Date:

DETERMINATION OF BIOCHEMICAL
OXYGEN DEMAND (BOD),
TOTAL SOLIDS AND
TOTAL SUSPENDED SOLIDS
Experiment No.

Aim:

6
Determination of biochemical oxygen demand (BOD), total solids, and total suspended
solids of given wastewater.

Learning objectives:
The student should understand following topics
 The concept of biological oxygen demand, chemical oxygen demand, dissolved
oxygen and their importance
 The assessment of BOD, COD, DO of the wastewater i.e. the strength of different
kinds of wastewater in relation to biological treatment processes.
 The mode of nutrition of different microorganisms with reference to the oxygen
consumption, especially bacteria
 Control tests and quality assurance procedures with reference to the BOD values of,
industrial wastewater (effluents), domestic wastewater
 The total solids, total filterable solids, total non filterable solids

A. DETERMINATION OF DISSOLVED OXYGEN (DO)

Introduction:
For the determination of DO, generally, Azide (Alsterberg) method is performed. It
dose not get affected by most common interference, nitrite, but most other oxidizing or
reducing agents should be absent. Fluoride (F ) eliminates the effect of Fe3+. Permanganate
(Rideal-Stewart) method is used in presence of Fe2+ but not organic matter. Pomeroy-
Kirshman-Alsterberg method is used for waters supersaturated with oxygen or containing
high organic matter content.
Under alkaline conditions (NaOH), manganese sulfate reacts with water to form
manganese hydroxide that precipitates at the bottom of the bottle. Oxygen is present in the
sample oxidizes it to produce higher oxides. Upon acidification (H 2SO4) of this hydroxide, in
the

The sequence of reactions occurring in the test is as follows:

1. MnSO4 + 2 NaOH Mn(OH)2 + Na2SO4

2. 2 Mn(OH)2 + (O) + H2O 2 Mn(OH)3
3. 2 Mn(OH)3 + 3 H2SO4 Mn2(SO4)3 + 6 H2O
4. Mn2(SO4)3 + 2 KI 2 MnSO4 + K2SO4 + I2
5. 2 Na2S2O3 + I2 Na2S4O6 + 2 NaI (titration)

presence of potassium iodide, iodine is liberated. The iodine liberated is chemically

equivalent to the amount of oxygen present in the sample. Iodine is estimated using sodium
thiosulfate (Na2S2O3) with starch solution as indicator. The method described in the following
text is Azide (Alsterberg) method for determination of dissolved oxygen.

Materials:
Sample

1.
2.
3.
Reagents, chemicals, and indicator
1. Alkaline iodide-sodium azide solution. Dissolve 500 g NaOH (or 700 g KOH) and
135 g NaI (or 150 g KI) in distilled water. Dilute to 950 ml, and allow to cool. Slowly,
with stirring, add solution of 10 g NaN3 in 40 ml water. Diluted and acidified solution
must not give colour with starch indicator. Store in dark bottle with rubber stopper.
2. Manganese sulfate solution. Dissolve 364 g MnSO4.H2O in water, filter and dilute to
1 liter. No more than trace of I should be liberated when solution is added to acidified
KI solution.
3. Potassium biiodate standard solution [0.025 N] Dissolve 0.8125 g KH(IO3)2 in
water in 1 liter volumetric flask and dilute to volume.
4. Potassium fluoride solution 40 g KF.2 H2O / 100 ml (Caution: KF is toxic and
corrosive)
 5. Sodium thiosulfate standard solutions a] 0.1 N – Dissolve 25 g Na2S2O3.5 H2O in
water, add I g NaOH or 5 ml CHCl 3, and dilute to 1 liter. Standardize against KH(IO 3)2
or K2Cr2O7. b] 0.025 N – Dilute 250 ml of 0.1 N to 1 liter (1 ml = 0.2 mg O).
6. Starch indicator solution Disperse 5 –6 g potato or arrowroot starch in mortar with
few ml of water. Pour into 1 liter boiling water, boil for a few minutes and let settle
overnight. Decant clear solution and preserve with 1.3 g salicylic acid or few drops
toluene.
7. Sulfuric acid (concentrated; sp. gr. = 1.84)
Glassware:
1. BOD bottles of 300 ml capacity
2. Pipette of 10 ml capacity

Method:
Add all reagents, except H2SO4, well below surface of sample from 10 ml pipette
graduated in 0.1 ml, with tips elongated about 50 mm.
1. Take the wastewater sample in the BOD bottle, fill to the brim, and seal with the
stopper.
2. Remove the stopper and add the reagents as follows.
3. Add 2.0 ml MnSO4 solution.
4. Add 2.0 ml alkaline I-NaN3 solution.
5. Replace the stopper, excluding air bubbles, and invert several times to mix.
6. Let floc settle and repeat mixing. Water with high chloride concentration requires 10
minutes contact with precipitate.
7. After floc has settled, leaving  100 ml clear supernate, remove stopper and add 2.0
ml H2SO4 down neck of bottle. If > 100 ppm Fe3+ is present, add 1.0 ml KF solution
before acidifying.
8. Re-stopper and mix by inversion until iodine is uniformly distributed.
9. Immediately titrate 203 ml (3 ml is allowance for added reagents) with 0.025 N
Na2S2O3 to pale straw yellow.
10. Add 1 – 2 ml starch indicator and titrate to disappearance of blue. Disregard
reappearance of blue.
11. Calculate the DO by using following equation.

DO (mg / liter) = (ml of 0.025 N Na2S2O3  0.2 / 200)  1000

12. Record the results and determine the quality of the wastewater by comparing the
recommended reference DO figures.
B. DETERMINATION OF BIOCHEMICAL OXYGEN DEMAND (BOD)
Introduction:
It is important to determine the activities of microorganisms and rate of
deoxygenation, the amount of organic matter in the water body receiving wastewater, for its
quality assurance and control tests. Increased introduction and accumulation of the organic
pollutants in the water body has many consequences such as decrease in the dissolved oxygen
(DO) concentration, lower availability of the oxygen to the organisms at comparatively high
organic matter concentration in the water body, elimination of the certain populations or
communities of organisms. In addition to this oxygen has extremely low solubility in water
(less than 12 mg / liter in the temperature range, significant for aquatic biology). As the
myriad variety of organic compounds enter the water body, it is not practical to measure, the
concentrations of each biodegradable organic compounds and the each population of
microorganisms involved in the depletion in the DO. Determination of the BOD is the method
devised to measure the pattern of the oxygen consumption while utilizing the organic matter.
For the determination of the BOD of wastewater, mathematical model has been
proposed but such kind of models are applicable under restricted conditions and do not met to
the actual situations in the wastewater. Among the several methods, the one used for
evaluation of wastewater treatment is the 5-day, 20 oC BOD (BOD5) bottle test. The chemical
oxygen demand (COD) test was developed because a BOD 5 test requires 5 days for
completion and therefore is not suitable either for real-time evaluation of the efficiency of
wastewater treatment or for operational control of the treatment processes. The total organic
carbon (TOC) test was developed as a third alternative for measuring the strength of
wastewater based on an assumption that the primary purpose of biological wastewater
treatment is to reduce the concentration of organic matter in the wastewater.
It is the bioassay in which, respiration (bio-oxidation of organic matter) of the
microorganisms present in the wastewater is measured. The sample is collected is sealed
during assay to prevent the interchange of the oxygen with surrounding. At a constant
temperature, the total amount of oxygen consumed varies with time. The selection of an
incubation time of 5 days at 20oC was a compromise from among many possibilities. The
temperature of 20oC is, roughly, a median summer temperature for surface waters in
temperate climates. Experience has shown that the microbiological oxidation of organic
matter in municipal wastewaters can be completed, for all practical purposes, in a period of 20
days at 20oC in a BOD bottle. However, 20 days is too long to wait for results, and the
empirically selected period of 5 days allows for exertion of about two-thirds of the 20-day
oxygen consumption of municipal wastewaters.
 Some points should be considered during this experiment such as the sufficient
dilution of the sample, DO of the diluted sample close to saturation, the test experiment
requires control experiment to run along with it, and addition compounds inhibiting the
microorganisms that gives false results.
The test involves the dilution of known quantity of wastewater to be tested.
Principally there should be sufficient organic matter in sample such that not all the oxygen in
the bottle is consumed during the biological oxidation of organic matter. After complete
oxidation of the organic matter the residual oxygen would be zero, therefore the BOD 5 cannot
be determined. Since the solubility of oxygen in water at 20 oC is about 9 mg / liter,
wastewaters have to be diluted 50- to 10,000-fold in order to have some DO remaining after 5
days of incubation. Dilution is important for the effluents with values less than 7 mg / liter,
usually two- to five- fold. The BOD5 is determined by the following equation.

BOD5 of the undiluted wastewater = (BOD5 of diluted sample)  (dilution factor)

Before making dilutions, the DO of the dilution water should be close to saturation,
because the respiration of some aerobic organisms is inhibited, if the DO drops below 1.0
mg./ liter during the BOD test. In order to provide a safety factor, the residual DO in the BOD
bottle after 5 days of incubation should be at least 2.0 mg / liter.
The test requires control experiment to run along with the test experiment, because the
dilution water is seeded with aerobic microorganisms capable of assimilating the organic
matter of the wastewater. In this situation even in the absence of wastewater, there may be a
small depletion of DO in 5 days. To account for this, for each BOD 5 test bottle, control bottle
containing only dilution water is incubated and a correction is made for the DO depletion in
the controls. The depletion of DO in the BOD test bottles should be at least 2 mg / liter during
the incubation period in order to be significantly greater than the DO depletion in the control.
To be sure of attaining this range of a DO depletion of at least 2 mg / liter with at least 2 mg /
liter remaining after the 5-day incubation period, it is necessary to prepare an incubate several
dilutions of the original sample.
The dilution water composition, therefore, becomes important. The dilution water
specified utilizes a phosphate buffer to provide a pH near neutrality during the incubation
period. The dilution water also contains the required mineral nutrients in case the wastewater
is deficient in any of them. Finally, to avoid confusing nitrogenous oxygen demand with
carbonaceous oxygen demand, the compounds inhibiting the nitrifying bacteria are
incorporated to the dilution water. It is important that the dilution water not contain any
constituents that are inhibitory to the microorganisms that exert the carbonaceous oxygen
demand. This needs to be demonstrated by running BOD 5 quality control standards prepared
from pure organic compounds, usually glucose and glutamic acid. The controls also protect
against the possibility of inhibitory material derived from the system for laboratory
purification of water used for making up solutions.
For measurement of the BOD5 of industrial wastewaters, the microorganisms that are
that are capable of degrading the waste organic compounds must be inoculated in the dilution
water. The seed may be obtained from a biological treatment process that has been acclimated
to the wastewater or from sediments downstream from the point at which the effluent is
discharged into the receiving waters. In some cases, development of a laboratory culture of
the seed organisms or purchase of a commercial seed may be necessary. Effluents from
biological treatment processes and municipal wastewaters are expected to contain the
microorganisms required for exertion of the carbonaceous oxygen demand, but dilution water
for testing these samples is seeded anyway
In very short time period, the COD test determines the oxygen equivalent of the
organic content of the wastewater. In the specified procedure, in the presence of catalyst
dichromate ion oxidizes the organic compounds by promoting the breakage of aromatic ring
structures. The dichromate solution is standardized in terms of equivalent molecular oxygen.
Most organic compounds are completely oxidized, or nearly so, but volatile compounds might
be driven out of the sample before they are oxidized, and some aromatic, nitrogen-containing
compounds are resistant to the oxidation. Inorganic compounds cause the interference with
the test.
Besides oxygen equivalent of the waste organic matter, COD test measures oxygen
equivalent of microbial cells. The oxygen demand associated with the microbial cells is only
partially exerted during the BOD5 test. Microorganisms, in either the BOD bottle or the
biological treatment process, may not metabolize some of the organic compounds, measured
by the COD determination. The COD, therefore, is usually higher than the BOD 5 both in
wastewater and in the effluent, but in many cases there is a consistent ratio between COD and
BOD5 although COD/BOD5 ratio for the untreated wastewater is different from the
COD/BOD5 ratio for effluent.
For adjusting the operation of a biological wastewater treatment process as the
strength of the waste varies from day to day, COD may be used as an index of BOD 5.
However, after around 50 years in general use, the COD test has not replaced the BOD 5 test
for evaluation of either the efficiency or the effectiveness of wastewater treatment, and there
is no indication that it is likely to do so.

Materials:
Sample
1.
2.
3.
Glassware and other equipments
1. BOD bottles (250 ml or 300 ml capacity)
2. Pipette of 10 ml capacity
3. BOD incubator (if it is not available, use the incubator adjusted to the 37oC)
Reagents, chemicals, and indicator
Refer to reagents, chemicals, and indicator used for the estimation of DO

Method:
As has been outlined earlier, BOD 5 is measured in terms of the difference in DO in
the sample after a period of 5 days incubation at 20oC.
1. Filter the sample to be tested through cotton wool. This will remove large sized particals
from the sample.
2. Take four sets of BOD bottles. Fill first set of bottles with the sample and dilutions of
the sample (Undiluted samples, 1:10 diluted, 1:100 diluted…, and 1:10,000) depending
on the extent of dilution required. The second set of bottles in made in exactly the same
manner as the first set. The third and fourth sets have one bottle each, containing the
dilution water [with seed (inoculum some aerobic bacteria)].
3. Fill the bottles as shown in the table to brim, stopper the bottles making sure that there
is bubble in the bottle.

Table No. 1: four sets of the BOD bottles used in the Azide (Alsterberg) method.
Set # 1 Undiluted 1:10 1:100 1:1000 1:10,000
(5 bottles) sample dilution of dilution of dilution of dilution of
sample sample sample sample
(S1-U) (S1-10) (S1-100) (S1-1000) (S1-10,000)
Set # 2 Undiluted 1:10 1:100 1:1000 1:10,000
(5 bottles) sample dilution of dilution of dilution of dilution of
 sample sample sample sample
(S5-U) (S5-10) (S5-100) (S5-1000) (S5-10,000)
Set # 3 ( 1 bottle) Dilution water (B1)
Set # 4 (1 bottle) Dilution water (B5)

4. Estimate the DO in the bottles of Set #1 (S1) and Set # 3 (B1) immediately, and note
the titration reading in ml.
5. Incubate the bottles of Set # 2 (S5) and Set # 4 (B5) at 37 oC (since a BOD incubator;
maintained at 20oC may not be available) for 5 days.
6. After 5 days incubation, take the bottles out and check for bubbles inside the bottle,
just under the stopper. If any air bubbles are present, DO NOT use these bottles for
the estimation of the DO. The presence of the air bubble indicates leakage of oxygen
or improper sealing of the stopper of the bottles.
7. After titration as described earlier, note the readings of these bottles. Tabulate all the
values as shown in table 2.
8. Select a suitable bottle(s) from the sets and calculate the BOD by using following
equation.

BOD (mg/liter) = (S1 – S5) – (B1 – B5)  0.2 x 1000  dilution factor
50 (volume of sample titrated)
 Table No.2: Titration of the acidified sample with 0.025 N Na 2S2O3.

DAY 1 READINGS DAY 5 READINGS

Sample in bottle Reading Mean Sample in bottle Reading Mean
reading reading
(ml) (ml) (ml) (ml)
Dilution water (B1) 1. Dilution water (B5) 1.
2. 2.
3. 3.
Undiluted (S1) 1. Undiluted (S5) 1.
2. 2.
3. 3.
Diluted 1:10 (S1) 1. Diluted 1:10 (S5) 1.
2. 2.
3. 3.
Diluted 1:100 (S1) 1. Diluted 1:100 (S5) 1.
2. 2.
3. 3.
Diluted 1:1000 (S1) 1. Diluted 1:1000 (S5) 1.
2. 2.
3. 3.
Diluted 1:10,000 (S1) 1. Diluted 1:10,000 (S5) 1.
2. 2.
3. 3.

C. DETERMINATION OF THE TOTAL SOLID (TOTAL FILTRABLE

SOLIDS AND TOTAL NON FILTRABLE SOLIDS) AND TOTAL
SUSPENDED SOLIDS

Introduction:
The “residue” generally refers to the solid matter suspended or dissolved in water or
wastewater. These residues may affect water or effluent quality adversely in a number of
ways. Waters with high residue generally are of inferior palatability and may induce an
unfavorable physiological reaction in the transient container. Highly mineralized waters also
are unsuitable for many industrial applications. For these reasons, a limit of 100 mg / L
residue is desirable for drinking waters. Waters with very high levels of non-filterable
residues may be aesthetically unsatisfactory for such purposes as bathing. “Total residue” is
the term applied to the material left in the vessel after evaporation of a sample and its
subsequent drying in an oven at a defined temperature. Total residue includes “non-filterable
residue” and “filterable residue”. The terms used earlier, i.e. “suspended” and “dissolved”
(residue) correspond to non-filterable and filterable residue, respectively.
Residues dried at 103-105oC may be expected to retain, not only water of
crystallization, but also some mechanically occluded water. Loss of organic matter by
volatilization will be very slight at this temperature, if it occurs at all. Because removal of
occluded water is marginal at 105oC, attainment of constant weight is very slow.
A well-mixed sample is evaporated in a weighed dish and dried to constant weight in
an oven at 103oC to 105oC. The increase in weight over that of the empty dish represents the
total residue, which is an arbitrary quantity, defined by the procedure followed. Although the
results of such procedures may not be completely accurate or reproducible and may not
represent the weight of the actual dissolved and suspended solids in wastewater samples, the
determination serves a useful purpose for wastewater treatment plant control.
Interference in estimation could come through large, floating particles or submerged
agglomerates of non-homogenous materials in the samples. These should be excluded prior to
drying. If there is visible floating oil and grease, the contents should be dispersed
homogeneously using a blender before withdrawing a sample portion for analysis.

Materials:
Sample:
1.
2.
3.
Glassware and other equipments:
1. Evaporating dishes of 100 ml capacity made of porcelain (90 mm diameter)
2. Muffle furnace (550  50oC)
3. Steam bath tub
4. Drying oven (thermostatically controlled for  1oC range)
5. Desiccator with a desiccant containing a color indicator of moisture concentration
6. Electronic analytical balance (200 g capacity capable of weighing to 0.1 mg).

Method:
1. Take a clean porcelain dish and heat it to 550  50oC for 1 hour in a muffle furnace.
2. Cool, desiccate and weigh the dish and store in a Desiccator until ready for use.
3. Transfer a measured sample to the pre-weighed dish and evaporate till it dries on a
steam bath or in a drying oven. Sample volume is chosen such that it will yield a
minimum residue of 25 mg to 250 mg. If the drying is being done in a drying oven, the
temperature should be lowered to 98oC to prevent boiling and splattering. Dry the
evaporated sample for at least 1 hour at 103 – 105oC.
 4. Cool the dish in a Desiccator and weigh on the electronic weighing balance. Repeat
the cycle of drying at 103 – 105oC, cooling, desiccating and weighing until a constant
weight is obtained, or until loss of weight is less than 4% of the previous weight, or
0.5 mg, which ever is less.
5. Using following equation, determine the total residue (in mg) per liter of the sample
taken.

(A - B)  1000
Total residue (mg / L) 
Sample taken in ml
Where A  Weight of the sample  weight of the dish
B  weight of the dish

Total Filterable Residue Dried at 180oC

Filterable residue is material that passes through a standard glass fiber filter disk and
remains after evaporation and drying to constant weight at 180oC. Highly mineralized waters
with a considerable calcium, magnesium, chloride and/or sulfate content may be hygroscopic
and require prolonged drying, proper desiccation and rapid weighing. Samples high in
bicarbonate require careful and possibly prolonged drying at 180 oC to ensure complete
conversion of bicarbonate to carbonate.

Materials:
1. All materials required for the estimation of the total solids.
2. Glass fiber filter disks without organic binder
3. Filtration apparatus (filter holder, Gooch crucible adapter)
4. Gooch crucible of 25 ml capacity
5. Suction flask.

Method:
1. The glass fiber disk is placed in filtration assembly and washed with 3 successive 20
ml volumes of distilled water. Suction is continued to remove all traces of water from
the disk. Washings are discarded.
2. The evaporating dish is prepared as in estimation for total solids.
3. 100 ml or more of a well-mixed sample is filtered under vacuum. The suction is
continued for about 3 minutes after filtration is complete to remove as much water as
possible.
 4. Transfer 100 ml of the filtrate to an evaporating dish and evaporate till it dries, on a
steam bath.
5. Dry the evaporated sample for at least 1 hour in an oven at 180  2 oC.
6. Cool in a Desiccator and weigh on the electronic balance.
7. Repeat the drying cycle until a constant weight is obtained or until the weight loss is
less than 0.5 mg.
8. using following formula determine the total

(A - B)  1000
Filterable residue at 180C (mg / L) 
Sample taken in ml
Where A  Weight of the dried residue  weight of the dish
B  weight of the dish

Total Filterable Residue Dried at 103 - 105oC

The procedure for this estimation is the same as described for total filterable residue at
180oC. The filtrate is dried at 103-105 oC instead of 190oC. Manipulate the above equation and
determine the filterable residue at 103-105oC.

D. DETERMINATION OF THE TOTAL NON-FILTERABLE RESIDUE

DRIED AT 103 - 105OC
Total non-filterable residue is the retained material on a standard glass fiber filter disk
after filtration of a well mixed sample of water or wastewater. The residue is dried at 103 -
105oC. If the suspended material clogs the filter and prolongs the filtration time, the difference
between the total residue and the total filterable residue provides an estimate of the total non-
filterable residue.

Materials:
Sample:
1.
2.
Glassware and other equipments:
 All materials required for estimation of total solids and total filterable solids (refer to
earlier text).

Method:
1. Prepare the glass fiber paper disk (refer to earlier text).
2. Filter the sample under vacuum (refer to earlier text).
3. Remove the filter and transfer it to an aluminium or stainless steel planchet as
supporting medium. If a Gooch crucible is used, remove the crucible-filter
combination.
4. Dry the filter in an oven at 103 – 105oC for 1 hour.
5. Cool the filter paper in a desiccator and weigh it on electronic balance. Repeat the
drying cycle until a constant weight is attained or until weight loss is less than 0.5 mg.
6. Determine the quantity of non – filterable residue by following equation.

(A - B)  1000
Non - Filterable residue (mg / L) 
Sample taken in ml
Where A  Weight of the filter paper disk  weight of the residue
B  weight of the filter