Efficacy of Ascorbic Acid Against Aluminum Chloride

Zag. Vet. J. (ISSN. 1110-1458) Vol. 40 No. 5 (2012) pp.115-127

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Efficacy of Ascorbic Acid Against Aluminum Chloride Toxicity in Catfish (Clarias garpinus) Ahmed NF Neamat-Allah1, Nabela I El-Sharkawy2, Hanaa MR Hegazy3, and Gamal A Elmowalid4 1

Department of Clinical Pathology, Fac. of Vet. Medicine, Zagazig University. Dept. of Forensic Medicine and Toxicology, Fac. of Vet. Medicine, Zagazig University. 3 Dept. of Forensic Medicine and Toxicology, Fac. of Vet. Medicine , Kafer El-Sheikh University. 4 Dept. of Bacteriology , Mycology and Immunology, Fac. of Vet.Medicine, Zagazig University. 2

ABSTRACT Few studies have been made in regard to the toxic effects of aluminum on fresh water fish at neutral pH; therefore, in the present study is focusing on evaluation the role of Ascorbic acid (AA) on aluminum chloride (AlCl3) exposed catfish on some clincopathological changes, innate immunity and histopathological alterations. This investigation was carried out using 48 Nile Catfish (Clarias garpinus), which were divided into four groups each of two replicates; 6 fish replicate. First group was kept as –ve control, second group exposed to AA at dose level 5ppm/ L of water ( +ve control) while third and fourth groups exposed to AlCl3 1/20 LC50 and AlCl3 concomitant with AA respectively, for 24 weeks at pH.7.2 At the end of the experiment blood was immediately harvested from the caudal vein then fish were humanly sacrificed. Hematological studies revealed a significant decrease in erythrocytic count, hemoglobin and packed cell volume in Al group associated with leucopenia while leucocytosis was detected in AA group. Moreover, Al exposed group showed a reduction of phagocytic cells capacity, bactericidal activity and nitric oxide production while biochemical analysis revealed panhypoproteinemia with an increase in ALT, AST activities and creatinine level, which were confirmed by histopathological changes in the related organs. These changes were improved by exposure to AA.

INTRODUCTION Domestic sewage, agricultural drainage water and other wastes heavily contaminate the drainage canals in the Egyptian Delta (1). Polluted drainage water heavily loaded with different contaminants such as pesticides and metals (2). Aluminum (Al) is one of the most abundant metals of the earth's crust representing about 8% of the total mineral components (3). It is a harmful metal to the aquatic ecosystem, being responsible for events of toxicity with serious ecological consequence. To date, no normal physiological functions in biological systems are attributed to this metal (4). Al is a

widely distributed metal in the air, water and soil; moreover, its presence in the environment has been increasing due to different anthropogenic activities such as its use in the manufacture of household utensils, cans, foods, cosmetics, medicaments, paper and herbicides (5). Besides, it is also used to precipitate solids in suspension during water treatment, a process that has shown a significant increase in the metal (6). European Commission considers the classification of a number of Al compounds as well as Al powder (not massive metal) as dangerous substances with the designation of R50/R53 (risk phrases according to the guideline 67/646/EEC, amendment 91/325/EEC) warning for very toxic effects

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Ahmed et al., on aquatic organisms and long-term harmful effects on water (Meeting of the Commission Working Group on the Classification and labelling of dangerous substances: Environmental Effects, Arona, 5-7 December 1995) (7). Aqueous Al is bioavailable and toxic to freshwater fish and invertebrates at neutral pH (8). In fish, high concentrations of Al may cause mortality, swim disturbances, endocrine disrupting, and alterations in various hematological parameters; besides, the metal can be deposited in their gills producing inflammation and mucus secretion as well as incorporated to the brain where it can give rise to severe ultrastructural changes (9,10).Moreover, Al disturbs immunity of freshwater crayfish (Pacifasticus leniusculus) (9). A number of studies demonstrated that supplementation with some vitamins and trace elements may reduce or protect against the harmful effects of Al in different animal species as AA, being antioxidant, ameliorated the adverse effect of Al administration in rabbits, by decreasing the free radicals, creatinine, and increased the activity of liver enzymes (11). Despite the high levels of the soluble metal at neutral pH immediately following neutralization of acidic Al-rich water and the potential contamination of water with Al (9), few reports about Al effects on aquatic organisms especially Clarias garpinus. Therefore, the aim of this work to study the effect of AlCl3 on hematological, immunological, biochemical and histopathological changes after long term exposure in catfish and their alleviation with AA at neutral pH.

MATERIAL AND METHODS Chemicals Aluminum chloride (AlCl3) and ascorbic acid (AA) were obtained from El-Goumhouria Company .Egypt.

Experimental fish This study was conducted on 48 live Nile Catfish ( Clarias garpinus) with an average 155 g body weight. Fish were purchased from Abbassa Fish Hatchery at Sharkia Province, Egypt. Fish were apparently healthy and free from any skin lesions or external parasites.They were fed on basal diet containing 35.4 % crude protein. The amount of food (on dry matter basis) per day was 3% of fish body weight and fed three times daily. Fish were maintained in glass aquaria (each, 80x40x30 cm capacity) having 96 liters of dechlorinated tap water with pH 7.2 and 25Cº and changed weekly . Each aquarium provided with aerator, thermostatically controlled heater and thermometer. Fish were acclimatized to laboratory environment for two weeks. They were divided equally into equal four groups each of two replicates (6 fish replicate). Group 1 was kept as –ve control, group 2 exposed to AA at dose level 5ppm in the water (12) and kept as +ve control, while 3rd and 4th groups exposed to AlCl3 1/20 LC50 according to (13) and AlCl3 concomitant with AA respectively for 24 weeks. Blood collection Three blood samples were collected from caudal vessels (14) of fish at the end of experimental period. First blood samples were collected in EDTA tubes for hematological investigations, second blood samples were collected without anticoagulant for separation of serum for biochemical analysis and finally blood samples were collected in heparinized tubes for immunological studies. Haematological studies The total erythrocytic and leukocytic count were performed using the improved Neubaur hemocytometer with Natt and Herrick solution as diluting fluid .The hemoglobin was estimated by cyanmethemoglobin method . Packed cell volume (PCV) was determined. Giemsa’s staining method was used for the differential leukocytic count (15). Immunological studies a.Phagocytic activity and nitric oxide production by immune cells

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Zag. Vet. J. Fish white blood cells were isolated and cultured in 24-well plates (16), then white blood cells phagocytic activity was assessed (17, 18), while nitric oxide production was estimated (19). b.Intracellular survival assay bactericidal activity Intracellular survival assay was used to measure the bactericidal activity of WBCs in all fish groups. The assay was performed on cells in 24-well culture plates (20) Percentage of survival = [1-absorbance after incubation for 120 min] x 100 Absorbance at time (0)

Biochemical analysis The serum total protein and albumin levels were measured (21,22).The activities of aspartate (AST) and alanine aminotransferases (ALT) (23), and serum creatinine (24) were determined colorimetrically. Histopathological examination Small specimens from the skin, brain, spleen and kidneys were collected from all groups, and then fixed in 10 % neutral buffered formalin for 48 h then washed overnight under running water. The samples were blocked in hard paraffin and then were cut into sections of 5 µ thickness, then stained with haematoxylin and eosin (H&E). The sections were mounted with Canada balsam and covered with cover slide to be ready for histopathological examination (25). Statistical analysis The data obtained from this investigation were statistically analyzed using F test (26). Means at the same column followed by different letters were significantly different and the highest value was represented with the letter (a).

RESULTS AND DISCUSSION The most characteristic lesions of AlCl3 in catfish are pale silver and chromatic, aberration with mucous secretion and corroded dorsal fin (Fig.1, a, b). Similar lesions were observed in zebra fish (27) which it may be developed either from anemic effect of Al (28) or any alteration of skin (29), whereas Al promote of skin cell damage, increased leukocyte invasion into the epidermis, tail muscle ischaemia and haemostasis within the caudal artery of free swimming zebrafish larvae (30). Also, skin lesions were confirmed by our histopathological findings in skin which showed hyperplastic activity of the epidermal layer especially the mucous secreting cells in the dermis especially around the blood vessels and in the upper surface of dermis at its junction with the epidermis. Moreover, Al caused necrosis in the basal layers of epidermis with edema visualized and represented by widely separated collagen fibers (Figs.1, c,d,e).Spongiosis and hydropic degenerative changes were also observed besides few lymphocytes infiltrations. The dermal skeletal muscles were rarely degenerated and necrotic. These lesions were completely vanished in fourth group which exposed to AA + Al which may be attributed to enhancement of Al excretion (31). Brain in Al exposed group revealed encephalomalacia and depletion of the granular cell layer besides degenerated neurons and neuronal edema .Intense submeningeal aggregations of lymphocytes were detected, congestion in the meningeal blood vessels, satellitosis and neuronophagia were also observed,(Fig.1,g) that could be due to the neurotoxic effect of Al on nervous system (32). On contrary these lesions were mild in the fourth group (AA+Al) (Fig.1,h) which showed a slight congestion in the meningeal blood vessels, lymphocytes aggregation and perivascular edema which may be attributed to AA attenuated the oxidative damage and morphological changes in the brain (33)

Ahmed et al.,

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Fig.1. Normal catfish of showing dark color (a). Catfish of Al group showing pale and silver color of skin and dorsal fin corrosions (b). Skin of control showing normal epidermis, dermis and dermal skeletal muscles, HE (Bar = 100 µm) (c). Skin of gp (3), showing hyperplastic activity of the epidermal layer especially the mucous secreting cells (arrow) besides activation of melanomacrophages (irregular arrows) and dermal edema,which represented by widely separated collagen fibers (arrowhead), HE (Bar = 100 µm) (d). Skin of gp (4), showing hyperplastic activity of the epidermal layer especially the club cells (arrow) besides few melanomacrophage infiltrations (arrowhead), HE (Bar = 100 µm) (e). Brain of control showing normal neuronal structure, HE (Bar = 100 µm) (f). Brain of gp (3) showing encephalomalacia and depletion of granular layer (arrow), HE (Bar = 100 µm) (g). Brain of gp (4), showing mild aggregation of lymphocytes (arrow), HE (Bar = 100 µm) (h).

Hematological results (Table 1), revealed highly significant decrease in erythrocytic count, hemoglobin and packed cell volume in Al group compared with the – ve control group. Similar results were obtained in Clarias batrachus (34), which might be due to the direct effect of Al on hematopoiesis causing anemia (35) either through decreased heme (36) or globin synthesis (37). Another possible explanations were increased rate of destruction or reduction in formation of erythrocyte after Al exposure (38) or a direct effect of Al on iron metabolism; by hindering iron’s transport in the serum ; and displaces iron’s binding to transferrin (39). It was also recorded that Al forms instable complexes with glutathione (GSH) (40) which is a major component of

RBCs (41) affecting the physiological responses of cells (42). Exposure to AA alleviates the anemic effect of Al toward the normal control which may be attributed to increase the activities of antioxidant enzymes in various tissues where the different blood cells are formed (43). Similarly there was a decrease in total leucocytic count in Al group which induced immunosuppressive effect (44) with lymphopenia which could be due to high affinity of T lymphocyte to Al ions (45), while there was heterophilia and monocytosis for helping removal of necrotic tissues (46). On contrary, the results showed leucocytosis, lymphocytosis, heterophilia and monocytosis in AA exposed group due to its activation of immune response (47).Our results were

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Zag. Vet. J. confirmed by histopathological finding of the spleen which revealed necrosis and depletion in the lymphoid cells of splenic pulps. Congestion and hemorrhages were seen besides moderate activation of the

melanomacrophages centers in Al group while exposure to AA with Al revealed normal spleen except with mild hemorrhages in some cases and focally congested splenic ellipsoids. (Figs.2,a,b.c).

Table 1. Effect of ascorbic acid, aluminium chloride and their combination on some haematological parameters (mean values±SE) in Catfish. Parameters WBCs Lymphocyte Heterophil Monocyte Oesinophil count (× 103/µl) (× 103/µl) (× 103/µl) (× 103/µl) 3 (× 10 /µl)

Groups

RBCs count (× 106/µl)

Control

1.94a±0.02 10.72a±0.35 34.60a±0.24 21.54b± 0.64 12.84a±0.56 6.80b±0.08 1.65d±0.04 0.23a±0.07

Hb (gm%)

PCV (%)

AA

2.01a±0.02 10.48a±0.57 35.00a±0.31 24.92a± 0.15 13.52a±0.28

7.73a±0.23 3.47b±0.12 0.19a±0.08

AlCl3

1.28c±0.09 6.04c±0.33 13.80c±0.37 15.57d± 0.21 8.61c±0.23

4.63c±0.06 2.12c±0.09 0.19a±2.07

AA + AlCl3

1.70b±0.05 7.72b±0.54 24.40b±0.60 18.49c± 0.48 10.50b± 0.33 3.58d±0.17 4.21a±0.15 0.18a±0.07

F test

**

**

**

**

**

**

**

N.S

LSD

0.16

1.38

1.20

1.25

1.11

0.45

0.33

-

** Highly significant difference at p≤0.01 LSD least significant difference N.S Non significant Means at the same column with different letters are significantly different

Fig. 2. Spleen of control showing normal lymphoid elements, HE (Bar = 100 µm) (a). Spleen of gp. (3) showing severe necrosis and depletion of lymphoid population (arrow), HE (Bar = 100 µm) (b). Spleen of gp (4), showing mild hemorrhage (arrow) and normal lymphoid cells, HE (Bar = 100 µm) (c).

Ahmed et al., The innate immune response is the first line of defense of the immune system of the host and is a prerequisite for potentiating the adaptive immune response. The cellular components of the innate immune system, namely macrophages and neutrophils (48) are critical for controlling microbial infection. These immune cells destroy the pathogen by phagocytosis and production of reactive oxygen species such as nitric oxide (49). In this study, the cells phagocytic capacity to engulf yeast (phagocytic cells containing > 5, > 15 and > 20 yeast cells), was significantly higher in fish group exposed to AA alone followed by that exposed to AA and AlCl3 compared to cells from fish exposed to AlCl3 alone, (Table 2) which showed a significant reduction in their capacity to engulf more

120 yeast (Figs.3,a,b,c,d).Our results are in agreement with the study of (50) who reported that AA enhance the immune cells phagocytic activity in Oreochromis niloticus fish which may be attributed to the ability of AA as antioxidant to improve immune cell function by reducing the harmful free radicals (51). The antioxidant mechanism of AA could in part, enhance immunity by maintaining the functional integrity of important immune cells (52) or increase cells ability to engulf and kill microbes. The reduction phagocytic cell capacity in AlCl3 exposed fish may be due to the reduction in phagocytic cell count which may be due to change in immune cells membrane integrity (53) or due to its generalized enhancing effect in fish health.

Fig. 3. The cell phagocytic capacity to engulf more yeast was higher in the control catfish cells (a); and cells from catfish exposed to AA (b); and cells from fish exposed to AA and AlCl3 (c) compared to the cells from catfish exposed to AlCl3 alone, that take few yeast (d) Microbicidal activity (bactericidal) was significantly low in fish cells received AlCl3

alone and the cells started to restore their bactericidal activity after receiving AA in

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Zag. Vet. J. combination with AlCl3 (table II). Since there is no supporting data in this regard, the reduction in cells microbicidal activity may be attributed to the ability of AlCl3 to reduce cells enzymatic activities; synthesis to toxic protein and reduction of synthesis of microbicidal agents (54). This immunosuppressive effect of AlCl3 (44) may be also associated with reduction of phagocytosis, and reduction of antioxidant enzyme level, which is necessary for maintenance of the bactericidal qualities of phagocyte (45). Which confirmed by decrease in total proteins, albumin and globulin (Table 3). In contrast, addition of AA to AlCl3 or using AA alone (Table 2) helped in enhancing phagocytosis and protection of the phagocytic cells (55) that might result in cells restoring to their bactericidal activity. The exact mechanism of impairment in cells

microbicidal investigation.

activity

needs

further

Nitric oxide production was significantly higher in immune cells culture of control fish and in fish groups exposed to AA alone or in combination with AlCl3 compared with AlCl3 exposed group (Table II). No available data on the relation between nitric oxide production by immune cells and Al toxicity. However, the reduction of nitric oxide due to Al intoxication may be due to reduction in enzymatic cascade of nitric oxide production, or due to interfering with the internal signals required to mitochondria to make this vital product. The increase in nitric oxide levels in groups received AA (Table 2) may be explained by its ability to potentiate the synthesis of nitric oxide (56).

Table 2. Effect of ascorbic acid, aluminium chloride and their combination on phagocytic cell capacity %, bactericidal activity % and nitric oxide production (mean values ±SE) in Catfish. parameters Phagocytic cell capacity (%) Groups Nitric oxide Bactericidal > 20 yeast activity (%) ( mol) > 5 yeast cells > 15 yeast cells cells Control

92.20b ±0.73

85.60b ±1.07

81.6b ±0.50

69.20b ±0.37

65.28b ±2.26

AA

97.40a ±0.58

90.40a ±0.50

85.2a ±3.17

74.60a ±0.92

71.33a ±0.30

AlCl3

68.80d ±0.53

42.00d ±0.94

21.8d ±0.80

33.40d ±0.92

16.95d ±0.87

AA + AlCl3

88.20c ±0.66

70.40c ±1.02

54.2c ±1.24

53.20c ±0.86

42.90c ±0.93

** 1.90

** 2.71

** 3.09

** 2.09

** 3.87

F test LSD

** Highly significant difference at p≤0.01 LSD least significant difference Means at the same column with different letters are significantly different

Al induced nephrotoxicity and hepatotoxicity (57), AlCl3 exposed fish showed a significant marker of renal dysfunction, as increased in creatinine (Table 3) which concomitant with pathological

lesions of kidneys (Figs.4,a,b,c) and AlCl3 intensifies acid secrete function of kidney and change the transport of sodium (58). In addition, Al has been implicated in the pathogenesis of several clinical disorders, such

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Ahmed et al., as renal dysfunction (59). Al is mainly excreted in urine by different mechanisms of renal Al elimination, such as glomerular filtration (60) tubular reabsorption of filtered Al (61) and excretion in the distal tubules (62). Moreover, it has also been reported that Al accumulates in the kidney promoting degeneration in renal tubular cells, and thus could induce nephrotoxicity (63). Histopathological findings in kidneys of Al group showed an areas of coagulative necrosis in the renal epithelia, hemorrhages and depletion of the hemopoietic tissue .The lesions were mostly alleviated or completely absent with exposure to AA. Also, Al group revealed a significant decrease in total protein, albumin and globulin, whereas this hypoproteinemia could be attributed to the inhibitory effect of Al to protein synthesis (64)

which was concomitant with chronic hepatic disease (46). Al significantly increase ALT and AST activities which could be attributed to liver damage and degenerative changes caused by Al intoxication (57) or Al induces the process of lipid peroxidation in the cell membrane (65),which is a principal cause of hepatotoxicity (66).AA plays an important role for improvement these biochemical alterations toward normal as decrease AST and ALT activities and creatinine level of groups treated with AA alone or in combination with Al which indicates protection of liver and kidney from Al toxicity (67) either by prevention Al accumulation in any tissue studied (31) or it acts against the toxic, mutagenic and carcinogenic effects of environmental pollutants by stimulating antioxidant enzymes (68).

Table 3. Effect of ascorbic acid, aluminium chloride and their combination on some biochemical parameters (mean values ±SE) in Catfish. Groups Parameters Total proteins (g/dl)

Albumin (g/dl)

Globulins (g/dl)

ALT Unit/l

AST Unit/l

Creatinine (mg/dl)

Control

4.20a± 0.09

1.60a±0.10 2.60a±0.17 18.88c±0.34 28.48b±0.44

0.78c±0.01

AA

4.24a± 0.11

1.48a±0.04 2.76a±0.11 18.70c±0.37 28.56b±0.37

0.76c±0.01

AlCl3

2.82c± 0.07

0.64c±0.06 2.18b±0.08 27.21a±0.58 40.23a±0.74

1.90a±0.03

AA + AlCl3 3.39b± 0.07 1.22b± 0.03 2.16b±0.09 21.63b±0.24 30.10b±0.81

1.34b±0.09

F test

**

**

**

**

**

**

LSD

0.26

0.19

0.35

1.19

1.83

0.14

** Highly significant difference at p≤0.01 LSD least significant difference Means at the same column with different letters are significantly different

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Fig. 4. Kidney of control showing normal glomerular and tubular structure, HE (Bar = 100 µm) (a). Kidney of gp (3) showing coagulative necrosis in the renal epithelium (arrow), hemorrhages and depletion of hemopoietic tissue besides shrunken of glomerular tuft (arrowhead), HE (Bar = 100 µm) (b). Kidney of gp (4), showing hydropic degeneration (arrows) and hemorrhages among the renal tubules (arrowhead), HE (Bar = 100 µm) (c). of brain toxicity. Arch. Toxicol., 82:789– 802.

Conclusions Ascorbic acid alleviates aluminum chloride toxicity on skin, kidney, spleen and brain of catfish (Clarias garpinus) and improves clinicopathological alterations and innate immunity. Acknowledgment The authors want to thank Prof. Dr. Mohamed Hamed Mohamed Professor of Pathology Faculty of Veterinary Medicine at Zagazig University for his help in Examining and reading Histopathological slides.

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‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ‬ ‫ﻛﻔﺎءة ﺣﻤﺾ اﻻﺳﻜﻮرﺑﯿﻚ ﺿﺪ اﻟﺘﺴﻤﻢ ﺑﻜﻠﻮرﯾﺪ اﻻﻟﻮﻣﻨﯿﻮم ﻓﻲ ﺳﻤﻚ اﻟﻘﺮﻣﻮط‬ ‫أﺣﻤﺪ ﻧﻌﻤﺖ ﷲ ﻓﺮﺣﺎت *‪،‬ﻧﺒﯿﻠﺔ إﻣﺎم اﻟﺸﺮﻗﺎوي**‪،‬ھﻨﺎء ﷴ ﺣﺠﺎزي***‪ ،‬ﺟﻤﺎل ﻋﺒﺪاﻟﻤﻨﻌﻢ اﻟﻤﻮﻟﺪ****‬ ‫*ﻗﺴﻢ اﻟﺒﺎﺛﻮﻟﻮﺟﯿﺎ اﻹﻛﻠﯿﻨﯿﻜﯿﺔ – ﻛﻠﯿﺔ اﻟﻄﺐ اﻟﺒﯿﻄﺮي – ﺟﺎﻣﻌﺔ اﻟﺰﻗﺎزﯾﻖ‬ ‫** ﻗﺴﻢ اﻟﻄﺐ اﻟﺸﺮﻋﻲ واﻟﺴﻤﻮم – ﻛﻠﯿﺔ اﻟﻄﺐ اﻟﺒﯿﻄﺮي – ﺟﺎﻣﻌﺔ اﻟﺰﻗﺎزﯾﻖ‬ ‫*** ﻗﺴﻢ اﻟﻄﺐ اﻟﺸﺮﻋﻲ واﻟﺴﻤﻮم – ﻛﻠﯿﺔ اﻟﻄﺐ اﻟﺒﯿﻄﺮي – ﺟﺎﻣﻌﺔ ﻛﻔﺮ اﻟﺸﯿﺦ‬ ‫**** ﻗﺴﻢ اﻟﺒﻜﺘﺮﯾﻮﻟﻮﺟﯿﺎ واﻟﻔﻄﺮﯾﺎت واﻟﻤﻨﺎﻋﺔ – ﻛﻠﯿﺔ اﻟﻄﺐ اﻟﺒﯿﻄﺮي – ﺟﺎﻣﻌﺔ اﻟﺰﻗﺎزﯾﻖ‬ ‫أﺟﺮﯾﺖ ھﺬه اﻟﺪراﺳﺔ ﻟﻤﻌﺮﻓﺔ ﻣﺪي ﺗﺄﺛﯿﺮاﺳﺘﺨﺪام ﺣﻤﺾ اﻻﺳﻜﻮرﺑﯿﻚ ﻟﺘﺨﻔﯿﻒ اﻻﺛﺎر اﻟﺴﻤﯿﺔ ﻟﻜﻠﻮرﯾﺪ‬ ‫اﻻﻟﻮﻣﻨﯿﻮم ‪.‬وﻗﺪ إﺳﺘﺨﺪم ﻋﺪد ﺛﻤﺎﻧﯿﺔ وارﺑﻌﻮن ﺳﻤﻜﺔ ﻗﺮﻣﻮط‪ .‬و ﻗﺪ ﻗﺴﻤﺖ إﻟﻰ أرﺑﻊ ﻣﺠﺎﻣﯿﻊ ﻣﺘﺴﺎوﯾﺔ‪.‬‬ ‫اﻟﻤﺠﻤﻮﻋﺔ اﻷوﻟﻰ‪ :‬ﺗﺮﻛﺖ ﻛﻤﺠﻤﻮﻋﺔ ﺿﺎﺑﻄﺔ‪ ،‬اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻧﯿﺔ ﺗﻢ ﺗﻌﺮﺿﮭﺎ ﻟﺤﻤﺾ اﻻﺳﻜﻮرﺑﯿﻚ ﺑﯿﻨﻤﺎ‬ ‫اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻟﺜﺔ ﺗﻢ ﺗﻌﺮﺿﮭﺎ ﻟﻜﻠﻮرﯾﺪ اﻻﻟﻮﻣﻮﻧﯿﻮم واﻟﻤﺠﻤﻮﻋﺔ اﻟﺮاﺑﻌﺔ ﺗﻢ ﺗﻌﺮﺿﮭﺎ ﻟﻼﺛﻨﯿﻦ ﻣﻌﺎ ﻓﻲ اﻟﻤﺎء ﻟﻤﺪة‬ ‫‪ ٢٤‬اﺳﺒﻮع ﻋﻠﻲ اﻟﺘﻮاﻟﻲ‪.‬‬ ‫ﻟﻘﺪ أظﮭﺮت اﻟﻤﺠﻤﻮﻋﺎت اﻟﻤﺘﻌﺮﺿﺔ ﻟﻜﻠﻮرﯾﺪ اﻻﻟﻮﻣﻮﻧﯿﻮم وﺟﻮد أﺛﺎر ﺟﺎﻧﺒﯿﺔ ﻋﻠﻲ اﻟﺠﻠﺪ ﻣﻊ ﺗﺂﻛﻼت ﻓﻲ‬ ‫اﻟﺰﻋﺎﻧﻒ واﻧﯿﻤﯿﺎ ﻣﻊ ﻧﻘﺺ ﺧﻼﯾﺎ اﻟﺪم اﻟﺒﯿﻀﺎء وﻛﺬﻟﻚ زﯾﺎدة ﻓﻰ إﻧﺰﯾﻤﺎت اﻟﻜﺒﺪ اﻷﻻﻧﯿﻦ أﻣﯿﻨﻮﺗﺮاﻧﺴﻔﯿﺮﯾﺰ و‬ ‫اﻻﺳﺒﺮﺗﯿﺖ أﻣﯿﻨﻮﺗﺮاﻧﺴﻔﯿﺮﯾﺰ و ﻧﻘﺺ اﻟﺒﺮوﺗﯿﻦ اﻟﻜﻠﻲ واﻻﻟﺒﯿﻮﻣﯿﻦ وزﯾﺎدة ﻧﺴﺒﺔ اﻟﻜﺮﯾﺎﺗﯿﻨﯿﻦ ‪ .‬إﻋﻄﺎء ﺣﻤﺾ‬ ‫اﻻﺳﻜﻮرﺑﯿﻚ ﺧﻔﻒ ﻣﻦ ھﺬه اﻻﺛﺎر ﻓﻲ اﺗﺠﺎه اﻟﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻄﺔ ﻣﻊ زﯾﺎدة ﻓﻲ ﻧﺘﺎﺋﺞ اﻟﻤﻨﺎﻋﺔ اﻟﺨﻠﻮﯾﺔ ‪.‬‬ ‫ﻧﺴﺘﺨﻠﺺ ﻣﻦ ھﺬا اﻟﺒﺤﺚ أن‪:‬‬ ‫‪ -١‬ﯾﻤﻜﻦ اﺳﺘﺨﺪام ﺣﻤﺾ اﻻﺳﻜﻮرﺑﯿﻚ ﻟﺘﺨﻔﯿﻒ اﻻﺛﺎر اﻟﺠﺎﻧﺒﯿﺔ ﻟﻜﻠﻮرﯾﺪ اﻻﻟﻮﻣﻨﯿﻮم ‪.‬‬ ‫‪ -٢‬اﺳﺘﺨﺪام ﺣﻤﺾ اﻻﺳﻜﻮرﺑﯿﻚ ﯾﺤﺴﻦ ﻣﻦ اﻟﺤﺎﻟﺔ اﻟﻤﻨﺎﻋﯿﺔ ﻻﺳﻤﺎك اﻟﻘﺮﻣﻮط‬