Ef ﬁ ciency of Cyperus alternifolius , Typha latifolia , and Juncus in ﬂ exus in the removal of nitrate from surface water

Nitrate is one of the most toxic and pathogenic substances in drinking water. Nitrate levels have increased signi ﬁ cantly in groundwater and surface water because of the high usage of nitrogenous fertilizers, which have lethal effects on human health. Therefore, the removal of this toxic material from surface water is absolutely essential. The goal of this paper is to evaluate the ef ﬁ ciency of three aquatic plant species, called Cyperus alternifolius , Typha latifolia , and Juncus in ﬂ exus , in the removal of nitrate from surface water. This experiment has been carried out in the laboratory scale using a hydroponics method. To do this, the water samples containing nitrate were collected and evaluated before and after the treatment to see the effects of these three plants on both the nitrate removal and the pH variation of water. The results show that all the plants can remove excessive levels of nitrate from the surface water. The comparison of the results has revealed that T. latifolia plant has higher ability to remove nitrate from water than C. alternifolius and J. in ﬂ exus . It was observed that the nitrate removal ef ﬁ ciency of T. latifolia was 95%, J. in ﬂ exus was 85%, and C. alternifolius was 70% after 10 days. Moreover, it was observed that the pH values of the treated water have been affected by these plants. The results of this study can be employed for further improvement of surface water quality, thereby lowering water puri ﬁ cation time and costs as well as decreasing the nitrate-related diseases signi ﬁ cantly.

INTRODUCTION natural remediation method. Phytoremediation is an economical, environmentally friendly, and multi-purpose treatment approach used for eliminating contaminants and impurities from surface water and wastewater (Sinha et al. ). The intrinsic capability of some plant species in absorbing nitrate from water for their growth can be applied as a technique for eliminating nitrate from ground and surface water sources (Li et al. ). Moreover, macrophytes are widely used in the field of phytoremediation because of their ability to absorb high amounts of pollutant in a short period of time, thus showing high rates of pollutant elimination (Bartucca et al. ). In addition, these plants enhance the growth of microbial communities in the vicinity of their root areas; thereby, a cumulative (synergistic) condition is created to eliminate the pollutants from water (Syngonium podophyllum) and money plant (Epiprennum aureum) in treating wastewater containing nitrate. They concluded that each process variable, such as the plant density, the growth period, and the initial nitrate concentration, has a significant effect on the nitrate removal process. Of all these variables, the growth period has the most considerable effect on nitrate removing using both arrowhead plant and money plant (Shyamala et al. ). Therefore, the identification of the easily accessible plants with high capability in removing nitrate can be of great importance.
Nitrate can be considered as the main source of groundwater contaminant (Goulding ) that is also regularly reported in Iran (Jalali & Kolahchi ). The average annual application rate of nitrate fertilizers is currently over 200 kg ha À1 in Iran (Jalali ). A survey conducted in well waters of western Iran showed that nitrate concentration is varied from 7 to 122 mg L À1 with an average of 41 mg L À1 .
Nitrate concentrations in 58% of samples were in the range of 25-50 mg L À1 , 18% of samples were in the range of 51-75 mg L À1 , and 6% of samples had concentrations above 75 mg L À1 . The survey shows that the concentration of nitrate in 24% of samples is above the recommended guidelines of the WHO (50 mg L À1 NO 3 ) (Jalali & Kolahchi ).
Therefore, it is rational to consider that we can achieve higher denitrification efficiency by using aquatic plants.
Most previous studies reported on the remediation efficiency of nitrate-contaminated water have used single or two plant species. However, the removal mechanisms of water nutrients using combinations of different aquatic plant species and the subsequent remediation of polluted water need to be more thoroughly explored. The main objective of this study is to investigate the potential efficiency of three plants called Cyperus alternifolius, Typha latifolia, and Juncus inflexus for the treatment of nitrate-containing water. J. inflexus is another perennial plant that has smooth and hairless leaves. To examine the efficiency of these three plants in removing nitrate from water, a laboratory-scale experiment has been conducted under uniform controlled environmental conditions. The paper also presents the effect of these plants on the variation of pH and temperature of water.

MATERIALS AND METHODS
This study was conducted to evaluate the potential of Among three salts (i.e. nickel nitrate, sodium nitrate, and silver nitrate), the nickel nitrate has been used for contaminating the water sample because it has low toxicity and a high solubility in water compared with the other two salts.
The experimental study was designed and conducted as All stages of work and tests were carried out in the laboratory conditions at an ambient temperature of 25 C. An aquarium pump was used for continuous aeration of plants with the air hose placed inside each aquarium. The water sampling was carried out in three steps from the aquariums to maintain the plants, using 1 L containers for specified volumes. The required parameters were determined before the actual tests. After 10 days, the water samples were collected from the aquariums.
The desired variables of the study such as nitrate, concentration, pH, and temperature were assessed before the actual experiments. By performing the tests and determining the variables from both before and after the treatment, the efficiency of nitrate removal was determined. Lastly, we compared the removal of nitrate for the three aforementioned plants. In this study, the nitrate, pH, and temperature parameters were measured in water samples.
The test time was 10 days and the parameters (such as nitrate, pH, and temperature) were measured daily.

SAMPLING AND ANALYSIS OF WATER SAMPLES
In this test, a 50 ml water sample was taken from the subsurface of plants (a total of 4 water samples were taken). At each stage, the main controlling parameters in the system were examined. Ambient temperature and water pH were measured daily to maintain environmental conditions. Ambient temperature during the experiments was controlled based on the desired thermal level of plants, i.e. 23-25 C. According to the physiology of the plant, neutral pH was also controlled in their tolerance range. To analyze the water    To measure the amount of nitrate removal, firstly, 10 ml water sample with special powder (NitraVer X), used for measuring nitrate, were poured inside the tube, and then, it was shaken vigorously to mix well. Then, the spectrophotometer was set at the wavelength of 500 nm as well as at the time of 1 min. After the time is up, we waited more than 5 min for the reaction to stop, and then, we read the number of nitrate. In the next step, we used the distilled water as a benchmark (blank) and read the nitrate value.
To obtain the value of nitrate, we used Equation (1). The reduction of nitrate levels can be attributed to the process of denitrification that can be occurred in the groundwater/root interface. Denitrification has a significant effect on the bioremediation of nitrates leaching into the soil.

RESULTS AND DISCUSSION
The process of denitrification is a stepwise reduction as bellow: The reduction of nitrate to nitrogen gas is conducted by enzymes. The main process of nitrate assimilation is located in leaves, while roots serve as the place for nitrate deposition (Melvani et al. ).
In   variation in pH water. The figure illustrates that C. alternifolius slightly decreased the pH value of water after 10 days.
However, J. inflexus and T. latifolia increased the pH value of water from 6.84 and 6.9 to 7.2 and 7.3, respectively.

Nitrate levels in different plants and days
To compare the amount of residual nitrate in the tested       In contrast, the amount of nitrate remaining in the water of C. alternifolius plant, with a significant difference, is always higher than T. latifolia plant; however, the rate of nitrate removal increases gradually so that the amount of nitrate remaining in the water of the two plants is almost the same after 9 days. This indicates the existence of an interaction between the two variables of plant type and day, which based on the performed test in Table 4; this interaction was found at the significant level of 0.01.

The relationship between nitrate and pH in the tested plants
To investigate the correlation coefficients of residual nitrate and pH in the tested plants, a Pearson correlation coefficient has been used. In Table 5, the positive and negative correlation coefficients, respectively, indicate that there are direct and inverse relations between the factors. Also, the evaluation of the intensity of the calculated correlation coefficients is taken place based on Table 6. This interpretation is also applicable to the negative values of the correlation coefficients. According to Table 5, there is a significant