Heavy Metal and Microbial Contamination of Top Soil and Water around Wadata Abattoir, Makurdi Metropolis

Odo Joel Inya^2* , Ebah Esther Eneyi¹ , Joseph Yitmwa Gontur¹ , Ode Omenka Samuel³

¹Department of Microbiology, University of Agriculture, Makurdi, Nigeria

²Department of Fisheries and Aquaculture University of Agriculture, Makurdi, Nigeria

³Department of Physics, Benue State University, Makurdi, Nigeria

Corresponding Author Email: odojoel@gmail.com

DOI : http://dx.doi.org/10.46890/SL.2022.v03i05.003

Abstract

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Introduction

Environmental pollution by heavy metals which adversely affect soil quality and pose a threat for human health requires a rapid and comprehensive solution. Numerous anthropogenic sources of pol­lutants can contaminate the soil and water environ­ment, including inputs from waste waters flowing from mines and waste storage, runoff of pesticides from agricultural land or atmospheric deposition (Song et al., 2010). Increasing industrialization has been accompanied throughout the world by the extraction and distribution of mineral substances from their natural deposits. The spread of heavy metals in the terrestrial environment is largely attributed to the disposal of waste products, mining activity and wastewater irrigation (Jillian et al., 2015). Heavy metal pollution is becoming an increasingly serious environmental problem for soils. For example, the cadmium (Cd) content can reach 446 mg kg^–1 in Japanese paddy soils (Herawati et al., 2000), and 10,000 hectares of farmland are polluted and cannot be used for food production in Germany (Lewandowski et al., 2006). Rice tends to accumulate Cd and chromium (Cr) in its grains, the consumption of which can have toxic effects on living organisms. Therefore, the Cd content in rice is of great concern (Nagajyoti et al., 2010; Soderland et al., 2010). To date, investigations of the effects of Cd on rice plant ecosystems have mainly focused on the relationship between Cd pollution in paddy soils and Cd contents in rice. Microbial communities play important roles in soil because of the many functions they perform in nutrient cycling, plant symbiosis, and the detoxification of noxious chemicals used to control plant pests and plant growth (Nannipieri et al., 2003). High heavy metal levels in soil are a problem due to their toxicity to soil microorganisms and impairment of ecosystem functions. The short, medium, and long-term responses of microbial communities to heavy metal contamination have been investigated (Ranjard et al., 2000; Sandaa et al., 2001; Shi et al. 2002; Renella et al., 2004). Heavy metals generally reduce the amount of soil microbial biomass (Brookes and McGrath 1984; Chander et al.,1995), leading to a decrease in functional diversity (Kandeler et al.,1996) and changes in microbial community structure (Frostegård et al., 1993; Gadd 1993; Elsgaard et al., 2001; Sandaa et al., 2001; Akmal et al., 2005). However, metal exposure may also lead to the development of metal-tolerant microbial populations (Ellis et al., 2003). Due to their relationship to soil functionality, soil microbial populations and activity can serve as useful indicators of soil improvement and soil degradation (Ebah et al., 2022; Pankhurst et al., 1995; Dick et al., 1996; Shi et al., 2002; Zhou 2003; Verma and Singh 2005; Ivshina et al., 2014). Many studies have focused on the effects of heavy metals on root exudations, soil microbial activity, and soil enzyme activity and so on. However, little information is currently available about how heavy metals regulate the entire microbial community in the soil. Rice is the most important food crop in China. Recently, safe rice production became the key attention point due to the increasing heavy metal contamination problem. The aim of this study is to investigate the heavy metals and microbial contamination of top soil and surface water around Wadata abattoir in Makurdi metropolis.

Materials and methods

3.1 Study Area

This research was carried out in Makurdi, the capital of Benue state. Nigeria, West Africa. Its geographical coordinate are 7⁰ 14’0 North, 8⁰ 32’0” East and its original name Makurdi. The area is characterized by two seasons, the dry season (October to April) and rainy season (April to October) about 45% the total population are civil servant and business people. While about 25% are famers, 30% vocational workers and students.

3.1 Materials 

The materials used in this research were; Soil samples, water samples, media [Nutrient Agar (NA), Petri dishes, wire loops, measuring cylinders, conical flask, spatula, etc.

3.2 Equipment

The following equipment were used during the course of the laboratory analysis of the samples, they include; Atomic Absorption Spectrophotometer (AAS), Microscope, Weighing balance, incubator, autoclave, refrigerator etc.

3.3 Reagents

The following reagents were used at the course of the laboratory analysis of the samples, these were; Gram reagents, urease reagents, Kovac’s reagent, oil immersion, distilled water, citrate and 90% ethanol.

3.4 Sample Collection

3.4.1 Soil Sample Collection

The soil samples were collected from and around Wadata abattoir, Makurdi metropolis. They were placed in small lather bags and were sealed tightly; they were then transported to the Research Laboratory, Department of Microbiology Federal University of Agriculture Makurdi for analysis.

3.4.2 Water sample collection

Water samples were obtained in sample containers at the abattoir sites from and around Wadata, Makurdi metropolis, each sample was directly collected into a factory-fresh 1.5 L plastic bottle, with cap securely tightened. After collection the bottles were immediately transported to the research Laboratory Federal University of Agriculture Makurdi for analysis.

3.5 Physicochemical Analysis of Soil and Water Samples

Physicochemical parameters such as temperature, pH, conductivity, turbidity, nitrate content were determined using standard instruments as described by APHA (2005)

Temperature was determined using a thermometer.

1. PH was measured using a pH meter.
2. Nitrate content was determined using nitrate meter
3. Electrical conductivity was measured using EC-meter
4. Turbidity was measured using sper turbidity meter

3.5.1 Heavy Metal Analysis in Soil

The soil were  spread on a clean plastic sheet, placed on a flat surface and were air-dried in open air in the laboratory under room conditions for 24 h. Afterwards, the soil was sieved on a 2 mm sieve and 5 g. A sample was taken from the sieved soil and was added in a beaker. 10 ml of nitric/perchloric acid, ratio 2:1 was added to the sample.

This sample was digested at 105°C. Next, HCl and distilled water, ratio 1:1 was added to the digested sample and the mixture was transferred to the digester again for 30 min. The digestate was then removed from the digester and allowed to cool to room temperature. The cooled digestate was washed into a standard volumetric flask and was made up to the mark with distilled water. Determination of the heavy metals was done in an atomic absorption spectrophotometer (AAS model 210 VGP).

3.6 Sterilization and Disinfection of Materials

Work benches were properly disinfected with sodium hypochloride. All glass wares (petri plates, test tubes, conical flasks) were washed during the bench work with detergents and rinsed with clean water and will be sterilized at 121⁰C.

3.7 Media Preparation

All media (Nutrient Agar (NA), Potato Dextrose Agar (PDA), and were prepared in accordance with the manufacturer’s specification, homogenized and thereafter sterilized by autoclaving at 121⁰C for 15 minutes. The media were then allowed to cool down to about 45⁰C before use. Streptomycin (0.1%) was added to the Potato Dextrose Agar to prevent bacterial contamination of the media.

3.7 Bacteria and Fungi Isolation

Bacteria and fungi were isolated from the soil and water samples by serial dilution and pour plate technique on NA for bacteria and PDA for fungi. The isolates were macroscopically examined for morphology and colony characteristics such as the shape, surface, elevation, pigment, edge and opacity for bacteria and growth patterns, spore and mycelia coloration, distribution of spores for fungi. The microscopic examination was done by Gram staining and then viewed under oil immersion objective (x100 magnification) to see the Color, Shape, etc. for bacteria while the fungi were examined by lacto-phenol cotton blue staining and viewed under x10 magnification, then x40 magnifications to see the hyphae, spores, spore arrangement.

3.8 Identification and Characterization of Bacteria Isolates

The identification of bacteria was based on morphological characteristics and biochemical tests that were carried out on the isolates. Morphological characteristics such as colony appearance, consistency, colony surface and pigmentation were observed after 24 hours of growth.

Characterization was done according to the method proposed   by Fawole and Oso (2004).

3.9 Gram Staining Technique

A thin smear of each of the 24hour old cultures was prepared on clean grease free slides, they were fixed by passing over flame gently. They were stained by the addition of 2 drops of crystal violet solution for 60seconds and were rinsed with water. The smears were flooded again with Lugols’ iodine for 30seconds and were rinsed with water, they were decolorized with 70% alcohol for 15seconds and were rinsed with distilled water. They were counter stained with two drops of safranin for 60seconds and finally were rinsed with water, they were then allowed to air dry. The smears were mounted on a microscope and were observed under oil immersion objective lens. Result was read according to color appearance.  Gram negative cells appeared pink/red while Gram positive organisms appeared purple (Fawole and Oso, 2004).

3.10 Catalase test

A small quantity of the 24hour old culture was transferred into a drop of 3% hydrogen peroxide solution on a clean slide with the aid of a sterile inoculating loop. Result was interpreted based on the appearance of gas seen as white froth (Cheesbrough, 2006).

3.11 Coagulase Test

A loopful of the isolates was emulsified with normal saline solution on a microscopic slide. A drop of undiluted plasma was added to the suspension and stirred for five seconds. A coagulase-positive enzyme is indicated by the clumping of colonies together (Olutiola et al., 2000)

3.12 Indole Test

Tryptone broth (5mL) was placed into different test tubes, after which a loopful of the bacterial isolates was inoculated into the test tubes, leaving one of the test tubes was not innoculated to serve as the control. The test tubes was then incubated at 37⁰C for 48 hours, after incubation, 0.5 mL of Kovacs’ reagent was added and shaken gently, it was allowed to stand for 20min to permit the reagent to rise. A red or red-violet color at the top surface of the tube indicates a positive result, while yellow coloration indicates a negative result (Cheesbrough, 2006).

3.13 Citrate Test

This test detects the ability of an organism to utilize citrate as a sole source of carbon and energy. About 2.4g citrate agar was dissolved in 100ml of distilled water. About 10ml of citrate medium was dispensed into each tube and covered, it was sterilized and allowed to cool in a slanted form. The tubes were inoculated by streaking the organisms once across the surface. A change from green to blue indicates utilization of citrate (Cheesbrough, 2006).

3.14 Urease Test

The surfaces of the urea agar slant were streaked with a portion of a well-isolated colony.  The caps were leaved on loosely and the tubes were incubated at 35-37°C in ambient air for 48 hours to 7 days. The development of pink color was examined for as long as 7 days (Olutiola et al., 2000).

 

RESULTS

4.1 Physicochemical Parameters of Surface Water and Top Soil at Wadata Abattoir

The physicochemical analysis of surface water and top soil from Wadata Abattoir was done and recorded in Table 1. The analysis shows levels and ranges of parameters such as temperature, turbidity, conductivity, nitrate content and pH.  The analysis of the soil shows that the soil sample SS had a temperature of 23^οC; the turbidity was 12 NTU with pH 9.0. The conductivity and nitrate content were 300µS/Cm and 15mg/l respectively as shown in Table 1.  The water sample WS has a turbid appearance and the temperature was 24^οC, turbidity was 13 NTU, pH was 7.6, conductivity and the nitrate content were 400 µS/Cm and 11mg/L respectively.

The colony morphologies of bacteria isolates in both the soil and water samples were obtained and recorded in Table 2, the Gram stain and biochemical test results were as recorded in Table 3. The morphologies and microscopic appearances of fungal isolates were also obtained and recorded in Table 4.

Table 1: Physicochemical Parameters of Surface Water and Top Soil at Wadata Abattoir

Samples	Appearance	Temperature (οC)	Turbidity (NTU)	pH	Conductivity (µS/cm)	Nitrate (mg/l)
SS	–	23	12	9.0	300	15
WS	Turbid	24	13	7.6	400	11
SB	–	20	16	8.5	500	14
WB	Turbid	15	14	7.7	400	13

Key:

SS:  Soil sample from slaughtering

SB: Water sample from slaughtering

WS: Soil sample from butchering

WB: Water sample from butchering

 

Table 2: Heavy Metal Analysis of Top Soil and Surface Water at Wadata Abattoir

                                                                                                              

Sample	Lead (ppm)	Copper (ppm)
SS	0.64	0.16
SB	0.65	0.17
WS	0.67	0.18
WB	0.68	0.19

Key:

SS:  Soil sample from slaughtering

SB: Water sample from slaughtering

WS: Soil sample from butchering

WB: Water sample from butchering

Table 3 Colony Morphology and Appearances of Isolates from Surface Water and Top Soil at Wadata Abattoir

SAMPLES	CFU/ml	COLOR	ELEVATION	MARGIN	FORM	Texture
SS10-2	5.2×104	Creamy	Raised	Entire	Circular	Smooth
SS10-4	4.4×106	Creamy	Raised	Entire	Circular	Smooth
WS10-2	7.24×104	Creamy	Raised	Entire	Circular	Smooth
WS10-4	5.0×106	Creamy	Undulate	Entire	Circular	Smooth
SB10-2	7.2×104	Creamy	Raised	Entire	Circular	Smooth
SB10-4	4.12×106	Creamy	Raised	Entire	Circular	Smooth
WB10-2	5.0×104	Creamy	Undulate	Entire	Circular	Smooth
WB10-4	3.0×106	Creamy	Raised	Entire	Circular	Smooth

Key:

SS:  Soil sample from slaughtering

SB: Water sample from slaughtering

WS: Soil sample from butchering

WB: Water sample from butchering

Table 4: Gram Stain and Biochemical Test of Isolates from Surface Water and Top Soil at

               Wadata Abattoir

Samples	Gram stain	Indole test	Catalase test	Citrate test	Urease test	Coagulase test	Suspected organisms
SS10-2	–	–	+	–	–	+	Klebsiella spp
SS10-4	+	–	+	–	–	+	Staphylococcus spp
WS10-2	–	–	+	–	–	+	Klebsiella spp
WS10-4	+	–	+	–	–	+	Staphylococcus spp
SB10-2	–	+	–	+	–	–	Escherichia coli
SB10-4	+	–	+	–	–	+	Staphylococcus spp
WB10-2	+	–	+	–	–	+	Staphylococcus spp
WB10-4	–	+	–	+	–	–	Escherichia coli

Key:

SS:  Soil sample from slaughtering

SB: Water sample from slaughtering

WS: Soil sample from butchering

WB: Water sample from butchering

Table 5: Microscopic and Macroscopic Appearances of Fungi Isolates from Surface Water and Top Soil from Wadata Abattoir

Samples	Microscopic Appearance	Macroscopic Appearance	Suspected Fungi
WS	Presence of septated hyphae, colourless long unbranched sporangiospore  with round radiate head of vesicle and brownish sclerotia	Dark brown powdery mycelium with dark spores on the surface and white to yellow colour on the reverse side.	Aspergillus niger
SBa	Septate hyphae, colourless and brownish conidiophores with round radiate ,  lead biseriate phialides	Yellow-green colour with a creamy edge appearance on the surface, which appears golden to reddish brown on the reverse side.	Aspergillus flavus
SBb	Septate and fruity mycelium and branched conidiophores with round radiate head vesicle and biseriate phialides	A rapid dark -green coloured growth with the edge surrounded by whitish margin on the surface and pale yellow on the reverse side.	Penicillium spp
SBc	Presence of non-septate hyphae with a visible spore and short sporangiospore , mycelium branched	Creamy colonies that covers the entire medium. Irregular shape.	Mucor spp
SSa	Presence of non septate branched mycelium with round shaped sporangia and filamentous hyphae.	Long hyphael growth cotton-candy like from the front white colonies which initially turns turns green to yellowish brown in lime the reverse is white to pale.	Rhizopus spp
SSb	Presence of septate hyphae usually yeast and aleuriconidia are oval, unicellular truncate with a broad base and round apex.	The front colour is white cream or brown and the reverse colour is yellow brown brown.	Paracoccidoides

Key:

SS= Soil sample from slaughtering

SB= Water sample from slaughtering

WS= Soil sample from butchering

WB= Water sample from butchering

Table 6: Colony Frequencies and Percentages of Bacteria Isolates in the Samples

Isolates	Frequencies	Percentages (%)
Klebsiella sp	2	25
E. coli	2	25
Staphylococcus aureus	4	50
Total	8	100

5.1 Discussion

 It is very essential and important to test the water before it is used for drinking, domestic, agricultural or industrial purpose. Water must be tested with different physic-chemical parameters, likewise soil.  This is because of the heavy metal contamination, microbial contamination and exceeding levels of physicochemical parameters (Sawane et al., 2006).  Selection of parameters for testing of water is solely depends upon for what purpose we are going to use that water and what extent we need its quality and purity. Water does contain different types of floating, dissolved, suspended and microbiological as well as bacteriological impurities (Saravanakumar et al., 2011).  Groundwater is the most important source of water supply for drinking, irrigation and industrial purposes. Increasing population and its necessities have lead to the deterioration of surface and sub surface water. The modern civilization and urbanization frequently discharging industrial effluent, domestic sewage and solid waste dump. The cause of ground water gets pollute and create health problems. Once the groundwater is contaminated, its quality cannot be restored by stopping the pollutants from the source it therefore becomes imperative to regularly monitor the quality of groundwater and to device ways and means to protect it (Aftab et al., 2005)

In this study, the analysis of the physicochemical parameters, heavy metal (lead and copper) and microbial contamination of top soil and water was both carried out on samples from abattoir sides of Wadata, Makurdi metropolis. The analysis of the physicochemical parameters carried out on the soil samples at slaughtering side shows a slightly alkaline pH, the analysis of nitrates shows a level that was slightly above normal range and a high level of turbidity, conductivity was within a normal range. However the analysis shows that the soil samples have some levels of nitrates together with water samples. The conductivity values of both the soil and water at slaughtering and butchering sides falls within normal ranges, but the appearances were both turbid for the water samples. This result is in line with the standard by APHA (2005). The heavy metal analysis of the soil and water samples shows a minute quantity of lead and copper in part per million (ppm) the values for lead in the samples were 0.64ppm for sample SS, 0.65ppm for soil sample at butchering side , 0.67ppm for water sample WS and 0.68ppm for WB respectively. These values are not significant as they fall far below normal ranges. The heavy metal analysis of copper also shows a minute quantity of copper in part per million (ppm) in the samples. The values range from 0.16ppm -0.17ppm for the soil samples while the water samples has values ranging from 0.18ppm-0.19ppm for the water samples both at slaughtering and at butchering sides. This is in agreement with the work of Kostal et al. (2004). The microbial analysis shows the presence of Klebsiella sp, Staphylococcus aureus and Escherichia coli. The colony forming units of the coliforms ranges from 3.0×10⁶ CFU/ml to 7.24×10⁴ CFU/ml for the water samples while that of the soil samples range from 4.12×10⁶  CFU/g to 7.2×10⁴ CFU/g this finding is in agreement with that of  Naseri et al. (2013).

     5.2 Conclusion

     The contamination of soil and water with heavy metals is a serious point of concern because of the effect on soil microorganisms and source of pollution. These metals contaminate the soil and water pose a threat to soil organism and aquatic lives. Apart from the danger on aquatic organisms, they are also detrimental to humans when ingested. Some of this metals are associated with serious ailments which brought about the need to investigate there soil and water contaminations. However the water and soil samples collected from abattoir sides in Wadata has minute and non significant levels of lead and copper. The conclusion made from the result of this research therefore is that the soil and water from abattoir sides of Wadata has minimal heavy metal contaminants and also restricted microbial species.

5.3 Recommendations

The recommendations made from the result of this study are;

1. The sitting of Abattoir sites away from water sources to avoid contaminants from animal remains.

2. Proper treatment of waste water before disposal to minimize heavy metal and microbial contamination.

3. Disposal of the water should be away from water sources to avoids mixing with contaminated water.

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