In this article we will discuss about the physical, chemical and microbiological examination of water.
Physical Examination of Water:
(i) Colour:
The colour is determined either by visual comparison of sample with known concentrations of coloured solution (by using Platinum Cobalt comparator) or by tintometric measurements.
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Platinum-Cobalt comparison method:
Take 1.246 gm. of potassium chloroplatinate K2PtCl6 and 1 gm of cobalt chloride (CoCI2. 6H2O) in 100 ml of conc. HC1 and dilute it to 1 litre by adding distilled water. Now the colour of this solution will be equal to 500 units. Different standards can be prepared by diluting the stock solution with distilled water.
Procedure:
Take Nessler tubes of 50 ml. mark and fill them with the sample and the standards. Place the tubes on a white filter paper and match the colour.
(ii) Odour and Taste:
Take a reagent bottle and fill it with about 2/3 of sample and shake it for 5 minutes. Now note the odour and frothing if produced in the bottle. The forming of water generally suggests the presence of detergents or surface active substances.
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Generally water may have chlorinous, fishy, mouldy, earthy or aromatic odour. The taste of the water also varies with the odour. The odourless water has a distinct taste.
(iii) Turbidity:
The turbidity is a measure of the interference caused due to the presence of suspended matter to the passage of light. Generally the water becomes turbid due to silt clay, organic and inorganic matter. The scale selected for the measurement of turbidity is 1 mg SiO2/Litre = 1 unit of turbidity.
The following three methods are used for measuring the turbidity in water:
(1) Standard Jacksons Candle Turbiditimeter 25 – 1000 units.
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(2) Baylis turbiditimeter 0-2 units and
(3) Helliges/Aplab turbiditimeter 0-150 units.
Procedure:
Jacksons Candle turbiditimeter:
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It consists of three parts:
(a) A candle,
(b) A holder and
(c) A calibrated glass tube.
The glass and the candle are supported in a vertical position so that the centre line of the tube may pass through centre line of candle. Now pour the sample into the glass tube slowly until the image of candle disappears. Measure the path of the light in cm from inside the bottom of glass tube. From the Table 4, the turbidity in units can be read directly.
Chemical Examination of Water:
(i) Total Solids:
The total solids include water soluble as well as insoluble matter.
Procedure:
Take about 200 – 500 ml. of water in a Pyrex beaker and evaporate to dryness in an oven at about 105 – 120°C. Cool it and weigh the residue.
Non-filtrable and filtrable residue:
Filter the sample through What-man filter paper and now dry in a beaker at 105 – 120°C, cool and weigh.
Non-filtrable residue in ppm = Total residue – filtrable residue.
(ii) Organic Matter:
A current of organic matter can be recorded by measuring the amount of ammonia given off by it provided the water is distilled with alkaline KMn04.
(iii) Alkalinity:
Generally the water shows alkalinity due to the presence of salts of weak acid and strong base.
The alkalinity in water is caused due to the presence of:
(a) Carbonates (CO3– –),
(b) Bi-carbonates (HCO3–) and
(c) Hydroxides (OH–).
The alkalinity can easily be determined by titration with phenolphthalein (pH 8.3) or methyl orange (pH 4.5).
Reagents:
(1) Phenolphthalein indicator,
(2) Methyl orange indicator,
(3) 0.02 NH2SO4 or HCL solution and
(4) 0.1 N sodium thiosulphate solution.
Procedure:
Take 100 ml. of sample each in two conical flasks. Add 0.5 ml. phenolphthalein indicator in one flask. If the sample gets pink titrate with 0.02 N H2SO4 until the pink colour disappears. Note the ml. of acid used in the titration.
Now add 0.5 ml of methyl orange in second flask and titrate with 0.02 N H2SO4 until the orange colour is arrived indicating the end point. Note again the ml. of acid used.
Suppose A = ml. of acid used for titration with phenolphthalein and
B = ml. of acid used for total titration (Phenolphthalein and methyl orange).
The observations can be summarised in five possible conditions:
(iv) Hardness:
Temporary hardness is due to the presence of bicarbonate of Ca++ and Mg++ while permanent hardness is due to sulphates and chlorides of Mg++ and Ca++.
In general term, hardness of water is due to the salts of calcium, magnesium, strontium, iron and manganese.
The following cations and anions are responsible for the hardness of water:
Reagents:
(1) Erich Rome Black T Indicator:
This is prepared by mixing together 0.5 g of Erich Rome Black T and about 100 gm. of NaCl in 20 ml. of water by warming. The solution is stable for 100 days.
(2) Ammonia buffer:
Take 16.9 gm. of NH4CI in 143 ml. of liquor ammonia and dilute to 250 ml. with distilled water. In presence of metallic ions, use borate buffer. Take 20 gm. of borax (Na2B4O7.10H2O) in 400 ml. of distilled water. Dissolve 5 gm. of NaOH and 2.5 gm. of sodium sulphide in 50 ml. of water, cool and mix with borax solution and dilute to 500 ml. with distilled water.
(3) Standard EDTA solution:
Procedure:
Take 100 ml. of sample in a conical flask and add 1 ml. of ammonia buffer and 2 drops of Erich Rome black T indicator. Shake the solution till the colour changes from wine red to blue. Note the end point.
(v) pH:
The pH is defined as the logarithm of the activity of hydrogen ions with negative sign.
pH = – log aH+
For a dilute solution, the activity coefficient is approximately equal to one, hence
Activity = Concentration
The pH is a measure of the relative acidity or alkalinity of water. For pure water at 25°C, (H+) = (OH–) = 10 -7, so pH = 7.
(i) If pH is = 7, the solution is neutral.
(ii) If pH is greater than 7, the solution is basic.
(iii) If pH is less than 7, the solution is acidic.
Procedure – The pH of any solution can easily be known with the help of pH meter.
(vi) Acidity:
Sometimes the water shows acidity due to presence of un-combined CO2, salts of strong acids and weak bases and mineral acids. For determining the acidity of water, it is titrated with standard solution of strong base by using a suitable indicator.
Reagents:
(1)Phenolphthalein & methyl orange indicators.
(2) (N/100) NaOH solution.
Procedure:
Take 100 ml. of sample in a conical flask and add 2 drops of phenolphthalein indicator, and titrate with (N/100) until a faint pink colour appears.
Total acidity as CaC03, mg/litre
(vii) Nitrogen:
a. As Nitrates:
Nitrate is found in some ground waters and also in surface water supplies. The high amount of nitrate may create an illness known as methemoglobinemia. It can be determined by colorimetric method using brucine or phenoldisulphonic acid.
Reagents:
Standard nitrate solution (1 ml. = 0.01 mg. of N)
0.028 N Sodium arsenite (Na3As03) solution.
Brucine sulphanilic acid reagent.
H2SO4 solution.
Procedure:
Prepare various nitrate standards in the range 0.10 mg. of nitrogen per litre. Pipette 2 ml. of each solution into 50 ml. beaker. Add 1 ml. of brucine – H2S04 in the beaker and keep the sample in the dark for 10 minutes.
Measure the colour of the standard in a Colorimeter and compare with reagent blank to 100% transmission. Draw the standard curve by plotting absorbance (Y-axis) against concentration (X-axis) and from the curve the concentration of an unknown sample can easily be calculated.
Pre-treatment of sample:
If the sample contains chlorine, it can be removed by adding 0.1 ml. of arsenite solution for each 0.01 mg. of chlorine present. Always add one or two drops of arsenite solution in excess in 50 ml. solution.
The concentration of nitrate nitrogen in the sample can be calculated with the help of the following formula:
Nitrate nitrogen mg/litre = mg. of nitrate nitrogen x 100/ ml. of sample taken
Or
Nitrate mg/litre = nitrate nitrogen x 4.43
b. As Nitrite:
The waters of ponds, tanks, rivers etc. have nitrite which is formed in the intermediate stage in the reduction or oxidation process either by plants, by the action of bacteria or by other organisms.
The nitrite concentration with range 0.005 – .05 mg/litre can be determined by colorimetric method through the formation of a reddish purple azodye produced at pH 2-5 by the action of a diazotized sulphanilic acid with naphthylamine hydrochloride. The experiments should be performed always with the fresh samples to prevent bacterial conversion of nitrite to nitrate or ammonia.
Reagents:
(1) Sulphanilic acid reagent.
(2) Naphtylamine hydrochloride reagent.
(3) 2M Sodium acetate buffer solution.
(4) Standard. Sodium Nitrate solution.
(5) Aluminium hydroxide solution.
(6) Nitrite free water obtained by distilling the water adding a small L crystal of each KMnO4 and alkali such as Ba(OH)2.
Procedure:
If the collected sample is not clear or contains suspension, add 2 ml. of Al(OH)3 in 100 ml. of water in a beaker. Allow to stand for 5-10 minutes and filter. Neutralize 50 ml. of filtrate till pH-7 is reached. Now add 1 ml. of sulphanilic acid and shake for 4 – 5 minutes, then add 1 ml. of naphthylamine hydrochloride and 1 ml of sodium acetate buffer solution.
Mix thoroughly and check the pH (it should be between 2 – 2.6). Allow to stand for 20 – 30 minutes and measure the reddish colour at 520 r nm in colorimeter using reagent blank to set the instrument to 100% transmission. Calculate the conc. of the sample from the standard curve.
Standard Curve:
Take 7-8 readings with standard sodium nitrite in colorimeter and plot a graph between absorbance against concentration.
The nitrite in mg/litre can be calculated from the following formula:
Or
Nitrite mg/litre = 3.29 x mg/litre nitrite nitrogen.
c. As Free Ammonia:
Almost all types of waters contain free ammonia nitrogen in combination with mineral acids and is a product of microbiological activity. Ammonia nitrogen causes pollution in surface and ground waters.
Reagents:
(1) Boric acid solution:
20 gm. of boric acid in 1000 ml. of distilled water,
(2) 0.02 NH2SO4 solution
(3) Mixed Indicator dis-solve 0.2 gm. Methyl red + 0.1 gm. of methylene blue in 300 ml. of 95% alcohol.
Boric acid method:
Take about 500 ml. of water in a distillation flask. Collect the distillate in 50 ml. of boric acid solution. Each milligram of additional ammonia nitrogen requires 50 ml. of boric acid solution. Titrate with 0.02 N H2SO4 using mixed indicator. The pale lavender colour indicates the end point
Ammonia nitrogen mg/litre
Nesslerization method:
In this method, Nessler reagent and phosphate buffer is used. Take 500 ml. sample, add 10 ml of phosphate buffer and distill it In 50 ml distillate, add 2 ml. Nessler solution and mix. After 10 – 12 minutes, compare with standard in a colorimeter. Calculate the conc. of unknown from the graph plotted between optical density as ordinate and conc. of ammonia as abscissa.
d. As Albuminoid Ammonia:
Albuminoid nitrogen indicates organic pollution in water supply. It is a measure of proteinaceous nitrogen present in waters of rivers, ponds etc. It is mainly derived from plants and animal life which are normal to aquatic environments. Generally, it is formed by the action of KMn04 on the un-substituted amino groups of many amino acids, proteins and polypeptides in an alkaline medium.
After determination of ammonia nitrogen, if a strong solution of alkaline KMn04 is added to the residual sample and distilled, any supplementary release of ammonia suggests the presence of albuminoid nitrogen.
Take 500 ml. of sample and when about 200 ml. has been distilled over for the estimation of ammonia nitrogen, distill off again 50 to 100 ml. Now add 50 ml. of alkaline KMnO4 (of 0.533%) and distill again. Collect the distillate in 50 ml. proportions and estimate ammonia by nesslerization method as described earlier.
The formula for calculation is same as in case of ammonia nitrogen.
Total nitrogen:
The estimation of total nitrogen as nitrite, nitrate, ammonia and albuminoid is of great importance in sewage and polluted waters as all of these forms cause pollution in water.
Reagents:
(1) Nessler reagent,
(2) Sulphuric acid,
(3) Phenolphthalein,
(4) Standard ammonium chloride solution,
(5) 12N Sodium hydroxide solution and
(6) 10% copper sulphate solution.
Procedure:
Take 500 ml. of sample water (or 50 ml. sewage dilute to 100 ml). Now add 1 ml. of copper sulphate solution and 10 ml. conc. H2SO4. Digest the mixture until it becomes straw coloured. Add a few drops of phenolphthalein and make it alkaline by adding NaOH solution. Now distill the sample till all ammonia is distilled (check with Nessler reagent). Measure the ml. of distillate.
Take 5 ml. of distillate, add 2 ml. of Nessler reagent and make the volume to 100 ml. After sometime a colour will be developed.
Take 5 Nessler tubes and pour 0.5, 1.0, 2.0, 2.5 and 3 ml. of standard ammonium chloride solution. Add 75 ml. distilled water and 2 ml. of Nessler reagent in each of five tubes and add water to make the volume to 100 ml.
After 10-18 minutes, compare the colours with the sample.
(viii) Sulphates:
Sulphates are generally found in hard waters. The sulphates also indicate the pollution in water. They can be determined gravimetrically as barium sulphate by the addition of an excess of barium chloride. They can also be determined volumetrically by titrating with barium chloride solution using tetrahydroxyquinone as indicator.
At the end point the colour is changed from yellow to red. Phosphates interfere in this procedure. For the estimation of sulphates, it is necessary that the alkalinity and the hardness of the sample must be determined before.
Reagents:
(1) Standard Barium chloride. Dissolve 2.44 gm. of barium chloride in water and make up to 1 litre.
(2) Ammonia-Ammonium chloride buffer:
Take 670 ml. ammonia and add 67.5 gm. of NH4CI and dilute to 1 litre.
(3) Erichrome Black T Indicator:
Mix 100 gm. NaCl and 0.5 gm. of chrome Black T to prepare a dry powder mixture.
(4) 0.01 M standard EDTA:
Dissolve 5 gm. of disodium salt of EDTA in 1 litre of water and standardize against BaCl2.
Procedure:
The method for the determination of sulphates is based on the fact that a sufficient amount of standard BaCl2 solution is added to the sample and back titrating the excess of barium left un-precipitated. Take 100 ml. of sample and add a few drops of methyl orange indicator and slight excess of HNO3. Boil the mixture to remove dissolved CO2.
Add 10 ml (or more) of standard BaCl2 solution in the boiling solution. Allow to cool down and make the volume up to 150 ml. of clear supernatant liquid into a beaker, add 1 ml. of buffer solution and some amount of Erichrome Black T indicator. Titrate with EDTA solution until a permanent blue colour is produced indicating end point.
Calculation:
Suppose 25 ml. of sample is taken for estimation of total hardness by EDTA titration and 10 ml. of BaCl2 solution is taken for precipitation of sulphates then the following formula can be used.
10 ml. of BaCl2 solution = 10 mg of CaCO3 = 10ml of EDTA solution.
Note:
If the quantity of sulphates in water is more than 100 mg/litre then volume of barium chloride solution should be increased.
(ix) Chlorides:
Chlorides are one of the major constituents found in all natural waters in different concentrations. Human excrete and industrial wastes etc. are rich in chlorides. For public health, chlorides up to 250 mg/litre are not harmful but increase of chlorides beyond this is indication of organic pollution.
Reagents:
(1) 0.0141 N Standard silver nitrate solution.
(2) Potassium chromate Indicator-Prepare 5% solution
Procedure:
Take 100 ml. of sample in each conical flask and add 2- 3 drops of potassium chromate in each of them. Titrate the solution in one flask with 0.014IN standard silver nitrate solution until a brick red colour is obtained (compare with second flask). Now note the ml. of AgNO3 used for the end point. Repeat the experiment with blank. The difference of the two observations is the ml. of AgNO3 used.
Calculation – Chlorides mg/litre
(x) Dissolved Oxygen:
The low values of dissolved oxygen affect the portability of water and can cause killing of fish and other animals of sea kingdom. It is a test which indicates the sanitary status of a water. The dissolved oxygen also suggests whether the processes undergoing a change are aerobic or anaerobic. A good water should have solubility of oxygen about 15 mg/litre at 0°C and 7 mg/litre at 35°C.
Reagents:
(1) Alkaline Potassium iodide – Dissolve 100 gm. of KOH and 150 gm. of KI in 1 litre of water.
(2) 48% MnSO4 solution.
(3) 0.0125N Sodium thiosulphate solution.
(4) Starch indicator.
(5) Cone. Sulphuric acid.
Procedure:
Take 200 ml. of sample in a conical flask. Add 1 ml. of MnSO4 solution (by pipette dipping the end below the surface) and 1 ml. of alkaline KI. Put the stopper and mix the solution thoroughly (avoid passage of air). After 10-15 minutes when the ppt settles down, add 2 ml. conc H2SO4.
Dissolve the ppt by shaking. Now titrate the solution with sodium thiosulphate using starch as indicator. Note the ml. of titrant used in getting the end point Perform the blank titration. The difference of the two should be regarded as ml. of sample titre.
Calculation:
1ml. of 0.0125 N Na2S2O3 solution = 0.1 mg of O2
Dissolved oxygen, mg/litre
ml. of sample titre x Normality of Na2 S 2 O3 x 8 x 1000/Volume of water in the conical flask
It indicates the organic pollution in water. It is a measure of the strength of sewage or polluted water.
(xi) Biochemical Oxygen Demand (B.OD.):
The B.O.D. is the amount of oxygen required by bacteria while stabilising decomposable organic matter under aerobic conditions. The decomposition of organic impurities in presence of bacteria results in utilisation of a part of the dissolved oxygen by the bacteria during their respiratory and metabolic activities. This depletion of oxygen is considered as a measure of the strength of water.
All organic constituents of sewage degrade under aerobic conditions.
The organics in sewage can be divided into three major groups:
(1) Carbohydrates (starches, sugars and cellulose),
(2) Proteins and
(3) Fats.
The approximate distribution of organics being 40 to 50% Carbohydrates, 40 to 50% Protein and 5 to 10% is Fat. The starches and sugars are easily metabolized by microorganisms while cellulose decomposes at a slower rate.
The proteins are complexes of amino acids which form major source of microbial nutrients.
The biochemical oxidation of organic matter in sewage can be considered as a monomolecular reaction, given by linear rate equation:
The L and D both serve as an effective means of gauging the qualitative and quantitative changes in the decomposable material in un-chlorinated and chlorinated samples. A reduction in k implies that as a result of chlorination, a quantitative change in the character of sewage constituents had taken place so that they had become less readily decomposable by aerobic organisms.
Thus k value of 0.2 will exert 90% of its ultimate first stage BOD in 5 days at 20°C while k value of 0.15 will have exerted only 32% of its BOD in the same 5 days at 20°C. The chlorination of sewage always results in the reduction of L value which indicates that there is a quantitative change in the decomposable fraction of the sewage.
Reagents:
(1) Cone. H2SO4.
(2) 48% MnSO4 solution in water.
(3) Alkaline KI – Add 700 gm. of KOH and 150 gms of KI in 1 litre.
(4) 0.0125 N standard sodium thiosulphate solution.
(5) Starch indicator.
(6) Sodium sulphite.
If the sample of sewage or water is alkaline or acidic then neutralize at pH 7 with IN H2SO4 or IN NaOH using pH meter. Excess of chlorine will be removed if sample is allowed to stand for 1 to 2 hrs. in open. If residual chlorine is too high titrate it with this solution and add the required amount of sodium sulphate.
Now take three bottles, A, B and C of 500 ml. capacity and fill the bottle with aerated water and stopper without leaving any air bubble. Determine the dissolved oxygen in A immediately by adding 2 ml. of MnSO4 + 2ml of conc. H2SO4 (Avoid air passage).
The (B – C) suggests loss of oxygen during incubation.
(xii) Chemical Oxygen Demand (COD):
Chemical oxygen demand is used for measuring the pollutional strength of waste water. Most of the organic compounds can be oxidised to carbon dioxide and water by the action of strong oxidising agents regardless of the biological assimilability of the substances.
Reagents:
(1) Standard ferrous ammonium sulphate solution.
(2) Standard potassium dichromate N/4.
(3) Sulphuric acid (with 1 gm. of silver sulphate in every 75 ml. acid)
(4) Ferroin indicator.
Procedure:
Take 50 ml. of the sample (A) in a conical flask. Add 100 ml. of distilled water and 15 ml. of standard potassium dichromate solution slowly and slowly and add 75 ml. conc H2SO4. Reflux the mixture for 2 hours, cool and wash down the condensate with distilled water.
Transfer the contents to 500 ml. flask. Dilute the mixture to about 300 ml. Titrate the excess dichromate with standard ferrous ammonium sulphate using ferroin indicator.
Now perform the blank experiment (B) by taking 100 ml. distilled water, 75 ml. acid and 25 ml. potassium dichromate solution. Reflux for 2 hours and titrate the excess dichromate with ferrous ammonium sulphate.
Calculations:
When A = ml. of ferrous ammonium sulphate used with sample,
B = ml. of ferrous ammonium sulphate used with distilled water,
C = Normality of ferrous ammonium sulphate.
(xiii) Free Carbon Dioxide:
The CO2 which is found in well waters and surface waters to a great extent can cause corrosion. The CO2 present in water in excess of carbonates and bicarbonates is known as free CO2.
It can be determined by titration with NaOH using phenolphthalein as indicator.
Reagents:
0.0454 N Standard Na2CO3.
0.0227 N Standard NaOH.
Phenolphthalein indicator.
Procedure:
Take 100 ml. of sample and keep it cool with ice until taken for estimation. Add 2.3 drops of phenolphthalein indicator. If the sample does not become red, it indicates the presence of free CO2. Titrate immediately with NaOH solution stirring with glass rod until a pink colour (pH 8.3) appears for about 1 minute.
Calculations:
CO2 mg/litre = ml. of NaOH x Normality of NaOH x 44 x 1000/ ml. of sample taken
(xiv) Free Available and Combined Available Chlorine:
The chlorine present in water as Cl2, HOCL, OC1–, H2OCl+, Cl3_ is called as free available chlorine. When chlorine in water combines with ammonia to form chloramines and other chloroderivatives then the mixture of chloramines and other derivatives is called available chlorine. Both free and combined chlorine may be present simultaneously in the chlorinated water.
Chlorine can be determined either by iodometric or by orthotolidine method. The iodometric method can be used to determine total residual chlorine only but not the free residual chlorine or the combined residual chlorine.
The orthotolidine method can be used to measure the total residual chlorine (free and combined). When nitrate, iron, or manganese is present in water, they interfere to give false colours for chlorine. It can, however, be applied if nitrite nitrogen contents are less than 2 ppm and manganic manganese are not more than 0.01 ppm.
The orthotolidine arsenite (OTA) method is the only one which gives differentiation of free available chlorine, combined available chlorine and the colour due to interfering substances present in water.
Way of sampling:
As chlorine is sensitive to light and also evaporates rapidly, hence chlorine in aqueous solution is not stable. It is, therefore, necessary that chlorine determinations should be performed immediately after sampling.
Iodometric method:
When KI and acetic acid are added in chlorine solution, it liberates iodine which can be titrated with sodium thiosulphate using starch as indicator. The minimum detectable concentration is approximately 0.04 mg of chlorine per litre. The reaction is mostly carried out at pH 3 to 4 in absence of light.
Reagents:
1. KI
2. Acetic acid (glacial).
3. Starch indicator.
4. 0.005 N Na2S2O3 solutions. A few drops of chloroform are added to increase the storage life for more than a month.
Procedure:
Avoid the presence of direct sunlight when performing the experiment. Take 200 ml. of the sample. Add 5 ml. of glacial acetic acid to reduce the pH between 3 and 4. Add about 1 gm. of KI in the sample and titrate with 0.005 N thiosulphate solution from burette until yellow colour of liberated iodine is almost discharged. Now add 1 ml. of starch solution and titrate until the blue colour is discharged.
Blank titration:
Perform the experiment with blank titration (without chlorine water) by mixing KI in acetic acid and distilled water with same quantity of the sample).
The actual titre value = (Titrate value with sample – titre value with blank).
Calculation:
Chlorine in water, mg/litre = Actual titre x normality of thio x 35.46 x 1000/ ml. of sample
Orthotolidine – Arsenite method:
This method is used in the determination of free available and combined available chlorine and colour due to interfering substances. Orthotolidine gives yellow colour in presence of chlorine.
The most important point is that all the experiments must be performed at low temperature (preferably 1°C as at room temperature some of combined available chlorine can react with orthotolidine giving a high free available chlorine value.
Reagents:
(1) Sodium arsenite reagent:
Dissolve 5 gm. of sodium arsenite in 1 litre distilled water.
(2) Orthotolidine reagent:
Dissolve 1.35 gm. of orthotolidine di-hydrochloride in 500 ml. distilled water. Now add in this solution, 350 ml. of distilled water and 150 ml. of conc. HCL. The total solution is 1 litre. Keep it in ambered bottle. The solution is stable for 6 months.
(3) 05M phosphate buffer solution:
Take 22.86 gm. of Na2HP04 and 46.16 gm. of KH2P04 in 1 litre flask and make up with distilled water.
(4) Permanent chlorine standard using chromate-dichromate stock solution:
Add 1.55 gm. of K2Cr2O7 and 4.65 gm. of K2CrO4 in a litre flask and make up with 0.1M standard buffer solution. The colour produced will be equal to 20 mg of chlorine per hue (10 mg/litre).
For getting the range between 0.01 – 1.0 mg of chlorine per litre, take 100 ml. of chromate – dichromate stock solution to 1 litre with 0.1 M phosphate buffer.
The colour develops is equal to 1 mg/litre. Take out 1, 3, 5, 7, 10, 15, 20, 40, 50, 60, 70, 80, 90 and 100 ml. of chromate- dichromate stock solution to 100 ml. flask and make up with 0.1M phosphate buffer to get the required range (between 0.01 and 1 mg chlorine per litre) of standards.
The whole analysis can be summarized in the following way:
Take three comparator cells A, B and C and perform the experiment as follows:
(xv) Chlorine Demand:
The chlorine demand is the difference between the amount of chlorine applied and the amount of free combined or total available residual chlorine remaining at the end of contact period.
Chlorine demand = Amount of chlorine – Total residual
or consumed applied chlorine
or chlorine dose
Chlorine demand determinations are very important as they determine the amount of chlorine needed to give a specific free, combined or total available residual chlorine after a selected period of treatment.
If sufficient chlorine is added to reach the breakpoint, depending on:
(1) Temperature,
(2) pH,
(3) Time of contact,
(4) Ratio of chlorine to nitrogen compound and
(5) Concentration of chlorine, subsequent addition of chlorine remains in free available state.
Generally the cooling tower should have about 2-3 ppm of free available chlorine or about 10 ppm of total residual.
Reagent:
(1) 11% bleaching powder solution.
(2) 10% hypochlorite solution.
(3) 1% chlorine solution.
(4) 0.025 N sodium thiosulphate solution.
(5) Starch indicator.
(6) Orthotolidine reagent.
(7) Sodium arsenite reagent.
(8) Glacial acetic acid.
(9) Potassium iodide.
Adjust the concentration of chlorine in such a way that 1 drop in 500 ml. water = 1 mg of chlorine per litre.
Procedure:
Take 500 ml. of sample each into ten conical flasks. Add 1 drop of chlorine solution to the first, 2 drops of chlorine solution to the second, 3 to the third and so on with mixing gently. Now determine chlorine concentrations after 10, 20, 40, 60 and 80 minutes to know the stability of residual chlorine as related to contact time. Note the time of contact when estimating the residual chlorine.
During contact time, all samples should be protected from sunlight. The total residual chlorine is estimated by iodometric method while the free and combined residual available chlorine can be estimated by orthotolidine-arsenite method.
Calculation:
Chlorine demand = Chlorine added – Residual chlorine
mg/litre mg/litre mg/litre
A residual chlorine of 10-20 ppm gives an effective germicidal treatment to the water and also produces no corrosion as the contact time is very short.
(xvi) Chlorine Dosage:
For chlorination of water, gaseous chlorine, bleaching powder, hypochlorite’s, chlorine dioxide etc. are used. Hypochlorite’s are used when only a small amount of chlorine is needed. Generally phenols, cresols, fly sprays, marking inks etc. should be removed from water plant because chlorine reacts with them even in minute quantities to give compounds of strong flavours and harmful to the human beings.
Hypochlorite’s can be added by pumping or by aspirating the solution into water. The gaseous chlorine is added to water by mixing the gas with water and then injecting this water into the supply line.
Calculation:
The amount of chlorine gas in kg/day can be estimated by the following formula:
Example – For 6 ppm of residual chlorine delivering 5,00,000 litres per 24 hours,
The wt. of chlorine required = 6 × 500000/1000000 = 3.0 kg per day.
This calculation is based on the assumption that the chlorine demand of water is Zero. If chlorine demand for water is 2 ppm then a dosage of 8 ppm would be needed for a residual of 6 ppm.
The hypochlorite’s used in water have a stock solution (0.5 – 1%) containing 5000-10,000 ppm chlorine.
The wt. or volume of hypochlorite can be calculated as follows:
To calculate volume of stock solution added to given water supply (assuming zero chlorine demand), the following formula is used:
(C) Microbiological Examination of Water:
All glassware used in microbiological examination should be neutral as cheaper varieties give off free alkali. For testing the glassware, fill the container with distilled water at pH 7 & add 0.4% phenol red to give yellow colour.
Now put the container in autoclave for 25 minutes at 100°C and then cool to room temperature. If the colour of water remains yellow, the glassware is pure. A pink or magenta colour clearly suggests that alkali is liberated from the glass.
The glassware should be cleaned first with hot water and soap and then with tap water and finally with distilled water. Generally washing soda is used instead of soap. Sterilize all glassware by passing them through Bunsen flame.
Preparation of Cultural Media:
The substance in which microorganisms grow are called cultural media. The preparations of cultural media are very necessary for the study of micro-organisms. Generally peptone, beef extract, agar, salts etc. are selected for the preparation of bacteriological media as given in Table 6.
Preparation of Nutrient Agar:
Take 5 gms of peptone in 1 litre of distilled water and add 3 gms beef extract. Heat on water bath until dissolve. Now adjust the pH of the solution at 7.0 by adding a few drops of (N/10)NaOH in it. Add 15 gms. of Agar to the solution and boil. Distribute 10 ml. each in test tube and sterilize.
Now dissolve 10 gm. Lactose, 20 gm. peptone and 5 gm. Sodium Chloride in 600 ml. distilled water. Take 5 gms. of bile salt in 200 ml. distilled water separately. Mix the two and make up the volume to 975 ml. by adding distilled water.
Adjust the pH to 7.4. Add 1 ml. of 1% alcoholic solution of Bron Cresol purple and make up the volume to 1 litre. Now distribute in test tubes 10 ml. each with fermentation tubes, plug with cotton and sterilise.
Testing of (i) Total Count and (ii) Coliform MPN:
Pathogenic and non-pathogenic bacteria, when in combination, form a group which is designated as Bacillus coli, abbreviated in short B-coli group. This group of bacteria is present in intestines of all living warm-blooded animals. The presence of bacteria of B-coli group may be used as one of the methods of biological examinations of water.
For Bacteriological examination of water following two tests are generally carried out:
(i) B-coli test or coliform test
(ii) Total count test.
Water Borne Diseases:
The most important water borne bacteria responsible for diseases are S. typhi, S. paratyphi, Shigella dysentrial and Vibrio cholera. If the source of such bacteria is known and if water treatment plant is properly operated, it is possible to detect and destroy such a bacteria and thus the danger of waterborne disease may be avoided easily.
(i) Total count:
Take four nutrient agar tubes and heat until they melt. Cool to 45°C. Pipette 1 ml. of a dilution into the centre of the Petri dish, transfer the contents of one agar tube to the dish and mix thoroughly. Allow the medium to set, invert and incubate for 24-40 hrs. Count plates having 30-300 colonies using a magnifier.
Colony count per ml = Number of colonies per plate × Reciprocal of × dilution
(ii) Coliform MPN:
The Most Probable Number (MPN) is a good measure of biological contaminants present in water. The allowable MPN/100 ml. in the effluent may vary from 100 to 500 MPN/100 ml. The coliform index is in general a measure of viral as well as bacterial pollution. The chlorination inactivates viruses.
Besides colour and odour generally water contains protozoa, algae etc. The numbers present are generally so few that it necessitates the use of a large volume of water which can be concentrated into a small volume before an accurate tabulation can be made.
Generally organisms are reported and counted in standard units or cubic standard units. A standard unit is the area of a visible surface of 400 square microns. A cubic standard unit is a volume of 8,000 cubic microns.
Procedure for Microbiological Examination of Water:
(1) Sedgwick – Rafter filter funnel:
The water (100 ml) under test is filtered through the filter. Concentrate the filtered water into 20 ml.
(2) Thoroughly mix the organisms by blowing air into the liquid through a pipette. Remove one ml. and place it in a Sedgwick-Rafter Counter cell.
(3) Now examine and record units with the help of a Whipple ocular micrometer. The examination of organisms consists of (1) survey and (2) total count.
(4) The microscope is adjusted in such a way that the area covered by the ruled square of the ocular micrometer is exactly one square millimeter and since the depth is one millimeter, the volume outlined by the ruled square is one cubic millimeter.
In the survey, the whole cell is examined for large organisms. In the total count, the organisms within the ruled area are counted. The cell is then moved and another area is counted and the process repeated until the representative areas are counted.
The average of the readings then gives the number of units in one cubic millimeter. As there are variations in the specific gravity of water organisms hence special care is needed to change the focus of the microscope in order that both upper and lower strata be examined.
From the data the number of organisms per ml. in the original sample is calculated with the help of the following formula:
when N = number of organisms per ml. in original sample;
W = ml. concentrate;
f = ml. filtered;
S = number of organisms found in the survey;
V = Volume in ml. of counting cell;
t = total number of organisms in the total count of all squares counted, and
n = number of squares counted.
A method of calculating the MPN surviving chlorination was suggested from pilot plant study of White.
His data fit the following equations:
Y = Y0 (1 + 0.23 Ct)-3
When Y = MPN in chlorinated effluent after time, t;
Yo = MPN in effluent prior to chlorination;
C = total chlorine residual, mg/1;
t = contact time, minutes.
If Y0 = 3.5 x 106/100 ml; C = 5 mg/1; t = 30 min, then
Y = 3.5 x 106 (1 + 0.23 x 5 x 30) 3 = 78.2 MPN/100 ml.
A recent development in testing for coliforms is the use of the membrane filter. The water sample is filtered through a sterilized membrane which will retain bacteria. After filtration the membrane is placed on a sterile pad which has absorbed nutrients.
Both membrane and pad are placed in an incubator for 2 hrs. and then membrane is transferred to another pad which contains a medium which allows to grow the coliforms. After 20 ± 2 hours, the membrane is examined for Colonies of Coliforms.
The membrane method is simple and takes shorter time than the previous one. It also gives a direct count of coliforms.
Expression of Results/Units:
Generally the chemical components are often referred as ions and are expressed as milligram per litre (mg/1). The pH is mentioned in pH scale (0-14). Alkalinity, Hardness and Acidity are measured as CaCO3 and are expressed as mg/1.
Turbidity is expressed as units in silica-scale one unit of turbidity are equal to 1 mg SiO2 dissolved in 1.0 litre of distilled water.
Estimates of the numbers of coliform are given as “most probable number” per 100 ml (MPN/100 ml) or as a determined number of direct plating procedures.