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Vol.:(0123456789)
Environment, Development and Sustainability
https://doi.org/10.1007/s10668-023-02914-w
1 3
Identification ofmicrobiogeochemical factors responsible
forarsenic release andmobilization, andisolation ofheavy
metal hyper‑tolerant bacterium fromirrigation well water:
acase study inRural Bengal
SandipanBarman1· DebjaniMandal2· PinakiGhosh3· AyanDas1·
MadhurinaMajumder1· DebankurChatterjee4· DebashisChatterjee1·
IndranilSaha5· AbhishekBasu2
Received: 17 March 2022 / Accepted: 2 January 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2023
Abstract
In Bengal Delta Plain (West Bengal and Bangladesh), shallow aquifer (< 50m) groundwa-
ter is often used in irrigation for paddy cultivation. The present study highlights the role
of anthropogenic activities on natural process and focuses on arsenic mobilization in the
shallow irrigation well water of rural Bengal. The major focus is to examine the role of
geochemistry, arsenic mobilization and their association with local microbial community
in irrigation well water. The results suggest that the groundwater of monitored wells is usu-
ally devoid of dissolved oxygen and consists of high concentration of dissolved redox ele-
ments like arsenic (AsT) (mean value 58.7 µgL−1) and iron (FeT) (mean value 2.6 mgL−1),
with low amount of oxyanions like sulphate (SO4
2−) and nitrate (NO3
−) (mean value 8.3
mgL−1 and 1.8 mgL−1, respectively). High concentration of alkaline earth metals like cal-
cium (Ca) (mean value 252.0 mgL−1) and magnesium (Mg) (mean value 96.9 mgL−1) with
high alkalinity (mean value 400.3 mgL−1) suggests that carbonate dissolution (calcite and
dolomite) is the key process in these monitored wells. The factor analysis reveals a positive
and strong correlation (r2 = 0.716) between arsenic (As) and iron (Fe). Some of the wells
are contaminated with high concentration of chloride (up to 128.5 mgL−1), total coliforms
(no. of wells contaminated with coliforms = 7) and faecal coliforms (no. of wells contam-
inated with faecal coliforms = 5), which point towards the local anthropogenic influence
(leakage of sewage). Some irrigation wells with non-permissible arsenic concentration also
harbour arsenic (As3+) and iron (Fe2+) hyper-tolerant bacteria. The microenvironment of
the irrigation wells consisting of suitable local reducing condition together advocates the
dominance of existing microbial community, particularly arsenic and iron hyper-tolerant
bacteria in them. In this study, an arsenic and iron hyper-tolerant bacterium has been iso-
lated from irrigation well water. Scanning electron microscopy and 16S rDNA sequencing
further established the identity of this bacterium as Enterobacter sp., which is a faculta-
tive anaerobe. The addition of fresh organic matter through local anthropogenic activities
could enhance the microbial activity in these monitored wells. The silver nitrate test reveals
Sandipan Barman and Debjani Mandal have contributed equally.
Extended author information available on the last page of the article
S.Barman et al.
1 3
the biotransformation potential of this bacterium from arsenite (As3+) to arsenate (As5+).
Altogether, these results point towards the dependence of arsenic mobilization process on
multiple microbial and geochemical factors, where bacterium like Enterobacter sp. might
also play an important role. Such biogeochemical processes are not only responsible for
the unsafe nature of irrigation well water (for both domestic and irrigation purposes) in
rural Bengal, but also convert it into a major potential source for soil arsenic accumulation,
thereby contaminating thefood chain.
Keywords Groundwater hydrochemistry· Arsenic mobilization· Irrigation well water·
Faecal coliform· Arsenic hyper-tolerant bacteria
1 Introduction
Groundwater is one of the most important sources of arsenic (As) exposure in human pop-
ulation. Arsenic (As) contamination of groundwater has emerged as a major quality prob-
lem, and it is associated with public health issues throughout the globe, notably in Ben-
gal Delta Plain (BDP) of South East Asia (Bhattacharyya etal., 2002a; Chatterjee etal.,
2005; Nriagu etal., 2007; Mukherjee and Bromssen 2008). In addition to geogenic origin
of arsenic-contaminated Holocene alluvial soil and deltaic sediments of BDP, uncontrolled
anthropogenic application of arsenical compounds as effective herbicides, insecticides and
fertilizers adds unprecedented level of arsenic in irrigation fields. Arsenic is also added to
natural water bodies in the form of cement, metallurgical, glass and pulp wastes. Although
arsenic is released from the sediments by natural biogeochemical processes, anthropogenic
activities enhance the rate, severity and extent of arsenic contamination of groundwater and
soil. West Bengal is one of the major arsenic-contaminated states of India. The groundwa-
ter samples from various parts of West Bengal were found to contain arsenic (As) beyond
the national permissible limit (0.05 mgL−1) in drinking water (BIS, 2012; Chakraborti
etal., 2009; Singh, 2006).
In West Bengal, summer season rice (boro) cultivation has greatly increased over the
past few decades and now contributes to 50% of national rice production. Due to the irreg-
ular monsoon pattern, the farmers are compelled to execute the irrigation demand by using
groundwater from shallow tube wells, where the groundwater are often contaminated with
high concentration of arsenic (As) (~ 200 µgL−1). The prolonged application of arsenic
contaminated groundwater for irrigation in the paddy field may increase arsenic (As) con-
tent in the soil and enhance its uptake in the staple crop (Chatterjee and Halder 2010).
Therefore, food crops and vegetables cultivated in arsenic contaminated soil or irrigated
with arsenic-contaminated groundwater play a significant role in biomagnification of arse-
nic in the food chain, which leads to serious health hazards when consumed (Roychowd-
hury et al. 2005; Roy chowdhury et al., 2002; Santra et al., 2013). Hence, scale of the
arsenic toxicity is grave and unprecedented, in terms of both human exposure (~ 60–80
million) and geographical area coverage of West Bengal (173 × 103 km2) (Bhattacharya
etal., 2003).
Arsenic chemistry and mobilization depend on multiple factors like physicochemical
parameters (temperature, pH, redox conditions, salinity, etc.) of soil and water, adsorption
and desorption of arsenic (As) species present, organic content and oxides present, bio-
chemical processes, microbial communities, etc. Aquifers can have oxidizing or reducing
environment, which, consequently, regulate the physicochemical properties (adsorption,
Identification ofmicrobiogeochemical factors responsible…
1 3
desorption, ionic charge, solubility, etc.) of chemical species present in them. Different
chemical constituents predominate in different aquifers, which in turn govern the release
and mobilization of arsenic species in aquifers and its adjacent area (Shrivastava et al.,
2014). Arsenite (As3+) is 5–10 times more mobile and soluble than arsenate (As5+). The
mobility and solubility of arsenite (As3+) increase in reducing alkaline environment. On
the other hand, arsenate (As5+) is more strongly adsorbed on iron oxides and is less soluble
and mobile than arsenite (As3+). Therefore, in alkaline-reducing aquifer, arsenate tends to
get precipitated with ferric hydroxide, whereas arsenite might stay in the solution due to
its increased solubility and mobility. In contrast to this, in alkaline-oxidizing aquifer, the
adsorption of arsenate on iron oxides weakens and arsenate is easily desorbed from active
surfaces of iron oxides (Singh, 2006).
The microbial communities present in the aquifer also influence the physicochemical
parameters and the rate of biochemical reactions, involving arsenic and other predominant
ions of the aquifer. Microbes participate in oxidation, reduction, adsorption and desorption
of various ions on active mineral surfaces either directly or indirectly, which in turn affect
the arsenic cycle in aquifer and the environment. Sewage contamination of the groundwa-
ter is a predominant worldwide phenomenon. This phenomenon not only alters the chemi-
cal composition and organic content of the groundwater, but also adds harmful pathogenic
bacteria like faecal coliforms to it. These bacteria could further enter into the food chain
through irrigation water in crops and vegetables and may cause fatal gastroenteric diseases.
Anthropogenic processes like open defecation, open dumping, leakage of sanitation pits,
mixing of sewage water and faulty construction of wells are responsible for microbial con-
tamination of groundwater and shallow aquifers, which is a predominant worldwide health
problem. Microbe mediated decomposition of organic matter results in a reducing envi-
ronment in the water system, thereby altering or enhancing the biogeochemical process
of arsenic release and mobilization (Vengosh and Keren 1996; Kass etal., 2005; Singh,
2006; Onwuka et al., 2019). Organic matter serves as energy source for the increased
microbial activity. Organic matter also enhances the metabolic processes of arsenic-oxi-
dizing and arsenic-reducing bacteria inhabiting the water system. Arsenic-oxidizing and
arsenic-reducing bacteria are resistant to different species of arsenic. These bacteria are
responsible for enzymatic transformation of some arsenic species, thereby influencing the
status of predominant arsenic species in aquifers. Arsenic-oxidizing bacteria oxidize arsen-
ite (As3+) into arsenate (As5+), whereas arsenic-reducing bacteria reduce arsenate (As5+)
to arsenite (As3+). Many arsenic-resistant bacteria accumulate (bioaccumulation) or adsorb
(bioadsorption) arsenic in them or on their membrane, respectively, which in turn reduces
the concentration of arsenic species in the aquifer. Various iron and sulphate-reducing bac-
teria also play a vital role in arsenic mobilization, in groundwater and shallow aquifers.
These microbial processes along with nature and type of organic matter, physicochemical
reactions and geochemical processes, aid in arsenic cycling in the aquifer. In a nutshell,
both microbial and geochemical factors are important for studying arsenic release and
mobilization in aquifers, especially in rural Bengal (Alegbeleye etal., 2018; Jiang et al.,
2019; Mandal etal., 2021; Singh, 2006).
In Bengal Delta Plain (BDP), extensive research was carried out for understanding the
geochemical processing of arsenic (As) and its release mechanism (Chatterjee and Halder
2010; Halder etal., 2013; Bhattacharya et al., 2003). However, the interdependence and
coordination of microbial and geochemical factors and processes received scant research
attention and hence form the major scientific gap in understanding high arsenic concentra-
tion, its release and mobilization in irrigation well water. Also, the correlation between
arsenic distribution, mobilization, and microbiogeochemical environment, depth and year
S.Barman et al.
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of installation of irrigation wells, has not yet been investigated thoroughly. Role of anthro-
pogenic activities in alteration of microbiogeochemical factors and the effect of such alter-
ation in arsenic distribution, release and mobilization remains unexplored. In this study,
we hypothesize that arsenic release and mobilization/immobilization are mediated by the
coordination and interplay of geochemical parameters and microbial factors in the irriga-
tion wells of Chakdaha block, a well-known arsenic-contaminated region of West Bengal.
The study focuses on important geochemical factors and their correlation with variation
of arsenic (As) level, its distribution pattern, groundwater composition and hydrochemis-
try. A relationship has been established between microbial quality parameters (most prob-
able number (MPN) index and faecal coliform (Fc)) and a conservative chemical parameter
(chloride) of the groundwater. Also, an arsenic and iron hyper-tolerant bacterium has been
isolated in this study. The role of this bacterium in biotransformation of arsenic has been
predicted, which might affect the geochemical processes of the aquifer and regulate arsenic
release and mobilization/immobilization. Finally, the presence of faecal coliform bacteria
in irrigation well water strongly argues for policy regulation to prevent unplanned human
habitation and associated anthropogenic activities to safeguard human health and micro-
scale rural environment.
2 Materials andmethods
2.1 Selection ofstudy area
The study area (Chakdaha block; latitude: 23000´20´´ N-23005´20´´ N; longitude:
88031´40´´ E-88049´00´´ E) is an integral part of world’s largest delta (Ganges–Brahma-
putra–Meghna deltaic alluvium, GBM system), located in the eastern side of the regional
river Ganga (Fig. 1). The lower stretch of the river Ganga (Hooghly river) is the main
drainage system of the region, flowing up to the Bay of Bengal from North-West to
Fig. 1 Location map of the study area (Chakdaha Block, Nadia District, West Bengal). The location and
distribution of the irrigation wells were shown by red spots
Identification ofmicrobiogeochemical factors responsible…
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Southward direction. The area is comparatively flat and intersected with major and minor
rivers, small streams and abundant channels (Nath etal., 2005). Several drinking water
wells contaminated with high concentration of arsenic (As) (up to 500 mgL−1) have been
already reported from this area (Bandyopadhyay, 2002; Chatterjee etal., 2005; Gault etal.,
2005; Nath etal., 2005). Irrigation tube wells are usually privately owned and have a depth
of 25–40m. These wells are generally not considered for arsenic (As)-monitoring program
during the campaign by the local government and other monitoring agencies. The large
numbers of irrigation wells with a density up to 20/km2 are used mostly during summer
months (March–July) for agricultural purpose (Chatterjee and Halder 2010). The change
in sanitation practice (use of pit latrine) and installation of many shallow irrigation tube
wells can be correlated to the improvement in public health status and Green Revolution,
respectively. Earlier, researchers predominantly focused on groundwater chemistry and
mitigation approaches (Srivastava etal., 2014; Mitra etal., 2017; A. Das, 2020; S. Koley,
2021). However, the microbial and geochemical factors and their relationship with arsenic
release and mobilization remain less explored in Chakdah block and therefore, need further
investigation.
2.2 Sampling andanalytical methods
The sampling and estimation of various chemical parameters were carried out in accord-
ance with themanual developed by American Public Health Association (APHA) and US
Environmental Protection Agency (USEPA), and methods used by Maity etal., Saywell
and Cunningham, and Saalidong et al. (Saywell and Cunningham, 1937; USEPA, 1999;
Maity etal., 2004; APHA, 2017; Saalidong etal., 2022). Some field parameters were esti-
mated insitu. Precautions were taken to minimize microbial growth, unwanted precipita-
tion and adsorption of ions on container surface.
2.2.1 Sampling strategy
The samples were collected in frequent interval of time (once in a week) during the month
of March to May (pre-monsoon time), when the boro-rice cultivation is the main practice
in the agricultural field. Groundwater samples were taken three times from each irrigation
well, to measure the on-site parameters precisely at the well head. Before the sampling,
irrigation wells were allowed to discharge (continuously) the standing volume of ground-
water (depending on the depth) in order to collect fresh groundwater from the aquifer. Con-
ductivity, pH and Eh values of water samples were measured at the well head using a pre-
calibrated multimeter (WTW, Germany).
2.2.2 Sample collection andanalysis
For chemical analysis, groundwater samples were collected from 40 irrigation wells in
pre-cleaned plastic bottles and filtered at the site using 0.45 µm membrane. The stand-
ard amount of discharge from the tube wells is 12,000–15,000 cubic litre/hour. Samples
used for the analysis of total arsenic (AsT) and total iron (FeT) were acidified with nitric
acid (0.25% v/v, Suprapur, MERCK). Arsenic (As) concentration was estimated by HG/GF
mode Atomic Absorption Spectrophotometer (AAS-240, Varian Inc.), while major cations
were analysed by ion chromatography (761 Compact IC, Metrohm). Unacidified samples
were used for the analysis of major anions using IC (761 Compact IC, Metrohm). Field
S.Barman et al.
1 3
parameters like pH, Eh, electrical conductivity, temperature and alkalinity (titration with
0.02M H2SO4) were measured in situ. FeT concentration was measured colorimetrically
following the 1, 10 o-phenanthroline method using Perkin Elmer (Lambda-20) spectropho-
tometer. Dissolved oxygen (DO) and total dissolved solid (TDS) are two important param-
eters which are the indicators of water quality. DO was measured by modified Winkler’s
method and TDS was estimated using standard probe electrodes.
2.2.3 Statistical analysis
As a statistical technique, factor analysis was done to investigate the relationship among
the variables obtained from the analysis of groundwater samples. For the interpretation
of the factor analysis (loadings), the factor axis was rotated by the normalized varimax
method. The extracted factors were rotated in a way, so that the variance of the factor load-
ings tended to a maximum. A high loading close to ± 1.0 indicates a strong relationship
between factor and variable, while a low loading close to zero (or negative) indicates the
lack of correlation.
2.2.4 Coliform analysis
Coliform bacteria in the groundwater of the irrigation wells were detected by culturing the
samples in lactose broth medium. Bacterial fermentation of lactose was detected byusing
pH indicator bromothymol blue and formation of gas bubbles in the Durham’s tube. The
presence of coliform bacteria was further confirmed by plating the groundwater samples in
Eosin Methylene Blue (EMB)-Agar selective medium. For detection of coliform bacteria,
culture tubes containing lactose broth (pH—7.2) and irrigation well water were incubated
at 37°C for 48h. For detection of faecal coliform (thermo-tolerant), culture tubes were
incubated at 44.5°C for 48h. The most probable number (MPN) is an important biological
parameter estimated to check the quality of water. For determination of MPN index irri-
gation well water were incubated at the abovementioned conditions in lactose broth. The
experiment was done in 3 sets (with 5 tubes per set). In the first set, double-strength (2X)
lactose broth was used as the medium, followed by single-strength (1X) lactose broth in
other two sets. 10ml, 1ml and 0.1ml of groundwater sample was used as inoculums in the
first set (with 2X lactose broth), second set (with 1X lactose broth) and the third set (with
1X lactose broth), respectively. The change of colour of the medium from green to yellow
and formation of gas bubbles in Durham’s tube indicate the presence of coliform and a
positive score. Five tubes were cultured for each set and from the number of positive scores
MPN index was determined.
2.3 Isolation, characterization andidentification ofarsenic hyper‑tolerant bacteria
Isolation, characterization and identification of arsenic hyper-tolerant bacteria were done
by well-established methods. Isolation of arsenic hyper-tolerant bacteria was done accord-
ing to the method described by Dey etal., (2016) and Mandal etal., (2021). Characteriza-
tion and identification of arsenic hyper-tolerant bacteria were performed by standard meth-
ods like scanning electron microscopy, 16S rDNA sequencing and subsequent phylogenetic
analysis. The biotransformation potential of the bacterium was analysed by the method
proposed by Dey etal., (2016), Krumova etal., (2008) and Mandal etal., (2021).
Identification ofmicrobiogeochemical factors responsible…
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2.3.1 Isolation ofarsenic hyper‑tolerant bacteria
Water samples collected from different irrigation wells were tested for the presence of
arsenic hyper-tolerant bacteria. Water sample collected from well no. 14 of Parari vil-
lage was found to be contaminated with 126ppb of total arsenic and harbours arsenic
hyper-tolerant bacteria. Arsenic-resistant bacteria were isolated by serial dilution (up to
104 times) and subsequent inoculation of the water sample on LB-agar medium in the
presence of arsenite. Spread plate method was used as the inoculation method of choice
in the presence of 0.077 mM arsenite (10 ppm sodium arsenite). The plate was incu-
bated at 37 ºC for 24h. Morphologically different colonies were selected and further
tested for their arsenic hyper-tolerance potential in the presence of increasing concen-
tration of arsenite (up to 1.924 mM arsenite (250 ppm sodium arsenite)). The inocu-
lated LB-agar and LB liquid medium were incubated at 37 ºC for 24h and shaking was
done at 160rpm for all subsequent experiments with LB liquid cultures. The positive
bacterial growth was determined by turbidimetric analysis and bacterial colony forma-
tion, in LB liquid and LB-agar medium, respectively. One arsenic hyper-tolerant isolate
(DACW8) was able to grow profoundly in the presence of 1.924mM arsenite (250ppm
sodium arsenite) and therefore, was selected for the downstream work.
2.3.2 Scanning electron microscope imaging oftheisolated bacteria
The bacterial suspension culture was fixed in 3% glutaraldehyde (in 0.1M phosphate
buffer, pH 7.2) for 1h. The fixed cells were washed at least three times with buffer. Post-
fixation of the fixed cells was done with 1% osmium tetroxide for 1h. Subsequent dehy-
dration of cells was carried out in increasing concentration of alcohol (30%, 50%, 75%,
90% and 100%) for 5min in each solution. 0.2-μ black polycarbonate filter was used to
filter the cells and subsequently,mounting was done onto SEM stub. Cells were sputter-
coated with 10nm gold and observed under 15kV scanning electron microscope.
2.3.3 16S rDNA sequencing andphylogenetic analysis foridentification ofthe isolate
From DACW8 pure culture pellet, the genomic DNA was isolated and subjected to
quality checking by agarose gel electrophoresis. The genomic DNA was taken as tem-
plate for amplification of 1.5kb rDNA using highfidelity DNA polymerase and 27F and
1492R universal forward and reverse primer sequence. The PCR amplicon was purified
to remove contaminants. Forward and reverse DNA sequencing reaction of PCR ampli-
con was carried out with 27F and 1492R primers using BDT v3.1 Cycle sequencing kit
on ABI 3730xl Genetic Analyzer. Consensus sequence of 16S rDNA gene was gener-
ated from forward and reverse sequence data using aligner software. The 16S rDNA
gene sequence was used to carry out BLAST with the ‘nr’ database of NCBI GenBank
database. The phylogenetic tree was constructed using hits with the lowest E-value and
the highest query coverage. The evolutionary history and evolutionary distances were
determined using the neighbour joining method and Jukes–Cantor method, respectively.
Evolutionary analysis was conducted in MEGA X (Jukes & Cantor, 1969; Saitou and
Nei 1987; Kumar etal., 2018).
S.Barman et al.
1 3
2.3.4 Determination ofmaximum tolerance limit ofheavy metals forarsenic
hyper‑tolerant bacteria
The maximum tolerance limit was determined by inoculating and streaking bacterial
culture in LB-agar and LB-liquid medium, with increasing concentration of arsenite
and arsenate. In both the cases, bacterial culture at log phase (with O.D. 0.4) was used
as inoculum. The concentration of sodium arsenite and sodium arsenate was increased
starting from 500ppm (3.848mM arsenite) and 1000ppm (7.697mM arsenate), respec-
tively, in LB-agar and LB-liquid medium. The bacterium DACW8 was also cultured
in increasing concentration of ferrous sulphate starting from 1mM. The cultures were
incubated at 37 ºC for 24h. The positive bacterial growth was determined by turbi-
dimetric analysis and bacterial colony formation, in LB-liquid and LB-agar medium,
respectively.
2.3.5 Growth curve study oftheisolated arsenic hyper‑tolerant bacteria
The growth curve study of DACW8 was done in the absence of arsenite and in the pres-
ence of four different concentrations of arsenite, 0.77mM arsenite (100ppm sodium arsen-
ite), 3.848mM arsenite (500ppm sodium arsenite), 7.697mM arsenite (1000ppm sodium
arsenite) and 15.395mM arsenite (2000ppm sodium arsenite). The LB medium with or
without arsenite was inoculated with DACW8 pure culture. The optical density as growth
indicator was measured at 600nm. Microsoft Excel was used to plot the graph with mean
and the standard deviation of three sets of same experiment.
2.3.6 Biotransformation ofarsenite byisolated arsenic hyper‑tolerant bacteria
The biotransformation potential of the isolate was tested by silver nitrate test in LB-agar
medium supplemented with 7.697 mM arsenite (1000 ppm sodium arsenite) (Dey et al.,
2016; Krumova etal., 2008; Mandal etal., 2021). The bacterium was inoculated and incu-
bated in LB-agar medium with (experimental set) or without sodium arsenite (control set)
at 37 ºC for 48h. In the absence of light, subsequent addition of silver nitrate solution was
done on LB-agar medium. After 72h of incubation, the colonies were observed for change
in colour (Dey etal., 2016; Krumova etal., 2008; Mandal etal., 2021). The oxidation of
arsenite to arsenate and subsequent formation of silver arsenate turn the bacterial colonies
brown, which indicate the biotransformation potential of the bacterial strain.
3 Results
The study area (Chakdaha block) is a part of Ganges–Brahmaputra–Meghna deltaic allu-
vium located in the eastern side of the Ganges (Fig. 1). The study area is adorned with
numerous geo-morphological features like series of meander scars of varied wavelength
and amplitude, oxbow lakes, abandoned channels, inter-distributary swamps, levees and
flood basins with steady southern slopes. The land-use pattern of the study area consists
of farming land (up to ~ 75%), where rice cultivation is the foremost agricultural practice.
The climate is characteristically warm and humid (temperature ranges between 10 and 40
Identification ofmicrobiogeochemical factors responsible…
1 3
0C, average relative humidity > 60%) with yearly precipitation ranging between 1172 and
1635mm (mean 1436mm) (Majumder etal., 2016; Nath etal., 2005).
3.1 Characteristics oftheirrigation wells
Sahishpur, Mathurgachi, Banamalipara, Chakudanga, Bishnupur, Neulia, Silinda, Dubra,
Tangra village of Chakdaha blocks were selected for survey of 40 irrigation wells (Fig.1).
Local farmers of these villages practise the cultivation of boro-rice (summer rice variety).
The irrigation wells not monitored during groundwater quality surveillance programme
and/or arsenic (As) awareness campaign were selected for this study. Characteristics of
the irrigation wells are summarized in Table 1. The installation year of irrigation wells
varies from 1985 to 2010. The installation process has shown significant increase (58%)
during 1995–2005, which could be attributed to the ‘Green Revolution’ campaign by the
local government. Financial assistance was provided to the local farmers for drilling shal-
low irrigation wells (< 50 m) for summer paddy cultivation using groundwater of BDP.
The depth of the irrigation wells varies between 15 and 42m depending upon the local
aquifer condition. Shallow near-surface aquifers (< 20m) were often trapped for irrigation
of summer paddy, where the groundwater table is high (Bandyopadhyay, 2002; Chatterjee
and Halder 2010; Gault etal., 2005; Nath etal., 2005; Bhattacharya etal., 2002b; Bhat-
tacharyya etal., 2003; Ahmed etal., 2004). Nearly 52% of the monitored wells are found
within the depth of 8–20m. Only 30% tube wells are installed in relatively deeper aquifer
(> 25m). These wells are used for round the year supply of irrigation water for cultivation
of crops, vegetables (like pumpkin, tomato, cabbage, chilli, cucumber, cauliflower, green
bean, spinach, carrot, purple onion, eggplant, beet, etc.) and seasonal flowers (marigold,
tuberose, sunflower, etc.). The arsenic (As) levels in these relatively deeper wells are often
high in comparison with near-surface (shallow aquifer) irrigation wells.
3.2 Groundwater composition andhydrochemistry
3.2.1 Physicochemical parameters ofthegroundwater used forirrigation
Physicochemical parameters like pH, EC, DO, Eh and alkalinity of the groundwater sam-
ples were measured. The results are summarized in Table2. The pH of the groundwater
ranges from 6.88 to 7.6 with a neutral mean value of 7.07, which indicates that the pH is
well buffered (low SD value) and the groundwater is suitable for human and agricultural
use. The conductivity of the groundwater is in medium-to-high range (516–894 µscm−2)
with a mean value of 643.52 µscm−2. This suggests that the groundwater is fresh in nature
and enriched with dissolved ions responsible for the maintenance of sediment–water inter-
action. The Eh of the groundwater indicates the nature of aquifer. A positive Eh value shows
that the aquifer is oxic, whereas negative value point towards anoxic character. The pre-
sent study reveals that the Eh of the groundwater is throughout negative with the mean
value of -6.18 and low SD value. It has been reported that iron (Fe)minerals are extremely
important for the release of arsenic (As). The most probable and accepted mechanism of
arsenic mobilization is reductive dissolution of Fe-oxyhydroxides along with metal-reduc-
ing microbial activity, which desorbs arsenic (As) into the groundwater and subsequently,
enhance the arsenic mobilization (Sutton etal., 2009; Shankar etal., 2014). DO value was
recorded to be very low or absent (regardless of the discharge pattern of the tube wells or
the land-use pattern) again indicating towards the reducing condition of the aquifer. The
S.Barman et al.
1 3
Table 1 Summary of monitored irrigation wells
Sample No. Villages Location Installation year Depth (m) Irrigated area
(hectares)
Cultivation
1 Mathurgachi 2304′35.8"N 88036′35.3"E 2001 18 3.7 Paddy
2 Mathurgachi 2304′43.2"N 88036′35.7"E 2000 21 5.0 Paddy
3 Mathurgachi 2304′36.2"N 88036′37.4"E 2000 18 5.0 Paddy/vegetables
4 Mathurgachi 2304′36.8"N 88036′37.3"E 2002 19 5.3 Paddy
5 Mathurgachi 2304′36.8"N 88036′37.4"E 2001 20 2.0 Paddy/vegetables
6 Neulia 2304′50.8"N 88036′1.5"E 2000 19 5.0 Paddy
7 Neulia 2304′49.6"N 88036′58.7"E 2000 21 6.3 Paddy
8 Neulia 2304′54"N
88036′0.2"E
2008 18 2.2 Paddy/vegetables
9 Neulia 2304′50.1"N 88036′36.9"E 2000 28 5.3 Paddy
10 Neulia 2304′35.5"N 88036′21.2"E 2000 36 4.0 Paddy
11 Neulia 2304′32"N
88036′19.7"E
2000 28 2.5 Paddy/vegetables
12 Parari 2303′56.2"N 88035′51.2"E 1997 15 3.7 Paddy/vegetables
13 Parari 2303′54.7"N 88035′52.6"E 2006 18 5.0 Paddy
14 Parari 23°05′05.8"N
88035′58.2"E
2005 42 5.3 Paddy
15 Parari 2304′5.3"N 88036′32.8"E 2009 15 3.7 Paddy/vegetables
16 Parari 2304′14.8"N
88036′37"E
1999 26 4.2 Paddy
17 Parari 2303′37.6"N 88037′27.7"E 2009 18 4.8 Paddy
18 Parari 2303′37.7"N 88037′32.9"E 2002 42 3.7 Paddy
19 Banamalipara 2303′40.9"N 88037′33.5"E 1988 15 4.0 Paddy
20 Banamalipara 2303′35.3"N 88037′35.2"E 2007 21 5.3 Paddy/vegetables
21 Banamalipara 2304′18.4"N 88036′26.8"E 1985 27 6.3 Paddy
22 Banamalipara 2304′20.1"N 88036′26.8"E 2001 28 4.8 Paddy
23 Banamalipara 2303′48.5"N 88037′14.7"E 1987 16 2.2 Paddy
Identification ofmicrobiogeochemical factors responsible…
1 3
Table 1 (continued)
Sample No. Villages Location Installation year Depth (m) Irrigated area
(hectares)
Cultivation
24 Banamalipara 2303′44.2"N 88037′16.5"E 1996 18 1.2 Paddy
25 Santra 2303′57.1"N 88037′23.2"E 1985 25 3.7 Paddy/vegetables
26 Santra 2303′54.5"N 88037′32.8"E 1996 24 5.3 Paddy/vegetables
27 Santra 2304′1.0"N 88037′11.9"E 1999 24 1.7 Paddy
28 Santra 2305′10.2"N
8037′4.5"E
2010 18 2.7 Paddy
29 Sahishpur 2303′42.8"N 88036′15.3"E 1992 19 5.0 Paddy/vegetables
30 Sahishpur 2303′37.7"N 88037′32.9"E 2002 20 3.7 Paddy
31 Sahishpur 2303′40.9"N 88037′33.5"E 1988 19 2.5 Paddy/vegetables
32 Sahishpur 2303′35.3"N 88037′35.2"E 2007 21 3.0 Paddy
33 Sahishpur 2304′18.4"N 88036′26.8"E 1985 18 2.0 Paddy
34 Tangra 2304′20.1"N 88036′26.8"E 2001 28 2 Paddy/vegetables
35 Tangra 2303′48.5"N 88037′14.7"E 1987 36 2.2 Paddy
36 Tangra 2303′44.2"N 88037′16.5"E 1996 28 5.3 Paddy
37 Tangra 2303′57.1"N 88037′23.2"E 1985 15 6.3 Paddy
38 Bishnupur 2303′54.5"N 88037′32.8"E 1996 18 5.0 Paddy/vegetables
39 Bishnupur 2304′1.0"N 88037′11.9"E 1999 42 4.5 Paddy
40 Bishnupur 2305′10.2"N 88037′4.5"E 2010 15 4.0 Paddy
S.Barman et al.
1 3
Table 2 Summary of physical parameters and major element concentration of the groundwater samples
Sample
No.
pH Conduc-
tivity
(µscm−2)
EhDO
(mgL−1)
Alkalin-
ity
(mgL−1)
TDS
(mgL−1)
Total
hardness
(mgL−1)
Ca+2
(mgL−1)
Mg+2
(mgL−1)
Na+
(mgL−1)
K+
(mgL−1)
SO4
2−
(mgL−1)
PO4
3−
(mgL−1)
NO3
−
(mgL−1)
Cl−
(mgL−1)
FeT
(mgL−1)
AsT
(µgL−1)
1 7.06 689 − 3.6 0.2 424 440.96 351.92 247.08 104.84 16.2 9.3 6.3 2.9 0.08 44.9 3 58.7
2 7.6 574 − 4.8 BDL 429 367.36 272.99 207.08 65.91 18.5 10.1 8.1 1.6 0.05 39.5 2.8 62.3
3 7.06 756 − 5.9 BDL 502 355.84 389.62 276.26 113.36 25.2 5.6 5.7 2.1 0.06 50.7 3.1 65
4 7.28 696 − 12 0.3 384 381.44 357.86 267.82 90.04 17.6 7.5 11.2 3.01 0.07 50.9 2.2 49
5 7.12 615 − 9.1 BDL 351 393.6 339.67 214.74 124.93 20.5 4.8 10.6 2.0 1.8 43.7 1.8 36.4
6 7.06 516 − 8.2 0.2 367 330.24 365.91 233.04 132.87 15.1 6.7 7.5 2.2 1.2 34.4 2.8 52
7 7.16 629 − 8.4 BDL 401 338.56 374.35 229.21 145.14 14.9 8.9 6.8 1.6 1.9 47.5 2.9 58
8 7.05 604 − 3.0 BDL 396 386.56 308.08 233.21 74.87 22.5 7.8 5.2 2.2 2.6 44.4 3.4 55
9 7.02 590 − 5.6 BDL 354 377.6 317.64 230.83 86.81 19.2 5.2 8.3 1.6 0.9 36.1 0.9 32.7
10 7.08 610 − 6.5 0.4 354 390.4 379.94 243.87 136.07 18.6 6.9 10.2 2.1 1.6 39.1 2.6 42.7
11 7.13 685 − 4.3 BDL 385 374.4 393.81 279.38 114.43 20.4 10.0 11.5 2.8 0.05 47.8 2.6 58
12 7.01 634 − 1.7 BDL 419 405.76 352.11 297.11 55 17.2 9.2 8.2 3.05 1.1 43 3.4 76.6
13 7.05 615 − 4.1 0.3 542 393.6 340.9 264.04 76.86 14.8 8.5 6.4 2.1 0.3 44.8 4.6 143
14 7.11 647 − 5.3 BDL 516 414.08 358.83 254.42 104.41 15.4 7.6 4.9 2.5 3.4 49.4 4.1 126
15 6.95 852 − 4.6 BDL 465 353.28 317.83 218.02 99.81 15.0 11.3 12.1 1.2 3.8 118.7 3.4 75.8
16 6.97 894 − 7.5 BDL 410 380.16 338.86 225.73 113.13 20.4 4.2 10.4 1.6 3.4 128.5 2.6 61
17 6.88 583 − 8.8 BDL 405 373.12 370.2 238.1 132.1 17.3 5.7 9.1 1.8 4.8 29.1 1.9 53
18 7.03 656 − 6.6 0.4 385 355.84 309.15 230.4 78.75 18.5 6.8 7.2 2.4 0.08 46.7 1.8 38.4
19 6.98 545 − 4.1 BDL 336 348.8 379.9 323.3 56.6 18.6 8.5 11.6 0.4 3.1 33.1 1.6 30.2
20 7.12 564 − 2.9 0.3 349 360.96 373.1 247.2 125.9 16.7 7.2 7.2 1.6 2.2 29.1 1.1 29.3
21 7.05 535 − 9.8 BDL 406 342.4 345.08 243.13 101.95 17.2 5.2 5.4 2.5 1.6 33.1 3.8 110
22 7.10 744 − 5.7 BDL 398 348.16 339.6 254.21 85.39 14.8 7.2 5.9 3.01 0.7 49 2 59
23 7.06 689 − 9.9 0.2 358 376.96 349.26 255.67 93.59 15.4 11.1 10.3 2.6 3.0 46.7 1.6 53.8
24 7.10 553 − 5.6 BDL 341 353.92 365.9 247.3 118.6 22.8 10.3 4.8 2.8 2.1 27.1 2.6 61.5
25 7.02 609 − 4.9 0.3 331 389.76 348.28 286.12 62.16 17.6 8.4 7.6 2.0 1.6 43.8 1.5 28.9
26 6.96 588 − 5.1 BDL 312 376.32 358.8 297.3 61.5 15.2 3.2 11.8 1.6 0.06 37.6 1.3 36.4
27 7.06 665 − 12.8 BDL 344 361.6 346.6 274.2 72.4 25.2 4.8 12.4 2.1 1.1 49.3 1.7 36
Identification ofmicrobiogeochemical factors responsible…
1 3
Table 2 (continued)
Sample
No.
pH Conduc-
tivity
(µscm−2)
EhDO
(mgL−1)
Alkalin-
ity
(mgL−1)
TDS
(mgL−1)
Total
hardness
(mgL−1)
Ca+2
(mgL−1)
Mg+2
(mgL−1)
Na+
(mgL−1)
K+
(mgL−1)
SO4
2−
(mgL−1)
PO4
3−
(mgL−1)
NO3
−
(mgL−1)
Cl−
(mgL−1)
FeT
(mgL−1)
AsT
(µgL−1)
28 7.18 545 − 11.2 0.1 338 348.8 337.9 230 107.9 14.3 7.6 7.6 0.9 0.8 35.5 2.1 35.3
29 7.08 658 − 8.3 BDL 402 357.12 348.9 237.4 111.5 20.5 6.1 3.8 3.02 3.8 44.8 2.4 45.1
30 6.98 616 − 7.8 BDL 367 330.24 352.11 297.11 55 15.1 5.4 10.2 2.5 4.1 43.7 3.5 53.6
31 7.12 545 − 4.6 0.3 401 348.8 340.9 264.04 76.86 14.9 3.7 7.3 3.06 2.9 34.4 1.4 39.2
32 7.05 712 − 5.1 BDL 396 391.68 358.83 254.42 104.41 22.5 9.5 8.1 2.3 1.6 47.5 3.2 62.8
33 7.10 698 − 5.9 BDL 354 382.72 317.83 218.02 99.81 19.2 8.2 11.2 0.5 2.8 44.4 2.6 59.4
34 7.06 558 − 2.6 0.4 354 357.12 338.86 225.73 113.13 18.6 6.4 8.1 1.2 0.7 36.1 2.8 76.8
35 7.10 575 − 4.7 0.1 385 368 370.2 238.1 132.1 20.4 10.1 7.6 2.3 3.7 39.1 4.3 91.7
36 7.02 649 − 3.9 BDL 419 415.36 309.15 230.4 78.75 15.2 9.0 9.2 2.4 4.2 47.8 3.9 76.2
37 6.96 635 − 3.6 BDL 542 406.4 379.9 323.3 56.6 18.7 5.1 11.3 1.6 2.3 43 3.5 73.1
38 7.06 608 − 6.9 0.3 516 389.12 373.1 247.2 125.9 20.4 4.6 5.6 3.0 1.4 44.8 2.8 49.5
39 7.18 717 − 2.1 BDL 465 330.88 345.08 243.13 101.95 19.5 7.0 7.8 2.2 0.6 49.4 2.1 41.6
40 7.08 888 − 9.8 0.1 410 376.32 339.6 254.21 85.39 16.4 7.9 7.5 1.3 1.2 118.7 2.4 56.2
MINI-
MUM
6.88 516 − 12.8 0.1 312 330.24 272.99 207.08 55 14.3 3.2 3.8 3.06 0.05 27.1 0.9 28.9
MAXI-
MUM
7.6 894 − 1.7 0.4 542 440.96 393.81 323.3 145.14 25.2 11.3 12.4 0.4 4.8 128.5 4.6 143
MEAN 7.076 643.525 − 6.18 0.26 400.3 371.856 348.9 252.0 96.9 18.1 9.6 8.3 2.09 1.8 47.93 2.6 58.7
SD 0.11 90.197 2.74 0.105 58.60 25.23 25.06 28.17 25.52 2.8 15.2 2.3 0.69 1.3 22.31 0.9 24.8
S.Barman et al.
1 3
mean value of TDS is 371.88 mgL−1 with a low range of variation (371.85–440.96 mgL−1),
indicating towards strongtomoderate sediment–water interaction in the monitored shallow
aquifer (Table 2) (Smith et al., 2000; Chatterjee et al., 2003; Harvey and Swartz 2005;
Nriagu et al., 2007; Polizotto etal., 2008; Mukherjee and Bromssen 2008). This study
emphasizes on the prevailing physicochemical nature of the shallow aquifer, which favours
the mineral dissolution process.
3.2.2 Chemical composition ofthegroundwater
Apart from few field parameters, a large number of chemical parameters (alkalinity, total
hardness, concentration of Cl−, SO4
2−, Ca2+, Mg2+, Na+, K+, FeT, AsT) have been meas-
ured in the laboratory. Among the cations (Ca2+, Mg2+, Na+, K+), bivalent alkaline earth
metal ion Ca2+ was predominantly found in the groundwater, followed by Mg2+, Na+, K+,
FeT and AsT (Table2). Both the bivalent alkaline earth metals (calcium and magnesium)
along with redox elements (iron and arsenic) are the important chemical constituents of
the monitored groundwater. The occurrence of redox elements indicates the dissolution
of iron minerals and hence desorption of arsenic (As) in the groundwater. The hardness
of the water has also been measured (mean—348.9 mgL−1 and range—272.99–393.81
mgL−1), which revealed that the irrigation water is usually hard (> 300 mgL−1) and very
hard (> 400 mgL−1) in some occasions. The presence of calcium (Ca) and magnesium
(Mg) ions in dissolved form at a relatively high concentration (Ca2+ -mean: 252.0 mgL−1,
range: 207.08–323.3 mgL−1 and Mg2+—mean: 96.9 mgL−1, range: 55–145.14 mgL−1)
in groundwater is due to the dissolution of carbonate minerals (Table2). Moreover, the
mixed carbonate (dolomite) dissolution is responsible for high level of dissolved magne-
sium (Mg) in the groundwater. Calcium (Ca) and magnesium (Mg) ions have relatively
high minimum concentration (Ca: 207.08 mgL−1; Mg: 55 mgL−1), as well and a basal level
of calcium (Ca) and magnesium (Mg) is always present in the groundwater. Calcium (Ca)
and magnesium (Mg) form calcium arsenate and magnesium arsenate, respectively, which
are less soluble (Ca3(AsO4)2) or insoluble (Mg3(AsO4)2) in water. These two cations (Ca2+
and Mg2+) can influence arsenic (As) release and mobilization in groundwater, by altering
the precipitation and adsorption equilibriums. Azam etal. and Fakhreddine etal. revealed
that calcium (Ca) and magnesium (Mg) can decrease the leachability of arsenic (As) by
increasing the anion sorption, by indirectly escalating the availability of positively charged
surfaces (Azam etal., 2010; Fakhreddine et al., 2015). The mean values of alkali metals
are relatively low (Na+: 18.1 mgL−1 and K+: 9.6 mgL−1) compared to dissolved bivalent
ions (Ca2+: mean 252.0 mgL−1, Mg2+: mean 96.9 mgL−1), suggesting that clay/silicate is
the possible source for Na+ and K+ ions rather than their common minerals (Table2).
3.3 Ion chemistry ofthegroundwater used forirrigation
The concentration of important redox elements iron (Fe) and arsenic (As) varies between
0.9–4.6 mgL−1 and 25.3–143 µgL−1, respectively, in the groundwater of irrigation wells.
Therefore, iron (Fe) occurs at a much higher concentration (in ppm range) compared to
arsenic (As) (in ppb range) (Table2). The concentration of arsenic (As) and iron (Fe) in
these shallow irrigation wells is greater than the WHO prescribed limit of arsenic (As)
(10ppb) and iron (Fe) (300ppb) in drinking water. Such high concentration of arsenic
(As) and iron (Fe) increases the possibility of correlation between them in terms of their
release, distribution and transformation. The occurrence of both iron (Fe) and arsenic
Identification ofmicrobiogeochemical factors responsible…
1 3
(As) in the groundwater follows a distinct trend because iron-bearing minerals in deltaic
sediments also host arsenic (As). Several anions (Cl−, HCO3
−) including few oxyanions
(SO4
2− and NO3
−) are also measured. Among the anions, HCO3
− predominates (mean
value 400.3 mgL−1) followed by Cl− (47.93 mgL−1), SO4
2−(8.3 mgL−1) and NO3
−(1.8
mgL−1) (Table2). The concentration of HCO3
− is variable with the highest mean value
among the cations and the anions indicating towards multiple sources of HCO3
−, such
as dissolution of carbonate and mixed carbonate minerals and breakdown of organic
matter. Continuous dissolution of carbonate might maintain the high minimum value
(312 mgL−1) of HCO3
− in the groundwater. HCO3
− also shows the highest maximum
value (542 mgL−1) amongst all cations and anions. The concentration of phosphate
(PO4
3−) (mean-2.09 mgL−1) is very low in these monitored wells. Gao etal. (2019) sug-
gested the role of carbonate in release, mobilization and transformation of arsenic (As).
Carbonate and phosphate both compete with arsenic for adsorption on iron oxide sur-
faces. Although the effect of carbonate on arsenic (As) adsorption is much lower than
the effect of phosphate, the cumulative effect of carbonate on arsenic (As) adsorption
will be much higher than phosphate (mean-2.09 mgL−1), due to its high concentration
in monitored wells of Chakdaha block. Therefore, higher carbonate concentration will
lead to higher total arsenic (As) release in groundwater of irrigation wells (Liu etal.,
2014; Radu etal., 2005). Other anions like Cl− and SO4
2− have minor effect on leaching
of arsenic (As) (Radu etal., 2005). Chloride is a conservative ion, showing large varia-
tion in concentration in the monitored wells (ranges 27–128 mgL−1, mean 47.93 mgL−1)
(Table2). Variation in the chloride concentration is an important finding because it can
have natural as well as anthropogenic sources. The irrigation wells containing high Cl−
also showed the presence of coliform bacteria (Chatterjee etal., 2003; Mc Arthur etal.,
2004; Neumann and Ashfaque 2010; Biswas etal., 2011).
Table 3 Factor loadings of
different physicochemical
parameters of the monitored
groundwater samples
Parameters Component
123
pH − 0.062 − 0.549 − 0.350
EC 0.665 0.399 − 0.382
Eh 0.487 0.139 − 0.009
DO − 0.069 − 0.067 − 0.029
Alkalinity 0.732 0.096 0.287
TDS 0.665 0.399 − 0.382
Hardness − 0.079 0.357 0.762
Ca+2 0.044 0.802 0.560
Mg+2 − 0.126 − 0.535 0.551
Na+ − 0.093 0.074 0.123
K+0.356 − 0.288 − 0.267
SO4
2− − 0.256 − 0.531 − 0.317
Cl−0.187 − 0.055 − 0.223
FeT0.786 − 0.245 − 0.260
AsT0.669 − 0.289 − 0.345
S.Barman et al.
1 3
3.4 Factor analysis andgroundwater hydrochemistry
Factor analysis has been adapted to address the association of geochemical environ-
ment with groundwater chemistry, notably arsenic (As) concentration. The analysis
was viewed in three domains of factors and is expressed by the data matrix variation of
10.8% (Table3). The factor 1 explains 20% of the total variant with strong loading of
conductivity, Eh, alkalinity, TDS, FeT and AsT. The combination of these factors usu-
ally represents the dissolved ion load of groundwater that might have enriched during
the sediment–water interaction in the aquifer (Chatterjee etal., 2005; Mukherjee etal.,
2007). Furthermore, (geological) formation of the sediment assemblage is another con-
trolling factor to explain the high loading value. The association of arsenic (As) and
iron (Fe) is the most important event and reflects the presence of arsenic (As) in the
groundwater along with iron (Fe). The relatively strong correlation between arsenic
(As) and iron (Fe) (r2 = 0.716) suggests that Fe-oxides/hydroxides could be the possi-
ble host for arsenic (As) and release arsenic (As) in the groundwater under the local
reducing condition (Fig.2, Table3). Factor 2 explains 15% of total variance with strong
loading of Ca2+ and SO4
2− (Table3). This suggests that gypsum (CaSO4) is also present
in the sedimentary environment. The presence of gypsum is significant because it can
also host arsenic (As) in the mineral surface, which can come out into the groundwater
during mineral dissolution (Chatterjee etal., 2005; Harvey and Swartz 2005; Mukherjee
etal., 2007). Factor 3 explains 12% of the total variant with strong loading of hardness
and very weak loading of calcium (Ca), magnesium (Mg) and alkalinity (Table3). This
further suggests that carbonate (calcite) and mixed carbonate (dolomite) are present in
the aquifer. The dissolution of carbonate is important because they can also contribute
towardsarsenic (As) in the groundwater during the desorption process (Manning and
Goldberg, 1996; Bhattacharya etal., 2002a).
Fig. 2 Relatively strong
correlation between arsenic
(As) and iron (Fe) concentra-
tion (r2 = 0.716) suggests that
Fe-oxides/hydroxides could be
the possible host for arsenic
(As). Dissolution of iron (Fe)
minerals release arsenic (As) in
the groundwater under the local
reducing condition
Identification ofmicrobiogeochemical factors responsible…
1 3
3.5 Presence ofcoliforms inthegroundwater ofirrigation well
The MPN index is usually estimated per 100ml of water and gives an estimate of number
of coliform bacteria present in the groundwater. Coliform bacteria are gram-negative rod-
shaped bacteria that are commonly found in soil and water contaminated by surface runoffs
and wastes of warm blooded animals (including human). The estimation of MPN index and
the presence of coliform bacteria were detected in the groundwater of irrigation wells by
culturing the same in lactose broth medium. These coliform bacteria could ferment lactose
and turn the culture medium acidic. The change in pH of the medium was detected by
the change in colour of the medium, supplemented with the pH indicator bromothymol
blue. Fermentation process results in the production of CO2 gas (bubbles) in the culture
tube. Both total and faecal coliforms are indicators of microbial contamination of water.
For detection of coliforms, groundwater was cultured in lactose broth at 37°C for 48h. For
specific determination of thermo-tolerant faecal coliform, culture tubes were incubated at
44.5°C. The cultured bacteria were grown on EMB-agar selective medium, which allows
the growth of gram-negative bacteria only. The MPN index (in terms of total coliform and
faecal coliform) per 100ml of water ranged between 4–110 (total coliform, Tc) and 2–34
(faecal coliform, Fc). Most of the monitored wells showed the absence of coliform bacteria
(both Tc and Fc). In the groundwater of irrigation well number 4, 15, 16, 32, 36, 39 and
40, coliform bacteria were present, whereas in well number 15, 16, 32, 36 and 40, fae-
cal coliform bacteria were present as well. Therefore, only 17.5% and 12.5% of monitored
wells showed the presence of total coliform (Tc) and faecal coliform (Fc), respectively.
The typical faecal coliform bacteria detected were thermo-tolerant E. coli, which produced
Fig. 3 Presence of coliform and faecal coliform in the water of irrigation wells. a) Fermentation of lactose
and production of gas bubbles, b) Green colony with metallic sheen indicates the presence of faecal coli-
form E. Coli, c) Purple/pink colony indicates the presence of Enterobacter aerogenes
S.Barman et al.
1 3
Table 4 Estimation of total
coliform and faecal coliform in
theirrigation wells
Irrigation Well
Number
Cl−(ppm) Total coliform
MPN index per
100ml
Faecal
coliform
MPN index
per 100ml
1 44.9 0 0
2 39.5 0 0
3 50.7 0 0
4 50.9 6 0
5 43.7 0 0
6 34.4 0 0
7 47.5 0 0
8 44.4 0 0
9 36.1 0 0
10 39.1 0 0
11 47.8 0 0
12 43 0 0
13 44.8 0 0
14 49.4 0 0
15 118.7 110 34
16 128.5 80 23
17 29.1 0 0
18 46.7 0 0
19 33.1 0 0
20 29.1 0 0
21 33.1 0 0
22 49 0 0
23 46.7 0 0
24 27.1 0 0
25 43.8 0 0
26 37.6 0 0
27 49.3 0 0
28 35.5 0 0
29 44.8 0 0
30 43.7 0 0
31 34.4 0 0
32 47.5 8 2
33 44.4 0 0
34 36.1 0 0
35 39.1 0 0
36 47.8 27 7
37 43 0 0
38 44.8 0 0
39 49.4 4 0
40 118.7 60 21
Identification ofmicrobiogeochemical factors responsible…
1 3
colony with green metallic sheen on EMB-agar medium. Purple/pink colony on EMB-agar
indicated towards the presence of Enterobacter aerogenes, which can be considered as a
faecal coliform (Fig.3a, b & c). When faecal coliform-contaminated groundwater is used
for irrigation, it adds coliforms to the agricultural soil. These bacteria might enter the veg-
etables and crops and cause food-borne diseases if consumed raw or in a less processed
form(Alegbeleye etal., 2018). Also, the high concentration of chloride ion in some of the
irrigation wells has direct correlation with the presence of coliforms in general and faecal
coliforms in particular (Table4 and Fig.4). This indicates towards anthropogenic source of
chloride ion-like leakage from septic tanks, sanitation pits, open dumping, etc., and faulty
construction of wells (Vengosh and Keren 1996; Kass etal., 2005; Onwuka etal., 2019).
Septic tank or sewage water leakage adds total and faecal coliforms, and other pathogenic
microorganism to the groundwater. Septic tank and sewage water leakage also add organic
Fig. 4 Variation of total and faecal coliform with chloride ion concentration in the irrigation wells. The
horizontal axis shows the number of irrigation well and the vertical axis shows the concentration of total
coliform, faecal coliform and chloride ion
Fig. 5 Isolated bacterium
DACW8 forms round-shaped,
white, mucoid colonies
S.Barman et al.
1 3
matter in the groundwater, thereby increasing the microbial metabolic activity, which sub-
sequently, play a significant role in arsenic release, distribution and mobilization.
3.6 Presence ofarsenic hyper‑tolerant bacterium inthegroundwater ofirrigation
well
Arsenic hyper-tolerant bacterium DACW8 was isolated from irrigation well water of Parari
village using increasing arsenic stress. The isolated bacterium was gram negative in nature
with round-shaped white smooth mucoid colonies (Fig.5). The bacterium is hyper-tolerant
to arsenite, arsenate and iron (Fe2+) as it can survive in 16mM arsenite, 241mM arsenate
and 20mM iron (Fe2+) (Table5). 16S rDNA sequencing followed by phylogenetic analysis
established the identity of the isolate (DACW8) as Enterobacter sp. Enterobacter sichuan-
ensis WCHECL 1597, Enterobacter sp. NA11309 and different strains of Enterobacter
bugandensis, Enterobacter chuandaensis and Enterobacter kobei were found to be close
Table 5 Isolated bacterial strain
DACW8 (Enterobacter sp.)
showing hyper-tolerance to some
heavy metals
Heavy metal Maximum
tolerance limit
of DACW8
(Enterobacter
sp.) (mM)
Arsenite (As3+) 16
Arsenate (As5+) 241
Ferrous (Fe2+) 20
Fig. 6 Phylogenetic tree (generated byNeighbour-joining method) of isolated arsenic-resistant bacterium
DACW8 (Enterobacter sp.) with 100 bootstrap replications. The evolutionary distances were determined
using the JukesCantor method. The branch length and bootstrap values are shown next to the branches
Identification ofmicrobiogeochemical factors responsible…
1 3
neighbours of this isolate (Fig.6). There are previous studies emphasizing on the arsenic
resistance and bioremediation potential of Enterobacter sp. (Selvi and Sasikumar 2014).
Few studies have also pointed towards the arsenic resistant potential of different strains
of Enterobacter bugandensis (Pati et al., 2018; Singh et al., 2018). Scanning electron
microscopic analysis exhibited that DACW8 is rod-shaped with a size of ~ 1.2µM, which
Fig. 7 Scanning electron micro-
scope image of DACW8 showing
its length to be ~ 1.2µM
Fig. 8 Growth pattern of the bacterial isolate DACW8 in increasing concentration of sodium arsenite
(100ppm, 500ppm, 1000ppm and 2000ppm). The optical density was measured at 600nm, as an indica-
tor of thegrowth rate. The graph shows the mean optical density (of three replicates) with standard devia-
tion as the error bar
S.Barman et al.
1 3
resembles the size and shape of Enterobacter in general (Kus, 2014) (Fig.7). DACW8 is
a fast-growing bacterium, which shows similar growth patterns in LB medium amended
with 0.77mM arsenite (100ppm sodium arsenite), 3.848mM arsenite (500 ppm sodium
arsenite) and 7.697mM arsenite (1000ppm sodium arsenite), owing to its hyper-tolerance
to arsenic stress. At 15.395mM arsenite (2000ppm sodium arsenite) (which is close to
the MIC), the growth of the bacterium slows down (Fig.8). DACW8 also shows biotrans-
formation potential as it can convert arsenite to arsenate, which is the less toxic form. The
colonies of bacterial strain DACW8 turned brown in LB-agar medium supplemented with
7.697mM arsenite (1000ppm sodium arsenite), when incubated with silver nitrate solu-
tion. The brown colour of the colonies indicated the formation of silver arsenate, which
further indicated towards the biotransformation of arsenite (As3+) to arsenate (As5+) by the
arsenic-resistant bacteria DACW8 (Fig.9). The biotransformation ability of this bacterium
might increase the concentration of arsenate (As5+) in the local environment of the aquifer.
However, such oxidation process is kinetically very slow, and therefore, arsenite (As3+) and
arsenate (As5+) usually coexist in the local environment. Both arsenite (As3+) and arse-
nate (As5+) are sorbed, sequestered and precipitated by iron oxides, but arsenite (As3+) is
released more easily from the iron surfaces (Darma etal., 2022; Lopez-Adams etal., 2021).
4 Discussion
The previous studies conducted in BDP (in West Bengal and Bangladesh) have dealt with
geochemistry of groundwater and its effect on arsenic (As) concentration, release and
mobilization in multi-depth drinking wells (Mukherjee and Bhattacharya, 2001; Bhat-
tacharya etal., 2002b; Bhattacharyya etal., 2003; Ahmed et al., 2004; Nath et al., 2007,
2008; Mukherjee and bromssen 2008; Chatterjee and Halder 2010). The current study is an
attempt to analyse the variation in arsenic (As) concentration and mobilization in irrigation
wells of BDP, in the presence of anthropogenic and microbial influences. The groundwater
samples have been collected only from those irrigation wells of Chakdaha block, which
are not selected during the groundwater quality surveillance programme/ arsenic awareness
Fig. 9 Biotransformation of
arsenite (As3+) to arsenate (As5+)
by arsenic-resistant bacterium
DACW8 (Enterobacter sp.) was
depicted by formation of brown
coloured bacterial colonies in
LB-agar medium containing
arsenite, upon incubation with
silver nitrate solution
Identification ofmicrobiogeochemical factors responsible…
1 3
campaign (Fig.1 and Table1). The groundwater of these wells is used throughout the year,
especially in summer time for paddy cultivation. The groundwater of the monitored wells
is well buffered and reducing in nature. The prevailing reducing condition of the shallow
aquifer (< 50m) has been indicated by negative Eh values and very low level of DO. This
study reveals that the conductivity of the monitored irrigation wells is medium to high
(516–894 µscm−2), suggesting long sediment–water interaction time (Table 2). These
results corroborate with the findings of Srivastava etal., highlighting the reducing nature
of the monitored aquifer in Chakdaha block (Srivastava etal., 2014). Findings of Anawar
etal. suggested a strong positive correlation between arsenic concentration and electrical
conductivity and a negative correlation of arsenic concentration with Eh of the ground-
water, in Ganga-Meghna delta plain of Bangladesh (Anawar etal., 2011). The reducing
nature of the shallow aquifers of Chakdaha block indicates the significance of anoxic
nature of the monitored aquifers in dissolution of Fe/Mn-bearing oxides/hydroxides (Ana-
war etal., 2011; Biswas etal., 2011; Srivastava etal., 2014; Middleberg etal., 1987). This
is a notable signature of the groundwater collected from shallow aquifer (< 50m) of BDP.
Therefore, mobilization/immobilization and biogeochemical processing of arsenic from its
mineral bound form are regulated by pH, oxic/anoxic condition and microbial redox reac-
tions. An important role is played by the bacteria in desorption of arsenic from iron (Fe)
and manganese (Mn)-oxide/hydroxide/oxyhydroxide phases, under anoxic condition and
near-neutral pH. Bacteria like Geobacter sulfurreducens, Shewanella putrifaciens, She-
wanella alga, Geobacter metallireducens, Geothrix fermentans, etc., are known for their
significant role in anaerobic dissimilatory iron (Fe) reduction. Some of these bacteria (for
example, Geobacter sulfurreducens, Shewanella putrifaciens, etc.) can reduce ferric iron
(Fe3+) directly by employing peripheral proteins ferric reductase and c-type cytochromes
present on their outer membrane, whereas other bacteria (like Shewanella alga, Geobacter
metallireducens, etc.) couple oxidation of organic compounds (complex organic polymers,
humic substances) with reduction of ferric iron oxides. Shallow aquifers of West Bengal
showing high rate of arsenic release and iron reduction are often found to be the habitat
of iron (Fe)- and manganese (Mn)-reducing microbes like Geobacter sulfurreducens and
Geothrix fermentans. Shewanella putrifaciens and Geobacter metallireducens reduce arse-
nicbearing ferrihydrite and enhance arsenic mobilization (Straub etal., 2001; Nevin and
Lovley, 2002; Islam etal., 2005; Tadanier etal., 2005; Aklujkar etal., 2013; Jiang etal.,
2013). These processes significantly release arsenic (As), iron (Fe) and manganese (Mn)
in the groundwater. In this reduction process, the organic matter supply necessary energy
to drive thermodynamically favoured redox reactions. The multi-step ladder-type redox
process began with the consumption of DO along with an increase in bicarbonate con-
centration due to breakdown of organic matter, where the nature, type and characteristics
of natural organic matter (NOM) are important for driving the process (Middleberg etal.,
1987; Smedley and Kinniburg, 2002; Bhattacharyya etal., 2003; Harvey and Swartz 2005;
Neumann and Ashfaque 2010; Biswas et al., 2011; Neidhardt et al., 2013). The reduc-
tion process further reduces metal oxides and hydroxides and shifts them from insoluble
to soluble phases [Fe (III)s → Fe (II)aq; Mn (IV)s → Mn (II)aq], resulting in increase in the
concentration of redox species [Fe (III) → Fe (II); Mn (IV) → Mn (II)] and bicarbonate in
the groundwater (Fig.10). The adsorbed arsenic (As) on metal oxide (hydroxide) phases
subsequently gets desorbed with change in redox pattern [As (V) → As (III)]. The concen-
trations of oxyanions (NO3
− and SO4
2−) are usually low, further suggesting the aquifer is
under reducing condition and the NO3
− reduction has occurred in a usual manner in the
shallow aquifers of BDP (Table2).
S.Barman et al.
1 3
The groundwater chemistry suggests that the groundwater is calcium bicarbonate
type. The slow movement of groundwater in BDP supports larger residence time and
more sediment–water interaction, which results in dissolution of carbonate/mixed car-
bonate minerals (Smith etal., 2000; Bhattacharya etal., 2002a&b; Harvey and Swartz
2002; Bhattacharyya et al., 2003; Mc Arthur et al., 2004; Harvey and Swartz 2005;
Mukherjee and Bromssen 2008). In BDP, significance of carbonate dissolution has been
reported by several research groups. The availability and amount of carbonate, sediment
dissolution are critical factors for groundwater quality and composition. Desorption of
arsenic (As) from carbonate may contribute towards increase in arsenic (As) concen-
tration in the groundwater. In addition to this, carbonate competes with arsenic (As)
for adsorption on the iron oxide surfaces. Therefore, high carbonate concentration in
groundwater of the monitored wells might increase arsenic (As) release and mobiliza-
tion (Liu etal., 2014). The secondary pumping, high discharge from irrigation wells and
supplies of fresh organic matter from nearby ecosystems are important factors influenc-
ing the aquifers with cross contamination (Nickson etal., 2000; McArthur etal., 2004;
Harvey and Swartz 2005; Van Geen etal., 2008; Neumann and Ashfaque 2010).
The field-scale investigation reveals that both iron (Fe) and arsenic (As) concentra-
tions are often beyond WHO-prescribed limit (As: 0.01 mgL−1; Fe: 0.3 mgL−1) and
national standard (As: 0.01 mgL−1; Fe: 0.3 mgL−1) in drinking water (Table2). How-
ever, the prescribed limit of iron and arsenic in irrigation well water is not specified by
BIS. The limits of heavy metals in irrigation well water are essential, since these heavy
metals could get bio-magnified in the food chain and might lead to fatal diseases (WHO,
Fig. 10 Redox ladder
Identification ofmicrobiogeochemical factors responsible…
1 3
2011; BIS, 2012). Among the several arsenic (As) release mechanisms (oxidation
model, reduction model, ion exchange model, infiltration model and microbial model),
the iron reduction model is widely accepted for high concentration of arsenic (As), arse-
nic desorption and mobilization in the groundwater (Sultan etal., 2009; Shankar etal.,
2014). In this model, iron (Fe)-oxides/hydroxide/oxyhydroxide dissolution occurs under
local reducing condition. The process usually desorbs arsenic (As) from the sediment
and releases it into the groundwater. In such circumstances, iron (Fe) (minerals) will
dissolute and have much higher concentration in comparison to arsenic (As). These
also suggest that most of the iron (up to 99%) will be in dissolved phase [Fe (II)]. Fe
(II) is highly soluble in water under neutral pH. Due to the neutral pH of the monitored
groundwater, iron (Fe) mostly stays in aqueous phase and provides necessary chemical
environment to release arsenic (As) (Biswas etal., 2011; Charlet and Chakraborty 2007;
Nath etal., 2008; Harvey and Swartz 2005; Polizzotto etal., 2008; Bhattacharya etal.,
2002a; Van Geen etal., 2008; Bhattacharyya etal., 2003). Leakage of sewage from pit
latrine can lead to increase in chloride (Cl−) ion concentration in the wells, which is
corroborated by the presence of faecal coliform bacteria in the irrigation wells (Figs.3,
4 and Table4). Overabundance of chloride ion decreases soil fertility and increases tox-
icity of crops (Geilfus, 2018). The presence of coliform in general and faecal coliform
in particular has a direct correlation with the chloride ion concentration of the ground-
water of irrigation wells. Consumption of water containing faecal coliform bacteria in
direct or indirect form by humans or animals (cattle) could have serious adverse health
effects like gastroenteritis and even death. When faecal coliform-contaminated ground-
water is used for irrigation, it adds coliforms to the agricultural soil. These bacteria
might enter the vegetables and crops and could causefood-borne diseases if consumed
raw or in a less processed form. WHO recommended ≤ 1000 CFU (colony-forming
unit)/ 100ml, as the permissible limit of coliforms in water used for irrigation purpose.
However, USEPA recommended the absence of faecal coliform (Fc) in water used for
irrigation of raw food crops and ≤ 200 Fc/ 100ml of water used for irrigation of com-
mercially processed crops and fodder crops (WHO, 2006; Kgopa etal., 2021). There-
fore, the chances of gastroenteritis caused by pathogenic coliform cannot be ignored in
case of intake of crops and vegetables (in raw or less processed form) from our study
area. In addition to this, it might increase the risk of bacterial infection in farm workers
and children playing in irrigation fields as suggested by Balkhair (2016). The pit latrines
contribute towards supply of fresh organic matter, which can also enhance the arsenic
(As) level in the drinking wells, by increasing microbe-mediated reductive dissolution
of arsenic-bearing iron minerals.
Some irrigation well with non-permissible arsenic concentration also harbours arsenic-
resistant bacteria, which is also hyper-tolerant to iron (Fe2+), the co-existing metal in the
aquifer (Fig.5 and Table 5). Scanning electron microscopy and 16S rDNA sequencing
identified this bacterium to be Enterobacter sp., which is also capable of biotransforma-
tion of arsenite (As3+) to arsenate (As5+) as a dissimilatory process (Figs.6, 7 and 9). The
energy derived during the oxidation process is metabolically used for unhindered growth of
the bacteria at high arsenic concentration. The oxidative transformation of arsenite to arse-
nate carried out by these bacteria might play a significant role in geochemical processes of
the aquifers. The anoxic condition of the aquifer favours the growth of Enterobacter sp.,
since it is a facultative anaerobe. The hyper-tolerance of the isolate to both iron and arsenic
might lead to regulation of arsenic mobilization from iron–arsenic-bearing minerals. Both
arsenite and arsenate act as substrate for arsenic-oxidizing and arsenic-reducing bacteria,
respectively. On the other hand, iron-oxidizing and iron-reducing bacteria act on the iron
S.Barman et al.
1 3
surfaces and lead to biotransformation of iron species. Therefore, in these shallow aquifers
anthropogenic factors might affect the geochemistry. Also, the role of microbial communi-
ties of an area on geochemical processes of arsenic release and mobilization could be con-
sidered as a future line of research (Vengosh and Keren 1996; Kass etal., 2005; Onwuka
etal., 2019; Alegbeleye etal., 2018; Selvi and Sasikumar 2014).
Factor analysis reveals an association between arsenic (As) and iron (Fe), alkalinity,
Eh and TDS. Among these associations, a strong correlation has been observed between
AsT (0.669), FeT (0.786) and alkalinity (0.732) (Fig.2). The positive correlation between
arsenic (As) and iron (Fe) indicates towards the reduction of iron (Fe) phase to observe
the increase in concentration of redox-sensitive species (both arsenic and iron). The factor
analysis also indicates that calcium (Ca) and magnesium (Mg) have an association with
alkalinity and hardness, which further suggests that carbonate dissolution (calcite as well
as dolomite) may contribute to overall composition of groundwater, including arsenic (As)
release in the groundwater (Table3). (Mukherjee and Bromssen 2008; Biswas etal., 2011;
Neumann and Ashfaque 2010; Bhattacharyya etal., 2002, Harvey and Swartz 2002; Mc
Arthur et al., 2004; Harvey and Swartz 2005; Majumder et al., 2016). Therefore, when
the groundwater of shallow aquifers of Chakdah block is used for irrigation purpose, it
adds more arsenic and pathogenic microorganisms to the irrigation field. Accumulation of
arsenic in paddy and its subsequent entry in food chain will impart a negative impact on
human health. Also, the arsenic added to the irrigation field can contaminate the natural
water bodies by surface run-off during the monsoon season. The time of current research
corresponds to the time of maximal usage of the irrigation wells. However, the effect of
recharge after post-monsoon on irrigation wells could be studied and related to the micro-
bial and geochemical parameters. The molecular mechanism of microbial heavy metal
hyper-tolerance and bioaccumulation, bioadsorption and biotransformation of arsenic
(As) and iron (Fe) species could be investigated. The sample size should be substantially
increased to decipher the entire microbiogeochemical phenomenon in the aquifers of a typ-
ical geographical area. In addition to this, the risk of bacterial infections in farm workers
and nearby residents could be studied as a future line of research.
5 Conclusion
At present, a large number of shallow tube wells (< 50m) are being used to meet the need
of the irrigation water for cultivation of paddy and various other crops. In this study, irriga-
tion wells are regularly monitored for analysing the groundwater composition and chemis-
try, which suggested that the groundwater is usually anoxic with high amount of dissolved
redox elements (arsenic and iron). The presence of high concentration of alkaline earth
metals and high alkalinity suggested that the carbonate dissolution is one of the principal
mineral dissolution processes in the shallow aquifers of BDP. Among the redox elements,
iron (Fe) has much higher concentration compared to arsenic (As). Presence of both iron
(Fe) and arsenic (As) in groundwater is significant because the sedimentary source of iron
(Fe) can also host arsenic (As). The hydrochemistry followed by the statistical approach
further support the association of arsenic (As) and iron (Fe) (r2 = 0.716) and suggested
that iron oxide/hydroxide can be the possible source for hosting arsenic (As) in the sedi-
mentary environment. The installation of sanitation can supply fresh organic matter, which
will enhance the reduction process under local reducing condition, resulting in release of
arsenic (As) in the groundwater. Also, under anaerobic condition, metal-reducing bacteria
Identification ofmicrobiogeochemical factors responsible…
1 3
gain energy through degradation of organic matter and catalyse the processing of iron oxy-
hydroxide, which also releases arsenic (As) in the groundwater. Besides, anthropogenic
processes add pathogenic faecal coliform bacteria to the groundwater of irrigation wells,
which might get incorporated in the food chain and cause fatal diseases. Some irrigation
well with non-permissible arsenic concentration also harbours arsenic-resistant bacterium
Enterobacter sp., which is also hyper-tolerant to iron (Fe2+) and capable of biotransforma-
tion of arsenite (As3+) to arsenate (As5+). In this context, a national guideline should be
established with respect to heavy metal and faecal coliform concentrations in irrigation
well water. These bacteria might play a significant role in geochemical processes of the
aquifers. United Nations Organisation mentioned in its Sustainable Development Goal 6
“Ensure availability and sustainable management of water and sanitation for all”. How-
ever, consumption of water in any form, containing poisonous heavy metals and pathogenic
bacteria, can lead to serious health hazards in humans and animals. Presence of arsenic
in the groundwater of irrigation wells may lead to its biomagnification in the rice crop as
observed in the rice fields of Murshidabad district, India and parts of Bangladesh. There-
fore, local government should take the initiative to train the farmers against the usage of
contaminated irrigation well water for crop production. It is essential that this kind of stud-
ies ensure safe-marking of irrigation wells as a translational outcome. Also, the indiscrimi-
nate usage of shallow aquifer for harvesting of groundwater should be regulated by policy
under an aquifer monitoring program.
Acknowledgements We are duly acknowledging the financial support from the University of Kalyani and
University Grants Commission (UGC), India. The authors also acknowledge Department of Science &
Technology and Biotechnology, Government of West Bengal, India, for funding the Research by its R&D
Project Scheme – ‘Gobeshonay Bangla’. The authors especially thank the owners of the irrigation wells for
the permission to access their agricultural land.
Funding Support for this study is provided by University of Kalyani, India and University Grants Commis-
sion (UGC), India. This work was also supported by Department of Science & Technology and Biotechnol-
ogy, Government of West Bengal, India [Grant No. STBT-11012(15)/26/2019-ST SEC].
Availability of data and materials Not applicable.
Declarations
Conflict of interest The authors declare no conflicts of interest/competing interests.
Ethical Approval Not applicable for this study.
Consent to Participate Not applicable
Consent to publish All the authors have given the consent for publication
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Authors and Aliations
SandipanBarman1· DebjaniMandal2· PinakiGhosh3· AyanDas1·
MadhurinaMajumder1· DebankurChatterjee4· DebashisChatterjee1·
IndranilSaha5· AbhishekBasu2
* Debashis Chatterjee
dbchat2001@yahoo.co.in
* Abhishek Basu
abhishek@mbbtsripatsinghcollege.in
1 Department ofChemistry, University ofKalyani, Nadia, WestBengal741235, India
2 Department ofMolecular Biology andBiotechnology, Sripat Singh College, P.O. Jiaganj,
Murshidabad, WestBengalWB-742123, India
3 Department ofMicrobiology, University ofKalyani, Nadia, WestBengal741235, India
4 T.A. Pai Management Institute, 80, Badagabettu Manipal Udupi, Manipal576104, India
5 Department ofChemistry, Sripat Singh College, P.O. Jiaganj, Murshidabad,
WestBengalWB-742123, India
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