Table of Contents

SL. No 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Method Description Measurement of pH for Water Estimation of Conductivity in Water Samples Estimation of Turbidity Estimation of Alkalinity Estimation of Total Suspended Solids Estimation of Dissolved Solids Estimation of Hardness Estimation of Calcium Estimation of Magnesium Estimation of Sodium Estimation of Potassium Estimation of Phosphate Estimation of Phosphate (Ortho or Dissolved) Calculation of Residual Sodium Carbonate, Sodium Absorption Ratio, Percent Sodium Estimation of Total Residual Chlorine Estimation of Chloride Estimation of Sulphate Estimation of Fluoride Estimation of Total Kjeldhal Nitrogen Estimation of Ammonical Nitrogen Calculation of Free Ammonia Estimation of Nitrate Estimation of Sulfide Estimation of Cyanide Estimation of Hexavalent Chromium Estimation of Biological Oxygen Demand Estimation of Chemical Oxygen Demand Estimation of Oil and Grease Estimation of Phenol in Water & Waste Waters Estimation of Boron Bioassay Test for Evaluating Acute Toxicity of Industrial Effluents and Waste Waters Using Common Carp Bioassay Test for Evaluating Acute Toxicity of Industrial Effluents and Waste Waters - Part 2 using Toxicity Factor to Zebra Fish Method Number SOPIWTD/01 SOPIWTD/02 SOPIWTD/03 SOPIWTD/04 SOPIWTD/OS SOPIWTD/06 SOPIWTD/07 SOPIWTD/08 SOPIWTD/09 SOPIWTD/10 SOPIWTD/11 SOPIWTD/12 SOPIWTD/13 SOPIWTD/14 SOPIWTD/15 SOPIWTD/16 SOPIWTD/17 SOPIWTD/18 SOPIWTD/20 SOPIWTD/19 SOPIWTD121 SOPIWTDI22 SOPIWTD124 SOPIWTD/23 SOPIWTD/25 SOPIWTD126 SOPIWTD127 SOPIWTD128 SOPIWTD/29 SOPIWTD/30 SOP/BTD/01 SOP/BTD/02 Revision 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

Determination of Total Coli forms and Fecal Coli Forms Determination of Total Coliforms and Fecal Coliforms (MPN Method) Methods for Gaseous Air Sampling Determination of Suspended Particulate Matter in Ambient Air Measurement of Respirable Suspended Particulate Matter (PM 10) in Ambient Air Determination of Sulphur Dioxide in Ambient Air Determination of Nitrogen Dioxide in Ambient Air Estimation of Lead in Ambient Air Determination of pH for Soil and Sludge Samples Estimation of Conductivity in soil and sludge samples. Determination of organic carbon. Determination of Total Water Soluble Solids Determination of Total Kjeldhal Nitrogen for soil and sludge samples Estimation of Potassium for soil and sludge samples Estimation of Total Organic Carbon

SOP/BTD/03 SOP/BTD/04 SOP/ATDI01 SOP/ATD/02 SOP/ATD/03 SO PlAT D/04 SOP/ATD/05 SOP/ATD/06 SOP/HWTD/01 SOP/HWTD/02 SOP/HWTD/03 SOP/HWTD/04 SOP/HWTD/05 SOP/HWTD/06 SOP/HWTD/07

01 01 01 01 01 01 01 01 01 01 01 01 01 01 00

Measurement of pH
1.0 Introduction: Measurement of pH is one of the most important and frequently used
tests in water chemistry. Practically every phase of waster supply and waste water treatment such as acid-base neutralization, water softening, precipitation, coagulation, disinfection and corrosion control depend on pH.

At a given temperature the intensity of the acidic or basic character of a

solution is indicated by pH or hydrogen ion activity. pH is defined by Sorenson as -log [H+]. It is the intensity factor of acidity.

2.0 Method: Electrometrical

3.0 Principle: The basic principle lies in determination of activity of hydrogen ions by potentiometric measurement using combination of glass and reference electrodes with temperature correction which responds selectively to hydrogen ions. Pure water dissociates to yield 10-7 moles/L of H+ at 25c.
H2O <=> H+ + HSince water dissociates to produce one OH- ion for each H+ ion, it is obvious that at 10-7 OH- ions are produced simultaneously. The product of [H+] and [OH-] always remains constant even if the value for one of the species changes. [H] x [OH] = 10-14 The large bracket sign, [ ], indicates molar concentration. Expression of the molar concentration of hydrogen ions is rather cumbersome because of the extremely small values and large variations. To overcome this difficulty, the concentration is expressed in terms of pH value, which is negative logarithm of the concentration in moles/L. pH = log [H+]

or

[H+]

10

-pH

The pH scale is usually represented from 0 to 14, with pH 7 at 25c representing neutrality. Acidic conditions increase as pH values decrease and alkaline conditions increase as pH values increase.

4.0Time Limit: pH Measurement for the sample must be done immediately in the
field itself or within 2 hours of collection in the laboratory.

5.0 Calibration of Instrument: 5.1. Prior to any use, calibrate the instrument daily. 5.2. Switch on the instrument and leave it for about 20 minutes to get stabilized. Calibration must be perfonned with atleast two buffer solutions, usually with pH 4. and pH 7 buffer solutions. 5.3. Follow the operation manual for detailed calibration of the instrument.

6.0 Reagents and Preparation

6.1. Commercial buffer solutions are supplied along with the instrument. Using these buffer solutions the instrument is calibrated. If these solutions are not available, buffer solutions are prepared from tablets, powder and concentrated solution forms. 6.2. These must be dissolved and made up to stated volume with distilled water. These buffer solutions should be prepared fresh as they may deteriorate as the result of mould growth or contamination and must be stored in polythene containers.
7.1. Before using instrument, remove electrodes from storage solution, rinse with distilled water and blot dry with soft tissue. 7.2. Calibrate the instrument with pH 4 and pH 7 buffer solutions. 7.3. Rinse the electrode with distilled water, shake off excess water. Immerse the electrode into the sample, wait till stable reading attained. Record the reading 7.4. Rinse the probe with distilled water between samples and before storing.

7.0 Procedure

8.0 Reference:
APHA, AWWA, WEF [For the Examination of WATER and Waste Water] standard methods Book 20th Edition 1995 Washington, DC. Hydrology Program Hydrology Project Training Module file: " 06 understanding Hydrogen Ion Concentration.doc" World Bank & Government of The Netherlands funded.

Measurement of Conductivity
1.0 Introduction: Conductivity is a measure of the ability of an aqueous solution to carry an
electric current. This ability depends on the presence of ions, on their total concentration, mobility and valence and on the temperature of measurement. Solutions of most inorganic compounds are relatively good conductors. Conversely molecules of organic compounds that do not dissociate in aqueous solution conduct current very poorly, if at all.

2.0 Method: Conductivity Meter.

3.0 Principle: Electrical conductivity (EC) of the sample indicates the concentration of
ionisable constituents of the solution and is measured using a conductivity meter.

4.0 Apparatus:
4.1. Conductivity meter 4.2. Beaker, 100 ml

5.0 Reagents: Standard KCL (0.01 M): Dissolve 0.7456 gm. KCI in distilled water and
make up to 1/iter. This solution has an electrical Conductivity of 1412 !-Is/em @ 25c.

6.0 Procedure:
6.1. Calibrate the Conductivity meter using standard 0.01 M KCI solution to 1412 !-Is/em. If the meter is not having temperature compensation; then it has to be calibrated to the conductivity value according to the table depending upon the temperature of KCI standard and the sample conductivity must be converted to 25C. 6.2. Take about 50 ml of water sample in a beaker. 6.3. Immerse the Conductivity cell in the solution and measure Conductivity as per the procedure given in the instrument manual.

7.0 Calculation: Report the Conductivity in !-Is/em.

8.0 Reference: . APHA, AVVWA, WEF [For the Examination of WATER and Waste water] standard methods Book 20th Edition 1995 Washington, DC.

Estimation of Turbidity
1.0

Introduction: Suspension of particles in water interfering the passage of light is called

Turbidity. Turbidity is caused by wide variety of suspended matter which range in size from colloidal to coarse dispersion depending upon the degree of turbulence. It also ranges from inorganic substance to those that are organic in nature. Turbidity is measured to evaluate the performance of water treatment plant.

2.0 3.0

Method: Nephelometric method. Principle: This method is based on a comparison of the intensity of light scattered by
the sample under defined conditions with the intensity of light scattered by a standard reference suspension under the same conditions. Higher the intensity of scattered light, higher the Turbidity.

4.0

Time Limit: 24 hours /48 hours

5.0

Interference: Turbidity can be determined for any water sample that is free of
debris and rapidly settling coarse sediment. Dirty glassware and the presence of air bubbles give false results.

6.0

Preservation: Analyze same day; store in dark up to 24 hours under

refrigeration.

7.0

Reagents and Preparation:

7.1

Solution - I: - Dissolve 1.000 gram of Hydrazine sulfate (NH2h H2SO4 in distilled water and dilute to 100 ml in a volumetric flask. Solution- II: - Dissolve 10 grams of Hexa-methylene tetra-amine (CH2)6 N4 in distilled water and dilute to 100 ml in a volumetric flask. Solution - III: - In a volumetric flask mix 5ml portions of solution I and II, allow to stand for 24 hours and dilute to 100 ml. This solution III will have turbidity of 400 NTU. Transfer stock suspension to an amber glass (or) other UV - light blocking bottle for storage. Make dilutions from this stock ~uspension. Standard Turbidity suspension: -Dilute 10 ml of solution III (400 NTU) to 100 ml in distilled water to have 40 NTU solution of turbidity.

7.2 7.3

7.4

8.0

Equipment:

Estimation of Alkalinity

1.0 Introduction: Alkalinity of water is its Acid-neutralizing capacity. It is the sum of the entire 8.1 Digital Nepheloturbidity meter. titratable alkaline groups. Alkalinity is significant in many uses and treatments of natural and 8.2 Light source: Tungsten - primarily a function of carbonate, bicarbonate and waste waters. Alkalinity of waters is filament lamp. 9.0 hydroxide content. It is taken as an indication of the concentration of these constituents.The Calibration: measured values also may include contributions from borates, phosphates, silicates or other basic ions if these are present. 9.1 Switch ON the instrument and allow warm up for 10 - 15 minutes. 9.2 9.3

2.0 Method: Titrimetric Method knobs. "COARSE and FINE"

Take distilled water in a cuvet and set the instrument to "zero" by using the Next take the standard turbidity suspension (40 NTU) and calibrate the

3.0 Principal: instrument to "40" digital display. Now a result of dissociation or hydrolysis NTU. Hydroxyl ions present in a sample as the instrument is calibrated for "40" of

solutes, react with additions of standard acid. Alkalinity thus depends on end point pH used. Take the sample in a cuvet and note down the direct reading. Report the reading 9.4 in Turbidity unit (NTU).

4.0 Time Limit: 24hrs If instrument reading is exceeding "40", the sample should be diluted 9.5 appropriately and the instrument reading should be multiplied by the dilution factor 5.0 Apparatus: to get the sample turbidity. Calculation. 9.6 Dosimeter 5.1 Turbidity in NTU = Instrument Reading x D

D = Sample 6.0 Reagents and Preparation dilution factor if applicable.

5.2 General Glass- wares

6.1 Sodium Carbonate Solution: Weigh 1.06 gms Sodium carbonate initially
10.0 Reference: APHA, AWNA, WEF [For the Examination of WATER and W~ste make up to the mark with distill water. This gives 0.02 N Na2C03 solution. Water] standard methods Book 20th Edition 1995 Washington, DC.

desiccated for about 3 hours .Transfer to a 1-lt volumetric flask, dissolve &

6.2 Standard stock sulfuric acid 0.1 N- Dilute 2.8ml of con H2SO4, make up to

1000 ml with distilled water. This solution gives 0.1 N H2SO4 6.3 Standard sulfuric acid 0.02N.Dilute 200ml of 0.1 N stock H2SO4 to 1000 ml with distilled water. Standardize against Na2C03 using methyl orange as indicator. End point is colour change to orange. 6.4 Methyl Orange Indicator: -0.5 grams Methyl orange powder in 1000ml of distilled water (CO2 free distilled water) 6.5 Phenolphthalein Indicator: - Dissolve 0.5 gms phenolphthalein in 50ml ethyl or Iso-propyl alcohol + 50ml distilled water.

7.0 Procedure 7.1 Phenolphthalein Alkalinity:

colour

7.1 . 1 To 25 ml or portion of the sample diluted to 25 ml taken in conical flask, add 2 to 3 drops of phenolphthalein indicator. If no pink appears, there is no phenolphthalein alkalinity. 7.1.2 If pink colour appears colourless (pH 8.3)
titrate with 0.02N H2SO4 until the solution becomes

7.2 Total Alkalinity: 7.2.1 Add 3 drops methyl orange indicator to the solution in which phenolphthalein alkalinity has been determined & continue titration against 0.02N H2SO4. (pH 4.5) till the colour changes from yellow to orange end point. 8.0 Calculation:

Phenolphthalein Alkalinity as CaC03, Mg/lt = A X N X 1000

Ml of sample taken

Total Alkalinity as CaC03, Mg/lt

= B X N X 1000

MI of sample taken

Where A = ml of H2SO4 consumed for first end point, B = ml of H2SO4 consumed till the second end point. N = Normality of H2SO4 9.0 Reference: APHA, A\NWA, WEF [For the Examination of WATER and Waste
Water] standard methods Book 20th Edition 1995 Washington, DC.

Estimation of Total Suspended Solids

1.0 Introduction: The solids refer to matter suspended or dissolved in water and
waste water. Solids may affect water or waste water quality adversely in a number of ways. Water high in suspended solids may be aesthetically unsatisfactory for such purposes as bathing. Suspended Solid analysis is important in the control of biological and physical waste water treatment processes and for assessing compliance with regulatory agency's effluent limitations. The total suspended solids are the portion of total solids retained by a filter of 2.0 Ilm (or smaller) pore size under specified conditions.

2.0 Method: Gravimetric method. 3.0 Principle: A well mixed sample is filtered through a weighed standard glass fiber
filter, dried to a constant weight at 103 to 105c. The increase in weight of the filter paper represents suspended solids. 4.0Time limit: Begin analysis as soon as possible. Refrigerate samples at 4c up to the time of analysis. Preferably do not hold samples more than 24 hrs and in no case more than 7 days.

5.0 Apparatus:
5.1. Glass fiber filter papers. 5.2. Filtration apparatus 5.3. Vacuum pump 5.4. Filtration Flask 5.5. Drying oven at 103 - 105C (Hot Air Oven) 5.6. Analytical balance

6.0 Procedure:
6.1. Condition the glass fiber filter papers in oven at 103 - 105C & weigh immediately before use. 6.2. Prepare filtration apparatus. Wash with distilled water before use. 6.3. Place pre-weighed filter paper on the filter holder. Transfer well mixed 50 ml or aliquot quantity of sample diluted to 50 ml to filtration unit & switch on the suction pump.

6.4. Confirm the complete filtration and wash the residue on the paper with another 50 ml portion of distilled water and remove the filter paper carefully with the help of a needle. 6.5. Place the filter paper for drying in oven at 103 - 105c for at least 1 hr. 6.6. After drying cool it, place in desiccator for constant temperature and weigh immediately. 7.0Calculation:
TSS mg/lt = (A-B) x 1000 x 1000 ml of sample taken Where, A = Final weight of filter paper in gms. B = Initial weight of filter paper in gms.

8.0 Method Performance: This method is suitable for the determination of suspended solids in surface water & industrial water

9.0 Reference: APHA, AWNA, WEF [For the Examination of WATER and Waste
Water] standard methods Book 20th Edition 1995 Washington, DC.

Estimation of Total Dissolved Solids

1.0
Introduction: The solids refer to matter suspended or dissolved in water or
wastewater. Solids may affect waste or effluent quality adversely in a number of ways. Water with high dissolved solids generally is non-potable and may induce an unfavorable physiological reaction in the transient consumer. For these reasons a limit of 500mg TDSII is desirable for drinking water. Dissolved solids is the portion of solids that passes through a filter of 2.0 \-1m (or smaller) pore size under specified conditions.

2.0
3.0

Method: Gravimetric method.

Principle: A well mixed sample is filtered through a standard glass-fiber filter

and the filtrate is evaporated to dryness in a pre-weighed dish & dried at 180c. The increase in dish weight represents the total dissolved solids.

4.0

Time limit: Begin analysis as soon as possible. Preferably do not hold samples more than 24 hrs and in no case more than 7 days. Refrigerate at 4c up to the time of analysis to minimize microbial decomposition of solids.

5.0

Apparatus:
5.1 Evaporating dishes. 5.2 Steam bath 5.3 Desiccators

5.4 Drying oven, 180 :I: 2c

5.5 Glass-fibre filter paper 5.6 Analytical Balance. 5.7 Filtration unit

6.0

Procedure:
6.1 Heat clean dish to 180 :I: 2c for 1 hour in an oven. Store in desiccators until needed. Weigh immediately before use. 6.2 Filter the aliquot quantity of well mixed sample diluted to 50 ml by applying suction. Filtered sample is transferred to pre-weighed dish. Wash the filtration assembly with 3 successive 10 ml volumes of reagent grade water allowing complete drainage between washings. Transfer all the filtrate portions to evaporation dish. 6.3 Evaporate to dryness on a steam bath. Dry evaporated sample at least for 1 hr in an oven at 180 :I: 2c. Remove the dish from oven, cool in a desiccator to balance temperature and weigh. Repeat this drying, cooling desiccating cycle until a constant weight is obtained.

7.0

Calculation: T.D.S mgll = (A-B) x 1000 x 1000 ml of sample Where A = Final weight of the dish with sample residue in gms B = Initial weight of the dish (empty dish) in gms

8.0

Method performance: This method is suitable for the determination of dissolved solids in potable, surface, domestic wastes and Industrial waste waters in the range up to 20,000 mg/Lt. Reference: APHA, AWWA, WEF [For the Examination of WATER and Waste Water] standard methods Book 20th Edition 1995 Washington, DC.

9.0

Estimation of Calcium Estimation of Total Hardness

1.0 Introduction: The hardness is defined as the sum of calcium and magnesium 1.0 Introduction: Total presence of calcium is from passage of water through or
over deposits both expressed as calcium carbonate in milligrams per liter. When concentrations,of limestone, dolomite, gypsum and gypriferrous and hale. Calcium contributes to the total greater than the sum of softening and bicarbonate hardness numerically ishardness of water. Chemical carbonate treatment, reverse osmosis, that amount of hardness equivalent to to total alkalinity alkalinity; electro dialysis or ion exchange is used the reduce calcium isand the called associated hardness"; "Carbonate hardness. the amount of hardness in excess of this is called "Non carbonate hardness".

2.0 Method: EDT A titrimetric method

3.0 Principle: When EDT A is Method. 2.0 Method: EDT A Titrimetric added to water

containing both calcium and magnesium, it combines first with the calcium. Calcium can be determined directly with EDT A when the 3.0 Principle: pH is made sufficiently high that the magnesium is largely precipitated as the hydroxide and an indicator is used that combines with calcium only.

4.0 Time Limit: of months metal cations. If a small amount of a dye such as Eriochrome solution 6 certain
Black- T is added to a solution containing calcium & magnesium, the solution becomes wine red. 5.0 Reagents and Preparation: 3.2. If EDT A added as a titrant, after calcium & magnesium ions have been complexed, the solution tums wine red to blue. Magnesium ion must be present 5.1.to yield a satisfactory end point. Preparation of EDTA solution (0.02N): Ethylene diaminetetraaceticaciddisodium salt (C1QH1Q011N2N02). Weigh 3.723gms of 4.0 Timeanalytical Months grade disodium ethylene diamine tetra acetic acid, dissolve in Limit: 6 reagent distilled water and dilute to 1000m!. Shake well in volumetric flask till salts dissolve 5.0 Reagents: giving clear solution. 5.2. 4% NaOH: Weigh 4 grams of Sodium Hydroxide salt and dissolve in 5.1. distilled water and dilute to 100m!. Ethylene di-amine tetra acetic acid. 5.3. Murexide Indicator: Murexide is the first indicator available for detecting 5.2. Erichrome Black - T the calcium end 5.3. Ammonia Buffer point. This indicator changes colour from pink to purple at the end point.

3.1. EDT A & its sodium salts form a chelated soluble complex when added to a

6.0 Preparation of Reagent: 6.0 Procedure:

6.1. EDTA: Weigh 3.723 grams of Ethylene di-amine tetra acetic acid di 6.1. sodium 50 ml or a in distilledportion& diluted to 50ml sample so that the Take salt, dissolve smaller water dilute t01000ml. calcium content T: O.5gms of Erichrome 2ml dye with 4.5gm of 6.2. Erichrome Black- is about 5 to 10mg. Add blackof 4% NaOH, 1 ml Propanol and pinch of Murexide indicator. hydroxylamine hydrochloride. Dissolve this mixture in 100ml of 95% of 6.2. isopropyl alcohol. against EDTA solution until the pink colour changes Titrate this solution to purple colour.

7.0 Procedure: 7.0 Calculation: 7.1. Take 50ml mgllt as CaC03 = to 50 ml of sample depending upon the Ca or portion diluted Burette readinq x 1000 concentration. Add 2ml of Ammonia buffer, thentaken or 2 drops of ml of sample add 1

Erichrome black- T indicator, titrate against EDTA (0.02N). The end point of the titration is pink to blue. 8.Q Reference: APHA, AVVWA, WEF [For the Examination of WATER and Waste 8.0 Calculation: WaJer] stanclF=1rd methods Book 20th Edition 1995 Washington, DC. Total Hardness Mg/lt as CaC03 = Burette Readinq X 1000 ml of sample

9.0 Reference: APHA, AWNA, WEF [For the Examination of WATER and Waste
Water] standard methods Book 20th Edition 1995 Washington, DC.

Estimation of Magnesium
1.0 Introduction: Magnesium is essential element in chlorophyll and in red blood cells.
It is used in alloys, pyrotechnics, flash photography, drying agents, refractories, fertilizers, phannaceutitals and foods. It is an important contributor to the hardness of water. Magnesium salts break down when heated causing scaling in boilers.

2.0 Method: Calculation Method

Estimation of Calcium and Hardness has to be conducted earlier. Magnesium may be estimated as the difference between hardness and calcium as CaC03. Mg mg/L as Mg = Total hardness - Calcium hardness X 0.243
(as CaC03mg/L) (as CaC03mg/L)

3.0 Reference: APHA, AWNA, WEF [For the Examination of Water and Waste
Water] Standard Methods Book 20th Edition 1995 Washington, DC.

Estimation of Sodium
1.0 Introduction: Sodium ranks sixth among the elements in order of abundance and is
present in most natural waters. The levels may vary from less than 1 mg NaIL to more than 500 mg NaIL. Soil permeability can be harmed by a high sodium ratio. The ratio of sodium to total cations is important in agriculture and human pathology.

2.0 Method: Flame photometric method 3.0 Principle: Trace amounts of sodium can be determined by direct reading type of flame
photometer. The sample is sprayed into a gas mixture flame and excitation is carried out under carefully controlled and reproducible conditions. The intensity of light is measured by a phototube. The intensity of light is proportional to the concentration of the element. The calibration curve may be linear but has a tendency to level off at higher concentration.

4.0 Apparatus:
4.1. Flame photometer 4.2. Glasswares. 5.0 Reagents 5.1. Stock sodium solution: Dissolve 2.542 g NaCI and dilute to 1000 ml with distilled water; 1.00 ml=1.00 mg of Sodium. 5.2 Standard sodium solutions: Prepare a series of working standard solutions in the range of 20, 40, 60, 80, and 100 mg/l by diluting 2,4,6,8, &10 ml of stock Sodium solution to 100 ml using distilled water. 6.0 Procedure: 6.1. Select Sodium filter with the help of filter selector of the burner unit of flame photometer. Ignite the burner and adjust the air supply pressure between 0.3 0.6 Kg/cm2 and gas supply so as to get blue cone flame in the burner. 6.2. Feed distilled water to the atomizer, wait at least for 30 seconds and adjust meter reading to Zero. 6.3. Run the standard solutions first adjusting the meter reading to 100 by using 100 mg/l standard solution. Feed 20, 40, 60 & 80 mg/l standard solutions, taking care to run distilled water between standard runs and ensuring meter shows zero reading. 6.4. Note the respective readings for 20,40, 60, & 80 mg/l standard solutions. 6.5. Run the filtered water samples, diluting if necessary with distilled water and note down both dilution factor and reading. 6.6. A calibration curve is plotted using the standard readings. 6.7. Sodium values for samples are calculated using the calibration curve

7.0Calculation: .
Direct reference to the calibration curve Total Sodium as mg of Na/L= Corresponding concentration from the graph x D Where: D= Dilution factor if applicable. 8.0 Reference:

. APHA, AWWA, WEF [For the Examination of WATER and Waste Water] standard
methods Book 20th Edition 1995 Washington, DC. . Flame Photometry Manual

Estimation of Potassium
1.0 Introduction: Potassium ranks seventh among the elements in order of abundance. It is
essential element in both plant and human nutrition and occurs in ground water as a result of mineral dissolution from decomposing plant materials, agriculture run off, solid waste etc. 7.0Calculation:

2.0 Method: Flame photometric method.

Potassium mgs/L= A x D

and reproducible conditions. The intensity of emitted light is measured by a phototube. The intensity of light is proportional to the concentration of the element. The calibration curve may be linear but has a tendency to level off at higher concentration. 8.0 Reference:

3.0Principle: = Concentration in ppm from direct reading type of flame photometer. The sample is Where: A Potassium is determined by the graph corresponding to instrument reading. sprayed D = Dilution factor if sampleanddiluted. into a air-fuel mixture flame is excitation is carried out under carefully controlled

. APHA, AWWA, WEF [For the Examination of WATER and Waste Water] standard
4.0 methods Book 20th Edition 1995 Washington, DC. . Apparatus:
4.1. Flame photometer 4.2. Glasswares

5.0 Reagents: 5.1. Stock potassium solution: Dissolve 1.91 gm KCI and dilute to 1000 ml with distilled water. This solution is; 1 ml = 1 mg of potassium. 5.2. Standard Potassium solutions: . Prepare a series of working standard solutions in the range of 20, 40, 60, 80, and 100 mg/L by diluting 2, 4, 6, 8, & 10 ml of stock Potassium solution to 100 ml using distilled water.

6.0 Procedure:
6.1. Select Potassium filter with the help of filter selector of the burner unit of flame photometer. Ignite the burner and adjust the air supply pressure between 0.3 - 0.6 Kg/cm2 and gas supply so as to get blue cone flame in the burner. 6.2. Feed distilled water to the atomizer; wait at least for 30 seconds and adjust Meter reading to Zero. 6.3. Run the highest concentration standard solution first adjusting the meter reading to 100 by using 100 mg/L standard solution. Feed 20, 40, 60, & 80 mg/L standard solutions, taking care to run distilled water between standard runs and ensuring meter shows zero reading. 6.4. Note the respective readings for 20, 40, 60, & 80 mg/L standard solutions. 6.5. Run the filtered water samples, diluting if necessary with distilled water and note down both dilution factor and reading. 6.6. A calibration curve is plotted using the standard readings. 6.7. Potassium values for samples are calculated using the calibration' curve.

Estimation of Phosphate (Total)

1.0 Introduction: Phosphate occurs in natural waters and in waste waters in various
forms. They are commonly classified as orthophosphates, condensed phosphates and organically bound phosphate. These various forms of phosphorous may occur in soluble form or in particulate form. Phosphorus is essential for the growth of organisms and can be the nutrient that limits the primary productivity of water body.

2.0 Method: Stannous Chloride Method 3.0 Principle: Molybdophosphoric acid is formed & reduced by stannous chloride to
intensely colored molybdenum blue. This method is more sensitive.

4.0 Time Limit: 48 hours

5.0 Interference: Positive interference is caused by silica & arsenate only if the
sample is heated. Negative interference is caused by arsenate, fluoride, thorium, bismuth, sulfide, thiosulphate, thiocynate etc. 6.0 Equipment: Digital Spectrophotometer.

7.0 Reagents: 7.1 Standard Phosphate: Dissolve 219.5 mgs anhydrous Potassium dihydrogen
ortho phosphate (KH2P04) in Distilled Water & dilute to 1000 ml. 1 ml = 50.0 I-Ig pO-l-p 7.2 Ammonium Molybdate: Dissolve 25gms (NH4)6 Mo? 024 4H20 in 175 ml distilled water. Then add 280 ml of H2SO4, 400ml distilled water, cool and dilute to 1000ml. 7.3 Stannous Chloride: Dissolve 2.5 gms of fresh [SnCI2.2H2O] stannous chloride in 100 ml Glycerol. Heat on a water bath & stir with glass rod to hasten dissolution. This reagent is stable for 6 months. 7.4 Strong Acid Reagent: Add 300 ml cone. H2SO4 to 600 ml distilled water. Add 4 ml cone. HN03, cool, dilute to 1000 ml. 7.5 Sodium hydroxide (3 N): Dissolve 12.0 gms NaOH and dilute to 100 ml.

cator

8.0 Sample pretreatment: Take 50 ml or portion diluted to 50 ml of well mixed

sample in a conical flask. Add one drop of Phenolphthalein indicator. If red colour appears, add sulphuric acid solution drop wise to just discharge the

colour. Then add 1 ml sulphuric acid solution in excess. Boil gently for at least 90 minutes. Cool and neutralize with Sodium hydroxide solution. Filter if necessary and make up the volume to 50 ml. Now proceed as described in the next step.

9.0 Procedure:
9.1 Take 50 ml or portion diluted to 50 ml of pretreated sample in 50 ml Nessler's tube, add 2ml of ammonium molybdate reagent and mix thoroughly. 9.2 Add 0.2 ml of stannous chloride (approximately 5 drops) reagent. 9.3 Prepare reagent blank by taking 50 ml 0 W along with reagents. 9.4 Prepare series of standards by pi petting 0.2, 0.4, 0.6, 0.8 and 1.0 ml of standard phosphate solution and made up to 50 ml. These contain 0.2, 0.4, 0.6, 0.8, and 1.0 ppm phosphate-P respectively. Add appropriate volumes of reagents. 9.5 Measure the intensity of blue color in absorbance mode at 690nm, for standards as well as samples, by setting the spectrophotometer to "ZERO" absorbance with blank. 9.6 Take readings with in 10 to 12 minutes. 9.7 Plot the graph of concentration in ppm against 00. Find out concentration factor from the graph.

10.0 Calculation: Mgllt POd -P = Sample 00 x F x 0 Where, "F" is concentration factor from the graph. "0" is dilution factor if portion of sample is diluted to 50 ml.

Reference: APHA, A WWA, WEF [For the Examination of WATER and Waste Water]
standard methods Book 19th Edition 1995 Washington, DC.

Estimation of Phosphate (Ortho OR Dissolved)

1.0 Introduction: Phosphate occurs in natural waters and in waste waters in various
forms. They are commonly classified as Orthophosphates, condensed phosphates and organically bound phosphate. These various forms of phosphorous may occur in soluble form or in particulate form. Phosphorus is essential for the growth of organisms and can be the nutrient that limits the primary productivity of water body.

2.0 Method: Stannous Chloride Method. 3.0 Principle: Molybdo phosphoric acid is formed & reduced by stannous
chloride to intensely colored molybdenum blue. This method is more sensitive.

4.0 Time Limit: 48 hours 5.0 Interference: Positive interference is caused by silica & arsenate only if the
sample is heated. Negative interference is caused by arsenic, fluoride, thorium, bismuth, sulfide, thiosulfate etc. 6.0 Equipment: Digital Spectrophotometer.

7.0 Reagents:
7.1. Standard Phosphate: 7.2. Ammonium Molybdate 7.3. Stannous chloride. 8.0 Preparation of Reagents:
8.1.

Standard Phosphate: Dissolve 219.5 mgs anhydrous Potassium dihydrogen ortho

phosphate (KH2P04) in Distilled Water & dilute to 1000 ml. 1 ml = 50.0 I-Ig P04-3 -Po 8.2. Ammonium Molybdate: Dissolve 25 gms (NH4)6Mo7024 4H2O in 175ml distilled water. Then add 280 ml of H2SO4, 400ml distilled water, cool and dilute to 1000ml. 8.3. Stannous Chloride: Dissolve 2.5 gms of fresh [SnCh.2H20] stannous chloride in 100 ml glycerol. Heat on a water bath & stir with glass rod to hasten dissolution. This reagent is stable for 6 months.

9.0 Procedure: 9.1. Take 50 ml or portion diluted to 50 ml of filtered sample in 50 ml Nessler's tube, add 2ml of ammonium molybdate reagent and mix thoroughly. 9.2. Add 0.2 ml of stannous chloride (approximately 5 drops) reagent. 9.3. Prepare reagent blank by taking 50 ml 0 W along with reagents. 9.4. Prepare series of standards by pipetting 0.2, 0.4, 0.6, 0.8 and 1.0 ml of standard phosphate solution and made up to 50 ml. These contain 0.2, OA, 0.6, 0.8, and 1.0 ppm phosphate-P respectively. Add appropriate volumes of reagents. 9.5. Measure the intensity of blue color in absorbance mode at 690nm, for standards as well as samples, by setting the spectrophotometer to "ZERO" absorbance with blank. 9.6. Take readings with in 10 to 12 minutes. 9.7. Plot the graph of concentration in ppm against 00. Find out concentration factor from the graph. 10.0 Calculation:

Mg/lt P04-P = Sample 00 x F x 0 Where, "F" is concentration factor from the graph. "0" is dilution factor if portion of sample is diluted to 50 ml.
11.0 Reference: APHA, AWWA, WEF [For the Examination of WATER and Waste Water] standard methods Book 19th Edition 1995 Washington, DC.

4.4. DETAILS of Residual Sodium Carbonate (RSC), CalculationOF IONS/MOLECULES CONCERNED Sodium Absorption Ratio (SAR) & Percent Sodium At. wtIMole. wt EQ.wt. (% Ions/molecules Na) Valency
Alkalinity CaC03 2 100 50 Total Hardness as CaC03 50 1.0 Introduction: These are secondary 2 calculations 100 based on the results of some Calcium as Ca 2 20 primary parameters applicable to effluents used for 40 irrigation purpose. If the values are exceeding the stipulated standard limits, they 24 may have adverse affects on MaQnesium as MQ 2 12 crops. Sodium as Na 1 23 23 39 39 1 Potassium as K 2.0 Method: Calculation. 3.04.5. EXAMPLE: A sample on analysis found to contain the parameters as Procedure: Estimations for Alkalinity, Total hardness, Calcium, Sodium and Potassium have to be done prior to proceeding for calculations. below.

4.0 Calculation: Alkalinity

- 240 mgs/L as CaC03 Total Hardness - 200 mgs/L as CaC03 4.1. BASIC PARAMETERSmgs/L as CaC03 Calcium - 160 REQUIRED Magnesium - 40 mgs/L as CaC03 4.1.1. Alkalinity ( in m.eqs/L ) as as Na Sodium - 90 mgs/L CaC03 4.1.2. Total Hardness (mgs/L as K as CaC03 Potassium - 20 in m.eqs/L) 4.1.3. Calcium ( in m.eqs/L) as Ca 4.1.4. Magnesium ( in m.eqs/L) as Mg (A) R S C: 4.1.5. Sodium ( in m.eqs/L) 4.1.6. Potassium ( in m.eqs/L) The basic formula is, Magnesium concentration is obtained from subtracting Calcium from = Alkalinity - Total Hardness Total hardness Residual Sodium and converting in to m.eqs/L. Carobonate in m.eqs/L in m.eqs/L in m.eqs/L On 4.2. EQUIVALENT WEIGHT: Atomic or molecular weight of any ion/molecule substitutingby its valency gives its equivalent weight. divided the values from the example, it becomes,

4.3. MILLI EQUIVALENTS: Milligrams concentration of any molecule/ion RSC = 40 - 20 divided by its Equivalent weight gives its concentration in milli 50 50 equivalents. Take out common divisor. ie. m.eqs/L = mQs/L

Eq.wt. RSC = (240 - 200) x 1/50 = 40 x 0.02 = 0.8 m.eqs/L

(B) S A R: The basic formula is, Sodium Absorption Ratio = Na [Ca + mg]1/2
2

All in m.eqs/L.
.

Substituting the values from the example: 90/23

SA R =

( 160 x 0.4 ) + ( 40 x 0.24 ) 20 12 2

~ q1

L (160XO.02)+(?40XO.02) J
= 3.91

112

r
=

( 160 + 4_0) x 0.02

J '"

3.91/1.41 = 2.77

Sodium Absorption Ratio is a ratio. Hence it has no unit.

IC) % SODIUM: The basic formula is, % Sodium = Na x 100 All in m.eqs/L Na + K + Ca + Mg Substituting the values from above example, %Na = 3.91 x 100 3.91 + 0.51 + 4

= 391 8.42 = 46.44 % NOTE: "Ions combine in terms of their equivalent weights."

Estimation of Total Residual Chlorine

1.0Introduction: Chlorine applied to water in its molecular or hypochlorite form initially
undergoes hydrolysis to form free chlorine consisting of aqueous molecular chlorine, hypochlorous acid and hypochlorite ion. These few Chlorine forms are pH and temperature dependant. Free Chlorine reacts readily with Ammonia and certain nitrogenous compounds to form combined chlorine. The Chlorination of water supplies and polluted waters serves primarily to destroy or deactivate disease producing micro-organisms. A secondary benefit particularly in treating drinking water is the overall improvement in water quality resulting from the reaction of chlorine with ammonia, iron, manganese and some organic substances.

2.0 Method: lodometric Method

3.0 Principle: Chlorine liberates free Iodine from Potassium Iodide solution at pH 8 or
less. The liberated iodine is titrated with standard solution of Sodium thiosulfate with starch as indicator. Titrate at pH 3-4 because the reaction is not Stoichiometric at neutral pH due to partial oxidation of thiosulphate to sulphate.

4.0Time Limit: Analyze the samples immediately. 5.0 Reagents and Preparation:
5.1. Acetic acid concentrated (Glacial) 5.2. Potassium Iodide (KI) crystals.

5.3. Standard Sodium thiosulphate 0.025N: Dissolve 6.205gms

Na2S203.5H2O in freshly boiled distilled water and dilute to 1 L or prepare it from ampoule provided. Standardize against Potassium dichromate.

5.4. Starch Indicator Solution: To 5gms Starch, add little cold water and make
paste. Pour it in to 1 L of boiling distilled water, stir and let settle overnight. Use clear supernatant. 5.5. Standard Potassium di-chromate (0.025N): Dissolve 1.226gms of anhydrous Potassium Dichromate of primary standard quality in distilled water and dilute to 1000m!.

5.6.Standardization of Sodium thiosulphate: Take 10ml potassium

dichromate solution in a conical flask. Add 30ml distilled water and 10ml

concentrated H2S04.Then add about 1 gm of potassium iodide and leave it for some time for complete liberation of iodine. Titrate against sodium thiosulphate using starch as indicator. Adjust the volume of sodium thiosulphate according to the titration result if required, to have correct normality.

6.0 Procedure:
6.1. Volume of sample: Select sample volume that will require not more than 10ml and not less than 0.2ml of 0.025N Sodium thiosulphate for starch
.

end point.
6.2. Preparation for titration: Place 5ml glacial acetic acid in a flask; add about 1 gm Potassium iodide estimated on a spatula. Pour sample into the conical flask and mix with stirring. Selection of sample volume is dependent on Chlorine concentration. 6.3. Titration: Titrate away from direct sun light. Add 0.025N Sodium thio sulphate from burette until yellow colour of liberated iodine is almost discharged. Add 1 ml starch solution and titrate until blue colour is discharged. End point is blue to colourless.

7.0 Calculation:
mgs Cb/L = Titre Readinq x N x 35450 ml of sample taken
Where N = Normality of Sodium thiosulphate

8.0 Reference: APHA, A'MNA, WEF ~For the Examination of WATER and Waste
Water] standard methods Book 20 h Edition 1995 Washington, DC.

Estimation of Chloride
1.0 Introduction: Chloride in the form of chloride ion (Cr) is one of the major inorganic
anions in water and waste water. The chloride concentration is higher in waste water than in raw water because sodium chloride (NaCI) is a common article of diet and passes unchanged through the digestive system. High chloride content may harm metallic pipes and structures as well as growing plants.

2.0 Method: Argentometric Method

3.0 Principle: In Neutral or slightly alkaline solution, Potassium chromate can indicate
the end point of the silver nitrate titration with chloride. Silver chloride is precipitated quantitatively before red silver chromate is formed.

4.0 Time limit: Preservation is not required; maximum storage recommended up

to 28 days.

5.0 Apparatus:
5.1. Erlenmeyer flask 5.2. Burette 5.3. General glass wares

6.0 Reagents and preparation

6.1. Standard Silver Nitrate titrant: 0.0282 N. 6.2. Standard Sodium Chloride: 0.0282 N. 6.3. Potassium Chromate indicator. 6.4. Calcium Carbonate powder. 6.5. Aluminum hydroxide suspension: AI(OHh 6:6. Preparation of Standard Silver Nitrate titrant( 0.0282 N): Dissolve 4.791 gm Silver nitrate in distilled water and make up to 1000 ml in volumetric flask. Store in an amber bottle. 1 ml of 0.0282 N AgN03 = 1 mg Chloride. 6.7. Standardization of Silver Nitrate titrant (0.028 N): Standardize AgN03 against 0.0282 N NaCI using K2Cr04 indicator. 6.8. Preparation of Standard Sodium Chloride (0.0282 N): Dissolve 1.648 gm of NaCI in distilled water and make up to 1000 ml in volumetric flask. 1 ml = 1 mg CI. 6.9. Potassium Chromate Indicator Solution: Dissolve 50 mg of K2Cr04 in distilled water and add AgN03 solution until definite red precipitate is formed. Let the solution stand for 12 hours, filter and dilute to 1 litre with distilled water in volumetric flask.

6.10. Preparation of Aluminum hydroxide suspension: Dissolve 125 gms Aluminum potassium sulfate or Aluminum ammonium sulfate in 1 L distilled water. Warm to 60c & add 55 ml of concentrated Ammonium hydroxide slowly with stirring. Let stand about 1 hr, transfer to a large bottle and wash precipitate by successive addition with thorough mixing and decanting with distilled water until free from chloride.

7.0 Procedure: 7.1. Sample Preparation: Take 50 ml or suitable portion of sample diluted to
50 ml with distilled water. If the samples are highly coloured, remove. colour with Charcoal or AI(OHh

7.2. Titration: Directly titrate samples in the pH range 7.0 t9 10.0. Adjust sample pH between 7 and 10 with CaC03 powder when the sample is acidic. Add 3 to 5 drops K2Cr04 indicator solution. Titrate with Standard AgN03 titrant to brick red end point.

8.0 Calculation: Mgs Cr/L =

Burette readinq X 1000 ml. of sample taken.

9.0 Method Performance: The Argentometric method is suitable for use in relatively clear waters when 0.152 to 10 mgs cr are present in the portion titrated. 10.0 Reference: APHA, AWWA, WEF [For the Examination of WATER and Waste Water] Standard Methods Book 20th Edition 1995 Washington, DC

Estimation of Sulphate
1.0 Introduction: Sulphate is widely distributed in nature and may be present in natural
waters in concentrations ranging from few to a thousand milligrams per liter. Mining drainage wastes may contribute large amounts of sulphate through pyrite oxidation. In the presence of organic matter certain bacteria may reduce sulphate to sulphur. To avoid this, heavily polluted or contaminated samples must be stored at 4C.

2.0 Method: Turbidimetric method.

3.0 Principle: 3.1. Sulfate ion (S04-2) is precipitated in an acetic acid medium with barium chloride (BaCI2) so as to form Barium sulfate (BaS04) crystals of uniform size. 3.2. Turbidity is the measure of the scattering of light in all directions by undissolved substances. Nephelometers determine the ratio of the intensity of scattered light at right angles to the main light path. This scattered light intensity is proportional to the concentration of the

suspension. .

4.0 Time: 28 days maximum storage time with refrigeration.

5.0 Equipment and Calibration:

5.1. Equipment application: Digital Nephelo-turbidity meter is used to
measure the turbidity caused by the precipitation of Barium sulphate.

5.2. Calibration: Prior to any use calibrate the instrument daily. 5.3. Switch on the instrument and leave for 10-20 minutes forwarm up.
5.4. Set zero with distilled water or reagent blank. 5.5. Calibrate the instrument with standard sulfate to 40 or 100 units (division).

6.0 Apparatus:
6.1. Nephelo turbidity meter 6.2. Magnetic stirrer 6.3. Glass wares.

7.0 Reagents and preparation: 7.1. Barium chloride mesh. 7.2. Acetate buffer: Dissolve 30 g Magnesium chloride (MgCI2.6H2O), 5 g Sodium acetate (CH3COONa.3H20), 1.0 g Potassium nitrate( KN03) and 20 ml of Acetic acid glacial (CH3COOH) (99%) in 500 ml distilled water and make up to 1000 ml. 7.3. Standard sulfate solution: Dissolve 0.1479 gm anhydrous Na2S04 in distilled water and dilute to 100 ml. This solution gives 1 ml = 1 mg of S04-2 7.4. Standard working sulfate solution for calibration: Take 2 ml standard sulfate (from 1 ml = 1 mg S04-2 solution) and make up to 50 ml, add 10 ml acetate buffer and 10 to 15 mesh BaCb. This solution gives 40 ppm S04-2 standard. Using this standard, calibrate the instrument to 40 or 100 (divisions) units.

8.0 Procedure: 8.1. Sample preparation: Take 10 ml of acetate buffer and 10 to 15 mesh BaCI2 crystals and add aliquot quantity of sample. 8.2. While adding sample, obseNe the formation of turbidity or precipitation and compare with standard. The final volume should be made to 60 ml in case sample volume is less than 50 ml. 8.3. Mix vigorously or with the help of magnetic stirrer just before introducing the standard or sample in to the instrument. 8.4. Measurement of barium sulfate turbidity: After stirring period had ended, pour solution in to sample container of Nephlometer and measure turbidity. 9.0 Calculation When the instrument is calibrated to 40 (divisions) units, S04-2 mgll = Instrument reading x sample dilution factor. When the instrument is calibrated to .100 (divisions) units, S04-2 mg/l = instrument reading x sample dilution factor x 0.4 10.0 Method performance: The turbidimetric method is applicable in the range of 1 to 40 mg S04-2 I It. The minimum detectable concentration is approximately 1 mg S04-2 II. 11.0 Reference: APHA, A WWA, WEF [For the Examination of WATER and Waste Water] standard methods Book 20th Edition 1995 Washington, DC.

Estimation of Fluoride
1.0.lntroduction: Fluoride may occur naturally in water or it may be added in
controlled amounts. A fluoride concentration of approximately 1.0 mg/L in drinking water effectively reduces dental carries without harmful effects on health.

2.0.Method: SPADNS colorimetric method. 3.0.Principle: The SPADNS colorimetric method is based on the reaction between
Fluoride and Zirconium- SPADNS dye. Fluoride reacts with the dye lake, dissociating a portion of it into a colorless complex ion (ZVF62-) and the dye. As the amount of Fluoride increases, the solution color becomes progressively lighter.

4.0.Time Limit: 28 days. 5.0.Equipment : Digital Spectrophotometer at 570 nm wave length.

6.0. Reagents and Preparation: 6.1. Zirconium Solution (Zirconyl Chloride): Dissolve 0.266gms Zirconyl chloride octohydrate (zrocb. 8H20) in 50ml water, add 700ml of cmc HCI and dilute to One liter. 6.2. SPADNS Solution: 1.916 gms SPADNS reagent dissolved in distilled 6.3. Zirconyl-SPADNS reagent: Mix equal volumes of SPADNS solution and Zirconyl Solution (Zirconyl Chloride). The combined reagent is stable for at least 2 years. 6.4. Standard Fluoride solution: Dissolve 0.0221gms of anhydrous NaF in 100ml distilled water. This gives 100 ppm Fluoride solution. 6.5. Sodium Arsenite solution: Dissolve 5.0 gms NaAs02 and dilute to One liter with distilled water.

water and made up to One liter (1 OOOml).

7.0 Preparation of Blank and Standards:

7.1 Take 25ml of D Wand add 5ml of Zirconyl-SPADNS reagent. This becomes blank for further calibration of the instrument. 7.2. Pipette out 0.1 ml, 0.2 ml, 0.3 ml, and 0.4 ml from stock standard of 100 ppm into 25 ml volumetric flasks, make up to mark with D W. These give

series of Fluoride standards of 0.4, 0.8, 1.2, and 1.6 ppm. To each of these add 5 ml Zirconyl-SPAONS reagent. 8.0 Calibration: 8.1. Switch on the instrument and leave it for 15 minutes to warm up. 8.2. Set the instrument to 570 nm wave length in Absorbance mode. 8.3. Adjust the instrument to "ZERO" Absorbance with OW. 8.4. Leave the first cuvette within the light path, fill the second with "BLANK" solution and set the instrument to 0.5 00. Then read absorbance for standards in increasing order of their concentration and note down the respective ODs. Here the abs. values are in decreasing order compared to their respective concentrations. 8.5. Similarly read abs. for samples prepared by taking 25 ml or portion diluted to 25 ml and adding 5 ml reagent. 8.6.Subtract abs. value for each standard from Blank abs. ( i.e. 0.5). Plot the graph of concentration against 00 (Blank 00 - STO 00) and find out concentration FACTOR from the graph. Colored and turbid samples should be distilled in acidic condition before adding reagent. If the sample contains residual chlorine, remove it by adding few drops of NaAs02 (Sodium arsenite of 1300 mg/l).

9.0 Calculations: Fluoride mgs/L = (Blank 0 0 - Sample 00) x F x 0

Where, F = Concentration factor from the graph. 0 = Dilution factor for sample if applicable.
10.0Reference: APHA, AWWA, WEF [For the Examination of WATER and Waste Water] standard methods Book 19th Edition 1995 Washington, DC.

Estimation of Total Kjeldahl Nitrogen-N

1.0Introduction: Total Kjeldahl nitrogen is the sum of ammonia nitrogen and organic
nitrogen which are converted to ammonium sulfate during the digestion process. Organic nitrogen is the difference obtained by subtracting the ammonia nitrogen value from the total Kjeldahl nitrogen value.

2.0 Method: Kjeldhal digestion Method.

3.0 Principle: Total Kjeldhal Nitrogen is the sum of Organic Nitrogen and Ammonical
nitrogen. This does not include Nitrate Nitrogen & Nitrite Nitrogen. In the presence of H2SO4, potassium sulfate (K2S04) and cupric sulfate (CUS04) catalyst, amino nitrogen of many organic materials is converted to ammonium. Free ammonia is also converted to ammonium. After addition of base, the ammonia is distilled from an Alkaline medium and absorbed in boric acid. The ammonia may be determined calorimetrically or by titration with a standard mineral acid.

4.0Time Limit: 7days minimum /28 days maximum

5.0Apparatus:
5.1. Kjeldhal Flask 5.2. Heating device with temp range of 375 - 380c. 5.3. Fume hood 5.4. Distillation set with ground glass joints. 5.5. Spiral condenser with ground glass joints. 5.6. General glass-wares.

6.0Reagents and Preparation: Prepare all reagents and dilutions in

ammonia-free water. 6.1 Copper Sulphate crystals (CUS04) 6.2 Potassium Sulphate crystals (K2S04) 6.3 Absorbent Solution:

add dilute to 1 litre. 6.4Mixed Indicator: Dissolve 200 mg methyl red indicator in 100 ml 95% ethyl or Isopropyl alcohol. Dissolve 100 mg methylene blue in 50 ml 95% ethyl or Isopropyl alcohol. Combine solutions. Prepare monthly. 6.5 Methyl red indicator: Dissolve 50mg methyl red in 100ml ethyl alcohol. 6.6 Sodium hydroxide (6 N): Dissolve 24 gms NaOH in OW and make up to 100 ml.

Boric Acid Solution(Absorbent Solution} - Dissolve 20 gms of H3B03 in water and

7.0Procedure: 7.1 Digestion: Take suitable volume of well mixed sample in kjeldhal flask. Add 2 ml Cone. H2SO4 and add pinch of CUS04 and K2S04 and keep on hot plate. Digest it around 380c till the solution appears clear to ensure complete decomposition/destruction of organic matter. . Remove the flask from digestion chamber and allow to cool. 7.2 Distillation: Take digested sample, add NaOH(6N) to raise pH above 11 and' make up to 200 ml by adding distilled water. Immediately place the flask in its proper position in distillation apparatus and turn on heat. Distill and collect the distillate in 10 ml Boric acid. Extend the tip of the condenser well below the level of Boric acid solution. Collect about 100 ml distillate, remove the flask containing distillate first and then put off heat to avoid back suction. Measure the distillate collected and note down. Titrate the distillate with 0.02 N H2SO4 using Mixed indicator. End point shows dark green to pale lavender color. Carry a blank through all above steps, with distilled water.

8.0 Calculations:
Total Kjeldahl Nitrogen-N mg /It = (A-B) X 280 ml of sample taken A = ml of 0.02 N H2So4 required for Sample. B = ml of 0.02 N H2So4 required for blank If Normality of H2SO4 is not 0.02 N, then use, Total Kjeldahl Nitrogen-N mg /It = (A-B) N X 14 X 1000 ml of sample

6.0 Reference:

- APHA, A WWA, WEF (For the Examination of WATER and WASTE WATER) Standard Methods Book 19th Edition 1995, Washington, DC.
38

Estimation of Ammonical Nitrogen

1.0

Introduction: The Nitrogen present in saline constituents is called Ammonical Nitrogen.

Ammonia is produced by the microbiological activity on organic nitrogenous matter. It appears in ground as well as surface waters.

2.0
3.0

Method: Titrimetric Method.(NH3-N concentration greater than 5 mk/L) Principle: The sample is buffered to pH 9.5 with a Borate Buffer to decrease hydrolysis
of cyanates and organic Nitrogen compounds. It is distilled in to a solution of Boric acid and titrated against standard sulfuric acid.

4.0 5.0
6.0

Time Iimit:7d /28 d Preservation: Analyze as soon as possible or add H2SO4 to pH < 2, Refrigerate. Apparatus: 6.1 Distillation apparatus 6.2 General Glass Wares Reagents and Preparation: 7.1 7.2

7.0

- Prepare all Reagents & dilutions in Ammonia free water.

H2S04(1 N) : Take 28 ml of conc. H2SO4 and make up to 1 liter by adding distilled water. This gives 1 N solution. Sodium Hydroxide Solution (1 N): Dissolve 40 gms. of Sodium hydroxide in distilled water and make up to 1000 ml. Absorbent Solution: Boric acid solution: - Dissolve 20 grams of H3B03 in water and add 10 ml of mixed indicator, dilute to 1 Liter. Mixed Indicator: Dissolve 200 mg methyl red indictor in 100 ml 95% Ethyl (or) Isopropyl alcohol. Dissolve 100 mg Methylene Blue in 50 ml 95% Ethyl (or) Isopropyl alcohol and mix both the solutions. Standard Sulphuric acid solution - 0.02 N: Dilute 20 ml volume of 1 N H2SO4 to 1000ml with distilled water. Standardize it against 0.02N sodium carbonate (Na2C03) using Methyl orange as indicator. At the end point a faint orange color will appear. Sodium hydroxide 6 N: Dissolve 24 gms in OW and make up to 100 ml.

7.3

7.4

7.5

7.6

8 Procedure: 8.5 Distillation: 8.5.1 Take suitable volume of sample in distillation flask and dilute to 200 ml. Neutralize it if necessary by using 1 N NaOH or H2SO4. Add 10 ml borate buffer and adjust the pH to 9.5 using 6 N NaOH. 8.5.2 Place the flask in its proper position in distillation apparatus and turn on heating. 8.5.3 Distill at the rate of 6 to 10 ml/min and collect the distillate in 10 ml Boric acid. Extend the tip of the condenser well below the level of Boric acid solution. 8.5.4 Collect about 100 ml distillate and remove the flask containing distillate first and then put of heating to avoid back suction. 8.5.5 Measure the distillate collected and note down the volume. Titrate the distillate with 0.02N H2SO4 using mixed Indicator. End point shows dark Green to pale Lavender Color. 8.5.6 Carry a Blank through all above steps. 9 Calculations: Mg NH3-N IL = (A - B ) x 280 MI sample A = ml of 0.02N H2SO4 required for sample. B = ml of 0.02N H2SO4 required for blank. If Normality of H2SO4 is not 0.02N, then use, Mg NH3-N IL = ( A - B ) N x 14 x 1000 MI of sample 10 Reference: APHA, AWNA, WEF [For the Examination of WATER and Waste Water] standard methods Book 19th Edition 1995 Washington, DC

3.1. The free ammonia concentration present in the water can be calculated by noting the temperature, pH and total ammonical nitrogen of the 1.0 Introduction: Ammonia is an important factor to be considered for water quality effluent criteria in industrial effluents, especially so in case of the fertiliser water. industry. It is a known fact that ammonia has a harmful effect on marine life. 3.2. The estimation of free ammonia by this type of calculation may not be The ecosystem of the receiving water may get upset due to the presence of strictly accurate in view of the fact that the equilibrium constants vary ammonia.' It acts as a nutrient to algae and bacteria causing eutrification. On the with the total ionic impurities present in the water. However, except with other hand, it is toxic to fish and other organisms. It may increase the oxygen the use of ion-selective electrode, there is no other chemical means demand and may cause depletion of dissolved oxygen. possible to determine free ammonia without disturbing the chemical Ammonia released into the water takes one of two forms, (1 )Free ammonia (unequilibrium. ionised ammonia, chemical symbol NH3). This form of ammonia is highly toxic to Mole percentage ammonia, chemical and bound ammOnia fish. (2) Ammonium (ionisedof free ammonia symbol NH/). This form of ammonia pH 350 C is virtually non-toxic to fish. 250 C NH40H NH/ 0.009 99.991 5.0 NH40H NH/ 2.0 Method:5.5 Calculation using 99.991 estimated values of0.030 99.970 Ammonical Nitrogen. 0.000

Calculation:

Calculation of Free Ammonia

3.0 Principle: Whenever NH3 is present in water the following equilibria exist:

0.081 99.919 -6.0 0.019 99.981 0.255 99.745 6.5 0.057 99.943 0.180 7.0 99.820 0.566 NH3(g) + H2O ~ NH40H (1)99.434 0.810 99.190 2.607 ......(1) 97.393 7.680 92.532 7.5 1.766 OH 98.234 5.384 NH40H NH4 + + ....... 20.406 79.594 44.660 8.0 + (ionized) 94.616 (unionized) ......(2) 8.5 free bound 15.250 84.750 36.286 55.340 71.841 28.159 63.734 64.224 35.776 88.975 11.025 96.231 9.0 Besides this, water molecules also03.769 98.776 01.224 ionize resulting in the following 85.053 14.947 9.5 equilibrium: 99.610 00.390 99.876 94.732 5.268 98.273 10.0 00.124 10.5 (3) H2O ~ 1.727 99.448 0.552 H+ +OH 99.825 0.175 11.0 The11.5 equilibria (2) and (3) are governed by the constants as follows: 12.0 Kb = (NH/l rOH-l

-.

The values of mole percentage of free ammonia are calculated at various pH and temperature [OH-l and given in Table for use. Kw = [H+] values (5) Free Ammonia mg/L as Nitrogen = corresponding free ammonia x Estimated ammonical concentration from the chart nitrogen concentration
Taking into consideration (4) and (5):
1

[NH3] [H2O]

(4)

= rNH4OH] = K H~ (6) [Bound ammonia] Eg: If Sample Ammonical[NH/] Nitrogen is [H+] mg/L at pH 7 and 25C, 1.5 Ko
-""""

r Free ammonia

100

The mole fraction of free ammonia, therefore, depends on pH and the equilibrium constants which are temperature dependent.

then

7.0 Procedure:

Estimation of Boron

1.0 Introduction: 4 ml of curcumin reagent to it. for plant growth. But in excess of 2.0 and add Boron is an essential element

7.1. Pipette 1.0ml or portion diluted to 1.0 ml sample in to an evaporating dish

mg/L in blank, instead of it is deleterious 1 ml distilled water with 4ml of 7.2. For irrigation water, sample, pipette to certain plants and some plants may be affected adversely by concentration as low as 1.0 mg/L (or even less in commercial curcumin reagent. green houses). 0.2, 0.4, 0.6, 0.8, and containportions of 1 ppm Boron inandfive 7.3. Pipette out Drinking water rarely 1.0 ml more than 1 mg/L Boron to generally less evaporating dishes and 4 ml curcumin reagent to each. for human consumption. than 0.1 mg/L. concentration is considered innocuous Boron mayall the dishes inin some waters or may find its way :tinto atemperature 7.4. Float occur naturally water bath for evaporation at 55 2C water course through80cleaning After drying cool to room temperature. for minutes. compounds and industrial effluents. Sea water contains approximately 5 add 10 ml of 95% and this element to each dish and stir estuaries in 7.5. Now mg of Boron /L isopropyl alcohol is found in saline thoroughly association withthe residue and make up with isopropyl alcohol in of Boron can affect to dissolve other seawater salts. Ingestion of large amount 25 ml the central nervous system and may result in a clinical syndrome known as BORISM volumetric flak. If the final solution is turbid filter through filter paper

before taking absorbance readings. 7.6. Switch on Spectrophotometer and allow warm up time. Set it to 540 nm wave length and adjust it to "ZERO" absorbance using Blank. 2.0 7.7. Read absorbance readings for standards in their increasing order of Method: Curcumin Method concentration and also for samples. 3.0 7.8. Plot graph of ppm concentration of Boron against 00 of seriesof curcumin under Principle: Boron is acidified and evaporated in the presence of controlled conditions. A concentration factor called Rosocyamine is formed. This red standards. Find out red colored product from the graph. color intensity varies with concentration of boron present in sample & is compared with standards photometrically.

4.0 Time Limit: 28 days /6 Months.

Boron mgs/L = 00 x F x 0 5.0 Equipment: Digital Spectrophotometer at 540 nm wave length. Where, 6.0 Reagents: "F" is concentration factor from the graph. "0" is dilution factor for sample portion diluted to 1 ml before evaporation. 6.1. Stock Born Solution: Dissolve 571.6 mg anhydrous Boric acid (H3B03), in distilled water and dilute to 1000 ml; 1.00.m\ = 100 ~g Boron. 6.2. StandardAPHA, AWWA, WEF [For the Examination of WATER and Waste 9.0 Reference: Boron Solution: Dilute 1.00 ml stock Boron solution to 100 ml in distilled water, 1.00 ml = 1.00 I-Ig Boron. Water] standard methods Book 20th Edition 1995 Washington, DC. 6.3. Curcumin Reagent: Dissolve 0.04 gm of curcumin & 5.0gm of oxalic acid in 80ml 95% ethyl or isopropyl alcohol and add 4.2 ml of cone. Hydrochloric acid and makeup to 100ml with ethyl alcohol in a 100ml volumetric flask. Filter if reagent is turbid. This reagent is stable for several days if stored in a refrigerator. 6.4. Ethyl or Isopropyl alcohol 95%.

8.0Calculation:

Determination of Nitrate-Nitrogen
1.0
Introduction: Synthetic fertilizer wastes contain enormous quantities of nitrates. Nitrates are the end products of the aerobic stabilization of organic nitrogen.

2.0
3.0

Method: Phenol di-sulphonic acid method: Principle: The basic reaction between nitrates and 1,2,4 Phenol-disulphonic acid
produces 6-nitro 1,2,4 Phenol-di-sulphonic acid which upon conversion to alkaline salt yields yellow coloured solution.

4.0 5.0

Time Limit: 48 hrs (28 days for Chlorinated sample)

Reagents & Preparation: 5.1 Phenol-di-sulphonic acid: Mix 25 gms of Phenol crystals in 225 ml of concentrated H2SO4 and heat for 2 hrs on a water bath. 5.2 50% NaOH: Dissolve 50 gms of Sodium hydroxide pelts in distilled water and make up to 100 ml. 5.3 Nitrate Standard: Dissolve 0.722 gms of anhydrous Potassium nitrate and dilute to 1000 ml. 1 ml = 100 I-Ig N.

6.0

Procedure:
6.1 Take suitable quantity of well mixed sample in glass dish & evaporate on water bath till the complete evaporation of water. Add 2ml of Phenol-di-sulphonic acid and ensure complete dissolution of the residue. 6.2 Add 50% NaOH with caution till the red litmus changes to blue (alkaline), filter & make up to 50ml in Nessler's tube. 6.3 Prepare a blank by treating 50 ml OW in the same manner as sample. 6.4 Prepare a series of standards containing 0.4, 0.8, 1.2, 1.6, and 2.0 ppm Nitrate-Nitrogen in final volume, by pipetting 0.2, 0.4, 0.6, 0.8, and 1.0 ml volumes from the standard and treating them in the same way as blank and samples. 6.5 Read absorbance at 450 nm wave length for standards and samples by adjusting instrument "ZERO" with blank. 6.6 Plot the graph of concentration against 00 and find out concentration factor. 7.0 Calculation mg/lt Nitrate as N = Sample OD x F x D Where, F = Concentration factor from the graph D = Dilution factor if applicable 8.0 Reference: APHA, AWWA, WEF [t=or the Examination of WATER and Waste Water] standard methods.

Estimation of Sulfide
1.0 Introduction: Sulfide often is present in ground water especially in hot springs. Its common
presence in waste water comes from the decomposition of organic matter and mostly from the bacterial reduction of sulfate. The hydrogen sulfide (H2S) escaping into the air from sulfide containing waste water causes odour nuisances. Gaseous H2S is very toxic to humans as it interferes with olfactory system. Dissolved H2S is toxic to fishes and other aquatic organisms. It attacks metals directly and indirectly causing serious corrosion of concrete sewers because it is oxidized biologically to sulphuric acid (H2SO4) on pipe wall.

2.0 Method: lodometric method.

3.0 Principle: Iodine oxidizes sulfides in acid solution. Iodine in excess quantity to that of expected sulfide content in a given sample in acidic condition is made to react with sulfide. Remaining quantity of Iodine is estimated against standard sodium thiosulfate using starch as indicator. Sulfide content is calculated on the basis of Iodine consumed.

4.0 Time Limit: Determine in the field or add 4 m] of 2N Zinc acetate preservative/100
ml sample & add NaOH to pH > 9 & refrigerate. The maximum holding time is 28 d.

5.0 Reagents & Preparation:

5.1 Zinc acetate solution 2N: Dissolve 220g Zinc acetate (ZnCC2H302h 2H20) in

870 ml distilled water. Shake it thoroughly and make up to 1 litre. 5.2 Standard iodine solution (0.025N): Dissolve 25g potassium iodide in distilled water and add 3.175g iodine to it. Make up to 1 L after all the iodine is dissolved in volumetric flask. Standardise against 0.025N sodium thiosulfate solution using starch as indicator. 5.3 Sodium thiosulfate titrant (0.025N): Dissolve 6.205g of Na2S203.5H2O with OAgms NaOH panets in distilled water and dilute to 1 litre. Standardize against. K2Cr207. (5ml of K2Cr207 + 50 ml distilled water +1 Oml H2SO4 + 1 gm of KI & leave some time for the liberation of iodine. Titrate against Na2S203 solution using starch as indicator). 5.4 Hydrochloric acid (6 N): (Normality of concentrated HCI is 12) Volume (ml) of conc reagent to prepare 1 litre of 6N HCI solution is 500 (1 + 1) i. e., 500 ml conc HCI + 500 ml distilled water. 5.5 Starch Indicator: Dissolve 1 gm of soluble laboratory starch in 100 ml distilled water. Add 0.1 g salicylic acid for preservation

6.0 Procedure: 6.1 Take 50 ml (or Estimation ofsample in conical flask. suitable quantity) of Cyanide 6.2Add 2ml Cyanide refers to an the (this step should be avoided for samples 1.0 Introduction:of Zinc acetate solution, CN- groups in cyanide compounds that preserved in the field),cyanideitions (CN} can be determined as shake thoroughly. 6.3 Leave the solution forrepresented by the formula A(CN)x where 'A' is an alkali or few minutes for precipitate formation. 1.1 Simple cyanides are

2.0

6.4 Filter the sample using glass fiber filter the number of CN- groups. Complex a metal and 'x' the valence of 'A', is paper. 6.5Transfer the pptaalong withformulae. cyanides have variety of filter paper to iodine flask. Add about 50 ml of distilled water. 1.2 The great toxicity of molecular HCN to aquatic life is well known. It is 6.6Add 10 ml 0.025N iodine solution + 2 ml of 6N HC!. Shake it water. formed in solutions of cyanide by hydrolytic reaction of CN- with vigorously. If iodine colour disappears add more iodine until colour persists. 6.7Titrate against standard sodium thiosulfate (0.025N) solution using starch as Method: indicator. End point is blue to colourless. Note down the burette reading. 2.1 Cyanide is determined by titrimetric, colorimetric or byblank without sample, using 6.8 Follow the above steps in the procedure for cyanide-ion electrodewater. Iodine solution volume must be the same for both blank and distilled methods. 2.2 The titration method is suitable for cyanide concentration above 1 mg/L. 2.3 The sample.
colorimetric method is suitable for cyanide concentrations to a lower

limit of 5 1-1911. Analyze higher concentration by diluting either the sample 7.0 Calculation: before distillation or absorbed solution before colorimetric measurement. 1 ml of 0.025N iodine solution is equal to 0.4 mg S-2
2.4 lon-selective electrode method using the cyanide ion-electrode is applicable in concentration range of 0.05 to 10 mgll. S-2 mgs/L = (A-B) x N x 16000 ml of sample taken

3.0

Where,

3.1 Most Cyanides are very reactive and unstable. Analysis of samples must be done as soon as possible. If samples cannot be analyzed immediately, A add NaOH pellets for blank = Burette reading or a strong NaOH solution to raise sample pH to 12B 12.5, add reading for sample = Burette dechlorinating agent if sample is disinfected and store in a N= Normality of Sodium thiosulfate solution closed, dark bottle in a cool place.

Principle:

8.0 Method Performance: The iodometric method is suitable for analyzing samples freshly Preliminary treatment of samples will vary according to interfering substances 3.2
taken from wellsThese are removed byfor wastewater and partly oxidized water. In & springs and used distillation. present. this method, sulfide concentration above 1 mg/l can be estimated.
Water] standard methods Book 20th Edition 1995 Washington, DC. 4.1 NaOH solution: Dissolve 40 gms NaOH in water and dilute to 1 L. 4.2 Magnesium chloride reagent: Dissolve 510 gms. Mgcb.6H20 in water and dilute to 1 L. 4.3 Sulphuric acid (H2SO4) 1+1:

4.0 Reagents for distillation: 9.0 Reference: APHA, AWWA, WEF [For the ~xamination of WATER and Waste

5.0

Procedure for Distillation:

5.1 Take 100 ml sample and dilute to 200 ml in a distillation flask. Add 10 ml of 1:1 H2SO4 to bring down the pH < 2.0 and add 10 ml of magnesium chloride reagent. Start heating and collect 100 ml distillate in a beaker containing 10 ml NaOH solution.

5.2

HCN is liberated from acidified sample by distillation. HCN gas is absorbed by passing it through NaOH solution. CN- concentration in NaOH solution is determined by any of the following methods.
Colourimetric Method: Principle: 6.1.1 Cyanide in the alkaline distillate from preliminary distillation is converted to CNCI by reaction with Cholramine-T at pH< 8 without hydrolyzing to CNO. After the reaction is complete, CNCI develops red-blue color on addition of pyridine-barbituric acid reagent. Maximum absorbance in aqueous solution is observed between 575 to 582 nm.

6.0 6.1

6.1.2 Apparatus: Spectrophotometer for use at 578 nm, providing a light path of 10 mm or longer. 6.2

Reagents & preparation:

6.2.1 Chloramine-T-solution: Dissolve 1.0 gm white, soluble powder in 100 ml water. Prepare weekly and store in refrigerator. 6.2.2 Acetate Buffer: Dissolve 410 gm Sodium acetate trihydrate in 500 mIDW. Add glacial acetic acid to adjust pH to 4.5. 6.2.3 Pyridine Barbituric acid reagent: Take 15 gms of Barbituric acid in 250 ml volumetric flask, add just enough water to wash sides of flask and wet Barbituric acid. Add 75ml pyridine and mix. Add 15 ml concentrated HCI, mix and cool to room temperature. Dilute to volume and mix until Barbituric acid is dissolved. Solution is stable for approximately 6 months if stored in amber bottle under refrigeration. Discard if precipitation develops. 6.2.4 NaOH(dilution solution): Dissolve 1.6 gm NaOH in 1 L distilled water.

6.3

Procedure:
6.3.1 Pipette a portion of distillate (absorbed solution) into 50 ml volumetric flask and dilute to 50 ml with NaOH dilution solution. 6.3.2 Add 1 ml acetate buffer, 2 ml chloramine-T solution, stopper and mix by inversion twice. Let stand exactly 2 min. 6.3.3 Add 5 ml Pyridine-barbituric acid reagent, mix thoroughly. Let stand exactly for 8 mins. 6.3.4 Prepare series of standard cyanide solutions of 50 ml each in the range from 0.04 to 0.20 ppm. 6.3.5 Process the standards and a 50 ml NaOH blank as samples for colour development. Measure absorbance of standards and

5.2

HCN is liberated from acidified sample by distillation. HCN gas is absorbed by passing it through NaOH solution. CN- concentration in NaOH solution is determined by any of the following methods.

6.0
6.1

Colourimetric Method:
Principle: 6.1.1 Cyanide in the alkaline distillate from preliminary distillation is converted to CNCI by reaction with Cholramine-T at pH< 8 without hydrolyzing to CNO. After the reaction is complete, CNCI develops red-blue color on addition of pyridine-barbituric acid reagent. Maximum absorbance in aqueous solution is observed between 575 to 582 nm. 6.1.2 Apparatus: Spectrophotometer for use at 578 nm, providing a light path of 10 mm or longer.

6.2

Reagents & preparation:

6.2.1 Chloramine-T-solution: Dissolve 1.0 gm white, soluble powder in 100 ml water. Prepare weekly and store in refrigerator. 6.2.2 Acetate Buffer: Dissolve 410 gm Sodium acetate trihydrate in 500 ml DW. Add glacial acetic acid to adjust pH to 4.5. 6.2.3 Pyridine Barbituric acid reagent: Take 15 gms of Barbituric acid in 250 ml volumetric flask, add just enough water to wash sides of flask and wet Barbituric acid. Add 75ml pyridine and mix. Add 15 ml concentrated HCI, mix and cool to room temperature. Dilute to volume and mix until Barbituric acid is dissolved. Solution is stable for approximately 6 months if stored in amber bottle under refrigeration. Discard if precipitation develops. 6.2.4 NaOH(dilution solution): Dissolve 1.6 gm NaOH in 1 L distilled water.

6.3

Procedure: 6.3.1 Pipette a portion of distillate (absorbed solution) into 50 ml volumetric flask and dilute to 50 ml with NaOH dilution solution. 6.3.2 Add 1 ml acetate buffer, 2 ml chloramine-T solution, stopper and mix by inversion twice. Let stand exactly 2 min. 6.3.3 Add 5 ml Pyridine-barbituric acid reagent, mix thoroughly. Let stand exactly for 8 mins. 6.3.4 Prepare series of standard cyanide solutions of 50 ml each in the range from 0.04 to 0.20 ppm. 6.3.5 Process the standards and a 50 ml NaOH blank as samples for colour development. Measure absorbance of standards and samples at 578 nm with instrument adjusted to "zero" with reagents blank. 6.3.6 Plot graph of concentration against absorbance and find concentration factor in ppm.

6.4

Calculations
CN- mgs/L = Sample 00 x F x C 0 Where, F = Concentration factor in ppm from the graph. C = Volume distillate collected. 0 = Volume of distillate processed.

7.0

TITRIMETRIC METHOD 7.1 Principle: 7.1.1 CN- in the alkaline distillate from the preliminary treatment procedure is titrated with standard AgN03 to form soluble cyanide complex, NaAg(CNh.

7.1.2 As soon as all CN has been complexed and a small excess of Ag+ has been added, the excess Ag+ is detected by silver sensitive indicator "p-dimethylaminobenzalrhodanine" which immediately turns from a yellow to a salmon-colour. The indicator is sensitive to about 0.1 mg Ag+ II.
7.2 Reagents: 7.2.1 Indicator solution: Dissolve 20 mg p-dimethyl- amino-benzal rhodanine in 100 ml acetone. 7.2.2 Standard silver nitrate titrant: Dissolve 3.27 9 AgN03 (0.0192N) in 1 L D.W. Standardize against standard NaCI solution, using argentometric method with K2Cr04 indicator. Dilute 500 ml AgN03 solution according to the titer value so that 1.00 ml is equivalent t01.00 mg CN-. 7.2.3 NaOH solution: Dissolve 1.6 9 NaOH in 1 liter Distilled Water. Procedure: 7.3.1 From the distillate (absorbed solution), take a measured volume so that titration will require between 1 to 10 ml AgN03 titrant. 7.3.2 Dilute to 100 ml using NaOH dilution or to some other convenient volume to be used for all titrations. For samples with low cyanide concentrations (~ 5 mg/L), do not dilute. Add 0.2mL indicator solution. 7.3.3 Titrate with standard AgN03 titrant to the first change in color from canary yellow to a salmon hue. Titrate a blank containing the same amount of alkali and water.

7.3

'

7.4

Calculation:

xCD Mg CN IL = (A-B) x 1000 m\ original sample Where, A = ml standard AgN03 for sample B = ml standard AgN03 for blank C = ml vol. of distillate collected. D = ml vol. of distillate used for titration.
8.0 Reference: APHA, AWWA, WEF [For the Examination of WATER and Waste Water] standard methods Book 20th Edition 1995 Washington, DC.

Determination of Hexavalent Chromium

1.0 Introduction: Chromium is used in alloys, electroplating and in pigments. Chromate
compounds are added to cooling waters for corrosion control. Chromium exists in water both as trivalent and hexavalent state though trivalent form rarely occurs in potable water. It is considered as non-essential to plants but an essential trace element in animals. Hexavalent compounds have been shown to be carcinogenic by inhalation and are corrosive to.tissue. Th.e chromium guidelines for natural water are linked to the hardness or alkalinity of water ( i.e. the softer the water, the lower the permitted level for chromium).

2.0 Method: Diphenyl Carbazide

3.0 Principle: Soluble Hexavalent Chromium (ct6) in the absence of interfering metals is determined colorimetrically by reaction with Diphenyl Carbazide.

4.0 Reagents:

4.1. Sulphuric Acid (H2SO4) 1 + 1 4.2. Ortho-phosphoric Acid 85% 4.3. Ammonia Solution 4.4. Diphenyl Carbazide: Dissolve 0.25 gms of Diphenyl Carbazide in 50 ml Acetone. Store in a amber glass in a cool and dark place. 4.5. Standard Chromium Stock Solution: Dissolve 0.14 gms potassium dichromate in 1000 ml distilled water. 1 ml of this solution contains 50jJg of Chromium.

5.0 Procedure: This method has linearity up to 0.5 ppm of Chromium. Prepare series of
standards of 50 ml volume having concentrations 0.1, 0.2, 0.3, 0.4 and 0.5 ppm ct6 along with blank with OW. Add 1 ml of dilute H2SO4 (1 +1) and 0.3 ml of Orthophosphoric acid and 1 ml of Diphenyl Carbazide Solutions to each with proper mixing after each addition. Allow to stand for about 5 minutes for colour development.Take an aliquot quantity of sample containing 0.1 - 0.5 ppm of chromium made up to 50 ml. Filter the sample 'If necessary. Add Ammonia or dilute H2SO4 and make it neutral. Treat it in the same manner as standards and allow to stand for colour development, (Pink colour). Measure the optical density at 540 nm wavelength using blank for instrument "ZERO" setting. Plot the graph of ppm concentration against 00 and find out concentration factor from the graph. 6.0 Calculation:

ct6 in mg/L = Sample 00 x F x 0 Where: F= Concentration factor from the graph in ppm. 0 = Sample dilution factor if applicable.

7.0 Reference: APHA, AWWA, WEF [For the Examination of WATER and Waste

Water] Standard Methods Book 20th Edition 1995 Washington, DC.

Estimation of Biological Oxygen Demand

1.0 Introduction: The Biological Oxygen Demand (BOD) is an empirical test in which
standardized laboratory procedures are used to determine the relative oxygen requirements of water and waste water. A number of factors may affect the accuracy and precision of BOD measurements. For instance soluble and floatable solids, oxidation of reduced ions and sulfur compounds, lack of mixing etc. the BOD test is dependent of dissolved oxygen (DO) available in the incubated sample. Therefore it is necessary to dilute the sample before incubation, to bring the oxygen demand and supply in to appropriate balance.

2.0 Method: Winkler method: The basic principle of Winkler's method is estimation of dissolved oxygen uptake at 27C temperature under incubation for 3 days. It has been modified to remove Nitrate interference by adding sodium azide along with Alkali-Iodide.
3.0 Principal: 3.1. The method consists of filling with samples to overflowing, airtight bottles of 300 ml size and incubating for 3 days at 2rC temperature. 3.2. Dissolved oxygen is measured initially and after incubation, and the BOD is computed from the difference between initial and final DO. 3.3. When the manganous sulfate is added to the solution containing sodium or potassium hydroxide, manganous hydroxide is formed which is oxidized by the dissolved oxygen of the sample to basic manganic oxi- hydroxide. 3.4. On addition of concentrated H2SO4 the basic manganic oxi-hydroxide forms manganic sulfate which further reacts with Iodide liberating Iodine equivalent to that of DO originally present in the sample. 3.5. The liberated iodine is titrated with standard solution of sodium thiosulfate using starch indicator. MnS04 + 2 NaOH ~ Mn(OHb+Na2S04 (White colored ppt)

2Mn(OHb + 02 (Dissolved Oxygen)

. 2 MnO (OHh (Basic maganic Oxi-hydroxide) (Brown colored ppt)

MnO(OH)2 + 2H2S04 Mn(S04b + 2Nal 2Na2S203 + b

.. Mn (S04b + 3H20 Manganic sulfate .. MnS04 + Na2S04 + 12

.. Na2S406 + 2Nal

4.0 Time limit: Start analysis within 6 h~urs of collection. When the sampling site is
distant from the lab, store at or below 4 C and start analysis with in 24 hours.

5.0 Apparatus:
5.1.300ml (BOD) bottles 5.2. Pipettes, measuring cylinders and conical flasks 5.3. Incubators with specified temperature.

6.0 Reagents and preparation:

6.1. Reagents for dilution water: 6.1.1.1. Phosphate buffer solution: Dissolve 8.5 9 KH2P04, 21.75 9 K2HP04, 33.4 9 N~2HP04 7H20 and 1.7 9 NH4CI in about 500ml 01 water and dilute to 1 It. The pH should be 7.2 without any further adjustment. 6.1.1.2. Magnesium sulfate solution: Dissolve 22.5 9 MgS04.7H20 in distilled water and dilute t01 It. 6.1.1.3. Calcium Chloride solution: Dissolve 27.5g CaCh in distilled water and dilute to 1 It. 6.1.1.4. Ferric Chloride solution: Dissolve 0.25g FeCb.6H20 in distilled water and dilute to 1 It. Note: If biological growth is noticed in any of the above reagents during storage, discard and prepare freshly.

6.2. Preparation Of dilution water: 6.2.1.1. Aerate required volume of water with supply of clean compressed air in a suitable container and add 1 ml each of phosphate buffer, MgS04 soln, CaCb solution and FeCb solution per one liter aerated water and mix thoroughly. Prepare this dilution water just before use.
6.3. Preparation

of Reagents for determination of DO :

6.3.1.1. Manganous sulfate solution: Dissolve 480g MnS04. 4H20 Or 400 9 MnS04. 2H2O or 364 9 MnS04. H2O in distilled water, filter and dilute to 1 It. This solution should not give color with starch when added to an acidified Potassium iodide (KI) solution. 6.3.1.2. Alkali - iodide - azide solution: Dissolve 500 gm of NaOH (or 700 gm KOH) and 135 gm Nal (or 150gms KI) in distilled water and dilute to 11t. Add 10 gms Sodium azide (NaN3) dissolved in 40 ml distilled water. This reagent should not give color with starch when diluted and acidified. 6.3.1.3. Concentrated H2SO4 6.3.1.4. Starch: Dissolve 2 gms laboratory grade starch powder and 0.2gm salicylic acid as a preservative in 100ml hot distilled water. 6.3.1.5. Standard sodium thiosulfate 0.025N: Dissolve 6.205 gms Na2S203. 5H2O in distilled water. Add OA gms NaOH pallet and dilute to 1000m!. Standardize against standard K2Cr207. 6.3.1.6. Standardization: 5 ml of std. K2Cr207 + 50ml 01 water + 10ml H2SO4 + 1 gm of KI and leave some time to liberate Iodine. Titrate against Na2S203 solution using starch as indicator

7.0 Seeding: Seeding is the addition of small measured volume water containing a good
bacterial population or micro organisms to the dilution water. For samples collected from untreated industrial wastes, disinfected wastes, high temp wastes or wastes with extreme pH value and chlorinated water, seed the dilution water by adding a population of micro organism~ by adding 2ml of supernatant from domestic waste water per 1000 ml dilution water.

8.0 Procedure:
8.1. Take aliquot sample in duplicate in 300ml BOD bottles and fill with dilution water. Incubate one bottle at 2?oC for 3days. 8.2. To the other bottle, add 2ml of MnS04 solution followed by the addition of 2 ml of alkali-Iodide azide solution. 8.3. Stopper carefully to exclude air bubbles and mix by inverting bottle few times. 8A. Allow the precipitate to settle (to approximately half the bottle volume) to leave clear supernatant above the brown colored manganese oxi-hydroxide floc. 8.5. Carefully remove the stopper and add 2ml concentrated H2SO4 by the sides of the bottle. 8.6. Re-stopper and mix by inverting several times until dissolution is complete. 8.7 Measure 200ml from the bottle and titrate against 0.025N Na2S203 solution to a pale yellow color. Add few drops of starch indicator & continue titration to first disappearance of blue color to colorless. 8.8. For blank, same above procedure is followed by taking 300ml dilution Water without sample, is carried out to estimate zero day DO. 8.9. After 3days incubation, same above procedure is repeated to estimate Third day DO

9.0Calculation: -When dilution water is not seeded,

BOD, mg/l

= ill1-021
P.

- When dilution water is seeded,

BOD, mg/l P

= ill1-D2)-(B1-B2)f

Where

01- Initial DO in sample. 02- Sample DO after 3 days incubation. B1- I nitial DO in Blank. B2- Blank DO after 3 days incubation. P -Oecimal volumetric fraction of sample used. F - Ratio of seed in diluted sample to seed in Blank. 10.0 Reference: APHA, A\N\NA, WEF [For the Examination of WATER and Waste Water] standard methods Book 19th Edition 1995 Washington, DC.

Estimation of Chemical Oxygen Demand

1.0 Introduction: The Chemica! Oxygen Demand (COD) is used as a measure of oxygen
equivalent of organic matter content of a sample that is susceptible to oxidation by a strong chemical oxidant. For samples from a specific source, COD can be related empirically to Biological Oxygen Demand, Organic Carbon or Organic matter. The test is useful for monitoring and control after

correlation has been established. 2.0 Method: Open Reflux method.

'

3.0 Principle: A sample is refluxed in strongly acid solution with a known excess of
Potassium di-chromate. After digestion the remaining unreduced K2Cr207 is titrated with standard Ferrous Ammonium Sulphate (FAS) to determine the amount of K2Cr207 consumed and the oxidizable organic matter is calculated in terms of oxygen equivalent. AgS04 is added as a catalyst to promote oxidation of certain compounds such as straight chain aliphatic compounds & HgS04 is added to eliminate the interference due to chloride.

4.0 Time Limit: Analyse immediately or refrigerate and add H3P04 or H2SO4 to
pH <2. Maximum storage time is 28 days.

5.0 Apparatus: Reflex apparatus consisting of 100 ml capacity COD tubes with air
condensers and a heating block which can maintain temperature around 150C.

6.0 Reagent & Preparation:

6.1. Mercury sulfate 6.2. Silver sulfate 6.3. Sulfuric acid conc. 6.4. Standard Potassium dichromate (0.25N): Dissolve 12.259 g K2Cr207 primary standard grade previously dried at 103C for 2 hours, in distilled water and dilute to 1000 ml. 6.5. Standard ferrous ammonium sulfate titrant Dissolve 19.5g Fe(NH4h (S04h to 100 ml, Add 10 ml conc. H2SO4 cool and dilute to 1000ml. 6.6. Ferroin indicator: Dissolve 1.485g 1, 10 Phenanthroline monohydrate and 695 mg FeS047H2O in distilled water and dilute to 100 ml. 6.7. Standardisation Of FAS: Mix 5 ml standard K2Cr207 with 15 ml conc. H2SO4 and cool. Add 50 ml DW and titrate with F AS titrant using 2 to 3 drops of Ferroin indicator.

7.0 Procedure: 7.1. Shake the sample well so that the contents are mixed thoroughly. 7.2. Place 10 ml or an aliquot sample diluted to 10 ml in the reflux tube. Add mercuric sulfate according to the chloride concentration with well mixing to dissolve HgS04. 7.3. Add pinch of AgS04 followed by addition of 15 ml of cone H2SO4 along with sides and coo/. 7.4. Pipette out 5ml of K2Cr207 (0.25N) solution into the flask and mix wel/. Use 0.025N K2Cr207 solution for samples having a COD of less than 50 mg/l 7.5. Place the reflux tube in reflux apparatus attached with condenser. Cover open end of condenser with a small beaker to prevent foreign material from entering refluxing mixture. Reflex the mixture for 2 hours at around 1500C temperature. 7.6. After 2 hours allow the flask to cool and wash down the condenser into the tube, add 40ml distilled water. The final volume of the mixture in the tube must be around 70m/. 7.7. Cool to room temperature and titrate against FAS (0.05N) using 2-3drops Ferroin indicator. The end point of the titration is the first sharp color change from blue green to reddish brown. 7.8.ln the same manner reflux and titrate a blank containing the reagents and 10 ml of distilled water and making final volume to 70 m/. 8.0 Calculation: COD as mg02/1 = (A-B) x FAS Normalitv x 8 x 1000 ml of sample taken Where, A = ml FAS consumed for reagents blank. B = ml F AS consumed for sample 8 = Equivalent weight of 02 9.0 Method performance: The open reflux method is suitable for a wide range of wastes where a large sample size is preferred. In this method determine COD values of > 50 mg 02/1t 10.0 Reference: APHA, AWWA, WEF [For the Examination of WATER and Waste Water] standard methods Book 20th Edition 1995 Washington, DC.

Estimation of Oil and Grease

1.0Introduction: Oil and Grease present in excessive amounts may interfere with
aerobic and anaerobic biological processes and lead to decreased waste water treatment efficiency. It may cause surface films and shoreline deposits leading to environmental degradation.

2.0 Method: By Partition-Gravimetric method. 3.0 Principle: Dissolved or Emulsified Oil and Grease is extracted from water by
intimate contact with an extracting solvent. Groups of substances with similar physical characteristics are determined quantitatively on the basis of their common solubility in an organic extracting solvent. The weight of the residue after solvent evaporation constitutes for Oil and Grease content.

4.0 Time Limit: Maximum holding time is 28 days with sample pH adjusted <2
and refrigerated 5.0 Apparatus: 5.1. Separating funnel, 2-L with TFE stop cork. 5.2. Pipettes. 5.3. Filter Papers. 5.4. Funnel. . 5.5. Evaporation dish. 5.6. Water Bath. 6.0 Reagents: 6.1. Hydrochloric acid, 1+1. 6.2. Diethyl Ether or Petroleum Ether. 6.3. Sodium Sulfate,(Na2S04, anhydrous crystals) 7.0 Procedure: 7.1. Mark sample level on the bottle for later determination of sample volume. Acidify the whole sample with 1: 1 HCI to pH 2 or lower if it is not initially acidified. 7.2. Transfer sample to a separating funnel, rinse sample bottle with 30 ml of extracting solvent and add solvent washings to separating funnel. 7.3. Shake vigorously for 2 minutes, let layers separate, drain out the lower aqueous layer. 7.4. Transfer the solvent extract in to a pre-weighed evaporating dish through a funnel containing about 10 gms anhydrous sodium sulfate in a filter paper.

1.0

7.5. Evaporate the solvent on a waterof Phenol80C. Cool and keep it in Estimation bath at about a desiccator for about 30 minutes. 7.6. Record the final weight of the evaporated dish.

Introduction: Phenols defined as hydroxy derivatives of Benzene and its

condensed nuclei, may occur in domestic and industrial waste waters, natural waters and potable water supplies. Chlorination of such waters may produce 8.0Calculation: odorous and objectionable tasting chlorophenols. Phenols removal processes in water treatment include super chlorination, chlorine dioxide or chloramines O&G mgs/L 0f:!.2-W1) x 1000 x 1000 treatment, ozonation and activated carbon adsorption.

Volume of sample taken

2.0

Method: Chloroform Extraction Method

W2 - Final weight of the evaporating dish in gms. W1 - Initial weight of the evaporating dish in gms.

Principle: Steam distillable phenols react with 4-Aminoantipyrine at pH 7.9 ::I: 3.0 9.0 Reference: APHA, AWWA, WEF [For theferricyanide to WATERcolored anitpyrine 0.1 in the presence of Potassium Examination of form a and Waste Water] Standard Methods Book. aqueous solution with CHCb & the absorbance is dye. This dye is extracted from
measured at 460 nm. This method covers the phenol concentration range from 1.0 I-Ig/l to 250 I-Igs/l with a sensitivity of 11-19/1.

4.0 5.0

Time Limit: 28days [add H2SO4. pH < 2]

Apparatus:
5.1 Distillation unit 5.2 Separating Funnel. 5.3 General Glass wares.

6.0 7.0

Equipment: Digital Spectrophotometer

Reagents and preparation:

7.1 7.2

7.3 7.4

7.5 7.6

Ammonium hydroxide (O.5N): Dilute 35ml fresh con NH40H to 1 liter with water. Phosphate Buffer Solution: Dissolve 104.5 gm K2HP04 (dipottasium hydrogen phosphate) & 72.3 gm KH2P04 (pottasium dihydrogen orthophosphate) in water & dilute to 1 liter. The pH should be 6.8. Amino Antipyrine Solution: Dissolve 2.0 gm 4-Aminoanitpyrine in water & dilute to 100ml. Prepare daily. Potassium ferry cyanide: Dissolve 8.0 gms K3Fe(CN)6 in water & dilute to 100 ml. Store in a brown glass bottle. Bromate-Bromide Solution: (0.1 N) Dissolve 2.784 gms of anhydrous KBr03 in water, add 10 gms KBr crystals, dissolve & dilute to 1 liter. Copper Sulphate Solution: Dissolve 100 gms Copper sUlphate & dilute to 1000 ml by distilled water.

7.7. Phosphoric acid: Dilute 10 ml cone. Phosphoric acid to 100 ml with OW. 7.8. Sulphuric acid: 1 N. 7.9. Sodium chloride: Solid. 7.10 Sodium hydroxide: 2.5 N. 7.11 Chloroform: 7.12 Hydrochloric acid: Cone. 7.13 Standard Sodium thiosulphate : 0.025 N. 7.14 Starch solution: 7.15 Sodium sulphate: Anhydrous (solid). 7.16 Potassium Iodide: Solid crystals. 7.17 Stock Phenol solution: Dissolve 1.00 gm Phenol in freshly boiled and cooled OW and dilute to 1 L. 1 ml = 1 mg Phenol. 7.18 Intermidiate Phenol solution: Dilute 10 ml stock in OW and make up to 1 L. 1 ml = 10 .0 ~g Phenol. 7.19 Working standard Phenol: Dilute 50 ml intermediate standard to 500 ml. 1 ml = 1 ~g Phenol.

8.0

Standardization of stock Phenol: 8.1 Take 100 ml OW in a conical flask. 8.2 Add 50 ml stock Phenol solution and 10 ml Bromate-bromide solution followed by 1 ml cone. HCI and swirl gently. 8.3 If brown colour of free bromine does not persist, add 10 ml portions of bromate-bromide solution until it does. 8.4 Cover the flask and keep for 10 minutes. 8.5 Then add 1 gm KI, swirl the flask and leave it for another 10 minutes. 8.6 Prepare a blank in the same manner using OW and 10 ml of 0.1 N Bromate-bromide solution. 8.7 Titrate blank and standard with 0.025 N Sodium thiosulphate using starch as indicator. 8.8 Calculate the concentration of Phenol solution as follows. mg/L Phenol = 7.842 x (AxB)-C Where: A = ml thiosulphate for blank B = ml bromate bromide solution used for sample divided by 10 C = ml thiosulphate used for sample

9.0

Procedure: 9.1 Procedure for distillation: Take suitable volume of sample containing not more than 50 I-Igs Phenol and dilute t0100 ml with distilled water. Adjust the pH approximately to 4.0 with phosphoric acid (H3P04), add 5ml of copper sulphate solution. Do not add them if the sample was preserved with CUS04 & H3P04, instead directly proceed for distillation. Collect about 50 ml distillate & continue to extraction method.

9.2

Dilute the distillate t0100 ml with OW in separating funnel & adjust the pH to 7.9 with phosphate buffer. Add 3 ml of 4-Aminoantipyrine solution. Mix well & add 3ml of potassium ferry cyanide solution [K3Fe(CN)6], mix well and let to color develop for 3 minutes. The solution should be clear & light yellow. Then add 25ml of chloroform & shake it thoroughly & allow for few minutes to phase separation & s~parate the chloroform layer & discard the aqueous layer.

9.3 9.4 9.5

Then collect the chloroform layer in clean & dry test tube.
Follow the above procedure for blank with only 100 ml OW and reagents. Repeat this extraction with 10, 20, 30, 40 & 50 I-Ig Phenol standards in 100 ml OW. Read absorbance at 460 nm wave length by setting instrument "ZERO" with blank. Plot the graph of concentration against 00 and find out concentration factor from the graph.

9.6 9.7

10.0 Calculation:
Mg/L Phenol = Sample OD x Concentration factor from the Qraph. ml sample taken for distillation

11.0 Reference: APHA, AVVWA, WEF [For the Examination of WATER and Waste Water] standard methods Book 19th Edition 1995 Washington, DC.

Table of Contents
SI. No
1. 2. 3. 4. 5. 6.

Method Description
Methods for Gaseous Air Sampling Determination of Suspended Particulate Matter in Ambient Air Measurement of Respirable Suspended Particulate Matter (PM1 0) in Ambient Air Determination of Sulphur Dioxide in Ambient Air Determination of Nitrogen Dioxide in Ambient Air Estimation of Lead in Ambient Air

Method Number
SOP/ATD/01 SOP/ATD/02 SOP/ATD/03 SOP/ATD/04 SOP/ATD/05 SOP/ATD/06

Page Number
1 7 9 11 15 19

Methods for Gaseous Air Sampling

1.0 PURPOSE: The purpose is to lay down a uniform and reliable method for the sampling of gaseous pollutants. Air monitoring normally involves sampling of gaseous pollutant and particulate matter in air. It is necessary to obtain representative samples of air at a number of strategic points. The concentrations are likely to vary with time and distance from the source of pollution depending upon the nature of release, meteorological factors and local conditions such as topography and presence of buildings and vegetation. The measured concentrations are also known to depend upon the average time namely, the period of sampling (24 hours). These aspects are required to be taken into Clccount for sampling and interpretation of measured values.

2.0 SCOPE: This method of sampling is applicable to the gaseous pollutants in the air such as oxides of Nitrogen and Sulphur, Ozone and other pollutants, which react with liquid absorbing reagents at atmospheric temperature and pressure when air is bubbled through the absorbing solution in the impinger. 3.0 INTERFERENCES: The interferences are governed by the following factors. 3.1. Chemical changes such as chemical interactions among the components of the collected sample or photochemical decomposition. 3.2. Absorption of the gases from the sample on to the walls of the container and leaks. 3.3. The interval between collection and analysis of sample should be as soon as possible. 3.4. Protect the samples from light and heat.

4.0 SAMPLE PRESERVATION: After sample collection, the solutions must

be stored at 5C in a refrigerator.

5.0 APPARATUS:

5.1. Sampling equipments: The sampling equipments to be used for air sampling
will consists of a standard impinger of 35-ml capacity, a trap, a flow meter or critical orifice device and a suction pump.

5.2. General instructions for the Care of the Instruments

5.2.1.1. The positions of the instruments must never be changed
once installed.

5.2.1.2. Tall grass or shrubs should not be allowed to grow around the rain gauge, as these would vitiate its exposure. 5.2.1.3. Sampling location should be approachable at any time during each season 5.2.1.4. No obstruction should be present near sampling station. 5.2.1.5. Try to select points where electricity is easily available. 5.2.1.6. If surface winds prevail in the area, put high volume sampler on rooftop or 3 m above ground. 5.2.1.7.If sampling station is at hill region, then it is necessary to see previous meteorological data to decide exact sampling location because its should be such that air is not obstructed by mountains, most of the time. 5.2.1.8. For Ambient Air Monitoring each station should have the following items: 5.2.1.9. RDS-1 5.2.1.10. Tool kit (ice box, 4 air sample tube, screw driver, pliers, bulb holder, wire, tester, carbon brush (two pair), electric board and wire (as per required length), one folder with log sheet (2), one hard board may be provided for holding log sheet). 7.0 Documenting observationsmarking samples. 5.2.1.11. Stickers for 7.1. The register or log sheet should be maintained at each station. 7.2. A special column should be provided for remarks and signature. 7.3. lt is & observer's duty to 6.0 REAGENTStheFIL TERPAPERS: record the reading immediately after taking 6.1. observation. All the reagents should be of A. RIG. R.grade. 7.4. GFIA Whatman glass neatly fiber filter papers or EPM 2000 filterbe avoided. Readings should be micro entered. Over-writing etc. should paper 6.2. 7.5. Readings should be written in a diary I pocket book I data sheet. 6.3. Absorbing Reagents: 7.6. After entering the readings neatly and faithfully, the observer should sign in the For NOx : Dissolve 4.0gm of Sodium Hydroxide in distilled water add 6.3.1. corresponding column. 7.7. Any unusual or remarkable weather phenomenon, if ml with distilled water. 1.0gm of Sodium Arsenate and dilute to 1000 noticed, its time of commencement, duration and cessation should be recorded and, if possible, its influence over the parameters being recorded. 6.3.1.1. REAGENTS: 6.3.1.2. Sulfanilamide Solution: Dissolve 20 gm Sulfanilamide in 700 7.7.1. General character of the weather with mixing theml ofshould be ml of distilled water add throughout 50 day 85% Ortho noted (windy, sunny,acid and rainy, etc) Phosphoric cloudy, dilute to 1000 ml with distilled water. 7.7.2. Regular and carefulPeroxide Solution: Dilute made punctually at the 6.3.1.3. Hydrogen observations should be 0.2 ml of 30% Hydrogen prescribed hours of observations.distilled water. This solution may be Peroxide to 250 ml with 7.7.3. The readings of each observed meteorological elementsfrom light. used for 1 month, if refrigerated and protection should be entered NEDAregister, neatly and 0.5 gm of NEDA in 500 ml of in the Solution: Dissolve eligibly, after each observation. 6.3.1.4. 7.7.4. Keep the instruments clean solution is stable for one month, if distilled water. This and free from dust. 7.7.5. Recording paper of the self recording instruments should be refrigerated and protection from light. changed as required. 7.7.6. Clock or watch by which the observer is guided should be kept correct according to 1ST. To avoid delay and irregularity, the observer should be ready near the instrument a few minutes before the prescribed time of observation. Time should be expressed

6.3.2. For S02: Dissolve 10.86 gm mercuric chloride + 0.066 gm EDTA + and 6.0 gm potassium chloride or 4.68 gm Sodium solution chloride in Distilled water and dilute to 1 Litre. 6.3.2.1. REAGENTS: 6.3.2.2. Sulphamic acid (0.6%):Dissolve 0.6 gm Sulphamic acid in 100 ml Distilled water. 6.3.2.3. Formaldehyde (0.2%) :Dilute 5 ml formaldehyde solution (36-38%)to 1 liter with distilled water. 6.3.2.4. Pararosoline Solution: . 6.3.2.4.1. Step I: Stock Solution: Dissolve 0.5 gm of purified PRA in 100 ml distilled water and keep for 2 days (48 hours) than filter 6.3.2.4.2. Step II: Working Solution: 4ml of stock PRA in a 100 ml volumetric flask, add 6 ml of conc. HCL and make up to volume 100 ml with distilled water.

according to the 24 hours clock. The 24 hours from midnight to the next midnight are numbered consecutively as 00, 01, 02, 03-23. 7.7.7. Every observation should be recorded faithfully as read. In case of doubt, observations should be repeated. If any observation is not made, then the space in the register allotted for it should be left blank and the reason should be stated clearly. 7.7.8. Register should be maintained regularly; name and signature of the observer also should be entered simultaneously.

8.0 Instruction for Respirable Dust Sampler (RDS) operations:

8.1. RDS should be operated in the following sequence: The equipment to be used for air sampling will consist of 8.1.1. Blower assembly 8.1.2. Time totalizer 8.1.3. Stabilizer 8.1.4. Monometer 8.1.5. Rotameter 8.1.6. Ice trap 8.1.7. 2 number of 35 ml impingers. (They may be cleaned by surging the appropriate cleaning solution back and forth through the frit and then rinsing with distilled water in the same fashion.) 8.2. Put the machine on a smooth surface that should be at least 1.5m, above the ground level. 8.3. Put the roof over the sample. 8.4. Connect the sampler with voltage stabilizer and then connect voltage stabilizer with the main switch. 8.5. Check the operation by just switching on the power switch. 8.6. Equipment fitted with a manometer should be fill with distilled water in manometer (up to 50mm). 8.7. Put filter paper of the rough side upward on wire mesh and tighten the bolts. 8.8. Note the following: 8.8.1. Filter paper number 8.8.2. Time totalizer reading (initial) 8.8.3. Manometer reading (read keeping the eye parallel) 8.8.4. Put on the switch and note the following: 8.8.5. Manometer reading [initial (or level) and final or (upper level)]. 8.8.6. From manometer reading and calibration curve check the flow rate. It should not be less than 1.1 m3/min, preferably between 1.2 or 1.3 m3/min. 8.8.7. Note the manometer reading in between the monitoring period to check variation in flow rate once in a hour. 8.8.8. After completion of monitoring, Time totalizer (final) and Manometer reading should be noted.. 8.8.9. Fold the filter paper into half with clean hands and put in a clean case polythene cover. Label the case mentioning the date and location of sampling.

9.0PROCEDURE: 9.1. Sampling period: Sampling period and rate of sampling shall vary with the type of sampling programme and its purpose. Normally the sampling periods are 30 minutes, one to four hours and eight hours depending upon the expected concentration of the pollutant, its nature and the investigation patterns. Based on practical experience the air sampling rates with respect to sampling period are as follows: Period of sampling 30 minutes 1 hour 1-4 hours 8 hours 8-24 hours Rate of sampling 2 1 0.5 0.2-0 0.1-0.2

9.2. Sample collection: Place 10 to 30 ml of the absorbing media in the impinger in an ice box and run the instrument as per the expected concentration of the pollutant and accordingly select the sampling period and rate of sampling (Lit/min.) After completion of the sampling, remove the impingers, measure the sample volume. Either make up the sample volum~ or note the final volume of the absorbing solution. 10.0 Collection of Samples 10.1. Collection of Respirable Suspended Particulate Matter (RSPM): The RDS should be operated at an average flow rate of 0.6 - 1.4m3 cum/min for collection of RSPM on pre weighed GF/A Whatman glass micro fibre filter paper or EPM-2000 filter paper. Sample should be collected for 8 hours/24hours as per requirement (normally 24 hours, sampling is done). After sampling period is over, filter paper is neatly folded and preserved in polythene bag and labeled. Simultaneously pre weighed plastic cup should be fixed at the bottom of the cyclone. After sampling period is over the cup has to be removed from the cyclone and covered with a cap and labeled. Particulate matter collected on filter paper represents RSPM (size < 10 mm).Particulate matter collected in cup below cyclone represents Non Respirable Suspended particulate Matter (NRSPM size >10mm)

'"

10.2. Collection of Suspended Particulate Matter (SPM): Sum of particulate matter collected in cup below cyclone and filter paper gives an indications of SPM 10.3.Collection of S02 Sample: S02 samples are collected by bubbling air through the absorbing solution as per the standard method. The absorbing solution should be covered from direct sunlight and kept in ice trap during and after sampling to prevent deterioration. Sample should be collected for 4 hours/8 hours/24 hours as per requirement. After required period, sample is collected in plastic bottles, kept in icebox at site, and transported to the laboratory for immediate analysis. 10.4. Collection of NOx Sample: NOx samples are collected by bubbling air through the absorbing solution as per the standard method. The absorbing solution should be covered from direct sunlight and kept in ice trap during and after sampling to prevent deterioration. Sample should be collected in plastic bottles and kept in icebox at site transported to the laboratory for immediate analysis.

11.0 CALCULATIONS:
The following equation is used for the calculation of gaseous pollutants in the ambient air. Concentration (l-lg/m3) = (A-B) x G.F. x Tv x 1000 T x Fr x Va A B GF Tr 1000 T Fr V = Absorbance of exposed sample at a particular wavelength = Absorbance of reagent blank solution = Graph factor of the concerned pollutant. (lJg/abs.) (Graph factor.is the inverse of the slope) = Total volume of the exposed sam~le (ml) = Conversion factor from liters to m = Total sampling time (min.) = Sampling flow rate (Litre/min.) = Volume taken for analysis (ml).

Determination of Suspended Particulate Matter (SPM) in Ambient Air

1.0 PURPOSE: The purpose is to lay down an uniform and reliable method for measurement of Suspended Particulate Matter (SPM) in the ambient air. 2.0 PRINCIPLE: Air is drawn through a size-selective inlet. Particles with aerodynamic diameters less than the cut-point of the inlet are collected by filter and cup. The mass of these particles is determined by the difference in filter weights prior to and after sampling. The concentration of suspended particulate matter in the designated size range is calculated by dividing the weight gain of the filter and cup by the volume of air sampled.

3.0 INTERFERENCES:
3.1. passive Deposition - Passive deposition occurs when windblown Dust deposits on a filter both prior to and after sampling. 3.2. To avoid particle loss during transport, filters are heavily loaded with large dry aerosols. It is more prevalent on membrane than on glass fiber filters. Particle loss is minimized by shorter sample duration in heavily polluted environments, folding the filter prior to transport, and by wrapping the filter paper with polythene cover. The filter paper should be folded into half with clean hands and put in a clean polythene cover. The exposed part of the filter paper should be folded inward to avoid sample loss. 4.0 APPARATUS: The RDS is a compact unit consisting of a protective covering blower, voltage stabilizer, time and rotameter.

5.0 Laboratory quipment:

5.1. Analytical Balance: The balance must be equipped with an expanded weighing chamber to accommodate 20.3 x 25.4 cm (8 x 10 in) filters and must have a sensitivity of 0.1 mg, this should be calibrated before weighing. Analytical balances can be calibrated by the operator while others require specialized skills to re-calibrate. any time the balance is moved, at least every twelve months, or whenever an NBS traceable 3.0000 g weight registers outside 1: 0.5 mg of its designated weight. 6.0 PROCEDURE: After sampling period is over, filter paper is neatly folded and preserved in polythene bag and cup 'has to be removed from the cyclone and covered with a cap and labeled. The final weight of the filter paper and cup particulate matter collected on filter paper represents RSPM (size less than 10 microns) particulate matter collected in cup below cyclone represents Non Respirable Suspended particulate matter (NRSPM size> 10 microns)

7.0Calculation of Suspended Particulate Matter in Ambient Air Sum of particulate matter collected in cup below cyclone and filter paper gives an indication of SPM. Calculation of volume of air sampled: Q = average flow rate in m3f min T= total sampling time in minute.

V=QxT

SPM Where: SPM Wi Wr V 106

= (Wr-Wi) x 106 V = = = = =
Mass concentration of suspended particles in I-Ig I m3 initial weight of filter in g. Final weight of filter in g. Volume of air sampled in m3 Conversion of g to I-Ig.

Measurement of Respirable Suspended Particulate Matter (PM1 0) in Ambient Air

1.0
PURPOSE: The purpose is to lay down uniform and reliable method for measurement of PM10 (particulate matter less than 10l-lm diameter) in ambient air
PRINCIPLE: 2.1 Air is drawn through a size-selective inlet and through a 20.3 X 25.4 cm (8X 10 in) filter that the cut-point of the inlet are collected by the filter, the mass of these particles is determined by the difference in filter weights prior to and after sampling. 2.2 The concentration of PM 10 in the designed size range is calculated by dividing the weight gain of the filter by the volume of air sampled. 3.0

2.0

INTERFERENCE: Passive Deposition- Passive deposition occurs when windblown dust deposits on a filter both prior to end after sampling. 3.1 Shipping Losses- Particle loss during transport occurs when filters are heavily loaded with large dry aerosols. It is more prevalent on membrane than on glass fiber filters. Particle loss is minimized by shorter sample duration in heavily polluted environments, use of fiber as opposed to membrane filters, folding the filter prior to transport, and careful shipping procedures. APPARATUS: The Respirable Dust Sampler is a compact unit consisting a protective housing, blower, voltage stabilizer, time rotameter and filter holder.
4.1 Inlet for PM10 Sampling:

4.0

4.1.1 Cyclonic Flow Inlet Cyclones use centrifugal force to remove dust. A particle in a rotating air stream is subjected to a centrifugal force that accelerates it towards a surface where it will impact and lose momentum, thus being removed from air stream. 4.1.2 In a typical cyclone pre-collector, the air enters tangentially at its Side and swirls around inside. 4.1.3 Particles above 10 I-Im are thrown to the cyclone walls and collected at its base ("grit-pot"). The air containing the Respirable dust leaves through the central exit at the top of the cyclone and is filtered to collect the dust on a filter paper.

5.0 Laboratory Equipment: 5.1 Analytical Balance: The balance must be equipped with an expanded weighing chamber to accommodate 20.3 x 25.4 cm (8 x 10 in) filters and must have a sensitivity of 0.1 mg, this should be calibrated before weighing. Analytical balances can be calibrated by the operator while others require specialized skills to re-calibrate. any time the balance is moved, at least every twelve months, or whenever an NBS traceable 3.0000 g weight registers outside :t 0.5 mg of its designated weight. 6.0 PROCEDURE: After sampling period is over, filter paper is neatly folded and preserved in polythene bag and cup has to be removed from the cyclone and covered with a cap and labeled. The final weight of the filter paper and cup particulate matter collected on filter paper represents RSPM (size less than 10 microns) particulate matter collected in cup below cyclone represents Non Respirable Suspended particulate matter (NRSPM size> 10 microns)

7.0

CALCULATIONS: Calculation of Volume of Air Sampled V = QT V = Volume of air sampled in m3 Q = Average flow rate in m3jminute T = Total sampling time in minute Calculation of PM10 in Ambient Air
PM10 =
. fJl"if

- Wi) X 106

V Where PM1Q =
Wi
Wf

V 106

= = = =

Mass concentration of particulate matter less than 10 Micron diameter in grams Initial weight of filter in g. Final weight of filter in 9. Volume of air sampled in m3 Conversion of 9 to 1-'9.

Determination of Sulphur di Oxide in Ambient Air

1.0
INTRODUCTION: S02 is a major contributor of air pollutant. Volcanos are a major source of natural air pollutant. Thermal power plants, smelting industries, fossil fuel combustion etc. Inhaling excess of sulphur causes bronchitis, emphysema and other lung related diseases. It causes Sulphrous Acid Smog. Taj Mahal which is a marble building faces stone leprosy due to refineries in Agra. It also causes acid rain. Plants are extremely sensitive S02 excess of it can cause leaf necrosis, bleaching of pigments.

2.0

PRINCIPLE: 2.1 Sulphur dioxide from air is absorbed in a solution of potassium tetrachloro-mercurate (TCM). A dichlorosulphitomercurate complex, which resists oxidation by the oxygen in the air, is formed. Once formed, this complex is stable to strong oxidants such as ozone and oxides of nitrogen and therefore, the absorber solution may be stored for some time prior to analysis. The complex is made to react with pararosaniline acid, and formaldehyde to form the intensely colored pararosaniline methylsulphonic acid. The absorbance of the solution is measured by means of a suitable spectrophotometer.

3.0

SCOPE: This method is applicable for the measurement of concentration of Sulphur dioxide present in ambient air. INTERFERENCES: The interference of trace metals may be eliminated by the addition of eythlendiaminetetraacetic acid (EDTA) to the absorbing solution prior to sampling. REAGENTS: 5.1 Sulphamic acid (0.6%) : Dissolve 0.6 gm Sulphamic acid in 100 ml Distilled water. 5.2 Formaldehyde (0.2%) :Dilute 5 ml formaldehyde solution (36 38%)to 1 liter with distilled water. 5.3 Pararosoline Solution: 5.3.1 Step I: Stock Solution: Dissolve 0.5 gm of purified PRA in 100 ml distilled water and keep for 2 days (48 hours) than filter 5.3.2 Step II: Working Solution: 4ml of stock PRA in a 100 ml volumetric flask, add 6 ml of conc. HCL and make up to volume 100 ml with distilled water

4.0

5.0

5.4

ABSORBING REAGENT: Dissolve 10.86 gm mercuric chloride + 0.066 gm EDTA + and 6.0 gm potassium chloride or 4.68 gm Sodium solution chloride in Distilled water and dilute to 1 Litre.

6.0

STANDARDISATION: 6.1.1 Sulphamic Acid (0.6%) - Dissolve 0.6 9 sulphamic acid in 100 ml distilled water. Prepare fresh daily. 6.1.2 Formaldehyde (0.2%) - Dilute 5 ml formaldehyde solution (36 38%) to 1 litre with distilled water. Prepare fresh daily.
6.1.3 Stock Iodine Solution (0.1 N) - Place 12.7 iodine in a 250 ml beaker, add 40 9 potassium iodide and 25 ml water. Stir until all is dissolved, then dilute to 1 litre with distilled water.

6.1.4 Iodine solution (0.01 N) Prepare approximately 0.01 N iodine solution by diluting 50 ml of stock solution to 500 ml with distilled water.

6.1.5

Starch Indicator solution: 0.4 gm soluble starch and 0.002 9 mercuric iodide preservative with a little water and adQ the paste slowly to 200 ml boiling water Continue boiling until the solution is clear, cool, and transfer to a glass-stoppered bottle.

6.1.6 Stock Sodium Thiosulfate Solution (0.1 N) Prepare a stock solution by placing 25 9 sodium thiosulfate pentahydrate in a beaker, add 0.1 9 sodium carbonate and dissolve using boiled, cooled distilled water making the solution up to a final volume of 1 liter. Allow the solution to stand one day before standardizing. 6.1.7 To standardize, accurately weigh to the nearest 0.1 . g, 1.5 9 primary standard potassium iodite dried at 180C, dissolve, and dilute to volume in a 500 ml volumetric flask. In to a transfer 500 ml Iodine flask. transfer 50 ml of iodated solution by pipette. Add 2 9 potassium iodide and 10 ml of N hydrochloric acid and stopper the flask. After 5 min titrate with stock thiosulfate solution to a pale yellow. Add 5 ml starch indicator solution and continue the titration until the blue color disappears, Calculate the normality of the stock solution.

6.1.8

Sodium Thiosulphate Titrant (0.01 N) dilute 100 ml of the stock thiosulfate solution to 1 liter with freshly boiled and cooled distilled water. The calibration curve, section 8.5.2 should have a slope of 0.030 + 0.002 absorbance unitlllg S02 at this path length when the dye is pure and the 6.1.9 sulphate solution is properly standardized. Pararosaniline Stock Sulphite Standardized sulphate solution for preparation of Working Solution - Dissolve 0.500 gm of specially purified pararosaniline (PRA) in TCM Solution Dissolve 0.30 g sodium metabisulphite (Na2S205) or 0.40 100ml of distilled water and keep 5002 days (48 hours). g sodium sulphate (Na2S205) in for ml of Pararosaniline cooled, distilled water, Sulphate solution is taken in a 250 recently boiled, Working Solution - 10 ml of stock PRA is unstable; it is ml volumetric flask. Adduse ml cone. HCL and make up toto minimize this therefore important to 15 water of the highest purity volume with distilled water. solution contains the equivalent of 320 -400 flg Iml of S02 instability. This The actual concentration of the solution is determined by adding excess iodine and back- titrating with standard sodium thiosulfate solution. To back-titrate, measure, by pipette, 50 ml of the 0.01 N iodine solution into each of two 500 ml iodine flasks A And B. To flask A (blank) add 25 ml distilled water and into flask B (sample) measure 25 ml Sulphite solution by pipette, Stopper the flasks and allow to react for 5 minutes. Prepare the working Sulphite- TCM solution (section 7.2.9) at the same time iodine solution is added to the flasks. By means of a burette containing standardized 0.01 N thiosulfate, titrate each flask in turn to a pale yellow. Then add 5 ml starch solution and continue the titration until the blue color disappears.

6.2

Working Sulphite - TCM Solution: - Measure 2 ml of the standard solution into a 100ml volumetric flask by pipette and bring to mark with 0.04 M TCM. Calculate the concentration of Sulphur dioxide in the working solution in micrograms of Sulphur dioxide per milliter. This solution is stable for 30 days if kept in the refrigerator at 5C. If no kept at 5'C, prepare fresh daily.
Purified Para-rosaniline Stock Solution (0.2% Nominal) Dye Specifications: The parosaniline dye must meet the following specification: The dye must have a wavelength of maximum absorbance at 540 nm when assayed in a buffered solution of 0.1 M sodium acetate acetic acid. The absorbance of the reagent blank, which is temperature sensitive to the extent of 0.015 absorbance unitfC, should not exceed 0.170 absorbance unit at 22C with a 1 em optical path length, when the blank is prepared according to the prescribed analytical procedure and to the specified concentration of the dye.

6.3 6.4

6.5

6.6

6.7

6.8 6.9

7.0

Analysis: 7.1 A spectrophotometer suitable for measurement of absorbance at 560 nm with an effective spectral band width of less than 15 nm is required. Reagent blank problems may occur with spectrophotometer having greater spectral band widths. The wavelength calibration of the instrument should be verified. If, transmittance is measured, this can be converted to absorbance by the formula: A = 2 - log 10 T PROCEDURE: Take 15 ml of sample in a 100 ml or 50 ml test tube then add 1 ml of Sulpha~c aci.9 + 2 ml formaldehyde + 2 ml of step II Pararosaniline;-"haRe vkll ~eave it for 30 min for colour development (Purple Colour). Carry a Blank simultaneously using absorbing media instead of sample and then read the % transmission or 0.0 at 560 nm.

8.0

9.0

CALCULATION: Ilg/m3 = 100- %T x Slope x 30 Volume of x Air Sample in m3 Sample taken for estimation

Method for determination of Nitrogen Dioxide in the Ambient Air 1.0 PRINCIPLE: 1.1. Ambient nitrogen dioxide (N02) is collected by bubbling air through a solution of sodium hydroxide and sodium arsenite. The concentration of nitrite ion (N02) produced during sampling is determined colorimetrically by reacting the nitrite ion with phosphoric acid, sulfanilamide, and N_(1naphthyl) - ethylenediamine dihydrochloride (NEDA) and measuring the absorbance of the highly colored azo dye at 540 n m. 1.2. The nominal range of the method is 9 to 750 ~g N02/m3) The range of the analysis is 0.04 to 2.0 ~g N02/ml, following Beer's Law throughout this range (0 to 1.0 absorbance units). 2.0 SCOPE: This method is applicable to 24 hours integrated sampling of N02 in ambient air. 3.0 INTERFERENCES: 3.1. Nitric oxide (NO) is a positive interferant and carbon dioxide (CO2) is a negative interferant. The average error resulting from normal ambient concentrations on NO and CO2) is small for most monitoring situations and does not necessitate applying a correction to measurement obtained with the method3). 3.2. Potential interference from sulfur dioxide (S02) is eliminated by converting any S02 to sulfate with hydrogen peroxide during analysis

4.0 SAMPLING PRESERVATION: Collected samples are stable for at least six weeks at room temperature. Stored samples should be tightly sealed to prevent absorption of N02from the atmosphere. 5.0 Apparatus of Equipment: 5.1. Volumetric Flasks - 100, 250, 500, 1, 000 ml. 5.2. Pipets - 1, 2, 5, 10, 20, 50 ml volumetric; 2ml, graduated in 1/10 ml intervals. 5.3. Test Tubes - Approximately 150 mm long x20 mm diameter. 5.4. Spectrophotometer Capable of measuring absorbance at 540 nm; equipped with 1 cm optical path length cells.

6.0 REAGENTS: 6.1. Sodium Hydroxide:

6.2. Sodium Arsenate 6.3. Absorbing Reagents - Dissolve 4.0 g of sodium hydroxide in Distilled water, add 1.0 g of sodium arsenite, and dilute to 1,000 ml with distilled water. 6.4. Sulfanilamide Melting point 165 to 167'C. 6.5. N-(Naphthyl)-ethylenediamine Di-hydrochloride (NEDA) -1 % Aqueous solution should have only one absorption peak at 320 nm over the range of 260-400 nm. NEDA showing more than one absorption peak over this range is impure and should not be used.

6.6. Hydrogen Peroxide, 30%. 6.7. Phosphoric Acid, 85%. 6.8. Sulfanilamide, Solution - Dissolve 20 g of sulfanilamide in 700 ml of distilled water. Add, with mixing, 50 ml of 85% phosphoric acid and dilute to 1,000 m!. This solution is stable for one month, if refrigerated. 6.9. NEDA Solution Dissolve 0.5g of NEDA in 500 ml of distilled water. This solution is stable for one month, if refrigerated and protected from light. 6.10. Hydrogen Peroxide Solution - Dilute 0.2 ml of 30% hydrogen peroxide to 250 ml with distilled water. This solution may be used for one month, if, refrigerated and protected from light.

7.0 PROCEDURE: 7.1. Preparation of Calibration Graph 7.2. Sodium Nitrite Assay of 97% NaN02 or greater. 7.3. Sodium Nitrite Stock Solution (1000 J.1g N02 Iml) Dissolve 1.5 g of desiccated sodium nitrite in distilled water and dilute to 1,000 ml such that a solution containing 1000 J.1g N02/ml is obtained. The amount of NAN02 to be used if, the assay percent is less than 100% is calculated as follows:

G = 1.500 A Where: G = Amount of NaN02. grams 1.500 = Gravimetric conversion factor A = Assay, percent (should be 97 or greater)

This stock solution can be stored for six weeks, if refrigerated. 7.4. Sodium Nitrite Working Standard (1.0 J.1g N02/ml) 7.5. Solution A - Pipet 5 ml of the stock solution into a 500 ml volumetric flask and dilute to volume with distilled water. This contains 10 flg N02 1m!.

7.6. Solution B Pipet 25 ml of solution A into a 250 volumetric flask and dilute to volume with absorbing solution. This contains 1 flg N02 1m!. Prepare fresh daily.

8.0CALIBRATION: 8.1. Spectrophotometer: Prepare calibration curve using 1 flg/ml working standards. 8.2. In accordance with the analytical procedure given in 9.4, measure and record the absorbance for each calibration standard (0,1,2,3,4,5,6,7,8,9,10,11115,20 flg N02. 8.3. Plot absorbance (y-axis) versus the corresponding concentration in flg N02/50 ml final solution (x-axis). Draw or compute the straight line best fitting the data to obtain the calibration curve.

9.0PROCEDURE: Take 10 ml of Sample in a 100 or 50 ml capacity test tube then

add 1 ml of Hydrogen Peroxide Solution + 10 ml of Sulfanilamide Solution, + 1.4 ml of NEDA Solution, with through mixing after the addition of each reagent and make up to 30 ml with distilled water or Absorbing Reagent, leave it for 10 min for colour development (Pink Colour) measure & record the absorbing or % T at 540 nm against blank. 10.0 CALCULATION:

100-% T Slope x 30 flg/m3 = Volume of x Sample taken

Air Sample in m3 for estimation

N02 Concentration in Analyzed Sample Determine flg N02 Iml graphically from the calibration curve or compute from the slope and intercept values N02 Concentration in Air Sample

as follows:

- Calculate as ~g of N02 per cubic meter of

~g/N02/m3= UQ/NO? X Vs X 0 Va X 0.82 X Vt Where: ~g/N02 = N02 concentration in analysed sample Va = Volume of air sample, m3 = Sampling efficiency 0 Vs Vt = Dilution factor (0 = 1 for no dilution; 0 = 2 for 1: 1 dilution). = Final volume of sampling solution = Aliquot taken for analysis

The N02 concentration may be calculated as ppm using: ppm N02 = (lJg/N02/m3)x 5.32 x 10-4

Determination of Sulphur di Oxide in Ambient Air

INTRODUCTION: S02 is a major contributor of air pollutant. Volcanos are a major source of natural air pollutant. Thermal power plants, smelting industries, fossil fuel combustion etc. Inhaling excess of sulphur causes bronchitis, emphysema and other lung related diseases. It causes Sulphrous Acid Smog. Taj Mahal which is a marble building faces stone leprosy due to refineries in Agra. It also causes acid rain. Plants are extremely sensitive S02 excess of it can cause leaf necrosis, bleaching of pigments.

PRINCIPLE: 2.1 Sulphur dioxide from air is absorbed in a solution of potassium tetrachloro-mercurate (TCM). A dichlorosulphitomercurate complex, which resists oxidation by the oxygen in the air, is formed. Once formed, this complex is stable to strong oxidants such as ozone and oxides of nitrogen and therefore, the absorber solution may be stored for some time prior to analysis. The complex is made to react with pararosaniline acid, and formaldehyde to form the intensely colored pararosaniline methylsulphonic acid. The absorbance of the solution is measured by means of a suitable spectrophotometer.

SCOPE: This method is applicable for the measurement of concentration of Sulphur dioxide present in ambient air. INTERFERENCES: The interference of trace metals may be eliminated by the addition of eythlendiaminetetraacetic acid (EDTA) to the absorbing solution prior to sampling. REAGENTS: 5.1 Sulphamic acid (0.6%) : Dissolve 0.6 gm Sulphamic acid in 100 ml Distilled water. 5.2 Formaldehyde (0.2%) :Dilute 5 ml formaldehyde solution (36 38%)to 1 liter with distilled water. 5.3 Pararosoline Solution: 5.3.1 Step I: Stock Solution: Dissolve 0.5 gm of purified PRA in 100 ml distilled water and keep for 2 days (48 hours) than filter 5.3.2 Step II: Working Solution: 4ml of stock PRA in a 100 ml volumetric flask, add 6 ml of conc. HCL and make up to volume 100 ml with distilled water

5.4

ABSORBING REAGENT: Dissolve 10.86 gm mercuric chloride + 0.066 gm EDTA + and 6.0 gm potassium chloride or 4.68 gm Sodium solution chloride in Distilled water and dilute to 1 Litre.

6.0

STANDARDISATION: 6.1.1 Sulphamic Acid (0.6%) - Dissolve 0.6 9 sulphamic acid in 100 ml distilled water. Prepare fresh daily. 6.1.2 Formaldehyde (0.2%) - Dilute 5 ml formaldehyde solution (36 38%) to 1 litre with distilled water. Prepare fresh daily.
6.1.3 Stock Iodine Solution (0.1 N) - Place 12.7 iodine in a 250 ml beaker, add 40 9 potassium iodide and 25 ml water. Stir until all is dissolved, then dilute to 1 litre with distilled water.

6.1.4 Iodine solution (0.01 N) Prepare approximately 0.01 N iodine solution by diluting 50 ml of stock solution to 500 ml with distilled water.

6.1.5

Starch Indicator solution: 0.4 gm soluble starch and 0.002 9 mercuric iodide preservative with a little water and adQ the paste slowly to 200 ml boiling water Continue boiling until the solution is clear, cool, and transfer to a glass-stoppered bottle.

6.1.6 Stock Sodium Thiosulfate Solution (0.1 N) Prepare a stock solution by placing 25 9 sodium thiosulfate pentahydrate in a beaker, add 0.1 9 sodium carbonate and dissolve using boiled, cooled distilled water making the solution up to a final volume of 1 liter. Allow the solution to stand one day before standardizing. 6.1.7 To standardize, accurately weigh to the nearest 0.1 . g, 1.5 9 primary standard potassium iodite dried at 180C, dissolve, and dilute to volume in a 500 ml volumetric flask. In to a transfer 500 ml Iodine flask. transfer 50 ml of iodated solution by pipette. Add 2 9 potassium iodide and 10 ml of N hydrochloric acid and stopper the flask. After 5 min titrate with stock thiosulfate solution to a pale yellow. Add 5 ml starch indicator solution and continue the titration until the blue color disappears, Calculate the normality of the stock solution.

6.1.8

Sodium Thiosulphate Titrant (0.01 N) dilute 100 ml of the stock thiosulfate solution to 1 liter with freshly boiled and cooled distilled water. The calibration curve, section 8.5.2 should have a slope of 0.030 + 0.002 absorbance unitlllg S02 at this path length when the dye is pure and the 6.1.9 sulphate solution is properly standardized. Pararosaniline Stock Sulphite Standardized sulphate solution for preparation of Working Solution - Dissolve 0.500 gm of specially purified pararosaniline (PRA) in TCM Solution Dissolve 0.30 g sodium metabisulphite (Na2S205) or 0.40 100ml of distilled water and keep 5002 days (48 hours). g sodium sulphate (Na2S205) in for ml of Pararosaniline cooled, distilled water, Sulphate solution is taken in a 250 recently boiled, Working Solution - 10 ml of stock PRA is unstable; it is ml volumetric flask. Adduse ml cone. HCL and make up toto minimize this therefore important to 15 water of the highest purity volume with distilled water. solution contains the equivalent of 320 -400 flg Iml of S02 instability. This The actual concentration of the solution is determined by adding excess iodine and back- titrating with standard sodium thiosulfate solution. To back-titrate, measure, by pipette, 50 ml of the 0.01 N iodine solution into each of two 500 ml iodine flasks A And B. To flask A (blank) add 25 ml distilled water and into flask B (sample) measure 25 ml Sulphite solution by pipette, Stopper the flasks and allow to react for 5 minutes. Prepare the working Sulphite- TCM solution (section 7.2.9) at the same time iodine solution is added to the flasks. By means of a burette containing standardized 0.01 N thiosulfate, titrate each flask in turn to a pale yellow. Then add 5 ml starch solution and continue the titration until the blue color disappears.

6.2

Working Sulphite - TCM Solution: - Measure 2 ml of the standard solution into a 100ml volumetric flask by pipette and bring to mark with 0.04 M TCM. Calculate the concentration of Sulphur dioxide in the working solution in micrograms of Sulphur dioxide per milliter. This solution is stable for 30 days if kept in the refrigerator at 5C. If no kept at 5'C, prepare fresh daily.
Purified Para-rosaniline Stock Solution (0.2% Nominal) Dye Specifications: The parosaniline dye must meet the following specification: The dye must have a wavelength of maximum absorbance at 540 nm when assayed in a buffered solution of 0.1 M sodium acetate acetic acid. The absorbance of the reagent blank, which is temperature sensitive to the extent of 0.015 absorbance unitfC, should not exceed 0.170 absorbance unit at 22C with a 1 em optical path length, when the blank is prepared according to the prescribed analytical procedure and to the specified concentration of the dye.

6.3 6.4

6.5

6.6

6.7

6.8 6.9

7.0

Analysis: 7.1 A spectrophotometer suitable for measurement of absorbance at 560 nm with an effective spectral band width of less than 15 nm is required. Reagent blank problems may occur with spectrophotometer having greater spectral band widths. The wavelength calibration of the instrument should be verified. If, transmittance is measured, this can be converted to absorbance by the formula: A = 2 - log 10 T PROCEDURE: Take 15 ml of sample in a 100 ml or 50 ml test tube then add 1 ml of Sulpha~c aci.9 + 2 ml formaldehyde + 2 ml of step II Pararosaniline;-"haRe vkll ~eave it for 30 min for colour development (Purple Colour). Carry a Blank simultaneously using absorbing media instead of sample and then read the % transmission or 0.0 at 560 nm.

8.0

9.0

CALCULATION: Ilg/m3 = 100- %T x Slope x 30 Volume of x Air Sample in m3 Sample taken for estimation

Method for determination of Nitrogen Dioxide in the Ambient Air 1.0 PRINCIPLE: 1.1. Ambient nitrogen dioxide (N02) is collected by bubbling air through a solution of sodium hydroxide and sodium arsenite. The concentration of nitrite ion (N02) produced during sampling is determined colorimetrically by reacting the nitrite ion with phosphoric acid, sulfanilamide, and N_(1naphthyl) - ethylenediamine dihydrochloride (NEDA) and measuring the absorbance of the highly colored azo dye at 540 n m. 1.2. The nominal range of the method is 9 to 750 ~g N02/m3) The range of the analysis is 0.04 to 2.0 ~g N02/ml, following Beer's Law throughout this range (0 to 1.0 absorbance units). 2.0 SCOPE: This method is applicable to 24 hours integrated sampling of N02 in ambient air. 3.0 INTERFERENCES: 3.1. Nitric oxide (NO) is a positive interferant and carbon dioxide (CO2) is a negative interferant. The average error resulting from normal ambient concentrations on NO and CO2) is small for most monitoring situations and does not necessitate applying a correction to measurement obtained with the method3). 3.2. Potential interference from sulfur dioxide (S02) is eliminated by converting any S02 to sulfate with hydrogen peroxide during analysis

4.0 SAMPLING PRESERVATION: Collected samples are stable for at least six weeks at room temperature. Stored samples should be tightly sealed to prevent absorption of N02from the atmosphere. 5.0 Apparatus of Equipment: 5.1. Volumetric Flasks - 100, 250, 500, 1, 000 ml. 5.2. Pipets - 1, 2, 5, 10, 20, 50 ml volumetric; 2ml, graduated in 1/10 ml intervals. 5.3. Test Tubes - Approximately 150 mm long x20 mm diameter. 5.4. Spectrophotometer Capable of measuring absorbance at 540 nm; equipped with 1 cm optical path length cells.

6.0 REAGENTS: 6.1. Sodium Hydroxide:

6.2. Sodium Arsenate 6.3. Absorbing Reagents - Dissolve 4.0 g of sodium hydroxide in Distilled water, add 1.0 g of sodium arsenite, and dilute to 1,000 ml with distilled water. 6.4. Sulfanilamide Melting point 165 to 167'C. 6.5. N-(Naphthyl)-ethylenediamine Di-hydrochloride (NEDA) -1 % Aqueous solution should have only one absorption peak at 320 nm over the range of 260-400 nm. NEDA showing more than one absorption peak over this range is impure and should not be used.

6.6. Hydrogen Peroxide, 30%. 6.7. Phosphoric Acid, 85%. 6.8. Sulfanilamide, Solution - Dissolve 20 g of sulfanilamide in 700 ml of distilled water. Add, with mixing, 50 ml of 85% phosphoric acid and dilute to 1,000 m!. This solution is stable for one month, if refrigerated. 6.9. NEDA Solution Dissolve 0.5g of NEDA in 500 ml of distilled water. This solution is stable for one month, if refrigerated and protected from light. 6.10. Hydrogen Peroxide Solution - Dilute 0.2 ml of 30% hydrogen peroxide to 250 ml with distilled water. This solution may be used for one month, if, refrigerated and protected from light.

7.0 PROCEDURE: 7.1. Preparation of Calibration Graph 7.2. Sodium Nitrite Assay of 97% NaN02 or greater. 7.3. Sodium Nitrite Stock Solution (1000 J.1g N02 Iml) Dissolve 1.5 g of desiccated sodium nitrite in distilled water and dilute to 1,000 ml such that a solution containing 1000 J.1g N02/ml is obtained. The amount of NAN02 to be used if, the assay percent is less than 100% is calculated as follows:

G = 1.500 A Where: G = Amount of NaN02. grams 1.500 = Gravimetric conversion factor A = Assay, percent (should be 97 or greater)

This stock solution can be stored for six weeks, if refrigerated. 7.4. Sodium Nitrite Working Standard (1.0 J.1g N02/ml) 7.5. Solution A - Pipet 5 ml of the stock solution into a 500 ml volumetric flask and dilute to volume with distilled water. This contains 10 flg N02 1m!.

7.6. Solution B Pipet 25 ml of solution A into a 250 volumetric flask and dilute to volume with absorbing solution. This contains 1 flg N02 1m!. Prepare fresh daily.

8.0CALIBRATION: 8.1. Spectrophotometer: Prepare calibration curve using 1 flg/ml working standards. 8.2. In accordance with the analytical procedure given in 9.4, measure and record the absorbance for each calibration standard (0,1,2,3,4,5,6,7,8,9,10,11115,20 flg N02. 8.3. Plot absorbance (y-axis) versus the corresponding concentration in flg N02/50 ml final solution (x-axis). Draw or compute the straight line best fitting the data to obtain the calibration curve.

9.0PROCEDURE: Take 10 ml of Sample in a 100 or 50 ml capacity test tube then

add 1 ml of Hydrogen Peroxide Solution + 10 ml of Sulfanilamide Solution, + 1.4 ml of NEDA Solution, with through mixing after the addition of each reagent and make up to 30 ml with distilled water or Absorbing Reagent, leave it for 10 min for colour development (Pink Colour) measure & record the absorbing or % T at 540 nm against blank. 10.0 CALCULATION:

100-% T Slope x 30 flg/m3 = Volume of x Sample taken

Air Sample in m3 for estimation

N02 Concentration in Analyzed Sample Determine flg N02 Iml graphically from the calibration curve or compute from the slope and intercept values N02 Concentration in Air Sample

as follows:

- Calculate as ~g of N02 per cubic meter of

~g/N02/m3= UQ/NO? X Vs X 0 Va X 0.82 X Vt Where: ~g/N02 Va m3 0 Vs Vt = N02 concentration in analysed sample = Volume of air sample, = Sampling efficiency = Dilution factor (0 = 1 for no dilution; 0 = 2 for 1: 1 dilution). = Final volume of sampling solution = Aliquot taken for analysis

The N02 concentration may be calculated as ppm using: ppm N02 = (lJg/N02/m3)x 5.32 x 10-4

Table of Contents
SL.
No. Method Description
Bioassay Test for Evaluating Acute Toxicity of

Method Number
SOP/BTD/01

Page Number
1

1 2

Industrial Effluents and Waste Waters Using Common Carp Bioassay Test for Evaluating Acute Toxicity of Industrial Effluents and Waste Waters - Part 2 using Toxicity Factor to Zebra Fish Determination of Total Coli forms and Fecal Coli

SOP/BTD/02

3 4

Forms Determination of Total Coliforms and Fecal Coliforms (MPN Method)

SOP/BTD/03 SOP/BTD/04

12 14

Bioassay Test for Evaluating Acute Toxicity of Industrial Effluents and Waste Water Using Cyprinus Carpio (Common Carp)
1.0 Introduction: The Bio-assay is very useful in connection with the control of effluent disposal. Bioassay test is the measurement of adverse effects of Industrial effluent against certain species. This test is necessary in water pollution evaluations because chemical and physical tests are not sufficient to assess potential effects on aquatic organisms. The interaction of chemical factors or toxic effects of complex materials cannot be determined with the help of analytical chemistry. The toxicity of effluents can be greatly influenced by interaction between their individual components and the dissolved minerals present in widely varying amounts in the receiving waters. The specific biological tests enable us to examine the effect of water components on test organisms under defined test conditions.

2.0 Methods: 2.1. The Indian Standard !S-6582-1971 lays down the Bio-Assay methods for evaluating the acute toxicity of industrial effluents and waste water to fish. The toxic effect is determined in terms of death of test organism in waste water samples with standard dilution water (Control). 3.0 Principle of Bioassay test using Fish: 3.1. The fish is the terminal member of aquatic food chain. It reacts very sensitively to changes in their environment. They suffer damage, if the water quality deteriorates. 3.2. Fish is affected in presence of toxic substances mainly in two ways: 3.2.1.1. Epithelia absorb the substances and are damaged, e.g. gills stick together or get congested with mucus and get destroyed 3.2.1.2. Besides these immediate effects, harmful substances are adsorbed through gills, skin or intestine and the physiological functions get impaired. These effects may ultimately lead to the death of fish.

4.0 Time Limit: 4.1. If all the test fishes survive in the test samples even after 96 hours the sample shall be reported as free from toxic substance. If there is no survival of fishes it is concluded that the waste water contains toxic substance as per EPAct 1986 standard.

5.0 Basic Requirements for Bioassay tests:

5.1. An abundant supply of water of desired quality. 5.2. An adequate space and well planned holding culturing and testing equipments (pH meter, conductivity meter, DO meter, Thermometer etc.) 5.3. An adequate source of healthy experimental organisms. 5.4. Stocking tanks - Tank should be made up of glass aquarium capable of holding approximately 2001trs. of water provided with arrangement for an aeration water 5.5. Acclimatization tanks - Tank should be made up of glass capable of holding approximately 50ltrs. of water provided with arrangement for aeration. 5.6. Test tanks - Tank should be made up of glass capable of holding approximately 20ltrs. of water provided with arrangement for aeration. 5.7. Glass aquarium- 20 Its capacity 5.8. Pipettes- 5, 10, 20 ml 5.9. Volumetric flask - 100 ml and 1 Itr 5.10. Measuring cylinders - 100 ml and 1 ml 5.11. Beakers - 100, 200, 500, and 1 Itr 5.12. Thermometer Aerators 5.13. Thermostats 5.14. Tanks for fish stock and digestion water 5.15. Hand nets 5.16. Water filter with activated charcoal

6.0 Equipments and Calibration:

6.1. DO meter: Application: Used to measure dissolved oxygen in water, waste water and industrial effluent sample. 6.2. pH meter: Application: Used to measure pH in water, waste water and industrial effluent sample. 6.3. Conductivity meter: Application: Used to measure conductivity in water, waste water and industrial effluent sample.

6.4. Calibration: 6.4.1.1. Prior to any use, calibrate the instrument daily and record the results in instrument calibration log. 6.4.1.2. See the manual for calibration of pH, Conductivity and DO. All calibration must be performed with standard provided with the instrument. 6.4.1.3. Record the slope of the instrument in the instrument calibration log book. 7.0 Reagents and Preparation: 7.1. Calcium chloride (CaCI2, 2 H2O) 7.2. Magnesium Sulphate (MgS04, 7 H2O) 7.3. Sodium Bicarbonate (NaHC03) 7.4. Potassium Chloride (KCI) 8.0 Quality of Reagents:

8.1. Pure chemicals (chemicals that do not contain impurities which affect the result of analysis) and distilled water or demineralized water shall be employed in the tests.
9.0 Preparation of dilution water: 9.1. Water to be used as dilution water shall be uncontaminated filtered chlorine free water of similar quality, with respect to its dissolved mineral content or prepared artificial from distilled or demineralized water as follows 9.2. The pH Dilution water is prepared by mixing 25ml each of the following four stock solutions and diluting to 1 litre with water. Thereafter the pH is adjusted using sodium hydroxide or hydrochloride acid solution. 9.3. Calcium chloride solution- Dissolve 11.76g of calcium chloride dehydrate (CaCI2, 2 H2O) in water and dilute to 1 liter. 9.4. Magnesium sulphate solution- Dissolve 4.93g of magnesium sulphate heptahydrate (MgS04, 7H20) in water and dilute to 1 litre. 9.5. Sodium bicarbonate solution- Dissolve 2.59 g of sodium bicarbonate (NaHC03) in water and dilute to 1 litre. 9.6. Potassium chloride- Dissolve 0.23 g of potassium chloride (KCI) in water and dilute to 1 litre. 9.7. Temperature, alkalinity, conductivity and hardness of this dilution water shall be adjusted to those of the natural water (pH of 7.8 :i: 0.2 and 'hardness of approximately 200 mg/l as CaC03). The dilution water shall be aerated for 24 h prior to the test. The dissolved oxygen content of the dilution water shall not be below 4 mgll and shall not contain residual chlorine

. 10.0 Procedure: 10.1. As per the IS 6582: 1971 recommendations, cyprinus carpio [common carp] fish of family cyprinidae and of size around 5 to 7.5 cm is used for the bio assay test. 11.0 Stock of Fish: 11.1. Fish should be brought to the laboratory and stocked in the stocking tank and maintained in good conditions by aerating and feeding at temperature of approximately 25c. 11.2. They should be fed fairly regularly with fish food (dried pellets) or Tubifex worms during the acclimatization period in aerated chlorine free potable water of roughly similar characteristics as the dilution water. The population density of fish shall not exceed 1 g per litre. 11.3. The minimum acclimatization period shall be 10 days prior to test under lab conditions. Mortality shall not exceed 1 percent per week. The following condition is maintained: 11.3.1.1. 24 hr prior to the test, feeding is stopped. 11.3.1.2. Fish for a single test, is selected from a tank with population of the same stock. 11.3.1.3. After obtaining the correct temperature and DO>4.0mg/l, 10 fishes is placed in each aquaria which is filled with 10 Itrs of test solution (Effluent) in triplicates. For each test a control shall be maintained by placing 10 fishes in a aquaria which is filled with 10 liters of dilution water. 11.3.1.4. Any fish dropped or harmed during the transfer is discarded. All the fish, for a single test shall be introduced to test aquaria within a period of 30 min. 11.3.1.5. The DO content of the test solution or dilution water should not fall below 4 mgll throughout the experiment. 11.3.1.6. Duration of the test is 96 hrs. The dissolved oxygen concentration, pH and count of the dead fish in each aquaria is measured at the beginning of the test and each after 6hrs, 24

hrs, 48hrs, 72hrs & 96hrs.

11 .3.1.7. The dead fish is removed from the aquaria, in case of fish dying in the control aquaria the test is discarded and fresh testing should be repeated. 11.3.1.8. The handling of fish, solution and procedures is carried out in the premises free from harmful vapors. Any disturbance that may change the behavior of fish is avoided.

All tests is carried out under normal laboratory illumination with natural photoperiod. 11.3.1.10. The temperature is maintained at 25:t1.0C. 11,3.1.9. 12.0 REPORT: The test report should include the fonowing information's 12.1. The specification of the test effluent and full information for identification of test samples. 12.2 .Any deviation from the procedure specified in this standard and the reason for this, including a description of the circumstances which could have influenced the results. 12.3. Standards is specified as per E(P)A 1986 depending upon the categories of industries. 13.0 REFERENCES: 13.1. Standard methods for the examination of water and waste water APHA publication. 13.2. Bureau of Indian Standards 6582: 1971.

Bioassay Test for Evaluating Acute Toxicity of Industrial Effluents and Waste Water Using Toxicity Factor to Brachydanio Rerio (Zebra Fish)

1.0 Introduction: Toxicity tests provide results that are useful in protecting human
health and aquatic life from impacts caused by the release of contaminants into surface waters. Toxicity tests have been used to: 1.1. Assess the suitability of environmental conditions for aquatic life and establish acceptable receiving water concentrations for conventional parameters (such as DO, pH, temperature, salinity or turbidity). 1.2. To study the effects of water quality parameters on waste water toxicity and assess the toxicity of waste water to a variety of fresh, estuarine & marine test species. 1.3. Establish relative sensitivity of a group of standard aquatic organisms to effluent as well as standard toxicants. Assess the degree of wastewater treatment needed to meet water pollution control requirements. 1.4. Determine the effectiveness of wastewater treatment plants. Establish permissible effluents discharge rates, and Determine compliance water quality standards. 2.0 Methods: 2.1. The toxicity method is based on Toxicity Factor (TF) that is minimum number of times an effluent or waste water is required to be diluted for obtaining no mortality for Zebra Fish, 2.2. The Indian Standard IS-6582-Part 11-2001 lays down the Bio-Assay methods for evaluating the acute toxicity of industrial effluents and waste water to zebra fish. The toxic effect is determined in terms of death of test organism in waste water samples with standard dilution water (Control). 2.3. The standard prescribe an alternative method for determination of acute lethal toxicity of waste water and industrial effluent to Zebra fish fbradchvdanio rerio] under specified conditions. 3.0 Principal of Toxicity Test Using Fish: 3.1. The fish is the terminal member of aquatic food chain. It reacts very sensitive to changes in their environment. They suffer damage, if the water quality deteriorates. 3.2. Fish is affected in presence of toxic substances mainly in two ways:

3.2.1. Epithelia adsorb the substances and are damaged, e.g. gills stick together or get congested with mucus and get destroyed 3.2.2. Besides these immediate effects, harmful substances are adsorbed through gills, skin or intestine and the physiological functions get impaired. These effects may ultimately lead to the death of fish. 4.0Time Limit: If all the test fishes survive in the test samples even after 48 hours the sample shall be reported as free from toxic substance. If there is no survival of fishes it is concluded that the waste water contains toxic substance as per E(P)A 1986 standard. 5.0 Basic Requirements for Toxicity Tests: 5.1. An abundant supply of water of desired quality. 5.2. An adequate space and well planned holding culturing and testing equipments (pH meter, conductivity meter, DO meter, Thermometer etc.) 5.3. An adequate source of healthy experimental organisms. 5.4. Stocking tanks - Tank should be made up of glass capable of holding approximately 2001trs. of water provided with arrangement for aeration. 5.5. Acclamatation tanks - Tank should be made up of glass capable of holding approximately 50ltrs. of water provided with arrangement for aeration. 5.6. Test beakers - Beakers should be made up of glass capable of holding approximately 5ltrs. of water. 5.7. Glass beaker- 5 Itrs capacity 5.8. Pipettes- 5, 10,20 ml 5.9. Volumetric pipettes- 10 ml, 25ml & 50 ml 5.10. Volumetric flask - 100 ml and 1 Itr 5.11. Measuring cylinders - 100 ml and 1 ml 5.12. Beakers - 100, 200, 500, and 1 Itr 5.13. Thermometer 5.14.Tanks for fish stock and dilution water 5.15. Hand nets 6.0 Equipments and Calibration: 6.1. DO meter: Application: Used to measure dissolved oxygen in water, waste water and industrial effluent sample. 6.2. pH meter: Application: Used to measure pH in water, waste water and industrial effluent sample. 6.3. Conductivity meter: Application:Used to measure conductivity in water, waste water and industrial effluent sample.

7.0 Calibration: 7.1. Prior to any use, calibrate the instrument daily and record the results in instrument calibration log. 7.2. See the manual for calibration of pH, Conductivity and DO. After calibrating, meter is left on for the remaining hours of the day. All calibration must be performed with standard provided with the instrument. 7.3. Record the slope of the instrument in the instrument calibration log book. 8.0 Reagents and Preparation 8.1. factor(T) Dilution Calcium chloride (CaCI2, 2waste water to Ratio of the H2O) Parts of dilution water to be added to 8.2. Magnesium Sulphate (MgS04, 7 H2O) test water (by volume) 1 part of waste water (by volume) 8.3. 1 Sodium Bicarbonate (NaHC03) 1:1 0 8.4. Potassium Chloride (KCI) 2 1:2 1 4 1:4 3 9.0 Quality of Reagents: Pure chemicals (chemicals that do not contain impurities which 8 1:8 7 affect the result of analysis) and distilled water or demineralized water shall be 16 1:16 15 employed in the tests. 32 1:32 31 64 1:64 63 10.0 Preparation of dilution water: 125 1:125 124 Water to be used as dilution water shall be uncontaminated filtered chlorine free 250 of similar quality, with1:250 249 water respect to its dissolved mineral content or prepared 500 1:500 499 artificial from distilled or demineralized water as follows 1000 1:1000 999 10.1. Dilution water is prepared by mixing 25ml each of the following four stock 11.3.solutions and diluting to 1 liter with has to be selectedthe pH is adjusted using Upto 5 consecutive concentration water. Thereafter based on previous experience with regarded potential toxicity of the sodium hydroxide or hydrochloride acid solution. effluent. Each test beaker is filled with 2 Itrs of test water. One 11.76g of calcium chloride dehydrate 10.2. Calcium chloride solution- Dissolvecontrol beaker containing only dilution water is provided for in water and test may be performed with one replicate. (CaCb, 2 H2O) the test. The dilute to 1 liter. 11.4.As per the IS 6582:2001 recommendations, brachvdanio ratio [zebra fish] 10.3. Magnesium sulphate solution- Dissolve 4.93g of magnesium sulphate should have (MgS04, 7H20) in water and dilute to 0.3gm heptahydrate30:t5mm size, corresponding to 0.2 to 1 liter. of mass is used for 1004. the toxicity test. Sodium bicarbonate solution- Dissolve 2.59 g of sodium bicarbonate (NaHC03) in water and dilute to 1 liter. 10.5. Potassium chloride- Dissolve 0.23 g of potassium chloride (KCI) in water and dilute to 1 liter. 10.6.The pH Temperature, alkalinity, conductivity and hardness of this dilution water shall be adjusted to those of the natural water (pH of 7.8 :i: 0.2 and hardness of approximately 200 mg/I as CaC03). The dilution water shall be aerated for 24 h prior to the test. The dissolved oxygen content of the dilution water shall not be below 4 mg/l and shall not contain residual chlorine.

11.0 Procedure: There are 2 steps to conduct the toxicity test: 12.4. The minimum acclimatization period shall be 10 days prior to test under lab conditions. 11.1. Range finding exceed 1 percent per of toxicity of an effluent is unknown, a 12.5. Mortality shall nottest: In case the rangeweek. range finding test should be performed prior to the standard test determine the concentration 13.0 Test Conditions: range within which 0 percent and 10 percent mortality is absorbed within the 24 shall 13.1. Following test conditionshour. be maintained: 11.2. Final test: The test water is prepared by adding the effluent to the 13.2. 24 hr prior to the test, feeding is stopped. dilution water in a rounded logarithmic tank with population given below 13.3. Fish for a single test, is selected from a progression scale as of the same stock. 13.4. After obtaining the correct temperature, 5 fishes is placed in each beaker. 13.5. Any fish dropped or harmed during the transfer is discarded. All the fish, for a single test is introduced to test beaker within a period of 30 min. 13.6. The samples and test solutions should not be aerated or treated; else BOD content or extreme pH may influence the result. 13.7. Duration of the test is 48 hrs. The dissolved oxygen concentration, pH and count of the dead fish in each beaker shall be measured of the beginning of the test and each after 2hrs, 6hrs, 24 hrs & 48hrs 13.8. The dead fish should be removed from the beaker, in case of fish dying in the control beaker the test is discarded and fresh testing is repeated. 13.9. The handling of fish, solution and procedures is carried out in the premises free from harmful vapours. Any disturbance that may change the behavior of fish is avoided. 13.10. All tests is carried out under normal laboratory illumination with natural photo period. 13.11. The temperature is maintained at 25 :i:1.0C.

14.0 Report: 14.1. The dilution factor of the test solution with the highest concentration of effluent in which all fish survive shall be recorded in rounded numbers as 12.0 Stock Tf. Fish: of 14.2.T = Dilution factor (T) is laboratory and stocked in the stocking tank 12.1. Fish should be brought to the the numerical expression of the volumetric proportion of the waste water in the test aerating and feeding atthe and maintained in good conditions by water. The 'T' signifies toxicity unit. of approximately 25c. temperature 14.3. Ex: If be 8, fairly, regularly or some of the fish pellets) or 12.2.They shouldTf =fed it shows that all with fish food (drieddie in the test solution for a T value, less than 8 and all are alivein aerated chlorine free Tubifex worms during the acclimatization period in test solutions for a T value of 8 and above, after 48 hr.characteristics as the dilution water. potable water of roughly similar 12.3. The population density of fish shall not exceed 1 g per lite~. 15.0 References: Standard methods for the examination of water and waste water APHA publication. Bureau of Indian Standards 6582:2001

Determination of Total Coli forms and Fecal Coli Forms

(Standard Plate Count)
1.0 Introduction: The standard plate count is a common procedure for the
enumeration of living bacteria. This method is used to determine the viable population of bacteria in a sample.

2.0 Method: Standard Plate Count

3.0 Principle: In plate count, Bacterial reproduction on solid medium results in the
formation of a macroscopic colony visible to the naked eye. The formation of visible colonies generally takes 16 to 24 hours. It is assumed that each colony arises from an individual bacterial cell. Therefore by counting the number of colonies that develop, forming units (DFUs) and by taking into account the dilution factors, the concentration of bacteria (number of bacteria/ml) in the original sample can be determined. Countable plates are those having colonies between 30 and 300 colonies. Fewer than 30 colonies are not acceptable for statistical reasons and more than 300 colonies on a plate are likely to produce colonies too close to each other to be distinguished as individual DFUs. 4.0 Time Limit: Analyse the samples immediately

5.0 Reagents and Preparation:

5.1. Preparation of Saline: Dissolve 8.5gms of Sodium Chloride in 1000ml distilled water. Transfer 9ml of saline to each test tube, plug with cotton and sterilize in Autoclave at 121C at 15 Ibs for 20 minutes. 5.2. Hitouch E colif Coli forms Flexi plate \

6.0 Procedure:
6.1. Arrange the dilution blanks serially in a test tube stand and label them with their dilution factor. 6.2. Transfer 1 ml of water sample to first tube containing 9 ml saline to Make 1 :10 dilution (10-1). Vigorously shake the dilution for 5 minutes to obtain uniform suspension of microorganisms. 6.3. Transfer 1 ml of suspension into the saline tube 2 with a sterile pipette

under aseptic conditions to make 1: 100 dilution (10-2) and shake it well for about 5 minutes. 6.4. Similarly, go on increasing dilutions up to 10-6 or more, by using a sterile
pipette every time. Transfer 1 ml of well shaken sample from each of the dilution tube to the three sterile flexi plates. 6.5. Mix the sample thoroughly with the medium and while mixing, rotate the plate in clockwise and anticlockwise directions. 6.6. Incubate the plates at 37C in an inverted position for 24 hours. Count the number of colonies 1m!.

7.0 Calculation: Calculate the number of organisms per ml of the water sample by applying the formula: No of viable cellsl ml of the = Averaqe No of colonies X Dilution factor original sample (CFUs) Volume of sample taken

8.0 Reference: . APHA, AWNA, WEF [For the Examination of WATER and Waste Water] standard methods Book 20th Edition 1995 Washington, DC. . Experiments in Microbiology, Plant pathology, Tissue culture and mushroom production technology, 3rd edition by K.R. Aneja, New age international publishers.

Determination ofsample Coliforms and Fecal Coliforms 5.8.1 Water Total 5.8.2 Lactose broth medium Technique) (Multiple Tube Fermentation or Mac-conkey broth medium
1.0
2.0
3.0

5.8 Requirements:

5.8.3 Durham tubes 15 5.8.4 1O-ml double-strength lactose broth test Introduction: Multiple Tube Fermentation tubes is used detect 5.8.5 6-ml single-strength lactose broth coliform bacteria by acid and gas productiontubes 5.8.6 Sterile pipettes, one each of 10-ml, 1-ml and 0.1-ml capacity Method: Multiple Tube Fermentation Technique, Most probable Number 5.8.7 Bunsen burner / spirit lamp 5.8.8 Glass organisms can ferment lactose present in the medium. Principle: Coliform marker pencil Hence they are lactose fermenters. Coliform organisms can be represented by acid and gas production. Acid production is indicated by color change in the medium from purple to yellow, which is a lower acidic pH. Gas production is indicated by gas bubble present in the durbanis tube; due to the fermentation of carbohydrates is lactose in the medium, to Carbon-di-oxide.

4.0 5.0

Time Limit: Analyse the samples immediately Presumptive coliform test 5.1 5.2 5.3 Principle The presumptive coliforms test is used to detect and estimate coliforms population of a water sample. In this test, known volumes of water (dilutions) are added to lactose fermentation tubes or Mac-conkey fermentation tubes and production of acid and gas from the fermentation of lactose is a positive test for Coliform bacteria. The lactose broth or Mac-conkey broth used in this test is selective for the isolation of coliforms because of the addition of bile and lauryl sulphate or brilliant green. A pH indicator such as Bromocresol purple is also added to lactose broth for the detection of acid. The colour of the indictor changes to yellow with the fermentation of acid from lactose. A statistical method is used to estimate the population of coliforms, which means that the result obtained is expressed as the most probable number (MPN) of coliforms. A count of number of lactose fermentation tubes showing production of gas following the incubation period is taken and MPN is found by matching the results with those provided in the statistical table.

5.4

5.5 5.6

5.7

6.0

Procedure: 6.1.1. Collect water sample. Prepare double strength [80gms in 1000ml distilled water] and single strength [40gms in 1000ml] of mac-conkey 6.1. broth. Dispense 10ml each of the double strength mac-conkey broth into 5 2 test tubes. Insert one Durham's tube to each test tube. 6.1.3 Dispense 6ml each of single - strength mac-conkey broth into 10 test tubes.Plug the test tubes with cotton and sterilize the tubes in
Autoclave at 121

6.1.4 Arrange fermentation tubes in rows of 15 in a test tube rack. Mix the water sample by thoroughly shaking. Aseptically inoculate 10ml of sample for 5test tubes of double 6.1.5 strength using 10ml sterile pipette. Aseptically inoculate the five tubes with 1 ml of water sample to single strength tubes using a 1 ml pipette 6.1.6 Using a 0.1 ml pipette, aseptically inoculate the five tubes with 0.1 ml of water sample. Incubate all the 15 inoculated tubes at 37C for 48hours. 7.0

C at 15 Ibs for 20 mins.

Confirmed Coliform test:

7.1 7.2 7.3 Principle This test is used to confirm the presence of coliforms in water samples showing positive or doubtful presumptive test. In the confirmed test, the samples from the positive presumptive macconkey broth tubes are inoculated into brilliant green bile lactose broth and streaked onto a eosin - methylene blue (EMB) agar plate. EMB agar is used because of the presence of the dye methylene blue which inhibits the growth of gram - positive bacteria, allowing the growth of gram - negative bacteria and lactose fermenting bacteria gives colored colonies (a positive confirmed test) lactose fermenters produce colorless - colonies on EMB agar.

7.4

8.2

7.5

Requirements:
7.5.1 Brilliant green bile lactose broth 7.5.2 EMB agar plate 7.5.3 24 - hour old positive mac-conkey broth culture 7.5.4 Spirit lamp 7.5.5 Inoculating Loop

7.6

Procedure:
7.6.1 Inoculate the BGB tubes and EMB agar plate with the positive 24 hour lactose broth culture with a sterile inoculating loop. 7.6.2 Incubate the tubes and plate for 24 - 48 hrs at 37C.

7.7

Observation: Examine the BGB tubes for total coliforms and presence or absence of E. Coli Colonies on EMB agar plate.

7.8

Results: Production of acid and gas in BGB tubes at 24hrs incubation

indicates a positive total Coliform test. The appearance of typical Coliform colonies with dark centers and metallic sheen on EMB agar plate indicates the presence of E.Coli.

8.0

Procedure No 3: Completed coliforms test

8.1 Principle: Completed test is used as a confirmatory test for the presence of E. coli in a water sample. In the test, lactose-positive colonies from EMB agar are isolated and inoculated into a lactose broth tube and streaked on a nutrient agar plate to perform gramstaining. If there is production of acid and gas in the inoculated lactose broth and there are rod-shaped bacteria showing gramnegative reaction, these confirm the presence of E. coli in the water sample and is considered a positive completed test.

Requirements: 8.2.1 24-hour Coliform-positive EMB agar culture (from the confirmed test) 8.2.2 Lactose - fermentation broth tube 8.2.3 Nutrient agar slant 8.2.4 Gram-stain reagents 8.2.5 Inoculating loop 8.2.6 Bunsen burner I spirit lamp 8.3

Procedure:
8.3.1 8.3.2 Inoculate lactose-fermentation broth tube with the isolated Coliform colony from the EMB agar plate using inoculating loop Streak nutrient agar slant with the colony from the EMB agar plate using inoculating loop. 8.3.3 Incubate both the inoculated broth and slant at 37C for 24 hours. 8.3.4 Gram-stain the organisms found on the slant.

8.4

Observations: Examine lactose fermentation broth tube for the

production of acid and gas. Observe the slide for positive or negative gram reaction and cell morphology.

8.5

Results: Production of acid and gas in an inoculated lactose broth and presence of gram-negative rods indicate a positive completed test and confirm the presence of Coliform bacteria in the water sample.

9.0

Procedure for Fecal coli form test:

9.1 Principle:
9.1.1 Prepare E.C Broth ( 37gms in 1000ml ) and dispense 6 ml of the medium in each test tube. 9.1.2 Insert one Durham's tube to each test tube. 9.1.3 Plug the test tubes with cotton and sterilize the tubes in Autoclave at 121 C at 151bs for 20 mins. 9.1.4 Aseptically inoculate a drop of sample from the positive mac conkey tubes into E. C broth tubes. 9.1.5 Incubate the tubes at 37C for 24hrs.

9.2 9.3

Observation: Observe the tubes for acid and gas production.

Results: Production of acid and gas in an E.C broth tubes confirms the presence of fecal coli forms.

9.4

Calculation:

Table 1: MPN Index and 95% confidence limits for various combinations of positive results when five tubes are used per dilution (10 ml, 1.0 ml, 0.1 ml)
Number of five tubes giving a positive reaction 1.0 ml 0.1 ml 0 0 1 2 0 0 1 1 2 0 0 1 1 2 3 0 0 1 1 2 2 0 0 1 1 1 0 1 0 0 0 1 0 1 0 0 1 0 1 0 0 0 1 0 1 0 1 0 1 0 1 2

10 ml 0 0 0 0 1 1 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4

MPN index / 100 ml <2 2 2 4 2 4 4 6 6 4 7 7 9 9 12 8 11 11 14 14 17 13 17 17 21 26

10 ml 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

1.0 ml 2 2 3 3 4 0 0 0 1 1 1 2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 5 5 5

0.1 ml 0 1 0 1 0 0 1 2 0 1 2 0 1 2 0 1 2 3 0 1 2 3 4 0 1 2 3 4 5

MPN index / 100 ml 22 26 27 33 34 23 30 40 30 50 60 50 70 90 80 110 140 170 130 170 220 280 350 240 300 500 900 1600 2: 1600

10.0 Reference: . Standard Method, 19th edition 1995. . Experiments in Microbiology, Plant pathology, Tissue culture and mushroom production technology, 3rd edition by K.R. Aneja, New age intemational publishers.

Table of Contents
SL. No
1.
2. 3. 4. 5.

Method Description
Determination of p for Soil and Sludge Samples Estimation of Conductivity in soil and sludge samples. Determination of organic carbon. Determination of Total Water Soluble Solids Determination of Total Kjeldhal Nitrogen for soil and sludge samples

Method Number
SOP/HWTD/01 SOP/HWTD/02 SOP/HWTD/03 SOP/HWTD/04 SOP/HWTD/05

Page Number
1 3 4 6 8

6. 7.

Estimation of Potassium for soil and sludge samples Estimation of Total Organic Carbon

SOP/HWTD/06 SOP/HWTD/07

11 13

Determination of pH for Soil and Sludge Samples

1.0 Introduction: pH of soil is an important physico chemical characteristic because it
influences: Availability of nutrients in soil, Microbial activity in soil, Physical properties like permeability and structure. Pure water has a pH of 7. As the hydrogen ion activity increases the pH value decreases and vice-versa. Thus this value is the measure of acidity and alkalinity of sailor any other substances in an aqueous solution. The soil pH can be measured on the extracted soil solution or on a suspension of soil in water.

2.0 Method: pH Meter.

3.0 Principle: 3.1. pH is a measure of Hydrogen ion concentration it is defined as the negative logarithm of H+ ion concentration or more rPrecisely Hydrogen ion activity of a solution and expressed by equation: p = -log ( H+) 3.2. It can be measured electrometrically using pH meter, This method is applicable to solid samples like soil, solid waste, sewage, sludge, municipal solid waste, compost.

4.0 Apparatus:

4.1. pH meter 4.2. Beaker, 100 ml

5.0 Reagents: Standard pH buffers pH 4.0, 7.0, 9.2

6.0 Procedure: 6.1. Take about 10 g. of the powdered and dried (at room condition around 25C for 24 hours) sample in a beaker. 6.2. Add 50 ml. of distilled water and stir for one hour ( 1:5 ratio, 1 part sample and 5 part distilled water) 6.3. Calibrate the pH meter using standard pH buffers of pH 4.0, 7.0 & 9.2. Measure the pH of the solid suspension using for standard pH meter following the procedure. 6.4. Record the pH value (1 :5) in 2 decimal units. Note: The Conductivity of the distilled water to be used for the preparation of the standard buffer solutions and KCL solution should be less than 2.0 MS/cm. 6.5. Discard the buffersolution if there is any mold growth or contamination.

7.0 Expression of Results: Express the result as pH (1 :50) in 2 deCimal units.

8.0 Reference: CPCB Manual for Analysis of Municipal Solid Waste

Estimation of Conductivity in Soil and Sludge samples

1.0 Introduction: conductivity is a measure of the ability of an aqueous solution to carry an
electric current. This ability depends on the presence of ions on their total concentration, mobility and valence and on the temperature of measurement solution of most inorganic compounds are relatively good conductors. Conversely molecules of organic compounds that do not dissociate in aqueous solution conduct current very poorly, if at all. 2.0 Method: Conductivity Meter. 3.0 Principle: 3.1. Electrical conductivity (EC) of the sample saturation indicates ionisable constituents of the solution and is measured using a conductivity meter. This method is applicable to solid samples like soil, sewage sludge, Municipal solid waste, compost.

4.0 Apparatus: 4.1. Conductivity meter 4.2. Beaker, 100 ml 5.0 Reagents: 5.1. Standard KCL (0.01 m) : Dissolve 0.7456 g. dry KCL in 1 liter of distilled water. 5.2. This solution has on electrical Conductivity of 1412 MS/cm @ 25 C.
6.0 Procedure: 6.1. Take about 10 g. of the air dried sample in a beaker, add 50 ml of distilled water and stir for one hour (1:5 ratio) 6.2. Calibrate the Conductivity meter using standard 0.01 KCL solution. 6.3. Immerse the Conductivity cell in the solution and measure Conductivity as per the procedure given in the instrument manual.

7.0 Calculation :Report the Conductivity in MS/cm (1:5 ratio) 8.0 Expression of results: Express the result as Conductivity (1 :5) in 2 decimal units. 9.0 Reference : Jackson M.L. (1967): Soil Chemical Analysis. Prentice - Hill of India
Pvt. Ltd., New Delhi.

Determination of Organic Carbon

1.0
2.0 Method: Walkley and Black Method Principle 2.1 Organic carbon is determined by treating the solid sample with a known
amount of potassium dichromate (K2Cr207) .in presence of concentrated sulphuric acid (H2 S04) Oxidation of organic carbon by K2Cr207 takes place as per the given reaction. 2K2Cr207 + 8H2S04 ---+ 2K2 S04 + 2Cr2 (S04h + 8H20 + 6 (0) 3C + 6 (0) ---+ 3 CO2 (organic Carbon)

2.2

The excess chromic acid left unutilized by the organic matter is back titrated with standard Ferrous Ammonium sulphate, Fe (NH4h (S04)2 6H20 (1.0N) solution using Diphenylamine indicator (Walkley and Black method) This method is applicable to solid samples like soil, solid waste, sewage sludge, municipal waste and Compost.

3.0

Apparatus:
3.1 3.2 3.3 3.4 3.5
Analytical balance Erlenmeyer conical flask (500 ml.) Pipette (10 ml) Burette (50 ml) Measuring cylinder (50 & 250 ml)

4.0

Reagents:
4.1 4.2 4.3
4.4 Potassium dichromate (1.0N): Dissolve 49.04 gm. of dried potassium dichromate (K2Cr207) in distilled water and make up to liter. Sulphuric acid (conc.) Diphenylamine indicator: Dissolve 0.5 gm. Diphenylamine in a mixture of 100 ml. conc. H2SO4 and 200 ml. distilled water. Ferrous ammonium sulphate (1.0N) : Dissolve 392.2 gm. of Fe (NH4h (S04)2. 6H20 in 1000 ml. of distilled water containing 20 ml. of conc. H2 S04. Sodium Fluoride Ortho phosphoric acid (85 %) : Mix 85 ml. of ortho-phosphoric acid (100 %) and 15 ml. distilled water.

4.5 4.6

5.0

Procedure: 5.1 5.2 5.3 5.4 5.5 5.6 Take 0.25 gm. or suitable quantity of oven dried (105C) sample thoroughly ground and sieved through 0.2 mm sieve. Transfer the weighed sample to a 500 ml. conical flask and add 10 ml. 1 N K2Cr207 solution. Then add 20 ml. Conc. H2 S04 and mix gently by rotation to ensure complete contact. Allow the mixture to stand for 30 minutes. Add 200 ml. distilled water, 10 ml. of H3 P04 (85%), 0.2 gm. of Sodium
fluoride and 1 ml. of diphenylamine indicator. Titrate the solution with 1 N Fe (NH4h (S04h 6H2O solution. The color is dull green at the beginning and then shifts to turbid blue as the titration proceeds at the end point the color sharply turns to brilliant green. Keep a blank titration without sample and follow the same procedure.

6.0

Calculation: % Organic carbon (%C) =

S

{ 10- (10 X~) } X 0.003 X 100

B 0.77

Wt

= ml. of 1.0 N Ferrous Ammonium Sulphate solution used for sample titration. B = ml of 1.0 N ferrous Ammonium sulphate solution used for blank titration. Wt = Weight of the solid sample taken for analysis in gm. 0.77 = It is the recovery factor; i.e recovery of 77% of organic matter is considered in his method.

Note: One ml of 1 N potassium dichloromate is equivalent to 3 mg of Carbon.The value of % carbon can be expressed as % organic matter by multiplying % carbon with the factor 1. 724*as follows: % organic matter = % C x 1 .724, * The factor 1.724 is based on the assumption that carbon is only 58% organic matter.

7.0 8.0

Expression of Result: Express the result as organic carbon % in 2 decimal units on oven dry weight basis. Reference: Jackson, M.L.(1967) . Soil Chemical Analysis prentice-Hill of India Pvt. Ltd. New Delhi.

6.0 Calculation:

Estimation of Total Water Soluble Solids Total water soluble solids (TWSS) Mg/gm = (A-B) x 1000 Wt 1.0 Introduction: The term dissolved solids replaces the filterable solids or ions dissolved in water. Water constitutes the soil solution. The concentration and composition of this solution changes due to: , 1.1. mineralization of soil organic matter releasing nutrients 1.2. fertilizers and application of pesticides 1.3. rainfall or irrigation 1.4. evaporation or transportation 1.5. uptake of ions by plants
The result of these processes leads to variation in concentration and composition of soil solution. 2.0 Method: Gravimetric Method 3.0 Principle: Soluble salts which may be present in solid samples consists of ions like calcium, Magnesium, Sodium, Potassium, Chlorides, Sulphates, Nitrate, Carbonates, Bicarbonates etc. A well mixed sample extract obtained using distilled water is filtered through whatmann filter paper and the filtrate is evaporated to dryness in a beaker (or silica glass dish) the weight of the residue remaining in the beaker represents the Total water Soluble Solids (TWSS) 4.0Apparatus : 4.1. Chemical flask, 500 ml. Capacity. 4.2. Glass beaker or evaporation dishes (or high-silical glass), 100 ml. Capacity. 4.3. Stirrer or shaker 4.4. Whatmann filter paper, No. 42 4.5. Filtration assembly 4.6. Hot air oven 4.7. Dessicator 4.8. Analytical balance 5.0 Procedure: 5.1. Dry clean dish in an oven at 110 C for 1 hour, cool it in a desiccators and then take the initial weight of the dish. Repeat till a constant weight is obtained. 5.2. Take 100 g. or suitable quantity of oven dried (105 C) thoroughly grounded sample in a conical flask and mix with 50 ml. of distilled water. 5.3. (1:5 ratio) using a stirrer or shaker for 2 hours. 5.4. Filter the sample and transfer the whole volume of the extract into the evaporation dish already weighed. Evaporate dryness. 5.5. After complete evaporation keep the dish for cooling. Weight the dish and note the final weight.

Where A = Weight of the dish + Residue in gm. B = Weight of the empty dish in gm. Wt. = Weight of the solid sample taken for extraction in gm. 7.0 Expression of Results : Express the result as Total water soluble solids (1 :5) mg/gm. In 2 decimal units. 8.0 Reference: CPCB Manual for Analysis of Municipal Solid Waste. Soils and the Environment - Alan Wild

Estimation of Total Kjeldhal Nitrogen

1.0 Introduction: Total Kjeldhal Nitrogen is the sum of ammonical nitrogen and organic nitrogen. Ammonia and organic nitrogen determinations are important in determining whether sufficient available nitrogen is present as nutrient for biological treatment system. Nitrogen is an important nutrient for growth of plants. Nitrogen is added to the soil system in many forms of fertilizers. 2.0 Principal: Total Kjeldahl Nitrogen (TKN) is the sum of ammonia nitrogen and
organic nitrogen present "is the sample. It does not include nitrate nitrogen. In the presence of sulphuric acid, potassium sulphate and cupric sulphate (catalysis), the nitrogen of organic matter as well as free ammonia is converted to ammonia sulphate on digestion at 360 to 410C. An excess of alkali is then added to liberate ammonia and is distilled. The librated ammonia is absorbed in boric acid solution (mixed with indicator) and TKN is determined titrimetrically with standard sulphuric acid. This method is applicable to solid samples like soil, solids waste sewages sludge, municipal solid waste, compost

3.0 Method: Titrimetric method 4.0 Apparatus:

4.1. Kjeldahl tube or flask, 500 ml capacity 4.2. heating device with temperature range of 360-41 OOC 4.3. Fume hood or scrubber unit 4.4. kjeldhal distillation unit.

5.0 Reagents:
5.1. sulphuric acid (cone.) sp.gs.1.84 5.2. potassium sulphate - copper sulphate mixture 5.3. (10:1ratio) : prepare a mixture 10 gm. of potassium sulphate (K2S04) and 1 gm. of copper sulphate (CUS04. 5H2O) Alternatively use Kjeldahl tablet. 5.4. Phenolphthalein indicator solution: Dissolve 0.5 gm. of Phenolphthalein in 50m\. ethyl or isopropyl alcohol and add 50 ml distilled water. 5.5. Mixed Indicator solution: Dissolve 0.2 gm. methyl red in 100 ml. 95 % ethyl or isopropyl alcohol and 0.1 methylene blue in 50 ml. of 95% ethyl or isopropyl alcohol Mix the two solutions. 5.6. Boric acid + mixed indicator solution: Dissolve 20gm. boric acid (H3B03) in ammonia free distilled water add 10 ml. of mixed indicator solution and dilute to 1 liter. 5.7. Standard sulphuric acid solution (0.02N) : Dilute 20 ml. of 1N H2 S04 to 1000 ml. with distilled water Standardize it against 0.02 N sodium carbonate solution (1.06 gm. of Na2 C03l'Litre) using methyl red indicator (0.2% solution in 95% ethyl or isopropyl alcohol). A faint orange color will appear at the end point.

6.0 Procedure:

6.1. Digestion:
6.1.1.1. Take 2.0 gm. or suitable quantity of oven dried (105C) sample thoroughly ground and sieved through 0.2 mm sieve in a kjeldahl digestion tube or flask. 6.1.1.2. Add 10 gm of potassium sulphate - copper sulphate mixture (10 : 1 ratio) or kjeldahl tablet into the tube and 35 ml. of cone. H2SO4. 6.1.1.3. Heat initially at low temperature for first 10 to 30 min. until frothing stops and then raise the temperature gradually to 360 - 410C. 6.1.1.4. Continue the digestion until the contents become light yellow color. 6.2. Distillation: 6.2.1.1. Cool the digested sample and add 50 ml. of distilled water. Mix thoroughly, let it stand for few minutes and transfer the content in 1 liter distillation flask 6.2.1.2. Carry out 4-5 washings with 50 ml. of distilled water and transfer the content of every washing with 50 ml of distilled water and transfer the content of every washing into the same distillation flask making the final volume of about 300 ml. 6.2.1.3. The solution is made alkaline (pH >11) with 40% sodium hydroxide solution using phenolphthalein indicator 6.2.1.4. Immediately attach the distillation flask or tube to the distillation unit. . Start distillation after immersing the tip of the condenser in 50 ml. boric acid solution (with mixed indicator) in a conical flask. Collect about 150 ml. of the distillate.

6.3. Titration:

6.3.1.1. Titrate the boric acid solution against the standardized

H2SO4 (0.02 N) 6.3.1.2. The end point) is the appearance of purple color. 6.3.1.3. Carry out blank titration similarly using distilled water as blank starting from the digestion step to final titration . Note: In case of laboratory has automated or semiautomatic TKN assembly the same may be used as per the procedure given in the operation manual.

7.0 Calculation:
% TKN = (S-8) x N x 1.4 Wt Where, S = ml. of standard H2SO4 acid used for sample B = ml. of standard H2SO4 acid used for blank N = Normality of standard H2SO4 acid Wt = Weight of the sample in gm. 8.0 Expression of results: Express the result as TKN % in 2 decimal units on oven dry weight basis. 9.0 Reference: Jackson M.L. (1967) Soil Chemical Analysis Prentice - Hill of India Pvt. Ltd., NEW DELHI.

Estimation of Potassium for Soil and Sludge

1.0 Introduction: Potassium is an essential element in a both plant and human nutrition and
occurs in ground waters as a result of mineral dissolution from decomposing plant material, agricultural runoff, solid waste etc.

2.0 Method: Flame Photometric

3.0 Principle: Trace amount of potassium can be determined in direct reading type of
flame photometer. The extract is aspirated into gas mixture flame and excitation is carried out under carefully controlled and reproducible conditions. The intensity of light is measured by a phototube . The intensity of light is proportional to the concentration of the element. The calibration curve may be linear but has a tendency to level off at higher concentration. Minimum detectable concentration of 0.1 mg/gm solid waste can be determined by this method

4.0Apparatus
4.1. Conical flask,250 ml 4.2. Hot Plate 4.3. Glasswares 4.4. Flame photometer 5.0 Reagents 5.1. Nitric acid (HN03) cone 5.2. Triacid mixture: Mix 10MI of Nitric acid (HN03),1 ml of sulphuric acid (H2SO4) and 4ml of 60% prechloric acid (HCI03) in a conical flask 5.3. Where Stock Potassium solution: Dissolve 1.907 gm KCI dried at 1100 C and dilute to A = conc. of water, 1 ml = 1 'K' in mg/l obtained from the calibration graph. 1000 ml with potassium as mg of K. Wt = Weight potassium solution: Dilute digestion potassium with water to 5.4. Intermediate of the sample taken for 10 ml stock V = Total volume of K. Use this solution to prepare calibration curve in 100ml 1 ml=0.10 mg the digested solution potassium range of 1 to 10 mg/L 5.5. Standards potassium solution: dilute 10 ml intermediate potassium solution with water to 100ml, 1 ml = 0.01 mg of K. Use this solution to prepare calibration curve in potassium range of 0.1 to 1 mg/l Note: Use de-ionised distilled water for preparing all reagents calibration standards and dilution water.

6.0 Procedure:
6.1. Take 1.0 gm or suitable quantity of oven dried (1050C)sample thoroughly ground and sieved through 0.2 nm sieve in a conical flask and add 5ml of Conc. HN03 (5ml per gm of sample) 6.2. Swirl the flask to moisten the entire mass of the sample and place it on a hot plate at a temperature of 180-200 C in a fume cupboard and allow to boil to dryness 6.3. Cool the flask and add 5ml (5ml per gram of Sam~le) of triacid mixture (HN03 H2S04-HCI04) to the sample digest at 180-200 C degrees till the sample appears whitish or light yellow in colour. 6.4. Add 20ml distilled water and phenolphthalein indicator drop by drop. Neutralized the solution by adding 1 N NaOH drop as until a faint pink colour appears. 6.5. Filter the whole content through whatmann No .42 filter paper and make up the volume to 100ml with distilled water in a volumetric flask. This account the total volume of digested solution. 6.6. Prepare calibration standard in stepped amounts in any of the following applicable ranges: 0 to 1.0, 0 to 10 or 100mgll K. Aspirate calibration standard and sample given enough time to secure a reliable average reading for each. Construct a calibration curve from potassium standards. 6.7. Determine potassium concentration of samples from calibration curve.

7.0 Calculation Total potassium as K mg/gm = Ax V 1000xwt

8.0 Expression of Result: Express the result as total potassium mg/gm on dry weight
basis in 2 decimal units.

9.0 Reference: Jackson M.L (1967); Soil chemical analysis, Prentice - Hill of India
Pvt Itd, New Delhi.

Estimation of Total Organic Carbon

1.0 Principle
1.1.1 Photo catalytic Oxidation
.

In the Presence of near UV light and Oxygen, titanium dioxide will catalyze the oxidation of organic compounds in an aqueous medium. This
reaction yields carbon dioxide, water and either the acid, base or salt of any organically bound elements (see Equation 1. Oxidation of Organic Carbon). 4CxHy + (4X+y)02 --+ 4XC02(g) + 2yH20 For example, 2,4,6-Trichlorophenol in solution would react as follows:

2CbC6H2OH + 1102 --+ 12C02(g) + 6HCI(aq.)

Equatlon-1. Oxidation of Organ'lc Carbon The reaction proceeds at room temperature and is very efficient and clean compared to other oxidation methods typically utilized for TOC measurements. Various theories have been proposed for the mechanism by which titanium dioxide catalyzes the oxidation of organic compounds. The simplest and most widely accepted is that the oxidation is catalyzed predominantly through the formation of hydroxyl radicals at the surface of the titanium dioxide. These hydroxyl radicals are formed when titanium dioxide is exposed to near UV light in the presence of water and oxygen. The hydroxyl radical is a very strong oxidizing agent so it can generally be said that the speed of CO2 formation is oxidation rate dependent.

2.0 Reaqent preparation

2.1 Standard peparation 2.1.1. 200 ppm carbon standard solution using benzoic acid.
Accurately weigh 0.1453 gm of pure benxoic acid (68.4% carbon) into a clean 250 ml beaker. Add 150ml of high purity water and gently heat or ultrasonicate to aid to dissolution. Transfer to 500 ml volumetric flask rinse beaker-into flask, allow to cool and make up to approximately 480 ml volume. Adjust the pH of the solution using perchloric acid to 3.0. Make up to 500 ml.

2.1.2. 200 ppm carbon standard solution using potassium hydrogen phthalate. Accurately weigh 0.2125 gm of pure potassium hydrogen phthalate (47.05% carbon) into a clean 250 ml beaker add 150 ml of high purity water and dissolve. Transfer to 500 ml volumetric flask, rinse beaker into flask and make up to approximately 480 ml volume. Adjust the solution to pH 3.0 using perchloric acid make up to 500 ml volume. 2.1.3 Preparation of titanium dioxide (Ti02) catalyst suspension. For normal catalyst suspension add 1 gm of Ti02 to 500 ml of high purity water. For concentrated catalyst suspension add 3 gm of Ti02 to 500 ml of high purity water. To assist the suspension of the titanium dioxide the suspension should be stirred and or shaken vigorously for 10-15 minutes. Adjust the pH 3.0 using 0.1 M perchloric acid solution.

2.2 Perchloric acid Solution (0.1 M)

Dilute 9 ml of concentrated (70%) perchloric acid in 500 ml of high purity water and make up to 1 liter. Use this solution to adjust pH where sample pH is greater then pH 3.0. 2.3 Sodium hydroxide solution (0.1M) Weigh out 4 gms of NaOH and add 1 liter of high purity water. Use this solution to adjust pH where sample pH is lower than 3 units. 3.0 Procedure

. Switch on the instrument and computer

. Add Ti02 catalyst to catalyst reservoir.

. Click on REMOTE .

icon for establishment of communication between

instrument and computer. Click on Stir icon. Ti02 catalyst in the reservoir is stirred for 30 seconds.

. Click on

Fill icon. Three levels are indicated. We have to choose the

level according to the TOC content of the sample.

. When baseline is stabilized, press Vent

icon. The base line will fall as

C O2 in the system is vented out. After attaining a stable base line injection (Forboth calibration and sample analysis) can be carried out.

4.0 Standard calibration

. Click standard calibration icon on the tool bar. . Enter fhe identification, volume of standard chosen to be injected and
concentration of the standard. Click INJECT button.

. When all green appears on the colour display below the detector response display
box (dynamic end point indicator) instrument is ready for standard or sample injection.

. Dialogue box, asking for standard is injection appears on the screen. At

this point standard injected into the port.

. After injection of the standard, CliclfOkl button on the dialogue box. . Ci and Cf (initial and final CO2 cOn~ion) values are recorded by the
instrument and calibration Co-efficient (Ko) is determined by the instrument's built in software system.

. Like wise, more injections of different volumes ( different carbon mass) are
injected for multipoint calibration. 5.0 Sample Analysis

. There are two types of sample analysis procedure. . Using the sample analysis panel. . Using the sample process table.
6.0 Using the sample analysis panel

. Press the sample analysis icon on the tool bar. Sample analysis panel will
be opened. UNK(1) C-24

Type

Identification

Operation

TOC -------Manual iniect(60) In this enter the sample name or code or any other

.
.
.
.

In this enter the volume of sample, which is going For TOC analysis enter TOC .
~

Dilution factor

enter 1 for no dilution.

When base line is Stable system message asking for manual injection is displayed. Click Inject button. Take specified volume of the sample, whose pH has been adjusted to 3.0 to 3.5 into svtnge and inject into reactor through sample injection port.

. Click OK button. Sample analysis will commence.

7.0 Using sample process table.

. Click the new table icon

in the tool bar or select the file New option from the menu. A new run window will be opened.

. =This icon is used to identify run table, chick on the icon. . Enter the following parameters in the sample analysis form.
. Type-unk(1 )

. Identification- used for entering an identifying table that will help to identify
the sample analysis.

. Injection volume. . Analysis type (TOC, TIC, NPOC etc)

Example:

. When base line is Stable i.e all green is the dynamic endpoint indicator,
click start button

on the tool bar or by selecting start run command from the menu bar.

. When system message asking for manual inject appears on the screen,
inject know volume taken in the syringe into reactor through injection port.

8.0 Operation The near-UV lamp is switched ON to allow photocalalytic oxidation of the organic carbon in the sample. The organic carbon present in the injected sample is oxidized to CO2 and the CO2 evolved is measured and reported.