The Analysis Of Nitrate Biology Essay

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Nitrate is one of the sources of nutrient that can occur naturally or by means from anthropogenic activities. Its natural occurrence does not have any impact or brings forth danger; however the flow of nitrate from groundwater or from effluents containing high concentrations of nitrates will effect human health and enhance the eutrophication. Nitrification takes place when the Nitrosomonas bacteria oxidise ammonium ions to nitrate (Radojevic and Bashkin, 2006), this is what is considered as a natural process of natural presence of nitrate in bodies of water, and however, there are also sources due to anthropogenic activities such as industrial and agricultural effluents. Below shows the reaction of ammonium to nitrite where the Nitrosomonas bacteria oxidise;

NH4+ + OH- + 1.5 O2  H+ + NO2- + 2H2O

and from that another bacterial reaction will oxidise the nitrite to nitrate;

NO2- + 0.502  NO3-

To reverse the affect or to reduce the amount of nitrate concentration in water bodies, denitrification uptake is usually applied (McMahon and Bohlke, 1996). Nitrate concentration however varies with season, amount of input soil leaching (Ruiz et al., 2002). OH in the equation stands for the hydroxyl radical, and it is thought that the photolysis of nitrate and nitrite ions are the main sources of the OH radical (Zuo and Deng, 1998).

The most major concern of nitrate from excessive input is eutrophication and health effecting both human and other living organisms within the water bodies. High concentration of nitrate may lead to the growth of algae and reduce the dissolved oxygen concentration thus affecting the biological state of the body of water (Houser and Richardson, 2010) With this, there has been concern about the concentration of nitrate around the world (Worrall and Burt, 1998).

The standard guideline for nitrate in drinking water is 50 mg NO3- L-1 which was set by WHO (World Health Organization) (Hespanhol and Prost, 1994), and the EC directive specified 11.3 mg NO3-N L-1 to be the maximum amount allowed in potable waters (Hooda et al., 1997). However nitrate concentration that can was found in drinking water that contains a reading of 10 mg NO3-N L-1 may cause defect or bring forth danger to children, this is because, nitrate turns to nitrite due to bacterial action and this affects the intestines of the children where it is less acidic unlike adults, the where it takes place in a higher digestive systems (Radojevic and Bashkin, 2006).

The aim of this study is to investigate nitrate concentration from different bodies of water and from different times (of when samples were taken). The most recent amongst the samples are dated from the 23/2/2010 and the most back dated samples were from December 2009.



Samples were collected from different places and at different times. Samples that were analysed were taken from Filey, Scalby (December 2009, 23.02.2010), Codbeck (I, 10)[taken at a different site, where 1 is from the where the river starts and 10 is where the mouth of the river is situated), Tees Lake, Tees River (December, 2009), and Tees River (23.02.2010) where the area is surrounded by mainly agricultural activities or/and industrial area and tap water was also used for the purpose of analysis.

Samples were then kept in a refrigerator at a temperature of 4°C or lower in order not to let any bacterial growth.


All methods will be based on methodology by Radojevic and Bashkin (2006). In order to prepare for calibration standard a standard nitrate solution was prepared by pipetting 1, 5, 10, 20, 40, and 50 mL of working nitrate solution in 100mL volumetric flasks. The flasks filled with nitrate solution were then filled up with water to make up to the 100mL mark.

Water samples taken were filtered before tests were carried out. In a 10mL volumetric flask, 2mL of samples were added and a drop of sulphite-urea reagent was added to each sample flasks. The samples were later placed in tray filled with water and were kept in a temperature between 10 - 20°C. 2mL of antimony reagent were added in each flask whilst swirling and was left for 4 minutes and later 1mL of chromotrophic reagent were added and while swirling and left for 3 minutes. When the flasks were put in the tray of water for the purpose of cooling, the samples may reduce due to evaporation, thus, to make it up to the 10mL mark, concentrated H2SO4 were added. The cap were placed flasks were inverted 4 times in order for the substances to be well mixed and the samples were left to stand for 45 minutes in a room temperature. Concentrated H2SO4 were added to some samples to make it up to the 10mL mark and again was left for 15 minutes.

UV spectrophotometer was used to measure the absorbance. The absorbance was measured at 410nm (visible region) and as for a blank sample, water was used. Each sample was then put in a 1cm absorption cell and absorbance was measured. Measurements of the absorbance of the sample were carried out using the Helios Unicam Gamma.


After reading and recording the absorbance, absorbance of water blank is subtract with absorbance of samples and standards.

e.g. blank = 0.024

First standard calibration read

0.1 mg/L

0.078 - 0.024 = 0.054

Concentration (mg NO3 - N)

absorbance - blank















Table 1 shows the standard calibration of nitrate with absorbance subtracting the water blank (e.g. 0.078 - 0.024

Figure 1 show the calibration curve derived from the calibration standards where the absorbance of the standards is subtracted with the blank. Linear equation was generated from the scattered graph and with that equation the concentration of nitrate will be determined.

Nitrate concentration calculation (NO3-)

Linear equation (y = mx + b) derived from the regression analysis is used to determine the concentration of nitrate. Calculations of the concentration are as follows. With this equation, concentration (x) will be determined.

y = 0.1874x + b


y = absorbance

m = 0.1874

x = concentration

b = intercept (which is 0)

y = 0.1874 (x)

0.017 = 0.1874 (x)

x = 0.017


= 0.090715048 mg L-1



Absorbance - blank (0.024)

Concentration mg/L

Tap water








Scalby 2




Scalby 23/2/2010




Codbeck 1




Codbeck 10




Tees lake




Tees river '09




Tees river 23/2/2010








Table 2 shows the different sites and the concentration and the absorbance of the each site. Absorbance of each samples were subtracted with the absorption of blank and using the equation above, the concentration was calculated.

Figure 2 show the absorbance of the samples subtract with the blank (0.024). The figure shows that Scalby has the lowest absorbance and Codbeck 1 has a higher absorbance than other samples.

Figure 3 show the concentration of each site measured in mg L-1. According to the figure, the concentration of Codbeck 1 is higher than other samples.

Quality assurance and quality control

Whilst conducting the analysis, the amount of each solutions or chemical put in has to be at the right amount, and has to be carried out according to the directions given. This is ensuring the result that is obtained will be accurate. Quality assurance in terms of this experiment is to collect fresh samples, or keeping samples at a temperature where bacteria will not be active. This is to ensure that samples are fresh so that the true nitrate content can be measured.

Before conducting or adding in any reagents, samples needed to be filtered, this is to get clear water without containing any suspended matter. During the experiment, the samples were added with reagents and H2SO4 and left for a 45 minutes to allow proper colouring, so that the absorbance can be measured using the spectrophotometer. This is because, if the substance were to be left at a short time and proper colouring is not developed then the absorbance may not be able to read. When putting the 1cm cell into the spectrophotometer, the sides with a clear surface, should not be touched, because that is where the light will go through to measure the absorbance.

The reason as to why the absorbance is read at 410nm is because that is the measure that the nitrate can be read at and where the intensity of the light can be absorbed by the substance.


Water samples that were analysed were found to have low nitrate concentration. The highest concentration of nitrate was found at the Codbeck samples (Codbeck I = 1.66 mg L-1, Codbeck 10 = 1.4mg L-1) and the Tees river dated 23/2/2010 (1.38mg L-1), whereas Filey has no reading of nitrate at all, this is because it has the same absorption with the blank. Tees lake (0.16mg L-1) and Scalby 2 (0.09mg L-1) has the lowest reading for both concentration and absorption. Even though Tees lake is near civil and a busy environment, the area still has a low nitrate concentration, the presence of nitrate at a low amount is probably due to the natural process and not so much from anthropogenic activities. Even though the number is high amongst the sample, it has not exceeded the EC Directive concentration limit. Codbeck is stream water where it is near agricultural land, and stream concentrations are normally high in concentration depending on the farming area (Hooda et al., 1997). Filey and Scalby has a low concentration is probably due to both areas are sea water samples. Sea water has less nitrate concentration because effluents do not fall directly towards the sea.

Although the substances have a light colouring, it still absorbs lighting. This is really shows that nitrate concentration in high at certain places. Beer lambert law can be applied in this experiment, however, there were samples that falls above 1, because absorbance of samples needs to fall between 0 -1 according to the law, where 1 being high in absorption and 0 no absorption or low absorbance. According to the law, the higher the absorbance the lower the intensity of light that passes through. Low absorbance indicates that there is low nitrate in the area, and in most of the area, the readings of absorbance and concentration are both low.

However according to the results, it can be seen that nitrate concentration in some areas are controlled and has a low reading. However, nitrate is still a concern, because with the current climate and the depletion of the ozone in the stratosphere, there are chances of the radiation of be absorbed by bodies of water and thus increasing the photochemical processes (Zao and Deng, 1998) and increase the bacterial activities and oxidising ammonia and nitrite to nitrate, instead of just having the agricultural and industrial activities to increase nitrate, increase of radiation may lead to the increase in natural processes. There has been a suggestion of nitrification and dentrification as ways or methods to reduce nitrogen in water and that it is one of the efficient ways to do it (Stenstrom, 1979). Although the concentration of samples were fairly low, it is still essential know the amount or the concentration of these chemicals in the river, so that standards can be done to limit the amount of effluents going in to the water bodies (Stalnacke et al., 2003).