin-B₁ contamination of fingerlings diet fed for 8 weeks.

The effects of aflatoxin-B₁ (AFB₁) were severe clinical lesions in the external organs and postmortem symptoms in the internal organs of the aflatoxicated fish. Significant (P≤ 0.01) decrease in body weight, average weight gain, average daily gain, relative growth rate, specific growth rate, survival rate and feed intake of fish. Significant (P≤ 0.01) increase in corrected mortality rate, organs indices and feed conversion ratio. Significant (P≤ 0.01) decrease in protein efficiency ratio, protein productive value and energy utilization. Significant (P≤ 0.01) reduction in dray matter, crude protein and energy content and significant (P≤ 0.01) increase in ether extract and ash of fish carcass.Decrease in dray matter and increase in ether extract of the fish liver.Significant (P≤ 0.01) decrease in hemoglobin concentration, red blood cells count and uric acid and significantly (P≤ 0.01) increase in white blood cells count and alkaline phosphatase and transaminases activity. Residues of AFB₁ were found in the whole body of the aflatoxicated fish directly at the end of the experiment and tended to decrease after freezing periods. Severe histological alterations were recorded in livers, kidneys, intestines and gills of the aflatoxicated fish.These alterations in all tested organs were increased by increasing level of aflatoxin (200 ppb AFB₁).The effects of either adsorbents at levels of 1 and 2%, respectively were useful in reducing the toxic effects of AFB₁ on fish via adsorbing the toxin from the fish diets, where it increased significantly (P≤ 0.01) the body weight, average weight gain, average daily gain, relative growth rate, specific growth rate, survival rate, feed intake, protein efficiency ratio, protein productive value, energy utilization, hemoglobin concentration, red blood cells count and uric acid in fish blood. Yet, it decreased significantly (P≤ 0.01) white blood cells count, alkaline phosphatase and transaminases activity, corrected mortality rate and most of the organs indices. However, these adsorbents (at levels of 1% egg shells and 2% shrimp wastes) alleviated the toxic effects of AFB₁ on feed conversion ratio, carcass composition and the liver composition (DM and EE) of the fish. Also, the dietary addition of these adsorbent to the aflatoxicated fish (100 and 200 ppb AFB₁) led to adsorption effects of the dietary aflatoxin-B₁. Moreover, using these adsorbents at these levels (1% ES and 2% SW) alleviated the adverse effects of AFB₁ on the histopathological changes in different internal organs (livers, kidneys, intestines, and gills) of the aflatoxicated fish.

INTRODUCTION

Mycotoxins are highly toxic secondary metabolic products of various toxigenic moulds, mainly those belonging to the genera Fusarium, Aspergillus and Penicillium. It has been estimated that at least 300 of these fungal metabolites are potentially toxic to animals and humans. However, the most notorious - from the agricultural point of view - and thus extensively investigated mycotoxin is aflatoxin B₁. Its global occurrence is considered to be a major risk factor. It is the silent enemy. Mycotoxins affect as much as 25% of the world’s feed crops each year (Yegany et al., 2002 and Heidler and Schatzmayr 2003). Aflatoxin B₁ (AFB₁) is a wide spreading hepato- carcinogen in fish diets. It is the most toxic mycotoxin which very often occurs all over the world in various commodities causing foodborne intoxications named aflatoxicosis by animal (Abdelhamid , 2000a) and human being (Fink-Gremmels,1999). Therefore, it is a major contaminant in aquafeeds and considered as a causative agent for fish mortality, morbidity and low productivity besides its residues in fish carcass leading to economic losses, human toxicity and affects public health (Abdelhamid et al., 1998 and 1999).

The Nile tilapia is one of the most important warm water fish species on accounts of its recognized advantages as an aquaculture species. Yet, tilapias still contribute little more than 5 % of the total world farmed freshwater fish supply (FAO, 1998). However, tilapia fishes, particularly O. niloticus are sensitive to aflatoxin (Abdelhamid et al., 1998 and Hemeda, 1999). The most applied method for protecting animals against mycotoxicoses is the utilization of adsorbents, which are mixed with a contamated feed. These materials supposed to bind the mycotoxins efficiently in the gastro-intestinal tract. The efficiency of mycotoxin binder, however, differs considerably depending mainly on the chemical structure of both adsorbent and toxin (Alexander et al.,2001). Different agents have been used for detoxification process (Abdelhamid et al., 2002 c, d & e and 2004 b).

Therefore, the aim of this study was to give light on the drastic effects of aflatoxin-B₁ on O. niloticus fingerlings and the inhibiting effects of two adsorbent agents, namely egg shell and shrimp waste against aflatoxin-B₁ contamination of the fish diet fed for 8 weeks.

MATERIALS AND METHODS

The present study was carried out in season 2003, for investigating the best source and level of two natural, cheap and available adsorbent materials, namely egg shells(ES) and shrimp wastes(SW), which can detoxify aflatoxin-B₁ contamination of O. niloticus fingerlings diet fed for 8 weeks. The experiment was carried out in in-door wet lab. A total number of 450 healthy fingerlings were purchased from Al-Manzalah Integrated Fish Farm, General Authority for Fisheries Resources Development, with an average initial body weight of about 13 gram. After an adaptation period of one week, the fishes were randomly divided into 15 treatments, each treatment at three replicates (each contained 10 fingerlings in a 40 L cylindrical plastic aquarium). Each aquarium was supplied with 35 L dechlorinated tap water and an air-stone connected with an electric compressor and covered with a fishing net. The replacement of the aquaria waters was done partially every 2 days to re-new the water and to remove the wastes. Electric light was used to complete the day light to 14 hours.

The experimental fishes received the tested diets twice daily at 9.00 a.m. and 3 p.m., six days a week. The daily feeding rate was 3% of the live body weight of the fish. The feed quantity was readjusted biweekly on the basis of the actual average biomass of the fish in each replicate. A ground basal floating diet was obtained from Joe Trade Company, Cairo. It consisted of fish meal, soybean meal, meat meal, wheat bran, rice bran, yellow corn, fish oil, dicalcium phosphate and vitamins and minerals mixture. Proximate analysis of the experimental diet is DM 89.5, CP 25.1, EE 13.1, ash 7.18, NFE 54.6, GE 490 kcal/100g DM and P/E ratio 51.3 mg CP/kcal GE . GE (Kcal/100 g DM) = CP x 5.64 + EE x 9.44 + Carbohydrates x 4.11 Calculated according to (Macdonald et al., 1973).The diet was supplemented with aflatoxin B₁ (prepared as described in Abdelhamid et al., 2004) at concentrations of 0.0, 100 and 200 ppb without or with additives (ES and SW) at rates of 0.0, 1 and 2% of each as shown in Table (1). Egg shells and shrimp wastes were purchased from the local market from Al-Mansourah and Ezbet El-Borg, Domiatta, respectively.

At the end of the experiment, the remained fish were sampled from each aquarium and kept frozen for chemical analysis. The chemical analyses of the basal diet, the whole body fish (at the start, 4^th and 8^th week of the experiment), and the liver of the fish (at the 4^th and 8^th week of the experiment) were carried out according to the AOAC (2000). Aflatoxin B₁ determinations in the media extract, basil diet and fish carcass were determined as described by Abdelhamid (1996). Water quality parameters were measured weekly (Abdelhamid, 1996) including temperature (via a thermometer), pH (using Jenway Ltd, Model 350 – pH-meter), dissolved oxygen (using Jenway Ltd., Model 970-dissolved oxygen meter), and conductivity (using Jenway Ltd., Model 470-portable conductivity meter). Body weight of individual fish was measured biweekly to point feed quantity and to calculate growth performance and feed utilization (Abdelhamid, 2000b) in form of average weight gain (g/fish) AWG = Average final weight (g) – Average initial weight (g). Average daily gain, (mg/fish/day) ADG = [AWG (g)/Experimental period (days)] x 1000. Relative growth rate, (RGR) = Average weight gain (g)/ Average initial weight (g). Specific growth rate (SGR, %/day) = [In final weight – In initial weight]x100/Experimental period (d). Feed conversion ratio (FCR) = Feed intake (g)/Live weight gain (g). Protein efficiency ratio (PER) = Live weight gain (g)/Protein intake (g). Protein productive value (PPV%) = [Retained protein (g)/Protein intake (g)] x 100. Energy utilization (EU%) = [Retained energy (Kcal)/Energy intake (Kcal)] x 100. Survival rate (SR%) = [End number of the alive fish/The beginning number of the fish] x 100. Corrected mortality rate (CMR%) (Abbot, 1925) = [Mortality rate in each treatment – Mortality rate in the control group] x 100/[100–Mortality rate in the control group].

Table (1) The experimental design.

Treatment No.	Aflatoxin- B1 level (ppb)	Adsorbent (%)
1	0.00	0.00 ES and 0.0 SW
2	0.00	1.00 ES
3	0.00	2.00 ES
4	100.00	0.00 ES and 0.0 SW
5	100.00	1.00 ES
6	100.00	2.00 ES
7	200.00	0.00 ES and 0.0 SW
8	200.00	1.00 ES
9	200.00	2.00 ES
10	0.00	1.00 SW
11	0.00	2.00 SW
12	100.00	1.00 SW
13	100.00	2.00 SW
14	200.00	1.00 SW
15	200.00	2.00 SW

At the end of the experiments , for all fish, the liver, spleen, kidneys and gonads were removed and weighted at once. The liver, spleen, kidneys and gonads indices were calculated, where: Hepato-somatic index (HSI) = Liver weight (g) x 100/Gutted fish weight (g) (Jangaard et al., 1967). Spleeno-somatic index (SSI) = Spleen weight (g) x 100/fish weight (g). Kidney-somatic index(KSI)=Kidneys weight (g) x 100/fish weight (g) (Alabaster and Lioyd, 1982). Gonado-somatic index (GSI) = Gonads weight (g) x 100/fish weight (g) (Tseng and Chan, 1982). Blood samples from each fish of the different groups were collected from the caudal peduncle. Whole blood was used for the determination of hemoglobin (Hb) by using commercial kits (Diamond Diagnostic, Egypt). Also, total erythrocytes count (RBCs) and total leucocytic count (WBCs) were estimated by Haemocytometer. Plasma samples were used for biochemical analysis. Uric acid concentration and activity of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined calorimetrically using commercial kits supplied by Diamond, Diagnostic, Egypt. For histopathological examination, representative samples from liver, kidneys, intestine and gills were proceeded (Bancroft et al., 1990). The obtained data were statistically analyzed using SAS (1996) procedures for personal computer. When F-test was positive, least significant difference (Duncan, 1955) was calculated for the comparisons among means.

RESULTS AND DISCUSSION

1 - Quality parameters of the rearing water:

All tested water quality criteria were suitable for rearing Nile tilapia fingerlings as cited by Abdelhamid (2000b) and Abd El-Hakim et al. (2002). Since water temperature ranged between 25 and 28^oC, pH values 7.6 – 8.7, conductivity 249 – 286 ms/cm and dissolved oxygen 6.9 – 12.7 mg/l. Also, Abdelhamid et al. (2002c) measured these water parameters and suggested their suitability for rearing Nile tilapia fish.

2 - The clinical lesions and postmortem examination of aflatoxicated fish:

External clinical symptoms (protrusive eyes, abdominal distension, hardening of the body, discarding viscera, fins erosion, discarded scales, hemorrhage, discoloration of skin, abdominal shrinkage, operculum erosion and cataract) and postmortem signs (enlarged gall bladder and stomach, distended yellowish liver, viscera covered by a thick layer of mucus and uncharacterized liver and viscera) of aflatoxicated fish were recorded from the 1^st week and continued throughout the experiment. However, at the end of the 4^th week of the experiment, all groups fed on the diets containing 200 ppb AFB₁ with and without additives died. These findings are in agreement with those mentioned by Hussein et al.(2000) and Abdelhamid et al. (2002 b&c). Moreover, Soliman et al.(2000) reported that the earliest symptoms of disease were seen 48 hours after oral administration of 1/10, 1/20 and 1/30 of the AFB₁ - LC₅₀.

3 - Growth performance and survival rate:

3.1- Body weight (BW):

Results in Table 2 show no significant (P ≥ 0.05) differences on BW at the start of the experiment. However, significant decreases were recorded in BW of the aflatoxicated fish without additives (T₄, 100 ppb AFB₁ and T₇, 200 ppb AFB₁). These decreases in BW increased significantly by increasing AFB₁ concentration comparing with the control groups (T₁, 0 ppb AFB₁, T₂, 0 ppb AFB₁+ 1% ES, T₃, 0 ppb AFB₁+2% ES, T₁₀, 0 ppb AFB₁ +1% SW and T₁₁, 0 ppb AFB₁ +2% SW) at different experimental intervals. Yet, the addition of ES (T₅, 100 ppb AFB₁ +1% ES, and T₈, 200 ppb AFB₁ +1% ES) and addition of SW (T₁₃, 100 ppb AFB₁ +2% SW, and T₁₅, 200 ppb AFB₁ +2% SW) to the aflatoxicated diets recorded significant increases in BW of fish comparing with the aflatoxicated fish without additives (T₄ and T₇) at different intervals of the experiment . T₇ was the worst treatment in BW followed by T₄ at different experimental intervals. Data presented in Table 3 show that the type of additives recorded no significant (P ≥ 0.05) effects on BW at different experimental intervals. Moreover, the increasing levels of additives caused significant increases in BW at different experimental intervals. While, there were no significant (P ≥ 0.05) differences recorded in BW between the two concentrations of additives (1 and 2%). Yet, addition of aflatoxin to fish diets at concentrations of 100 and 200 ppb AFB₁ led to significant decreases in BW comparing with the untreated fish (zero ppb, AFB₁) at different experimental intervals. These decreases in BW increased significantly by increasing the dietary level of AFB₁ comparing with the untreated fish at the 4^th week (W₄) of the experiment. Similar negative effects of AFB₁ on BW of fish were recorded in other works ( Abdelhamid et al.,2002 bc and Shehata et al.,2003).

In this context, AFB₁ treatment led to a clear reduction in growth rate in a direct relation to the dietary aflatoxin level (Marzouk et al.,1994).However, Abdelhamid et al.(2002a) suggested that adsorbents, e.g. Antitox plus, Fix-a-tox and tafla did not significantly reduce the aflatoxicity. However, egg shell (including egg shell membrane which contains 10% collage) can be used as an adsorbent (Gittins and Drakley, 2002). Its fibers show the source of the unique and highly valued type of collagen present in the membrane (Healy et al.,2003a). Moreover, prawn and egg shell wastes could be utilized as adsorbents (Healy et al., 2003b) to prevent the drastic effects of AFB₁ on the fish.

Table (2) Effect of aflatoxin B₁ (AFB₁) on body weight (g), body weight gain (g/fish) and average daily body weight gain (mg/fish) of the fish at different intervals of the experiment.

Treat.	Body weight	Body weight gain	Daily body weight gain
Weeks	W0	W4	W8	W0-4	W0-8	W0-4	W0-8
1	13.89	17.35 ab	22.56 a	3.46 ab	8.67 a	123.51ab	154.82a
2	13.88	17.31 ab	22.66 a	3.43 ab	8.78 a	122.62ab	156.79a
3	13.88	17.49 a	22.65 a	3.61 a	8.77 a	129.17a	156.67a
4	13.87	15.71 gh	17.94 c	1.83 gh	4.06 c	65.59gh	72.56c
5	13.88	16.30 ef	21.02 b	2.42 ef	7.14 b	86.55ef	127.56b
6	13.88	15.97 gf	20.41 b	2.08 fg	6.52 b	74.52fg	116.55b
7	13.88	15.09 i	-------	1.21 i	--------	43.21i	--------
8	13.89	15.76 g	-------	1.88 g	--------	67.14g	--------
9	13.87	15.37 hi	-------	1.48 hi	--------	52.86hi	--------
10	13.87	17.17 abc	22.43 a	3.29 abc	8.56 a	117.74abc	152.86a
11	13.88	17.28 ab	22.72 a	3.40 ab	8.84 a	121.55ab	157.86a
12	13.88	16.52 de	14.72 b	2.63 de	5.84 b	94.16de	104.35b
13	13.88	16.84 cd	22.38 b	2.96 cd	6.50 b	105.71cd	116.07b
14	13.87	16.56 ed	-------	2.69 de	---------	96.07de	---------
15	13.88	16.97 bc	-------	3.08 bc	---------	110.24bc	---------

a-i: Means in the same column having different letters differ significantly (P ≤ 0.01).

Table (3) Main effects on body weight (g),body weight gain (g/fish) and average daily body weight gain (mg/fish) of the fish at different intervals of the experiment.

Items	Body weight	Body weight gain	Daily body weight gain
Weeks	W0	W4	W8	W0-4	W0-8	W0-4	W0-8
E.S	13.88	16.27	21.21	2.38 b	7.33	85.03 b	130.82
S.W	13.88	16.61	20.96	2.73 a	7.08	97.54a	126.42
AF0 ppb	13.89	17.33 a	22.60 a	3.45 a	8.72 a	123.04a	155.64a
AF100 ppb	13.88	16.18 b	19.57 b	2.30 b	5.69 b	82.02b	101.61b
AF200 ppb	13.89	15.81 c	-----------	1.93 c	---------	68.79c	---------
0 %Add.	13.88	16.05 b	20.25 b	2.17 b	6.37 b	77.46b	113.69b
1 %Add.	13.88	16.61 a	21.46 a	2.73 a	7.58 a	97.38 a	135.39a
2 %Add.	13.89	16.66 a	21.54 a	2.77 a	7.66 a	99.00a	136.79a

a-c: Means in the same column having different letters differ significantly (P ≤ 0.01).

3.2 - Average weight gain (AWG) and average daily gain (ADG):

Average weight gain (g/fish) and average daily gain (mg/fish/day) were calculated and illustrated in Tables 2 and 3. The results indicated significant decreases in AWG and ADG of the aflatoxicated fish without additives (T₄ and T₇). These decreases in AWG and ADG were increased significantly by increasing AFB₁ levels (T₇) comparing with the control groups (T₁, T₂, T₃, T₁₀ and T₁₁) at different experimental intervals.Yet, the addition of ES at a level of 1% (T₅ and T₈) and the addition of 2% SW (T₁₃ and T₁₅) led to significant increases in AWG and ADG comparing with the aflatoxicated fish without additives (T₄ and T₇) at different experimental intervals. Yet, T₇ was the worst treatment in AWG and ADG followed by T₄ at the different experimental intervals.

The reduction in body weight gain in the present study may be attributed to the loss of appetite and reduction of feed intake . Table 3 shows that there were no significant (P ≥ 0.05 ) differences in AWG and ADG in all fish groups concerned with type of additives throughout W_0-8. However, the AWG and ADG were increased significantly by increasing levels of additives comparing with the untreated fish (zero% additives) at different experimental intervals. Yet, average weight gain and average daily gain were decreased significantly by increasing the levels of aflatoxin-B₁ at all intervals of the experiment. Many workers recorded the same negative effects of AFB₁on AWG and ADG ( Abdelhamid et al.,2002 b&c ; Nuguny et al., 2002 and Shehata et al.,2003). Soliman et al. (1998) indicated that the presence of Fix-A- tox accompanied with aflatoxin in the experimental diets, caused an alleviative effect towards the adverse effect of aflatoxin. However, prawn and egg shell wastes could be utilized as adsorbents (Healy et al., 2003b).

3.4 - Relative growth rate (RGR) and specific growth rate (SGR):

Tables 4 and 5 illustrate the relative growth rate and specific growth rate (%/day).The results indicated significant decreases in RGR and SGR of the aflatoxicated fish without additives (T₄ and T₇). These decreases in RGR and SGR were increased significantly by increasing AFB₁ level (T₇) comparing with the control groups (T₁, T₂, T₃, T₁₀ and T₁₁) at different experimental intervals. Yet, the addition of ES at 1% level (T₅ and T₈) and 2% of SW (T₁₃ and T₁₅) led to significant increases in RGR and SGR comparing with the aflatoxicated fish without additives (T₄ and T₇) at different experimental intervals. Yet, T₇ was the worst treatment in RGR and SGR followed by T₄ at different experimental intervals . The reduction in growth rates in the present study may be attributed to the loss of appetite and feed intake reduction . However, the RGR and SGR were increased significantly by increasing levels of the additives comparing with the untreated fish (zero% additives) at different experimental intervals. Yet, the RGR and SGR were decreased significantly by increasing the levels of aflatoxin-B₁ at all intervals of the experiment. Many workers recorded the same negative effects of AFB₁on growth rates of tilapia fish (Hussein et al.,2000 ; Abdelhamid et al.,2002 bc and Shehata et al.,2003). Yet, Abdelhamid et al. (2002a) suggested that adsorbents, e.g. Antitox plus, Fix-a-tox and tafla did not significantly reduce AFB₁. However, egg shell can be used as an adsorbent (Gittins and Drakley, 2002). Moreover, prawn and egg shell wastes could be utilized as adsorbents (Healy et al., 2003b).

Table (4) Effect of aflatoxin B₁ (AFB₁) and adsorbents on specific growth rate (%/day) and relative growth rate of the fish at different intervals of the experiment.

Treat.	Specific growth rates	Relative growth rates
Weeks	W0-4	W0-8	W0-4	W0-8
1	0.79ab	0.87a	0.25ab	0.62a
2	0.79ab	0.87a	0.25ab	0.63a
3	0.83a	0.87a	0.26a	0.63a
4	0.44g	0.46d	0.13gh	0.29c
5	0.57ef	0.74b	0.17ef	0.51b
6	0.50fg	0.69bc	0.15fg	0.47b
7	0.30h	--------	0.08i	--------
8	0.45g	--------	0.14g	--------
9	0.36h	--------	0.11hi	--------
10	0.76abc	0.86a	0.23abc	0.62a
11	0.78ab	0.88a	0.24ab	0.64a
12	0.62de	0.63c	0.19de	0.42b
13	0.69cd	0.69bc	0.21cd	0.47b
14	0.63de	--------	0.19de	---------
15	0.72bc	--------	0.22bc	---------

a-i : Means in the same column having different small letters differ significantly (P ≤ 0.01).

Table (5) Main effects on specific growth rate (%/day) and relative growth rate of the fish at different intervals of the experiment.

Items	Specific growth rates	Relative growth rates
Weeks	W0-4	W0-8	W0-4	W0-8
E.S	0.560 b	0.750	0.171 b	0.53
S.W	0.638 a	0.730	0.196a	0.51
AF0 ppb	0.791 a	0.870 a	0.248a	0.63 a
AF100 ppb	0.545 b	0.610 b	0.165b	0.41 b
AF200 ppb	0.460 c	---------	0.138 c	---------
0 %Add.	0.512b	0.660 b	0.156 b	0.46 b
1 %Add.	0.639 a	0.770 a	0.196a	0.55 a
2 %Add.	0.646 a	0.780a	0.199a	0.55 a

a-c: Means in the same column having different letters differ significantly (P ≤ 0.01).

3.5 - Survival rate (SR) and corrected mortality rate (CMR):

Data in Tables 6 - 9 show that there were significant decreases in SR and increases in CMR of thefish treated with aflatoxin-B₁ without additives (T₄ and T₇) by increasing AFB₁ levels (T₇) compared with the control groups (T₁, T₂ and T₃) at different experimental intervals. However, the dietary addition of ES (1%, T₅ and T₈) and SW (2%, T₁₃) to the aflatoxicated fish diets led to significant increases in SR and decreases in CMR compared with T₄ and T₇ at different experimental intervals. So, the T₇ was the worst treatment in SR and CMR followed by T₄ at all experimental intervals. From the results in Table 7 there were no significant (P ≥ 0.05) differences in SR in all fish treatments concerning with the type and concentrations of the additives (ES and SW) at different experimental intervals. However, the SR was decreased significantly by increasing levels of AFB₁ comparing with the untreated fish (zero ppb AFB₁) at different experimental intervals. However, data in Tables 8 and 9 show that there were no significant (P ≥ 0.05) differences in CMR in all fish treatments concerning with the type of additives (ES and SW) at different experimental intervals. Yet, increasing levels of additives led to significant decreases in CMR of fish comparing with the control group (zero% additives). On the contrary, AFB₁ caused significant increases in CMR of the aflatoxicated fish (comparing with the control group, zero ppb AFB₁) which were increased by increasing level of AFB₁. Similar reduction in survival rate by AFB₁ was recorded (Hussein et al.,2000; Abdelhamid et al., 2002 bc and Salem 2002). Shehata et al.(2003) suggested also that aflatoxin-B₁ caused significant increases in the mortality rate of O. niloticus fish . They added that using adsorbents significantly reduced the toxic effect of aflatoxin on SR of fish. The increased survival rate by the adsorbents used herein may be due to there ability for adsorption of mycotoxin in the gastrointestinal tract and thereby decrease the toxic effects on animals (Galvano et al., 2001). On the other side, Abdelhamid et al., (2002a) mentioned that adsorbents, e.g. Antitox plus, Fix-a-tox and tafla did not significantly reduce AFB₁.

Table (6) Effect of aflatoxin B₁ (AFB₁) and adsorbents on survival rate (%) of the fish at different intervals of the experiment.

Treat.	SR4	SR8
1	96.66 a	86.66 a
2	96.66 a	86.66 a
3	96.66 a	86.66 a
4	76.66 bc	60.00 d
5	90.00 ab	76.66 ab
6	90.00 ab	73.33 bc
7	56.66 d	---------
8	63.33 cd	---------
9	63.33 cd	---------
10	93.33 a	83.33 ab
11	96.66 a	83.33 ab
12	76.66 bc	63.33 cd
13	83.33 ab	76.66 ab
14	60.00 d	---------
15	66.00 cd	---------

a-d: Means in the same column having different letters differ significantly (P ≤0.01).

Table (7) Main effects on survival rate (%) of O. niloticus at different intervals of the experiment.

Items	SR4	SR8
E.S	87.22	78.33
S.W	83.33	75.56
AF0 ppb	92.78 a	……..
AF100 ppb	82.22 b	68.33 b
AF200 ppb	61.11 c	---------
0 %Add.	81.67	73.33
1 %Add.	85.83	77.50
2 %Add.	88.33	80.00

a-c: Means in the same column having different letters differ significantly (P ≤ 0.01).

Table (8) Effect of aflatoxin B1 (AFB1) and adsorbents on corrected mortality rate (%) of O. niloticus at different intervals of the experiment.

Treat.	CMR 4	CMR 8
1	3.33 ef	10.00 c
2	3.33 ef	3.33 c
3	0.00 f	0.00 c
4	20.74bcd	65.55a
5	7.04 def	24.07 bc
6	10.00def	23.33 bc
7	41.48 a	---------
8	34.07ab	---------
9	34.07ab	---------
10	0.00f	3.70 c
11	0.00f	0.00 c
12	17.41cde	55.28 ab
13	13.33def	47.06 ab
14	38.15 a	---------
15	30.74abc	---------

a-f: Means in the same column having different letters differ significantly (P ≤ 0.01).

Table (9) Main effects on corrected mortality rate (%) of O. niloticus at different intervals of the experiment.

Items	CMR 4	CMR 8
Additives (E.S)	17.12	35.95
(S.W)	18.35	43.61
0(AF0)	2.84b	……..
100(AF100)	14.88 b	46.31a
200(AF200)	36.67 a	---------
0	21.85a	52.03a
1	16.66ab	36.87b
2	14.69b	30.44b

a-b: Means in the same column having different letters differ significantly (P ≤ 0.01).

4 - Organs indices:

Indices of organs of fish fed on different levels of AFB₁ with and without additives were presented in Tables 10 and 11.The results showed that there were significant increases in indices of aflatoxicated fish (100 ppb AFB₁) with and without additives comparing with the control fish group (zero ppb AFB₁) at the end of the experiment. However, the addition of 1% ES (T₅) and 2% SW (T₁₃) led to significant decreases in HSI, KSI and SSI and insignificant (P ≥ 0.05) decreases in GSI comparing with the aflatoxicated fish without additives (T₄). Data in Table 16 show the main effects on indices (%) at the end of the experiment. The results showed that there were no significant (P ≥ 0.05) differences in indices in all fish groups concerning with the type of additives (ES and SW). However, increasing the concentration of the used additives led to significant decreases in different indices of fish at the end of the experiment. Yet, significant increases were recorded in indices of aflatoxic fish (100ppb AFB₁) comparing with the untreated fish (zero ppb AFB₁). However, in the present study, the positive effects of ES and SW may be due to their adsorbative characteristics as mentioned before, so prevent or reduce absorption of AFB₁ and hence hide its negative effects on indices of fish. Similarly, negative effects of AFB₁ on indices of fish were recorded by Hussein et al.(2000) and Abdelhamid et al.(2002c and 2004a). Anyhow, AFB₁ is a strong hepatic mycotoxin (Nguyen et al., 2002), it has also nephritic (Abdelhamid and Saleh, 1996) as well as sexual negative effects (Constantini et al., 1999), therefore, it affected either of the tested indices. However, Abdelhamid et al. (2002a) found that adsorbents, e.g. Antitox plus, Fix-a-tox and tafla did not significantly reduce aflatoxicosis. Recently, Abdelhamid et al. (2004a) added that the additives (tafla, ammonia and hydrogen peroxide) did not alter the organs weight; yet, they slightly diminished- to some extent- the negative effect of dietary aflatoxin inclusion on the relative weights of all tested organs.

Table (10) Effect of aflatoxin B₁ (AFB₁) and adsorbents on indices (%) of O. niloticus at the end of the experiment.

Treat.	Indices
HSI	KSI	SSI	GSI
1	2.68 c	0.18 c	0.14 c	0.97 b
2	2.66 c	0.21 c	0.20 c	0.96b
3	2.63 c	0.19 c	0.14 c	0.97a
4	3.97a	0.30a	0.30a	1.63b
5	3.22 b	0.25b	0.25b	1.06b
6	3.16 b	0.25b	0.25b	1.08 b
7	---------	---------	---------	---------
8	---------	---------	---------	---------
9	---------	---------	---------	---------
10	2.62 c	0.19c	0.14 c	0.44 b
11	2.89bc	0.14c	0.14 c	0.97 b
12	3.38b	0.26b	0.27b	1.01 b
13	3.22b	0.24b	0.27b	1.00 b
14	---------	---------	---------	---------
15	---------	---------	---------	---------

a-c: Means in the same column having different letters differ significantly (P ≤ 0.01)

Table (11) Main effects on indices (%) of O. niloticus at the end of the experiment.

Items	HSI	KSI	SSI	GSI
E.S	3.06 ±0.13	0.23 ±0.01	0.23 ±0.01	1.11 ±0.06
S.W	3.13 ±0.12	0.23 ±0.01	0.23 ±0.01	1.09 ±0.06
AF0 ppb	2.69 b ±0.06	0.19 b ±0.005	0.19 b ±0.002	0.97 b ±0.009
AF100 ppb	3.49 a ±0.09	0.27 a ±0.007	0.27 a ±0.004	1.24 a ±0.07
AF200 ppb	----------	----------	----------	----------
0 %Add.	3.33 a ±0.20	0.24 a ±0.02	0.24 a ±0.02	1.30 a ±0.10
1 %Add.	2.97 b ±0.12	0.23 ab ±0.009	0.23 b ±0.01	1.00 b ±0.02
2 %Add.	2.98 b ±0.09	0.22 b ±0.01	0.22 b ±0.01	1.00 b ±0.02

a-b: Means in the same column having different small letters differ significantly (P ≤ 0.01).

5. Feed intake and feed conversion:

5.1 - Feed intake (FI):

Results in Tables 12 and 13 show significant (P ≤ 0.01) decreases in FI of the aflatoxicated fish without additives (T4, 100 ppb AFB1 and T7, 200 ppb AFB1). These decreases increased significantly by increasing AFB1 concentration comparing with the control groups (T1, 0 ppb AFB1, T2, 0 ppb AFB1+ 1% ES, T3, 0 ppb AFB1+2% ES, T10, 0 ppb AFB1 +1% SW and T11, 0 ppb AFB1 +2% SW) at different experimental intervals. Yet, the addition of ES to the aflatoxicated diets (T5, 100 ppb AFB1 +1% ES, and T8, 200 ppb AFB1 +1% ES) and the addition of SW (T13, 100 ppb AFB1 +2% SW, and T15, 200 ppb AFB1 +2% SW) recorded significant increases in FI of fish comparing with the aflatoxicated fish without additives (T4 and T7) at different intervals of the experiment. From Table 18, the results show that there were no significant (P ≥ 0.05) differences in FI in all fish treated with both types of additives (ES and SW) at different experimental intervals. Yet, increasing the concentration of these additives caused significant increases in FI, but no significant (P ≥ 0.05) differences were recorded between both concentrations of the additives (1 and 2%) at different intervals of the experiment. However, significant decreases in FI were recorded for the aflatoxicated fish, which increased by increasing the level of aflatoxin (200 ppb) comparing with the control group (zero ppb AFB1) at different experimental intervals. The similar negative effects of AFB1 on FI were recorded in other researches (Abdelhamid et al.,2002b and Salem, 2002). Also, in the same trend, Nguyen et al. (2002) suggested that fish fed diets containing 10 and 100 mg AFB1/kg expel feed after ingestion

Table (12) Effect of aflatoxin B1 (AFB1) and adsorbents on feed intake (g/fish) of O. niloticus at different intervals of the experiment.

Treat.	W0-4	W0-8
1	9.36ab	20.97a
2	9.33abc	20.94a
3	9.34abc	20.99a
4	9.24bcd	14.66d
5	9.38ab	20.38bc
6	9.32abc	20.09c
7	9.09e	--------
8	9.23bcd	--------
9	9.17de	--------
10	9.31bcd	20.88a
11	9.30abcd	20.93a
12	9.20cde	20.42bc
13	9.43a	20.74ab
14	9.24bcd	---------
15	9.36ab	---------

a-f :Means in the same column having different letters differ significantly (P ≤ 0.01).

Table (13) Main effects on feed intake (g/fish) of O. niloticus at different intervals of the experiment.

Items	W0-4	W0-8
E.S	9.27	20.51
S.W	9.28	20.60
AF0 ppb	9.34a	20.95a
AF100 ppb	9.30a	20.16b
AF200 ppb	9.20b	---------
0 %Add.	9.23b	20.32b
1 %Add.	9.28a	20.66a
2 %Add.	9.32a	20.69a

a-b: Means in the same column having different letters differ significantly (P ≤ 0.01).

5.2 - Feed conversion ratio (FCR):

Results in Tables 14 and 15 present that there were significant increases in FCR of the aflatoxicated fish comparing with the control ones (zero ppb AFB₁) at different experimental intervals. These increases of FCR were increased significantly by increasing the dietary AFB₁ level. This increase in FCR may be attributed to the very low weight gain values which were recorded in aflatoxicosis, where FCR calculation depends on weight gain besides FI. So, T₇ was the worst treatment in FCR at different intervals of the experiment. Results show that there were no significant ( P ≥ 0.05) differences in FCR of the fish treated with both types of additives (egg shell and shrimp waste) at the 2^nd week (W_0-2), during 4^th to 6^th week (W_4-6) and throughout the experiment (W_0-8). However, the addition of egg shell led to significant ( P ≤ 0.01) increases in FCR at 2^nd to 4^th week (W_2-4) and from the start to 4^th week (W_0-4) of the experiment comparing with the fish treated with shrimp waste. While, at the 6^th to 8^th week (W_6-8), the addition of egg shell caused significant ( P ≤ 0.01) decreases in FCR comparing with the shrimp waste supplementation. Anyhow, there were no clear effects on FCR concerning with both types of additives in the treated fish groups. Yet, the levels (1 and 2%) of these additives (egg shell and shrimp waste) caused significant ( P ≤ 0.01) decreases in FCR comparing with the control group (zero % additives), while there were no significant ( P ≥ 0.05) differences in FCR between both concentrations (1 and 2%) of the additives at different experimental intervals. However, feed conversion ratios were increased significantly ( P ≤ 0.01) by increasing the level of AFB₁ at all intervals of the experiment, except during the interval (W_4-6), during which there were no significant ( P ≥ 0.05) differences in FCR.

Similar negative effects of AFB₁ on FCR of O. niloticus fish were recorded in other studies ( Abdelhamid et al.,2002 b&c and Salem, 2002). The aflatoxicosis negatively affected feed conversion efficiency and nutrients utilization by O. niloticus and the effect was proportional to the level of dietary contamination with aflatoxin. Salem (2002) reported also that the feed conversion ratio was significantly increased in all aflatoxin treated fish groups in comparison with the non-aflatoxin treated groups. Recently, Abdelhamid et al.(2002b ) suggested that feed conversion ratio (FCR) were significantly differed in their values between the control group and the groups treated with AFB₁. Generally, there were no clear trend for these differences in all groups whether with or without Biogen^®. However, El-Saidy and Gaber (1997) reported that fish fed on diet containing 4% garlic showed low feed conversion ratio during the experimental period.

Table (14) Effect of aflatoxin B₁ (AFB₁) and adsorbents on feed conversion ratio of O. niloticus at different intervals of the experiment.

Treat.	Experimental intervals in weeks
W0-2	W2-4	W0-4	W4-6	W6-8	W0-8
1	3.64 d ±0.42	2.31 c ±0.34	2.71 fg ±0.10	4.42abc ±0.62	1.58 c ±0.16	2.43 de ±0.13
2	3.78 d ±0.27	2.20 c ±0.14	2.72 fg ±0.08	4.09 bc ±0.08	1.57c ±0.21	2.40 e ±0.13
3	3.77 d ±0.41	2.04 c ±0.12	2.58g ±0.04	4.88 ab ±0.39	1.57 c ±0.19	2.42 de ±0.17
4	4.84 cd ±0.29	5.29 a ±0.28	5.06 c ±0.25	5.87 a ±0.88	4.10 a ±0.25	4.85 a ±0.14
5	3.42d ±0.39	4.58ab ±0.34	3.93 de ±0.34	3.55bc ±0.48	1.83c ±0.16	2.86 cd ±0.05
6	3.94 d ±0.25	5.30 a ±0.69	4.50cd ±0.28	3.64bc ±0.59	1. 93 c ±0.10	3.08 c ±0.07
7	10.15a ±0.83	6.11 a ±0.43	7.60 a ±0.56	--------	--------	--------
8	5.12cd ±0.27	5.12 a ±1.03	4.99c ±0.46	--------	--------	--------
9	7.69b ±1.81	6.08 a ±1.15	6.20 b ±0.11	--------	--------	--------
10	3.93 d ±0.08	2.25c ±0.07	2.83fg ±0.04	3.62 bc ±0.30	1.71 c ±0.29	2.47 de ±0.19
11	4.11d ±0.18	2.10c ±0.09	2.74 fg ±0.05	3.72 bc ±0.29	1.63 c ±0.29	2.39 e ±0.14
12	7.20bc ±2.07	2.61c ±0.23	3.54ef ±0.28	3.10 c ±0.35	4.14 a ±0.36	3.51 b ±0.14
13	3.03d ±0.16	3.39 bc ±0.21	3.20efg ±0.13	4.15 bc ±0.66	2.85 b ±0.44	3.20 bc ±0.14
14	4.88cd ±0.42	2.71c ±0.05	3.44ef ±0.05	---------	---------	---------
15	3.77d ±0.77	2.76c ±0.27	3.04 fg ±0.09	---------	---------	---------

a -g: Means in the same column having different letters differ significantly (P ≤ 0.01).

Table (15) Main effects on feed conversion ratio of O. niloticus at different intervals of the 2nd experiment (means ± standard errors).

Items	W0-2	W2-4	W0-4	W4-6	W6-8	W0-8
Additives (E.S)	5.15 ±0.47	4.34 a ±0.35	4.48 a ±0.33	4.41 ±0.27	2.09 b ±0.23	3.00 ±0.21
(S.W)	5.06 ±0.47	3.28 b ±0.27	3.79 b ±0.30	4.15 ±0.29	2.67 a ±0.29	3.14 ±0.22
AFB1, ppb
0 (AF0)	3.81 b ±0.12	2.20 b ±0.08	2.72 c ±0.03	4.19 ±0.18	1.61b ±0.08	2.42 b ±0.05
100(AF100)	4.55 b ±0.45	4.41 a ±0.29	4.21 b ±0.20	4.36 ±0.35	3.16 a ±0.26	3.72 a ±0.20
200(AF200)	6.96 a ±0.69	4.81 a ±0.43	5.48 a ±0.46	---------	---------	---------
Additives(%)
0	6.21 a ±0.71	4.57 a ±0.41	5.12 a ±0.50	5.14 a ±0.39	2.84 a ±0.39	3.64 a ±0.37
1	4.72 b ±0.43	3.24 b ±0.41	3.58 b ±0.20	3.59 b ±0.18	2.31 b ±0.34	2.81 b ±0.14
2	4.38 b ±0.46	3.61 b ±0.42	3.71 b ±0.31	4.09 b ±0.26	1.44 b ±0.20	2.77 b ±0.12

a-c: Means in the same column having different small letters differ significantly (P ≤ 0.01).

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