To lay down a standard procedure for analysis of Effluent water.

2.0   SCOPE:

This SOP is applicable for analysis of Effluent water in Quality Control Laboratory at Sano-Cito Therapeutics Inc. Unit-II, Vill. Loharan, P.O. Ghatti, Solan-173211 (H.P.)


Officers / Executive – Quality Control shall be responsible for follow that procedure.


Manager- Quality Control and Head QA shall be accountable for compliance of this SOP.


Report of Effluent water                                                       Attachment-I


Collect the sample

6.1    Chemical Analysis:

6.1.1 Description:

Examine the water physically such as color, odor.

6.1.2 pH:

Check pH of solution using standardize pH meter.

6.1.3 Total dissolved solids (TDS):

After stirring the sample, transfer accurately measured 25 ml of the solution with pipette in a 100 ml pre-weighted beaker. Evaporate the solution upto the dryness. Put the beaker in oven at 105° for 30 minutes. Again weigh the beaker and calculate the residue weight(X).

Wt. of residue in gm (X) X 1000 X 1000

Total dissolved solids ——————————————————-


V= volume of the sample taken (25 ml)

X= Difference of weight residue in mg

6.1.4 Determination of Suspended solids:

After stirring the sample, filter about 50 ml solutions in separate beaker. Transfer accurately measured 25 ml of the filtrate with pipette in a 100 ml pre- weighed beaker. Evaporate the solution upto the dryness. Put the beaker in oven at 105°C for 30 minutes. Again weigh the beaker and calculate the residue weight (Y)

Wt. of residue in gm (X) X 1000 X 1000

Total dissolved solids: —————————————————————-


Y= Weight of residue in mg

V= Volume taken

Mg/l of total suspended solids= Total solids-Total dissolved solids

6.1.5 Determination of Oil and Grease:

Transfer accurately measured volume if 250 ml of effluent sample to the separating funnel and add 50 ml of n-Hexane and shake it thoroughly so all oily matter to get dissolved in n-hexane. Allow the mixture to settle and the separate the hexane layer. Filter the hexane in pre-weighed beaker. Wash residue on filter paper with 10 ml n-hexane. Evaporate the hexane up to dryness on a water bath at 60° to 65°C. When hexane is evaporated then cool the beaker and weighed. Take the difference to get the oil content in 250 ml of effluent sample.

Wt. of residue in gm (W) X 1000 X1000

Oil & grease in mg/l:———————————————————


W= Weight of residue in gm

V= Volume of sample

6.1.6 Chemical Oxygen Demand (COD):


Std. Potassium Dichromate solution (0.25N) Dissolve 12.259 gm of  K2Cr2O7 primary std. Grade previously dried at 103° C for 2 hours in distilled water and dilute to 1000 ml.

Sulphuric Acid Reagent: Take 500 ml concentrate H2SO4 and 5 gm Ag2SO4 Stirred it very well and keep overnight.

Procedure: Take 0.4 gm (approx.) HgSO4 in around bottom flask. Add 10 ml std. K2Cr2O7 solution, few glass beads & 10 ml distilled water. Add 30 ml H2SO4 reagent, slowly with constant stirring. Add sample drop by drop with constant stirring. If light green colour (nearly to green) is obtained stop adding of solution. Measure how much sample is added.

Attach a refllux condenser and reflux for nearly 11/2 hours. Wash the reflux condenser with distilled water. Cool to room temperature and titrate against Fe(NH4)(SO4)2 using ferroin indicator. Colour changes from blue green to wine red indicates end point. Carry out a blank determination.

(A-B) X N X 8000


  1. of sample

A= Blank reading

B=Sample reading

N=Normality of Fe(NH4)(SO4)2

6.1.7 Determination of (BOD):


Phosphate Buffer: Dissolve 8.5 gm KH2PO4. 21.75 g of KH2PO4, 33.4 gm. NaHPO4 and 1.7 gm NH4Cl  in distilled water and dilute to 1 liter pH=7.2.

Magnesium sulphate solution: Dissolve 22.5 gm MgSO4 7H2O in distilled water and dilute to 1 liter.

Calcium chloride solution: Dissolve 27.5 gm anhydrous caCl2 in distilled water and dilute to 1 liter.

Ferric chloride solution: Dissolve 0.25 gm FeCl3 6H2O in distilled water and dilute to 1 liter.

Seeding Material: A proper seeding material must be used in B.O.D. determination usually raw, settled sewage is used. But at time acclimate seed or soil or receiving water can be used as seed.

Acid and alkali solution: To neutralize the samples, hence any strength can be used.


The sample either highly acidic or highly alkali must be neutralized to about 7.0. The pH of dilution water should not change with lowest dilution of sample. Samples which has residual chlorine, either they should be kept for about 2 hours to dissipate the residual chlorine or should be treated with equivalent quantity of sodium sulphate solution. Samples containing toxic metals etc. should be carefully treated. The metals must be precipitated out. If super saturation of D.O. is there in water, then D.O. should be brought down to saturation value at 20°C by vigorously shaking it.

Dilution suggested:

Waste quality                                         % dilution

Strongly industrial effluents               0.1-1.0

Raw & settled sewage                           1.0-5.0

Oxidized effluents                                 5.0-25.0

Polluted river water sample               25.0-100.0

The dilution water is prepared by adding 1ml/l of each phosphate buffer, MgSO4 solution, CaCl2 solution and FeCl3 solution. Seed, if necessary, is also added in the same amount i.e, 1 ml/l. Then the required dilution of sample is prepared in graduated cylinders. The diluted sample is then simphoned into B.O.D. bottles. One is tested for initial D.O. By acid modification method and the other is incubated for 5 days at 20°C. A blank is also tested for initial D.O. and after incubation. After incubation for 5 days, the D.O. value is found out using azide modification method.


Biochemical Oxygen demand:

D1: Initial D.O. of diluted sample taken after 15 minutes of preparation of diluted sample.

D2: Final D.O. of diluted sample after 5 days incubation at 20°C

P: % dilution taken


B.O.D . mg/l: —————-X100


6.1.8 Determination of Dissolved Oxygen:


Manganese sulphate solution: Dissolve 480 gm of MnSo4 4H2O or 400 gm of MnSo4 2H2O or 364 gm MnSo4 4H2O in distilled water, filter and dilute to 1 litre. If Uncertainly exists regarding the water of crystallization, a solution of specific gravity of 1.270 at 20°C should be prepared. This solution should not liberate more than trace of Iodine when added to used solution of K.I.

Alkali-Iodine-Azide Reagent: Dissolve 500 gm of NaOH and 13.5 gm of NaI ( or 700 gm of KOH, dissolve separately 150 gm of K.I.) in distilled water and dilute to 1 Litre. To this solution, add solution obtained by dissolved 10 gm of sodium azide (NaN3) in 40 ml of distilled water. This solution, should not given colour with starch when dilute and acidified (sodium and potassium salts may be used interchangeably).

Conc. H2SO4-Approx. 36N

Starch solution 0.5% solution: Make a paste or 0.5 of starch in little distilled water, pour this paste in 200 ml. of hot distilled water and allow boil for 1 min.

Sodium Thiosulphate stock solution (0.1 N): Dissolve 24.82 gm Na2S2O3 5H2O in boiled and cooled distilled water and dilute to 1 litre. Preserve by adding 5 ml chloroform or 1 gm NaOH per litre.

Sodium Thiosulphate standard solution 0.0125N: Dilute 125 ml of stock solution to 1 litre.


To the sample collected in a BOD (Capacity 250-300 ml) bottles and 2 ml MnSO4 solution and 2 ml iodine azide reagent. Shake and allow the floc to settle down when ¾ supernatant is clear above the floc, add 2 ml conc. H2SO4 shake and dissolve the floc completely 100 ml. solution is titrated against 0.0125 N Na2S2O3 solution using starch as indicator.



B.O.D. in mg/lit: ———————————

% dilution in faction

D1= Initial dissolve oxygen

D2=Final dissolve oxygen




   SOP: Standard Operating Procedure

QC: Quality Control

QA: Quality Assurance

Ppb: Parts per billion

M:           Molarity

°C: Degree centigrade

mg: Miligram

TDS: Total dissolved solids

ppm: Parts per million


Quality Assurance Department

         Quality Control Department

Version Number Effective Date Reason for Revision