Limnology

1. Determination of chemical characteristics of inland (lentic and lotic) waters

Practical No. : 4 & 5

Determination of chemical characteristics of inland (lentic and lotic) waters
Chemically, pure water does not exist on the earth but the natural waters differ in their chemical content. As discussed under physical characteristics of inland waters, the chemical properties are also equally important because the existence and continuance of life in water depend upon the presence of substances which natural water contains, thereby greater the biological productivity. Therefore it is concerned in a way not only with the water itself but also with its large and varied chemical content. Hence, the most important chemical characteristics of inland waters are considered here for their analyses as per the standard methods.
1. Determination of pH
It is the measure of the relative acidity or alkalinity and represents the negative logarithm of the concentration of free hydrogen ions in a solution. The ‘p’ of pH denotes the power of hydrogen ion activity in mole per litre.
pH = log₁₀ [H+] = log₁₀ 1/H+
Neutral
|______Acid_______|______Alkaline_______|

0 7 14

For convenience, only the logarithm of the number of hydrogen ions is used. pH scale ranges from0 to 14 with 7 as neutral point, below and above which it is acidic or alkaline respectively. In natural waters, the pH extremes and may reach 3 and 12. Most of the Indian lakes show pH normally between 6 and 9. Increase in pH during day time is largely due to photosynthetic activity, whereas, decrease at night is the result of catabolic process.
The pH in water is a measure of hydrogen ion concentration present in water. If the pH value is 7 it is expressed as neutral while pH values below 7 indicates acidity whereas values above 7 indicates the alkalinity of the water sample. The pH probe measures pH as the activity of hydrogen ions surrounding a thin-walled glass bulb at its tip. The probe produces a small voltage (about 0.06 volt per pH unit) that is measured and displayed as pH units by the meter.

Water pH meter

Principle: The basic principle of the electrometric pH measurement is determination of the activity of the hydrogen ion by potentiometric measurement using a standard hydrogen electrode and a reference electrode.
Apparatus: pH meter consisting of potentiometer, a glass electrode, a reference electrode and a temperature-compensating device.
Glass electrode: The sensor electrode is bulb of special glass containing a fixed concentration of HCl and a buffered chloride solution in contact with an internal reference electrode.
Procedure:
• Before use, remove electrode, rinse, and blot, dry with a soft tissue paper.
• Calibrate the instrument with standard buffer solution. [eg, KCl solution of pH 7.0]
• Once the instrument is calibrated remove the electrode from standard solution; rinse, blot and dry.
• Dip the electrode in the sample whose pH has to be measured.
• Stir the sample to ensure homogeneity and to minimize CO₂ entrainment.
• Note down the reading (pH) from the pH meter.
2. Determination of Dissolved oxygen by Winkler’s method
Principle:
Manganous sulphate reacts with the alkali to form white precipitate of Manganese hydroxide which is the precipitate of O₂ and get oxidized to a brown color of higher hydroxide, which on acidification liberate iodine equivalent to that of O₂ fixed. The iodine is titrated against sodium thiosulphate using starch as indicator.
Reagents
a. Manganous sulphate solution : Dissolve 480 g of MnSO₄.4H₂0 or 400 g of MnSO₄.2H₂0 or 363 g of MnSO₄.H₂0 in 1000 ml distilled water.
b. Alkaline iodide solution : Dissolve 500 g NaOH (or 700 g KOH) and 135 g NaI (or 150 g KI) in 1000 ml distilled water (or dissolve 100 g KOH and 50 g KI in 200 ml distilled water).
c. Concentrated Sulphuric acid
d. Starch solution : Dissolve 2gm laboratory grade soluble starch and 0.2 g salicylic acid in 100 ml hot distilled water.
e. Sodium thiosulphate solution (0.025 N): Dissolve 6.203 g of Na₂S₂O₂.5H₂O in 1000 ml distilled water.
Procedure :
• Water sample is collected from surface in 125 ml Dissolved Oxygen bottle avoiding formation of bubbles.
• Add 1 ml alkaline iodide and 1 ml MnSO₄ into the bottle.
• Mix well and the precipitate formed is allowed to settle for few minutes.
• Add 1 ml of conc. H₂SO₄ and shake well to dissolve the precipitate.
• Take 50 ml solution in a conical flask.
• Titrate against std. Na₂S₂O₃ using starch as indicator (2-3drops).
• The end point is blue to colorless and titer value is measured.
Calculation

A x N x V x 1000 x 22.4

DO (mg/l) = ______________________

B (A-L) x 0.4 x 0.698

Where,

A= Volume of the DO bottle

N= Normality of Na₂S₂O₃

V= Titrate value

B= Volume of sample taken

L= Volume of reagent used

3. Estimation of free Carbon dioxide
Titrimetric method
Principle: Free CO₂ reacts with NaOH or Na₂CO₃ to form Na(HCO₃)₂, the completion of the reaction is indicated by the appearance of pink color in the presence of phenolphthalein indicator at pH of 8.3.
Reagents:
a. Stock NaOH solution : Dissolve 4 g NaOH in 1000 ml distilled water.
b. Std. NaOH solution (0.02N) : Dissolve 200 ml of stock solution to 1000 ml with distilled water.
c. Phenolphthalein indicator : Dissolve 0.5 g of phenolphthalein powder in 50ml of 95% C₂H₂OH and add 50ml distilled water.
Procedure:
• Take 50 ml of sample in a conical flask.
• Add 4 to 5 drops of phenolphthalein indicator
Upon addition of the indicator if the colour changes to pink it indicates absence of CO₂ and if no change, then titrate the sample against standard 0.02 N Sodium hydroxide until the pale pink color develop and remain for 30 sec.
• Note down the burette reading
Calculation :

V x N x 44 x 1000

Free CO₂ (mg/l) = _________________

Volume of sample

V = Volume of 0.02N NaOH
N= Normality of NaOH
4. Determination of Alkalinity in water
Titration Method
Principle:
Hydroxide ions present in a sample react with acid at a specific end point pH. Alkalinity is primarily due to anionic carbonate and bicarbonate. The pH equivalent at the end point of titration is determined by the concentration of CO₂ at that point and it is in turn is regulated by concentration of carbonate. Phenolphthalein alkalinity is a term generally used for titration at a pH of 8.3 when hydroxide and CO₃^- are present the end point is indicated by disappearance of pink colour. Similarly, methyl orange alkalinity is the alkalinity due to the presence of CO^-₃ and HCO₃^- and the sample aquires yellow colour and at the end point of titration changes to orange red or red at a pH of 4.4.
Reagents :
• Phenolphthalein indicator : Dissolve 0.5 g of phenolphthalein powder in solution of 95% ethyl alcohol and add to it 50ml distilled water.
• Methyl orange indicator : Dissolve 0.5 g methyl orange in 100 ml distilled water.
• Std. (0.02 N) Sulphuric acid
Procedure :
• Take 100 ml water sample in a 250 ml conical flask.
• Add 3 drops of phenolphthalein indicator.
• If turns pink, titrate with 0.02 N H₂SO₄ until the pink color just disappears.
• Note down volume of acid used.
• Then add 3 drops of methyl orange indicator to the same water sample.
• If the water turns yellow, titrate acid until a faint orange end point is obtained.
• Note the volume of the acid used decreasing this titration.
5. Determination of reactive Phosphate – Phosphorus
Principle :
The method depends on the formation of phosphomolybdate complex and its subsequent reduction to highly coloured blue compounds. The water sample is allowed to react with a mixed reagent containing molybdic acid; ascorbic acid and trivalent antimony. The resulting complex heteropolyacid is reduced to give a blue solution, the extinction of which is measured at 8850 A^o or 885 nm using a spectrophotometer.
Reagents :
• Ammonium molybdate : Dissolve 15g of ammonium molybdate in 500ml of distilled water.
• Sulphuric acid : Add 140 ml of conc. H₂SO₄ to 900 ml distilled water.
• Ascorbic acid solution : Dissolve 27g of ascorbic acid in 500 ml distilled water.
• Potassium Antimony Tartarate (PAT) : Dissolve 0.34 of PAT in 250ml of distilled water.
• Mixed reagent : To prepare 500 ml of mixed reagent, mix together 100 ml ammonium molybdate, 250ml H₂SO₄, 100ml ascorbic acid and 50 ml PAT. Prepare this reagent for use and discard any excess.
Procedure:
• Take 100ml of sample in conical flask.
• Add 10+0.5ml of mixed reagent.
• After 5 min and preferably within 2-3 hrs, measure the extinction of the solution at 885 nm.
Calculation :

µg at. P/l = Extinction x F

Where, F=Factor value

6. Determination of Nitrate - Nitrogen in water
Cadmium reduction method
Principle : Nitrate is the reduced almost quantitatively to nitrite, when a sample is run through a column containing Cadmium fillings loosely coated with metallic copper. The nitrite thus produced is determined by diazotizing with sulphanilamide and coupling with NNED [N-(1-naphthyl ethylene diamine dihydrochloride) to forma highly colored azo dye, the extinction of which is measured at 543 nm. A correction may be made for any nitrate initially present in the sample. The nitrate in the sample can also be reduced to nitrate by an overnight reduction method.
Reagents :
• Ammonium chloride solution (concentrated) I : Dissolve 125g of AR quality ammonium chloride in 500 ml distilled water.
• Ammonium chloride solution II: Dilute 50 ml of conc. Ammonium chloride solution to 200 ml with distilled water.
• Sulphanilamide solution : Dissolve 5g of sulphanilamide in a mixture of 50 ml of conc. H Cl and about 300ml of distilled water.
• NNED [N-(l-Naphthyl) – ethylene diamine dihydrochloride solution] : Dissolve 0.5g of NNED in 500ml distilled water.
Procedure :
• Add 1-2 of ammonium chloride to the 50 ml sample in the conical flask and mix it.
• Add the sample to the column and allowed to pass through it.
• Collect 25 ml of the reduced solution in measuring cylinder.
• As soon as possible after reduction, add 0.5 ml sulphanilamide and add 0.5 ml of NNED solution and mix.
• Measure the extinction between 10 min to 2 hrs at 543 nm wave length.
Calculation:
µg at. NO₃ –N/l = (E x F) – C
Where,

F = Factor value

C = Concentration of nitrite present in sample (mg at. N0₂ –N/lt).

7. Estimation of chemical oxygen demand (COD)
The chemical oxygen demand or permanganate value of water is normally estimated by adopting alkaline oxidation with permanganate. Chromic and permanganate in acidic conditions are not suitable for oxidizing the organic matter present in water, these oxidants are known to oxidize the chloride ions of the water into free chlorine keeping this in view, the oxidation of organic matter with permanganate in alkaline condition is preferred for the analysis of COD in water.
Principle
Under alkaline condition, permanganate oxidizes only the organic matter present in water without oxidizing Cl, Br and I to Cl₂, Br₂ and I₂ respectively when all such organic matter is oxidized permanganate is allowed to liberate iodine, from potassium iodide under acidic condition. Iodine so liberated is titrated against thiosulphate using starch as an indicator. By running a blank, the quantity of thiosulphate required to react with all the iodine liberated by the unreduced permanganate is estimated. From those 2 litre values, the chemical oxygen demand of the sample is calculated.
Reagents
1. 0.1N potassium permanganate stock solution : 3.16g of KMnO₄ is dissolved in a little distilled water and the volume is made up to 1 litre with distilled water. this solution is stable when kept in a amber, coloured bottle.
2. 0.01N potassium permanganate working solution : 100ml of the above stock solution is made up to 1lit with distilled water.
3. 25% sulphuric acid solution : 100ml of concentrated sulphuric acid is carefully mixed with distilled water and is made up to 400ml.
4. 5% sodium hydroxide solution : 5g of sodium hydroxide is dissolved in 100ml of distilled water.
5. 0.1m potassium iodide solution
16.6g of KI is dissolved in distilled water and the volume is made up to 1 litre with distilled water.
6. 0.02N sodium thiosulphate solution : 4.964g of sodium thiosulphate pentahydrate is dissolved in 1 litre of distilled water.
7. 1% starch solution (indicator) : 1g of soluble starch is added to 100ml of boiling distilled water. to this 0.5ml of phenol is added as preservative.
Procedure

The blank value is estimated by using 100ml of distilled water instead of the water sample. All over the treatments and chemical analysis of the blank should be carried out as per the procedure – adopted for the sample.
Calculation :
Chemical oxygen demand or permanganate value is calculated using the formula
COD (mg/l) = 8 x N x 1000 (b – s) / 100 (volume of the sample)
N – Normality of sodium thiosulphate (0.01 N)
b – ml of sodium thiosulphate solution used for titrating the blank
s – ml of sodium thiosulphate solution used for titrating the sample.