الفهرس 
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Abstract
In the present study 514 specimens of Clarias gariepinus were used to determine LC50 of HgCl2 and the adverse effects of two sublethal concentrations of mercury chloride (0.04 and 0.12 mg/l) on behavioral changes, growth performance, blood hematology (red blood cells, hemoglobin, haematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cells, lymphocytes, monocytes and neutrophils), biochemistry (ALT, AST, ALP, Albumin, globulin, total protein, cholesterol, triglycerides, HDL and LDL) and Hormones (T3, T4, TSH, Testosterone and E2). The histopathological characteristics of the liver and kidney were also studied. The validity of selenium (0.1 ppm) and vitamin E (100 mg/kg of the fish weight) for inhibition of such effects was studied.
The results can be summarized in the following points:
LC50 Determination
Estimation of mercury LC50 showed that LC50 values of HgCl2 were 0.637, 0.569, 0.501 and 0.413 (mg/L) at 24, 48, 72 and 96 hrs respectively.
Behavioral, mortality and morphological changes
After 28 days of treatment, fish specimens exposed to mercury showed abnormal behavior such as partial loss of equilibrium, rapid pectoral and opercula movements, reduction in the feeding activity, fins hemorrhage, loss of some skin parts and changes in color. Fishes treated with selenium and vitamin E and their combination were in better conditions.
Growth performance
Growth performance of Clarias gariepinus exposed to Hg, Selenium and/ or vitamin E and their interaction was recorded in terms of CF and HSI (Table 3). There was a significant difference between control and different treatments by the addition of selenium and selenium with vitamin E. On the other hand, a decrease was recorded in both parameters in treated groups with mercury in low and high doses. The main effect of Hg, selenium, vitamin E and their interaction was significant for the condition factor and HSI (P<0.05). One can conclude that HSI and CF as a rough measurement of energy reserve are sensitive parameters to reflect sublethal stress of Hg and the protective role of Se and vitamin E.
Blood constituents
A- Hematological parameters
Mercury main effect was significant in the four periods for red-blood-cells (RBCs) count, haemoglobin (Hb) content and haematocrit (Hct) value of Clarias gariepinus. Selenium main effect was significant in all periods for the previous parameters except RBCs. Vitamin E main effect was significant in all exposure periods for RBCs, Hb and Hct. Se-VE interaction effect was not significant in all exposure periods for RBCs, Hb and Hct. Hg- Se- VE interaction effect was only significant in Hb and Hct in all exposure periods.
Mercury main effect was highly significant (P<0.0001) in all periods for MCV, MCH and MCHC. Selenium main effect was significant for the three previous parameters in all periods. Vitamin E and its interaction with mercury were also significant in all exposure periods for MCV, MCH and MCHC. SE-VE interaction effect was significant for MCV, MCH and MCHC. Hg- Se -VE interaction showed a significant effect for the three parameters. Se- VE- period of exposure interaction effect was significant only for MCH.
Mercury main effect was highly significant (P<0.0001) in all exposure periods for WBC, NEUT, LYM and MONO. Selenium main effect was significant in all exposure periods for the four previous parameters. Vitamin E main effect was significant in all exposure periods for WBC, NEUT, LYM and MONO. Hg- Se- VE interaction was not significant in NEUT. The period of exposure main effect was highly significant for the four previous parameters. Se-VE- Period of exposure interaction effect was not significant in NEUT and LYM.
Mercury main effect was highly significant (P<0.0001) in all exposure periods for erythrocytes alertation and micronucleus percentage. Selenium and Vitamin E main effect was significant in all exposure periods for both of them. Vitamin E and its interactions main effect was significant in all exposure periods for the previous parameters. The period of exposure main effect was significant in erythrocytes alteration and micronucleus percentage. The percentage of micronucleus was increased significantly at (p≤0.05) with increase of mercury dose treatments and exposure periods.
Exposure of Clarias gariepinus to sublethal concentrations of mercury chloride (0.04 ppm and 0.12 ppm), 0.1-ppm sodium selenite and 100 mg/kg of Vitamin E resulted in morphological changes in the red blood cells and appearance of some pathologic types of cells. The major alterations of RBC’s are damage of cell membrane (DR) with pale cytoplasm (Pc), cells have prominent vacuoles (Vac), distinct micronucleus (Mn), nuclei lose some of their central dye (A sign of karyolysis, also crenated cells; echinocytes (Cr), tear DROP like cells (Tr), acanthocyte (Ac) sickle cells (Sk), Swelled cells (Sc), pale cytoplasm (Pc), lysis of some red cells and some cells appear to be stacked together
B- Biochemical parameters
Kidney functions
Mercury main effect was highly significant (P<0.0001) in all exposure periods for Na+ and K+. Selenium main effect was significant in all exposure periods for the two previous parameters. Vitamin E main effect was significant in all exposure periods for Na+ and K+. Se-VE interaction effect was highly significant in all exposure periods (P<0.0001) for Na+ and K+.
Mercury main effect was highly significant (P<0.0001) in all periods for both of Urea and Creatinine. Selenium main effect was significant for the two previous parameters. Vitamin E main effect was only significant for Creatinine in all exposure periods. SE-VE interaction effect was significant for Urea only. Hg- Se -VE interaction effect was not significant for the two parameters. Se-VE-period of exposure interaction effect was not significant for Urea and Creatinine.
Liver functions
Mercury main effect was highly significant (P<0.0001) in all exposure periods for ALP, ALT and AST. Selenium main effect was significant in all exposure periods for the three previous parameters. Vitamin E main effect was significant in all exposure periods for ALP, ALT and AST. Se-VE interaction effect was highly significant in all exposure periods for ALP, ALT and AST (P<0.0001).
Mercury main effect was highly significant (P<0.0001) in all exposure periods for TP, Alb, Glo and alb/glo ratio. Selenium main effect was significant in all exposure periods for the four previous parameters. Vitamin E main effect was significant in all exposure periods for Tp, Alb, Glo and Alb/Glo. Se-VE interaction effect was not significant only for Tp in all exposure periods. Hg- Se- VE interaction effect was significant in all exposure periods for the previous parameters.
Mercury main effect was highly significant (P<0.0001) in all exposure periods for TB, DB and IndB. Selenium main effect was significant in all exposure periods for the previous parameters. Vitamin E main effect was significant in all exposure periods for TB, DB and IndB. Se-VE interaction effect was highly significant in all exposure periods (P<0.0001) for the previous parameters. Hg- Se- VE interaction effect was significant in all exposure periods for TB, DB and IndB.
Glucose and Lipogram
Mercury reflected highly significant effects in Glu, CHO, TRI, HDL and LDL (P<0.0001) in the four periods of exposure. Selenium main effect was significant for all parameters in all exposure periods. Vitamin E main effect was significant in all exposure periods for Glu, CHO, TRI, HDL and LDL. Se-VE interaction effect was highly significant in all exposure periods (P<0.0001) for the previous parameters. VE-Periods interactions were not significant for HDL in all exposure periods. Hg-Se-Period interaction effect was significant in all parameters except CHO and HDL in all exposure periods.C- Hormonal parameters
T3 level through weeks decreased with mercury exposure doses. The interactions with Se and Se with Vitamin E in low mercury dose (0.04 Ppm) after 2 weeks of exposure were not significantly different for T3 level.
The main effects of high mercury dose (0.12 ppm) and exposure periods were significantly different (P<0.05) for T3 level. The interaction effect between mercury, selenium and vitamin E after 4 weeks of exposure was significant (P < 0.05) for T3 level. After 2 weeks of exposure there was no significant in T4 level in low mercury dose and its treatments. However, such patterns are questionable since their interaction and week Se and Hg-Se-week were significant. However, the period of exposure and mercury make the effect of selenium significantly different. Only high mercury dose treatment effects on T4 level. T4 level decreased through weeks in high mercury dose. But, the interactions with VE, Se and Se with Vitamin E in low mercury dose were not significantly different after 2 weeks of exposure for T4 level.
T3/T4 ratio is used in screen thyroid gland disorders. After 2 weeks of exposure, the addition of selenium to mercury treatments resist the significant effect of high and low doses of mercury, whereas VE was unable to resist that decrease of ratio.
The main effects of mercury and exposure periods were significantly different at (P<0.05) for TSH level.However such effects were different since there were significant interactions effects between mercury, selenium and period of exposure for TSH level. Weekly factorial analysis reveals that Mercury reflects its significant difference action in weeks of exposure at (P < 0.05). The interactions effect of mercury, selenium, vitamin E and weeks were also significant (P<0.05) for TSH level.
The main effects of mercury and exposure periods were significant (P<0.05) for Testosterone level. The interactions between mercury, selenium, vitamin E and weeks were also significantly different (P < 0.05) for Testosterone level. TES level through weeks decreased by mercury addition except for its interaction with Vitamin E. The interaction between mercury and Vitamin E was reversible to that of mercury and Se.
The main effects of mercury and period of exposure were significant (P<0.05) for 17β-Estradaiols level. The interactions between mercury, selenium, vitamin E and weeks were also significant (P < 0.05). E2 level through weeks decreased in mercury addition. The interactions between mercury with Se and Vitamin E were significantly decreased for 17β-Estradaiols level. However such effects were different since there were significant for 17β-Estradaiols level. Interactions between mercury, selenium and period of exposure was significant for 17β-Estradaiols level.Histopathology
Liver
Treatment with (0.04 and 0.12 mg/l) mercury for 14 and 28 days showed histopathological changes in the liver:
• Dilatations of blood vessels which often congested with blood.
• Wall thickening of blood vessels.
• Degeneration of hepatocytes.
• Dissociation of hepatocytes.
• Rupture in hepatocytes membranes
• Presence of pyknotic cells.
• Presence of necrotic areas.
• Increase of melanomacrophages.
• Lymphocytic aggregation.
• Hypertrophy and hyperplasia of küpffer cells.
Examination of liver sections of selenium (0.1 ppm) and/or vitamin E (100 mg/kg of the fish weight) administered fish in the four periods showed improvement in the hepatic tissues when compared with those of mercury- treated fish with presence of histopathological changes as:
• Degenerated hepatocytes.
• Hyperplasia of küpffer cells.
• Dilatation of blood vessels and blood sinusoids.
• Blood vessels Congestion.
• Rupturig in central vein.• Small lymphocytic aggregation.
• Presence of melanomacrophages.
Kidney
Treatment with (0.04 and 0.12 mg/l) mercury for 14 and 28 days showed histopathological changes in the kidney:
• Edema in renal tubules.
• HyDROPic and hyaline degeneration of renal tubules.
• Dissociation in the cells of renal tubules.
• Hypertrophy of the glomerulus.
• Dilated blood capillaries of the glomerulus.
• Observed pale stained hyDROPic cytoplasm.
• Proliferation of hemopoietic tissue.
• Dilated blood vessels congested with blood.
• Increased of melanomacrophages.
• Presence of necrosis.
Examination of kidney sections of selenium (0.1 ppm) and/or vitamin E (100 mg/kg of the fish weight) administered fish in the four periods showed little improvement in the renal tissues when compared with those of mercury- treated fish with presence of histopathological changes as:
• Shrinkage of the glomerulus.
• Hypertrophy of the glomerulus.• Hyaline and hyDROPic degeneration in the renal tubules.
• Obliterated Bowman’s space.
• Necrotic areas in the hemopoietic tissue.
• Edema in renal tubules.
• Presence of melanomacrophages.
Ultrastructural changes
TEM examination of the liver revealed the following changes in treated fish (0.12 ppm mercury) as compared to control.
• Transformation of the rough endoplasmic reticulum into concentric arrays.
• Increased amount of lysosomes of varying densities and sizes.
• Degranulation and fragmentation and loss of ribosomes from the surface of the rough endoplasmic reticulum.
• Alterations in rough and smooth endoplasmic reticulum.
• Dilatation of some parts of rough endoplasmic reticulum.• Concentric arrays of rough endoplasmic reticulum were observed.
• Swelling in some hepatocytes with large rarefied areas in the cytoplasm.
• Proliferation of the smooth endoplasmic reticulum.
• The mitochondria were abundant and heterogenous.
• Some mitochondria tended to take dumbbell-like shapes.
• Residual bodies (lipofuscin pigment) were also encountered.
• Granulocytes were occasionally observed among degenerated hepatocytes.
• Pyknotic nuclei were visible within the outer and inner nuclear layers.
• Fragmentation of the rough endoplasmic reticulum after the high dose.
Many ultrastructural alterations were observed in fish exposed to selenium (0.1 ppm) plus mercury chlorid (0.12 ppm):
• Swelling in some hepatocytes.
• Highly abundance of mitochondria with variable shapes and sizes.
• Dilated and fragmented rough endoplasmic reticulum.
• Disarrangement of the rough endoplasmic reticulum.• Swollen of mitochondria with destructed cristae.
• Small myelinated figures.
The ultrastructaral changes in the liver of Fish exposed to Vitamin E (0.1 mg/Kg) plus mercury chloride (0.12 ppm) are:
• Appearance of swelling in some hepatocytes.
• Large lipid DROPlets were detected.
• Many lysosomes of different sizes and shapes.
• The rough endoplasmic reticulum cisternae were disarranged.
• Mitochondrial calcification was observed.
• Large amounts of heterogenous materials.
The ultrastructaral changes in the liver of Fish exposed to mercury chloride (0.12 ppm) plus Selenium (0.1 ppm) and Vitamin E (0.1 mg/Kg) are:
• Abundance of Mitochondrial count was observed.
• Some Mitochondria were swollen with destructed cristae.
• The cytoplasm contained numerous lysosomes of varying densities.