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2349-8870 Europeanof Journal of Biomedical and Pharmaceutical ISSN Sciences European Journal Biomedical Volume: 4 Issue: 9 AND Pharmaceutical sciences

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444-448 Year: 2017

ANALYSIS OF BIOCHEMICAL PROPERTIES OF SOIL AND GROUNDWATER IN ARSENIC AFFECTED BLOCKS OF MURSHIDABAD DISTRICT AND ISOLATION OF POTENTIAL ARSENIC RESISTANT BACTERIA Dr. Abhishek Basu1*, Debjani Mandal1, Bibhas Bhattacharyya1, Manali Biswas1, Indranil Saha2, Dr. Gunjan Dhar3 and Dr. Shamsuzzaman Ahmed2 1

Department of Molecular Biology and Biotechnology, Sripat Singh College, Jiaganj, Murshidabad, Under University of Kalyani, West Bengal, India. 2 Department of Chemistry, Sripat Singh College, Jiaganj, Murshidabad, Under University of Kalyani, West Bengal, India. 3 Department of Zoology, Sripat Singh College, Jiaganj, Murshidabad, Under University of Kalyani, West Bengal, India. *Corresponding Author: Dr. Abhishek Basu Department of Molecular Biology and Biotechnology, Sripat Singh College, Jiaganj, Murshidabad, Under University of Kalyani, West Bengal, India. Article Received on 25/06/2017

Article Revised on 15/07/2017

Article Accepted on 05/08/2017

ABSTRACT Arsenic is one of the major contaminants of soil and groundwater, responsible for a number of health hazards. Various blocks of Murshidabad district show arsenic concentration above the maximum permissible limit in soil and water samples. In the present study, we have focused on some highly arsenic contaminated regions of Murshidabad district. We have analyzed various biochemical parameters of soil and water samples of this district. The soils of these regions show high alkalinity and the groundwater samples also exhibits a basic pH. The total dissolved solids of these water samples varied from 200 mg to 300 mg. Also, the bacterial load in the water samples was extremely high. Since, groundwater is a source of drinking water in the blocks of Murshidabad district, consumption of such high amount of total dissolved solids would take a toll on the detoxifying and excretory system of the body i.e., on the hepatic and renal systems. The uncharacterized microorganisms in the soil could be faecal coliforms or other pathogenic bacteria, and their consumption would have serious health consequences. After serial dilutions (106 to 109 folds) of the groundwater and the soil samples, we could still isolate some bacteria thriving in these samples. Since, the soil and groundwater of these regions are highly arsenic contaminated, bacterial colonies isolated after serial dilutions could be potentially arsenic resistant bacteria. KEYWORDS: Murshidabad district, Arsenic toxicity, Soil and groundwater, pH, Total dissolved solutes, Phosphate content, Arsenic resistant bacteria. INTRODUCTION Arsenic toxicity is one of the serious problems both from national and global perspective. Arsenic is a toxic metal, which contaminates soil and groundwater. In India arsenic contamination of soil and groundwater is a severe problem in lower Gangetic plain and GangaBrahmaputra deltaic region (SOES, 2006). U.S. Environmental protection agency prescribed the maximum permissible limit of arsenic in drinking water to be 10 µg/l (EPA, 2006). However, World Health Organization notified that in absence of any alternate source of drinking water, the maximum permissible limit of arsenic would be 50 µg/l (WHO, 2011). In countries like Canada and Australia, this permissible limit is 5 µg/L and 7 µg/l, respectively (Kapaj S et al., 2006). Arsenic is released in the soil by natural biogeochemical cycles, from there it leaches into the groundwater by natural processes. However, anthropogenic activities like

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use of arsenic in herbicides, disinfectants, wood preservatives and medicines, release high amount of arsenic into the soil (Pais IJ and Benton Jons JR, 1997). Arsenic occurs in inorganic and organic form, and also as arsine gas. Inorganic arsenic occurs predominantly as Arsenite (III) +3, and Arsenate (V) +5 state. Inorganic arsenic also occurs as Arsenide (-3) in intermetallic alloys (Yang HC and Rosen BP, 2016, Dey U et al., 2016). Arsenic also occurs in organic form as Arsenobetaine and Arsenocholine (Saha JC et al., 1999).The inorganic form of arsenic is more toxic for human. Also, Arsenite is the most toxic form of arsenic due to its higher solubility, mobility and bioavailability. Arsenic causes various health hazards in a dosedependent and time-dependent manner. The predominant health disorders include diseases of pulmonary, respiratory, circulatory and gastrointestinal system (Saha

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JC et al., 1999, Hendryx M, 2009, Ratnaike RN, 2003). It also causes severe harm to hepatic and renal system. Arsenic toxicity has neurological, dermal and hematological effects. Also, exposure to arsenic over a considerable period of time leads to genetic mutation and chromosomal aberration, making a person pre- disposed to cancers of liver, lungs, skin, intestine, kidney etc (Smith AH et al., 1992, Saha JC et al., 1999). Arsenic enters the body through water and food. There are evidences that staple food crops like rice have accumulated arsenic. This leads to biomagnification of arsenic within the food chain (Huq Imamul SM et al., 2006, Chen Y et al., 2017).

mg soil/1 ml of distilled water. It was then mixed well and allowed to settle as mentioned above. The groundwater and the soil solutions were mixed with 1:1 ammonium molybdate reagent i.e. 5 ml of sample was mixed thoroughly with 5 ml ammonium molybdate solution. Then 200 µl of stannous chloride solution was added and mixed. The whole procedure was carried out at room temperature. Absorbance was measured at 690 nm wavelength using UV-Visible spectrophotometer. A standard curve was prepared using known phosphate concentrations and the concentration of phosphate present in the sample was estimated from the standard curve.

West Bengal is one of the severely arsenic affected states of India. Within West Bengal nine districts are affected by arsenic toxicity. Murshidabad is one of the severely arsenic affected districts of West Bengal. The river Ganga separates it from Bangladesh. Eastern bank of Bhagirathi river constitutes more arsenic contaminated blocks of Murshidabad district (64.7% above 10 μg/l and 32.5% above 50 μg/l) compared to the western bank of the river (30.1% above 10 μg/l and 11.7% above 50 μg/l) (Rahman MM et al., 2005). In this study, our main aim is to analyze the conditions of soil and groundwater in highly arsenic contaminated regions of Murshidabad district. For this purpose, we have qualitatively and quantitatively checked various biochemical and microbiological parameters of soil and groundwater samples from these affected regions. Also, we have made an attempt for isolation of potential arsenic resistant bacteria from these regions.

Estimation of bacterial load in the soil and groundwater samples and isolation of potential arsenic resistant bacteria. Water samples, and soil samples dissolved in water was plated in a LB-agar plate to estimate the bacterial load in the soil and groundwater samples. Further, serial dilutions of water and soil samples were made and these diluted stocks were plated in LB- agar plates for isolation of single colonies of potential arsenic resistant bacteria.

METHODS Measurement of pH of soil and groundwater samples Soil samples were diluted to obtain a concentration of 1 mg soil /1 ml distilled water. Soil samples were mixed well with water by constant stirring so, that the soluble solutes i.e. ions and electrolytes come in the polar phase. The particulate matter in the soil (soil sediments) was allowed to settle down. The pH of the solution was measured at 25⁰C. Similarly, the pH of the water samples was measured directly at 25⁰C. Estimation of total dissolved solids in the water samples The weight of the conical flask was measured. Then 100 ml of water sample was taken in the conical flask and allowed to evaporate by application of heat. The conical flask was dried and weighed again. The difference between the second weight and the first weight is the TDS in 100 ml of water sample. Subsequently, we can calculate the TDS per liter of the water sample. Estimation of Phosphate in the groundwater and soil samples Phosphate present in the soil and groundwater samples was measured Spectrophotometrically. Soil samples were dissolved in water to make final concentration of 1

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RESULT Estimation of pH and Total Dissolved Solids in the soil and groundwater samples from severely arsenic affected regions of Murshidabad district Water samples were collected from Bhagobangola I, Hariharpara, Chunakhali and Asrampara, which are the regions amongst the severely arsenic affected blocks of Murshidabad district. Soil samples were also collected from Bhagobangola I, Hariharpara and Chunakhali (Figure 1). pH of the soil and water samples was measured at 25⁰C. Both soil and water samples from the four arsenic contaminated blocks were found to be alkaline in nature. Soil sample of Hariharpara showed the maximum pH (8.37) amongst all the other soil samples. Among the water samples, water sample from Bhagobangola I showed the maximum pH (7.60). All the soil samples showed pH above 8 (Table 1). Total Dissolved solids (TDS) in the water samples were found to be very high. TDS in water samples from Hariharpara was 200mg/l. The other three sources showed TDS concentration of 300mg/l (Table 2).

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c) Figure 1: Soil samples collected from a) Mahisasthali village, Bhagobangola I b) Asrampara and c) Chunakhali block. Table 1: pH of soil and water samples from arsenic affected blocks of Murshidabad district. pH of soil and water samples from arsenic affected blocks at 25⁰C Soil sample pH Water sample pH Bhagobangola I 8.30 Bhagobangola I 7.60 Chunakhali 8.2 Chunakhali 7.20 Asrampara Not done Asrampara 7.10 Hariharpara 8.37 Hariharpara 7.30 Table 2: TDS in water samples from four arsenic affected blocks of Murshidabad district. Total Dissolved Solutes in water samples ( in mg/l) Bhagobangola I Chunakhali Asrampara Hariharpara 300 300 300 200 Estimation of Phosphate content in the soil and groundwater samples from severely arsenic affected regions of Murshidabad district The next aim was to estimate the total phosphate content in soil and water samples of these blocks using spectrophotometry. Various studies suggested the common uptake mechanism of arsenic and phosphate by plants, i.e. through phosphate transporters. As affinity of phosphate transporters toward phosphate is higher than their affinity towards arsenic, estimation of total phosphate content in a sample will indicate indirectly the

chance of uptake of arsenic by plants of that area. Therefore, total phosphate was measured by an assay using a spectrophotometer (Figure 2). The total phosphate content in water samples of Bhagobangola I, Hariharpara, Chunakhali and Asrampara was 780 μg/l, 60.5 μg/l, 4.5 μg/l and 10.5 μg/l, respectively. Soil samples from Bhagobangola I, Hariharapara and Chunakhali contained phosphate concentration at 17 μg/l, 90 μg/l and 40.5 μg/l, respectively (Table 3).

Table 3: Estimated total phosphate content in soil and water samples from four arsenic affected blocks of Murshidabad district. Total phosphate content of soil and water samples from arsenic affected blocks Soil sample Total Phosphate Water sample Total Phosphate Bhagobangola I 17 μg/l Bhagobangola I 780 μg/l Chunakhali 40.5 μg/l Chunakhali 4.5 μg/l Asrampara Not done Asrampara 10.5 μg/l Hariharpara 90 μg/l Hariharpara 60.5 μg/l

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Figure 3: Bacterial plates showing isolated colonies from soil and water samples.

Figure 2: Test tubes showing five standard phosphate solutions and soil and water samples after reaction with ammonium molybdate and stannous chloride. Isolation of potential arsenic resistant bacteria from the soil and groundwater samples Next, with an aim to isolate arsenic resistant bacteria from these samples, soil and water samples from aforementioned arsenic affected blocks were plated with and without dilutions on LB agar plate. Huge number of bacterial colonies was observed when undiluted soil and water samples were plated. As PHED had marked these blocks as severely arsenic contaminated, we wanted to identify arsenic resistant microorganisms from soil and water samples of these blocks. Therefore, we isolated bacteria from these soil and water samples by serially diluting the samples and spreading them on LB agar plate. Single bacterial colonies could be observed in the plates (Figure 3). And considering the huge arsenic toxicity of the aforementioned regions, these colonies could be categorized as colonies of potential arsenic resistant bacteria.

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DISCUSSION Arsenic is a naturally occurring element which recycles mainly by water in the environment. Arsenic contamination of soil and groundwater is a global problem and millions of people are at risk of diseases associated with long term intake of arsenic via food and drinking water. Contamination of groundwater by arsenic occurs mainly by natural processes. But nowadays anthropogenic activities (use of arsenic in disinfectants, pesticides, wood preservatives and others) are responsible for increasing concentration of arsenic in soil and groundwater (Pais IJ and Benton Jons JR, 1997). Bangladesh and the state of West Bengal in India are severely affected by arsenic contamination in groundwater and soil. Nine districts of West Bengal are affected by arsenic toxicity. Murshidabad is one of the most arsenic affected districts of West Bengal (Rahman MM et al., 2005, SOES, 2006). We have concentrated our study on four blocks of Murshidabad district. These were Bhagobangola I, Hariharpara, Chunakhali and Asrampara. All of these blocks had been marked by PHED (Public Health and Engineering Department) as severely arsenic affected blocks. We collected soil and water samples from these areas and measured the pH and total dissolved solids (TDS). Both soil and water samples of these areas were found to be very alkaline. Amongst the soil samples tested, soil sample of Hariharpara was found to be most alkaline with a pH of 8.36 at 25⁰C. The maximum pH amongst the water samples was observed for water sample from Bhagobangola I with a pH of 7.60. This goes with the general idea that arsenic is found more commonly in alkaline soil and water samples. The TDS in water sample of Hariharpara was 200 mg/l. Bhagobangola I, Chunakhali and Asrampara water samples showed 300 mg/l TDS. From this, it could be estimated that a daily intake of 4 litres of drinking water

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would introduce 800 and 1200 mg/l of TDS, respectively, in people of these blocks. Such a huge amount of TDS would put a strain on the liver for detoxification. Therefore, residents of these blocks might suffer from liver diseases along with diseases associated with arsenic intake. They might also show symptoms of bone diseases and dental problems. Arsenic and phosphate are taken up by plants through same transport system, i.e. using phosphate transporters. These phosphate transporters show more affinity towards phosphate compared to arsenic. Therefore, it can be assumed that plants growing in areas with low phosphate content will take up more arsenic compared to those growing in areas with high phosphate content. So, the estimation of total phosphate content in water and soil samples from Bhagobangola I, Hariharpara, Chunakhali and Asrampara will give an indirect idea of arsenic uptake by plants growing in these soils or utilizing water from these water sources. Soil sample from Bhagobangola I showed less concentration of phosphate compared to its water sample, possibly because of excessive rainfall and water logged condition of that area. The soil sample of Chunakhali block showed 40.5 μg/l of phosphate. On the other hand, lesser amount of phosphate was found in water sample of this region. The reason behind this could involve excessive application of phosphate containing fertilizers (like NPK) and pesticides in the soil of this region. In Hariharpara, both soil and water samples were heavily contaminated with phosphate, 90 μg/l and 60.5 μg/l, respectively. Deposition of organic phosphate followed by decomposition and mineralisation might also give rise to high level of phosphate in the soil. Phosphate in soil dissolves with rain water and by leaching it is introduced into the water bodies. The permissible limit of phosphate in drinking water as recommended by U.S. Environmental Protection Agency is 25 μg/l. Therefore, water samples from Bhagobangola I and Hariharpara blocks were contaminated with phosphate at a concentration above the permissible limit. So, these waters if consumed could affect bone density with an increasing risk of osteoporosis in residents of these blocks. Also, phosphate could associate with calcium and get deposited in muscles and soft tissues causing them to harden. People residing in these blocks might also suffer from digestive problems due to excessive intake of phosphate. Soil and water samples from the above mentioned arsenic affected blocks were plated with and without dilutions on LB-agar plate. With serial dilutions single bacterial colonies could be obtained on the LB-agar plate. Considering the huge arsenic toxicity of these regions, the soil and water microbiota obtained from these regions could be potentially arsenic resistant. Huge number of bacterial colonies was observed when undiluted soil and water samples were plated. Therefore, people of these blocks were drinking water contaminated with millions of bacteria. As characterisation of these bacteria was not done therefore, it could only be assumed

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that these water samples might be contaminated with toxic bacteria including faecal coliforms and other pathogenic bacteria. ACKNOWLEDGEMENT We would like to acknowledge the students of Department of molecular Biology and Biotechnology, Sripat Singh College, who have voluntarily agreed to help us in sample collection. We are grateful to the Department of Biotechnology, Government of West Bengal for the financial assistance. REFERENCE 1. Chen Y, Han Y-H, Cao Y, Zhu Y-G, Rathinasabapathi B and Ma LQ. Arsenic Transport in Rice and Biolological Solutions to Reduce Arsenic Risk from Rice. Frontiers in Plant Science, 2017; 8: 268. 2. Dey U, Chatterjee S, Mondal NK. Isolation and characterization of arsenic-resistant bacteria and possible application in bioremediation. Biotechnology Reports, 2016; 10: 1–7. 3. EPA. Drinking Water Requirements for States and Public Water System, EPA, 2006. 4. Hendryx M. Mortality from heart, respiratory and kidney disease in coal mining areas of Appalachia. Int Arch Occup Environ Health, 2009; 82(2): 243-9. 5. Huq Imamul SM, Joardar JC, Parvin S, Correll R and Naidu R. Arsenic contamination in food chain: Transfer of arsenic into food materials through groundwater irrigation. J Health Popul Nutr, 2006; 24(3): 305-316. 6. Kapaj S, Peterson H, Liber K and Bhattacharya P. Human Health Effects From Chronic Arsenic Poisoning–A Review, 2006; 41(10): 2399-2428. 7. Pais IJ and Benton Jons JR. The hand book of trace elements. Publishing by: St. Luice press Boca Rrton Florida, 1997. 8. Rahman MM, Sengupta MK et al. Murshidabad— One of the Nine Groundwater Arsenic-Affected Districts of West Bengal, India. Part I: Magnitude of Contamination and Population at Risk. Clinical Toxicology, 2005; 43: 823–834. 9. Ratnaike RN. Acute and Chronic Arsenic Toxicity. Postgraduate Medical Journal, 2003; 79(933): 391396. 10. Smith AH, Hopenhayn- Rich C, Bates MN, et al. Cancer risks for arsenic in drinking water. Environ Health Perspect, 1992; 97: 259-67. 11. Saha JC, Dikshit AK, Bandyopadhyay M and Saha KC. A review of arsenic poisoning and its effects on human health. Critical Reviews in Environmental Science and Technology, 1999; 29(3): 281-313. 12. SOES. Groundwater arsenic contamination in West Bengal- India (20 years study), SOES, 2006. 13. WHO. Guidelines for drinking water quality, WHO, 4th edition, 2011.

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