GENERAL:- In the presence of free oxygen, aerobic bacteria use the organic matter found in wastewater as “food”.  The BOD test is an estimate of the “food” available in the sample.  The more “food” present in the waste, the more Dissolved Oxygen (DO) will be required. The BOD test measures the strength of the wastewater by measuring the amount of oxygen used by the bacteria as they stabilize the organic matter under controlled conditions of time and temperature.
BOD INTRODUCTION:- The BOD test is used to measure waste loads to treatment plants, determine plant efficiency (in terms of BOD removal), and control plant processes.  It is also used to determine the effects of discharges on receiving waters.  A major disadvantage of the BOD test is the amount of time (5 days) required to obtain the results.
When a measurement is made of all oxygen consuming materials in a sample, the result is termed “Total Biochemical Oxygen Demand” (TBOD), or often just simply “Biochemical Oxygen Demand” (BOD).  Because the test is performed over a five day period, it is often referred to as a “Five Day BOD”, or a BOD5.
In many biological treatment plants, the facility effluent contains large numbers of nitrifying organisms which are developed during the treatment process.  These organisms can exert an oxygen demand as they convert nitrogenous compounds (ammonia and organic nitrogen) to more stable forms (nitrites and nitrates).  At least part of this oxygen demand is normally measured in a five day BOD.
Sometimes it is advantageous to measure just the oxygen demand exerted by organic (carbonaceous) compounds, excluding the oxygen demand exerted by the nitrogenous compounds.  To accomplish this, the nitrifying organisms can be inhibited from using oxygen by the addition of a nitrification inhibitor to the samples.  The result is termed “Carbonaceous Biochemical Oxygen Demand”, or CBOD.
TYPES OF SAMPLES:- Samples used for the BOD test can be either grab or composite.  A composite sample  is usually specified in most NPDES Permits and will be more representative of the wastestream over a period of time than will a grab sample.  The type and location of sample taken will depend on  your facility NPDES Permit.  Samples should be the type which best fits the capabilities of and requirements for each individual plant.  Samples should be taken at a point where they will be well-mixed and proportional to the amount of the flow.
SAMPLE PRESERVATION:- Samples for BOD analysis may change greatly during handling and storage.  Testing should be started as quickly as possible.  To reduce the changes in those samples which must be held, keep the samples at or below 4°C.  Do not allow samples to freeze.  Samples may be kept for no more than 48 hours before beginning the BOD test.
NOTE:  The 48 hours starts when the very first aliquot of a composite sample is collected (i.e. when the composite sampler starts collecting a composite sample).
SAMPLE CONTAINERS:- Special sampling devices and storage containers are not necessary for collecting samples for BOD determinations. Sampling devices should be capable of collecting samples from well mixed areas of tanks and/or pipes, made of resistant materials that will not rust or corrode, capable of taking samples that are proportional to the plant’s flow and easily cleaned (including acid cleaning).
NOTE:  A long handled aluminum dipper attached to a wooden handle, or an equivalent device, is acceptable for collecting samples.  Do not use containers such as coffee cans.
Storage containers should be made of corrosion resistant material (such as plastic), which can stand repeated refrigeration.  These containers should have leak proof caps or lids.
SAMPLE CONTAINER PREPARATION:- All collection containers should be cleaned thoroughly on a regular basis (preferably at the end of each day’s sampling) with soap and water, and rinsed well with distilled water.  This will prevent buildup (such as grease and scum) from contaminating samples.  Between sample collections, the sampling containers should be rinsed thoroughly and allowed to dry.  This is especially important for containers used for samples which are high in solid and/or grease content.  It is recommended that each collection point have its own sampling container.  One sampling container should not be used throughout the plant.  If separate containers are not possible, be sure to clean sampling containers thoroughly between collections.
Sample storage containers should also be cleaned thoroughly between samples.  It is recommended that they be acid cleaned on a regular basis to prevent residue buildup which occurs over time.  It is also recommended that each sampling point have its own storage container.  (For example, if influent and effluent samples are taken, always use the same containers for influent and do not use a container for influent one day and then effluent the next.)  The containers should be clean and dry before a new set of grab or composite samples are stored in them.
BOD – DESCRIPTION OF METHOD:- A sample is pipetted into a BOD bottle containing aerated dilution water.  The DO content is determined and recorded and the bottle is incubated in the dark for five days at 20°C.  At the end of five days, the final DO content is determined and the difference between the final DO reading and the initial DO reading is calculated. The decrease in DO is corrected for sample dilution, and represents the biochemical oxygen demand of the sample.
Test reagents are as follows:

  1. Phosphate buffer solution
  2. Magnesium sulfate solution (MgSO4.7H2O)
  3. Calcium chloride solution (CaCl2)
  4. Ferric chloride solution (FeCl3)
  5. Sodium hydroxide (NaOH), 1 N
  6. Sulfuric acid (H2SO4), 1 N
  7. Sodium sulfite (Na2SO3), 0.025 N
  8. Potassium iodide solution (KI), 10%
  9. Acetic acid solution (CH3CO2H), (1:1)
  10. Sulfuric acid solution (H2SO4), (1:50)
  11. Starch indicator solution
  12. Glucose glutamic acid solution
  13. Nitrification inhibitor (2 chloro 6 (trichloromethyl) pyridine)
  14. Distilled water.

NOTE:  Use only high grade distilled or deionized water. The water must contain less than 0.01 mg/L copper, and be free of chlorine, chloramines, caustic alkalinity, organic material, or acids.

  1. BOD meter with probe for measurement of dissolved oxygen in 300 mL BOD bottles
  2. 300 mL BOD bottles
  1. Incubator, capable of maintaining 20±1°C
  1. 250 mL graduated cylinders
  2. 100 mL graduated cylinders
  3. 25 mL measuring pipettes (wide mouth)
  4. 10 mL measuring pipettes (wide mouth)
  5. 100 mL beaker
  6. 1000 mL beaker
  7. 250 mL Erlenmeyer flask
  8. Burette graduated to 0.1 mL
  9. Dilution water bottle of suitable volume for the number of tests to be performed
  10. Pipette bulb
  11. Equipment for pH measurements
  12. Magnetic stirrer and stirring bars

The BOD test relies on a measurable depletion of DO over a specified period of time.  Because most samples of wastewater will have a BOD higher that the amount of oxygen available in the BOD bottle during the incubation period, the samples must be diluted.  This dilution is done by adding dilution water to the sample in the BOD bottle.  If the sample is not diluted, the biological activity of the microorganisms will use up the DO in the BOD bottle before the five day incubation time is up.  If the final DO is too low, the BOD cannot be determined.  There is no way of knowing at what point during the five days the DO reached zero.
One of the most difficult steps in the BOD procedure is deciding how much sample to place in the BOD bottles for incubation.  Some plants have influent and effluent BOD’s that do not vary greatly over time, while others fluctuate greatly from day to day.  In all cases, several different dilutions of each sample should be prepared to obtain the desired DO depletions.
Once a general range for the BOD of a sample has been determined, the dilutions can be established which will ensure that at least one dilution will meet the criteria for valid BOD results.  The following procedure can be used to calculate volumes for sample dilution from the estimated BOD.
the formulas to calculate the minimum and maximum estimated dilution are as follows:

  1. mL sample added to BOD bottle = (minimum allowable depletion, mg/L x Volume of BOD bottle, mL)/estimated BOD, mg/L
  2. mL sample added to BOD bottle = (maximum allowable depletion, mg/L x Volume of BOD bottle, mL)/estimated BOD, mg/L

Since the BOD value used is only an estimate, and BOD bottles do not always have a volume of exactly 300 mL, several bottles with different volumes of sample are set up to ensure that test requirements are met.
NOTE:  Those sample dilutions which deplete less than 2 mg/L, or have a final DO of less than 1 mg/L would not be used in the calculation of the average sample BOD.
It is very important that the distilled water used for dilution water be of high grade and free from contaminants (such as copper and chlorine) which could inhibit the growth of bacteria.  For this reason, it is recommended that ordinary commercial distilled water (i.e. for use in car batteries) not be used.
Prepare dilution water as follows:

  1. If necessary, aerate a bottle of distilled water long enough to allow the water to become saturated with dissolved oxygen (approximately 8 mg/L at room temperature). This can be accomplished by aerating with clean compressed air.  If 5 gallon water bottles are used, aerate for 24 hours with a small aquarium pump.
  2. If less than the entire bottle will be used in a single day, siphon into a separate container an amount slightly greater than will be needed for the sample dilutions.
  3. Add 1 mg/L each of phosphate buffer, magnesium sulfate solution, calcium chloride solution and ferric chloride solution.
  4. For those samples which require seeding, the analyst may add a sufficient volume of seed directly to the dilution water, or a small amount of seed directly to the sample dilutions.
  5. If nitrification inhibition is used, store seeded dilution water at 20°C long enough for the dilution water depletion to meet the quality criteria (depletion of no more that 0.2 mg/L DO). Storage is not recommended when nitrification inhibition is not going to be used because nitrifying bacteria can develop in the dilution water during storage.
  6. If nitrification inhibition is to be used, add enough nitrification inhibitor to the dilution water to produce a final concentration of 10 mg/L. As an alternative, 3.33 mg of nitrification inhibitor can be added to each BOD bottle for inhibition.

PRETREATMENT OF SAMPLE:- Samples with extreme pH values and samples containing disinfectants such as residual chlorine must be treated prior to testing.
PRETREATING SAMPLES THAT CONTAIN CAUSTIC ALKALINITY OR ACIDITY:- Caustic alkalinity or acidity can prevent bacteria from growing during the course of the BOD test.  To prevent this, samples which have pH values higher than pH 8.0 or lower than pH 6.0 must be neutralized to pH 7.0 before the test is performed.
NOTE:  Neutralized samples must be seeded for the BOD test.
Procedure for neutralizing samples

  1. Pour 50 mL of sample into a 100 mL beaker.
  2. Measure the pH of the sample using a pH meter. If the pH is out of the range of pH 6.0 to pH 8.0 continue with steps 3 6, otherwise perform the BOD test on the untreated sample.
  3. Add 1 N sulfuric acid if the sample is alkaline, or 1N sodium hydroxide if the sample is acidic, until the pH reaches 7.0.
  4. Calculate the amount of sulfuric acid or sodium hydroxide needed to neutralize 1000 mL of the sample.
  5. Add the calculated amount of acid or base to the sample.
  6. Repeat steps 1 5 until the pH test shows pH 7.0.


  1. Calculate the amount of 1 N sodium hydroxide or 1 N sulfuric acid needed to neutralize the sample to pH 7.0 using the following formula:

mL needed = (mL acid or base used x mL total test sample)/mL sample portion used for neutralization.
Because chlorine is such a strong oxidizing agent, it will inhibit the growth of living bacteria in the BOD test.  Any samples containing residual chlorine must be pretreated to remove chlorine before the test is run.  This is done by adding sodium sulfite to the sample.
NOTE:  Those samples which are dechlorinated must be seeded for the BOD test.
Procedure for dechlorinating samples:

  1. To a 250 mL Erlenmeyer flask, add 100 mL of a well mixed portion of the sample to be dechlorinated.
  2. Add 10 mL of either 1:1 acetic acid solution or 1:50 sulfuric acid solution to the flask and swirl to mix.
  3. Add 10 mL of potassium iodide (KI) solution, and 1 mL of starch indicator solution. Swirl to mix and let stand for 15 minutes.
  4. If a blue color does not appear, there is no chlorine in the sample and it does not require further treatment prior to the BOD test.

NOTE:  Do not assume that the sample was not chlorinated simply because there is no reaction.  Chlorine can disappear from the sample while it sits in the sample container.  The only way to be sure a sample is not chlorinated is to know exactly where the sample was collected.

  1. If a blue color appears, titrate the treated portion of sample with 0.025 N sodium sulfite (Na2SO3) until the blue color first disappears. Record this amount on a lab sheet.
  2. Calculate the amount of sodium sulfite solution needed to dechlorinate the selected BOD sample volume.
  3. Add the calculated volume of sodium sulfite to the BOD sample and mix thoroughly.
  4. Allow the sample to stand for 10 to 20 minutes, then repeat steps 1 3.
  5. If no chlorine is detected, continue with the BOD test procedure, otherwise continue with steps 5 8 until the sample is dechlorinated.


  1. Calculate the amount of sodium sulfite needed to dechlorinate the BOD sample using the following formula:

mL Na2SO3 needed = (mL Na2SO3 used x mL total test sample)/mL sample portion used for dechlorination.

  1. Completely fill two BOD bottles with dilution water.
  2. Into additional BOD bottles, partially filled with dilution water, carefully measure out the proper volume of sample. Add dilution water until the bottles are completely filled.

NOTE:  If the modified Winkler procedure is to be used for DO measurements, two BOD bottles should be prepared for each dilution; one for determination of the initial DO and one for incubation and final DO measurement.  If the meter method is used for DO measurements the initial and final DO determinations can be performed on the same bottle.
ADDITIONAL NOTE:-  If the nitrification inhibition is to be used to determine the carbonaceous BOD fraction (CBOD) of the sample, a separate dilution series of uninhibited sample can be prepared to determine the combined nitrogenous and carbonaceous BOD for the sample.  To inhibit the nitrifying bacteria in the sample, add 3.33 mg of nitrification inhibitor to one set of sample dilutions, while the second set of dilutions remains untreated.  Continue with the remaining procedural steps with both sets of dilutions.

  1. Stopper each bottle taking care to avoid trapping air bubbles inside the bottles as the bottle stoppers are inserted.
  2. Fill the top of each bottle neck around the stopper with dilution water.
  3. Determine the initial DO content on one of each set of duplicate bottles, including the dilution water blank by one of the approved methods and record data on the lab sheet.
  4. Place the remaining bottles in the incubator at 20°C and incubate for five days.
  5. At the end of exactly five days (±3 hours), test the DO content of the incubated bottles.
  6. Calculate the BOD for each dilution. The most accurate BOD will be obtained from those dilutions that have a depletion of at least 2 mg/L DO and at least 1.0 mg/L DO residual.  If there is more than one dilution that meets these criteria, the BOD results should be averaged to obtain a final BOD value.
  7. The dilution water blanks are used only to check the quality of the dilution water. If the quality of the water is good and free from impurities, the depletion of DO should be less than 0.2 mg/L.  In any event, do not use the depletion obtained as a blank correction.
  8. If nitrification inhibition is used, the BOD test must also be performed on a series of sample dilutions which have not been inhibited.
  9. Report the results of the nitrification inhibited samples as CBOD5 and uninhibited samples as BOD5.

To determine the value of the BOD in mg/L, use the following formula:
BOD, mg/L = [(Initial DO   Final DO) x 300]/mL sample
Whenever a sample is dechlorinated, it must be seeded.  If the sample is seeded, a correction factor must be calculated to determine the effects that the seed material has on the DO depletion.  A number of BOD’s must be run on the seed material to determine the seed correction factor.
INTERFERENCES:- Since the BOD test is dependent on biological activity, the major interferences will be those substances which inhibit the growth of the microorganisms.  These will include chlorine, caustic alkalinity or acidity, mineral acids, and heavy metals (such as copper, zinc, chromium, and lead).
Excessive nitrites can interfere with the BOD determination.  Growth of algae in the presence of light can cause problems by actually increasing the DO of the sample before testing, which must be removed by deaeration.
A common problem encountered in BOD testing results from residues building up in the BOD and dilution water bottles.  To prevent this, all glassware should be acid cleaned on a regular basis.
PRECISION AND ACCURACY:- While precission is sometimes tough with the BOD test, a check of dilution water quality, seed effectiveness, and analytical technique can be made using a glucose glutamic acid solution.  A 2% dilution (6 mL per 300 mL BOD bottle) should yield 200 +/ 37 mg/L BOD, after five days incubation at 20°C.  To ensure valid results for this “standard” check, the glucose glutamic acid dilutions must be seeded since the solution is essentially sterile and does not contain any microorganisms.
The BOD test is a biological test, dependent on the actions of the microorganisms found in the wastewater and, as such, is subject to a number of variations.  These variations can be caused by a number of factors, including changes in temperature, weather, composition of incoming sewage, in plant operations, and  sampling points.  Results can vary widely from day to day, or even hour to hour.  One of the major disadvantages of the BOD test is the time lag between the collection of samples and the final calculation of results.  This makes the BOD test a poor test for determining whether or not operational changes are needed.
In addition, the rate and degree that organic matter in wastewater is decomposed (or oxidized) by the normal bacteria present in a sample is largely dependent on the characteristics of the organic matter.  For example, some organic matter (like sugars or starches) are oxidized very easily and rapidly, and will almost always result in measurable “BOD”.  Other organic matter, however, is sometimes resistant to biological oxidation, and may require special “acclimated” bacteria to oxidize the material and to show a “BOD”.  Although this is what actually happens in nature, it causes significant variation in BOD results from sample to sample.
Some common ranges of BOD results are as follows, in mg/L:

Influent           150 400
Primary Effluent         60 160
Secondary Effluent     10 60
Digester Supernatant  1000 4000+
Industrial Wastes        100 3000+
CBOD results are almost always lower than TBOD results.  For a highly nitrified effluent sample, the difference can be as great as 50%.
SEED CORRECTION PROCEDURE:- The BOD test relies on the presence of healthy organisms. If the samples tested contain materials which could kill or injure the microorganisms (such as chlorine, high or low pH, toxic materials), the condition must be corrected and healthy active organisms added.  This process is known as seeding.  The following step by step description is one technique for seeding samples and the seed correction procedure.  There are other techniques which can be used.
NOTE:  The appropriate pretreatment step (dechlorination, pH adjustment, acclimation, etc.) should be performed prior to preparation of the sample dilutions for this test.
PREPARATION OF SEED MATERIAL:- Select a material to be used for seeding which will have a BOD of at least 180 mg/L. This will help ensure that the seed correction meets the 0.6 mg/L minimum specified in “Standard Methods”, current Edition.  Place the material in a suitable container and incubate at 20°C for 24 36 hours. Usually, settled raw domestic sewage prepared in the manner above will have sufficient BOD for use as a seed material.  If not, small quantities of digester supernatant, return activated sludge, or an acclimated seed material can be used to increase the potency of the seed material used for the test.  As an alternative, commercially available seed material may be used. The seed correction should not exceed 1.0 mg/L BOD, therefore care should be taken not to use too strong a seed material for the test.  The key to a good seed correction is a relatively stable seed material which produces a good seed correction in every test situation.
SEED BOD DETERMINATION:- This step requires preparation of a dilution series using the seed material and unseeded dilution water.  Prepare two bottles of each dilution for the seed control series.  If the meter method is used for DO measurements, only one bottle for each dilution needs to be prepared.  The percentage of seed used in each dilution and the number of dilutions is optional, but sufficient dilutions should be used to ensure that at least one dilution gives a depletion of 2.0 mg/L with at least 1.0 mg/L DO residual.
Determine the initial DO of each dilution, then incubate the dilutions for five days at 20°C.  At the end of the incubation period, determine the final DO of the dilutions.  Calculate the depletion of each seed dilution using formula #1 below.
#1  DO depletion = Initial DO   Final DO
Select the seed dilution(s) which meet the required criteria and calculate the BOD of the seed material using formula #2 below.  (If more than one dilution meets the criteria, calculate the BOD of each such dilution and average the results for the seed material BOD.)
#2  Seed BOD = (DO depletion x 300)/Seed dilution, mL
The calculated seed BOD represents the BOD exerted by 300 mL of undiluted seed material.  The ratio of the seed BOD to 300 mL will be used to calculate the seed correction for seeded samples.
DETERMINATION OF SEED VOLUME:- The most common methods for introducing the seed material into the sample dilutions are
(a) addition of the seed to the dilution water and
(b) addition of the seed directly to the sample BOD bottles.  Method (a) requires a calculation to determine the volume of seed for each dilution since the amount of seed will vary with the volume dilution water used for each sample dilution.  Method (b) is somewhat easier to use as the volume of seed for each dilution is constant.
When Method (a) is used to introduce the seed material, calculate the volume of seed in each sample dilution using formula #3 below.
#3  Volume of seed in sample dilution = (Volume of seed in dilution water x dilution water in sample, mL)/Total volume of dilution water.
SEED CORRECTION:- The calculation of the seed correction is based on the BOD of the seed material and the volume of seed used in each dilution.  The seed correction is actually the oxygen demand exerted by the oxidation of the small amount of organic matter in the seed material in the sample dilutions.  If the BOD exerted by 300 mL of seed material and the volume of seed material in each sample dilution are known, the seed correction can be calculated using formula #4 below.
#4  Seed Correction = (Seed BOD x mL seed in sample dilution)/300
It should be noted that the seed correction for each sample dilution must be calculated when the seed is added to the dilution water.  When the seed is added directly to each BOD bottle, the seed correction is the same for all seeded dilutions using the same seed volume and material.
DETERMINATION OF SAMPLE BOD:- The calculated seed correction is subtracted from the DO depletion in the determination of the BOD for each valid sample dilution.  It should be noted that there are two criteria specified in “Standard Methods” which should be checked before the seed correction is used to determine the sample BOD.  Those sample dilutions meeting these criteria should yield the most valid results.  These criteria are as follows:

  1. The sample dilutions should deplete at least 2.0 mg/L DO after five days incubation at 20°C.
  2. The sample dilutions should have a final DO of at least 1.0 mg/L after five days incubation at 20°C.

The BOD, using the seed correction, should be calculated for the sample dilutions which meet both criteria.  If more than one sample dilution meets the criteria, the final BOD should be an average of the individual BOD results for the sample dilutions.  If none of the sample dilutions meet both of the criteria, the one dilution which comes closest should be used to calculate the final BOD of the sample.
NOTE:  If this is the case, a notation should be made on the sample bench sheet that potentially invalid data has been used to determine the noted value.  Sample dilution volumes should be carefully selected to ensure that at least one dilution meets both criteria.
Calculate the seeded sample BOD using formula #5 below.
#5  BOD mg/L = (DO depletion   Seed correction) x 300/mL of sample
Phosphate buffer solution
Dissolve 8.5 g potassium dihydrogen phosphate (KH2PO4), 21.75 g dipotassium hydrogen phosphate (K2HPO4), 33.4 g disodium hydrogen phosphate heptahydrate (Na2HPO4.7H2O), and 1.7 g ammonium chloride (NH4Cl) in about 500 mL of distilled water and dilute to 1 liter.  The pH of this buffer should be 7.2 and should be checked with a pH meter.  Discard this reagent if there is any sign of biological growth in the storage bottle.
Magnesium sulfate solution
Dissolve 22.5 g magnesium sulfate heptahydrate (MgSO4.7H2O) in distilled water and dilute to 1 liter.  Discard this reagent if there is any sign of biological growth in the storage bottle.
Calcium chloride solution
Dissolve 27.5 g anhydrous calcium chloride (CaCl2) is distilled water and dilute to 1 liter.  Discard this reagent if there is any sign of biological growth in the storage bottle.
Ferric chloride solution
Dissolve 0.25 g ferric chloride hexahydrate (FeCl3.6H2O) in distilled water and dilute to 1 liter.  Discard this reagent if there is any sign of biological growth in the storage bottle.
Sodium hydroxide solution, 1 N
Dissolve 40 g solid sodium hydroxide (NaOH) in approximately 800 mL of carbon dioxide (CO2) free distilled water.  Cool and dilute to 1 liter.
Sulfuric acid solution, 1 N
Cautiously add 28 mL of concentrated sulfuric acid (H2SO4), with mixing, to 800 mL of distilled water.  Allow to cool and dilute to 1 liter.
Sodium sulfite solution, 0.0250 N
Dissolve 1.575 g anhydrous sodium sulfite (Na2SO3) in distilled water and dilute to 1 liter.
NOTE:  This solution is not stable and must be prepared daily.
Potassium iodide solution, 10%
Dissolve 10 g potassium iodide (KI) in 100 mL of distilled water.  Discard if solution turns yellow.
Acetic acid solution, 1:1
Carefully pour 50 mL of glacial acetic acid (CH3COOH) into 50 mL distilled water with mixing.
Sulfuric acid solution, 1:50
Cautiously add 5 mL of concentrated sulfuric acid (H2SO4) with mixing to 250 mL of distilled water.
Glucose glutamic acid solution
Dry reagent grade glucose and reagent grade glutamic acid at 103°C for 1 hour.  Add 150 mg glucose and 150 mg glutamic acid to distilled water and dilute to 1 liter.  Prepare this solution fresh immediately before use.
Nitrification inhibitor
The Hach Chemical Company’s Nitrification inhibitor 2533(2 chloro 6 (trichloro methyl) pyridine) or equivalent can be used for inhibition during carbonaceous BOD testing.


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