VPP 322: Aquatic Animal Diseases, Health Care and Management (1+1)

Chemical parameters- Nitrite

DETERMINATION OF NITRITE (NO₂) IN CULTURE SYSTEMS

• The NO₂ is formed as an intermediate product of nitrification. NO₂-N is also formed when the NO₃-N is denitrated under very low level of oxygen. NO₂-N is also known to be excreted by phytoplankton under light limiting conditions.

Principle

• Acidification of the water samples containing NO_2-N leads to the formation of nitrous acid. By the addition of an aromatic amine (sulphanilamide), a diazonium salt is formed. This quantitatively couples with a second aromatic amine, N (1-naphthyl) ethyl diamine dihydrochloride. This coupling yields a pink azo dye with absorption maximum at 543 nm.

Reagents

Sulphanilamide reagent (NH₂ C₆H₄SO₂NH₂)

• 8 g of this salt is dissolved in a mixture of 80 ml of conc. HCl and 400 ml of distilled water. The solution is then made up to 500 ml. This reagent is stable for several months when kept in refrigerator.

Diamine solution

• 0.8 g of N-(1- Napthyl)-ethylene diamine dihydrochloride is dissolved in 500 ml of distilled water. This solution is discarded if it develops brownish colour at any stage.

Procedure

• 25 ml of filtered sample is taken in a conical flask. To this solution, 0.5 ml of sulphanilamide solution is added and the solution is stirred well. After 3-8 min, 0.5 ml of diamine solution is added and the content of the conical flask is thoroughly mixed. After 10 min the OD of the red azo dye developed is measured in a spectrophotometer at 543 nm. The colour is stable for 2 hrs. The values of OD recorded for triplicate samples are recorded.

Preparation of NO₂- N working solution

• 0.345 g of anhydrous NaNO₂ is dissolved in water and the volume is made up to 1 litre. 1 ml of chloroform is added as preservative. 1 ml of this solution contains 70 mg at NO_2-N. The NO_2- N working solution is prepared from the stock solution by adding 1 ml of the solution to 100ml. 1ml of this solution contains 0.7 mg or 0.05 mg-at N.

Formation of calibration curve

• The working solution is further diluted with distilled water to get the following standard solutions.
  • 1ml of working solution made up to 100 ml with distilled water = 0.05 mg-at NO-N/I
  • 1ml working solution made up to 100 ml with distilled water = 0.1 mg at NO₂-N/I
  • 0.5 ml of working solution made up to 100 ml with distilled water = 0.25mg-at NO₂^-N/I
  • 1ml of working solution made up to 100 ml with distilled water
  • 2ml of working solution made up to 100 ml with distilled water = 1 mg-at NO₂- N/I
• 25 ml of the above-diluted working solution is treated with sulphanilamide and diamine solution to get an azo dye. The values of OD recorded for the different concentrations are tabulated. The OD is taken on the ‘X’ axis and concentrations plotted to the ‘Y’ axis. From the linear graph drawn between OD and concentration of NO₂-N it is possible to assess the NO₂-N content of the given water sample.