The Indian standards for water were originally published in 1983 and were adopted by the Bureau of Indian Standards (BIS). As per the standard “Drinking water is water intended for human consumption for drinking and cooking purposes from any source”. It includes water (treated or untreated) supplied by any means for human consumption.
The first revision of the original standards was undertaken in 1991 to take into account the up-to-date information available about the nature and effect of various contaminants. The second revision was undertaken in 2012 to upgrade the requirements of the standards and align with the internationally available specifications on drinking water.
A. Physio-chemical analysis of water samples
The temperature of the water samples can be recorded using a food-grade analytical/ digital thermometer while pH and turbidity can be measured using pH and turbidity meter, respectively. Water samples can also be analyzed for total hardness, TDS, alkalinity, calcium, magnesium, sodium, potassium, nitrate, total phosphate, ammonium, sulphate, and chloride by standard methods available in the literature (Mackereth et al., 1978 and APHA, 2005) in a standard laboratory. The methodology of analysis is explained as follows:
1. Estimation of pH
Apparatus: pH meter with glass electrodes and Thermometer
Standard buffer solution:Dissolve commercially available one buffer tablet each of pH 4.0, 7.0, and 9.2 in freshly prepared distilled water separately and make up the volume to 100 mL. Three to four drops of toluene need to be added in standard buffer solutions to prevent the growth of mold.
(i) Turn the pH meter ON and allow it to warm for 15 minutes.
(ii) Standardize the glass electrode using a standard buffer of pH 7.0 and calibrate with the buffer solutions of pH 4.0 and 9.2.
(iii) Take 50 mL of filtered water sample in 10 ml beaker and immerse the glass electrodes of the pH meter.
(iv) While taking the pH value of the sample, record the pH value to the nearest 0.1 unit, keeping the pH meter in standby mode immediately after each reading.
(v) The electrode needs to be removed after each determination and carefully blotted and dried with filter paper before the next determination.
(vi) When not in use the electrode should be kept in distilled water and ensure that the reference electrode always contains saturated potassium chloride solution in contact. with solid potassium chloride crystals.
2. Estimation of Turbidity
Apparatus: Turbidity tube
(i) Take a sample of water from the water source.
(ii) Hold the tube in one hand near the bottom and look into the open end with the head about 10 to 20 cm above the tube, so that one can clearly see the black circle, cross, or other marks on the bottom of the tube.
(iii) Slowly pour the water sample into the tube, waiting for air bubbles to rise if necessary until the mark on the bottom of the tube just disappears.
(iv) Stop pouring the water sample into the tube and look at the level of water in the tube. For turbidity tubes that have a turbidity scale marked on the side, read the number on the nearest line to the water level. This is the turbidity of the water.
(v) If the tube does not have a scale marked, measure the distance from the bottom of the tube to the water level with a tape measure and look up or calculate the turbidity of the water sample using the instructions provided with the tube.
(vi) After use, wash the tube in clean water and store the two parts of the tube where they cannot be damaged.
3. Estimation of Total Dissolved Solids (TDS)
Residue remained after the evaporation and subsequent drying of a known volume of water sample in an oven at a specific temperature of 100-150°C represents total dissolved solids.
Apparatus: Electrically heated temperature-controlled oven and Petri plate.
Procedure: Take 50 mL of filtered water sample in pre-weighed Petri-plate (W1) and place in oven at 150°C for 24 h. After 24 h, Petri-plate needs to be removed from the oven to room temperature for cooling and again weighed (W2). The weight of the Petri-plate must be in milligram (mg).
4. Estimation of Total Hardness
The total hardness of water is determined by the (Ethylene diamine tetraacetic acid ) EDTA method in alkaline conditions. EDTA and its sodium salts form a soluble chelated complex with certain metal ions. Calcium and magnesium ions develop wine red color with Erichrome Black T in aqueous solution at pH 10.00.1. When EDTA is added as a titrant, calcium and magnesium divalent ions get complexed resulting in a sharp change in color from wine red to blue which indicates the end-point of the titration.
(a) Buffer solution: Dissolve 16.9 g of NH4CI in 143 mL of NH2OH and then add 1.25 g of magnesium salt of EDTA into it, and dilute to 250 mL.
(b) Inhibitor: Dissolve 4.5 g of hydroxylamine hydrochloride in 100 mL of 95% ethyl alcohol.
(c) Erichrome Black T indicator: Mix 0.5 g of dye with 100 g NaCl.
(d) 0.01M EDTA: Dissolve 3.723 g of EDTA sodium salt in 100 mL distilled water
(e) Standard calcium carbonate solution (1ml = 1 mg CaCO3)
Procedure: Take a 50 mL filtered water sample in a 250 mL conical flask, add 2 mL of buffer solution followed by 1 mL of inhibitor, 0.2 g Erichrome Black T indicator, and mix it by shaking. Titrate with 0.01M EDTA till the pink color of solution changes to purplish-blue, the volume of EDTA solution used will be noted as ‘A’.
The blank should be titrated against 0.01 M EDTA solutions and the volume of EDTA used will be noted as ‘B’. Calcium carbonate solution will be used as standard and the volume of 0.01 m EDTA titrant used will be ‘D’.
5. Estimation of Total Alkalinity
The alkalinity of a water sample can be estimated by titrating the sample with a standard solution of H2SO4 at pH 8.3 using Phenolphthalein as an indicator (carbonate alkalinity) and then titrating further to the second endpoint at pH 4.5 using methyl orange as an indicator (total alkalinity)
(a) 0.02 N Sulphuric acid:Carefully add 2.8 mL sulphuric acid in a volumetric flask and dilute to 500 ml with distilled water to get the strength of 0.2 N sulphuric acid. Further, dilute 100 mL of this solution to 500 ml to get 0.02 N sulphuric acid.
(b) Phenolphthalein indicator:Add 0.5 g of phenolphthalein in a mix of 50 mL of 95% ethanol and 50 mL of distilled water, add 0.02 N NaOH to it dropwise until faint pink color appears.
(c) Methyl crange indicator:Dissolve 0.05 g of methyl orange in 100 mL of distilled water.
Procedure: Take a 50 mL filtered water sample in a 250 mL conical flask, to it add 0.2 mL of phenolphthalein indicator; after shaking color changes to pink. It should be titrated with 0.02 N H2SO4 until the pink color disappears, the disappearance of the pink color will be noted as endpoint ‘A’. This indicates phenolphthalein carbonate alkalinity.
To determine the total alkalinity, add 2-3 drops of methyl orange to the same sample and continue titration till the color changes from yellow to orange. The amount of titrant used will be noted as ‘B’.
6. Estimation of Calcium
Calcium content in water samples can be determined by titration with standard EDTA titrant. Murexide (ammonium purpurate) indicator forms pink solution with Cat, when titrated with EDTA, the pink solution is changed to purple.
(a) Murexide indicator: Mix with 5 g NaCl with 10 mg ammonium purpurate and grind it to a fine powder.
(b) 0.01 M EDTA titrant: Dissolve 7.446 g of the sodium salt of EDTA in 200 mL distilled water.
(c) 2 N Sodium hydroxide:Dissolve 8 g NaOH in 100 mL distilled water.
Procedure: Take 50 mL of water sample in a conical flask. To it add 2 mL of 2 N NaOH solutions and 0.2 g of murexide indicator and mixed by shaking, pink color appears. Titrate with 0.01 M EDTA and appearance of purple color will be noted as an endpoint.
7. Estimation of Magnesium
The concentration of magnesium in water can be determined as the difference between total hardness and amount of calcium in the water, multiplied by factor 0.244.
B = amount of calcium (mg L-¹)
8. Estimation of Sodium on Flame Photometer
(a) Standard Stock Solution (100 Me of Na/L): Take a small amount of NaCl and dry it in the oven. Dissolve 5.845 g of dried NaCl in water and made the volume 1 liter with distilled water.
(b) Working Standard Solution of Sodium (Na): Dilute 5, 10, 15, 20, 30, 40, and 50 mL portions of the stock solution (containing 100 mE of Na/L) to 100 mL in volumetric flasks to get working standards of 5, 10, 15, 20, 30, 40 and 50 mE of Na/litre concentrations.
(1) Filter a portion of the water sample because it is desirable as it prevents chocking of the capillary tube of the flame photometer.
(2) Take the different working standard solutions and record the flame photometer reading against each, after setting zero with distilled water and 100 with the highest concentration i.e. 50 mE of Na/L.
(3) Take readings of the test sample for calculating sodium content in the test sample.
9. Estimation of Potassium on Flame Photometer
(a) Standard Stock Solution (100 mE of K/L): Dissolve 5.845 g of dried KCL in water and make the volume to 1 liter with distilled water.
(b) Working Standard Solution of Potassium: Dilute 5, 10, 15, 20, 30, 40, and 50 mL portions of the stock solution (containing 100 mE of K/L) to 100 mL in volumetric flasks to get working standards of 5, 10, 15, 20, 30, 40 and 50 mE of K/litre concentrations.
(i) Filter a portion of the water sample as it prevents chocking of the capillary tube of the flame photometer.
(ii) Take the different working standard solutions and record the flame photometer reading against each, after setting zero with distilled water and 100 with the highest concentration. i.e. 50 mE of K/L.
(iii) Take readings of the test sample for calculating potassium content in the test sample.
10. Estimation of Nitrate Content
Nitrate reacts with phenol disulphonic acid and produces a nitro-derivative which in alkaline solution develops yellow color.
(a) Phenol disulphonic acid:Dissolve 12.5 g phenol was in 75 mL concentrated H₂SO
(b) 12 N KOH: Dissolve 33.65 g of KOH was in 50 mL distilled water.
(c) Standard KNO, solution
Procedure: Take a 20 mL filtered water sample was in a 50 mL test tube and evaporate at 100°C in an oven, cool at room temperature. Add 0.5 mL of phenol disulphonic acid and then dissolve the residue with the help of a glass rod. Then add 5 mL distilled water added to it followed by 2 mL of 12 N KOH, mix well by shaking, and keep it for 5-10 min, the yellow color will be developed. Record the absorbance at 410 nm. The concentration of nitrate can be calculated from the standard curve prepared with KNO₂.
11. Estimation of Total Phosphate
Organically combined phosphorus and all other forms of phosphates including polyphosphate first convert to orthophosphate by digestion with a strong acid (Perchloric acid). In acidic conditions, orthophosphate reacts with ammonium molybdate to form molybdophosphoric acid. The blue color developed after the addition of ammonium molybdate will be measured at 660 nm in Spectrophotometer.
(a) 5% Ammonium molybdate:Dissolve 5 g of ammonium molybdate in 100 mL distilled water.
(b) 0.2% Aminonaphthol sulphonic acid: Dissolve 0.5 g of 1,2,4-aminonaphthol sulphonic acid, 30 g sodium bisulphite, and 6 g crystalline sodium sulphite in distilled water by shaking and make the final volume 250 mL, keep it overnight and filter before use.
(c) Phenolphthalein indicator:Dissolve 0.5 g of Phenolphthalein in 500 mL of 95% ethyl alcohol and add 500 mL distilled water to it. Then add 0.02 N NaOH drop-wise till faint pink color appears.
(d) Perchloric acid
(e) 2 N NaOH: Dissolve 8 g NaOH in 100 mL Distilled water.
(f) Standard KH₂PO4: Dissolve 1 mL KH₂PO4 was in 9 mL of distilled water. Take 1 mL of this solution and add 9 mL of distilled water. Then again take 1ml of this solution and add 9 mL distilled water. This solution will be used as a standard solution.
Procedure: Evaporate 15 mL filtered water sample in 50 mL test tube at 100ºC, cool and add 1 mL of perchloric acid and mix well. Again keep at 100°C to evaporate the perchloric acid; residue will be allowed to cool, and then add 15 mL of distilled water to it, followed by one drop of phenolphthalein indicator. Titrate against 2 N NaOH until the faint pink color just restores. Then take 2 mL of the above solution and add 1 mL of ammonium molybdate and 0.5 mL of sulphonic acid to it. The reagents will be mixed thoroughly and allowed to stand for 5 minutes, a blue color develops. Record the absorbance of the solution at 660 nm. The concentration of phosphate can be calculated from a standard curve made with KH₂PO4.
12. Estimation of Ammonium
Ammonium produces a yellow-colored compound when it reacts with alkaline Nessler’s reagent.
(a) Nessler’s reagent: Mix 5 g HgCl, and 3.5 g KI and then dissolve it in a small quantity of water. Then add 25 mL chilled solution of 4 N NaOH and dilute with distilled water to 50 mL final volume. Keep it overnight and filter, supernatant must be stored in an amber-colored bottle.
(b) 4N NaOH: Dissolve 16 g of NaOH in 100 mL of distilled water.
(c) Standard NH CI solution
Procedure: Take a 50 mL filtered water sample in a conical flask, it add 2 mL Nessler’s reagent. Thoroughly mix the reagent and allow it to stand for 5 minutes, pale yellow color develops; record the absorbance at 640 nm. The concentration of ammonium can be calculated from the standard curve prepared with NH CI.
13. Estimation of Sulphate
Sulphate ions precipitated in an acetic acid medium with Barium chloride, so as to form uniform size crystals of Barium sulphate. The absorbance of the BaSO, suspension can be measured in a spectrophotometer.
(a) Buffer solution: Dissolve 30 g MgCl₂.6H2O, 5 g CH₂COONa.3H2O, 1 g KNO, and 20 mL acetic acid (99%) were in 500 mL distilled water and make the final volume 1000 mL.
(b) Barium chloride.
(c) Standard NaSO4 solution.
Procedure: Take a 25 mL filtered water sample in a 100 mL conical flask. Add 10 mL buffer solution to it and mix well. Then add 0.2 g of BaCl, crystals while the flask is constantly stirred with the help of a magnetic stirrer for one min. Appeared turbidity will be recorded against the blank. at 510 nm. The concentration of sulphate can be calculated from the standard curve prepared with NaSO4.
14. Estimation of Chloride
The chloride content in the water sample can be determined by titration with standard silver nitrate solution, using potassium chromate as an indicator.
(a) Potassium chromate indicator solution: Dissolve 2.5 g of K₂Cr₂O7 in 50 mL distilled water, add AgNO3 to it till definite red precipitate forms and then allow it to stand for 12 h, filter and diluted to 50 mL.
(b) 0.0141M Silver nitrate titrant: Dissolve 1.1975 g AgNO3 in 500 mL of distilled water.
Procedure: Take 50 mL of filtered water sample in a conical flask, add 1 mL of K₂Cr₂O, indicator solution and mix thoroughly. Titrate it against 0.0141M AgNO, solution. The appearance of pinkish-yellow colour is endpoint ‘A’. The volume of titrant used for the blank is ‘B’.
A = mL of titrant used for sample
B = mL of titrant used for blank
M = Molarity of AgNO3
B. Microbial Analysis Of Water Samples
Most Probable Number (MPN) is a method used to estimate the concentration of viable microorganisms in a sample. So, MPN is most commonly applied for quality testing of water i.e. to ensure whether the water is safe or not in terms of bacteria present in it. A group of bacteria commonly referred to as fecal coliforms act as an indicator for fecal contamination of water. Thus, the presence of very few fecal coliforms would indicate that water probably contains no disease-causing organisms, while the presence of large numbers of fecal coliform bacteria would indicate a very high probability that the water could contain disease-producing microorganisms making the water unsafe for consumption.
Ideally drinking water should be free from any visible biological organisms. It should also be free from microscopic organisms such as algae, zooplanktons, flagellates, parasites, and toxin-producing organisms.
Water to be tested is diluted serially and inoculated in lactose broth, coliforms if present in water utilizes the lactose present in the medium to produce acid and gas. The presence of acid is indicated by the color change of the medium and the presence of gas is detected as gas bubbles collected in the inverted Durham tube present in the medium. The number of total coliforms is determined by counting the number of tubes giving positive reaction i.e both color change and gas production and comparing the pattern of positive results (the number of tubes showing growth at each dilution) with standard statistical tables. MPN is performed in three steps viz.
First: The presumptive test, is a screening test to sample water for the presence of coliform organisms. If the presumptive test is negative, no further testing is performed, and the water source is considered microbiologically safe. If, however, any tube in the series shows acid and gas, the water is considered unsafe and the confirmed test is performed on the tube displaying a positive reaction.
Second: Some microorganisms other than coliforms also produce acid and gas from lactose fermentation. To confirm the presence of coliform, a confirmatory test is done.
(a) Lactose broth or Mac Conkey Broth or Lauryl tryptose (lactose) broth: Dissolve the stated amount of dehydrated medium in distilled water to obtain single and double strength concentration.
(b) Kovacs Reagent:Dissolve 10 g of p-dimethyl amino benzaldehyde in 150 mL of isoamyl alcohol and then slowly add 50 mL of concentrated hydrochloric acid.
(c) Gram Stain Solution:It is a collective name for the following three solutions:
Gram Iodine Solution – Dissolve 1 g of iodine, 2 g of potassium iodide, and 3 g of sodium bicarbonate in 300 mL of water.
Gram Decolorizer Solution – Mix equal volumes of 95% ethanol and acetone.
Gram Safranin Solution – Dissolve 2.5 g of safranin O in 100 mL of 95% ethanol to make a stock solution.
1. Dispensed the requisite volume into culture tubes containing an inverted Durham Tube, and cap the culture tubes. Take 5 tubes of double strength and 10 tubes of single strength for each water sample to be tested. Sterilise the media in autoclave at 115°C for 10 min.
2. Using a sterile pipette add 10 mL of water to 5 tubes containing 10 mL double strength medium.
3. Similarly add 1 mL of water to 5 tubes containing 10 mL double strength medium and 0.1 mL water to the remaining 5 tubes containing 10 mL double strength medium.
4. Incubate all the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48 hrs.
5. Compare the number of tubes giving a positive reaction to a standard chart and record the number of bacteria present in it.
1. From each of the fermentation tubes with positive results transfer one loopful of medium to 3 mL lactose broth or brilliant green lactose fermentation tube, to an agar slant and 3 mL tryptone water.
2. Incubate the inoculated lactose broth fermentation tube at 37°C and inspect gas formation after 24+ 2 hours. If no gas production is seen, further incubate up to a maximum of 48 ± 3 hours to check gas production.
3. The agar slants should be incubated at 37°C for 24+ 2 hours and Gram Stain Preparation made from the slants should be examined microscopically.
4. The formation of gas in lactose broth and the demonstration of Gram-negative, non-spore-forming bacilli in the corresponding agar indicates the presence of a member of the coliform group in the sample examined.
5. Incubate the tryptone water at (44.5 ± 0.2°C) for 18-24 hours. After it, add approximately 0.1 ml of Kovacs reagent and mix gently. The presence of indole is indicated by a red color in the Kovacs reagent, forming a film over the aqueous phase of the medium.