How much spatial and temporal variation in groundwater microbiology can occur following open dumping of municipal solid waste?

Worldwide proper disposal of municipal solid waste (MSW) is a major challenge, and this is more so in poor and developing countries due to un-scientific waste dumping in unlined landfills (Mukherjee et al. 2015). Open dumping of MSW is the most-followed practice in India mainly due to its simplicity and cost-effectiveness (Mohan & Joseph 2021). MSW disposal in unlined landfills is known to cause several environmental and health hazards and often groundwater is the most significantly affected system (Chakraborty & Kumar 2016; Samadder et al. 2017; Abiriga et al. 2020).

Open dumping exposes the waste to several environmental variables such as precipitation and moisture, which leads to the generation of complex landfill leachate (Abiriga et al. 2021a). Landfill leachate is complex effluent, which is known to contain dissolved organic substances, inorganic substances (ammonia, calcium, magnesium, sodium, potassium, iron, sulphate, chloride etc.), metals (cadmium, chromium, copper, lead, zinc, nickel etc.) and xenobiotic organic compounds (Thitame et al. 2010; Abiriga et al. 2020; Lee et al. 2020). Leachate has been reported to migrate from unsaturated zone to groundwater where it gets mixed and forms plume which spreads with groundwater movement (Shu et al. 2018; Abiriga et al., 2021b).

Pathogenic microbes in groundwater due to pollution can pose a severe risk to the human population. Microbial contamination of groundwater may lead to several water-borne diseases such as cholera, typhoid etc. (Giglio et al. 2016; Kapembo et al. 2019). Presence of microorganisms such as Escherichia coli, Enterobacteriaceae, Campylobacteriaceae, Cryptosporidium sp, Giardia sp, Citrobacter, Streptococcus sp, Staphylococcus sp, Salmonella sp, Klebsiella sp, Shigella sp etc. in groundwater is highly undesirable for human consumption (Aydin 2007; Hrudey & Hrudey 2007). Several water-borne diseases due to contaminated groundwater are constantly being reported across the globe irrespective of the economic status of the region (Zhang et al. 2014; Andrade et al. 2018). E. coli is regularly used as an indicator organism to check faecal discharges and ensure biological safety of water sources (Navab-Daneshmand et al. 2018; Gizaw et al. 2022).

Getting a scientific outlook on this important issue of groundwater quality in times of high rate of industrialization and urbanization will help make management decisions to protect the water resources and human health. Most of the studies on open MSW dumping and its impact on regional water resources have generally focused on the changes in physicochemical characteristics, comparatively fewer studies have focused on the presence of pathogenic bacteria and that too mostly as a one-off sampling event. In the current work, the impacts of open dumping of MSW were assessed on the groundwater bacterial density around the dumping site in Ranchi, Jharkhand, India. The major aim of this study was to gain firsthand information on the spatio-temporal variations in the abundance of heterotrophs, total coliforms and faecal coliforms in groundwater around the MSW dumpsite.

MSW dumpsite at Jhiri locality is situated 20 km away from Ranchi (23 °35′N and 85 °33′E), the capital of Jharkhand State in East India. MSW dumpsite covers an area of 8.9 ha and has been in operation since 2000. Ranchi is situated at an average elevation of 652 m from the mean sea level with an annual rainfall of 1,400 mm and the average number of rainy days is 75. The climate of Ranchi is subtropical with substantial rainfall occurring in the monsoon months (June to September). Summer season is from March to June and November to February is winter. The maximum mean annual temperature is 29.6̊C whereas the minimum is 18̊C.

The dumpsite (23° 22′N and 85° 20′ E) in Ranchi is divided into two parts, a larger area presents the old dumping yard which is also near a village, and another part is the new dumpsite where fresh waste is deposited daily which is close to agricultural land (Figure 1). Adjacent to the landfill area, there is a village with a population of 3,486 spread over an area of 258.6 ha (Census of India 2011). The landfill does not have any type of liner material at the base to contain the migration of leachate. Waste segregation and compaction at the landfill is not carried out and there is no practice of covering the deposited waste with soil cover, thereby exposing the waste to the environmental conditions. Approximately 700 tons/day of co-mingled waste are collected from the various sites of Ranchi, and it is deposited without any kind of processing/treatment. The dumpsite gets waste from residential sector, commercial sector, local vegetable and fruit markets, slaughterhouses and fish markets etc. Often, biomedical waste is co-disposed along with MSW and further, the waste collected from drains in the form of wet silts is also disposed of in a landfill.

Groundwater for bacteriological analyses was sampled in 50 mL sterilized high-density polyethene bottles. Samples were collected to ensure homogeneity and representativeness by flushing the source water till the point temperature was constant. Following collection, the groundwater samples for microbial analysis were kept in an ice box and immediately taken to the laboratory and stored at 4 °C for further analyses as per the standard methods (APHA 2017). Sampling locations were chosen around the MSW dumping site in a radius of 0.5 to 5 km from the periphery of the landfill to get representative data. Sampling points were selected based on the availability of groundwater samples throughout the year and the ones being used by the residents of the village near the dumpsite.

A total of eight groundwater sampling locations (dug wells and bore wells) already available were identified during the initial site survey with major selection criteria as year around water availability and site accessibility. Groundwater sampling locations were named as 2GW1, 2GW2, 3GW1, DW1, DW2 etc. GW indicates hand pump samples and DW indicates dug well samples. A control site (DW1) was also included in the sampling set that was far away from the MSW dumpsite. Sampling sites 2GW1, 2GW2, 2GW3, 2GW6 and 3GW1 are located around the old part of the dumpsite and sampling locations DW2 and DW3 are located around the new part of the dumpsite (Figure 1). Different zone of the sampling locations was identified by using the prefix number 2 or 3 based on their distance from the dumpsite. Sampling could not be carried out in the 1st zone due to the unavailability of groundwater sources at the periphery of the MSW dumpsite. Monthly sampling was carried out for one year to determine the seasonal variations (summer, monsoon, and post-monsoon) in groundwater microbiology. In this study, March to June is referred as summer, July to September as monsoon and October to February as the post-monsoon season.

Heterotrophic bacteria, coliforms and faecal coliforms were enumerated to assess the microbiological status of groundwater in the study area (APHA 2017). All the analyses were undertaken in <24 h of sampling. Groundwater samples were first serially diluted up to 10⁻¹ using sterile distilled water. 100 μL of each sample was plated on nutrient agar, MacConkey agar and Eosin methylene blue agar (EMB) using the spread plate technique to determine HPC, TC and FC, respectively. The inoculated plates were incubated for 24 h at 37 °C. A digital colony counter was used to count the number of bacterial colonies present in the groundwater samples as colony-forming units per millilitre (CFU/mL).

Molecular characterization was performed for faecal coliform colonies found on EMB agar plates, which were found to be morphologically identical during all the sampling events. A sample for molecular characterization was taken from 2GW2 (within 500 m from dumpsite), which presented the highest faecal coliforms count. 16S rDNA sequencing method was used to identify bacteria from the identical colonies. For this purpose, DNA was first isolated from the cultured sample by using a DNA isolation kit. The isolated DNA was amplified by PCR using 8F and 1492R primer and ran on 0.8% agarose gel to check the quality of the DNA. Amplicon from PCR were purified and processed for sequencing of nucleotide. Forward and reverse sequencing reactions of PCR amplicon was performed using 704F and 907R primers (BDT v3.1 sequencing kit – ABI 3730xl Genetic Analyzer). A consensus sequence of 1,486 bp 16S rDNA was produced from forward and reverse sequencing data by using the software. The 16S rDNA sequence was utilized to perform BLAST alignment search tool (NCBI gene-bank database). Maximum identity score of ten sequences were chosen and aligned with the multiple alignment software (Clustal W). Finally, the phylogenetic tree was constructed following the protocol detailed in the literature (Kumar et al. 2016).

Data generated from the study were analyzed using MS Office-Excel. Groundwater microbiology data were also analyzed to obtain correlation coefficients to determine the relationship between distance from the dumpsite and bacterial contamination. Single-factor ANOVA and two-factor ANOVA were applied to study the variance in the data. All the statistical tests were performed using the MS Office-Excel with Analysis Toolpak. Data on spatial variations in the microbial count was interpolated by applying the IDW method (inverse distance weighting) using ArcGIS (Reed et al. 2000; Adhikary & Dash 2017). GIS maps to show the spatial variations were also produced using the IDW process in ArcGIS.

Coliforms were present in most of the samples collected from various sites around the dumpsite. Heterotrophic bacterial density was from 4.2 × 10² to 4.5 × 10⁴ CFU/mL and total coliforms ranged from 2.0 × 10² to 3.7 × 10⁴ CFU/mL (Figure 2). Major reasons for such high bacterial contamination of groundwater could be due to poor sanitary conditions of human settlements, livestock faecal matter, in addition to open dumping of MSW that contains a variety of waste such as slaughterhouse waste biomedical waste, and kitchen waste. Faecal coliforms were also found in most of the samples, which is of major concern as the water is used for different domestic uses in the area. The highest faecal coliform count (2.1 × 10⁴ CFU/mL) was found from the hand pump (2GW2) located very close to a solid waste dumping site, coliform bacteria were detected at a higher range during monsoon and post-monsoon. During the monsoon, there is a higher discharge of leachate and surface runoff compared to the dry season, which could be one of the factors responsible for higher numbers of coliforms in groundwater in the vicinity of the dumpsite. Further, the water sources in the area are not protected by any measures to minimize pollution.

Landfill leachate often offers a conducive environment for bacterial growth and thereby may contain high bacterial density (Grisey et al. 2010; Abiriga et al. 2021b). Grisey et al. (2010) reported total coliform density as 20,000 CFU/100 mL, E. coli and Enterococci density as 15,199 CFU/100 mL and 3,290 CFU/100 mL, respectively in groundwater collected near an abandoned sanitary landfill. Their study demonstrated that the landfill was not a significant source of bacterial load in this case and the major reason for the high coliform count was faecal inputs from septic tanks. The faecal coliform count can be in the range of 10⁸ CFU/100 mL in regions significantly influenced by septic tank influents (Sinton 1982). Like the higher bacterial load found in this study, Gwimbi et al. (2019) also reported that the E. coli count was substantially lower (maximum 4,800 CFU/100 mL) in protected water sources (springs, open wells, streams) compared to water sources that were unprotected (4.35 × 10⁷ CFU/100 ml). The main reasons attributed to such high counts of enteric pathogens were due to faecal contamination due to open defecation, faecal matter from livestock, latrines near water sources and unhygienic routines.

Bacterial contamination in groundwater was found to be low during summer but the bacterial density increased considerably during the monsoon season. HPC in the summer season was in the range of 4.2 × 10² to 5.1 × 10³ CFU/mL and it increased to 3.9 × 10⁴ CFU/mL during the monsoon season. Similar results were also recorded for faecal coliforms (1.7 × 10⁴ CFU/mL) in the monsoon, and it was comparatively lower in summer (1.6 × 10³ CFU/mL). Contrary to the present study, Grisey et al. (2010) noted that the coliform count in groundwater influenced by MSW landfill and faecal inputs was higher in summer than in the monsoon season. However, the present results agree with the work of Pujari et al. (2007) who recorded that the bacterial population can be higher during the monsoon than in summer as the survival rate of pathogens is better due to higher water flux. They also concluded that during the monsoon the pathogens can move faster in the unsaturated zone.

Spatial variations in microbial populations were also assessed (Figure 3). The degree of bacterial load in various zones declined as the distance increased from the dumpsite. The zone-wise data showed that the nearest point to the dump site (within 0.5 km) where three samples (2GW1, 2GW2 and 2GW3) were taken, exhibited HPC of 1.3 × 10⁴ CFU/mL and faecal coliform of 6.8 × 10³ CFU/mL. The control site DW1 showed fewer numbers of faecal coliforms (3.9 × 10³ CFU/mL) when compared to other sites. Faecal contamination detection at the control site can be attributed to the poor sanitation conditions maintained by the residents resulting in easy contamination of the open dug well (Gwimbi et al. 2019). Low microbial contamination was recorded in DW2 and DW3 as they are far (∼5 km) from the dumpsite. 2GW6 and 3GW1 were identified as moderately affected areas as the level of microbial pollution was relatively lower than that in sites close to the dumpsite. HPC, TC and FC of these sites were 1.2 × 10⁴ CFU/mL, 8.2 × 10³ CFU/mL and 5.8 × 10³ CFU/mL, respectively. Suthar et al. (2009) also suggested that an increased bacterial population can be found more around nearby solid waste dumpsites. Bacterial load based on the distance from the dump site confirmed that the area is getting affected by the open MSW dumpsite.

The correlation coefficient was determined between the distance of sampling points with HPC, TC and FC which showed a negative correlation between distance and HPC (-0.998), distance and TC (-0.969) and distance with FC (-0.906) (Table 1). These data signify that groundwater samples around the MSW dumping site were impacted due to unscientific disposal of solid waste and landfill leachate generation where the degree of microbial contamination gradually lowered with increasing distance from the dump site. On the other hand, HPC showed a high degree of correlation with TC (0.976) and FC (0.921) while TC was significantly correlated with FC (0.981). Data were subjected to one-way ANOVA to test the differences in the mean of different types of bacteria zones wise. For instance, there was no significant difference between HPC, TC and FC at the nearest zone (0.5 km) and farthest zone (5 km) of the dumpsite. There was a significant difference between different bacterial groups (F_5.14 = 5.82, P < 0.05) at 1.5 km from the dumpsite. Data was also tested with two-factor ANOVA to find the statistical significance of means between the bacterial groups and the distance of the site from landfill. There was a significant difference between the types of bacteria detected (F_3.40 = 21.45, P < 0.05) and the distance from landfill was statistically significant in terms of influencing the existence of bacteria (F_3.00 = 5.29, P < 0.05) (see supplementary data for ANOVA tables). Another study at same site has shown that the groundwater quality in this region was poor and there was lateral movement of leachate up to 5 km from the boundaries of MSW dumpsite (Chakraborty & Kumar 2016).

The presence of faecal coliforms in groundwater is a major concern as it affects the potability of water, whereas the local population in developing countries mainly depends only on these water sources for drinking often without any type of treatment or minimal treatment. To know the major types of bacteria present, morphologically identical colonies were primarily screened visually which were members of the Enterobactoriaceae family as indicated by typical colony characteristics such as mucoid, smaller, pinkish colonies when cultured on EMB agar. EMB agar medium helps to differentiate E. coli and Enterobacter, where the presence of E. coli can be easily identified by greenish metallic sheen colonies. The secondary screening was carried out by using the 16S rDNA genome sequencing method to identify the species and establish the taxonomic position of the isolates. Figure 4 indicates that the isolate (Klebsiella ssnkbit, KU647674) was almost 99% similar (through NCBI BLAST) with K. pneumoniae, strain S001 (Accession No. KC524425).

K. pneumoniae is known to cause pneumonia, infant bronchitis, several pulmonary diseases etc. It must be noted that the main pathway for the spread of K. pneumoniae is person-to-person contact and it is typically not linked with waterborne diseases. NCBI BLAST tree view software was used to construct the phylogenetic tree (Query_64527) based on a neighbour-joining analysis of 1,000 replicates (Figure 4). Suthar et al. (2009) also reported the presence of K. pneumonia in 90% of the groundwater samples of rural areas in northern Rajasthan, India. The presence of faecal coliforms and subsequent detection of K. pneumoniae highlighted that the groundwater in the study area is seriously impacted due to open dumping of MSW. Open dumping appears to be the major source of bacterial contamination and it is not to be discounted on the fact that human settlements near dumpsites (with very poor sanitary conditions), livestock faecal matter and surface runoff may also contribute to bacterial density in groundwater that is completely unprotected. Further studies on identifying the contribution of landfill leachate and other sources of bacterial load will be interesting to gain detailed insights for site remediation and management.

Groundwater sources around the MSW dumpsite were evaluated to find the temporal and spatial variations in the bacterial population. Open MSW disposal is strongly affecting the groundwater in terms of bacterial contamination. Most of the groundwater sources in the study area exhibited a wide range of the bacterial population. The heterotrophic plate count of groundwater samples showed the highest bacterial population of 4.5 × 10⁴ CFU/mL and the maximum faecal coliform of 2.1 × 10⁴ CFU/mL was at a hand pump source near the MSW dumping area. Bacterial contamination was found to be very high during monsoon and the contamination gradually declined with increasing atmospheric temperature. In terms of spatial contamination, a nearby area of the MSW dumping site was most affected, whereas the impact was found to be dispersed up to 5 km from the dumping site. Molecular characterization of the faecal coliform colonies revealed the presence of K. pneumoniae in groundwater samples of the area, a causative agent for pneumoniosis, infant bronchitis, several pulmonary diseases etc. The groundwater microbiology indicates a high level of contamination, which requires disinfection before consumption and other domestic use. It will be better to avoid using untreated groundwater at least 5 km from the periphery of the dumpsite and groundwater from such areas must not be consumed directly without effective disinfection during any season. Further research on source identification to know the contribution of landfill leachate and other sources of bacterial load will be useful for site remediation and management.

Financial assistance provided by the University Grants Commission (UGC), New Delhi, India through a major research project (No. 42-426/2013) to carry out this work is thankfully acknowledged. We thank Dr Rajib Bandopadhyay, University of Burdwan, India and Dr Amit Kumar Gorai, NIT, Rourkela, India for the support provided in carrying out this study. The authors thank the reviewers for the useful comments on an earlier version, which helped to improve the manuscript.

Shubhrasekhar Chakraborty: Conceptualization, Data curation, Formal analysis, Methodology, Field survey, Writing – original draft. Mohini Verma: Methodology, Data curation, Field survey. Jawed Iqbal: Software, Validation, Supervision, Writing – review and editing. R. Naresh Kumar: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – review and editing.

All relevant data are included in the paper or its Supplementary Information.

The authors have no competing interests to declare.