PHYSIOLOGICAL EFFECTS OF LEAD ,CADMIUM AND THEIR MIXTURE ON SOME ORGANS OF Alburnus Mossulensis FISH

Alburnus mossulensis has been exposed to lead and cadmium for (96)h in order to determination lethal concentration 50 (LC 50 ) value. A total of 140 fish used in this experiment divided into two groups each group contain 70 fishes .The first group exposure to pbcl 2 while the second group exposure to cdcl 2 . Method of Finney Probit Analysis used to analysis data. LC 50 value for PbCl 2 and CdCl 2 was (57.8 and 29.6) mg/l respectively. Then used about 200 fishes divided into 4 groups, the first group exposure to 1/2LC 50 of PbCl 2 ,the second group exposure to 1/2LC 50 CdCl 2 while the third groups contained (1/2LC 50 PbCl 2 +1/2LC 50 of CdCl 2 ) as so as control group for peroids (1,4,7 and 14) days to determination level of antioxidants Glutathione (GSH) and the level of lipid peroxidation (Malondialdehyde MDA) . The testes were carried out as three replications, lead ,cadmium and their mixture induced oxidative stress . The results of this study explained significant decrease of the level Glutathione and significant increase (Tukey test, P˂0.05) of the level of Malondialdehye compared with control in all treatments . It is possible to conclude that lead and Cadmium have cytotoxic effects, Fish mortality rates increase with increasing concentrations used and increasing exposure periods.


INTRODUCTION
The important environmental heavy metal contaminants are lead and cadmium that has widespread distribution. Human and animal exposure were due to both natural and anthropogenic causes, such mining, smelting, and other chemical products. Most times, these contaminants are co pollutants, resulting in mutual damage to living creatures and complementary harmful effects on health (30). Lead has been one of the nutrient-free metals that causes contamination in the aquatic environment (4). Lead induces oxidative stress through a direct effect on membranes (28). The increased ROS level coincides with inhibition of Na +, K + -ATPase, which disrupts the regulation of osmosis as well as causes tissue damage (20).Cadmium becomes considered to be a significant industrial source of pollution in the aqueous medium and a serious threat to aquatic species, mainly fish (3). Ecological exposure to cadmium can increase the occurrence of the element being ingested, causing damage to the organ (25). Fish play a role as a physiological indicator of the quality of water (2) . So with their affinity to the accumulation of materials in their muscle tissue (32), they cause change in physical, biochemical as well as hereditary parameters in their body (16). Penetrating fish through direct water absorption via their gills and skin, or via ingestion of contaminated food (6). Cadmium been regarded to be among the greatest harmful pollutants of contaminated water which cause toxicity to every ecological component (27). Cadmium doesn't really produce additional Reactive oxygen species (ROS), but also can change GSH levels. Alterations in Glutathion (GSH) may give rise to lipid peroxidation (LPO) of the cell membrane. Except for cadmium, lead can cause LPO and this is demonstrated by the outcome of several scientists (10). The aim of study determination of LC 50 for Pb and Cd and then Studying effects of cadmium, lead and their mixture on level of effectiveness of GSH and MDA.

MATERIALS AND METHODS Collecting fish samples:
The fish used during this study were collected from the waters of the Shalalat area in north of Mosul during the period from mid-October . A manual fishing net was used in the process of collecting fish, after which the fish were transported to the laboratory by a plastic basin in which water from the same aquatic environment was placed in it.

Specimens Preparation
Fish were placed in a glass tank containing chlorine-free water in addition to the water of the natural fish environment. The aquarium were provided with devices. Aeration and left for about two weeks for acclimatization, use Chinese-made commercial food.

Preparation of stock solution
The standard stock solution for lead was prepared at a concentration of 100 mg / l by dissolving 0.671 g of PbCl 2 with a quantity of distilled water and then the volume was completed to 5l , from which the concentrations (0,10, 30, 50, 70 and 90) mg/l used in the concentration determination experiment were prepared. As for cadmium, (0.812) g of CdCl 2 was dissolved in a quantity of distilled water and then the volume was completed to 5000 ml, from which the concentrations (0.10, 20, 40, 60 and 80) mg/l used in the concentration determination experiment were prepared.

Determination of LC 50 of lead and cadmium
The lethal concentration was determined for half the number of fish during the acute exposure period 4 days, by exposing the fish to lead concentrations (0, 10, 30, 50, 70, 90 and 100) mg / l. By placing 10 fish in each aquarium, the number of deaths was recorded within 4 days. The same applies to cadmium, as the concentrations (0, 10, 20, 40, 60, 80 and100) mg / liter were used during the acute exposure period (4 days). In the current experiment, 1/2 LC 50 for lead , 1/2LC 50 for cadmium and their mixtures were used.

Statical Analysis
Concentration was determined for LC 50 using a probit analysis software system, version 1.5 (12) The program used Graph padprism 5 to analyze the data statistically. The arithmetic mean was compared using the ANOVA One way program by means of a linear analysis of variance of the variables in the criteria under study for each group separately, in addition to the control group, using (Tukey's test) at a significant level (p ˂ 0.05) To compare the effect of lead on the members under study within different exposure periods, and letters have been placed in the tables to denote the significant differences (29). Similar letters demonstrate statistically significant differences in treatment and regulation. The values with different letters in the same row differ significantly.

Experimental design
The fish were divided into 4 groups: The first group: Control is represented by fish in aquarium containing dechlorinated water. The second group: is represented by fish in aquarium containing an aqueous solution of lead at a concentration of 28 mg /l (1 /2L C 50 ) for a period of 96 hours. The third group: is represented by fish in aquarium containing an aqueous solution of CdCl 2 at a concentration of 15 mg /l (1/2LC 50 ) for a period of 96 hours. Fourth group: is represented by fish in aquarium containing an aqueous solution of a mixture of lead (28 mg / l) and cadmium (15 mg / l).

RESULTS AND DISSCUSTION
Serious PbCl 2 toxicity demonstrates that the mortality rate dependent on the concentration of heavy metal lead, whereas the number of deaths is much absent from of the control (Tab. 1 and 2). Table 1. Relationship between lead concentration and mortality rate of A. mossulensis fish exposed for a period of (96) hours. Severe CdCl 2 poisoning demonstrates that the death rate is dependent on the concentration of heavy metal lead whereas the mortality rate is virtually absent from the control (Tab. 3 and 4).  The difference in the value of LC 50 in the studied fish varies with species and this may be due to the difference in the age, size, sex, feeding quality of fish, and the conditions of the experiment (23). Acute toxicity also differs due to different water quality such as hardness (8) and different pH and water temperature (21). The toxicity of minerals to fish also varies; Some minerals may be highly toxic and at low concentrations for species of fish, but they may be less toxic or non-toxic to other species of fish and when they are present in the same or even higher concentrations than when present in the ecosystem (14).

Antioxidant and lipid peroxidation level
The activity of antioxidant(GSH) in the various organs of A. mossulensis has decreased significantly (p>0.005) in the gills , brain, liver, intestine and muscles Compared to the control in all periods ,Also, Significant differences were observed between the control and Cd ,Pb and mixture groups. The decrease in the level of GSH in the group exposed to lead was greater than in the group exposed to cadmium , There was no significant difference in muscle compared to the other groups and to the control group . The lowest level of GSH was found in muscles in all periods. As for the effect of treatments, the lowest level were found in the mixture group, which differed significantly from other of treatments     The level of MDA significant increase in liver and intestine through(1 and 4) day Table(9 and  10), while it seen significant increase in all organs through in (7 and 14 days) Tables(11   and 12).The greatest higher level of MDA was found in brain and in mixture groups in all periods.  This study agree with Thi et al. (31)who observed significant increase of the level of MDA in brain of fish exposure to lead . Changes in GSH, and MDA were used as indicators of cadmium and lead toxic effects in fish (18). The results of the present study agree with the findings of Elarabany and Bahnasawy (9) whom they studied the effect of lead and cadmium on Clarias gariepinus and observed a decrease in the level of GSH in fish gills. It also agrees with Alfakheri et al (1) finding of low GSH in the catfish serum Clarias gariepinus. It also agree with Jing et al. (19) who found, in terms of a decrease in the level of glutathione in the liver and gills of Boleophthalmus pectinirostris after exposured to Pb and Cd. Heavy metals deplete antioxidants (7). Heavy metals impedes the activity of antioxidant (13). Oxidative stress begins through a decrease in the defense system of the antioxidants, and the decrease in GSH in fish reflects the ability of heavy metals (lead and cadmium) to bind to a covalent bond with a group of sulfhydryl group of several antioxidant enzymes, which causes inactivation of these enzymes and renders them susceptible to oxidation by free radicals (24) The presence of Glutathione S-Transferase (GST) in the cytosol of most cells stimulates the binding of GSH to foreign substances in xenobiotic biology. By forming a pinhole Thio -ether between the thiol group of GSH glutathione and substance for detoxification purpose (11). Glutathione is an antioxidant rich in Sulfhydryl group, which gives it a distinctive electron-giving property and thus gives an electron to active oxygen species / free radicals which easily oxidizes GSSG. This rapid use of both GST and GSH leads to a decrease in their level (17). Glutathione binds to the metal lead. Thus, it works to protect cells from their negative effects ( 15). The results of the current study are in agreement by Thi et al. (31), as they found that when exposure to Zebra fish with the chronic effect of lead causes a significant increase in the level of MDA in the brain, the results of the current study also agree with what researchers Elarabany and Bahnasawy (9)whom found When exposed to Pbcl 2 , Clarias gariepinus raised MDA in the liver and gills. The results of the current study are in agreement with the recent study conducted by Thi et al. (31), as they found that when exposure to Zebra fish with the chronic effect of lead causes a significant increase in the level of maloonaldehyde in the brain. And Alfakheri (1)found an elevated level of MDA in the blood serum of the catfish Clarias gariepinus after exposure to lead. Also it was in agreement with that found by Zheng (33) who exposed fish to cadmium and found an increase in MDA level in the organs under study. The results of this study also agree with Ayoola (5) who exposed the fish Hemichromis fasciatus and Chrysichthys nigrodigitus they found high levels of malondaldehyde in the liver and gills. It is also inferred from the results of the current study that the effect of lead is greater than the effect of cadmium , This agrees with Mahjoub (22) and Rajeshkumar et al. (26) CONCLUSIONS Lead and cadmium has a harmful effect on the cells of living organisms, even at low concentrations. Fish mortality increases with increasing concentration and increasing duration of exposure. Exposure to lead and cadmium causes a change in the level of antioxidants. In addition, there was a significant increase in the level of MDA which indicates the occurrence of the lipid peroxidation process in the membranes and the increase of ROS. ACKNOWLEDGMENTS Authors are very grateful to the the President of the University, Professor Dr. Qusay Kamal Al-Din Al-Ahmadi. Also the authors thank to University of Mosul, Collage of Education for Pure Science, Department of Biology for providing support materials and laboratory facility for conducting this study.

CONFLICTS OF INTEREST
There are no conflicts of interest declared by the authors in relation to the publication of this manuscript.