Heavy Metal Toxicity In Wistar Rats Exposed To Leachates From Awotan Municipal Solid Waste Dumpsite In Ibadan City, Nigeria

Heavy metal toxicity in Wistar rats exposed to leachates from Awotan municipal solid waste dumpsite in Ibadan city, Nigeria.

ABSTRACT

Inefficient municipal waste disposal system is one of the major sources of direct contamination of ground water and food sources with heavy metal toxicant which often result in exposure of plants and animals tissues with risk of bioaccumulation in man. Increased urbanization and rapid pace of technological advancement have led to increase in electronic wastes which are major sources of heavy metal contamination. This study therefore was carried out to investigate the physicochemical properties of leachates from Awotan dumpsite (3.848oE, 7.461oN), Ibadan, Nigeria; its organosomatic, haematologic and organopathic effects in wistar rats following a sub-acute exposure. This was determined by evaluation of the heavy metal concentration in the leachates and exposing rats to graded concentrations (0%,1%,2.5%,5%, 10% & 25%) for 30 days orally. Clinical signs and parameters related to the above stated aims were evaluated during the study. Statistical analysis was done using one way ANOVA and Duncan multiple range test. Heavy metal concentration in the leachates were found to be higher (>250%) than the acceptable limits especially with regards to Lead (Pb).Concentration dependent severity in signs such as anorexia, alopecia, muscle stiffness, dyspnoea and diarrhea were observed within the experimental groups. There was also a concentration dependent significant decrease (P<0.05) in the organosomatic index with concomitant significant increase (P<0.05) in the levels of the heavy metals residue in the blood and liver of the rats. Decrease in red cell index values was observed concentration dependently also with a significant macrocytic hypochromic anaemia observed in the 25% group. Furthermore, there was concentration dependent Leucopenia most significant in the 25% group while lymphopenia and heterophilia were significant in the 5%, 10% and 25% groups. Aspartate Platelet ratio, an index of liver fibrosis, decreased as do serum albumin with increasing leachates concentration most significant in the 10% & 25% groups. Heterocyte lymphocyte ratio increased with increasing leachates concentration. Histology of the liver also revealed a progressive concentration dependent damage while the lesions of kidney damage were uniform. In conclusion, severity of hepatopathic changes increases with increasing exposure to heavy metal concentration while renopathic changes are uniform. This mirrors risks residents of such areas are exposed as they become final recipients of a multi-sourced exposure from contaminated ground water, bioaccumulated in plant tissues, and possible chemical Zoonoses from exposed animal tissues consumed as meats. Hence, proper municipal waste management program away from expanding residential areas should be provided with waste recycling programs encouraged.

Keywords: Heavy metals, Leachate, Wistar rats, Toxicity, Organosomatic, Liver.

INTRODUCTION

Waste is an inevitable byproduct of daily living which must be managed in an environmentally friendly and healthy manner. With efforts to increase standard of living by most developing countries, there would also be an inevitable increase in production of solid waste stemming from accelerated industrialization, urbanization, and population growth.

Solid wastes disposed off at designated sites such as landfills and dump sites undergo a combination of physical, chemical and microbial processes (Christensen et al., 2001). These processes transform the solid wastes into different water-soluble compounds and particulate matter which can percolate the underground water from the surface soil. (Bjerg et al., 2003). The contaminant-rich aqueous solution of pollutants formed is termed “leachate”. The mechanism of the percolating of contamination may include precipitation, irrigation, surface runoff, ground-water intrusion and the initial moisture content present within the wastes (El-Fadel et al., 1997).

Municipal solid waste landfills have been identified as one major threat to ground water and surface water resources. This is most especially important as residential areas expand toward formerly distant landfills which have greater possibility of ground water contamination from leachates originating from the nearby sites (Nixon et al., 1997, Aldecy et al, 2008). Such contamination of groundwater resource poses a substantial risk to local resource users and the natural environment.

Municipal landfill leachates are highly concentrated complex effluent which contain dissolved organic matter; inorganic compounds such as ammonium, calcium, magnesium, sodium, potassium, iron, sulphates, chlorides and heavy metals such as cadmium, nickel, chromium, copper, lead among others. (Lee, et al 1986).

Heavy metals are often problematic environmental pollutants, with well-known toxic effects on living systems. Nevertheless, because of certain useful physical and chemical properties, some heavy metals, including mercury, lead, and cadmium, are used in the manufacture of some consumer and industrial products such as batteries, switches, circuit boards, and some paint pigments (Haugen, 1994). In recent years, advances in technology has also brought about an increase in the use and disposable electronic devices such as cell phones, mp3 players, and computers, raising questions about the fate of these devices, and the metals they contain, in landfills. These products typically contain lead, cadmium, mercury, arsenic, copper, zinc and other heavy metals thus posing a great threat to the biotic components of the ecosystem around landfill sites.

Man, who interacts with both abiotic and biotic components of his ecosystem are exposed to greater risk from multiple sources. This is especially true as polluted air from automobile and industrial exhausts, contaminated ground water from leachates and industrial affluent, accumulated concentrations in plant tissues from ground uptake while bioaccumulation in animal tissues from grazing on plants and scavenging on wastes sites consumed as meat are potent sources exposure to varying concentrations of heavy metals in msn. Therefore this study is conducted to evaluate the physicochemical properties of leachates from Awotan usage have greatly over the years, while some are reducing, others are increasing. Cadmium usage for instance, has increased especially in the manufacture of rechargeable batteries. Although production and consumption of cadmium in the U.S. have declined, these reductions have been offset by increased uses in other regions. Globally, cadmium consumption is reported to have increased by about 1% in 2003 vs. 2002, with China the leading cadmium consumer (Plachy, 2003). It is estimated that the Zn-Mn batteries occupy over 90% of the total annual sales of portable batteries due to their low prices, especially in developing countries like Nigeria and China. They are usually rapidly run out and thrown away. Thus, despite efforts to increase the recycling of consumer items that have rechargeable batteries containing cadmium, it is likely that many such consumer items ultimately enter the municipal waste stream thus, posing a threat to the environment.

MATERIALS AND METHODS

Description of study site and leachates collection

Samples were taken from Awotan dumpsite, Ibadan, Nigeria which is located in Apete (3.848oE, 7.461oN), Ido Local government area in Oyo state. The dumpsite is an extensive expanse of land approximately covering 25 hectares with an altitude of 249m surrounded by a densely populated and rapidly expanding residential area with an altitude of 180m. It is situated along Akufo-Ibadan Polytechnic road and was opened in 1998. The dumpsite is managed by Ibadan Waste Management Authority. Raw leachates was collected from 15 different points on the dumpsite and was pooled together to get a homogenous representative sample for the site. The homogenate was thus transported to the laboratory and processed according to Alimba et al., (2012).Briefly, the leachate was filtered with Whatmann® No. 42 filter paper to remove the solid and suspended particles, and the filtrate was centrifuged at 3000rpm for 10min and kept in the refrigerator at 40 C until use.

Physico-chemical parameters and heavy metal analysis

The physical and chemical components of the leachate were analyzed according to AOAC (2004). Nitrate, ammonia, chloride, phosphate, sulphate, total alkalinity, biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total solids (TS) were determined. The concentrations of the Lead (Pb), Copper (Cu), Cadmium (Cd), Chromium (Cr) and Nickel (Ni) were also determined according to AOAC (2004). Briefly, 100mL of the leachates was digested by heating with concentrated perchloric/nitric acid in a ratio 1:2 on a hotplate in a fume cupboard. The digest was allowed to cool and made up to 25ml with distilled water. Heavy metal concentration was then determined using Buck scientific Atomic Absorption Spectrophotometer (model 210/211 VGP, United Kingdom).

Animals and experimental design

Wistar rats of about 4-5 weeks old were used for the experimental procedure. They were acclimatized for 2 weeks and maintained in laboratory conditions of 12 hours dark and light cycle, temperature of 26 ± 2oC, relative humidity of 70 ± 20%. They had access to drinking water and standard rodent chow (Ladokun feed Nigeria®) ad libitum.

A number of five rats of both sexes were in five treatment groups receiving 0.5mL of 1, 2.5, 5, 10 and 25% (leachates diluted with distilled water, v/v) of leachates and the sixth group received distilled water alone. Each rat was given 0.5mL of the treatment concentration for each group while the negative control group was also given distilled water for 30 consecutive days.

Observations and body weight measurement

Each of the rats were observed twice daily both before and after the exposure to the treatment concentrations. The body weight of each animal in the treatment and control groups was measured at the beginning of the experiment and once weekly during the duration of exposure using Acculab® USA, Model-vic-303 electronic analytical weighing balance.

Haematological analysis

At the end of exposure period, rats were fasted overnight and blood collected from the orbital plexus using heparinise micro-haematocrit capillary tubes into heparinised bottles. The recommended haemogram for the complete blood count includes: RBC count, haemoglobin content (Hb), packed cell volume(PCV), mean corpuscle haemoglobin concentration (MCHC), mean corpuscle volume (MCV), mean corpuscle haemoglobin (MCH), platelets, total white blood cell count (WBC) and differentials [lymphocytes, heterophils and mixed cell count (MXD; monocytes, eosinophils and basophils).

Organo-somatic indices

The weight of the liver and kidney were taken and calculated by the equation below. Organo-somatic index is one of the parameters used in ecological studies to monitor response of living tissues to changes in the environment. Organo-somatic indices such as hepato-somatic index (HSI), and reno-somatic index (RSI) is calculated as follows:

Organosomatic Index = Organ weight (g)/rat carcass weight (g) ×100.

Serum biochemical analysis

The blood collected was also used for serum biochemical analysis. The blood was allowed to clot and then centrifuged at 3000rpm for 10minutes in order to separate the serum which was stored under a cold temperature prior to the biochemical analysis (Alimba et al., 2012). Serum collection was done in order to check the serum biomarkers which indicate that the animals have been exposed to some kind of environmental stressors. An increase in the enzymes, Aspartate aminotransferase (AST) and Serum glutamate pyruvate transaminase (SGPT), also known as Alanine aminotransferase (ALT) is an indicator liver stress, and this was determined according to Reitman and Frankel, 1957. Blood urea was also determined according to Weatherburn (1967) while serum creatinine was measured as described by Henry et al. (1974) with slight modifications.

Heavy metal analysis in organs and blood

Heavy metals were analyzed in the liver and the blood using the digestion method according to AOAC (2004), the analysis of heavy metals in these organs was done to check for bioaccumulation of heavy metals from exposure to leachates.

Histology

After the experiment, organs (liver, kidney and brain) from euthanized rats were processed for histologic examination. They were fixed in 10% buffered formalin before trimming and sectioning for histology. Briefly formalinised samples were dehydrated in graded alcohol concentrations, cleared in xylene grades, and embedded in molten wax. On solidifying, sections of 5µm thick was made with microtome (model), floated in water bath and incubated at 60oc for 30 minutes. The sectioned tissue were cleared in grades of xylene and dehydrated in graded alcohol concentrations, sections were stained with routine Hematoxylin and Eosine stains. Examination was done using light microscope while photomicrograph was taken with Toupview® camera and software.

Statistical analysis

The heavy metal concentration in the blood and liver, haematological counts and the organo-somatic indices resulting from the experiment were subjected to one-way analysis of variance (ANOVA) using SPSS (Statistical Package for Social Sciences version 17.0). Duncan multiple range test was also used to compare differences among individual means.

RESULTS

The heavy metals concentrations of leachates sample from Awotan dumpsite (Table I) reveals an excess up to 205% for lead, (USEPA), 59% for cadmium, 17% for chromium while copper was within range. Various clinical signs of toxicity observed were presented in table II below with more rats showing signs in the 10% & 25% groups than the lower concentration or the control which presented with no clinical signs. Anorexia was observed at varying days during the procedure in the leachates groups but could not be quantified individually because the animals were jointly fed in groups. This however was absent in the control group. The hepato-somatic index and reno-somatic index (table III) showed a significantly decreasing ratio with increasing concentrations of the leachates indicative of progressive organ damage with increased exposure. The heavy metal residue in the liver and the blood (table IV) also increased with increasing leachates concentrations. The latter may be due to increasing toxicity to organs such as liver and kidneys hence decreased capacity for clearance. Red cell values presented a gradual decline with lower leachates concentrations (1%, 2.5%) although within lower boundary for the reference interval in rats. However in 25% group, significant macrocytic hypochromic anaemia was observed. Results also (table VI) showed a progressive concentration dependent leucopenia most significant in the 25% group. More specifically, concentration dependent lymphopenia and thrombocytopenia were observed. However, as the leachates concentration increased, significant heterophilia was observed across the groups most significant in the 10% and 25% groups. Hence, the Heterocyte Lymphocyte ratio, an index of toxicity was observed to increase in a concentration dependent manner most significant in the 25% group as a result of increased heterophilia. Aspartate Platelet ratio interval (APRI), an index often explored in human as an indicator for assessing degree of liver damage more specifically liver fibrosis is used in this study to describe the extent of fibrotic damage induced by toxicity of the heavy metal containing leachates. APRI ratio when compared to the control group showed a significant increase as leachates concentration increased most significantly different from the control in 5%. 10% and 25% groups respectively. This showed the extent of increasing liver damage in these groups significantly different from each other as opposed to what was observed in the control group. Reduced total protein was observed (table VII) concentration dependently across the experimental groups. This is as a result of decreased albumin concentration as leachates contration decrease. This is another indicator of liver amage as the protein is chiefly produced by the liver.

Histopathology

Kidneys

The histopathology of the kidneys groups exposed to graded doses of leachates concentration shows similar presentation with no clearly distinct concentration dependent severity in lesions. Group A which was the control showed no visible lesion (Plate A, Fig. A). Group B, exposed to 1% leachates concentration presented with renal tubular casts and interstitial congestion & haemorrhages (Plate A, Fig. B). Group C, exposed to 2.5% showed tubular epithelia and glomerular mesangial cellular degeneration and tubular cast formation (Plate A, Fig. C). Group D, exposed to 5% leachates concentration showed severe renal interstitial congestion and haemorrhages with tubular epithelia damage (Plate A, Fig. D). Group E, exposed to 10% leachates concentration showed severe renal tubular epithelia degeneration and tubular cast formation (Plate A, Fig. E) while Group F exposed to 25% leachates concentration also showed renal tubular epithelia degeneration and tubular cast formation (Plate A, Fig. F).

Liver

The liver histopathology revealed a mild concentration dependent damage which relates to the graded dosages of leachates administered to the groups. Group A which was the control showed no visible lesion (Plate B, Fig A). Group B exposed to 1% concentration showed sinusoidal widening and congestion (Plate B, Fig B), group C, exposed to 2.5% concentration showed severe vacuolar degeneration, kupffer cell hyperplasia and sinusoidal congestion (Plate B, Fig C). Group D, exposed to 5% concentration presented with sinusoidal congestion, hepatocellular necrosis, kupffer cell hyperplasia and some polymorphonuclear cell infiltration (Plate B, Fig D), group E exposed to 10% concentration presented with severe sinusoidal congestion, hepatocellular necrosis and kupffer cell hyperplasia (Plate B, Fig E), while Group F gavage with 25% concentration showed hepatocellular necrosis and kupffer cell hyperplasia (Plate B, Fig F).

Central nervous system

There were no observable lesions at histologic sections of cerebrum within the exposed groups compared to the control.

Table I: Heavy metal concentration in leachate sample.

USEPA: United States Environmental Protection Agency.

Table II: Clinical signs of toxicity

Fig 2: Diarrhea in rat exposed to 10% conc.

Table III: Organo-somatic indices

Superscripts a, b, c, d and e bearing the same alphabet along the same column are not significantly different.

Table IV: Heavy metal concentration residue in the liver and blood.

Superscripts a, b, c, d and e bearing the same alphabet along the same column are not significantly different.

Table V: Red Blood Cell Parameters

Superscripts a, b, c, d and e bearing the same alphabet along the same column are not significantly different.PCV- Packed Cell Volume, Hb-Haemoglobin, RBC- Red blood cell, MCHC- Mean corpuscle haemoglobin concentration, MCH- Mean corpuscle haemoglobin, MCV- Mean corpuscle volume.

Table VI: White Blood Cell counts

Superscripts a, b, c, d and e bearing the same alphabet along the same column are not significantly different. W.B.C- White blood cells, LYM- Lymphocytes, HET- Heterophils, APRI-Aspartate (ASP)-Platelet ratio interval, HLR- Heterocyte-Lymphocyte ratio.

Table VII: Total Protein

Superscripts a, b, c, d and e bearing the same alphabet along the same column are not significantly different.

Plate A: Photomicrograph of the Kidney.

Plate A. Fig A-F (Mag:X100) represents photomicrograph of Kidney of groups A-F fed orally with0%, 1%, 2.5%, 5%, 10% & 25% leachate concentration respectively.A: Control showing no visible lesion, B: Renal tubular casts (black arrows) and interstitial congestion & haemorrhages (white arrows), C: Tubular epithelia degeneration (black arrows) and congestion (white arrow), D: Severe renal interstitial congestion and haemorrhages (white arrows) with tubular epithelia damage (black arrows), E: Severe renal tubular epithelia degeneration (black arrows), F: Renal tubular epithelia degeneration (black arrows).

Plate B: Photomicrograph of the Liver

Plate B, Fig A-F (Mag:X100) represents photomicrograph of Liver of groups A-F fed orally with 0%, 1%, 2.5%, 5%, 10% & 25% leachate concentration respectively. .A: Control showing no visible lesion. B: Mild thinning of hepatic crds and congestion (white arrows), C: Kupffer cell hyperplasia (circle), severe vacuolar degeneration (Black arrows) and sinusoidal congestion (white arrows). D: kupffer cell hyperplasia, Hepatocellular necrosis and hepatocyte thinning (black arrows). E: Hepatocellular necrosis and thinning of hepatic cords (black arrows), sinusoidal congestion (white arrows). F: Hepatocellular necrosis and thinning of hepatic cords (black arrows).

DISCUSSION

Dumpsites are a major source of various environmental pollutants and health hazards, hence a great threat to public health if not properly managed. The various physic-chemical parameters analysed were all higher than the acceptable limits which could be as a result of the various waste materials at the dumpsite such as disposable electronic wastes which contributes greatly to the elevated concentration of the heavy metals observed. As compared to acceptable limits, some of the heavy metal concentrations were found to be over 250 times beyond recommended limits (NESREA, 2009). Therefore, infiltrates from these leachates into surrounding ground water the residents utilize would be extremely dangerous and presents a possible source of metallic poisoning. Also, at risk are plants and animals around the site. Reena et al 2011 listed the mechanisms proposed to be involved in metal accumulation in plants to be phtyoaccumulation, phytoextraction, phytoevaluation, phytodegradation and phytostabilization. Heavy metals accumulation in plants beyond acceptable limits hinders biological processes leading to oxidative stress in plants, subsequent bioaccumulation along the trophic in animals and human that consumes them. However, human populations are the most at risk because they occupy the topmost position on the food pyramid being the dead end host, thus concentrating heavy metals from all sources. These sources include air, water, plant sources and possible chemical zoonoses from animals (meat) source. (Jagun, 2015 PhD thesis)

During the exposure, various clinical signs were observed even as early as the third day of the exposure. Various signs of toxicity observed were alopecia, anorexia, muscle stiffness, nose bleeding, frequent sneezing and diarrhea. Heavy metals have being reported to interfere with the homeostatic regulatory mechanisms in different organs such as heart, brain, kidneys, bone and liver. They also effect the deleterious actions by displacing vital nutritional minerals from their cascade thereby hindering biological function (Reena et al., 2011). More specifically, heavy metals such as Mercury, Arsenic,Copper, Nickel, Chromium and Lead could interfere with vital process like nerve transmission, renal function, cellular division and blood clotting cascade to effect the observed changes seen in subjects of toxicity. These associated signs together with good history of occupational or environmental exposure as in the case of residents of Awotan landfill area could assist clinicians in faster tentative diagnosis of heavy metal toxicity in humans. These were corroborated in the report of (Oyeleye (2015) [unpublished] who stated various symptoms such as tiredness, muscle weakness, nasal irritation and slight abdominal pain observed in the residents around the dumpsite.

Organosomatic indices which can be described as the ratios of organs to the body weight (Ronald and Bruce, 1990), measured organ in relation to the body mass can be directly linked to the toxic effects of chemical on target organ. Organosomatic indices reflect the status of organ systems, which may change in size due to change in environmental factors or even exposure to environmental stressors such as leachates. The significant decrease (p<0.05) in both hepato-somatic index (HSI) and the reno-somatic index (RSI) in leachates treated rats indicates relative liver and kidney enlargement with a decrease in the relative body weight gain. Barton et al., (1987) reported that decrease in HSI may reflect glycogen loss in the liver as the energy stores were utilized, and this decrease could also be linked to histological changes in the liver, including hepatocyte damage and degeneration (Gabriel et al., 2009) The significant dose dependent reduction (p<0.05) in the HSI could result from the effects of the various leachate constituents. Also, the significant concentration dependent increase (p<0.05) in the RSI could also be a result of the exposure to the leachate constituents. Olivier et al., (2005) suggested that obstructions of these organs by the leachate constituents, likely the heavy metals, may be responsible for the change in organ weight, due to the role of these organs in breaking down and storage of these metals immediately following their entry into the body systems.

Concentration dependent significant increase (p<0.05) in the amount of heavy metal residue in the blood and the liver above the recommended levels could also be an indicator of organ damage resulting from a decreased capacity for clearance of these heavy metals from the body .

The haematological parameters of the leachate treated rats were altered compared to the control. Various researchers have used haematological parameters routinely to determine stress associated with environmental, nutritional, and/or pathological factors. The observed changes in the blood parameters of leachates exposed rats may be an adaptive response of the bone marrow or peripheral blood cells to physiological changes due to stress, and hypoxia induced by constituents of the leachates especially heavy metals. (Duthie and Tort, 1985, Alimba et al., 2012). The significant decrease in RBC indicates anaemia while the decrease in haemoglobin conconcentration indicates failure in oxygen carrying capacity of the red cells. This is not unconnected with the effects of heavy metal on red cells among which are increased fragility of the cells due to weakenend cell membrane, oxidative stress from outburst of free radicals and exhaustion of antioxidant stores from exposure to leachates, increased intravascular and extravascular hemolysis, bleeding disorders from disturbed clotting cascade, resultant splenomegaly from upgraded function of reticuloendothelial system to mention but a few. (Sharma et al., 2007, Mahour and Saxena, 2009, Alimba and Bakare, 2012).

MCHC is a measure of the concentration of hemoglobin in a given volume of packed red blood cells. The significant dose dependent decrease (P<0.05) in the MCHC suggest that the heamoglobin concentration in a given volume of packed red blood cells is lower than normal as well as poor haemoglobin carrying capacity of the erythrocytes (Eaton and Klaassen, 1998) thus; signifies hypochromic anemia. MCV is the average volume of red blood cell, the significant dose-dependent increase (p<0.05) in the MCV means that the volume of the red blood cells are increasing, and are becoming larger than normal, thus signifies macrocytic anemia. These effects are sequel effects of rapid extravascular red cell destruction such in the RES, loss of iron from such extravascular damage and rapid turnover rate of red cell production form the bone marrow which shortens erythropoetic process hence churns out macrocytic cells or immature red cells. This was corroborated in the reports of Sanchez-Chardi et al., (2008) where bioaccumulation of heavy metals (Pb, Cu and Cr) from landfills significantly correlated with altered haematological parameters in small mammals (Shrew). Also, Lead (Pb) has being known to target heme synthetic pathway, inhibiting heme and haemoglobin synthesis (Flora et al., 2008); which also precipitate a shortfall in heme concentration per red cell.

Significant thrombocytopenia could be attributed to decreased production of thrombopoietin by the liver and kidneys which could be resulting from progressive damage to these organs by leachates constituents. This thrombocytopenia could be responsible for the epistaxis observed in rats exposed to 10% and 25% concentration.

The potential for chemicals to cause damage to the immune system is of considerable public health significance and alterations to immune functions may lead to incidence of disorders, increased susceptibility to infectious diseases or neoplasia (Germolec, 2004). White blood cells are components of blood cells are useful in signaling clinically relevant haematologic changes that may result in clinical identifiable autoimmune disorders and various forms of leukemia (Lee et al., 1999). Hence, significant concentration dependent leucopenia observed indicates possible risk of immune suppression and could hence predispose to increased susceptibility to previous non-susceptible pathogens especially body microbial flora previous put in check by these cells. This could specifically be responsible for dose dependent increase in heterophilia observed while damage effects on lymphoid tissues could account for lymphopenia observed. This was validateby the reports Efuntoye et al., 2011 where he reported the presence of faecal coliforms in leachates from a landfill site in Ibadan which have been implicated in gastroenteritis. The production of enterotoxin by these diarrhea-causing bacteria may be responsible for the elevated neutrophils due to their phagocytic activities against bacterial infections in the damaged tissue. This is also responsible for the concentration dependent elevation in Heterocyte Lymphocyte ratio observed across treatment groups.

The Aspartate Platelet ratio interval (APRI), is an index for the assessment of degree of liver damage more specifically liver fibrosis in human related studies. Scanty data or none are available about its use in animal models hence absence of standardized reference intervals. However, comparing the APRI values of experimental groups with the control shows a statistically significant concentration dependent increase. This indicates an increase in Aspartate enzyme concentration, an indicator of early and progressive liver dysfunction as opposed to platelet counts. The latter could stem from reduced thrombopoetin production, a trigger for thrombocyte production and differentiation (Kaushansky 2006) as a result of progressive liver and kidney damage.

Concurrently, concentration dependent decrease in total protein count observed as a result of similar dependent decrease in Albumin concentration affirms the progressive degree of damage the liver in these groups are undergoing at different levels of exposure. Albumin, produced from the liver, is an important large molecular weight protein responsible for oncotic pressure of blood plasma in vessels. Any drop in required concentration could lead to derangement in homeostatic regulation of fluid between the intravascular and extravascular compartments resulting on fluid accumulation in tissue spaces. i.e edema.

Histologically, the effect of heavy metal exposed to graded concentration of the leachates showed more damaging effect with increasing heavy metal concentration on the liver than the kidneys. This might not be unconnected to the role of metabolism and detoxification the liver plays as it becomes the first parenchymatous organ to be exposed to these toxic concentrations after absorption from the intestine.

The Kidneys, being the clearance organ did not show concentration dependent damage but the results implicated that at concentration doses beyond the tolerable limits, glomerular epithelia cells could be damaged leading to seepage of large molecular weight components of blood such as protein which could be responsible for the cast observed formed in tubules. Also, congestion observed might not be unconnected with vascular endothelial damage caused by heavy metal toxicity.

Absence of observable lesions in sections of CNS might be due to sub-acute duration of exposure insufficient enough to precipitate observable histologic changes.

In conclusion, this study shows that leachate from Awotan dumpsite contains very high heavy metal concentrations when compared to the reference limits causing severe organopathic effects at different concentrations of exposure. This mirrors the risk residents of such areas as Awotan dumpsite in Ibadan, Nigeria are exposed to as leachates containing these heavy metal toxicants seeps into ground water, taken up and bioaccumulates in plant and animal tissues ultimately posing a possible chemical Zoonotic risk to humans feeding on meat from such animals and plant vegetables. Thus, an exposure to varying concentrations of these toxicants is possible from afore mentioned multiple sources. Hence, proper municipal waste management program away from expanding residential areas should be provided or waste recycling programs encouraged while further studies are recommended to juxtapose the frequency clinic visits and severity of complaints residents of such areas in concert with the concentration of heavy metal residue in blood and their nutritional lifestyle. Also nervous development or evaluation of brain function could be done as prolonged elevated heavy metal concentration in blood has being implicated to impair brain development in children (Jagun, 2015 PhD thesis). Although this could not be validated from the results of this study.

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