Arsenic, a highly toxic carcinogenic substance with diverse chemical properties was discovered by a German alchemist Alberts Magnas in1250 (Rahaman et al., 2020). Basically, it is a metalloid that has raised serious environmental and human health concerns recently (Bag et al., 2019). Most abundant forms of arsenic in the environment are As⁺³ and As⁺⁵, of which As⁺³ is more toxic and relatively more mobile than As⁺⁵ (Jones, 2007). Among inorganic forms under reduced condition, As⁺³ is the dominant form, while As⁺⁵, is prevalent in oxygenated environment and they can change their valence states depending on soil pH and redox conditions. The toxicity pattern of different forms and oxidation states of Arsenic is  TMA (-3)>MMA(3) >DMA(3) >As⁺³>> As⁺⁵>MMA(5) >DMA(5) (Gong et al., 2001).
       
Arsenic enters into terrestrial and aquatic ecosystem through a combination of geological and anthropogenic processes. Arsenic contamination is reported in over 70 countries throughout the World in which, 20 countries are severely affected including Bangladesh, India, China, Brazil, Chili, United States of America (Sanyal., 2019). In the severity of Arsenic toxicity, Bangladesh ranks first followed by India. Among the 28 states of India, lower Gangetic plane especially West Bengal is most severely affected by arsenic contamination (Rahman et al., 2001). Analysing inner scenario Sanyal, (2019) reported arsenic contamination in ground water of West Bengal is distributed over 3417 villages in 111 blocks of 12 districts. Among these 12 districts, Malda, Murshidabad, North 24-parganas, South 24-parganas, Nadia, Howrah, Hooghly and Kolkata have Arsenic levels above 300 μg l^-1, which is much higher than the safe limits of Arsenic in drinking water stipulated as per WHO guidelines (50 μg L^-1).
       
Not only for drinking water, arsenic became even a threat for irrigation water also, because of continuous use of arsenic contaminated irrigation water may enhance its concentration in edible parts of crops (Khanam et al., 2021).  Latter consequences are little bit horrifying, because it will certainly enter in our food chain through edible parts of plant or water and ultimately increase the content of arsenic in our body leading to different types of health issues like keratosis, melanosis, backfoot and ultimately cancer (Rahman et al., 2001).
       
On the other hand, one of the most important components of soil is microorganisms, which make it living and dynamic (Jacoby et al., 2017). Microorganisms primarily enhance the plant growth by decomposing organic matter, increasing the availability of nutrients and in so many ways. Among different types of microorganisms, bacteria is most dominated one followed by fungi in soil (Rousk et al., 2010). It is observed that high concentration of arsenic may affect microbial population, whereas reverse result also found, where arsenic concentration is reduced by native microbial population. Researches also proved that among all these microorganisms, bacteria preformed best in reducing As content of soil and plant. Present investigation on study on arsenic tolerance level of bacteria was conducted to know the tolerance level of soil bacteria upto which it can withstand As toxicity.

Study area
 
Nadia district of West Bengal is recently considered as the highest arsenic affected zone of our country (Fig 1). Four different blocks viz., Haringhata, Krishnanagar, Ranaghat, Chakda of Nadia district, WB, India had been selected for the present study Surface soil samples (0 to 15 cm ) taken from five different sites of each four blocks. Soil samples were collected from low land rice fields with GPS. 


       
Soil samples collected from the experimental sites, properly levelled and brought into the directorate of research laboratory, Bidhan Chandra Krishi Viswavidyalaya (BCKV). Half portion of each soil samples was stored in the refrigerator for microbial work and other half were air-dried, ground to pass through a 2-mm sieve, stored in polythene packets for analysis,. Counting of bacterial population was carried out just after collection of soil samples.
 
Physical and chemical analysis of soil samples
 
Some physical properties like particle size distribution, textural class, bulk density and water holding capacity were.
       
The soil pH and EC were determined in soil-water suspension (soil: water :: 1: 2.5) (Jackson, 1973).
       
Oxidizable soil organic carbon (OC) was following the Walkley-Black wet digestion method (Walkley and Black, 1934). The available nitrogen content of soil was determined through modified Kjeldhal method (Subbiah and Asija, 1956). Available phosphorus by Olsen method. Available potassium by neutral normal Amonium acetate (soil: extractent :: 1:10 ) (Brown and Warncke 1988).
       
Soils are extracted with Olsen reagent (0.5M NaHCO₃, pH 8.5; Soil: extractant : : 1: 10) and filtered for estimating available As in soil (Khanam et al., 2021). HNO₃ acid digestion method (Rahman et al., 2007) was followed to estimate total As content in soil.
 
Bacterial population study
 
Isolation of Bactria from soil is done by Thronton’s Agar media (Thornton,1922).
       
Serial dilution and pore plate technique (Sanders, 2012) is followed for isolating bacteria from soil. Bacterial growth was determined by the turbidity of bacterial broth in spectrophotometer for their turbidity in 600nm wavelength. Then after five days plating was done with Throntons ager medium for counting of colony forming unit.

Physical properties of the soil
 
Results (Table 1) of mechanical analysis revealed that all the soils collected from Nadia district have high  clay and textural class varies from silty clay to sandy loam. Soils collected from Chakda have highest clay (49.8%) but lowest sand (3.5%),whereas soils collected from Haringhata were high in sand (61.2%) but poor in clay (18.2%). Similar results were found by Bhattacharya et al. (2009), which showed high clay content (70%) in Chakda block making it suitable for rice cultivation. Bulk density (BD) was found highest in case of Haringhata soils (1.49 g cm^-3) may be due to high sand content, while lowest BD was recorded in the soils of Chakda block (1.23 g cm^{3 -1}) probably due to higher clay content and diversified farming (Mashiane et al., 2023). water holding capacity (WHC) varies between 48% to 38.3% and found highest in Chakda block due to high clay content (Table 2).




 
Chemical properties of soil
 
The chemical properties of the experimental soils have been analysed and tabulated in Table 3. Chemical properties viz., pH, EC, oxidizable organic carbon, available Nitrogen, Phosphorus and Potassium, which may controls the availability of arsenic, have been analysed following standard procedure. pH of soil samples varied from 6.34 to 7.53, which is more or less in neutral range, which aligns with results of Ghosh et al., (2021). According to Borah et al. (2020) pH of soil in this lower Gangetic plain is neural (6.90), which also supports the magnitude of pH in the current research. Electrical conductivity of soil varies between 0.14 to 0.36 dS m^-1, which is much lower than critical value (4dS m^-1) and similar to Ghosh et al. (2021) finding showing non-saline property of soil. The mean oxidizable organic carbon (OC) content was 0.48%, which is low to medium, with a range of 0.43 to 0.56%. Nitrogen content varies from 147 to 222 Kg ha^-1, showing low content of nitrogen in soil and phosphorus content varied from 19 to 36 Kg ha^-1, which represents the magnitude of P is usually high. Potassium content in the tested soils was medium, which ranged from 133 to 195 kg ha^-1 with average of 161.20 kg ha^-1. Available N, P, K content of this area was similar to the findings of Mondal et al., (2022).


 
Olsen extractable (available) and total As in soil
 
Olsen extractable or available arsenic directly affect plants and microorganisms (Brandt et al., 2023). Soil collected from arsenic affected belts of Nadia district showed high Olsen extractable arsenic content (Fig 2) with mean value 1.89±0.62 mg kg^-1. Mishra et al. (2022) reported that mean concentration of available As in Nadia district is 1.45 mg kg^-1, which is similar to the current study. Among four different blocks, Chakda showed highest available. As ranged from 1.74 to 3.28 mg kg^-1 with a mean value 2.57±0.73 mg kg^-1, which is similar to the findings of Sinha and Bhattacharyya, (2012) whereas, lowest magnitude recorded in Krishnagar with average As content 1.18±0.55 mg kg^-1. Total soil arsenic content in soil shows similar pattern (Fig 3) , where highest As content recorded in Chakda (13.34±3.63 mg kg^-1), while lowest magnitude was recorded in Krishnanagar (4.20±2.01 mg kg^-1). Total As concentration in soil generally varies from 0.1 to 10 mg kg^-1 but Shrivastava et al., (2014) recorded average magnitude 9.5 mg kg^-1 for the specific area, which is quite similar to that of  present finding.




 
Corelation of As with different parameters
 
Values from Table 3 is a significant positive correlation between soil available As and pH (r²=0.708**). According to Singh and Srivastava (2020) bio-availability and mobility of arsenic depends on soil pH. Results revealed that (Table 4) there was positive corelations between Olsen extractable As and EC (r²=0.318), OC (r²=0.301), N (r²=0.296), P (r²=0.205), K (r²=0.359) though these values are not statistically significant. Total as in soil showed positive and significant corelation with soil pH (r²=0.575) and Olsen extractable As (r²=0.875**) content at significance level p<0.01. Strong positive corelation also found between total As and organic carbon content in soil (r²=0.502) with available N content (r²=0.463) also but at 0.05 level of significance (p<0.05). Dobran and Zagury (2006) also found similar types of correlation between As content and organic carbon present in soil. Corelation coefficient (r²) between total soil As and EC, P and K observed positive but not significant with correlation coefficient (r²) magnitudes 0.356, 0.351 and 0.313 respectively.
 
Available arsenic content and bacterial population of soil and their relationship
 
Available arsenic content in soil and bacterial cfu population has been represented in  Fig 4 and 5. Bacterial population has been represented by colony forming units (cfu) of bacteria and the readings are taken after 3 days of incubation. Highest bacterial population of incubation was observed in K₅ soil sample of Krishnagar block (44×10⁵ /g of dry soil), which might be due to low available As ultimately not hampering the natural proliferation of bacteria. Lowest bacterial population recorded in soil sample C₅ of Chakda (12×10⁵ g^-1 of dry soil) , where high contamination of arsenic of 3.275 mg kg^-1. Similar type of result  shown by Majumder et al. (2013), where they have found decrease in bacterial population due to high nutrient content. Results also showed that bacterial population after 3 days of incubation and Olsen arsenic content was negatively correlated. A decreasing trend in bacterial population with increasing inherent arsenic loading was observed and  equation, which shows relation between Olsen extractable As content and bacterial growth y = -4.983x₃ + 32.352x₂ - 68.309x + 56.545 with r² value 0.62 (Fig 4), where x is available As content in soil. The equation represents a third order polynomial or a cubical relationship of Olsen extractable As with bacterial growth. Hassan et al. (2022) also found similar type of polynomial correlation between bacterial growth and heavy metal concentration. From lower As to comparatively higher As values, bacterial growth drastically reduces due to high toxic effects of As, after that with higher As values, the bacterial population remains similar or constant might be due to development of arsenic resistance in bacteria. Highest concentration of As shows further gradual decrease of bacterial count signifying that registrant bacteria also cannot withstand such amount of As toxicity. Arsenic is a toxic metalloid that can inhibit the microbial growth, hence resulting low microbial population. A decrease in microbial population with increase in As content has been recorded by Van Zwieten et al. (2003). Decrease in bacterial population with increasing As content in soil might be due to enzymatic inhibition, disruption of cellular respiration, DNA rupture and toxin exclusion mechanism of bacteria (Pal et al., 2022).





Tolerance level of bacteria
 
Bacterial broth treated with arsenic salt (As₂O₃) and kept for five days in incubation chamber and then OD value of solutions were taken under spectrophotometer (λ=600 nm). In lower As concentration of 100 ppm, the OD values were highest in bacterial broth isolated from soils of Krishnagar block i.e., K₁ gave the highest (1.427), which signifies that broth was totally turbid due to high bacterial growth. But for all soils, OD value sharply decreases with increase in As concentration in broth. At 2500 ppm arsenic broth for most of the soils except Chakda become transparent as the bacterial growth stops due to high as toxicity.
               
The OD value ranges from 0.036 to 0.018, which signifies there is no bacterial growth in the broth. In case of soils collected from Chakda block, OD values were low at lower arsenic concentration than Ranaghat and Krishnanagar but were not sharply decrease with increase in arsenic concentration. Results showed that for C₄ and C₅ sample the OD values were 0.228 and 0.211 respectively in 4000 ppm arsenic solution which signifies the bacteria in C₄ and C₅ sample can tolerate As concentration up to 4000 ppm. Regression equation between As content and OD value was y = -0.522ln(x) + 1.4292 with high negative regression coefficient (R² = 0.926), where y is the OD value measured in spectrophotometer and x represents As content in broth (Fig 5). It was observed that bacterial population decreases in a logarithmic rate with increasing As content which might be the of cellular toxicity due to higher concentration of arsenic (Mairaj et al., 2022). Tolerance level of bacteria is reported 10-100 µ mols for arsenite which is similar to the current study (Rehman et al., 2010).

Arsenic content both total and available recorded higher than permissible limit in most of the sites, though the highest concentration was recorded in the soils of Chakda district. It can be concluded that soil total arsenic has a direct positive correlation with Olsen extractable arsenic at 0.01 level of significance but with other physicochemical parameters no such strong relation was found. Number of bacterial showed a third order polynomial regression with soil available As having R² value -0.93. Deta further revealed that bacterial growth mostly restricted beyond 2500 mgl^-1 As concentration in broth due to enzymatic inhibition and genotoxicity though the bacteria isolated from Chakda block showed As tolerance upto 4000 mgl^-1 or ppm. Present study concluded that due to continuous exposure of soil arsenic, bacteria found in the Chakda block become somewhat registrant to As through natural selection or exclusion mechanism. Bacteria present in the soils of other blocks become suspectable to arsenic as limited exposure to this toxin and the growth was drastically reduced with increase in As content of the broth. Although after a certain concentration all type of bacteria is killed by arsenic toxicity.

This statement declares whether the authors do not have any conflict of interest (financial or personal) for the research.