Biochemical Oxygen Demand and

Carbonaceous Biochemical Oxygen

Demand
Written by: Sheree Gossett-Johnson
www.WaterWorldCE.com
 Biochemical Oxygen Demand
and Carbonaceous Biochemical
Oxygen Demand
Educational Objectives
Upon completion of this course, the operator should understand what BOD and CBOD analytical tests mea-
sure and how to perform those tests. The operator should also have a clear understanding of the appropriate
quality assurance checks used for this analytical test. In addition, a template depicting data arrangement is
provided for the operator to use for test result validity.

I. Abstract
This operator education course explains in a step by step method
how to perform the Biochemical Oxygen Demand (BOD) and
Carbonaceous Biochemical Oxygen Demand (Carb. BOD) ana-
lytical tests, including quality assurance and control measures.
This method can be used to report BOD and Carb. BOD results
to Ohio EPA as required in wastewater treatment plant National
Pollutant Discharge Elimination System (NPDES) Permits. All
needed equipment, chemicals, and glassware is listed. Data and
sample calculations are presented to provide templates for the
operator to use when performing this test.
II. Keywords
Take a glance through this list and be sure to read carefully
any terms or abbreviations with which you are not familiar.
BOD Nutrient Buffer solution: A solution of nutrients,
phosphorus, trace metals and “buffer” chemicals to ensure a
pH suitable for bacterial growth.
Seed Solution (for seeded BOD’s): Commercial seed, using
a quantifiable number of a micro-organism population.
Dissolved Oxygen (DO): The amount of dissolved oxygen
present within the wastewater sample.
Quality Assurance / Quality Control: To assure there was
not any known bias introduced to the test procedure and to
provide a control mechanism that diminishes any bias intro-
duced in the test procedure.

Biochemical Oxygen Demand (BOD): Determination of the III. Introduction

oxygen requirements wastewater, as related to the biochemi-
cal degradation of organic material (carbonaceous demand)
and the oxygen used to oxidize inorganic (sulfides and ferrous
iron) and reduced nitrogen.
Carbonaceous Biochemical Oxygen Demand (Carb. BOD):
The BOD test containing a nitrification inhibitor is termed a
carbonaceous BOD analytical test.
Glucose / Glutamic Acid: A “standard” check solution to
determine if the distilled water is contaminated.
KHP High and Low Standard: A “standard” Phosphate
check solution to determine if the Dilution water is contami-
nated.
The Biochemical Oxygen Demand (BOD) determination is a
test used to determine the oxygen requirements of wastewa-
ter. The measurements of oxygen are performed after 5-days
of incubation to determine the biochemical degradation of or-
ganic (carbonaceous demand) and the oxygen used to oxidize
inorganic materials (sulfides and ferrous iron) as well as re-
duced nitrogen. The analytical test procedure is computed by
determining the difference between initial and final Dissolved
Oxygen (D.O.) measurements of the wastewater sample over
a 5-day period. The sample’s carbonaceous BOD will be less
than or equal to the BOD result.
Historically, it has been found that the nitrogenous demand
interferes with determining the carbonaceous demand. The
BOD measurement is therefore generally not useful in assess-
ing the oxygen needed to satisfy the carbonaceous demand.

2 www.WaterWorldCE.com
To adjust the BOD to accurately determine the biochemical
degradation of only the carbonaceous demand, an inhibitor
can be added to prevent oxidation of the nitrogen compounds.
The BOD analytical test containing the inhibitor is
termed a carbonaceous BOD analytical test. Comparing the
BOD with the carbonaceous BOD is useful when developing
wastewater treatment trends with relation to time and seasons.
It should be understood that nitrogenous demand can not be
determined by subtracting the BOD from the carbonaceous
BOD because the BOD calculation includes the degradation
of inorganic materials (sulfides and ferrous iron). To deter-
mine the nitrogen content of the sample (ammonia) an am-
monia probe is used.
Due to the number of factors that may affect the accuracy
and precision, “quality assurance and control” checks are used
to assure the operator that the test results are not influenced by
contaminants within the distilled water or the BOD dilution
water. To assure that the human error factor does not cause a
bias, duplicate analysis is performed. To preserve the sample,
the wastewater samples must be maintained at or below 4
degrees Celsius (refrigeration), no chemical preservative is
used in addition, the test procedure for carbonaceous BOD
and BOD must begin within 6 hours of collection for both
24-Hour composite and Grab samples.
The purpose of a wastewater treatment plant is to take
in polluted water and remove the pollutants. One type of
wastewater treatment uses micro-organisms. The micro-
organisms biologically convert and stabilize the pollutants,
removing them from the water. The treated water may then
be discharged into a stream without deteriorating the receiv-
ing stream. To assure that the receiving stream is not being
adversely affected, the operator tests the treated discharged
water, effluent, for the biochemical oxygen demand (BOD).
The BOD analytical test is performed on the wastewater
treatment plant influent and effluent. The tests are performed
in a laboratory and their values are expressed in mg/l units
(ppm). The results of these tests indicate the affect the effluent
will have on the receiving stream. If the BOD of the effluent is
found to be high than that means the dissolved oxygen avail-
able for the stream biota is limited. Limited dissolved oxygen
causes impairment to the fish and macro-invertebrate habitat.
A high BOD causes excessive oxygen demands on the stream
which can cause the death, elimination or deformity of the
fish community. In addition, the macro-invertebrates (fish
food) may be eliminated due to the lack of sufficient dissolved
oxygen. The BOD results indicate the oxygen requirements to
metabolize and stabilize all of the organic compounds in the
wastewater. Due to the interference of different organic com-
pounds within this test method, the BOD test is modified to
reflect only the carbonaceous oxygen demand ( Carb. BOD).
Although both the BOD and Carb. BOD analytical test re-
sults indicate the effluent quality; the Carb. BOD can be used
by the operator to determine treatment efficiency and trend
analysis through out the year. The BOD analytical results are
www.WaterWorldCE.com 3
mainly used to balance the influent wastewater BOD load and
the mass of micro-organisms, termed F/M ratio.
In this course, the type of wastewater treatment analyti-
cal test examined uses a method that determines the oxygen
demand present after the micro-organisms metabolize and
stabilize the wastewater. This wastewater contains many
pollutants in the form of suspended and dissolved organic
compounds. The operator must be familiar with how his/her
individual wastewater treatment plant is designed and oper-
ates.
In general, wastewater is delivered from homes, public
entities, businesses and industries through a sewer collection
system to the wastewater treatment plant (WWTP), termed
the influent. The collection system can be a closed system,
relying on pumps to push or lift the sewage to the WWTP
or a gravity system that requires greater than or equal to a
2% slope or a combination of gravity and pumps. Once the
wastewater is delivered to the WWTP, the micro-organisms
in the treatment portion of the WWTP use the wastewater
compounds as a food source to reproduce.
The food source used first by the micro-organisms is the
carbon compounds, Car. BOD. The Carb. BOD in the waste-
water is metabolized by the micro-organisms lowering the
Carb. BOD in the effluent. The operator can determine the
percent of Carb. BOD removal the WWTP has achieved by
comparing the influent Carb. BOD to the effluent Carb. BOD.
The lower the oxygen demand found in the effluent the more
percent removal. In addition, low Carb. BOD results indicate
that the quality of the effluent is good and will not deterio-
rate the stream. After the Carb. BOD is lowered to less than
10 mg/L; other organic compounds are metabolized by the
micro-organisms.
Once the operator has achieved a low Car. BOD result in
the effluent, the challenge is to maintain it at this low con-
centration. To maintain the food with the number of micro-
organisms, the operator must “balance” both variables. To
determine the balance, the operator divides the influent BOD
by the mass of micro-organisms, measured as suspended sol-
ids. This ratio is called the F/M Ratio.
IV. Procedure
MATERIALS
1. BOD Bottles, stoppers and caps: BOD Buffer Pillow
2. Volumetric Pipettes: BOD Siphon
3. Graduated Cylinders: DO Meter
4. KHP High Standard: DO Probe
5. KHP Low Standard: BOD Bench Sheets
6. Polyseed BOD Capsules: 0-Toulidine
7. Sodium Sulfite: Acetic Acid
8. Erlenmeyer flask: Starch
9. Stir Plate: Distilled Water
10. Calcium Chloride: Phosphate Buffer
11. Magnesium Chloride: Ferric Chloride
12. Manganous Sulfate: Alkali Iodide Azide 2. Put in the bottle to be read, wait for it to stabilize,
PREPARATION OF MATERIALS
A. Glucose / Glutamic Acid - Dry the Glucose and Glutamic
Acid at 104 degrees Celsius for a minimum of one (1)
hour. Place in a desiccator for a minimum of one/half (½)
hour. Weigh 0.15 grams of each and add to a one (1) liter
volumetric. Using distilled water, bring the water up to
level appropriately (ensuring that the bottom of meniscus
is level with the line).
B. KHP High and Low Standard - Dry KHP for two (2)
hours at 104 degrees Celsius for two (2) hours. Place in
desiccator for one (1) hour. Weigh 0.8503 grams and add
to a one (1) liter volumetric. Using distilled water, bring
the water up to level mark appropriately (ensuring that
the bottom of meniscus is level with the line).
1. High Standard = 136 mg/I. Put 20 mI of KHP solu-
tion in a one-hundred milliliter (100 ml) volumetric.
Using distilled water, bring the water up to the level
mark appropriately (ensuring that the bottom of me-
niscus is level with the line).
2. Low Standard = 68 mg/l. Put 10 ml of KHP solution
in a one-hundred milliliter (100 ml) volumetric. Us-
ing distilled water, bring the water up to the level
mark appropriately (ensuring that the bottom of
meniscus is level with the line).
C. BOD Nutrient Buffer solution – Using a five (5) gallon
jug of distilled water, remove one (1) three-hundred mil-
liliter (300 ml) bottle full of distilled water, then add a
BOD Buffer pillow (poly seed) or the nutrient solutions
from the storage area and mix the contents thoroughly.
See below.
Nutrients:
• 1 ml - Calcium Chloride (Ca Cl) Dissolve 27.5 g
CaCl2 in distilled water and dilute to 1 L.
• 1 ml - Magnesium Sulfate (Mg SO) Dissolve 22.5 g
MgSO4*7H2O in distilled water and dilute to 1 L.
• 1 ml - Ferric Chloride (Fe Cl) Dissolve 27.5 g CaCl in
distilled water and dilute to 1 L.
• 1 ml - Phosphate Buffer, Dissolve 8.5 KHP2PO4,
21.75 g K2HPO4, 33.4 g Na2HPO4* 7 H20 and 1.7 g
NH4CL in 500 Ml distilled water and dilute to 1 L.
D. Seed Solution - Follow the directions on the manufac-
turer’s label; Example: Poly Seed BOD capsules.
SETTING UP SEEDED BODS
A. To properly read Initial Dissolved Oxygen (IDO) and
Final Dissolved Oxygen (FDO):
1. Turn the D.O. probe off and remove it from the cali-
bration bottle.
4 www.WaterWorldCE.com
record the reading in the correct space correspond-
ing to the numbered bottle.
3. Repeat the above being sure to rinse probe with
distilled water between bottles.
B. Line up BOD Bottles in rows 1-5 for a quality control
check of the test.
1. Row 1 - (1) distilled water, (1) Glucose/Glutamic
Acid
2. Row 2 - (1) Nutrient Buffer Solution
3. Row 3 - (3) Seed Standards
4. Row 4 - Low Standard Control 68 mg/L KHP Stan-
dard
5. Row 5 - High standard Control 136 mg/L KHP Stan-
dard.
a. Row 1 - Perform once for each distilled jug. Put
the BOD bottle full of distilled water first in
the row. To the next bottle add 6 ml Glucose/
Glutamic Acid, 2 ml seed solution and fill with
nutrient water.
Read and record IDO.
Stopper bottle, add distilled water on top of the stop-
per and cap.
b. Row 2 - Fill one (1) B.O.D bottle full of nutrient
water.
Read and record IDO.
Stopper bottle, add distilled water on top of the stop-
per and cap.
c. Set up three (3) BOD bottles, while using a
Volumetric and Class A pipette add 20 ml of
seed solution to the first bottle, then fill with
nutrient water, in the second bottle add 15 ml
of seed solution, then fill with nutrient water, in
the third bottle add 10 ml of seed solution, then
fill with nutrient water.
Read and record IDO.
Stopper bottle, add distilled water on top of the stop-
per and cap.
d. Row 4 - Set up three (3) BOD bottles, while
using a Volumetric and Class A pipette add 2
ml seed solution, 25 ml of low KHP standard to
the first bottle, then fill with nutrient solution,
in the second bottle add 2 ml seed solution, 15
ml of low KHP standard, then fill with nutrient
solution, in the third bottle add 2 ml seed solu-
 tion, 10 ml of low KHP standard, then fill with
nutrient solution.
Read and record IDO.
Stopper bottle, add distilled water on top of the top-
per and cap.
e. Row 5 - Set up three (3) BOD bottles, while us-
ing a Volumetric and Class A pipette add 2 ml
seed solution, 15 ml of high KHP standard to
the first bottle, then fill with nutrient solution,
in the second bottle add 2 ml seed solution, 7.5
ml of high KHP standard, then fill with nutrient
solution, in the third bottle add 2 ml seed solu-
tion, 3 ml of high KHP standard, then fill with
nutrient water.
Read and record IDO.
Stopper bottle, add distilled water on top of the stop-
per and cap.
C. Line up BOD Bottles in rows 6-9 for influent, effluent
B.O.D and Carb. BOD analysis the test.
1. Row 6 – (3) Effluent samples for BOD analysis
2. Row 7 - (3) Effluent samples for Carb. BOD analysis
3. Row 8 - (3) Influent samples for BOD analysis
4. Row 9 - (3) Influent samples for Carb. BOD analysis
a. Row 6 - Set up three (3) BOD bottles, while
using a graduated cylinder add the following
amount of effluent to the bottles,
1) If very clean samples 150, 60, and 25 ml.
2) If cloudy samples 100, 50, and 20 ml.
while using a wide mouth pipette add two (2) ml of
seed solution fill with nutrient water.
Read and record IDO
Stopper bottle, add distilled water on top of the stop-
per and cap.
b. Row 7 - Set up three (3) BOD bottles, add 0.16
grams of nitrification inhibitor then while using
a graduated cylinder add the following amount
of effluent to the bottles,
1) If very clean samples 150, 60, and 25 ml.
2) If cloudy samples 100, 50, and 20 ml.
while using a wide mouth pipette add two (2) ml of
seed solution, then fill with nutrient water.
Read and record IDO.
www.WaterWorldCE.com 5
Stopper bottle, add distilled water on top of the stop-
per and cap.
c. Row 8 - Set up three (3) BOD bottles, while
using a graduated cylinder add the following
amount of influent to the bottles,
1) If very clean samples 12, 7.5, and 3 ml.
2) If cloudy samples 7.5, 3, and 1 ml.
while using a wide mouth pipette add two (2) ml of
seed solution, then fill with nutrient water.
Read and record IDO
Stopper bottle, add distilled water on top of the stop-
per and cap.
d. Row 9 - Set up three (3) BOD bottles, add 0.16
grams of nitrification inhibitor then while using
a graduated cylinder add the following amount
of influent to the bottles,
1) If very clean samples 12, 7.5, and 3 ml.
2) If cloudy samples 7.5, 3, and 1 ml.
while using a wide mouth pipette add two (2) ml of
seed solution , then fill with nutrient water.
Read and record IDO
Stopper bottle, add distilled water on top of the
stopper and cap.
READING FINAL D.O.
1. Turn the D.O. meter on and allow it warm up for at least
15 minutes.
2. Remove the 5 Day BODs/Carb. BODs out of the incuba-
tor on the appropriate day. The BOD bottles are situated
in the incubator from the left to the right. Remove them
in the order that they are placed in the incubator.
3. Obtain the BOD work sheets with all of the necessary
information, and then see the two Example Tables shown
here (Example Tables : Rows 1 – 3; Rows 4 – 9).
4. Perform a Winkler Titration to calibrate the DO meter or
if applicable use Air Calibration.
5. Uncap all of the BOD bottles and place the plastic caps in
the incubator.
6. Remove the stopper from the last bottle in the first row in
measure the Final D.O.
7. Allow the probe to stabilize in the sample, after stabiliza-
tion has been achieved, record the value on the Bench
work sheet in the appropriate space and column.
8. Remove the probe from the last bottle of the first row and
place it in the middle bottle of the first row.
9. Allow the sample to be read until stabilization has been
achieved, and record the result. Proceed with the mea-
 surement and the recording of the final D.O. in the first
bottle of the first row.
10. Rinse the probe off with distilled water and place the
probe in the third bottle of the next row.
11. Allow the stabilization of the D.O. to be achieved and
record the reading on the worksheet in the appropriate
space and column.
12. Repeat steps described in items 7 - 11 until all of the final
D.O.s have been measured and recorded on the BOD
work sheets.
Note: It is not necessary to rinse from one bottle to the
next, because samples are read from an area of less concentra-
tion to an area of higher concentration. However the probe
must be rinsed off before beginning measurements in a new
row.
V. Calculations
1. Aliquot (Alq) = ml of sample.
2. Dilution (Dil.) = 300(BOD bottle volume)/Aliquot of
sample.
3. X = Means to multiply in all listed calculations below.
4. Rows one and two are the distilled water blank and the
nutrient blank rows.
NOTE * The D.O. on these two rows should not decrease
by more than 0.2 mg/L, if more than 0.2 mg/L decrease
the test is not valid.
5. Glucose/Glutamic Acid = F.D.O. X Dil = mg/L
In the Example Table, 4.45 X 50 = 222.5 rounded to 223.
NOTE * Acceptable range is 200 +/- 37 week.
6. Seed Control = (I.D.O. - F.D.O.) X (dilution factor seed
control).
After all three bottles have been calculated average the
three for the total seed control to be subtracted from all
seeded samples.
In the Example Table, (7.10 X 0.1) + (5.25 X 0.133) + (3.60
X 0.2)/3 = 0.710.
6 www.WaterWorldCE.com
7. The fourth and fifth row of bottles contains the high and
low KHP standards.
a. I.D.O. - F.D.O. = D.O.
b. D.O. - seed control = D.O. less the seed
c. D.O. (less the seed) X dilution factor = mg/L RE-
COV. of Standard
d. Average the recovery /Standard X 100 = % of Re-
covery
In the Example Table the three bottles “recovery” is found
under the column “BOD”. Average the three values 70 + 70 +
67 / 3 = 69 and divide by the standard used to determine %
Recovery.
69/68 X 100 = 101 %
NOTE* Acceptable Recovery range is 80 - 120 %. Repeat the
same steps with the high KHP standards.
8. The sixth through ninth rows, calculation = D.O. Less
Seed X Dil. = BOD or Carb. BOD.
VI. Quality Assurance and Quality Control
1. For each batch of nutrient water, one (1) blank distilled is
analyzed.
2. For each wastewater sample three (3) samples are deter-
mined then averaged.
3. For each sample date three (3) nutrient blanks are ana-
lyzed.
4. For each sample date three (3) glucose/glutamic bottles
are analyzed.
5. One set of KHP Standards are analyzed once (1) per
VII. Summary
Determining the Carb. BOD of the influent and effluent al-
lows the operator the ability to determine the percent of Carb.
BOD removal the WWTP has achieved. The lower the oxygen
demand found in the effluent the more percent removal, trans-
versely, the higher the oxygen demand found in the effluent
the less percent removal. To maintain treatment toward the
more efficient low effluent Carb. BOD, the operator needs to
balance the influent BOD with the number of micro-organism,
termed the F/M ratio. A trend analysis of the effluent Carb.
BOD can be established through out the year to assist the
operator with adjusting the range of the F/M ratio.
The F/M Ratio represents the pounds of BOD applied per
day per pound of micro-organisms, measured as suspended
solids in the treatment portion of the wastewater treatment
plant. The operator has control of the micro-organism popula-
tion, but generally not the influent BOD. To control of the level
of treatment the operator increases or decreases the removal
of micro-organisms from the treatment system.
 The best F/M value to be used for process control must
be determined for each wastewater treatment plant. There
is no perfect number, but, most wastewater treatment plants
operate best in the 0.2 – 0.5, lb influent BOD/lbs solid inven-
tory per day. The operator must determine the optimum F/M
ratio or the individual wastewater treatment plant to achieve
the lowest Carb. BOD analytical value within the discharged
effluent.
The operator’s goals are to operate the wastewater treat-
ment plant in such a manner to efficiently lower the Carb.
BOD, maintain that efficiency and therefore assure that the
receiving stream is not being adversely affected. Generally,
poorly treated wastewater effluent causes impairment to the
fish and macro-invertebrate habitat through placing a high
demand on the streams dissolved oxygen. In addition, the
macro-invertebrates (fish food) may be eliminated due to the
lack of sufficient dissolved oxygen. Both analytical tests re-
sults, from BOD and Carb. BOD, indicate the effluent quality;
the Carb. BOD can be used by the operator to determine treat-
ment efficiency and trend analysis through out the year. The
BOD analytical results are mainly used to balance the influ-
ent wastewater BOD load and the mass of micro-organisms,
termed F/M ratio.

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Online Completion
Use this page to review the questions and choose your answers. Return to www.waterworldce.com and sign in. If you have not previously purchased
the program select it from the “Online Courses” listing and complete the online purchase. Once purchased the exam will be added to your Archives
page where a Take Exam link will be provided. Click on the “Take Exam” link, complete all the program questions and submit your answers. An
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Questions
1. To be valid, a BOD sample dilution
must have a residual dissolved oxygen
(D.O.) of at least 1 mg/L after five
days incubation. True or false?
A. False
B. True

2. All National Pollutant Discharge Elimina-

tion System testing must be performed
within 6 hours of collection for only 24-
hour composite samples. True or false?
A. True
B. False

3. All samples tested for BOD must be

seeded with a commercial seed product
such as Polyseed. True or false?
A. False
B. True

4. In Standard Methods, the BOD

method recommends that the analyst
test a 2% glutamic acid with each batch
of samples. True or false?
A. True
B. False

5. The recommended maximum

storage time for the BOD test
is: Choose the best answer.
A. 24 hours
B. analyze immediately
C. 6 months
D. 6 hours
E. 7 days

6. What is the proper preservative used

for the Biochemical Oxygen Demand
test? Choose the best answer.
A. refrigeration
B. none, analyze immediately
C. add nitric acid to a pH
D. add sulfuric acid to a pH<2
E. add sodium hydroxide

7. A particular sample’s carbonaceous

BOD will be ___________ the BOD
result? Choose the best answer.
A. less than or equal to
B. greater than
C. less than
D. equal to

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 Biochemical Oxygen Demand and
Carbonaceous Biochemical Oxygen Demand

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