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Present Testing Methods
Various testing methods are available at present. In India, the test methods indicated in IS Codes
(developed by BIS) are considered most acceptable.
The following standards are in use for various tests-
IS 3025-15: Methods of sampling and test (physical and chemical) for water and wastewater,
Part 15: Total residue (total solids-dissolved and suspended)
IS 3025-16: Methods of sampling and test (physical and chemical) for water and wastewater,
Part 16: Filterable residue (total dissolved solids)
IS 3025-17: Methods of sampling and test (physical and chemical) for water and wastewater,
Part 17: Non-filterable residue (total suspended solids)
IS 3025-18: Methods of sampling and test (physical and chemical) for water and wastewater,
Part 18: Volatile and fixed residue (total filterable and non-filterable)
IS 3025-31 (1988): Methods of sampling and test (physical and chemical) for water and
wastewater, Part 31: Phosphorus
IS 3025-38 (1989): Methods of sampling and test (physical and chemical) for water and
wastewater, Part 38: Dissolved Oxygen (DO)
IS 3025-44 (1993): Methods of Sampling and Test (physical and chemical) for Water and
Wastewater, Part 44: Biochemical Oxygen Demand (BOD)
IS 3025-58 (2006): Methods of sampling and test (physical and chemical) for water and
wastewater, Part 58: Chemical oxygen demand (COD)
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Why This Manual?
Obviously, one may ask, “What is the need to having this manual when we have very useful and
authentic test standards available?” Well, the question is right. But, the test standards are too
lengthy and become difficult to be used by ordinary chemists. Let us take a few examples.
As per the standard IS:3025-44, BOD determination needs calculation of dilution factor,
particularly if the sample needs to be diluted due to having a high expected BOD. On discussion
with many chemists, they were using different ways to calculate dilution and getting different
results with the same measured values. Not only this, in case when the test sample is seeded
during the test, there is some printing mistake in the test method, which has been corrected by
adding ‘addendum’ at the beginning of the standard. Obviously, many chemists did not pay
attention to that.
Similarly, during COD testing, some chemists did not mix well Mercuric Sulphate and sample
thoroughly, as they were doing their jobs in a routine way. BIS standard clearly indicates that
these should be mixed thoroughly. This indicates that some more detailed knowledge should be
given to chemists in order to make them more concerned towards proper testing.
This manual is expected to serve as a handy and easy tool for the chemists.
In addition, a couple of simple thumb rule type tests have been included to help ETP chemists
evaluate if the operators are adding excess nutrients. A couple of ways are also highlighted to
check if the chemists are checking BOD and COD properly or not.
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Filter Paper:
Whatmann Filter Paper Comparison
Circle Dia. Reference mm 125 125 125 125 125 125
Alternate grade 617 640m 640de 1670 1640m
Grade # #4 #41 #42 #54 #113 #541
Particle Retention μm 20-25 20-25 2.5 20-25 30 20-25
Air Flow Rate s/100mL/in2
3.7 3.4 107 4.2 1.3 3.8
Ash % 0.06 0.007 0.007 0.015 NA 0.006
Basis Weight gsm 96 84 100 92 131 82
Net Ash mg 0.71 0.07 0.09 0.17 NA 0.06
Price, per 100 circles Rs. 1004 2514 2345 2795
Filter Paper Description
#541 Hardened Ashless Quantitative
#113 General purpose wet strengthened qualitative
#54 Hardened Low Ash Quantitative
#41, #42 Ashless Quantitative
#4 Qualitative
Filter papers are required for testing MLSS, MLVSS and SS.
You may notice that the #42 grade filter paper has a particle retention size of 2.5µm. That
indicates this would be too slow to filter for checking MLSS, MLVSS. For suspended solids
measurement, there could be some suspended particles of such low size having size in the range 2.5µm to
20µm, and hence it may be used for suspended solids. For most practical purposes, #4 or #41 work well,
and give fairly accurate results.
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Reagents Preparation
In this section, preparation of different reagents with estimated daily consumption of reagent
solutions is given considering daily testing of 2 samples each of COD, BOD and DO.
Alkali-Iodide-Azide Reagent: Dissolve 250 g NaOH, and 75g KI, in distilled water and dilute to
500ml. Add 10 g sodium azide, NaN3 dissolved in 40 ml distilled water. This reagent should not
give colour with starch solution when diluted and acidified.
Estimated Daily Consumption: 16 ml
Prepared Solution Adequate for: 1 month
In place of 250g NaOH, 350g KOH and in place of 75g KI, 67.5g NaI can also be used.
Ammonium Molybdate Reagent: Dissolve 12.5 g Ammonium Molybdate in 87.5 ml distilled
water. Continuously add 140 ml conc. Sulphuric Acid to 200mL distilled water in a separate
beaker. Cool, and mix the Ammonium Molybdate Solution to it, and make up the volume to
500mL.
Estimated Daily Consumption: 2-3 ml
Prepared Solution Adequate for: 6 months
Calcium Chloride Solution: Dissolve 13.75g calcium chloride (CaCl2) in distilled water and
dilute to 500ml.
Estimated Daily Consumption: 3 ml
Prepared Solution Adequate for: 4 months
Ferric Chloride Solution: Dissolve 0.125 g hydrated ferric chloride (FeCl3.6H2O) in distilled
water and dilute to 500ml.
Estimated Daily Consumption: 3 ml
Prepared Solution Adequate for: 4 months
FAS (Ferrous Ammonium Sulphate), 0.1 N: Dissolve 19.5g of Fe(NH4)2(SO4)2.6H2O in
distilled water. Add 10 ml. conc. H2SO4, cool and dilute to 500ml.
Estimated Daily Consumption: 75 ml
Prepared Solution Adequate for: 6 Days
Manganous Sulphate Solution: Dissolve 182g Manganese Sulphate (MnSO4.H2O) in freshly
boiled and cooled water, filter and make up to 500 mL by addition of distilled water. Check that
the solution does not give blue colour by addition of acidified potassium iodide solution and starch.
Estimated Daily Consumption: 30 ml
Prepared Solution Adequate for: 15 Days
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Manganous Sulphate is available in three different forms. If other form is being used, the
quantity to be used should be accordingly corrected as under-
MnSO4.4H2O 240g
MnSO4.2H2O 200g
MnSO4.H2O 182g
Mercuric Chloride Solution: Dissolve 6g. Mercuric (II) Chloride in distilled water and dilute to
100 ml.
Estimated Daily Consumption: few drops
Prepared Solution Adequate for: Several months
Potassium Dichromate Standard (0.25 N): Dissolve 6.129g. Potassium Dichromate
(previously dried at 105o
C for 24 hours) in distilled water and dilute to 500 ml. Add 60mg
Sulphamic Acid to stabilize.
Estimated Daily Consumption: 30 ml
Prepared Solution Adequate for: 15 Days
COD Acid: Add 25g Silver Sulphate to 2.47 liter conc. H2SO4. Keep overnight for dissolution.
Shake well after dissolution. Silver Sulphate acts as a catalyst.
Estimated Daily Consumption: 90 ml
Prepared Solution Adequate for: 27 Days
As per standard, 10.12g Silver Sulphate needs to be added to 1 Liter Sulphuric acid. However, the
Silver Sulphate is available in 25gm packing and it is one of the costliest laboratory reagents, so
the dilutions are accordingly scaled.
During COD test, Silver Sulphate acts as a catalyst, and its absence from the test may result
inadequate conversion of carboneous compounds thus resulting in a false low COD result.
Phosphate Buffer Solution: Dissolve 4.25g Potassium Dihydrogen Phosphate (KH2PO4),
10.875g Potassium Hydrogen Phosphate (K2HPO4), 16.7g Disodium Hydrogen Phosphate
(Na2HPO4.7H2O) and 0.85g Ammonium Chloride (NH4Cl) in about 250mL distilled water and
dilute to 500ml. The pH of the solution should be around 7.2 ± 0.1 when measured by pH meter
without any further adjustment.
Estimated Daily Consumption: 3 ml
Prepared Solution Adequate for: 4 Months
Magnesium Sulphate Solution: Dissolve 11.25g magnesium sulphate (MgSO4.7H2O) is
distilled water and dilute to 500ml.
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Estimated Daily Consumption: 3 ml
Prepared Solution Adequate for: 4 Months
Sodium Thiosulfate, 0.025N - Dissolve 3.103g Sodium Thiosulfate-Petahydrate, Na2S2O3.5H2O
in 500ml copper free, boiled and cooled distilled water. Add 0.125 g NaOH as preservative.
Estimated Daily Consumption: 60 ml
Prepared Solution Adequate for: 12-13 Days
Starch Indicator - Dissolve 2 g of Starch and 0.2 g of salicylic acid as preservative, in 100 ml hot
distilled water.
Estimated Daily Consumption: 1-2 ml
Prepared Solution Adequate for: 2-3 Months
Salicylic acid acts as preservative so that the solution can be used for longer time.
Stannous Chloride Solution - Dissolve 2.5 g of Stannous Chloride in 100 ml glycerol. Slow
heating may be done to dissolve faster.
Estimated Daily Consumption: Few Drops
Prepared Solution Adequate for: Several Months
Seed Solution (for BOD) – Use 1ml of treated effluent per 500ml of distilled water.
Estimated Daily Consumption: 6 ml
Prepared Solution Adequate for: 2 Months
Seed solution contains some bacteria, which would be useful in BOD testing procedure. However,
please do ensure that the treated effluent BOD is within 10-30 ppm only. In addition, in case any
biomass activity (like generation of green colour algae) appears in the seed solution; discard this
solution, and prepare a fresh one.
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MLSS Mixed Liquor Suspended Solids
1. Take a filter paper previously dried.
2. Whatmann filter paper #4, #41 or #54, #113 or #541 can be used for the same.
3. Take a sample from aeration tank
4. Stir well gently and measure 100ml of the same.
5. Filter through filter paper.
6. Dry the filter paper at 105 C in oven, and weigh again.
7. Calculate the weight difference in milligrams.
8. Multiplying this with 10 gives you MLSS.
Additional Information:
Do not use Whatmann filter paper #42 for MLSS. This is very slow draining.
Ashless filter papers are costly, and for MLSS only these are not generally required.
However, if you need to check MLVSS also, you may prefer Ashless grades.
Commercial filter papers may also be used for determination of MLSS only.
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MLVSS Mixed Liquor Volatile Suspended Solids
1. Take the filter paper containing dry MLSS from previous test.
2. Put this in a crucible and keep it in muffle furnace.
3. Muffle furnace temperature should be set at 450-550 C previously.
4. After all the contents have been incinerated properly, take out crucible.
5. Put crucible in a desecrator for cooling for 10 minutes.
6. Take out and weigh the contents of crucible.
7. MLVSS can be calculated as-
(Initial Solids Weight-Ash Weight) *100
MLVSS = ---------------------------------------------------
Initial Solids Weight
Additional Information:
Commercial filter papers contain generally around 7% ash and sometimes even more. Hence
results obtained indicate significantly lower MLVSS than actual. That is why these should not be
used.
The furnace temperature must not exceed 550°C as beyond this temperature the Calcium
Carbonate, which may be present as inorganic material in MLSS starts thermal degradation to
Calcium Oxide and Carbon Di-Oxide. That results in false high MLVSS.
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DO DISSOLVED OXYGEN IS:3025-38
1. Take the sample gently in a BOD Bottle.
2. Add gently 2ml Manganese Sulphate below the surface of liquid.
3. Then add 2ml Alkali-Iodide-Azide in the same way.
4. Replace cap and mix several times.
5. Let the bottle rest for some time.
6. When flocks settle down to less than half of the bottle, then-
7. Add 2ml concentrated Sulphuric Acid just above the surface of sample.
8. Replace cap, invert bottle and mix.
9. Take 201ml from bottle and add 2 drops of Starch Solution.
10. Titrate with Sodium Thiosulphate, 0.025N solution.
11. Burette reading will give DO in ppm directly.
Additional Information-
After sampling, try to complete the test as early as possible.
Occasionally, take around 1 liter of distilled water in a 2 liter can, replace cap, and shake well for
2 minutes. Remove cap, wait for 5 minutes and replace the cap. Then repeat the shaking again
for 2 minutes. Check DO after five minutes. Ideally, DO should be around what is given in the
following table-
Temperature, °C 10 15 20 25 30 35
DO, ppm 11.3 10.2 9.2 8.3 7.6 6.9
A high DO indicated from the above table means that the testing is not proper. Too low DO
result obtained also indicates that there was a need to check the test procedure for some error.
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COD CHEMICAL OXYGEN DEMAND IS:3025-58
1. Take 5 (inlet)/20(outlet) ml sample in COD Tube.
2. Add distilled water to make 20ml volume.
3. For blank, just take 20ml distilled water.
4. Add 0.4gm Mercuric Sulphate.
5. Add 10ml Potassium Dichromate.
6. Then add 30ml COD Acid, and mix well.
7. Place in COD digester for 150 ⁰C and 2 hours.
8. Take out sample and cool down.
9. Then add 60ml distilled water.
10. Transfer it to conical flask.
11. Add 2 drops of Ferroin Indicator, and titrate with 0.1N FAS.
12. Repeat the same with a blank without sample.
COD can be determined by the following formula-
(FASb-FASs) * 8000 * N FASb: FAS consumed for blank
COD: --------------------------------- mg/l FASs: FAS consumed for Sample
Volume of Sample N: Normality of FAS solution (Here 0.1)
Precautions:
1. After addition of 0.4gm Mercuric Sulphate, swirl the reflux tubes so that it is mixed with
the sample. Mercuric Sulphate acts to reduce interferences arising from the chloride ions.
If it is not mixed well, the results may be wrong.
2. Addition of COD acid is very important. Too fast addition may result in sudden heating
of the sample within the reflux tube thus some of the organic matter may escape out from
reflux tube, thus giving false low COD value. It is advisable to add 10ml Sulphuric Acid,
wait for 1 minute, and then repeat the same two times.
3. Use of Silver Sulphate makes COD testing a costly one, as it is a very costly reagent.
Silver Sulphate acts as a catalyst for the reaction, and some chemists feel that a smaller
quantity of the same may be tried. However, never try to reduce its dosage. The readings
with lesser Silver Sulphate are often false low and not reliable.
4. Check blank FAS reading. If it is not around 24.0-25.5, check the reagent again for its
purity.
5. If FAS reading with sample is below2.0, take smaller sample volume, and repeat the test.
6. After step 6 of the test method, if the contents of the reflux tube turn green, it is an early
indication that COD is high, and you need to repeat the test with smaller sample quantity.
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BOD BIOLOGICAL OXYGEN DEMAND IS:3025-44
1. Take 3 liter well aerated distilled water.
2. Add 3ml each of
a. Calcium Chloride,
b. Ferric Chloride,
c. Magnesium Sulphate and
d. Phosphate Buffer Solution.
Prepare two dilutions of sample as per estimated BOD, as given in the following table (if you
wish, you may use only one dilution also). Use three 2 Liter beakers and take the indicated
quantity of sample, and add dilution water as prepared in step 2 to make 1000ml each. Thus, the
beakers shall have (1) only dilution water, (2) sample with 1st
dilution & (3) sample with 2nd
dilution.
Estimated BOD 25 50 100 200 400
1st
Dilution, mL/L 100 50 25 10 5
2nd
Dilution, mL/L 200 100 50 20 10
3. Add 2ml seed solution in both distilled water as well as prepared BOD diluted sample.
4. Fill six BOD bottles, as under-
a. 2 Bottles of blank (using dilution water only)
b. 2 Bottles with 1st
Dilution
c. 2 Bottles with 2nd
Dilution
5. Now check the DO of one bottle each from these bottles.
6. DO testing is already described earlier.
7. Name the data as B0 & D0 for blank and diluted sample respectively.
8. As there are two dilutions, there shall be two different figures for D0.
9. Incubate the two bottles remaining at 27 ⁰C for 3 days.
10. After three days, check the DO of these.
11. Name these values as B3 & D3 for blank and diluted sample respectively.
BOD can be calculated as-
BOD: [ (D0-D3)-(B0-B3) ] * 1000 / Sample Vol.
Where,
D0: DO on 0th
day for Sample
D3: DO on 3rd
day for Sample
B0: DO on 0th
day for Blank
B3: DO on 3rd
day for Blank
Precautions:
1. Glassware cleaning plays a significant role in BOD test. Before the test, ensure that
all glassware are well clean and dry, otherwise the test results will certainly be wrong.
2. If D3 value is less than 1.0, just discard the readings and repeat the test taking a more
diluted sample.
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3. If (B0-B3) is more than 1.0, it indicates that the dilution water is contaminated.
Discard all test results and repeat the test.
4. Ensure that the final testing (after 3 days) should be done exactly after 72 Hours (71-
73 Hours). If you tested the initial DO at 3.00PM on Monday, the final testing should
be done on Thursday between 2.00 PM to 4.00 PM.
5. If you find any algal growth in any of the reagent, just discard the same and prepare a
fresh solution.
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N Nitrogen
This is a non-standard test, barely for indicative purpose.
1. Take 40-50ml sample in a 100mL beaker.
2. Put a black cardboard on its back side.
3. Add a couple of drops of strong NaOH solution in the center.
4. Let it wait for a minute.
5. Add a drop of Mercuric (II) Chloride solution.
6. Do not stir, and wait for 2-4 minutes.
7. A white cloud like formation seen indicates the presence of ammonia.
8. In case no such cloud formation appears, there is no nitrogen as nutrient.
Additional Information:
This procedure has been developed just by experience and no documentary evidence is yet
available about it. However, it has been observed that overdosing of nutrients (mainly N & P)
results in carryover of excess nutrients with treated effluent. This results in eutrophication and
hence generation of green colour algae development in treated effluent, particularly when the
treated effluent is kept standstill for some time.
In addition, it has also been observed that if a sample containing residual Ammonical Nitrogen,
and is tested or BOD after say 10-15 days, there becomes a possibility of getting a false high
BOD value. Hence, it is useful to conduct this test.
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P Phosphorus IS:3025-31
This is a non-standard test, barely for indicative purpose.
1. Take 25ml sample in a 100mL beaker.
2. Add 1mL Ammonium Molybdate Reagent.
3. Add 2-3 drops of Stannous Chloride Solution.
4. Wait for 5 minutes.
5. During 5-15 minutes, the solution turns blue.
6. The depth of blue colour can be visually compared to the standard solution already
prepared to find phosphorus content.
Additional Information:
BIS standard (IS:3025-31) is based upon measurement of absorbance of light at 470nm
wavelength through the prepared solution. However, as presently phosphate content is not
notified as a parameter, the aim is to ensure that the effluent does not create eutrophication, i.e.
development of algae in discharge streams, by ensuring that the phosphorus content is below
0.5ppm. That is why; simple comparison method has been used here.
To prepare standard solution, first prepare a 1gpl solution of DAP. Take 2.5mL and dilute to
1000mL. This will give a phosphorus content of 0.5ppm. Similarly taking 5mL, 10mL, and
25mL will give you standards of 1gpl, 2gpl and 5gpl.
Nitrogen and phosphorus are essential for the biomass to grow, and hence reducing BOD load.
However, too high (more than 5ppm) phosphorus often creates problems like high SVI,
increased effluent turbidity etc. Hence periodic measurement of the same is generally helpful in
maintaining good performance of ETP.
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Cross Checking of Chemists:
Cross checking is extremely important, and hence adequate steps need to be taken to ensure that
the chemists are really doing their job sincerely. Following steps can be taken to ensure the same.
GGA Test for BOD:
BOD is a bioassay test, which means the results are strongly influenced by the bacterial activity
and hence can vary significantly between repeated tests or laboratories. To check the same,
BIS:3025-44 indicates use of Glucose-Glutamic Acid solution prepared by dissolving 150mg
each of L-Glutamic Acid and Glucose per liter of distilled water, should give a BOD value of
200+37mg/l. This sample may be kept for months, but it is recommended that every month the
old sample should be discarded and when required, a fresh one is prepared.
Whenever desired or preferably once a month, a fresh sample of GGA is prepared and by the
management, a dilution of this (1:10; 1:9, 1:80 or something) is prepared in the absence of the
chemist. Thus the management knows the expected test result, but the chemist does not know.
Now, the chemist is asked to test the sample for BOD, and report the BOD value so obtained. If
the value matches with the expected value, the management can be sure that the chemist is
testing BOD in a right way. Typically, if obtained values are within +20% of the expected ones,
the testing may be considered as satisfactory.
KHP Test for COD:
Unlike BOD, Bureau of Indian Standards does not specify any standardization for COD.
However, it has been found from the literature that KHP (Potassium Hydrogen Phthalate)
solution of 425mg/l gives a COD value of 500mg/l. The prepared solution can be kept without
any problem for more than a month. This way, having a COD standard of 500, it becomes easier
for the management to prepare a known dilution, and ask the chemist to check the COD of the
same. Unlike BOD, the test results using KHP standard are fairly within range and a 10 sample
cross check indicated just 4-6% variation.