Introduction

Water is critical for all forms of biological life and its quality can have a main impact on human health. Consumption of contaminated water can cause several types of diseases, collectively called waterborne diseases, such as cholera, salmonellosis, typhoid, dysentery, hepatitis a, and poliomyelitis. According to the World Health Organization (WHO), such diseases cause about 1.5 million human deaths annually (Prüss-Üstün et al. 2008). Several studies demonstrated that some toxins in drinking water may increase the risk for cancer developing (Inoue-Choi et al. 2015, 2016; Chen et al. 2010).

Water pollution by nitrite and nitrate is a global issue, and it may cause health problems when occur at elevated levels (Bahadoran et al. 2015; Khademikia et al. 2013; Ward et al. 2008; WHO 2011; Inoue-Choi et al. 2015; Stayner et al. 2017). Nitrite and nitrate are occurring naturally as part of the nitrogen cycle. A significant amount of nitrite and nitrate in water are of anthropogenic origin. Their sources in water include leaching of nitrogen-containing fertilizers and animal waste, improperly treated septic and sewage discharges and decomposing living organisms. Nitrite may also be produced in distribution galvanized steel pipes by the action of Nitrosomonas bacteria during stagnation of nitrate-rich and oxygen-poor drinking water or if uncontrolled chloramination is used as disinfection process (WHO 2011).

To prevent methemoglobinemia (blue-baby syndrome) in infants, the WHO sets the maximum contaminant levels (MCLs) at 50 mg L−1 (or 11 mg L−1 as nitrate-nitrogen) for nitrate, and 3 mg/L (or 0.9 mg L−1 as nitrate-nitrogen) (short-term exposure) and 0.2 mg/L (0.06 mg L−1 as nitrate-nitrogen) (long-term exposure) for nitrite (WHO 2011). The Food and Drug Administration (FDA) lists 10 mg L−1 total nitrate and nitrite (as nitrogen) as a maximum acceptable level in bottled water (ATSDR 2016).

The noxious health impacts of nitrate are mostly due to its transformation to nitrite by the action of some types of bacteria in the gastrointestinal tract (Govoni et al. 2008). Methemoglobinemia is the major adverse health impact related to human exposure to nitrite (or nitrate), especially in infants. Nitrite oxidizes the Fe2+ in heme to Fe3+ converting hemoglobin to methemoglobin, which has no ability to carry oxygen. Concentrations of methemoglobin above 10% can lead to cyanosis, and that exceed 50% can be fatal (EPA 1990). Studies in experimental animals showed that both nitrite and nitrate are carcinogenic when administered in the presence of nitrosatable amines (or high protein intake), due to the formation of carcinogenic nitrosamines in the stomach (Cancer 2010; Furukawa et al. 2000). Studies among human are contradictory and provide inadequate data about the relation between nitrite or nitrate exposure and increased risk for cancer (DellaValle et al. 2014; 2013; Aschebrook-Kilfoy et al. 2012; Inoue-Choi et al. 2015).

To the best of our knowledge, there are no adequate studies about nitrite and nitrate levels in drinking water and its health risk assessment in Egypt. Therefore, the purpose of the present study is to determine the concentrations of nitrate and nitrite in drinking water of some cities and villages in Dakahlia governorate, Egypt, and to compare these concentrations with the standard levels. The obtained results were used for the determination of the hazard quotient (HQ) and the hazard index (HI) for nitrite and nitrate.

Materials and methods

Description of study area

Dakahlia is one of the 27 governorates of Egypt lying northeast of Cairo. It covers an area of about 3500 km2 and it has a population of approximately 6.2 million people. This area is characterized by mild climate with average annual temperature of 20.5 °C. The major cities of Dakahlia governorate are Mansoura, Talkha, Dikirnis, Aga, El Senbellawein, Mit Ghamr, Sherbin, Manzala and Bilqas. Like most of the governorates of Egypt, residents of this area are depending mainly on the Nile River as the main source of drinking water after treatment in water stations. Some houses in the small villages depend on groundwater for drinking. Seventeen cities and villages in Dakahlia governorate were involved in this study namely; Mansoura, Talkha, Aga, Dikirnis, Meet Khamis, Nawasah, Wish elhagar, Meniet Samanoud, Kafr Hassan, Serso, Meet elsarem, Elkhayriah, Meet Mazah, Mahalet Damanah, Salamoon, Elbaklyia and Telbanah (Fig. 1).

Fig. 1
figure 1

Map of the study area and location of the cities where sampling sites are located

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Reagents

All chemicals used in this study were of analytical grade, supplied from Sigma-Aldrich (St. Louis, MO, USA), unless stated otherwise, and all solutions were prepared using ultrapure water (UPW) obtained by Milli-Q water purification system (Millipore, Billerica, MA, USA). A stock standard solutions (1000 mg L−1) of nitrite and nitrate were prepared by dissolving 150 mg and 163 mg of pre-dried NaNO2 and KNO3, respectively, in UPW containing a small amount of NaOH to prevent decomposition. The resulting solution was diluted to 100 mL after adding 0.2 mL of chloroform to prevent the growth of bacteria. These solutions were stored in brown bottles at 4 °C, for a maximum period of 2 weeks. Working standard solutions were prepared daily by appropriate dilution with UPW. 0.1% solution of N-(1-naphthyl)-ethylenediaminedihydrochloride stock solution (reagent A for nitrite) was prepared by dissolving 50 mg of the compound in 50 mL of UPW. Five hundred milligrams of sulfanilamide was dissolved in 50 mL of 5% phosphoric acid to give 1% solution of sulfanilamide (reagent B for nitrite). Reagent A and B were stored at 4 °C in brown bottles. Griess reagent for nitrite was prepared immediately prior to the analytical run by mixing equal volumes of reagent A and B. This reagent is not used after 8 h of mixing. Brucine-sulfanilic acid reagent for measurement of nitrate was prepared by dissolving 1 g of brucine sulfate and 0.1 g of sulfanilic acid in 3 mL of concentrated HCl and the volume was then completed to 100 mL with UPW in a volumetric flask.

Water sampling and analysis

A total of 1291 drinking water samples were collected in polyethylene bottles from December 2015 to May 2016 (556 from urban areas, 563 from rural areas and 172 from ground water). Samples were immediately transported to the laboratory in ice-box and analyzed within 2–3 h after collection. Nitrite was determined spectrophotometry by a modified Griess reaction which involves diazotizing with sulfanilamide and coupling with N-(1-naphthyl)-ethylenediamine dihydrochloride (Jeffery et al. 1989). Briefly, 100 μL of Griess reagent, 300 μL of the sample and 2600 μL of UPW were mixed in a test tube and incubated for 30 min at room temperature. The absorbance was measured at 548 nm relative to the reagent blank using an UV-Visible spectrophotometer (UV-2550, Shimadzu, Japan).

Nitrate was analyzed using the EPA approved Brucine method (Method No. 352.1.) (Jenkins and Medsker 1964). In each test tube in the set, 100 μL of Brucine-sulfanilic acid reagent was mixed with 2.0 ml of sample followed by addition of 2.0 ml of concentrated sulfuric acid. The mixture was heated in a boiling water bath for 25 min. After cooling to room temperature, the absorbance was read at 410 nm. The concentration of nitrite and nitrate were calculated from calibration curves of the standards.

Quality assurance

The accuracy of the procedure was determined by analysis of spiked water samples at the beginning and the end of each run. The recoveries (expressed as mean ± S.D.) of known amounts of nitrite (0.03 mg L−1) and nitrate (2.0 mg L−1) spiked into water samples were 97.4 ± 3.2 and 95.8 ± 4.1%, respectively. Relative standard deviation (RSD) was used to measure the precision. The intra-day RSD was determined by measuring 3 replicate standard samples containing 0.03, 0.04 and 0.08 mg L−1 of nitrite or 1.5, 3.0 and 6.0 mg L−1 of nitrate. The within-day RSD was found to be 2.6, 3.3 and 3.7% for nitrite and 3.7, 2.9 and 3.5 for nitrate, respectively. The inter-day precision was assessed by measuring the same three concentrations for 5 different days. The obtained results providing RSD of 3.2%, 3.4%, and 3.9% for nitrite, and 2.9, 3.8 and 4.0% for nitrate, respectively.

Risk assessment

Risk assessment indices were calculated according to EPA (1992). Average daily intake (ADI) was calculated based on the measured levels of nitrite and nitrate from the following formula:

$$ \mathrm{ADI}\ \left(\mathrm{mg}\ {\mathrm{kg}}^{-1}{\mathrm{day}}^{-1}\right)=\frac{\mathrm{C}\ \mathrm{x}\ \mathrm{DI}}{\mathrm{BW}} $$
(1)

Where, C is concentration of nitrite or nitrate in drinking water (mg L−1), DI is an average daily intake rate of drinking water (L day−1), and BW is average body weight (kg).

Hazard quotient (HQ) is calculated separately for nitrite and nitrate by dividing the ADI of the ion by its reference dose (RfD).

$$ \mathrm{HQ}=\frac{\mathrm{ADI}}{\mathrm{RfD}}\kern0.5em $$
(2)

According to EPA, the RfD for nitrite and nitrate were taken as 0.33 and 7.0 mg/kg/day, respectively (EPA 2017).

Hazard Index (HI) is defined as the sum of HQ’s for nitrite and nitrate and is calculated from Eq. (3):

$$ \mathrm{HI}={\left(\mathrm{HQ}\right)}_{\mathrm{Nitrite}}+{\left(\mathrm{HQ}\right)}_{\mathrm{Nitrate}} $$
(3)

Statistical analysis

All the statistical calculations were performed by using the SPSS statistics package program (SPSS software, IBM SPSS product version 20). The data were checked for normality by using Kolmogorov-Smirnov test. Nitrite and nitrate concentrations between groups were compared by using Student’s t test or One-way ANOVA. Correlation between nitrite and nitrate concentrations was assessed with Pearson’s correlation coefficient (r). Linear regression was used to detect independent predictor of nitrite levels in water. A value of probability (p) < 0.05 was considered to be statistically significant.

Results and discussion

Nitrite and nitrate levels in drinking water for the general population

In the present study, the concentrations of nitrite and nitrate in 1291 drinking water samples from different localities of Dakahlia Governorate were determined. Table 1 shows the obtained results in comparison to WHO water quality guidelines. According to the results, the levels of nitrite and nitrate were significantly lower than the WHO guidelines that set out to prevent methemoglobinemia (p < 0.01). Nitrite and nitrate levels in water are depending on many factors such as the type of water (surface or ground), soil type, fertilizer used, level of contamination and geochemical conditions. Reviewing the data in literature showed that, in most countries, nitrite and nitrate concentrations in drinking water do not exceed 0.1 and 10 mg L−1, respectively, regardless the source of drinking water whether surface, ground or bottled water (Espejo-Herrera et al. 2013; Kermanshahi et al. 2010; Cidu et al. 2011). However, in some areas, the concentrations of nitrite and nitrate may exceed the MCL as a result of discharge of sewage and industrial wastes as well as the consequence use of nitrogenous fertilizers (D’Alessandro et al. 2012; Gulis et al. 2002).

Table 1 Nitrite and nitrate concentrations in drinking water in the studied area in comparison with the WHO water guidelines
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Correlation coefficient calculations showed a highly significant relationship between the concentrations of nitrite and nitrate, regardless of the sample sources (r = 0.622, p < 0.001). The results of multiple regression analysis show that the nitrate level [Odds ratio 0.006 95% C.I. (0.005–0.007), p < 0.001] is an independent predictor of nitrite regardless sample location [odds ratio 0.004 95% C.I. (0.0001–0.007), p < 0.05].

Figure 2 shows the scatter plot and distributions of nitrite and nitrate concentration, respectively, of rural and urban regions. As shown from the scatter plot, the rural samples are located in the top right corner, i.e., with high concentrations of nitrite and nitrate. On the other hand, the urban samples are located in the bottom left corner where the concentrations of both nitrite and nitrate are lower. Moreover, from the two distributions in the top and left panels, it is clear that the discrimination between samples of rural and urban using nitrate concentration is higher than using the nitrite concentration. Further, the range of nitrate variation for rural samples was higher than urban samples, while the ranges of nitrite variation for both rural and urban samples were approximately the same.

Fig. 2
figure 2

Scatter plot and distributions of rural and urban sampling stations. The top panel is the distribution of nitrite concentration of rural and urban regions. The left panel is the distribution of nitrate concentration of rural and urban regions

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When samples were classified according to the geographic origin as presented in Table 2 and the histograms in Fig. 3, the results showed significant regional differences for nitrite and nitrate (p < 0.001). Drinking water samples from Meniet Samanoud, Kafr Hassan, Meet El-Sarem and Mahalet Damanah showed the highest mean concentrations for nitrite, while Kafr Hassan, Serso, Mahalet Damanah and Salamoon displayed the highest levels of nitrate. All of these locations are agricultural areas.

Table 2 Regional differences for nitrite and nitrate as mean ± SD
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Fig. 3
figure 3

Regional differences of nitrite and nitrate levels. (1) Mansoura, (2) Talkha, (3) Aga, (4) Dikirnis, (5) Meet Khamis, (6) Nawasah, (7) Wish elhagar, (8) Meniet Samanoud, (9) Kafr Hassan, (10) Serso, (11) Meet elsarem, (12) Elkhayriah, (13) Meet Mazah, (14) Mahalet Damanah, (15) Salamoon, (16) Elbaklyia, (17) Telbanah

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The one-way ANOVA was used to examine the significant seasonal variations in nitrite and nitrate concentrations among all the studied locations. As presented in Table 3, no statistically significant monthly differences were observed for nitrite and nitrate. Similar findings were observed by Schullehner et al. (2017). However, contrast results were obtained by others (Pachero et al. 2001). We explained our results by the constant use of nitrogen fertilizers by farmers.

Table 3 Seasonal variations of nitrite and nitrate levels among the studied locations
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Table 4 presents a comparison of the nitrite and nitrate levels in drinking water in urban and rural sites. The results indicate that groundwater, which is used as the main source of drinking water in some villages, has the highest concentrations of both nitrite and nitrate. Moreover, water samples that collected from rural sites showed higher levels of nitrite and nitrate compared to those collected from urban areas. This indicates that leakage of fertilizers from the soil as well as the decay of agricultural traces may be responsible for higher concentrations of nitrite and nitrate in drinking water.

Table 4 Comparison between urban, rural and ground water in relation to nitrite and nitrate levels
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Daily intake and risk assessment of nitrite and nitrate from drinking water in the studied population

The mean values of our measurements for nitrite and nitrate were used to calculate ADI. The estimation of ADI also requires knowledge of daily water intake and body weight. Certainly, water intake depends on several factors including weight, age, gender, climate and some living habits (FNB 2004). For infants, a quantity between 100 and 190 mL kg body weight−1 day−1 is recommended by the European Food Safety Authority (EFSA) for infants up to 6 months (EFSA 2010). The EPA estimates water ingestion rate for infant below 3 months as 205 mL kg−1 day−1. The water intake from breastfeeding is included in these considerations (EPA 2011). Although nitrate level in breast milk did not reflect its concentration in drinking water (Dusdieker et al. 1996), infants still at risk for nitrite and nitrate exposure from drinking water that is used for the preparation of their formulas. This exposure becomes more significant in the presence of nitrite and nitrate in the powder of the formula (Sadler et al. 2016).

The Tropical Agriculture Association sets the minimum water requirement of 3 L day−1 and 4.1–6 L day−1 for a 70-kg human in a temperate and tropical zone, respectively (Tropical Agriculture Association 2017). The FNB recommended amount of 1.5 mL water/kcal of energy expenditure for the United States civilian population (FNB 1989). Otherwise, several online web tools are available to estimate daily water demand such as H4H Hydration calculator (H4H hydration calculator 2017).

In the present study, we used daily water intake values that have been established by the Food and Nutrition Board in 2004 (FNB 2004). These age and gender-specific values are presented in Table 5. The average body weights of infant and children groups were given by the Egyptian growth chart (Ghalli et al. 2008). We estimated the maximum recommended body weight for 25 years adult males and females from an online tool (Ideal Weight calculator 2017) based on the average height for Egyptian males and females of 170.3 and 158.9 cm, respectively (El-Zanaty and Way 2009).

Table 5 Specific exposure factors applied for exposure quantification
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The calculated ADI of nitrite and nitrate from drinking water in the current study are presented in Table 6. Interpretation of these results indicates that the daily intake of nitrate is very smaller than that associated with methemoglobinemia in infants (WHO 2011) and adults (Joint FAO/WHO Expert Committee on Food Additives 1996) which are in the range of 37.1–108.6 and 33–150 mg kg−1 day−1, respectively.

Table 6 Risk data for nitrite and nitrate in drinking water for different age groups
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Hazard indices of exposure to nitrite and nitrate from drinking water were calculated in terms of HQ and HI for non-carcinogenic effects. As one can see in Table 6, the values of HQ and HI for all localities and age groups are less than unity. This assigns that the risk of harmful health impact of nitrite and nitrate in drinking water among the studied population is acceptable. We should keep in mind that this accepted daily intake concerns with all routes of ingested nitrite and nitrate and not only drinking water. A complete assessment of exposure requires knowledge of the concentration of these ions in infant formula, the possible exposure from breastfeeding and the determination of nitrite and nitrate in foodstuff as meat, dairy products, vegetables and fruits. Infants and children, especially those under 3 months of age, are the most susceptible population to hazard of nitrite and nitrite in term of methemoglobinemia (WHO 2011). Few studies have demonstrated an association between the concentration of nitrites and/or nitrates in drinking water and adverse health effects in adults. There is a link between intake of high levels of nitrite or nitrate and gastric cancer mortality (Sandor et al. 2001) and thyroid cancer (Ward et al. 2010). Methemoglobinemia is also possible in adults with reduced gastric acidity (WHO 2011).

Table 7 shows a comparison of nitrite and nitrate concentrations in water resources found in the present study with those reported by other authors. As presented, the levels of nitrite and nitrate in samples collected in the under-studied area are low or comparable with the results obtained by other reports. In contrast with our findings, the exposure to nitrite and/or nitrate from drinking water in some areas may exceed the standard guidelines and health effects are possible (Sadler et al. 2016; Taneja et al. 2017; Su et al. 2013).

Table 7 Comparison of nitrite and nitrate levels and human health risk with other studies
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Conclusion

This study has suggested that nitrite and nitrate concentration in drinking water of Mansoura city and its suburbs are within the acceptable limits and health risk of the population in these areas is low. The ADI values for the studied population were lower than the WHO guideline values. The HQ and HI for all age groups were less than unity indicating a low level of risk for non-carcinogenic health impacts. Other potential sources of nitrite and nitrate, such as infant formulas, vegetables, fruit, meat, dairy products, etc., may increase hazard indices. For that reason, it would be recommended to determine nitrite and nitrate in the major foodstuff among Egyptian community to provide a complete profile about the possible impact of these nitrogenous ions on the health.