Arsenic Contamination of Groundwater and Its Clinical Impact on Inhabitants in Central East India

We have worked on the As concentrations present in the groundwater samples in the Rajnandgaon region of India, as well as the clinical assessment of the impact of Arsenicosis on the human population. Out of twenty groundwater samples from eight villages, arsenic was found to be above the permissible limit in eighteen samples. The people in the affected area were found suffering from diffused and spotted melanosis, diffused and nodular keratosis, leucomelanosis, hepatomegaly, and splenomegaly. The hematological parameters, viz. low Hemoglobin, high ESR, and low PCV, with RBC, WBC, and Platelet counts adversely altered and increased Prothrombin time. The change in biochemical parameters reects the toxic effect of prolonged exposure to arsenic viz. – low Serum Glucose, high LDL, HDL, and TG, decreased Total Protein, Albumin, and Globulin. Total Bilirubin, Creatinine, Urea, and SGPT were at higher levels, but the activity of Alkaline phosphatase was found lower. The study reveals the severe health impact on humans, especially on the liver, kidney, and skin caused by arsenic toxicity in Kaurikasa village and adjoining area. the study DT 18.10.2014). Blood was collected from the population showing symptoms of Arsenicosis with their kind consent and with the help of the medical technician. Volunteers were told not to take any food in the morning before drawing blood. Blood samples were drawn by sterile disposable syringe and needle and collected in a sterile vial containing EDTA. The hematological examination was carried by assessing the blood Hemoglobin by Sahli’s method with the help of a hemometer (Marieneld, Germany). The Total Red Blood Corpuscles (RBC), White Blood Corpuscles (WBC), and Platelet count were determined by the improved Neubauer Hemocytometer method. The Packed Cell Volume (PCV) was estimated by the Macrohematocrite method and while Erythrocyte Sedimentation Rate by the Wintrobe method. The biochemical examination was carried out by photometric determination for serum Glucose. At 505 nm, total cholesterol at 546 nm, Low-Density Lipoprotein, and High-Density Lipoprotein at 340 nm, triglyceride at 546 nm, total Protein at 546 nm, Creatinine at 510 nm, Serum Glutamate Oxaloacetate Transaminase at 340 nm, Serum Glutamate Pyruvate Transaminase at 340 nm and Alkaline Phosphate at 420 nm. The enzymatic kit for the above experiments was obtained from Merck Specialties Private Limited, Mumbai (India). Autoanalyser of Merck (Microlab 300) was used for the above estimations. both quantitatively qualitatively in liver disorder. In any disease-causing hepato-cellular damage, the concentration of serum albumin decreases. The dynamically changing levels of serum albumin, therefore, are a valuable indicator of severity, progress, and prognosis in hepatic diseases. In the present study, liver cell damage is attributed due to arsenic toxicity. The elevated levels of bilirubin were observed in the current study. In plasma, bilirubin is found as indirect reacting bilirubin which is insoluble in water. The direct reacting esteried bilirubin is water-soluble. At the end of the life, span erythrocytes are destroyed in the reticuloendothelial system and liberate hemoglobin. The globulin is separated from Hemoglobin, and the porphyrin ring is opened. The released iron part goes into the iron store and may be used further for hemoglobin synthesis. Green color biliverdin forms rst from the non-iron-containing residue of Hemoglobin (i.e. Protoporthyrin). Biliverdin gets reduced to yellow-colored bilirubin. Generally, total serum bilirubin is found to increase in case of hepato-cellular damages (toxic hepatopathy neoplasm, etc.), obstructions in intra and extrahepatic biliary tract, intravascular, and extravascular hemolysis processes. Excessive elevation of direct bilirubin is seen during cholestasis and late in the course of chronic liver diseases. In the present nding, an elevated level of bilirubin in 16 samples out of 20 suggests liver toxicity among the population due to Arsenicosis.


Introduction
Arsenic is a common element that is found in soil, air, water, and also in living organisms. In terms of abundance, it is ranked 20th in the earth's crust, and seawater, and the 14th most abundant element in the human body, it is the 12th most abundant element. The element is carcinogenic to human beings, and there is very little evidence available for arsenic-induced carcinogenicity in animals.
Essential normal wellsprings of arsenic incorporate deposits of minerals, hydro, geothermal, and volcanic activities. The leading anthropogenic roots of arsenic lie in wood additives, pesticides, mechanical uses, mining, and industrial puri cation processes. The most harmful type of arsenic is arsine gas, trailed by arsenite and arsenate, which are the species typically found in groundwater. Up to this point, the estimation of total element concentrations was considered to be adequate for clinical and ecological contemplations. Even though the total elemental determination gives valuable and basic logical data for scienti c information, the degree of individual species is common of more relevant. For instance, the precise determination of speciation of the toxic species is more signi cant in the setting of environmental and toxicological parameters as compared to the determination of total elemental concentration. Knowledge about speciation is crucial as the bioavailability and toxicity. Different arsenic compounds have varied toxicity levels. For instance, the inorganic arsenic species, As(III) and As(V) are more potent toxic than their pentavalent methylated counterparts. Arsenic is one of the signi cant components in huge numbers of sea kelp and the degree of toxicity depends upon the type of arsenic species present. (Environmental Health Criteria; 224 2001).
Arsenic is the most toxic environment-derived metal and the most potent source of arsenic toxicity in humans is its contamination in the drinking water derived from natural underground sources rather than from the agricultural, mining, or smelting processes. (Matschullat et al. 2000).
The working group of the World Health Organization (WHO) characterized Arsenicosis as an "interminable wellbeing condition emerging from lengthened arsenic ingestion (at least more than six months) for over a safe dose, which is generally characterized by trademark skin lesions, with or without affecting the internal organs" (WHO Regional O ce for South-East Asia, 2003). The most extreme allowable breaking point of arsenic levels in groundwater according to WHO proposals are 10 µg/L, yet India and Bangladesh have supported <50 µg/L as the acknowledged level in arsenic-de led zones, where elective hotspots for drinking water are not accessible (Smedley and Kinniburgh 2002). Arsenic exists in the form of a natural contaminant and is widely distributed in the biosphere as arsenite (As 3+ ) and arsenate (As 5+ ) (Hei et al. 2004). Thus Arsenate contamination of drinking water is a grave ecological issue around the globe, and large numbers of populations are at risk (Chowdhury et al. 2000).
The toxicological and human health effects caused by arsenic exposure is known for centuries. Yet, there are still many questions that are unanswered, particularly concerning the mechanisms of action of arsenic and the factors that may affect susceptibility to the damaging effects of these elements and their compounds. The toxicity of inorganic arsenic (As) relies upon its valence state (-3, +3, or +5), and also on the physical and chemical properties of the compound in which it is present. The trivalent (As +3 ) compounds are usually more toxic than it's pentavalent (As +5 ) form. More water-soluble compounds are usually more toxic and more likely to have systemic effects than the less soluble compounds and are more likely to cause chronic pulmonary effects if inhaled. In this connection, the arsine gas (AsH 3 ) is the most toxic inorganic compound. Generally, humans are more sensitive than laboratory animals to the toxic effects of inorganic arsenic. In this connection, the rodents exhibited a critical impact in the form of immuno-suppression and hepato-renal dysfunction, whereas in humans, the primary target areas of arsine gas are the skin, vascular system, and peripheral nervous system. Water-soluble inorganic arsenic compounds are assimilated through the G.I. tract (<90%) and lungs, dispersed predominantly to the liver, kidney, lungs, spleen, aorta, and skin; and excreted chie y in the urine at a higher rate of 80% in 61 hours following oral dose (U.S. EPA 1984; ATSDR 1989; Crecelius 1977). The pentavalent arsenic form is reduced to the trivalent form and then it is subjected to methylation in the liver to less toxic methylarsinic acid forms (ATSDR, 1989).
The world's largest arsenic-contaminated area is the Bengal Delta Plain (BDP). In this BDP, Bangladesh and West Bengal in India are the worst affected territories in the world. In 42 districts of Southern Bangladesh and nine adjacent districts of West Bengal, India, 79.0 million and 42.7 million people respectively are manifested to groundwater with arsenic level, way above the WHO's maximum permissible limit of 50µg/L (Choudhury et al. 2000). The source of arsenic is geological in origin, which primarily contaminates the aquifers that provide water for over one million tube wells (Nickson et al. 1998). In some of the tube wells of West Bengal, India, the arsenic contamination is very high (3400 µg/L) (Guha et al. 1998).
Sarkar in 1983 has reported uneven distribution of arsenic in central India. This region is a part of the Lower Proterozoic age formations popularly known as the Bailaidla group and is characterized by typical rock compositions of Phyllitic shales and haematitic quartzes. The is region located at Dalli -Rajhara, 25 km east of Kaurikasa, consists of Banded Haematite or Magnetite quartzes (BHQ) and iron ore mines. The western sector of the area shows broad volcanism, and the rocks of the Nandgaon bunch comprised of lower Bijli Rhylites and Pitepani Volcanics. This volcanic stage was trailed by the emplacement of Dongargarh granite rocks and the intrusion of meta-dolerite dykes and quartz veins. The arsenic-disinfested zone, which shapes the eastern outskirts of the Dongargarh granite batholiths, comprises metabasalt and metarhyolites. Along these lines, the source of arsenic in Kaurikasa territory is topographically in uenced by the Nandgaon Orogeny (Pandey et al. 2002).
In Central India, the contamination of arsenic in the groundwater was rst reported by Pandey et al. in 1999 in the village Kaurikasa of Chhattisgarh State. In this communication, we report the levels of total arsenic (and speciation) in groundwater in Kaurikasa and six adjoining villages of Ambagarh Chowki (Rajnandgaon) of Chhattisgarh state in Central East India ( Fig. 1), and for the rst time its toxicological and health impact on the human population.

Materials And Methods
The contaminated area in Chhattisgarh Rajnandgaon district is situated in the southeastern part of India and Chhattisgarh state. The region lies between 20°70' and 22°29' N latitude and 81°29' and 88°29' E longitudes. The total area of the region is 6396.28 km 2 out of which 2987 km 2 is forest area. The climate is tropical, and the average rainfall is 1275 mm. Sheonath is the key river of the Rajanadgaon district; it is a feeder of the Mahanadi River, the greatest waterway of Central east India. The river starts at Garhchiroli in Maharashtra crossing over more than 2000 km and streams northeastern way (Pandey et al. 2002).
Almost the entire district depends on tube wells and dug wells for drinking water. The tube wells are tted with either hand pumps or power pumps. In the Ambagarh Chowki block (20

Sampling of Groundwater
Twenty groundwater test samples were gathered from the hand siphons, tube wells, and open wells from Kaurikasa and connecting villages. Water is drawn for the rst 5 min. was disposed of and afterward cleaned plastic sampling bottle was lled to the top with the test samples. The sampling bottles were at rst washed with non-ionic detergent followed by an acid wash and then washed with clean lab water nally rinsed with distilled water. Two types of control test samples were collected i.e. duplicate samples and equipment blanks. The reason for the duplicated tests was to ensure the preciseness of samplings and analysis. The duplicate test samples were gathered for screening and laboratory analyses. After ltration of the examples with 0.45 µm membrane lters, a quickly concentrated HNO 3 of pH < 2 was added to preserve the samples for further arsenic analyses. Arsenic was estimated by Varian Cary 300 Atomic Absorption Spectrophotometer utilizing Carry Win UV programming software following the method prescribed by the American Public Health Association (APHA) (Greenberg et al. 1992). For the arsenic speciation examination, the standard arrangement was obtained from Sigma, USA. As (III) was directly analyzed by a generation of hydride at pH 5-7 and afterward As (V) was estimated at PH <1 by utilizing a prereductant, viz. potassium iodide-ascorbic acid technique (Francesconi et al. 1994).
Symptomatic, hematological, and biochemical tests of the affected population We carried a survey for the symptomatic study of the human population from the arsenic-contaminated Kaurikasa village of Ambagarh Chowki area of Rajnandagaon district of Chhattisgarh India availing the assistance of a physician. Prior permission from the Institutional Ethics Committee of our institute, Government V.Y.T.PG. Autonomous College, Durg, Chhattisgarh, India, was also availed before conducting the study (IEC/GVYTPGAC/07/DURG, DT 18.10.2014). Blood was collected from the population showing symptoms of Arsenicosis with their kind consent and with the help of the medical technician. Volunteers were told not to take any food in the morning before drawing blood. Blood samples were drawn by sterile disposable syringe and needle and collected in a sterile vial containing EDTA.

Results And Discussion
The extent of underground water contamination In the present study, the arsenic contamination in water was evaluated using the groundwater samples collected from various adjoining villages of Kaurikasa of Rajnandgaon district in Chhattisgarh. Twenty water samples were collected from eight villages, including Kaurikasa. Table 1 summarises the obtained results. Total arsenic was above the permissible limit in eighteen samples of ve villages. The geogenic distribution of arsenic was found variable in the region, which might be due to the variable geochemical system. An examination of the results shows that the water sample from Kaurikasa village recorded (AS III-980±126.08μg/L; AS V-1220±120.06) was the highest of all samples, which could be due to the greater depth of the borewell compared to the handpump samples from the same village. In the descending order, the arsenic levels (in μg/L) in groundwater samples were Bharritola (ASIII-432±28.  (Table-01). The arsenic level in groundwater samples in Kaurikasa and surrounding villages is far above the limit of 10 μg/L recommended by WHO for drinking water (WHO, 1999).

Symptomatic features of Arsenicosis among the population of Kaurikasa
The survey was carried in association with a physician among the population of Kaurikasa village who con rmed the highest degree of Arsenicosis. The study revealed a prevalence of various symptoms of Arsenicosis among people, as described below: 1. Diffused type of melanosis with darkening of the skin in various parts of the body, especially on the palm. . General weakness, sleeplessness, breathlessness, and indigestion are common complaints from the people in the affected area (Fig 1a, b, c & d).

Hematological alterations among the population suffering from Arsenicosis
Based on prevailing symptoms, twenty people (12-62 years) were selected for hematological and biochemical evaluation from Kaurikasa village. The Hemoglobin percentage was found below the normal range in nineteen samples and RBC count was found low in thirteen samples. WBC was lower in eleven samples, but ESR was found high (>20 mm/h) in sixteen samples in comparison to their normal range.
PCV and platelet count were lower in twelve than their standard limit, but prothrombin time was elevated in eighteen samples relative to their standard limit. (Fig. -3 of which 30% of male photolithography workers were found Leucopenic than 5-6% in control male workers. Our ndings for the twenty people exposed to arsenic-contaminated groundwater are almost in concurrence with the earlier reports from different parts of the world.

Biochemical alterations among the population suffering from Arsenicosis
The biochemical alterations noticed in the twenty blood samples were found signi cant. The serum glucose was below the normal range in ten samples (Fig. 4). Among lipid pro les the total cholesterol, LDL and HDL were found above the normal range in eight, eighteen, and thirteen samples respectively, but the TG was above the normal range in all twenty samples (Fig. 5). Protein metabolism was also disturbed. The total protein, albumin, and globulin levels were found below the normal range in nineteen, twenty, and sixteen samples, respectively (Fig. 6).
The total bilirubin and creatinine were higher than the normal range in fteen and sixteen samples, respectively (Fig. 7). The serum urea level was above the standard limit in all twenty samples (Fig. 8). SGOT was within the normal limit in all twenty samples, but SGPT was above the normal range in all twenty samples. Alkaline phosphatase was below the normal range in all twenty samples (Fig. 9). Pal  arsenite inhibits the enzyme pyruvate and alpha-ketoglutarate dehydrogenase (Aposphian 1989) which is necessary for glucogenesis and glycolysis processes. Krebs in early 1993 also described that arsenic interferes with pyruvic acid metabolism. Arsenate, on the other hand, can mimic phosphate during energy transfer pathways of phosphorylation and it is also involved in oxidative phosphorylation uncoupling (Kennedy and Lahninger 1949). However, it is unlikely that these toxic effects of acute arsenic exposure take place as a result of chronic exposure to environmentally relevant doses (Tseng 2004). Arsenic could in uence alteration in sugar metabolism by other mechanisms, including oxidative stress, in ammation, apoptosis, and the nonspeci c mechanism that has been involved in the pathogenesis of type-2 diabetes. Arsenic exposure can augment the production of reactive oxygen species (Chen et al. Overall the experimental and epidemiological evidence at present is insu cient to con rm the arsenic-induced hyperglycemia or hypoglycemia. In the present study, we found raised levels of total cholesterol, HDL, LDL, and TG in a majority of samples. In a study conducted in Bangladesh in the arsenic-contaminated region, overall 45% of residents were reported with a low range of total cholesterol, 54% with low range of HDL, and 20% with low range of LDL but triglyceride was above the normal range of 47% population (Nabi et al. 2005). Protein metabolism was also found disturbed in the present study (Fig. 6). In a study where arsenic contamination in drinking water was established, the total protein in serum was found higher in 43% population (Nabi et al. 2005). In an experiment conducted on the pigs, the total protein concentration was found to decrease than the normal range (Wang et al. 2006). The oxidative stress ensuing from arsenic toxicity causes damage to sulfur-containing enzymes and other proteins. This phenomenon ends up in the form of inactivation of protein, defective cross-linkages, and protein denaturation (Serhan et al. 1991). Serum albumin and a small fraction of globulin are synthesized in the liver, and the serum protein is a icted both quantitatively and qualitatively in liver disorder. In any disease-causing hepato-cellular damage, the concentration of serum albumin decreases. The dynamically changing levels of serum albumin, therefore, are a valuable indicator of severity, progress, and prognosis in hepatic diseases. In the present study, liver cell damage is attributed due to arsenic toxicity.
The elevated levels of bilirubin were observed in the current study. In plasma, bilirubin is found as indirect reacting bilirubin which is insoluble in water. The direct reacting esteri ed bilirubin is water-soluble. At the end of the life, span erythrocytes are destroyed in the reticuloendothelial system and liberate hemoglobin. The globulin is separated from Hemoglobin, and the porphyrin ring is opened. The released iron part goes into the iron store and may be used further for hemoglobin synthesis. Green color biliverdin forms rst from the non-iron-containing residue of Hemoglobin (i.e. Protoporthyrin). Biliverdin gets reduced to yellow-colored bilirubin. Generally, total serum bilirubin is found to increase in case of hepato-cellular damages (toxic hepatopathy neoplasm, etc.), obstructions in intra and extrahepatic biliary tract, intravascular, and extravascular hemolysis processes. Excessive elevation of direct bilirubin is seen during cholestasis and late in the course of chronic liver diseases. In the present nding, an elevated level of bilirubin in 16 samples out of 20 suggests liver toxicity among the population due to Arsenicosis.         Normal range-Total protein-6-8g/dl, Albumin-3.5-5 g/dl Gloobulin-2.5-3.5 g/dl. Showing alterations in the protein pro le of the human population affected by Arsenicosis.