Exp 3 Biochemical Oxygen Demand



To determine the amount of Biochemical Oxygen Demand (BOD) in domestic waste water.


Biochemical Oxygen Demand (BOD)

The  amount  of  oxygen  required  by  the  bacteria  while  stabilizing  decomposable organic  matter under aerobic conditions. Decomposable means that organic  matter can serve as food for the bacteria and energy is derived from its oxidation.

  • Biochemical oxygen demand is a measure of the quantity of oxygen used by microorganisms (e.g., aerobic bacteria) in the oxidation of organic matter.
  • Natural sources of organic matter include plant decay and leaf fall. However, plant  growth and decay may be unnaturally accelerated when nutrients and sunlight are overly abundant due to human influence.
  • Urban runoff  carries  pet wastes  from streets and sidewalks;  nutrients  from lawn  fertilizers;  leaves,  grass  clippings,  and  paper  from  residential  areas, which increase oxygen demand.
  • Oxygen consumed in the decomposition process robs other aquatic organisms of the  oxygen they need to live. Organisms that are more tolerant of lower dissolved oxygen levels may replace a diversity of more sensitive organisms.

BOD Level (in ppm)               Water Quality

1 – 2                                           Very Good-not much organic waste present

3 – 5                                           Moderately clean

6 – 9                                           Somewhat polluted

10+                                            Very polluted

Importance of BOD Test in Environmental Engineering

1 The BOD test is used to determine the relative oxygen requirements of wastewaters, effluents,  and  polluted  waters.  The  test  measures  the  oxygen  utilized  during  a specified incubation period for the biochemical degradation of organic material. It is also used to determine treatment plant efficiency.

Determination of BODexp3


The method consists of filling with sample, to overflowing, an airtight bottle of the specified  size and incubating it at the specified temperature for 5 days. Dissolved oxygen is measured initially and after incubation, and the BOD is computed from the difference between initial and final DO. Because the   initial DO is determined shortly after the dilution  is  made,  all  oxygen uptake occurring  after this  measurement  is included in the BOD measurement.

Sampling and Storage:

Sample  for  BOD  analysis  may   degrade   significantly   during   storage  between collection and analysis, resulting in low BOD values. Minimize reduction of BOD by analyzing  sample  promptly  or  by  cooling  it  to  near-freezing  temperature  during storage. However, even at low temperature, keep holding time to a minimum. Warm chilled samples to 20 ± 3°C before analysis.


a.         Incubation bottles: Use glass bottles having 60 mL or greater capacity (300mL bottles having ground-glass stopper and a flared mouth are preferred).
b.         Air  incubator  or  water  bath,  thermo-statistically  controlled  at  20  ±  1°C. Exclude all light to prevent possibility of photosynthetic production of DO.


Air Incubator


Prepare  reagents  in  advance  but  discard  if  there  is  any  sign  of  precipitation  or biological growth in the stock bottles.

a.         Phosphate buffer solution: Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4,  33.4 g

Na2HPO4.7H2O, and 1.7 g NH4CI in about 500 mL distilled water and dilute

2 to 1  Lit.  The  pH  should  be  7.2  without  further  adjustment.  Alternatively, dissolve 42.5 g KH2PO4   or 54.3 g K2HPO4   in about 700 mL distilled water.

Adjust pH to 7.2 with 30% NaOH and dilute to I Lit.

b.         Magnesium sulfate solution: Dissolve 22.5 g MgS04.7H20 in distilled water and dilute to 1 L.

c.         Calcium chloride solution: Dissolve 27.5 CaCl2  in distilled water and dilute to 1 L.

d.         Ferric Chloride solution: Dissolve 0.25 g FeCl3.6H2O in distilled water and dilute to 1 L.

e.         Acid  and  alkali  solution,  1N,  for neutralization  of  caustic  or  acidic  waste samples. 1) Acid-Slowly and while stirring, add 28 mL cone. Sulfuric acid to distilled Water.  Dilute to 1 L. 2) Alkali-Dissolve  40 g sodium hydroxide in distilled water. Dilute to 1 L.

f.         Sodium sulfate solution: Dissolve 1.575 g Na2SO3  in 1000 mL distilled water. This solution is not stable; prepare daily.

g.         Nitrification  inhibitor:  2-chloro-6-(trichloromethyl)  pyridine  (if  nitrification inhibition desired).

h.         Glucose-glutamic acid solution: Dry reagent-grade glucose and reagent-grade glutamic acid at 103°C for 1 h. Add 150 mg glucose and 150 mg glutamic acid to distilled water and dilute to 1 L. Prepare fresh immediately before use.

i.          Ammonium  chloride  solution:  Dissolve  1.15  g  NH4CI  in  about  500  mL distilled  water,  adjust  pH  to  7.2  with  NaOH  solution  and  dilute  to  1  L. Solution contains 0.3 mg N mL-1

j.          Dilution water: Use demineralized, distilled, tap, or natural water for making sample dilutions.

Procedure:  (with out seeding)exp3.3

1)  First of all it is important to know the amount of samples to be used for test.

For this purpose the source of sample is to be recorded which will indicate the approximate value of BOD5 for the sample.

(i)         Domestic sewage BOD5  =100-500mg/L

(ii)        Effluent from treatment plant= 20-80mg/L (iii)    River water = 2-4mg/L

2)  Take 9 BOD bottles note their numbers and arrange them in 3 groups.

3)  Fill each bottle half with dilution media ensuring that no air gets mixed with the media while fill in as in DO test.

4)  Add 2ml sample in each of the three bottles marked as first group; 5 ml in each bottle pf 2nd group and 10ml in each bottle of the 3rd  group.

5)  Fill the bottle completely with dilution media and place the stopper such that no air bubbles are  trapped.

6)  Now take one bottle from each set and estimate its DO. This will

be DO initial or DO 0days.

7)  For comparison prepare two more bottles with blank dilutions media (with out sewage sample) and find the DO from one bottle.

8)  Place the rest of the six bottles with sewage samples and one bottle for blank in the incubator at 200C

9)  After 5 days find out DO in all bottles.

10) That value of oxygen depletion should be considered correct which gives an oxygen  depletion of at least 2 mg/L. and which have at least 0.5 mg/L DO after 5 days of incubation.

11) Calculate BOD5  at 200C. for the sample using following relation ship.

BOD(mg/L) = (DO depletion (mg/L) *300 ) / (Volume of sample in bottle (ml))

Observations & Calculations

 At zero days.


Sample added (ml)

Volume of sample (ml)

Volume of Na2S2O3

DO (mg/L)





















After 5 days.






Sample added (ml)



Volume of sample (ml)


Volume of Na2S2O3


DO (mg/L)


Mean DO (mg/L)









































Sr #

Sample added (ml)

DO at Zero days (mg/L)

DO at 5 days (mg/L)

DO Depleted (mg/L)

DO Depleted  – Blank DO depleted (mg/L)

BOD5    (mg/L)



























                                                                                                               Mean BOD5  = 482 mg/L


DO depletion in case of blank should be equal to zero but in our case it is coming out to be 0.1 mg/L. This value should be subtracted from the readings and then the B.O.D should be calculated. The possibility of entrance of air can also be one reason because we dropped the extra amount of sample Over the cap and do depletion occurred in case of blank also which is an error &  should not be corrected. Our BOD value came out to be 482 mg/L which is much higher and this can be of untreated sewer.


 1). Define BOD.exp3.4

Biochemical oxygen demand or BOD is a chemical procedure for determining the amount of dissolved oxygen needed by aerobic biological organisms in a body of water to break down organic material present in a given water sample at certain temperature over a specific time period. It is not a precise quantitative test, although it is widely used as an indication of the organic quality of water.[1] . It is most commonly expressed in milligrams of oxygen consumed per litre of sample during 5 days of incubation at 20 C and is often used as a robust surrogate of the degree of organic pollution of water.

2) Why natural water and tap water cannot be used for preparing dilution media in the BOD test.?

In natural waters suspected of carrying large amounts of organic waste/ sewage, the oxygen demand may be so great that all oxygen is consumed before the 5-day period. The above approach would not reveal the true oxygen demand over the 5-day period.

  • Ø Urban runoff of rain and melting snow that carries sewage from illegal sanitary sewer connections into storm drains; pet wastes from streets and sidewalks; nutrients from lawn fertilizers; leaves, grass clippings, and paper from residential areas;
  • Ø Agricultural runoff that carries nutrients, like nitrogen and phosphates, from fields;
  • Ø Runoff from animal feedlots that carries faecal material into rivers.

Chlorine present in tap water can also affect BOD measurement by inhibiting or killing the microorganisms that decompose the organic and inorganic matter in a sample. If you are sampling in chlorinated waters, such as those below the effluent from a sewage treatment plant, it is necessary to neutralize the chlorine with sodium thiosulfate.

Various contaminants in the water may have an effect on BOD:


Biochemical oxygen demand is a measure of the quantity of oxygen used by microorganisms in the oxidation of organic matter. If organic matter is present in the dilution water, it may increase its oxygen demand.

Chlorine or other disinfectants

Chlorine is often present in tap water in order to control microbial contamination. As chlorine would interfere with the microorganisms used in the BOD test, it should be removed from the water used for the dilutions and the blanks. The same is true of other commonly used tap water disinfectants (chloramine, etc.)

Heavy metals (copper, mercury, cadmium…)

Water must be free of heavy metals toxic to microorganisms. Like for disinfectants, any compound which may inhibit the growth of microorganisms will have a deleterious effect on the BOD test.


While bacteria are a necessary component of the BOD test, it is best to minimize their levels in the dissolution water, as they may release organics during storage.

Distilled water may be used to prepare dilution water; however, chlorine may co-distil with water and interfere with the BOD test. This water would require an additional sodium thiosulfate treatment. Distillation from alkaline permanganate is sometimes recommended, but this purification procedure is quite cumbersome as well. Deionized water, purified with ion exchange resins, may contain organics and microorganisms, thereby causing BOD blank failure. It is therefore not recommended for this test.

Water purified with a combination of technologies, such as reverse-osmosis, ion exchange, activated carbon and ultra-violet photooxidation is extremely low in organics and contains no inorganic substances toxic to bacteria. It is best fitted for the preparation of BOD dilution water.

Examples illustrating the impact of water quality on the BOD test

In order to better understand the effect of water quality on BOD blanks, three types of water were evaluated:

  • Tap water
  • Deionized water (DI) obtained using ion-exchange resins
  • High purity water produced by a Direct-Q 3 UV water purification system

For each water type, seven blanks were prepared every week for seven weeks. Disposable BOD bottles were used (made of PET with an inner amorphous carbon coating that prevents oxygen transport), as well as a luminescent dissolved oxygen probe (LBOD) from Hach Company (Loveland, CO).

Figure 1: BOD blank averages and maximums for three water types.exp3.5
Disposable PET bottles and LBOD probe were used. Bars represent the average of 49 blanks, error bars represent standard deviations.

The same three types of water (tap, deionized (DI), and Direct-Q 3 UV high purity water) were evaluated using either the standard glass BOD bottles or disposable PET bottles. Glass BOD bottles should be carefully washed, cleaned with acid and rinsed, while disposable PET bottles do not require washing.

Figure 2: BOD blank averages for glass and PET bottles .Bars represent the average of 7 blanks, error bars represent standard deviations.

The high purity water produced by the Direct-Q 3 UV water purification system is extremely low in organics and contains no inorganic substances toxic to bacteria. This study confirmed that the water produced by this system reliably yields low blank BOD values and good test reproducibility.

3) What is the role of nitrifying bacteria in BOD test?

 Sometimes wastewaters do not contain significant levels of microorganisms or certain nutrients and may have to be supplemented with these additives. In other cases, the levels of organic matter are too high and the wastewater samples have to be diluted. If microorganisms are added or the wastewater sample is diluted, the measured BOD value must be corrected appropriately.

In addition to the organic matter, any ammonia present in a waste stream may also be oxidized by nitrifying bacteria in a process called nitrification. Nitrification also demands oxygen, which is referred to as nitrogenous BOD (NBOD). A general equation for the overall nitrification process is shown below.

ammonia + oxygen + carbon dioxide + nitrifying bacteria ==== nitrate + water + new cells + energyexp3.6

Nitrifying bacteria grow slowly, more slowly than the microorganisms that oxidize organic matter, and it normally takes from 6 to 10 days before they start to consume oxygen. However, as shown in the figure below, if a significant number of nitrifying bacteria are present in the wastewater, they might exert sufficient oxygen demand to introduce error even into the measurement of organic matter using the BOD5 test. In these

cases, the wastewater sample being tested should be pre-treated with an agent that suppresses nitrifying bacteria, and the results of the BOD test should be reported as CBOD (carbonaceous BOD).

4) What is the history of BOD test?

The Royal Commission on River Pollution, which was established in 1865 and the formation of the Royal Commission on Sewage Disposal in 1898 led to the selection in 1908 of BOD5 as the definitive test for organic pollution of rivers. Five days was chosen as an appropriate test period because this is supposedly the longest time that river water takes to travel from source to estuary in the U.K. In 1912, the commission also set a standard of 20 ppm BOD5 as the maximum concentration permitted in sewage works discharging to rivers, provided that there was at least an 8:1 dilution available at dry weather flow. This was contained in the famous 20:30 (BOD:Suspended Solids) + full nitrification standard which was used as a yardstick in the U.K. up to the 1970s for sewage works effluent quality.

The United States includes BOD effluent limitations in its secondary treatment regulations. Secondary sewage treatment is generally expected to remove 85 percent of the BOD measured in sewage and produce effluent BOD concentrations with a 30-day average of less than 30 mg/L and a 7-day average of less than 45 mg/L. The regulations also describe “treatment equivalent to secondary treatment” as removing 65 percent of the BOD and producing effluent BOD concentrations with a 30-day average less than 45 mg/L and a 7-day average less than 65 mg/L.

 Typical BOD values

Most pristine rivers will have a 5-day carbonaceous BOD below 1 mg/L. Moderately polluted rivers may have a BOD value in the range of 2 to 8 mg/L. Municipal sewage that is efficiently treated by a three-stage process would have a value of about 20 mg/L or less. Untreated sewage varies, but averages around 600 mg/L in Europe and as low as 200 mg/L in the U.S., or where there is severe groundwater or surface water infiltration. (The generally lower values in the U.S. derive from the much greater water use per capita than in other parts of the world.)

5) What is seeding?

The purpose of seeding is to introduce a biological population capable of oxidising the organic matter in the sample. Seeding would not be necessary for domestic and municipal sewage, unchlorinated treated effluents and surface waters. When there is a reason to believe that the sample contains very few micro-organisms, for example as a result of chlorination, high temperature, extreme pH or because of the specific composition of some industrial wastes, the dilution water should be seeded.

Most often, the supernatant of fresh, settled sewage may be used as a seed. In cases where the sample may contain organic matter, which is hard to degrade, the seed may be developed by adding a small amount of soil to a portion of the sample and aerating it for 24 to 48 hours. Soil is a medium that supports a wide variety of micro-organisms capable of metabolising many different types of organic matter. Alternatively, water from a body of water receiving the waste may be used as a source of seed. A small volume of seed added to the dilution water, 4 – 6 mL per litre, would contain a sufficient number of micro-organisms adapted to the waste to carry out the oxidation of the organic matter.

Correction must be carried out to account for the oxygen consumed in oxidation of organic matter carried with the seed. The volume of seed added to the dilution water should be recorded and parallel seed control test should be run to determine the BOD of the seed.

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