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

Chemical parameter-Ammonia

AMMONIA

• Increasing ammonia level of the water is mainly due to left-over feed and associated release of metabolic byproducts and by the decomposition of organic matter including faecal matter. The toxicity of ammonia nitrogen is attributed primarily to its un-ionized form. When un-ionized ammonia concentration increases, there is a reduction in the ammonia excretion of aquatic organisms and consequently the enzyme catalyzed reactions and membrane stability is affected.
• Further, ammonia is known to increase the oxygen consumption, by tissues, besides damaging gills and impairing the transport of oxygen through circulation. Due to ammonia toxicity, resistance to pathogens may be significantly reduced in freshwater and marine finfish and shellfish. Ammonia removal can be effected in freshwater fish ponds using Zeolite. Use of probiotics containing variable isolates of Nitrosomonas may quickly reduce ammonia level by converting it into nitrite. Water exchange in the pond would help in the instant reduction in ammonia levels.

Indophenol method of estimating ammonia

Principle

• Ammonia reacts with phenol and hypochlorite under alkaline condition in the presence of sodium nitroprusside which acts as a catalyzer in the colour development at room temperature to form indophenol blue. The colour intensity is measured in a spectrophotometer or ELISA reader at 630 nm. Intensity is measured in a spectrophotometer or ELISA reader at 630 nm. The intensity of colour is directly related to the concentration of ammonia in the sample.

Reagents

• Ammonia free water: Deionised water is usually recommended for the preparation of reagents for ammonia estimation. Ammonia free water may also be prepared by boiling distilled water containing 2-3 drops of bromine liquid. Ordinary distilled water can also be distilled with 2 ml of conc. H₂SO₄ and 1 g of K₂S₂O₈ (Potassium persulphate) to obtain ammonia free water.
• Phenol-nitroprusside solution: 27g of phenol and 0.30 g of sodium nitroprusside dehydrate are dissolved in ammonia free distilled water and the solution is made up to litre. This reagent is stable for many months if stored in refrigerator in dark glass bottles.
• Sodium hypochlorite solution: Commercial grade sodium hypochloride (NaOCl) or bleaching powder (8.25% solution) is used.
• Trisodium citrate solution (1.19 M): 350g of C₆H₅Na₃O_7. 2H₂0 is dissolved in distilled water and is made up to 1 litre.

Procedure

• 25 ml of the water sample is taken in a conical flask and to it are added 1 ml of citrate solution, 1 ml of phenol nitroprusside solution and 1 ml of NaOCl or 3 ml of bleaching powder solution. The contents are mixed well and the flask is covered with aluminium foil. The flask is then kept in a dark place for about 2 hrs. If the colour develops within 5 minutes, the analysis is repeated with fresh sample. After 2 hrs the optical density (OD) of the blue coloured complex is measured at 630 nm in a spectrophotometer against distilled water blank.

Preparation of standard solution

• 0.0382 g of NH₄CL is dissolved in NH₃ free distilled water and is made upto 100ml. A drop of chloroform is added as preservative. This solution contains 100 µg (7.01 µg at) of NH₃-N/ml with 14, the respective µg at NH₃-N values can be calculated. The optical density of the working standard solutions and the blank treated with different reagents to develop Indophenol blue are also measured in the spectrophotometer. The concentration of standard solution and OD values is plotted in the graph sheet on the X and Y-axis respectively. A linear graph is drawn and the same is kept ready for measuring the unknown concentration of NH₃ in the given water sample. Alternatively, the level of ammonia of a sample can be found out directly through the following calculation.

 Less of NH3 = ( Conc. of the standard solution  / OD value of corresponding stanard ) / OD value of the sample -EB

• Where, EB = Absorbance of distilled water plus reagents.
• If the strength of the hypochlorite is not satisfactory, fresh reagents should be used for the analysis. If balancing powder is used for the analysis a separate curve should be drawn different working solutions.

Note

• Samples should be analysed immediately
• Polluted waters may suitably be diluted before the analysis.
• The colour produced after 1-2 hours is stable for at least 24 hours.
• Blank extinction should not exceed 0.075, when it is read in 10 cm cuvettes of a spectrophotometer.
• While adding sodium hypochloride, the pH of the sample should not exceed 9.8.

Estimation of other physical and chemical parameters...