FIS3203

Water Quality Management for Aquaculture

Determination of Nitrite-Nitrogen (NO2- N)

Introduction
Nitrite (NO2-) is formed from the oxidation of ammonium in the aquatic environment like
aquaculture ponds or tanks by nitrifying bacteria under anaerobic condition. Nitrite tends to
bind to the red-blood cells and reduces their ability to transport oxygen. If nitrite is present
in high concentration, the fish blood will turn chocolate-brown in colour. This is known as
the brown-blood disease. The symptom of the brown-blood disease is easily seen in the fish
gills. However, the effect of nitrite can also occur even before the gills have turned into
obvious brown colour. Nitrite is commonly detected and analyzed using Griess Reaction,
which involves the formation of deep red-coloured azo dye upon treatment of the sample
with sulphanilic and napthyl-1-amine under acidic condition (Parsons et al. 1984).

Sampling:
Analyse the sample immediately. There is no suitable preservation for nitrite sample.
Acidifying the sample using concentrated acid will covert the nitrite into nitrate and this will
hinder the determination of individual nitrite.
Reagents
Nitrite standard solution:
Dissolve NaNO2 is distilled water according to the required concentration (0-1 mg NO 2- -
N/L). Dry the chemical at 105⁰ C for one hour to remove moisture.
Sulphanilamide solution:
Dissolve 1.0 g sulphanilamide, NH2C6H4SO2.NH2 in a mixture of 10 ml concentrated HCL
and 60 ml distilled water. Dilute the reagent to 100 ml with distilled water.
N-(1-naphtyl)-ethylenediamine dihydrochloride solution:
Dissolve 0.1 g N-(1- naphtyl)-ethylenediamine dihydrochloride, C12H4N2. 2HCL in 100 ml
distilled water

Methods
Prepare 100 ml of each standard solution using the standard solution (0.30 mg/l, 0.20 mg/l, 0.15
mg/l, 0.10 mg/l, 0.05 mg/l and 0.00 mg/l -blank reagent). Use deionized water or artificial seawater
(depend on sample) for dilution.

 Dissolve 4.929 gm of NaNO2 into a 1 L volumetric flask (Stock I), and from where
transfer 1 ml to a 100 ml Vol flask (Stock II).
 Prepare standard solution (0.30 mg/l, 0.20 mg/l, 0.15 mg/l, 0.10 mg/l, 0.05 mg/l)
transferring 3.0, 2.0, 1.5, 1.0 and 0.5 ml from stock II to 100 ml water.
 Pipette 10 ml of each standard solution and control (distilled water) into test tubes with 3
replicates.
 To each test tube, add 0.2 ml sulphanilamide and mix. Allow more than 2 minutes but not
less 10 minutes (A)
 Add 0.2 ml N-(1- naphtyl)-ethylenediamine dihydrochloride solution into the samples
and mix thoroughly (B).
 Measure the absorbance of the sample in a 1 cm cuvette after 1 hour suing UV-VIS
spectrophotometer at 543 nm (C).
 Plot the absorbance reading obtained from each standard solution against their
respective concentration. The liner curve obtained in the standard curve can later be
used for the determination of NO2-N concentration from the water samples.

0.35

0.3

0.25
y = 0.3363x - 0.0004
R² = 0.9992
Nitrite (mg/L)

0.2

0.15

0.1

0.05

0
0 0.2 0.4 0.6 0.8 1
Aborbance

Analysis of water sample:

 Filter water samples using nitrocellulose membrane filter paper prior to analysis and
transfer 10 ml of sample to the test tube and follow A to C mentioned above and
calculate the concentration of NO2-N from the above standard equation.
For example: If your sample absorbance is 0.65, then your NO2-N content will be
Y (Nitrite) = 0.3363 x (Absorbance = 0.68) – 0.0004 = 0.228 mg/l