Studies on counteracting the dietary mercury toxicity in nile tilapia (oreochromis nilotius)

 

Nile tilapia fish were divided into twelve groups, each group of fish was stocked into 3 aquaria and each contains 20 fishes. Fish of the first four groups were fed the normal basal diet, the next four groups were fed on the basal diet contaminated with 50 mg mercury/kg diet and the other four groups were fed on the same diet contaminated with 100 mg mercury/kg diet. Within each dietary mercury level, the first group was fed the diet without supplementation the second was fed on the diet supplemented with 200 mg magnesium sulfate /kg diet, the third group was fed on the diet supplemented 50 mg vitamin E per kg diet and the fourth group fed on the diet contained wheat grass.

Fish fed diets contaminated with 50 or 100 mg mercury/kg diet recorded lower final live body by 6.94 and 11.19%, respectively than those fed diets without mercury contamination after 4 months of the experimental period. Fish fed 50 or 100 mg mercury level recorded the lower feed intake by 1.26 and 3.15% and impaired feed conversion by 9.80 and 16.37%, respectively. Plasma total protein, albumin significantly (P<0.001) decreased, while urea-N and creatinine, AST and ALT significantly (P<0.001) increased with increasing mercury level in fish diets. Body moisture, protein and ash content were not affected by dietary mercury level. Ether extract content was decreased significantly (P<0.001) by increasing dietary mercury level. Fish group fed the diet containing 50 or 100 mg mercury recorded higher body mercury level with 700.0 and 800.0%, respectively when compared with those fed diet without mercury supplementation.

Fish fed on the diet supplemented with vitamin E or contained wheat grass (diet 2) increased final body weight by 7.65 and 7.98%, respectively, and increased daily body gain by 11.15 and 11.23%, respectively. Magnesium sulfate and vitamin E supplementation in Nile tilapia diets and wheat grass diet (diet 2) increased feed intake and improved feed conversion significantly (P<0.05) during the first 2 months after experiment start. Fish fed the diets supplemented with vitamin E recorded the best feed conversion during the whole experimental period. Serum albumin significantly (P< 0.01) increased, while urea-N, creatinine and AST were significantly (P<0.001, 0.01 or 0.05) decreased with magnesium sulfate or vitamin E supplementation in fish diets and those fed on wheat grass diet (diet 2). Body composition was not significantly effected with dietary treatment with additives. Magnesium sulfate or vitamin E supplementation as well as the diet containing wheat grass decreased mercury residues significantly (P<0.001) in fish bodies. In fish groups fed Magnesium sulfate or vitamin E supplementation or wheat grass recorded lower mercury residues by 65.94, 71.74 and 76.81%, respectively, when compared with those fed diet without supplementation.

The interaction between dietary mercury level and dietary supplementation did not show any significant differences in live body weight, daily body gain, daily feed intake or feed conversion during the whole experimental period. Within each mercury level, feed conversion improved with the supplementation of magnesium sulfate or Vitamin E or wheat grass. Plasma total protein, albumin, creatinine, AST and ALT values were insignificantly affected by the interaction between mercury level and dietary supplementation. Body composition was insignificantly affected by the interaction between mercury level and dietary supplementation. The interaction between mercury level and dietary supplementation affected significantly (P<0.001) body mercury residues

Fish is an excellent source of high biological value protein it contains low saturated fat and contains polyunsaturated fatty acids such as essential omega-3 polyunsaturated fatty acids. It is also a good source of some vitamins, particularly vitamin D. Fish is also an excellent source of iodine (Eastman 1999). Increased stress reduces the fish ability to ward off diseases and heal itself. The use of many chemicals in agriculture and industry, requires evaluation of their concentrations in the environment as well as in the food chain. Among these chemicals, chlorinated pesticides and toxic elements (Hg) are most extensively studied due to their toxicity and bioaccumulation (Spiric et al., 2001). Mercury is known to accumulate in the internal organs, especially in the liver (Holsbeek et al., 1998; Meador et al., 1999; Zhou et al., 2001). Mercury toxicity in environmental pollution is a major concern because of increased usage of fossil fuels and agricultural products, both of which contain mercury. Mercury occurs naturally in the environment as metallic mercury, inorganic mercury or organic mercury.  Mercury is readily absorbed from the gut following ingestion and is rapidly distributed via blood to the tissues (ATSDR 1999; NRC 2000). The major routes of excretion are through the bile and faeces, with lesser amounts in urine (NRC 2000). Saleh (1986) reported that the non-regulation of mercury in fish body causes a progressive increase of mercury content in its gills. Phillips and Buhler (1978) reported that nearly 70% of the mercury ingested and 10% of the mercury passed over the gills was assimilated.

Studies of mercury conentrations in Amazonian fish, have revealed levels that often surpass 0.5 ug/g fresh weight (Padovani et al., 1993). Lebel et al. (1996) obtained similar results with 25,8% of fish sampled having concentrations above  the 0.5 ug of Hg/g. Porvari (1995) found concentrations as high as 1.3 ug Hg/g in carnivorous species, 0.32 ug Hg/g in planktivores and omnivores; and 0.11 ug Hg/g in herbivorous species. According to Malm et al., (1995) in the control areas (that theoretically have no Hg contamination) levels on average were below 0,2 ug Hg/g , whereas in contaminated areas they can exceed 2 ug Hg/g. Mercury concentration ranged between 0.025 ug/g and 0.776 ug/g in pirarucu  Arapaima gigas fish in the lower Amazonian varzea (Crossa and Mc Grath, 1999). Mercury tends to accumulate in some types of fish more than others. This is due to a number of key factors, including age of the fish, natural environment, and food sources. Exposure to high levels of mercury can permanently damage the brain and kidneys (ATSDR, 1999). The maximum level of 0.5 ug/g (0.0005 ppm) of mercury in fish muscle tissue considered safe for human consumption which is accepted by the Brazilian Ministry of Health (Crossa and Mc Grath, 1999).

The objective of the present study was to investigate the effects of dietary mercury contamination and its amelioration by using dietary magnesium sulfate, vitamin E and wheat grass supplementation in fish diet on growth performance, feed efficiency, blood components and body composition of young Nile tilapia fish.

 

The present study was conducted at the Department of Animal Production, Faculty of Agriculture, Zagazig University. Young Nile tilapia (Oreochromis niloticus) averaged about 6.7 g in weight raised in Abbassa Fish Hatchery were used in this study. The fish were stocked in thirty six glass aquaria (70 X 40 X 60 cm) supplied with fresh dechlorinated and aerated tap water. Fish were divided into twelve groups, each group of fish was stocked into 3 aquaria and each contains 20 fishes. All fish groups were fed on a basal diet (Diet 1; Table 1)

The fish of the first four groups were fed the normal diet, the second four groups were fed on the basal diet contaminated with 50 mg mercury (mercuric chloride)/kg diet and the third four groups were fed on the same diet contaminated with 100 mg mercury/kg diet. Fish were fed two times daily at a feeding rate of 3% of body weight per day. The total duration of the experimental feeding trial was 4 months (from April to July, 2003). Within each dietary mercury level, the first group was fed the diet without supplementation the second was fed on the diet supplemented with 200 mg magnesium sulfate /kg diet, the third group fed on the diet supplemented 50 mg vitamin E per kg diet and the fourth group fed on the diet contained wheat grass (Diet 2; Table 1). Fish wastes were siphoned out and 25% of the water in each aquarium was removed daily and replaced with fresh new water. Fish were individually weighed to the nearest 0.1 gm at the beginning of the experiment and at biweekly intervals throughout the experimental period. Blood samples were taken from the caudal vein from three fish in each group which randomly selected for collecting blood samples. The blood samples were centrifuged at 3000 RPM for 20 min. to separate the serum. Total protein, albumin, urea-N, creatinine and serum transaminase enzymes (AST; aspartate amino transferase and ALT; alanine amino transferase) were determined in blood plasma by colormetric methods using commercial kits. Proximate chemical compositions of experimental diets and the whole fish body were determined according to AOAC (1980). Total mercury contents (T-Hg) in the whole fish body were analyzed using a flameless atomic absorption spectrophotometer after digestion of samples by a mixture of HNO₃, HClO₄ and H₂ SO ₄ according to the method described by Agrawal and Desai (1985).

The obtained data were statistically analyzed by 3 X 3 factorial design and evaluated by using the following model (Sendecor and Cochran, 1982):

Y_ijk = µ + L_i + V_j + LV_ij + E_ijk

Where, µ is the overall mean, L_i is the fixed effect of i^th dietary mercury level, V_jj is the fixed effect of j^th dietary supplementation, LV_ij is the interaction effect of i^th dietary mercury level and j^th dietary supplementation and E_ijk is the random error. Differences between treatments were statistical tested by Duncan'^,s multiple range test (Duncan, 1955).

 

Live body weight and daily body weight gain of Nile tilapia decreased significantly (P<0.001 or P<0.01) with increasing dietary mercury level (Table 2). Fish group fed diets contaminated with 100 mg mercury/kg diet recorded the lowest (P<0.05) live body weight and weight gain. Fish fed diets containing 50 or 100 mg mercury/kg diet recorded lower final live body by 6.94 and 11.19%, respectively than those fed diets without mercury contamination after 4 months of the experimental period. On the other hand, daily body weight gain during the whole experimental period (0-4 months) decreased by 10.39 and 16.88%, respectively. In this connection, Sivakami et al.  (1995) found that the growth rate of fish fed in diet contaminated with mercury was significantly lower than those fed uncontaminated diets. Decreased growth may be due to the greater expenditure of energy towards overcoming the stress caused by the heavy metal toxicity. Agrawal and Chansouria (1989) found a significant increase in adrenal and plasma corticosterone levels in rats exposed to mercuric chloride after 120 days of exposure. These findings cleared the mod of action of mercury in reducing the growth processors in fish fed diet contaminated with mercury. On the other hand, Zalups and Lash (1994) reported that mercury can promote oxidative stress, lipid perocxidation, mitochondrial dysfunction and changes in heme metabolism. Mercuric chloride (HgCl₂) has been shown to cause depolarization of the mitochondrial inner membrane, with a consequent increase in the formation of H₂O₂ (Lund et al., 1993).

Live body weight and daily body gain of Nile tilapia increased significantly P<0.001 or 0.01) with magnesium sulfate, vitamin E or wheat grass supplementation in diets (Table 2). Fish fed diet supplemented with vitamin E or that with wheat grass increased final body weight by 7.65 and 7.98%, respectively, and increased daily body gain by 11.15 and 11.23%, respectively, as compared with those fed diets without supplementation during the whole experimental period. Gustraunthaler et al. (1983) found that the treatment with mercury results in depletion of cellular defense mechanisms against oxidative damage such as glutathione, superoxide dismutase, catalase and glutathione peroxidase. Chemicals that protect against oxidative damage (such as vitamin E and wheat grass) may decrease the toxic effects of mercury. Anti-oxidants reduce cell mutations, artery damage, skin wrinkles, and other changes indicative of aging. Increased survival and decreased toxicity were observed when rats given vitamin E during treatment with mercury (Welsh, 1979). Bapu et al. (1994) found that therapy with vitamin E was found to mobilize a significant amount of mercury from all tissues examined (brain, spinal cord, liver and kidneys). Inclusion of a saline cathartic, such as magnesium sulfate speed removal of mercury from the gastrointestinal tract has been recommended (Haddad and Winchester, 1990).

The interaction between dietary mercury level and dietary supplementation did not show any significant effects in live body weight or daily body gain (Table 2). Within each mercury level, live body weight and daily body gain increased in fish groups fed diets supplemented with magnesium sulfate or vitamin E or diet contained wheat grass compared to those fed diets without supplementation. Fish fed diets supplemented with vitamin E or contained wheat grass recorded higher growth rate than the other experimental groups.

 

Daily feed intake decreased significantly (P<0.01 or 0.05) with increasing dietary mercury level and impaired significantly (P<0.001 or 0.05) the feed conversion during the all experimental periods (Table 3). Fish fed the 50 or 100 mg mercury level recorded the lower feed intake by 1.26 and 3.15% and impaired feed conversion by 9.80 and 16.37%, respectively, at the whole of the experimental period. These results are in line with those reported by Sivakami et al. (1995) who found that the exposure of fish to sublethal concentration of mercury led to a decrease in feeding rates. They also added that decreased conversion efficiency of feed may be due to the greater expenditure of energy towards overcoming the stress caused by the heavy metal.

Magnesium sulfate and vitamin E supplementation in Nile tilapia diets and wheat grass diet (diet 2) increased feed intake significantly (P<0.001) at 2-4 and 0-4 months of the experimental period and improved feed conversion significantly (P<0.05) at 0-2 months. Fish fed the diets supplemented with vitamin E recorded the best feed conversion during the whole experimental period (Table 3).

The interaction between dietary mercury level and dietary supplementation did not show any significant effects in daily feed intake or feed conversion during the whole experimental period (Table 3). Within each mercury level, daily feed intake was slightly increased and feed conversion improved with the supplementation of magnesium sulfate or Vitamin E or fish fed the diet containing wheat grass.

 

Plasma total protein, albumin significantly (P<0.001) decreased, while urea-N and creatinine (as indicator of kidney function), AST and ALT (as indicator of liver function) significantly (P<0.001) increased with increasing mercury level in fish diets (Table 4). Increasing the level of AST and ALT in the Plasma of fish fed diets contaminated with mercury may reflect the impairment of the liver functions due to its role in mercury detoxification. Also, the results obtained indicated that the kidney function (urea-N and creatinine level in the Plasma) increased with the increasing dietary mercury level and decreasing the protein synthesis in the liver. In this connection Berntssen et al. (2004) found that the intestinal and renal function in fish fed diet contaminated with 100 mg mercury / kg were reduced as seen from the significantly reduced protein and glycogen digestibility and increased plasma creatinine levels. Goldman and Blackburn (1979) and Sin et al. (1990) found that Plasma levels of thyroid hormones (triiodothyronine and/or thyroxin) in mice decreased after administration of mercuric chloride. These finding may explain the decreasing of growth rate as affected with the mercury toxicity. Also, Goldman and Blackburn (1979) found an increase in iodine release from the thyroid gland following the administration of 7.4 mg mercury/kg/day as mercuric chloride to rats for 6 days.

Plasma albumin significantly (P< 0.01) increased, while urea-N, creatinine and AST were significantly (P<0.001, 0.01 or 0.05) decreased with magnesium or vitamin E supplementation in fish diets or in fish fed on wheat grass diet (diet 2), while Plasma  total protein and ALT insignificantly affected (Table 4).

Plasma total protein, albumin, creatinine and ALT were insignificantly affected by the interaction between mercury level and dietary supplementation, while AST was significantly (P<0.05) affected (Table 4). The obtained results indicated that vitamin E, magnesium sulfate or wheat grass could modify the function of kidney and liver in fish exposed to the mercury toxicity.

 

Body moisture, protein and ash contents did not affected by dietary mercury  level (Table 5). Ether extract content was decreased significantly (P<0.001), while protein content decreased insignificantly by increasing dietary mercury level. Sivakami et al.  (1995) reported that decreased conversion efficiency of feed and low levels of proteins, carbohydrates, and lipids in different tissues may be due to the greater expenditure of energy towards overcoming the stress caused by the heavy metal

Body composition did not show any significant effect with dietary treatment (Table 5). Also, body composition was not significantly affected by the interaction between mercury level and dietary supplementation (Table 5).

Body mercury residues increased significantly (P<0.001) by increasing mercury level in fish diets (Table 5). Fish group fed diet containing 50 or 100 mg mercury recorded higher body mercury level with 700.0 and 800.0%, respectively when compared with those fed diet without mercury. Several investigators have reported high level of mercury in tissues after exposure to mercury (Magos and Butler, 1972 and WHO, 1990). Phillips and Buhler (1978) reported that nearly 70% of the mercury ingested and 10% of the mercury passed over the gills was assimilated in body tissues. Menasveta (1975) found in fish fed diets administered with mercuric chloride, and the highest percentage of Hg in the fish body was found in the miscellaneous parts (the composite of head, bone, skin, and visceral mass). Berntssen et al. (2004) reported that dietary inorganic mercury mainly accumulated in intestine (80% of body burden) and assimilation was low (6%). They added that highest accumulation of dietary inorganic mercury was observed in the gut and kidney. Fish fed 10 mg Hg kg-1 had an early (after 2 months) significant increase in renal metallothionein (MT) level and intestinal cell proliferation, followed by intestinal pathological changes after 4 months of exposure. At 100 mg Hg kg-1, intestinal and renal function were reduced as seen from the significantly reduced protein and glycogen digestibility and increased plasma creatinine levels.

Magnesium sulfate or vitamin E supplementation or diet containing wheat grass decreased significantly (P<0.001) mercury residues in fish bodies (Table 5). In fish groups fed Magnesium sulfate or vitamin E supplementation or diet containing wheat grass recorded lower mercury residues by 65.94, 71.74 and 76.81%, respectively, when compared with those fed diet without supplementation. Menasveta (1975) reported that orange peel and nitrilotriacetic acid (NTA) reduced the methylmercury accumulation in the fish (approx) 50%, and 30%, respectively, when compared with fish fed methylmercury without either additive. Orange peel and NTA also significantly reduced inorganic Hg accumulation.

The interaction between mercury level and dietary supplementation affected significantly (P<0.001) body mercury residues (Table 5). Based on the results obtained in this study it can be concluded that the vitamin E supplementation or fed on diet containing wheat grass reduced and ameliorate the hazards of mercury pollution in fish farms. In addition, results obtained revealed that fish get enough vitamin E will absorb less mercury.

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*** Nitrogen free extract

 

 

M= Month, *** P<0.001, ** P<0.01 and NS = Not significant.

Means in the same column within each classification with different letters differ significantly (P<0.05).

 

 

M= Month, *** P<0.001, ** P<0.01, * P<0.05 and NS = Not significant.

Means in the same column within each classification with different letters differ significantly (P<0.05).

 

 

*** P<0.001, ** P<0.01, * P<0.05 and NS = Not significant.

Means in the same column within each classification with different letters differ significantly (P<0.05).

 

 

*** P<0.001 and NS = Not significant.

Means in the same column within each classification with different letters differ significantly (P<0.05).