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Municipal Landfill Leachate Characterization and its. Induction of Glycogen Vacuolation in the Liver of. Clarias gariepinus. Adeolu O. Aderemi#1, Gbenga A.
International Journal of Environmental Protection

IJEP

Municipal Landfill Leachate Characterization and its Induction of Glycogen Vacuolation in the Liver of Clarias gariepinus Adeolu O. Aderemi#1 , Gbenga A. Adewumi*2 , Adebayo A. Otitoloju#3 #

Ecotoxicology Laboratory, Depart ment of Zoology, Facu lty of Science, University of Lagos, Akoka, Lagos *Department of Botany and Microbiology, Facu lty of Science, Un iversity of Lagos, Akoka, Lagos 1

2

[email protected] [email protected] 3 [email protected]

Abstract-In an effort to characterize leachate and evaluate its toxicity, physico-chemical and microbiological parameters were analyzed in leachate obtained from an unlined municipal solid waste landfill. Clarias gariepinus were exposed to it and its LC 50 and histopathological effects on the liver were determined. Heavy metals, Cd (8.8mg/l), Pb (10.2mg/l), Zn (9.0mg/l), and Fe (6.5mg/l) were observed in the leachate. A high population of Enterobacteriaceae (1.26 x 105 ± 37264 CFU/ml) was also detected. Behavioural responses in the form of erratic swimming and uncoordinated opercula movements as well as mortality were observed in the exposed fishes. The 96hr median lethal concentration LC 50 of the test leachate was 2.353%. Histopathological lesions in form of glycogen vacuolation were observed in the liver of fish exposed to sublethal concentrations of 0.19%, 0.39% and 0.78% of the test leachate. These observations are of prime health concern because there is no containment system for the leachate generated from the study site. The LC50 value obtained from the acute toxicity study indicates that the leachate is toxic and could be of assistance in the assessment of the hazardous effects of landfill leachate discharged into the environment. The observation of glycogen vacuolation emphasizes its usefulness as a histopathological biomarker of response to landfill leachate exposure. This is important for the monitoring of the environmental safety of landfills in a bid to protect wildlife, human health and the environment.

The physical and chemical characterizations of solid waste leachates have been reported widely in Literature [3]; [4]; [5]. Previous studies have also shown that leachates from M SW landfills are very similar in co mparison to those from hazardous waste landfills [1]; [6]; [7]. Municipal landfill leachate are highly concentrated comp lex effluents which contain dissolved organic matters; inorganic compounds such as ammoniu m, calciu m, magnesium, sodiu m, potassium, iron, sulphates, chlorides and heavy metals such as cadmiu m, chromiu m, copper, lead, zinc, nickel; and xenobiotic organic substances [8]; [9].

Keywords-Leachate; Landfill; Histopathology; Acute Toxicity; Biomarkers; Glycogen Vacuolation; Clarias Gariepinus

This leachate contamination of surface waters and groundwater may pose serious health risks to aquatic organisms as well as hu mans and farm an imals that drink these natural resources. The realization of the polluting and potential public effects of MSW leachate has led to a number of studies on the toxicity of leachates on bacteria [11], p lants [12]; [13], Drosophila melanogaster [14], Daphnia and fishes [15]; [16], mice [17], rats [18], and human cells [19].

I. INT RODUCTION One of the major environ mental issues in Nigeria is the improper management of solid waste. There is an increase in household waste generation and this is largely attributed to the rapid urbanization and population growth. In Nigeria like most developing nations, municipal solid wastes (MSW) are commonly d isposed of in unlined landfills or open dumps, many of which are located in public places surrounded by residential quarters. These MSW landfills hold discarded products many of wh ich are manufactured fro m to xic materials. These products create very similar hazards for the environment, wildlife and humans as hazardous wastes after they have been buried in the ground [1]. Wastes placed in landfills are subject to either groundwater underflow or infiltrat ion fro m precipitation and as water percolates through the waste, it picks up a variety of inorganic and organic compounds, flowing out of the wastes to accumulate at the bottom of the landfill. The resulting contaminated water is termed ‘leachate’ and can percolate through the soil [2].

The chemical co mpounds in leachate may leach or percolate via the soil into groundwater for a long time and pose serious risks to ecosystems and human health if the chemicals migrate to surface waters or drinking water wells. Such risks are very high when the landfill lacks an impermeab le liner and leachate collection system allowing the direct flow of leachate into groundwater. Un lined landfills have been reported to release large amounts of hazardous and deleterious chemicals in leachate to nearby groundwater [10]; [9]. Landfill leachate is considered one of the most potentially significant sources of groundwater pollution for waters that could be used for domestic water supply purposes [8].

The use of fish as a test organism in ecotoxicological studies is essential because of their link to the human in the food chain. The fish, as a bio indicator species, plays an increasing important role in the monitoring of water pollution because it responds with great sensitivity to the changes in the aquatic environment. The sudden death of fish also indicates heavy pollution and the effects of exposure to sublethal levels of pollutants can be measured in terms of biochemical, physiological or histopathological responses of the fish organism [20]. The induction of glycogen vacuolation in the liver cells as histological biomarkers of response to contaminant exposure in fish has been widely reported in literature [21]; [22]; [16].

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International Journal of Environmental Protection

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This study was undertaken to determine the physicochemical and microbial characteristics of leachate and to evaluate its toxicity by using a tropical catfish commonly eaten in Nigeria. In order to achieve this, Clarias gariep inus was exposed to leachate obtained fro m an unlined municipal solid waste landfill in Igando new town in the Lagos metropolis; and its LC50 and histopathological effects on the liver were investigated.

conductivity (EC), total dissolved solids (TDS), chemical oxygen demand (COD), total hardness (TH), sodiu m (Na+), sulphate (SO4 2-), nitrate (NO3 -), ammon iu m (NH4 +), iron (Fe), zinc (Zn), cadmiu m (Cd), and lead (Pb). The concentrations of heavy metals were determined by using an atomic absorption spectrophotometer. D. Microbial Enumeration Serial dilutions were carried out on the leachate sample collected. Appropriate dilutions were placed on MacCon key agar (Oxoid CM 3 Basingstoke, Hampshire, Eng land, UK) by using the spread plate technique [32], for the enumeration of Enterobacteriaceae. After the period of incubation the total viable count was expressed as the means of three determinants.

II. MATERIALS AND METHODS A. Study Site The study site is Soluos (II) landfill, located at the ext reme east-west area of metropolitan Lagos state (30 15. 160’E and 60 34. 300’N). The site, originally an excavation pit for laterit ic soil opened for the dumping of trash in 2008 and has been used for municipal solid waste disposal by Lagos waste management authority (LAWMA). The site spreads over an area of 7.8 hectares with wastes filling heights varying fro m 12-15 m. It receives mainly do mestic, household hazardous and commercial wastes. Due to its topographically depressed nature; in addition to rainfall infiltration, the site is prone to surface water runoff fro m surrounding areas and groundwater infiltrat ion fro m the sides and bottom. Th is results in the generation of a large volu me of leachate at the fill thus, increasing its potential to contaminate the environment. The site is bordered by residential houses and is not equipped with a leachate collection/removal system; thus, the leachate produced is freely discharged into the environment.

E. Toxicity Testing of Leachate by Using Clarias Gariepinus 1) Collection of Test Organisms, Laboratory Cultures and Acclimatization: Fingerlings of Clarias gariepinus obtained commercially (Yo mola fish farm in Ejigbo, Lagos) were utilized in this study. The fish is available, inexpensive and important in aquaculture in Nigeria. The care and use of these fishes was in accordance with international guidelines on the use of fishes for research [24]. They were acclimatized and maintained in plastic tank (10 L capacity) for 14 days, during which they were fed with co mmercial fish pellet until average weight of 2 ± 0.08 g was reached. The dechlorinated tap water was kept oxygen saturated with aeration. Mortality during the period of acclimat ization was less than 1% and each experiment was carried out in duplicate.

B. Sampling of Leachate Leachate samples collected in April, 2009 fro m the bottom of the landfill (Fig. 1) were mixed thoroughly and transferred to the laboratory in 500ml clean p lastic bottles, filtered to remove debris and stored at 4o C for analysis the same day. This was considered the stock solution. The sample for microbial analysis was aseptically taken in 50ml sterile universal containers.

F. Acute Toxicity Study Serial dilutions were prepared fro m the stock solution of leachate in accordance with standard procedures for short term static bioassay [25]. The fish were exposed to definitive concentrations of 1, 2, 3, and 5% of the test leachate and a control group (only dechlorinated water). The definit ive concentrations were selected after a concentration range finding experiment (data not shown). Ten fishes per test tank were used in this assay with 2 rep licates for each concentration and the control. The set up was left for 96 hr while the tested fishes were observed for behavioural/physiological changes and mortality count was carried out once every 24 hr for 96 hr. G. Assessment of Quantal Response (Mortality) A fish was considered dead when there was a lack of opercular movement when prodded with a glass probe [26].

Fig. 1 Leachate collects at the base of waste heap on Soluos landfill, 2009

C. Physico-chemical Analysis of Leachate The leachate samp le was analy zed for relevant physicochemical parameters according to internationally accepted procedures and standard methods [23]. The parameters analyzed in the leachate sample include pH, electrical

H. Histopathological Study A similar experiment as described above was carried out and Clarias gariepinus was exposed to sublethal concentrations of the leachate. Each treatment including control was replicated and 5 fishes (average weight 4.46 ± 0.40 g) were stocked in each group and were fed at 5% body weight once daily. Sublethal concentrations of leachate were extrapolated fro m the 96hr LC50 as fractions given by 1/3rd of 96hrLC50 , 1/6th of 96h rLC50 and 1/12th of 96hrLC50 . The concentrations used for the experiment are 0.19%, 0.39%, 0.78% of test leachate and control (only dechlorinated water). Each concentration and the control were renewed every 48 hr for 4 weeks to maintain a continuous exposure. On day 28 of the assays, 2 live fishes per concentration including control were randomly selected and the liver and g ills were surgically removed and fixed in Bouin’s fluid. The fixed samples were

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transferred to phosphate buffer (p H 6.8) after 7 hours of fixation in Bouin’s flu id. The tissues were then dehydrated in graded alcohol, cleared in xy lene before embedding in paraffin wax (melt ing point 56.0ºC). Serial sections of 2-5 u m thickness were cut in rotary microtome and then were passed through xylene fo llo wed by absolute alcohol and water. The sections were stained with Haemato xy lin and Eosin, dehydrated in graded alcohol, cleared in mo re xy lene and mounted in Canada balsam. The slides were left to dry on the hot plate for 2 hours before being observed under the microscope. I. Statistical Analysis The result of the microbial count was presented as mean ± SE. To xicological dose-response data involving quantal response (mortality) were analyzed by probit analysis [27], based on a computer programme written by Ge Le Pattourel, Imperial College, London, as adopted by Don-Pedro [28]. The index of to xicity measurement derived fro m this analysis is: LC50 - Lethal concentration that causes 50% responses (mortality) in exposed organisms. III. RESULT S A. Physico-chemical and Microbial Characteristics of Leachate The physico-chemical and microb ial characteristics of the leachate sample collected fro m the landfill as well as the drinking water standards recommended by WHO and FEPA are presented in Table 1. The pH of the leachate was alkaline (8.1) and very high concentrations of EC (124000µS/cm), TDS (62000mg/l) and Na+ (7863mg/l) were detected. COD (160mg/ L), SO4 2- (1.21mg/ L), NO3 - (1.2mg/L), NH4 + (4.5mg/ L) as well as high level of heavy metals (Fe (6.5mg/ L), Pb (10.2mg/ L), Cd (8.8mg/ L) and Zn (9.0mg/ L)) were likewise detected. The total v iable count of Enterobacteriaceae in the leachate was 1.26 x 105 ± 37264 CFU/ ml.

There was 20%, 40%, 60% and 80% mortality at the 1%, 2%, 3% and 5% concentrations of the leachate respectively. The 96hr LC50 of the leachate obtained by using the probit method is 2.353%. C. Histopathological Study The results of the histopathological effects of test leachate on the liver of Clarias gariepinus after 28 days of exposure are shown in Fig. 2(a-d). The histopathological investigations of the liver of the control fish showed no visible lesions after 28 days (Fig. 2a). However some degrees of histological alterations were observed in the liver of the exposed fishes. There was a prevalence of glycogen vacuolation in the liver of the fishes exposed to sublethal concentrations of the test leachate which was concentration dependent (Fig. 2b to d). At 28 days of exposure, focally extensive areas of fatty infiltrat ion and diffuse glycogen vacuolation were observed in the liver of fishes exposed to 0.19% of test leachate (Fig. 2b). A generalized glycogen vacuolation with the whole parenchyma affected was recorded in the fishes exposed to 0.39% of test leachate (Fig. 2c). A d iffuse and extensive glycogenic vacuolation was observed in the liver o f fishes exposed to the highest concentration of the test leachate (Fig. 2d).

( a)

( b)

TABLE I PHYSICO -CHEMICAL AND MICROBIAL CHARACTERISTICS OF LEACHATE AND DRINKING WATER STANDARDS

*Parameter pH TH TDS EC T CC SO4 2NO3 Na+ NH4 + Zn Fe Pb Cd

Leachate 8.1 9300 62000 124000 (100ml) 1.21 1.20 7863 4.5 9.0 6.5 10.2 8.8

FEPAa 6-9 2000 1.26 20 0.01 5.0 0.05 0.01 0.05

WHOb 6.5-9.2 300 500 300 c x 200 10 200 1.5 5.0 0.3 0.05 0.01

( c)

( d)

Fig. 2 a) Section of liver of fish showing no visible lesion; b) Section of liver of fish exposed to 0.19% test leachate showing focally extensive areas of fatty infiltration (double arrows) and diffuse glycogen vacuolation (single arrow); c) Section of liver of fish exposed to 0.39% test leachate showing generalized glycogen vacuolation with whole parenchyma affected; d) Section of liver of fish exposed to 0.78% test leachate showing diffuse and extensive glycogen vacuolation (arrowed).

*All values in mg/L, except pH, EC (µS/cm) and T CC-Total coliform count (CFU/ml); a [33], b [34], c [35]

B. Acute Toxicity of Leachate Against Clarias Gariepinus After the test leachate was admin istered into the test chambers, behavioural responses like rap id and erratic swimming, uncoordinated and rapid opercula movements were observed. These observations were more pronounced with increasing concentrations and mortality was directly proportional to the concentration of the test leachate. In the LC50 study, no death was recorded after 96 hr in the control.

IV. DISCUSSION The low COD and the alkaline pH observed in the leachate can be attributed to the methane fermentation phase of the landfill. In this phase, alkaline pH supports the growth of methanogens which converts much of the organic contaminants in leachate to methane gas [29]. An environmental consequence of this phase is the landfill fires that occur frequently on the landfill site in the dry season due to spontaneous combustion. The high level of inorganic contamination in the leachate as shown by the high concentrations of TDS, EC, and Na+ observed agrees with the

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report of Mor et al. [2]. The observation of a low level of NO3 - is likely to be a result of the occurrence of anaerobic processes such as nitrate reduction and denitrification in which most of the nitrate are converted to ammon iu m [30]. The presence of high levels of heavy metals in the leachate suggests their origin could be fro m the various wastes dumped in the landfill. The p resence of high concentration of Fe in the leachate indicates that Fe scraps are likely to be dumped in the landfill. Elevated levels of Pb in leachate had also been observed by Moturi et al. [31] and this may be attributed largely to the disposal of batteries, leadbased paints and lead pipes found at the site. The high value of Zn may be attributed to the presence of fluorescent tubes, batteries, and a variety of food wastes as well as the burning tyres at the site. The discarding of dry cell batteries and paint cans are the possible sources of cadmiu m. The Enterobacteriaceae is the total coliform and indicators of water quality. They are normal flora of the intestinal tract of many an imals and their presence in the leachate is likely to be influenced by wastes from the nearby abattoir and poultry farm as well as the waste pickers who defaecate at the site. The low LC50 and histopathological changes observed in the toxicity studies indicate that the leachate is to xic; producing dose-responsive increases in mortality as well as abnormalities in the liver of exposed Clarias gariep inus. The contaminants or constituents in the leachate are believed to have induced the observed effects or mortality. This agrees with earlier reports on the toxicity of leachate on fishes [15]; [16]. The glycogen vacuolation of the liver cells in the exposed fish can be attributed to the presence of heavy metals such as Pb, Zn, and Cd in the leachate and has been reported in previous studies [16]. V. CONCLUSION The findings fro m this study are of prime health concern as there is no collection system for the leachate generated fro m the study site, thus increasing its potential to contaminate the surrounding aquifer. The results obtained fro m the acute toxicity study show that the leachate is toxic and could be used in the risk assessment of leachate fro m landfills. This study also emphasizes that glycogen vacuolation of the liver cells are the useful b io markers of contaminant effects in fish and should be taken into account when evaluating histopathological bio markers of response to landfill leachate exposure. This is important for the monitoring of the environ mental safety of landfill sites in a bid to protect wildlife, human health and the environ ment. ACKNOWLEDGMENT

The authors are grateful to the officials of the Lagos Waste Management Authority (LAWMA) and Mr Bayo Adeyeye, a colleague in the Depart ment of Zoology for their assistance during the course of this study

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