ENVIS Technical Report: 25

ECOLOGICAL ASSESSMENT OF LENTIC WATER BODIES OF BANGALORE

PHYSICO – CHEMICAL ANALYSIS

Temperature

Temperature is one of the most important parameters that influence almost all the physical, chemical and biological properties and thus the water chemistry.

Apparatus required: The temperature is measured in the field using a thermometer with 0.1^oC division on a Celsius scale. For measuring the temperature of open waterbody, the thermometer is dipped directly into the water and the reading is taken while it is in the waterbody.

Procedure: Thermometer is immersed directly in the waterbody for a period of time sufficient to permit constant reading. While collecting the sample, care was taken that it is not exposed to heat or direct solar radiation.

Total dissolved solids

Dissolved solids are solids that are in dissolved state in solution. Waters with high dissolved solids generally are of inferior palatability and may induce an unfavourable physiological reaction in the transient consumer.

Apparatus required: An electronic probe, which also measures pH, and conductivity, is used to measure TDS. The values are expressed as mg/L of water.

Procedure: The probe is immersed directly in the water collected in a wide mouthed sampling bottle at the sampling site immediately after collection for a period of time sufficient to permit constant reading.

Transparency

Transparency is a characteristic of water that varies with the combined effect of colour and turbidity. It measures the depth to which light penetrates in the waterbody.

Apparatus required: Secchi disc, a metallic disc of 20 cm diameter with four quadrats of alternate black and white on the upper surface is used to measure transparency and the values are expressed in cm or mm.

Procedure: The disk is attached to a rope and lowered into the water until it is no longer visible. Secchi disk depth, then, is a measure of water clarity. Lower readings indicate turbid or coloured water. Transparency is measured by gradually lowering the Secchi disc at respective sampling points. The depth at which it disappears in the water (X₁) and reappears (X₂) is noted.

Estimation: The transparency of the waterbody is computed as follows:
                      Transparency (Secchi Disc Transparency)      =        (X₁ + X₂) / 2
              Where, X₁ = Depth at which Secchi disc disappears
                          X₂ = Depth at which Secchi disc reappears.

pH

pH – potential of hydrogen, is the measure of the concentration of hydrogen ions.  It provides the measure of the acidity or alkalinity of a solution.

Apparatus required: An electronic probe or a pH meter, which also measures, TDS and conductivity, and is used to measure pH at a scale of 0 – 14.

Procedure: Immerse the probe directly in the water collected in a wide mouthed sampling bottle at the sampling site immediately after collection for a period of time sufficient to permit constant reading.

Specific conductivity

Conductivity is the capacity of water to conduct electric current and varies both with number and types of ions in the solution. The values of conductivity and TDS are interrelated.

Apparatus required: Conductivity meter, is used to measure conductivity. Conductivity is reported in mmho or µ mhos/cm or as  µS/cm.

Procedure: the electrode of the conductivity meter is immersed directly in the water collected in a wide mouthed sampling bottle at the sampling site immediately for a period of time sufficient to permit constant reading.

Dissolved oxygen

DO content indicates the health and ability of the waterbody to purify itself through biochemical processes. DO is a very important parameter for the survival of fishes and other aquatic organisms.

Sampling: Samples from surface waters is collected in narrow mouthed bottles with glass-stopper. It is important not to let the sample remain in contact with air, air bubbles to be formed or to be agitated, because either condition can cause a change in its gaseous content. The bottles should be filled to overflow and stoppered. Entraining or dissolving atmospheric oxygen should be avoided. DO should be determined immediately on the sampling site.

Method: Winkler’s method. Titration.
Principle: Oxygen present in the sample oxidises the dispersed divalent manganous hydroxide to the higher valency to precipitate as a brown hydrated oxide after addition of potassium iodide and sodium hydroxide. Upon acidification, manganese reverts to its divalent state and liberates iodine from potassium iodide, equivalent to the original dissolved oxygen content of the sample. The liberated iodine is titrated against 0.025N sodium thiosulphate using fresh starch as an indicator.
Apparatus required: BOD bottles-125 ml capacity, lab glassware - measuring cylinder, conical flasks, analytical balance, glass rods and Bunsen burner.

Reagents:

• Manganese sulphate: 480 g of manganous sulphate tetra hydrate is dissolved and made up to 1000 ml with distilled water
• Conc. sulphuric acid
• Starch indicator: 0.5 g of starch is dissolved in glycerine and boiled for few minutes. Once cooled 2 drops of formaldehyde is added as a preservative.
• Stock sodium thiosulphate: 24.82 g of sodium thiosulphate pentahydrate (Na2S202. 5H2O) is dissolved in distilled water and made up to 1000ml.
• Standard sodium thiosulphate (0.025N): 250 ml of the stock sodium thiosulphate pentahydrate is made up to 1000 ml with distilled water to give 0.025N.

Procedure: The sample is collected in BOD bottles (125 ml), to which 2ml of manganous sulphate and 2ml of potassium iodide are added and sealed. This is mixed well and the precipitate allowed to settle down. At this stage 2ml of conc. sulphuric acid is added, and mixed well until all the precipitate dissolves. 25 ml of the sample is measured into the conical flask and titrated against 0.025N sodium thiosulphate using starch as an indicator. The end point is the change of colour from blue to colourless. The amount of titrant consumed gives the direct reading for DO in ppm. DO is calculated using the following formula.

Estimation:
                    DO (mg/L) = (ml * N) of titrant * 8 * 1000 / V₂ [(V₁-v)/V₁]
Where, V₂ = volume of the part of contents from the sample bottle titrated
            V₁ = volume of the sample bottle
            v =  volume of  added
            N = normalcy of sodium thiosulphate

Alkalinity

The alkalinity of water is a measure of its capacity to neutralise acids.  It is an anionic phenomenon.
Method: Sulphuric acid titrimetric method
Principle: Alkalinity of a water sample is estimated by titrating with standard sulphuric acid. Decolourisation of phenolphthalein indicator (phenolphthalein alkalinity) or a sharp change from yellow to pinkish orange (total alkalinity) will indicate the end point.
Apparatus:  Conical flasks, standard flask, measuring cylinder, burette, pipette and analytical balance
Reagents:

• Standard H₂SO₄ 0.02 N
• Phenolphthalein indicator: 0.5 g of phenolphthalein is dissolved in 50 ml (as per NEERI, it is 500) 95% ethyl alcohol. To this another 50 ml of distilled water is added.  0.02 N NaOH is added drop wise till a faint pink colour appears.
• Methyl orange indicator:  0.5 g of methyl orange is dissolved in 100 ml of distilled water.

• 25 ml of sample is taken in a conical flask, to which 2-3 drops of phenolphthalein indicator is added
• If pink colour develops, this is titrated with 0.02 N H₂SO₄ till the colour disappears. The volume of H₂SO₄ needed is noted.
• To the same flask 2-3 drops of methyl orange indicator is added and titration is continued with 0.02 N H₂SO₄ till the yellow colour changes to pinkish orange. Again the volume of H₂SO₄ needed is noted.
• In case the pink colour does not develop after addition of phenolphthalein, the titration is continued after adding methyl orange indicator.

• P (phenolphthalein alkalinity), mg/L= A * 1000 / ml of sample
• T (total alkalinity), mg/L= B * 1000 / ml of sample
• In case H₂SO₄ is not 0.02 N, then the following formula is applied
  Alkalinity, mg/L = A / B * N * 50000 / ml of sample
  Where,
  A = ml of H₂SO₄ required to change from pink to colourless with phenolphthalein indicator
  B = ml of H₂SO₄ required to change from yellow to pinkish orange with methyl orange indicator
  N = normality of H₂SO₄ used

Total hardness, Calcium hardness & Magnesium hardness

Water hardness is the traditional measure of the capacity of water to react with soap, hard water requiring a considerable amount of soap to lather. Hardness is generally caused by the calcium and magnesium ions (bivalent cations) present in water. The total hardness is defined as the sum of calcium and magnesium concentrations, both expressed as CaCO₃ in mg/L. Carbonates and bicarbonates of calcium and magnesium cause temporary hardness. Sulphates and chlorides cause permanent hardness.

Method: EDTA titrimetric method
Principle: In alkaline conditions EDTA (Ethylene-diamine tetra acetic acid) and its sodium salts react with cations forming a soluble chelated complex when added to a solution. If a small amount of dye such as Eriochrome black-T is added to an aqueous solution containing calcium and magnesium ions at alkaline pH of 10.0 ± 0.1, it forms wine red colour. When EDTA is added as a titrant, all the calcium and magnesium ions in the solution get complexed resulting in a sharp colour change from wine red to blue, marking the end point of the titration. Hardness of water prevents lather formation with soap rendering the water unsuitable for bathing and washing. It forms scales in boilers, making it unsuitable for industrial usage. At higher pH>12.0, Mg⁺⁺ ion precipitates with only Ca⁺⁺ in solution. At this pH, murexide indicator forms a pink color with Ca⁺⁺ ion. When EDTA is added, Ca⁺⁺ gets complexed resulting in a change from pink to purple indicating the end point of the reaction.

When EDTA (Ethylene-diamine tetra acetic acid) is added to the water containing calcium and magnesium, it combines first with calcium. Calcium can be determined directly with EDTA when pH is made sufficiently high such that the magnesium is largely precipitated as hydroxyl compound (by adding NaOH and iso-propyl alcohol). When murexide indicator is added to the solution containing calcium, all the calcium gets complexed by the EDTA at pH 12-13. The end point is indicated from a colour change from pink to purple. The difference between total hardness and calcium hardness is the magnesium hardness.
Apparatus:  conical flasks, measuring cylinder, burette, pipette, micropipette and analytical balance.

• EDTA solution 0.01 M: 3.723 g of disodium salt of EDTA is dissolved in distilled water and made up to 1000 ml.
• Buffer solution: 6.9 g of ammonium chloride and 1.25 g of magnesium salt of EDTA is dissolved in 143 ml of concentrated ammonium hydroxide and diluted to 250 ml with distilled water.
• Eriochrome black T indicator: 0.5 g of Eriochrome black-T indicator is dissolved in 100 g of triethanolamine.
• Sodium hydroxide 1 N (NaOH)
• Murexide indicator (ammonium purpurate):  0.2 g of murexide is ground well with 100 g of sodium chloride thoroughly.

• 1 ml each of buffer solution and inhibitor is added to 25 ml of the sample in a conical flask
• One pinch of EBT indicator is then added to this solution
• This is titrated against EDTA solution until the (wine red) pink colour changes to blue
• The amount of titrant used is noted.
• (Calcium hardness): 1 ml of NaOH and a pinch of murexide indicator is added to 25 ml of the sample in a conical flask
• This is titrated against EDTA solution until the dull pink colour changes to purple
• The amount of titrant used is noted.

Nitrates

Nitrates are the most oxidised forms of nitrogen and the end product of the aerobic decomposition of organic nitrogenous matter. Nitrogen along with phosphorus is termed as a bio stimulant. Nitrogen is an essential building block in the synthesis of protein. The evaluation of nitrogen is therefore of paramount importance in understanding the nutritional status of waterbodies.

Method: Phenyl disulphonic acid method
Principle: Nitrate reacts with phenol disulphonic acid to form a nitro derivative, which in an alkaline medium (liquid ammonia) develops a yellow colour. The concentration of NO₃ can be determined colorimetrically, since the colour so formed obeys the Beer’s law. (The concentration of the colour is directly proportional to the concentration of nitrates in the sample).
Apparatus:  porcelain crucibles, measuring cylinder, Nesslers tubes, standard flasks, pipette, micropipette, glass rods, spectro photometer and analytical balance.
Reagents:

• Phenol disulphonic acid
• Liquid ammonia, 30 %
• Standard nitrate solution: 0.7218 g of KNO₃ is dissolved in distilled water and made up to 1000 ml in a standard flask. This will be 100 mg N/L (100 ppm solution). From this an intermittent solution of 10 ppm is prepared by adding 25 ml of the stock standard and making it up to 250 ml in a standard flask with distilled water.

• 50 ml of sample is poured into the crucibles
• Standards of 0.1 – 0.5 mg N/L are also prepared and are poured into the crucibles
• These are kept in a steam water bath to evaporate
• The crucibles are cooled and the residue is dissolved in 2 ml of phenol disulphonic acid and the contents are diluted to 50 ml with distilled water in Nesslers tubes
• 6 ml of liquid ammonia is added to this which gives it a yellow colour
• The reading is then taken after thoroughly mixing the samples in a spectro photometer at 410 nanometer

Estimation: The standard curve between concentrations and absorbance for the standards is prepared. The concentration of nitrate nitrogen is calculated from the standard curve. The standard graph is plotted by taking concentration along X-axis and the spectrophotometric readings (absorbance) along Y-axis. The value of nitrate is found by comparing absorbance of sample with the standard curve and expressed in mg/L. The high concentration of nitrate in water is indicative of pollution.
Calculation: Nitrates  (mg/L) = Absorbance of sample  * Conc. of Std  * 1000 / Absorbance of Std. * Sample taken

 Phosphates

Phosphorus is essential to the growth of organisms and can be the nutrient that limits the primary productivity in water. Phosphorus occurs in natural waters and in wastewaters almost solely as phosphates.

Method: Stannous chloride and ammonium molybdate method
Principle: The phosphates in water react with ammonium molybdate and forms the complex molybdophosphoric acid, which gets reduced to a complex of blue colour in the presence of stannous chloride. The absorption of light by this blue colour can be measured at 690 nm to calculate the concentration of phosphates.

Apparatus: measuring cylinder, Nesslers tubes, standard flasks, pipette, micropipette, glass rods and analytical balance.
Reagents:

• Ammonium molybdate solution:
• 6.25 g of ammonium molybdate is dissolved in 44 ml of distilled water.
• 70 ml of concentrated sulphuric acid is slowly added to 100 ml of distilled water and cooled. Solutions i and  ii are mixed together. 
• Stannous chloride solution:  1.25 g of stannous chloride (SnCl₂) is mixed with 50 ml of glycerol by heating on a water bath for rapid dissolution.
• Standard phosphate solution: 0.2195 g of anhydrous potassium hydrogen phosphate (KH₂PO₄) is dissolved in distilled water and made up to 1000 ml. this gives us a 50 ppm solution. From this an intermittent solution of 5 ppm of 250 ml is prepared. From this intermittent solution standards of 0.1 – 0.5 concentrations are prepared

• 50 ml of samples are taken in Nesslers tubes along with one each of blank and the standard concentrations of 0.1 – 0.5 ppm
• To this 2 ml of ammonium molybdate is added followed by 5 drops of stannous chloride solution
• A blue colour will appear.

Estimation: Reading is taken at 690 nm on a spectrophotometer using distilled water as blank with the same amount of the chemicals. The readings need to be taken after 5 minutes but before 12 minutes of the addition of the last reagent and calibration curve is prepared. The concentration of phosphates in the sample is found with the help of the standard curve. A reagent blank is always run with same treatment with distilled water as sample. The value of phosphate is obtained by comparing absorbance of sample with the standard curve and expressed as mg/L.

Calculation: Phosphates  (mg/L) =
Absorbance of sample  * Conc. of Standard  * 1000 / Absorbance of Standard * Sample taken
High phosphorus content causes increased algal growth till nitrogen becomes limiting, although blue green algae continues to dominate because of its ability to utilise molecular nitrogen. Besides sedimentation, high uptake by phytoplankton is one of the reasons for the fast depletion of phosphorus in water.

Sodium

Sodium is one of the most abundant elements and is a common constituent of natural waters. The sodium concentration of water is of concern primarily when considering their solubility for agricultural uses or boiler feed water. The concentration ranges from very low in the surface waters and relatively high in deep groundwaters and highest in the marine waters. Sodium is present in a number of minerals, the principal one being rock salt (sodium chloride). The increased pollution of surface and groundwater during the past has resulted in a substantial increase in the sodium content of drinking water in many regions of the world.

Method: Flame photometric method
Principle: A characteristic light is produced due to excitation of electrons when the sample with sodium is sprayed into the flame. The intensity of this characteristic radiation is proportional to the concentration of sodium and can be read at 589 nm by using suitable filter devices. For determination of sodium, the samples need to be stored in polythene bottles to check the leaching of sodium from the glass.

Apparatus: Flame photometer, measuring cylinder, Nesslers tubes, standard flasks, pipette, glass rods and analytical balance
Reagents:

• Stock sodium solution: 0.2545 g of NaCl (anhydrous sodium chloride) is dissolved in distilled water and make up to 100 ml in a standard flask. This will be 100-ppm solution.
• Standard sodium solution: From the stock solution, standards of 10, 20, 40, 60-ppm concentrations of 50 ml solutions are prepared.

• Highly polluted samples and wastewaters are pretreated before determination of total sodium. For non-polluted samples where only sodium is to be determined, the sample needs to be filtered to avoid clogging of the capillary of the flame photometer.
• The concentration of sodium is determined using the flame photometer.
• The filter of the flame photometer is set to 589nm (marked for Sodium, Na). By feeding distilled water the scale is set to zero and maximum using the standard of highest value. A standard curve between concentration and emission is prepared by feeding the standard solutions. The sample is filtered through filter paper and fed into the flame photometer and the concentration is found from graph or by direct readings.
• Calibration curve is prepared in the ranges for the various standards of 10, 20, 40, 60  ppm and blank.
• If the sample is having higher concentrations of sodium, it can be diluted to come to in the range of determination and the dilution factor is taken into account during the estimation.

Estimation: The standard curve is a linear one at lower concentrations of sodium, however at higher concentrations it has got a tendency to level off. This curve is used to estimate the concentration of sodium in the sample.

 Potassium

Potassium ranks seventh among the elements in order of abundance, behaves similar to sodium and remains low. Though found in small quantities (<20mg/L) it plays a vital role in the metabolism.

Method: Flame photometric method
Principle:  Like sodium, potassium can also be determined accurately by flame photometer. The characteristic radiation for potassium is 768 nm, the intensity of which can be read on a scale by using a filter for this wavelength.
Apparatus: The estimation of potassium is based on the emission of spectroscopy. This is done using a flame photometer. Other apparatus include measuring cylinder, Nesslers tubes, standard flasks, pipette, glass rods and analytical balance.
Reagents:

• Stock potassium solution: Dissolve 0.1907 g of KCl (anhydrous potassium chloride) in distilled water and make up to 100 ml in a standard flask. This will be 100 ppm solution.
• Standard potassium solution: From the stock solution standards of different concentrations are prepared. Generally, 10, 20, 40, 60 ppm of 50 ml solutions are prepared.

• The concentration of potassium is determined using the flame photometer.
• The filter of the flame photometer is set at 768 nm (marked for Potassium, K) the flame is adjusted for blue colour. The scale is set to zero and maximum using the highest standard value. A standard curve of different concentrations is prepared by feeding the standard solutions. The sample is filtered through the filter paper and fed into the flame photometer.
• Calibration curve in the ranges of 10, 20, 40 and 60 ppm and for blank are prepared
• The concentration is found from the standard curve or as direct reading.
• If the sample is having higher concentrations of potassium, it can be diluted to come to in the range of determination and the dilution factor is taken into account during the estimation.

Estimation: The standard curve is used to estimate the concentration of potassium in the sample.

 COD

COD is the oxygen required by the organic substances in water to oxidise them by a strong chemical oxidant. This shows the oxygen equivalent of the organic substances in water that can be oxidised by a strong chemical oxidant such as potassium dichromate in acidic solution. The determination of COD values are of great importance where the BOD values cannot be determined accurately due to the presence of toxins and other such unfavourable conditions for growth of microorganisms the COD usually refers to the laboratory dichromate oxidation procedure.
Method: Open reflux method using potassium dichromate.
Principle: COD is the measure of oxygen consumed during the oxidation of the oxidisable organic matter by a strong oxidising agent. Potassium dichromate (K₂Cr₂O₇) in the presence of sulphuric acid is generally used as an oxidisng agent in the determination of COD. The sample is treated with potassium dichromate and sulphuric acid and titrated against ferrous ammonium sulphate (FAS) using ferroin as an indicator. The amount of K₂Cr₂O₇ used is proportional to the oxidisable organic mater present in the sample.
Apparatus: Conical flasks, measuring cylinder, Nesslers tubes, standard flasks, pipette, micropipette, titration burette, glass rods and analytical balance.
Reagents:

• FAS (ferrous ammonium sulphate or Mohr’s salt) 0.1 N: 8.07 g of ammonium ferrous sulphate is dissolved in a little distilled water, to which 5 ml conc. H₂SO₄ is added. This is allowed to cool and then diluted and made it up to 250 ml in a standard flask
• (K₂Cr₂O₇) Potassium dichromate 1 N (stock): 4.90 g of potassium dichromate, is dissolved in distilled water and made up to 100 ml in a standard flask
• Potassium dichromate 0.025 N: 25 ml of Potassium dichromate 1 N is taken and made up to 1000 ml with distilled water
• Ferroin indicator: 0.69 g of Ferrous sulphate and 1.4 g of phenonthroline monohydiate is dissolved in distilled water and made up to 100 ml.

• 0.1 N FAS is standardised and the normality value of the prepared reagent is calculated using the formula N (FAS) = V (K₂Cr₂O₇) * N (K₂Cr₂O₇) / V (FAS). The value thus got will be the normality of the FAS prepared and it is supposed to be closer to the normality required for the COD analyses, i.e. 0.1
• 10 ml of each sample and a blank is taken in conical flasks
• 10 ml K₂Cr₂O₇  is added to this
• 15 ml of conc. H₂SO₄ is added carefully from the sides of the flask, allowed to cool, digested for about 30 minutes.
• To this 50 ml distilled water and 5 drops of ferroin indicator is added
• This is titrated with FAS in a burette carefully till the orange turns bluish green to wine red (deeper wine red indicates high COD) and the amount of titrant consumed is noted

Estimation: COD value is calculated using the formula:
(Volume of titrant used in blank – volume of titrant used in sample) * N of FAS * 8 * 1000 / volume of sample taken.