A R T I C L E  I N F O		ABSTRACT
ORIGINAL ARTICLE		Introduction:  Several diseases, especially in infants such as some cancer and blue baby are related to the presence of nitrate in drinking water. The Environmental Protection Agency (EPA) specified the maximum contaminant level (MCL) of nitrate as 50 mg L-1 for regulated public water systems. This study aimed to evaluate the concentration of nitrate and to assess its probabilistic risk exposure in drinking water wells of Abarkouh city, Iran. Materials and Methods:  The average annual nitrate level was studied from 18 wells around Abarkouh in 2017. The Hazard Quotient (HQ) was also investigated as health risk assessment and sensitivity analysis was carried out for effective variables. Results: Average concentration of nitrate was 27.57 ± 6.80 mg L-1 and all measured concentrations were below the permitted maximum standard (50 mg L-1) according to the National Standard of Iran. The HQ value for children and adults were more than 1 (1.81) and less than 1, respectively. In calculating HQ for children, the most important variable was the concentration of nitrate in drinking water. Conclusion: According to the results, children health is highly at risk in these areas and exposure to nitrate should be reduced for at-risk populations.
Article History: Received: 24 January 2019 Accepted: 20 April 2019
*Corresponding Author: Nasrin Homayoni bezi Email: n.homayoni73@gmail.com Tel: +98 916 252 3893
Keywords: Nitrate, Geographic Information Systems, Risk Assessment, Drinking Water, Abarkouh City.

Introduction
Nitrate and nitrite are among the stable forms of nitrogen in aerobic systems ¹. Nitrate is widely used as a mineral fertilizer in agriculture. Furthermore, it is used as a food preservative. Sodium nitrate is considered as a preservative, especially in meat conserves ². The concentration of nitrate in surface water is naturally low and in the range of 0-18 mg L^-1. The entry of surface runoffs, especially runoff from agricultural land, can increase the concentration of nitrate in surface and groundwater ³. Nitrate concentrations in surface waters are usually variable with season changes. In many European countries, nitrate concentrations have risen in recent decades; in some cases nitrate concentrations doubled in the last 20 years. For example, in some rivers in England, nitrate concentration had an average annual increase of 0.7 mg L^{-1 4}.
Efforts to treat wastewater containing nitrate compounds were targeted at reducing its concentration in environment ⁵. The concentration of nitrate is usually low in groundwater under aerobic conditions and depends on the type of soil and its geological characteristics. In the United States, nitrate concentration in groundwater typically does not exceed 4-9 mg L^-1 for nitrate and 0.3 mg L^-1 for nitrite ⁶. However, with increasing uncontrolled agricultural activities, nitrate concentration can increase dramatically ⁷. For example, a concentration of more than 1500 mg L^-1 was observed for nitrate in groundwater areas of India that was good for agricultural activities ⁸. Considering nitrates exposure, carcinogenesis was not reported in laboratory animals, but increased tumor growth was reported in animals exposed to high levels of nitrite ⁹. Nitrate toxicity in humans depends on the reduction of nitrate to nitrite. The most important biological effect of nitrite in humans is conversion of hemoglobin to methemoglobin, which cannot carry oxygen to body tissues. Common clinical signs appear when more than 10 percent of hemoglobin is converted to methemoglobin, which is called methemoglobin anemia. High concentrations of methemoglobin may result in choking and death. Typically, methemoglobin concentration in body is less than 2 percent, but it is less than 3 percent among children of younger than three months old ². Many reasons, such as the entrance of agricultural water and sewage industries can increase the concentration of nitrate in groundwater ^{5, 10}. In some previous studies over nitrate concentration in ground water in Iran, the nitrate concentration was higher than the standard level in some areas. For example, in the study conducted by Mousavifazl and Fathi Hafshejani, the nitrate concentration was investigated in ground water in Mashhad and Shahrekord, respectively. The results showed that nitrate concentration was higher than the standard level (50 mg L^-1) in some areas ^{11, 12}.
Today, electronic systems and software are used to monitor groundwater and assess the risks of existing pollutants ¹³. One of the best ways to prevent groundwater contamination is to investigate the spatial distribution of groundwater quality and use its results in managing water resources and land use ¹⁴. Geographic Information System (GIS) is a new technology used to analyze and interpret the distribution of pollutants in environmental studies ^{15, 16}. Inverse distance weight (IDW) is one of ArcGIS's application techniques for spatial and pollutants' distribution, which simulates pollutant concentrations in other parts of the study area based on the distance between points and concentration of pollutants at each point ¹⁷. So far, various GIS software has been used to analyze, interpolate, and zone various pollutants ^18-22. Health risk assessment is a method that measures risk assessment based on the input data such as concentration of chemical and other parameters. This method can examine the real risk in areas where low risk is considered. Most recently, health risk assessment was used as a reasonable method to calculate risk potential of chemical pollutants ^{23, 24}. The aim of this study was to investigate the concentration of nitrate in drinking waters wells of Abarkouh. In this study, the health risk was studied for different groups.
Materials and Methods
Study area, sampling and analysis
In this study, the drinking water of Abarkouh city located in Yazd province in center of Iran was investigated. This city is located in the GPS coordinates of 31.1304 N, 53.2504 E has a population of 51552. In this area, drinking water is provided by underground water. Nitrate concentration data were obtained from water lab of Yazd health office.  This data were seasonally (Four seasons) sampled during 2017 and assessment was based on the average concentration of nitrate in the studied area in 2017. The concentration of nitrate was determined by spectrophotometer method with DR-5000. The demographic and geographic data of the studied area are presented in Table 1 and Figure 1 shows the location of wells in the city.

City	Population	City location	Study area (wells) location	Number of wells	Average flow (lit sec-1)
Abarkouh	51552	31° 7'N - 53°17'E	30˚52'N - 31˚12'N 52˚50'E - 53˚10'E	18	22.22 ± 2.63

Spatial distributions
In order to zone the nitrate concentration in drinking water, ESRI's ArcGIS 10.1 software was used. The IDW interpolation technique was used for zoning and providing an independent raster layer related to the concentration of contaminants in different points of the study area ²⁵. In many studies, IDW techniques were applied to zone pollutants, such as investigation of the heavy metals' presence in West Bokaro groundwater and its spatial variation ²⁶ as well as the survey of Eğirdir Lake Basin groundwater quality assessment and risk assessment used by GIS ²⁷. As mentioned, IDW is a non-statistical method using spatial prediction techniques for environmental studies to predict the concentration of pollutants at geographical points with unspecified concentrations ^{28, 29}.
In the IDW hypothesis, the predictive values have a linear relation with the available data. The IDW model is calculated by the following equation ^{30, 31}.

Where, W is the station weight i, Di is the distance between point i and place of unspecified values, α is the weight of power, and n is the total number of points used in zoning.
Health Risk Assessment
In this section, the risk of non-carcinogenicity associated with nitrate was studied to evaluate its health effects. Hazard Quotient or Non-carcinogenic hazards related to the nitrate was calculated by the following equation.

Where, RfD is the reference dose for nitrate that has been received by a specific exposure pathway in mg Kg^-1day^-1 based on the USEPA's Integrated Risk Information System (IRIS) database.
The Estimated Daily Intake (EDI) shows the daily intake of nitrate consumed by drinking water and is estimated using Equation 3 introduced by USEPA (1989) ³².

In this equation, Cw is the concentration of nitrate in drinking water in mg L^-1, IRw is the drinking water ingestion rate based on L day^-1, EF is the exposure frequency based on Day year^-1, ED is the exposure duration in terms of years, BW is the body weight in Kg, and AT is the averaging time in days.
In this study, the sensitivity analysis technique was used to determine how different values of input variables can effect risk assessment in the assumed conditions. In this study, risk assessment was conducted using the Monte Carlo Simulation technique which is provided by the Oracle Crystal Ball (ver 11.1.2.4) software ^33-35.
Ethical issues
This study was conducted with the approval of Shahid Sadoughi University of Medical Sciences and Health Services, Medical Ethics Committee. Code: IR.SSU.SPH.REC.1397.104  
Results
The results of nitrate concentration analysis within 18 wells of Abarkouh showed that nitrate concentration in the studied wells was less than the standard set by both the EPA guidelines and Industrial Research of Iran No. 1053, which is 50 mg L^{-1 36}. Figure 2 shows the concentrations and repetitions of nitrate in the wells. Based on Figure 2, the maximum concentration was 40 mg L^-1 and the most frequent concentration was 25 mg L^-1. Wells number 1 to 4 had the highest nitrate concentration. The RMSE was 0.09826 for zoning nitrate concentration. Based on the risk assessment results for children and adults, the children group had a HQ of higher than 1, which represents a high risk population. Sensitivity analysis showed that concentration of drinking water nitrate was the most important factor affecting health risk of both groups.

Figure 2: Histogram of nitrate concentration and frequency in the study area
 

Discussion
Spatial distributions
The spatial distribution of nitrate concentration was conducted by ArcGIS software using the IDW technique in the studied area. Based on the spatial variation, wells numbers of 1-4 had the highest concentrations of nitrate; whereas, wells numbers 13, 14, and 16 had the lowest nitrate concentrations. Figure 3 shows the map prepared based on the concentration of nitrate in the 18 studied wells. High concentrations of nitrate in these wells, which are near each other and in the same region, can indicate a source of regional pollution such as agriculture. Therefore, the area should be investigated in terms of the contamination source. The concentration of nitrate in groundwater under aerobic conditions is usually low and depends on the type of soil and its geological characteristics. In the United States, nitrate concentration in groundwater typically ranges from 4-9 mg L^-1 for nitrate and 0.3 mg L^-1 for nitrite ³⁷. However, with increasing uncontrolled agricultural activity, nitrate concentration can increase significantly ³⁸. For example, a concentration of more than 1500 mg L^-1 was observed for nitrate in groundwater of India that was used for agriculture ⁸. Mousavifazl et al. conducted a study to evaluate nitrate in 276 wells in Mashhad city, Iran. The nitrate plans showed that nitrate concentrations in a certain part of some areas are higher than the standard limit ³⁹. In another study Fathi Hafshejani et al. evaluated the spatial distributions of nitrate concentration in 100 groundwater wells of Shahrekord (Iran) from 2006 to 2011. The results showed that concentrations of nitrate were high in the south part of the studied area, which can be caused by the presence of municipality treatment plants and intensive cattle farming in this area ¹¹.

Figure 3: Zoning the concentration of nitrate in the studied area
 

Health Risk Assessment
An HQ non-carcinogenic risk assessment was conducted to assess health risk. According to this assessment, in the case that HQ is higher than 1, the target population is considered to be at high health risk and water consumption can cause illness. The population studied in this research was divided into two groups of children (0 to 7 years old) as well as teens and adults (over 7 years old). Figure 4 shows the calculated HQ for children and Figure 5 shows the HQ for teens and adults. The average non-carcinogenic risk for the adult group in the study area was estimated to be less than 1 and therefore, it is negligible. The HQ values for the 95th percentile in the adult age group was less than 1 and for children group was higher than 1, which indicates a high non-carcinogenic risk for the children age group. The reason of high risk for children is their low body weight ⁴⁰. The highest 95th percentile of the calculated HQ in the study areas was 1.81 for children, which shows a higher non-carcinogenic risk. For all studied regions, the non-carcinogenic risk of nitrate for the two exposed groups was Adults>Children. According to the results of health risk assessment, children are the population at risk, which is similar to the results reported by Zhang et al. ⁴¹ and Guissouma et al. ⁴²
Sensitivity analysis
Sensitivity analysis was conducted to determine the most effective variable in increasing the health risk. Figures 6 and 7 show the sensitivity analysis of variables in calculating HQ for children and adults, respectively. Based on the results, the amount of nitrate concentration in drinking water had the greatest impact on increasing non-carcinogenic risk for the two groups; so, decrease of nitrate concentration in water can reduce the risk of health. It should be noted that due to the high non-carcinogenic risk for children group, reducing the concentration of nitrate would have the most effect in reducing the health risk. In addition to drinking water, nitrate can also enter the body through other forms of contact, such as skin absorption ²³ and the consumption of various foods ³⁶. In this regard, the best way to reduce pollutant concentrations in this area is to investigate the whole region and eliminate the sources of contamination ⁴³. As a result, reduction of nitrate concentration would reduce the health risks in children group ²⁴.

Figure 4: The range of HQ for the children population

Figure 5: The range of HQ for the teens and adults population

Figure 6: The results of sensitivity analysis of the variables involved in calculating HQ for the children population
 
Figure 7: The results of sensitivity analysis of the variables involved in calculating HQ for teens and adults population
 

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