Appraisal of Heavy Metal Presence and Water Quality having Microbial Load and Associated Human Health Risk: A study on tube-well water in Nalitabari township of Sherpur district, Bangladesh

This article is based on a study aimed to determine physiochemical parameters, fecal coliform, total coliforms, heterotrophic plate count, arsenic, iron and lead of water to evaluate their effects on human health. Analysis was carried out on tube-well water collected from Nalitabari township of Sherpur District in Bangladesh. The dissolved oxygen (DO), total dissolved solids (TDS), salinity and electrical conductivity were in the ranges of 4.30 to 7.30 ppm, 350 to 792 mg/l, 0.2 to 0.5%, and 715 to 1,970 μS/cm. The pH values were slightly lesser or more than permissible value. Due to the vicinity to the latrines, 17 tube-wells’ water was contaminated by fecal coliforms. The hig hest heterotrophic plate count was 7.5×10 3 cfu/ml in ward-8 of the town. Eschericia coli and Vibrio cholerae were identified in ratio of 30.56% and 18.06%, respectively, in the tube-well water, resulting into diarrhea among children. About 6.94% of tube-well water was contaminated with arsenic. 3.25% and 4.5% respondents were suffering from skin diseases and headache, respectively. So, an alternative source of drinking water should be arranged for a better public health of present and next generations.


Introduction
Groundwater from quaternary to recent sediments is the principal source of water for domestic consumption and utilization in industry and irrigation system in Bangladesh.The shallow alluvial aquifers receive water through rainfall and flooding.The static water level in much of Bangladesh is due to its availability within 7 meter of the ground surface round the year.Simple suction hand pumps are the dominant water supply technology in Bangladesh (Luby et al., 2008).More than 90% of households in Bangladesh generally use tube-well water for domestic consumption such as drinking and cooking purposes.It is a matter of great concern that the drinking water is getting polluted with various organic and inorganic matters (Rezania et al., 2015).Depending on the availability and the level of groundwater, these tube-wells have been installed in Bangladesh at various depths.It may be insufficient to avoid contamination of the tube-well water with human-pathogenic bacteria due to unfavorable immediate environmental conditions (e.g., the distance of tube-wells from latrines or sewage-contaminated ponds or tanks).Despite regular use of tube-well water for drinking, Bangladesh has failed to protect the gastrointestinal diseases caused by water pollution (Islam et al., 2001).Diarrheal diseases are still a leading cause of death of children under 6 years and about 5.2% of all infant deaths occur in Bangladesh due to diarrheal diseases (Feachem and Koblinsky, 1983).Underground water systems of Bangladesh are increasingly vulnerable due to both microbiological contamination and heavy metal pollution, especially by arsenic and iron.Such problems have also been arisen even in developed countries (Hartley, Edwards and Lepp, 2004).
It was found that 41% water of tube-wells was contaminated by total coliforms, 29% by thermo-tolerant coliforms and 13% by fecal coliforms (Saha et al., 2018).About 40% water of shallow tube-wells in Bangladesh were contaminated with human fecal organisms (Knappett et al., 2011;Malla et al., 2018).Coliform bacteria indicate a pathway for more pathogenic bacteria, viruses and protozoans that can be introduced by anthropogenic activities and poor sanitation.According to World Health Organization, placing tube-wells at a safe distance from latrines, ensuring that the tube-well has a sound platform without cracks, and that the hand pump is firmly attached, prevent the contamination of fecal coliforms.Every year more than 3.4 million people die as a result of water related diseases, making it the leading cause of disease morbidity and mortality around the world, especially in South-Asia (Souter et al., 2003).From this point of public health, it is highly imperative that potable water supply system should be safe that prevents and controls diarrheal diseases (Motarjemi and Käferstein, 1999;Yager et al., 2006).Drinking water quality among the natural parameters, such as Fe, Mn and salinity, are matters of concern over large areas in deep and shallow aquifers, and in both urban and rural areas of Bangladesh (Ahmed et al., 2019).Arsenic exposure through groundwater has been a major public health problem in Taiwan, Mexico, USA, Mongolia, Argentina, Chile, India and Bangladesh.Worldwide, more than 100 million people have been estimated to be chronically exposed to arsenic from drinking water contamination of high levels of arsenic.The situation is devastating in Bangladesh.From about 7-11 million hand pumped tube-wells, approximately half of them have been estimated supplying groundwater with an arsenic concentration more than 50 micrograms/l, which is the maximum level of arsenic allowed in a drinking water (Rahman et al., 2018;Mukherjee et al., 2006).Up to 77 million people in Bangladesh have been exposed to toxic levels of arsenic from drinking water and one in ten has the probability of developing cancer from the arsenic poisoning (Smith, Lingas and Rahman, 2000).The iron contamination in groundwater is one of the most discussed issues because iron (Fe) contamination in groundwater is now a vital problem in Bangladesh (Hug, Leupin and Berg, 2008).It was estimated that about 80% of the diseases in developing countries are attributed to contaminated water and resulting death toll is as much as 10 million per year (Mara and Alabaster, 1995).The improvement of health is not possible without proper sanitation system.Sanitation is one of the major problems in Bangladesh that threat the public health.In this regard, water supply and sanitation facilities in terms of quality and quantity are utmost necessities for assessing the living condition of the urban and semi-urban areas of Bangladesh.Due to poor sanitation and unawareness about personal hygiene practices, drinking water is contaminated by some pathogenic bacteria and increases the risks of water-borne diseases (Suthar, Chhimpa and Singh, 2009).
Besides, the presence of heavy metals in drinking water is a matter of great concern due to their impacts on human life.Contamination of tube-well water with arsenic and heavy metal is hazardous for health.People are suffering from headaches, abdominal pain, cancer, kidney damage, nerve damages and skeletal damages due to the toxic effects of these metals (Rasool et al., 2016).Therefore, the present study was designed to evaluate tube-well water quality and to identify the presence of heavy metal contaminations in tube-well water.The objectives of this study were also to investigate the tube-well water's physiochemical parameters (such as dissolved oxygen (DO) electrical conductivity (EC), total dissolved solids (TDS) and pH), microbial load of tube-well water and their health impact on people of Nalitabari Township of Sherpur District in Bangladesh.

Study Area
Nalitabari is an Upazila (sub-district) of Sherpur District under the Division of Mymensingh in Bangladesh.It is located between 25°01' and 25°13' N latitudes and between 90°04' and 90°19' E longitudes.It is 174.9 kilometer away from Dhaka and situated on the bank of the river Bhogai in northern part of Bangladesh.Nalitabari municipality is one of the oldest municipalities in Bangladesh, established on 1st April in 1869.It has an area of 327.61 sq.km with 42,698 households.The study was carried out from June 2016 to April 2017.

Sampling
Total 72 water samples from 8 locations (9 samples from each ward) were randomly collected and analyzed.The tube-well was continuously pumped for one minute to clear the way of opening and the water samples were collected in a sterile container.All the samples were stored in ice box with proper aseptic technique and immediately transported to the laboratory for experimental analysis.Samples were collected in sterilized bottles and prior to filling, the sample bottles were rinsed two to three times with the water to be collected.The bottles used for collecting samples for metal analysis were filled with acid to keep the pH of the water samples low.Special caution was taken to restrict the overflow of sample water (with acid) from the bottle.The samples were transferred to the laboratory within the six hours of collection (Jidauna et al., 2013).

Analysis of Physiochemical Parameters
The water quality parameter such as pH was determined by the digital pH meter (Model: pH Scan WP 1, 2 and made in Malaysia).Buffer solution containing pH 4.0 and 7.0 was used to calibrate the digital pH meter.Digital Electrical Conductivity (EC) and Total Dissolved Solids (TDS) meters (Model: HM digital and made in Germany) were used to determine EC and TDS, respectively.Salinity was also measured by it.The Dissolved Oxygen (DO) was determined by digital DO meter (Model: D.46974 and made in Taiwan) where sodium thiosulphate (0.025N) was used as a reagent (Islam et al., 2014).

Determination of heavy metal
Arsenic, lead and iron were determined by test kit developed by HACH Company, USA (Reddy et al., 2020).

Heterotrophic Plate Count (HPC)
For determination of heterotrophic plate count, 100 micro liters of a tenfold serial dilution of bottled water and 100 micro liters of a tenfold serial dilution of tube-well water from samples were transferred and spread onto a plate count agar medium using micro pipette for each dilution.The diluted samples were spread as quickly as possible on the surface of plate with a sterile glass spreader.One sterile glass spreader was used for each plate.The plates were then incubated at 37ºC for 24-48 hours.Following incubation, plates exhibiting 30-300 colonies were counted.The heterotrophic plate count was calculated, and the result of total bacterial count was expressed as the number of organism or colony forming units per milliliter (CFU/ml) of water samples (Kabir et al., 2015).

Total Coliform Count
The most probable number (MPN) test for the presence of coliforms in water carried out according to the procedures described by Harley and Prescott (2002).An estimate of the number of coliforms (MPN) can also be done in the presumptive test.In this procedure, 15 lactose broth tubes were inoculated with the water samples.Five tubes received 10 ml of water, another 5 tubes received 1 ml of water and rest 5 tubes received 0.1 ml of water.A count of the number of tubes showing gas production was then made, and the figure was compared to a table developed by American Public Health Association.The number was the MPN of coliforms per 100 ml of the water sample (Hassan et al., 2018).

Detection of Fecal Coliforms
The positive presumptive cultures were transferred to lactose broth, which is specific for fecal coliform bacteria.Any presumptive tube which showed gas production after 24 (+/-2) hours incubation at 44.5°C (+/-0.2°C)confirmed the presence of fecal coliform bacteria in that tube and was recorded as positive (Manja, Maurya and Rao, 1982).

Isolation of Pathogenic Bacteria
To isolate specific pathogenic bacteria, the samples were enriched separately with alkaline peptone water (APW) for plating in thiosulfate citrate bile salts sucrose agar (TCBS) medium, with GN (Gram-Negative) broth for plating in Salmonella shigella (SS) agar, with Enterobacteria Enrichment broth -Mossel for plating in MacConkey medium.From each sample, 1 ml of water was added with 3 ml of respective enrichment media.All the samples were then incubated at 37°C for 24 hours.After overnight enrichment, the samples were plated in MacConkey, TCBS and SS agar plate separately.All the plates were incubated at 37°C for 24 hours.After overnight incubation, the plates were observed for selective pathogens.For the confirmation of Escherichia coli, red/pink colonies form MacConkey agar plates were plated in eosin methylene blue (EMB) agar plates and for the confirmation of Vibrio cholerae standard biochemical tests were performed from the yellow and green colonies in TCBS media, respectively (Pavlov et al., 2004).

Biochemical Test
Biochemical tests were performed to identify the bacterial flora from different water samples.In this study, different Biochemical tests (such as KIA, MIU, CITRATE, VP, OXIDASE, CATALAE, MANNITOL, STARCH, MR, GLUCOSE, LACTOSE, EMB) were performed according to Bergey's Manual of Determinative Bacteriology, 9th Edition, 1994 (Ewalt et al., 1994).

Antibiotic Sensitivity Test
Antibiotic susceptibility test was accomplished by disk diffusion method using the commercial antibiotic disk and MHB on Mullar-Hinton agar to assess the susceptibility and resistance pattern of the isolates.For this purpose, 13 different antibiotic discs were used from commercial sources (Oxoid Ltd., England).The selected antibiotics used were Ampicillin, Amoxicillin, Chloramphenicol, Erythromycin, Tetracycline, Gentamicin, Penicillin, Sulphomethoxazole, Kanamycin, Nalidixic Acid, Ciprofloxacin, Streptomycin, Norfloxacin and Azithromycin.The interpretation on susceptibility was done according to the guidelines of Clinical and Laboratory Standard Institute, formerly known as NCCLS (Liasi et al., 2009;Ali et al., 2020).

Assessment of Health Impact
A semi-structured questionnaire was prepared for field investigation to evaluate the health impact of the people who used these tube-well waters.Total 400 (200 children and 200 adults, among them 50% were male and 50% were female) respondents of the study area were interviewed to determine the health status of people in the study area (Rakib et al., 2019;Ali et al. 2020).

Physiochemical Properties of Water
Amounts of pH, DO, EC, TDS and salinity contained in the tube-well water of 8 different locations collected from Nalitabari Township of Sherpur district were summarized in Table 1.The pH value of all water samples was in normal range from 6 to 8.5.pH value observed for all the water samples were slightly less or more than 7 with the average value of 6.8.Lowest value of pH (6.01) was found in ward-8 at TW67 and the highest value (7.92) found in ward-3 at TW25.In other study, it was reported that the pH of 60% water samples collected from tube-wells in Matlab of Bangladesh was acidic and lower than recommended by the World Health Organization (Robinson et al., 2011).
The DO, TDS, salinity, and conductivity of water samples were in the ranges of 4.30 to 7.30 ppm, 350 to 792 mg/l, 0.2 to 0.5%, and 715 to 1970 μS/cm, respectively.The mean DO content of all water samples was 5.78 mg/l.The maximum concentration of DO was 7.30 mg/l in the water collected from TW22 (ward-3), whereas the minimum concentration was found 3.95 mg/l in TW15 belonging to ward-2.The value of DO of all water samples was not satisfactory, as the standard value is 6.00 mg/l or more for Bangladesh drinking water set by DoE (Alam et al., 2007).
According to International Organization for Standardization, the palatability of drinking water has been rated to its TDS level as follows: excellent, less than 300 mg/liter; good, between 300 and 600 mg/liter; fair, between 600 and 900 mg/liter; poor, between 900 and 1200 mg/liter; and unacceptable, greater than1200 mg/liter (Beyene, 2015).So, all the values of TDS were in an acceptable range.The result of the study showed that the electrical conductivity (EC) of 50% water samples was within the standard value of drinking water in Bangladesh.The maximum permissible limit of EC in Bangladesh is 1,200 μS/cm (Mebrahtu and Zerabruk, 2011).It was reported that electrical conductivity (EC) of the drinking water coolers of different teaching institutes in Lahore ranged from 185-362 μS/cm and was well within the permissible limit of 400 μS/cm as set by WHO guideline (Asif et al., 2015).All the tube-wells water of the study area was within the acceptable salinity range where salinity of the freshwater is 0 to 0.5%.There was a significant relationship among the salinity, TDS and EC.In this study, it was found when the salinity of tube-wells water was 0.5% or more, the TDS and EC values were also high.Another study suggested that most of the physicochemical parameters of groundwater in Rajshahi city were not at the alarming stage (Rasul and Jahan, 2010).

Presence of Heavy Metals
This study revealed that there was a significant association with the arsenic contamination of tube-wells water and deepness of the tube-wells.Among 72 tube-wells, all the tube-wells that contained excessive amount of arsenic have the depth within 90 feet.Out of 72 tube-wells water, 16 contained more iron (Fe) than the recommended limit set by Bangladesh; whereas in Bangladesh, permissible limit of Fe is 0.3-1.0mg/l, while WHO standard level is 0.1 mg/l.About 93.75% of the tube-wells contaminated with excessive iron in which deepness was within 90 feet (Roy et al., 2015).About 12% of the water samples contain moderate sediment and 76% samples contain no sediment after centrifuge at 10,000 rpm.Table 1 showed that there is no contamination of lead found in tube-wells water in the study area.A study reported that more than 60% of the groundwater in Bangladesh contained naturally occurring arsenic with concentration levels often significantly exceeding 10 µg/l (Bang, Viet and Kim, 2009).The National Drinking Water Quality Survey Report ( 2009) used an estimated national population of 164 million to estimate that 22 million and 5.6 million people are drinking a water with arsenic concentrations more than 50 μg/l and 200μg/l (George et al., 2012), respectively.Present study showed very little amount of arsenic contamination in the tubewells water in the study area.Only 6.94% of tube-wells water was contaminated with arsenic more than 50μg/l, which is the recommended limit set by DoE, Bangladesh.It was found that out of 330 tube-wells in Rajshahi city, 72 were found having arsenic levels above the WHO guideline value (0.01 ppm), of which 30 exceeded the Bangladesh drinking water standard (0.05 ppm) (Rasul and Jahan, 2010).

Determination of Microbial Load
This study also showed that all the tube-well water samples contained a variety of microorganisms (Table 2).Out of 72 tube-wells, water of 17 contained more fecal coliforms than the recommended limit set by WHO (23.61% of the samples) (Khan et al., 2013).There was a significant association found between tubewell water contamination with fecal coliforms and distance of tube-well from the latrine.All the tube-well waters that contained fecal coliforms were within 30 feet from the latrine with exception of TW69, which was found at a distance of 41 feet from the latrine.According to the results in Table 2, it was clear that the presence of fecal coliforms in the tube-well water was directly related to the surrounding latrine condition and distance from the latrine.In a similar study on analysis of tube-well water from Fulbaria pourasava in Mymensingh district of Bangladesh, it was reported that 32% water samples were contaminated by fecal coliforms of which 30% of samples were contaminated with total coliforms (TC) than the recommended limits (≤10 coliforms/100 ml water) (Islam et al., 2001).There was no significant relationship between deepness of the tube-well with the contamination of fecal coliforms.
Amount of heterotrophic plate count (HPC) and total coliform count (TCC) contained in the tube-well water samples of 8 different locations of Nalitabari township of Sherpur district were summarized in Table 3.It showed that all water sources (100%) contained total coliforms (TC) ranging from ≤2 cfu/100 ml to 130 cfu/100 ml and HPC ranging from 1.0×10 3 cfu/ ml up to 7.5×10 3 cfu/ ml.Twenty-six water samples contained more TCC than the permissible limit and 76.92% of these tube-wells were located within 30 feet away from latrine.Among them TW13, TW15, TW50, TW66, and TW67 were highly polluted with TCC which have 110 cfu/100 ml, 95 cfu/100 ml, 130 cfu/100 ml, 90 cfu/100 ml and 130 cfu/100 ml of TCC, respectively, against permissible limit in Bangladesh of up-to 10 coliforms/100 ml water (Kabir et al., 2015).Tube-well number TW13, TW15, TW50, TW66, and TW67 were only 11 feet, 6 feet, 15 feet, 26 feet and 8 feet away from the latrines, respectively.Highest TCC was found in the sample of TW67 in ward-8 and TW50 in ward-6 and the highest value was 130 cfu/100 ml.There was a significant association between tube-well water contamination with total coliforms or HPC and surrounding latrine condition.The highest HPC count was found in tube-well water sample of TW67, which was 7.5×10 3 cfu/ml.This tube-well was only 8 feet away from latrine.Water samples TW3, TW4, TW16, TW27, TW35, TW42, TW51 and TW61 contained very lowest amount of HPC count and that was 1×10 3 cfu/ml in each of the samples.The mean HPC was observed 1.78×10 3 cfu/ml in ward-1, 2.66×10 3 cfu/ml in ward-2, 2.83×10 3 cfu/ml in ward-3, 2.20×10 3 cfu/ml in ward-4, 2.99×10 3 cfu/ml in ward-5, 2.76×10 3 cfu/ml in ward-6, 3.01×10 3 cfu/ml in ward-7 and 3.24×10 3 cfu/ml in ward-8.It has been generally believed in Bangladesh that groundwater is relatively free of microorganisms and, therefore, suitable for human consumption without treatment.However, the results of this study clearly showed that all samples of tube-well water in Bangladesh, that were examined, contained different counts of bacteria, which are above permissible limit (Prosun et al., 2018).Among the isolates, two kinds of bacteria (E. coli and Vibrio cholerae) were confirmed based on biochemical experiments.The results of biochemical tests for isolates from water samples were summarized in Table 3.

Table 3: Biochemical analysis of the isolated bacteria Antibiotic Susceptibility Test
Nowadays antibiotic-resistant bacteria are a great threat to our health and environment as well as act as a culprit in medical health care.These bacteria might have gained resistance property due to the indiscriminate use of antibiotics.In this study, 20 Vibrio cholerae and 30 E. coli bacteria were taken under antibiogram experiment.These antibiogram experiments revealed that Vibrio cholerae was resistant to Ampicillin (91%), Nalidixic Acid (89%), Kanamycin (83%), and Amoxicillin (69%).Mobile genetic elements are responsible for the spreading of drug resistance genes in V. cholerae strains in response to quorum sensing signaling.Thus, tetracycline resistant El Tor strains of V. cholerae re-emerged in Bangladesh in 1991 (Fazil and Singh, 2011).On the other hand, E. coli showed higher resistance to Chloramphenicol (89%), Kanamycin (89%), Amoxicillin (83%), and Sulphomethoxazole (83%) (Figure 1).Other studies also showed that E. coli isolates were established antibiotic resistance upon commercially used antibiotic like as Streptomycin, Sulfamethoxazole, Tetracycline, Ampicillin, and so on (Singh et al., 2005;Tadesse et al., 2012).Survey analysis showed that some adults and children were affected by different diseases attributed to microbial infection (Table 4).Out of 400 individuals, 1.75% were suffering from diarrhea and 0.5% were suffering from dysentery for a long time (Table 4).Children were more frequently affected by diarrhea and were prone to these diseases than adults.According to some other study, children under the age of five are the more susceptible group accounting for major part in deaths due to diarrhea or diarrheal diseases (Thiam et al., 2017;Black, Morris and Bryce, 2003).Among 200 adults, 6.5% individuals were rarely affected by typhoid whereas 1% adults were affected within last month.6% adults and 4.5% children were rarely or sometime affected by Salmonellosis whereas within last month 2.5% adults and 0.5% children were affected.No respondents were found in this study affected by Campylobacteriosis (Table 4).Some respondents were also affected by diseases attributed to heavy metal toxicity such as scabies skin diseases, neurological problems, bad headache and anemia (Table 5).About 3.25% and 4.5% respondents were suffering from a long-time skin diseases and bad headache, respectively.Adults were more frequently affected by skin diseases and bad headache than children, only 1% and 1.5% individual children out of 200 children were frequently affected by skin diseases and bad headache, respectively.On the other hand, out of 200 adults 8.5% and 3% were frequently affected by skin diseases and bad headache, respectively.No respondents were reported in this study to be affected by lead poisoning and arsenicosis because the water of the study area was not contaminated by lead and rarely contaminated by arsenic (6.94%).

Conclusion
In this study, it was found that the tube-wells, which were close to latrine, were more susceptible to contamination with fecal coliform.When the surrounding area was more polluted, then there was more chance of contamination.Heterotrophic plate count (HPC) was high in some tube-well water, which may be due to polluted earth environment.Identification of E. coli and Vibrio cholerae in the tube-well water indicated poor sanitation condition in the study area.Maximum tube-well water samples were negative to arsenic, only a few, about 6.94%, had arsenic pollution.A proper sanitation and drainage network system in the township must get a priority in municipal functioning.All tube-wells should be far away from polluted earth environment and distance of tube-well from latrine should be minimum 40-50 feet.The tube-well water of the studied area in Bangladesh cannot be considered safe for drinking unless properly treated.For developing a modern township, drinking water must be free from hazards which are threatening the public health.

Table 1 :
Physiochemical properties and presence of heavy metal in tube-well water

Table 2 :
Microbiological analysis of tube-well water

Table 4 :
Diseases related to microbial contamination in drinking water

Table 5 :
Diseases related to heavy metal toxicity