Determination of the lethal concentration 50% (LC50) of lead chloride and its accumulation in different organs of Gambusia affinis fish

Article history: Received March 28, 2020 Accepted April 28, 2020 Available online March 15, 2021 In the present research, mosquito fish Gambusia affinis have been exposed to lead chloride during 24, 48, 72 and 96 hours in order to evaluate the lead chloride lethal 50 (LC50) concentration and the Its residue in certain organs of fish. Usage of the EPA computer software based on Finney Probit Analysis method has been statistically tested for the data collected LC50 values of G. affinis if 24, 48, 72 and 96 hours were found to be 59.4, 55.9, 51.1 and 49. 0 mg/L, respectively. LC50 decreased as mean exposure times. 20 fish were placed in each concentration of four sublethal concentrations 20 and 25 mg/L for two acute periods 24 and 96 hours as well as 10 and 5 mg/l for chronic periods 15 and 30 hours. The testes were carried out as three replications, the accumulation of lead in various fish organs was determined by Atomic Absorption Spectrophotometer. The finding revealed that the accumulation of PbCl2 on different organs of G. affinis be time dependent fashion and Pbcontent in organs increased significantly time dependent at chronic exposure as compared as acuteexposure.


Introduction
Owing to their toxicity, heavy metal is considered to be environmental contaminants, persistence in the ecosystem and bio accumulative existence (1). It is characterized as a metal with density is bigger than 5 g/cm 3 and their atomic weight is 63.5-200.5 g/mol (2). In the environment there are many sources of heavy metals for example lead, arsenic, mercury, cadmium, selenium, nickel, chromium, and copper lead, heavy metals in general, are commonly categorized as foundation and non-core minerals (3). Heavy metals are an important source of pollution because of their toxic nature and their capacity to accumulate (4). The potential of heavy metals to build up in the aquatic environment due to its failure to decompose (5). In different manufacturing methods Lead is a commonly used metal in various industrial processes and in the marine world is very persistent (6). Lead is a highly poisonous metal, and its routine use has caused excess contamination of the environment and associated health problems in many parts of the world (7). Fish have been recognized as the main aquatic organisms that accumulate considerable amounts of certain metals exceeding their concentrations in the aquatic ecosystem (8). Individual growth rates, physiological functions, reproduction and mortality in fish (9). Heavy metals penetrate fish bodies in 3 possible ways: through gills, through the digestive track and through the surface of the body. The gills are known to be the important site of direct absorption of water metal (10,11), Although it is generally calculated the body surface take a small part in absorption of heavy metals in fish (12). Levels in fish usually reflect levels found in sediment and water of the particular aquatic environment from which they are sourced (3) levels in fish usually reflect levels found in sediment and water of the particular aquatic environment from which they are sourced (13). At the end of the aquatic food chain fish may collect heavy metals and transfer them through the food chain to people who cause acute or chronic diseases (14). The purpose of the present study was to determine the LC50 of lead chloride in acute and chronic treatment of mosquito fish lead accumulation in various G. affinis organs.

Materials and methods
Fishes were collected from the Tigris River banks on the left side of Mosul, Iraq. Experimental fishes which measured an average length 2.025 cm and weighed 0.18 g experimental fish have been exposed to metal concentration. First phase of repair laboratories included a quarantine the duration during which the fish were acclimatized to the requirements of the laboratory for the experiment for two weeks at least. The animals were housed in glass aquariums filled with dechlorinated tap water. There were 16:8 pgotoperoids (16 h. light/8 h darkness) and fish were fed twice daily with commercially available fish food.
Ten fish have been placed in clean tap water, acting as monitors, and there were 10 fish put in aquariums 25*25*30 cm at 20 cm in depth; 20 and 25 mg/L for chronic exposure and chloride (PbCl2) for acute and every procedure had 10 replicates. Animals were then traded for 10and 5 mg/L PbCl2 as lead quantitative metal head, liver, gills, muscle, and, intestine dissected. In Pyrex test tubes, tissues of fish organs have been dried at a stable weight of 48 hrs at 60ºC. Analysis was carried out according to the procedure described by (15). Dried tissues with concentrated sulphoric acid and perochloric acid were weighed and digested at 350 rpm. When the gases were white and clear the solution was transparent, the specimens were cooled to room temperature and ten ml of tubes filled with ultra-pure water. All samples were analyzed a graphite furnace AAS technique is used to assess the PbCl2 concentration (ZEEnit700). Triplicate samples were analyzed. The coefficient of variance was generally less than 10 percent. Metal concentrations in the tissues there were measured on a dry basis of weight and express as dry weight of μg/g.

Conditions
Metal toxicity testing was carried out under laboratory conditions. This experiment was conducted with six metal treatments in a fully randomized system. A glass tank was for water substitution. Fish density was 10 fish per tank. Stock solutions of lead chloride a were prepared in double distilled water by dissolving the analytical grade PbCl2. 10 Fishes were per concentration.

LC50 determination
For determination of the LC50 values, the following ranges were tested, six PbCl2 10, 20, 40, 60, 80 and 100 mg/L concentrations were chosen for mosquito fish. Metal solutions were prepared by dilution of a stock solution with dechlorinated tap water. A control with dechlorinated tap water only was also used. The number of dead fish was counted every 12 hours and removed immediately from tanks. The mortality rate was determined at the end of 24, 48, 72 and 96 hours. The acute toxicity test was conducted in accordance with standard methods (16).

Statistical analysis
The acute toxic effect of lead chloride on mosquito fish was calculated in this study by the use of Finney's method of determining Probit Analysis LC50 (17). In order to Graphpadprism 5. The statistical significant differences between different treatments and control are reported by different letters a, b, c, d. The values with different letters in the same row are significantly different (Tukey test, P ≤ 0.05).

Result
Acute lead toxicity shows that mortality is directly proportional to the heavy metal lead chloride concentration while the mortality rate is virtually absent in the control (Tables 1-5).

LC50 of lead for mosquito fish
Mosquito fish susceptibility to the impact of lead toxicity was found to increase mortality with an increase in lead concentration, while mortality was virtually absent in the control (Table 1). Analytical results showed that the mean lethal concentration (LC50) of lead to mosquito fish for exposure was 24, 48, 72 and 96 hours 59.443, 55.978, 53.256 and 500.514 mg/L respectively. It soon became evident that an improvement in the duration of exposure led to a rise in mortality (Table 6). Fish are capable of acquiring and both active ingredients absorb metals from water and passive procedures in their bodies; the accumulation of metals in fish tissues is also based on metal absorption, tissue distribution and deposition. In our research, the amounts of lead accumulated in Gambusia affinis tissues varied dependent on the time of exposure, the concentrates and the form the tissue. Atomic absorption analysis showed that the Pb content in different measured organs increased significantly based on time and PbCl2 concentration. In this analysis, we observed variations in PbCl2 accumulation between control and treatment. Our results indicated that PbCl2 the accumulation in tissues of the brains > gill > intestine > liver > muscles in fish exposed to lead chloride 20 mg/L, period 96 hours of significant differences and more impact than 24 hours and control fall (Table 7). Similar letters show the statistically significant differences between different treatment and regulation. There are significantly different values of different letters in the same row (Tukey test, P≤0.05) Different letters. show statistically significant differences between different treatments and regulation. The values with different letters in the same row differ significantly. We also observed variations in PbCl2 accumulation between control and treatment. Our research found that PbCl2 collection in tissues in gills the order of the > liver > intestine > brain > muscle in fish exposure to lead chloride 25 mg/L, period 96hours significant differences and greater impact than 24hours and control fall ( Table 8). Whereas the concentration of lead chloride 5 mg/L shows the gills sequence > liver, intestine > brain > muscles. There is no significant difference between The period of 30 days and 15 days while both of two period differ from control (Table  9). Finally, the concentration of lead chloride 10 mg/L shows the sequence gills > intestine > liver > brain > muscles. The accumulation of lead chloride in the period 30 is greater and significant difference between The period 15 days control groups while both of two period differ from control (Table  10). Similar letters show the statistically significant differences between different treatment and regulation. There are significantly different values of different letters in the same row (Tukey test, P≤0.05) Different letters. show statistically significant differences between different treatments and regulation. The values with different letters in the same row differ significantly.

Discussion
Results of the present study showed that the LC50 value of 24, 48, 72 and 96 hours of lead exposure in Gambusia were 59.443, 55.978, 53.256 and 50.514 mg/L respectively, no death among the control group. The use of the LC50 test as a general indicator of chemical toxicity has become popular and has been questioned as inaccurate and in sightful criteria for decades. It is thus useful to reconsideration the repeated identification of the LC50 before conducting laboratory tests (9).     For all tissues, the PbCl2 concentration was lower in the control group than in the treatment group at the periods 24.96, 15 and 30 days. It is well known that heavy metals like Cd and Pb are potentially deposited in marine organisms and sediments, where they are subsequently passed to humans via the food chain (18). The influence of the metal depends on the animal, s size and the form of the species. Although the organisms survive the initial attack of pollutants due to their defense adaptations, the injuries caused by the gradual exposure will be observed even in small doses at later stages when the resistance of the organism is reduced due to ageing. In addition, the test organism's state and reaction to the amount of metal entering its body, the degree of retention and the rate of excretion affect the toxic effect of heavy metal (7). We studied lead accumulation in the brain, gill, liver, intestine, and muscles tissues of mosquito fish. The tendency of each organ to accumulate lead chloride in this study was brain > gills > intestine > liver > muscle in fish exposure to lead chloride 20 mg/L, period 24 and 96hours, while the concentration of PbCl2 in fish exposure to 24 mg/L gills > liver > intestine > brain > muscle. The accumulation of lead chloride in G. affinis exposure to 5 mg/L was gills sequence > liver, intestine > brain > muscles and in the concentration 10 mg/L was gills > intestine > liver > brain > muscles. The accumulation of metal in fish tissues depends on the concentration and time of exposure, as well as other factors such as temperature, age, contract with other metals, water chemistry and fish metabolic activity (19). Pb is a nonessential factor and in aquatic species close to anthropogenic sources, high concentration can occur. Even at low concentrations, it is toxic and has no known role in biochemical processes (7). Fish have the capacity to store heavy metals in their tissues to higher levels than environmental concentration through absorption along the gills surface and kidney, liver and gut tract wall (20). The present study revealed that the lead was collected in the various fish organs, the highest concentration of lead was detected in the gills tissue of G. affinis, while in the muscle tissue the lowest was found. Gills has the highest levels of lead accumulation, this finding was being contradictory to the work of Mahboob et al who reported lead was greater accumulation in gills, kidney, liver and muscles in Cyprinus carpio (21). Gills are important location for heavy metals to join (22); and it is the first target organ for fish exposure. The large concentration of metals in the gills is related to complexation of metals with the mucus, which is difficult to extract from the tissue completely prior to the analysis. The metal concentration in the gill shows the level of the metals in waters where fish live, while the concentration in kidney and liver contraction reflect the storage of metals. Therefore, gills in fish are more commonly recommended as environmental markers of fish organs (23), Of great significance is the deposition of Lead in fish tissues. Lead in the gills has genotoxic and cytotoxic damage (24).
Liver was the target organ for Pb accumulations (25). Many experiments have shown that the liver accumulates more metals than other tissues. The bioaccumulation of metals in liver may be linked to its function of metabolism, liver is the primary detoxifying organ and the target for the accumulation in fish of most metals (26,27). The accumulation of metals in the liver is a very rapid process suggesting the presence of a non-saturable ion channel and lysosomal system where metals usually arrived coupled to metallothioneins (7). Metallothioneins (MTs) are proteins bound to metal that are upregulated upon metal exposure. In contrast, MTs can attenuate the detrimental effects of the metal to the system of the organism to a certain extent and concentrations of exposure (28,29). Also, (30) reported the accumulation of Pb in the Pangus fish has the decreasing order of gill > liver > kidney > gonad > muscle.
Lead toxicity is targeted towards processes of brain memory and learning, and can be regulated by three processes. Lead can impair brain learning and memory by inhibiting the N-methyl-d aspartate (NMDAR) receptor and may block neurotransmission by inhibiting the release of neurotransmitters, block the neuronal voltage gated calcium (Ca2 + ) channels (VGCCs) and reduce brain derived neurotrophic factor (BDNF) (31), however it is apparent in this study that there is a relatively little accumulation of Pb in brain tissues compared to the Gills and liver. Similarly, (6) determined that bioaccumulation of lead was Pb liver > gills > kidney > brain > muscles in Cirrhina mrigala.
Fish intestines are charged with digestion, nutrient absorption, digested food excretion, and Metabolic active tissues such as gills, liver and kidneys absorb heavy metals in concentrations comparatively higher than the rest of the body's tissues such as skin and muscles (1,28). Muscles are not the primary target for accumulation (6,18). Furthermore, Das et al reported the effects of sublethal concentrations exposure of Pb in nervous system (32). After different exposure durations 3-42 days, the accumulation profile of Pb was brain > liver > kidney > gills > muscles > skin. The bioaccumulation of Pb in the Katla fish Gibelion catla has the decreasing order of liver > gill > kidney > gonad > muscle (30).
Among animals, muscle tissue doesn't play a very active role among metal accumulation. In our study the lowest rate of accumulation of lead was in muscle tissue, which is the fish's edible component. Many studies have found the same result (33,34). The low concentrations of metals in the muscle of fish species can indicate the low levels of binding proteins in the muscle (35).

Conclusions
Present investigation revealed variable toxicity of PbCl2 to the G. affinis in LC50 and bioaccumulation in some organs of mosquito fish. LC50 decreased as mean exposure times. All the fish tissues showed significantly variable exposure sublethal concentration and time-dependent greater accumulation of PbCl2 observed in gills while the trace PbCl2 was least in the muscles. lead have harmful health effects even at lower levels, and there is no known safe exposure level.