BIOCHEMICAL OXYEGEN DEMAND

(BOD)
GAYATHRI S MOHAN
CPET 712
BIOCHEMICAL OXYGEN DEMAND
The biochemical oxygen demand (BOD) determination is an empirical test in which
standardized laboratory procedures are used to determine the relative oxygen
requirements of wastewaters, effluents, and polluted waters.

Applications are in measuring waste loadings to treatment plants and in evaluating
the BOD-removal efficiency of such treatment systems.

The test measures the

Oxygen utilized during a specified incubation period for the biochemical
degradation of organic material (carbonaceous demand)
Oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless
their oxidation is prevented by an inhibitor.
Oxygen used to oxidize inorganic material such as sulfides and ferrous iron.
The seeding and dilution procedures provide an estimate of the BOD at pH 6.5 to
7.5.


Introduction
Interference: Oxidation of reduced forms of nitrogen, such as ammonia and organic
nitrogen, by microorganisms.
The interference from nitrogenous demand can be prevented by an inhibitory
chemical.

Ultimate BOD (UBOD)

The UBOD measures the oxygen required for the total degradation of organic material
(ultimate carbonaceous demand) and/or the oxygen to oxidize reduced nitrogen
compounds(ultimate nitrogenous demand).

UCBOD + UNBOD UBOD

Report results as carbonaceous biochemical oxygen demand (CBOD
5
) when
inhibiting the nitrogenous oxygen demand. When nitrification is not inhibited,
report results as BOD
5




Dilution Requirements
The BOD concentration in most wastewaters exceeds the concentration of
dissolved oxygen (DO) available in an air-saturated sample. Therefore, it is
necessary to dilute the sample before incubation to bring the oxygen demand and
supply into appropriate balance.

Because bacterial growth requires nutrients such as nitrogen, phosphorus, and
trace metals, these are added to the dilution water, which is buffered to ensure
that the pH of the incubated sample remains in a range of 6.5 to 7.5, suitable for
bacterial growth.

The source of dilution water is not restricted and may be distilled, tap, or
receiving-stream water free of biodegradable organics and bioinhibitory
substances such as chlorine or heavy metals.

5-Day BOD Test
Principle
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 d. Dissolved
oxygen is measured initially and after incubation, and the BOD is computed from
the difference between initial and final DO.
Sampling and storage
1) Grab samplesStore the sample at or below 4C from the time of collection, if
analysis is not begun within 2 h of collection.
Begin analysis within 6 h of collection; when this is not possible because the
sampling site is distant from the laboratory and report length and temperature of
storage with the results.
In no case start analysis more than 24 h after grab sample collection.

2) Composite samplesKeep samples at or below 4C during compositing. Limit
compositing period to 24 h.
Apparatus

Incubation bottles
Use glass bottles having 60 mL or greater capacity (300-mL bottles having a ground-
glass stopper and a flared mouth are preferred).
Clean bottles with a detergent, rinse thoroughly, and drain before use.
Use a water seal to prevent the drawing of air in to the bottle.
Place a paper or plastic cup or foil cap over flared mouth of bottle to reduce
evaporation of the water seal during incubation.

Air incubator or water bath
thermostatically controlled at 20 1C.
Exclude all light to prevent possibility of photosynthetic production of DO.
Reagents

a. Phosphate buffer solution

b. Magnesium sulfate solution

c. Calcium chloride solution

d. Ferric chloride solution

e. Acid and alkali solutions,1N

f. Sodium sulfite solution

g. Nitrification inhibitor,2-chloro-6-(trichloromethyl) pyridine

h. Glucose-glutamic acid solution

i. Ammonium chloride solution

j. Dilution water














a
Procedure
a. Preparation of dilution water
Place desired volume of water in a suitable bottle and add 1 mL each of
phosphate buffer, MgSO4, CaCl2, and FeCl3 Solution, Seed dilution water, if
desired.
Before use bring dilution water temperature to 20 3C.
Saturate with DO by shaking in a partially filled bottle or by aerating with
organic-free filtered air. Alternatively, store in cotton-plugged bottles long enough
for water to become saturated with DO.
b. Dilution water storage
Source water may be stored before use as long as the prepared dilution water
meets quality control criteria in the dilution water blank.
Discard stored source water if dilution water blank shows more than 0.2 mg/L DO
depletion in 5 d.


Procedure
c. Glucose-glutamic acid check
To check the dilution water quality.
Periodically check dilution water quality, seed effectiveness, and analytical
technique by making BOD measurements on a mixture of 150 mg glucose/L and
150 mg glutamic acid/L as a standard check solution.
Glucose has an exceptionally high and variable oxidation rate but when it is used
with glutamic acid, the oxidation rate is stabilized and is similar to that obtained
with many municipal wastes.
Determine the 5-d 20C BOD of a 2% dilution of the glucose-glutamic acid standard
check solution
Procedure
d. Seeding
Seed source: Some samples do not contain a sufficient microbial population (for
example, some untreated industrial wastes, disinfected wastes, high-temperature
wastes, or wastes with extreme pH values). For such wastes seed the dilution
water or sample by adding a population of microorganisms.

The preferred seed is effluent or mixed liquor from a biological treatment system
processing the waste.
Where such seed is not available, use supernatant from domestic wastewater after
settling at room temperature for at least 1 h but no longer than 36 h.

When effluent or mixed liquor from a biological treatment process is used,
inhibition of nitrification is recommended.

Some samples may contain materials not degraded at normal rates by the
microorganisms. In such cases develop an adapted seed in the laboratory by
continuously aerating a sample of settled domestic wastewater and adding small
daily increments of waste.

Procedure
Seed control Determine BOD of the seeding material as for any other sample. This is
the seed control.
To determine DO uptake for a test bottle, subtract DO uptake attributable to the
seed from total DO uptake

e. Sample pretreatment
Check pH of all samples before testing

1) Samples containing caustic alkalinity (pH >8.5) or acidity (pH <6.0)Neutralize
samples to pH 6.5 to 7.5 with a solution of sulfuric acid (H
2
SO
4
) or sodium
hydroxide (NaOH) of such strength that the quantity of reagent does not dilute
the sample by more than 0.5%.

2) Samples containing residual chlorine compoundsIf possible, avoid samples
containing residual chlorine by sampling ahead of chlorination processes.

If the sample has been chlorinated but no detectable chlorine residual is present,
seed the dilution water.



Sample treatment

In some samples chlorine will dissipate within 1 to 2 h of standing in the light. If
residual chlorine is present, dechlorinate sample and seed the dilution water using
Na
2
SO
3
.
Determine required volume of Na
2
SO
3
solution on a 100- to1000-mL portion of
neutralized sample by adding 10mL of 1 + 1 acetic acid or 1 + 50 H
2
SO
4
, 10 mL
potassium iodide (KI) solution (10 g/100 mL) per 1000 mL portion, and titrating
with Na
2
SO
3
solution to the starch-iodine end point for residual.
Samples containing other toxic substancesCertain industrial wastes, for example,
plating wastes, contain toxic metals. Such samples often require special study and
treatment.
Sample temperature adjustmentBring samples to 20 1C before making
dilutions.
Nitrification inhibitionIf nitrification inhibition is desired add 3 mg of 2-chloro-6-
(trichloro methyl) pyridine (TCMP) to each 300-mL bottle before capping or add
sufficient amounts to the dilution water to make a final concentration. Note the
use of nitrogen inhibition in reporting results.


Dilution technique
Make several dilutions of sample that will result in a residual DO of
atleast 1 mg/L and a DO uptake of at least 2 mg/L after a 5-d
incubation.
Five dilutions are recommended unless experience with a particular
sample shows that use of a smaller number of dilutions produces at
least two bottles giving acceptable minimum DO depletion and
residual limits.
In the absence of prior knowledge, use the following dilutions: 0.0 to 1.0% for
strong industrial wastes, 1 to 5% for raw and settled wastewater, 5 to25% for
biologically treated effluent, and 25 to 100% for polluted river waters.
When using graduated cylinders or volumetric flasks to prepare dilutions, and
when seeding is necessary, add seed either directly to dilution water or to
individual cylinders or flasks before dilution.

Dilution technique
Seeding of individual cylinders or flasks avoids a declining ratio of seed to sample
as increasing dilutions are made.
When dilutions are prepared directly in BOD bottles and when seeding is
necessary, add seed directly to dilution water or directly to the BOD bottles.

Using a wide-tip volumetric pipet, add the desired sample volume to individual BOD
bottles

Add appropriate amounts of seed material either to the individual BOD bottles or
to the dilution water

Fill bottles with enough dilution water, seeded if necessary

Determine initial DO on one bottle

Stopper tightly, water-seal, and incubate for 5 d at 20C.
Determination of initial DO
If the sample contains materials that react rapidly with DO, determine initial DO
immediately after filling BOD bottle with diluted sample.

If rapid initial DO uptake is insignificant, the time period between preparing
dilution and measuring initial DO is not critical but should not exceed 30 min.

Dilution water blank: Use a dilution water blank as a rough check on quality of
unseeded dilution water and cleanliness of incubation bottles.

Together with each batch of samples incubate a bottle of unseeded dilution water.
Determine initial and final DO. The DO uptake should not be more than 0.2 mg/L
and preferably not more than 0.1 mg/L

Incubation: Incubate at 20C 1C BOD bottles containing desired dilutions, seed
controls, dilution water blanks, and glucose-glutamic acid checks. Water-seal
bottles.

Determination of final DO: After 5 d incubation determine DO in sample dilutions,
Blanks.
Methods to find DO
Two methods for DO analysis are

The Winkler or iodometric method and its modifications -titrimetric
procedure based on the oxidizing property of DO

Electrometric method using membrane electrodes-rate of diffusion of
molecular oxygen across a membrane

The choice of procedure depends on the interferences present, the
accuracy desired, and, in some cases, convenience or expedience.
Iodometric Methods
Principle
It is based on the addition of divalent manganese solution, followed by strong
alkali, to the sample in a glass-stoppered bottle.
DO rapidly oxidizes an equivalent amount of the dispersed divalent manganous
hydroxide precipitate to oxides of higher valence state.
In the presence of iodide ions (I
-
) in an acidic solution, the oxidized manganese
reverts to the divalent state, with the liberation of iodine (I
2
)equivalent to the
original DO content
The released I
2
can then be titrated with standard solution of sodium thiosulfate
(Na
2
S
2
O
3
) using a starch indicator
The titration end point can be detected visually, with a starch indicator, or
electrometrically, with potentiometric or dead-stop techniques.
The liberated iodine also can be determined directly by simple absorption
spectrophotometers.
Interference
Certain oxidizing agents liberate iodine from iodides (positive interference) and some
reducing agents reduce iodine to iodide (negative interference).
Modifications of Iodometric
method
azide modification-removes interference caused by nitrite

the permanganate modification-removes the interference of ferrous iron

the alum flocculation modification- suspended solids interference

the copper sulfate-sulfamic acid flocculation modification-activated-sludge
mixed liquor interference.

The choice is based on the effect of interferences, particularly oxidizing or
reducing materials that may be present in the sample.
Azide Modification
Reagents
a. Manganous sulfate solution
b. Alkali-iodide-azide reagent
c. Sulfuric acid
d. Starch
e. Standard sodium thiosulfate titrant
f. Standard potassium bi-iodate solution

StandardizationDissolve approximately 2 g KI in 100 to 150 mL distilled water

few drops of conc H
2
SO
4
+ 20.00 mL standard bi-iodate solution

Dilute to 200 mL


Titrate the liberated iodine against Na
2
S
2
O
3
Starch indicator, pale straw color end point


Procedure
a. To the sample collected in a 250- to 300-mL bottle

1 ml of MnSO4

1 mL alkali-iodide-azide reagent

Stopper carefully to exclude air bubbles and mix by inverting bottle a few times.
When precipitate has settled sufficiently add 1.0 mL conc H
2
SO
4

Restopper and mix by inverting several times until dissolution is complete.
Titrate a volume corresponding to 200mL original sample after correction for
sample loss by displacement with reagents. Thus, for a total of 2mL (1 mL each) of
MnSO4 and alkali-iodide-azide reagents in a 300-mL bottle, titrate
200 300/(300 2) = 201 mL.

b. Titrate with 0.025M Na
2
S
2
O
3
solution to a pale straw color. Add a few drops of
starch solution and continue titration to first disappearance of blue color.
Permanganate Modification
only on samples containing ferrous iron
ineffective for oxidation of sulfite, thiosulfate, polythionate, or the organic matter
in wastewater.

Reagents

a. Potassium permanganate solution
b. Potassium oxalate solution
c. Potassium fluoride solution
All the reagents required for the azide modification
Procedure
To a sample collected in a 250- to 300-mL bottle

0.70 mL conc H
2
SO
4
+ 1 mL KMnO
4
solution + 1 mL KF solution

Stopper and mix by inverting

Remove permanganate color completely by adding 0.5 to 1.0 mL K
2
C
2
O
4
solution

Mix well and allow to stand in the dark to facilitate
the reaction.

1 ml of MnSO
4
+ 1 mL alkali-iodide-azide reagent
Stopper, mix, and let precipitate settle for short time

Acidify with 2 ml H
2
SO
4

take 200 300/(300 7.7) = 205 mL for titration
Alum Flocculation Modification
The interference due to solids may be removed by alum flocculation.

Reagents
a. Alum solution
b. Ammonium hydroxide
c. Manganous sulfate solution
d. Alkali-iodide-azide reagent
e. Sulfuric acid
f. Starch
g. Standard sodium thiosulfate titrant
All the reagents required for the azide modification


Procedure
To a sample collected in a 250- to 300-mL bottle

10 mL alum solution + 1 to 2 mL conc NH4OH

Stopper and invert gently for about 1 min settle for about 10 min


Continue sample treatment as same of azide modification.
Copper Sulfate-Sulfamic Acid
Flocculation Modification

Used for biological flocs such as activated sludge mixtures, which have high oxygen
utilization rates.
Reagents
a. Copper sulfate-sulfamic acid inhibitor solution
b. All the reagents required for the azide modification
Procedure
Add 10 mL CuSO4-NH
2
SO
2
OH inhibitor to a 1-L glass-stoppered bottle.

Insert bottle in a special sampler designed so that bottle fills from a tube near bottom
and overflows only 25 to50% of bottle capacity.

Collect sample, stopper, and mix by inverting

Let suspended solids settle and siphon relatively clear supernatant liquor into a 250-
to 300-mL DO bottle

Continue as per azide modification
Membrane Electrode Method
Being completely submersible, membrane electrodes are suited for analysis in situ.

Principle
Oxygen-sensitive membrane electrodes of the polarographic or galvanic type are
composed of two solid metal electrodes in contact with supporting electrolyte
separated from the test solution by a selective membrane.

In polarographic, electrode reaction is spontaneous
In galvanic types external source of applied voltage is needed to polarize the
indicator electrode.
Membranes like Polyethylene and fluorocarbon membranes are normally used as
they are permeable to molecular oxygen and are relatively rugged.
In the device diffusion current is linearly proportional to the concentration of
molecular oxygen.
The current can be converted to concentration units by calibration.
Apparatus

Oxygen-sensitive membrane electrode, polarographic or galvanic, with appropriate
meter.
Procedure
a. Calibration :calibrate membrane electrodes by reading against air or a
sample of known DO concentration (determined by iodometric method) as well as in a
sample with zero DO.

b. Sample measurement: Follow all precautions recommended by manufacturer to
insure acceptable results.

c. Validation of temperature effect: Check frequently one or two points to verify
temperature correction data
Calculation

Membrane electrodes exhibit a relatively high temperature coefficient largely due
to changes in the membrane permeability
The effect of temperature on the electrode sensitivity, (microamperes per
milligram per liter)
log = 0.43 mt+ b
where:
t= temperature, C,
m= constant that depends on the membrane material, and
b= constant that largely depends on membrane thickness.

sensitivity at any desired temperature ( and t)=
log = log
0
+ 0.43 m(t t
0
)

Temperature correction can be found from the nomographic charts

Electrode sensitivity varies with salt concentration according to the following
relationship:
log
s
= 0.43 mSCs+ log
0

Calculation

For each test bottle meeting the 2.0-mg/L minimum DO depletion and the
1.0-mg/L residual DO, calculate BOD5 as follows:
When dilution water is not seeded:

where:
D
1
= DO of diluted sample immediately after preparation, mg/L,
D
2
= DO of diluted sample after 5 d incubation at 20C, mg/L,
P= decimal volumetric fraction of sample used








Calculation

BOD (mg/L)= [(D
1
D
2
) f(B
1
B
2
)]/P
where:
D
1
= DO of diluted sample immediately after preparation, mg/L,
D
2
= DO of diluted sample after 5 d incubation at 20C, mg/L,
P= decimal volumetric fraction of sample used,
B
1
= DO of seed control before incubation, mg/L,
B
2
= DO of seed control after incubation mg/L, and

f = ratio of seed in diluted sample to seed in seed control = (%
seed in diluted sample)/(% seed in seed control).

If seed material is added directly to sample or to seed control
bottles:

f = (volume of seed in diluted sample)/(volume of seed in seed control)

Ultimate BOD Test
An extension of 5-day BOD test.
Principle
Placing a single sample dilution in full, airtight bottles and incubating under
specified conditions for an extended period depending on wastewater
effluent, river, or estuary quality.
Dissolved oxygen (DO) is measured (with probes) initially and intermittently
during the test. From the DO versus time series, UBOD is calculated.
Temp: 20C
When DO concentrations approach 2 mg/L, the sample should be reaerated.
If the result is being used to estimate the rate of oxidation of naturally
occurring surface waters, addition of nutrients and seed probably accelerates
the decay rate and produces misleading results.
If only UBOD is desired, it may be advantageous to add supplemental nutrients
that accelerate decay and reduce the test duration. When nutrients are used,
they also should be used in the dilution water blank.
Apparatus
Incubation bottles: Glass bottles with ground-glass stoppers 2-L (or larger)
capacity clean bottles with a detergent and wash with dilute HCl (3N) to
remove surface films and precipitated inorganic salts; rinse thoroughly
with DI water before use.
To prevent drawing air into the sample bottle during incubation, use a
water seal.
Place a clean magnetic stirring bar in each bottle to mix contents before
making DO
measurement or taking a subsample.
Reservoir bottle:4-L or larger glass bottle. Close with screw plastic cap or
non-rubber plug.
Incubator or water bath, thermostatically controlled at 20 1C. Exclude
all light to prevent the possibility of photosynthetic production of DO.

Procedure: River water samples
Fill large BOD bottle with sample at 20C. Add no nutrients, seed, or
nitrification inhibitor if in-bottle decay rates will be used to estimate in-
stream rates.
Measure DO in each bottle, stopper, and make an airtight seal. Incubate at
20C in the dark.
Measure DO in each bottle at intervals of at least 2 to 5 d over a period of
30 to 60 d(minimum of 6 to 8 readings) or longer under special
circumstances.
To avoid oxygen depletion in samples containing NH3-N, measure DO
more frequently until nitrification has taken place. If DO falls to about 2
mg/L, reaerate .
Pour a small amount of sample into a clean vessel and reaerate the
remainder directly in the bottle by vigorous shaking or bubbling with
purified air (medical grade). Refill bottle from the storage reservoir and
measure DO. This concentration becomes the initial DO for the next
measurement. If using 300-mL BOD bottles, pour all of the sample from
the several bottles used into a clean vessel, reaerate, and refill the small
bottles.
Analyze for nitrate plus nitrite nitrogen on Days 0, 5, 10, 15, 20, and 30.
Alternatively, determine NO2-N andNO3-N each time DO is determined,
thereby producing corresponding BOD and nitrogen determinations. If the
ultimate demand occurs at a time greater than 30 d, make additional
analyses at 30-d intervals.
If the purpose of the UBOD test is to assess the UBOD and not to provide
data for rate calculations, measure nitrate nitrogen concentration only at
Day 0 and on the last day of the test.
When using a dilution water blank, subtract DO uptake of the blank from
the total DO consumed.
High-quality reagent water without nutrients typically will consume a
maximum of 1mg DO/L in a 30- to 90-d period. If DO uptake of the dilution
water is greater than 0.5 mg/L for a 20-d period, or 1 mg/L for a 90-d
period, report the magnitude of the correction and try to obtain higher-
quality dilution water for use with subsequent UBOD tests.
Wastewater treatment plant samples
Use high-quality reagent water for dilution water.

Add no nitrification inhibitors if decay rates are desired.

If seed and nutrients are necessary, add the same amounts of each to the dilution
water blank the ultimate BOD of the diluted sample should be in the range of 20 to
30 mg/L.
Dilution to this level probably will require two or three sample reaerations during
the incubation period to avoid having dissolved oxygen concentrations fall below 2
mg/L.

Fill a BOD bottle with dilution water to serve as a dilution water blank

Use 2-L or larger BOD bottles (alternatively, multiple 300-mL BOD bottles) for each
dilution. Add desired volume of sample to each bottle and fill with dilution water.

Treat blank the same as all samples.
Calculations
UBOD can be estimated by using a first-order model described
as follows:
BOD
t
= UBOD(1 e
-Kt
)
where:
BOD
t
= oxygen uptake measured at time t, mg/L, and
k= first-order oxygen uptake rate.
Respirometric Method
Respirometric methods provide direct measurement of the oxygen consumed by
microorganisms from an air or oxygen-enriched environment in a closed vessel
under conditions of constant temperature and agitation.
Respirometry measures oxygen uptake more or less continuously over time.
Respirometric methods are useful for assessing
biodegradation of specific chemicals
treatability of organic industrial wastes
the effect of known amounts of toxic compounds on the oxygen-
uptake reaction of a test wastewater or organic chemical
the concentration at which a pollutant or a wastewater
measurably inhibits biological degradation
the effect of various treatments such as disinfection, nutrient
addition, and pH adjustment on oxidation rates
the oxygen requirement for essentially complete oxidation of
biologically oxidizable matter
the need for using adapted seed in other biochemical oxygen
uptake measurements, such as the dilution BOD test
Respirometric data typically will be used comparatively, that is, in a direct
comparison between oxygen uptakes from two test samples or from a test sample
and a control.
Types of Respirometers
Manometric respirometers relate oxygen uptake to the change in pressure
caused by oxygen consumption while maintaining a constant volume.
Volumetric respirometers measure oxygen uptake in incremental changes
in gas volume while maintaining a constant pressure at the time of
reading.
Electrolytic respirometers monitor the amount of oxygen produced by
electrolysis of water to maintain a constant oxygen pressure within the
reaction vessel.
Direct-input respirometers deliver oxygen to the sample from a pure
oxygen supply through metering on demand as detected by minute
pressure differences.
Reaction-vessel contents are mixed by using a magnetic or mechanical
stirring device or by bubbling the gaseous phase within the reaction vessel
through the liquid phase.
All respirometers remove carbon dioxide produced during biological
growth by suspending a concentrated adsorbent (granular or solution)
within the closed reaction chamber or by recirculating the gas phase
through an external scrubber.
Interferences
Evolution of gases other than CO
2
may introduce errors in pressure or volume
measurements; this is uncommon in the presence of dissolved oxygen.
Incomplete CO
2
absorption will introduce errors if appropriate amounts and
concentrations of alkaline absorbent are not used.
Temperature fluctuations or inadequate mixing.
Fluctuations in barometric pressure
Most commercial respirometers can detect oxygen demand in increments as small
as 0.1 mg.
Upper limits of oxygen uptake rate are determined by the ability to transfer oxygen
into the solution from the gas phase, which typically is related to mixing intensity.
The continuous readout of oxygen consumption in respirometric measurements
indicates lag, toxicity, or any abnormalities in the biodegradation reaction. The
change in the normal shape of an oxygen-uptake curve in the first few hours may
help to identify the effect of toxic or unusual wastes entering a treatment plant in
time to make operating corrections.
Apparatus
Respirometer system
Incubator or water bath

Reagents
a. Distilled water
b. Phosphate buffer solution
c. Ammonium chloride solution
d. Calcium chloride solution
e. Magnesium sulfate solution,
f. Ferric chloride solution
Reagents

Potassium hydroxide solution,6N
Acid solutions,1N
Alkali solution,1N
Nitrification inhibitor: Reagent-grade 2-chloro-6
(trichloromethyl) pyridine (TCMP) or equivalent.
Glucose-glutamic acid solution
Electrolyte solution
Sodium sulfite
Trace element solution
Yeast extract solution
Nutrient solution
Procedure
Instrument operation: Follow respirometer manufacturers instructions for
assembly, testing, calibration, and operation of the instrument.
Sample volume: Small volumes or low concentrations may be required for high-
strength wastes. Large volumes may be required for low-strength wastes to
improve accuracy.
Data recording interval: Set instrument to give data readings at suitable intervals.
Intervals of 15 min to 6 h typically are used.
Sample preparation
HomogenizationIf sample contains large settleable or floatable solids,
homogenize it with a blender and transfer representative test portions while all
solids are in suspension. If there is a concern for changing sample characteristics,
skip this step.
pH adjustmentNeutralize samples to pH 7.0with H
2
SO
4
or NaOH of such strength
that reagent that reagent quantity does not dilute the sample more than 0.5%.
DechlorinationAvoid analyzing samples containing residual chlorine by collecting
the samples ahead of chlorination processes. If residual chlorine is present, aerate
or let stand in light for 1 to 2 h. If a chlorine residual persists, add Na
2
SO
3
solution.
Procedure
Initial oxygen concentrationIf samples contain dissolved oxygen concentrations
above or below the desired concentration, agitate or aerate with clean and filtered
compressed air for about 1 h immediately before testing.
Temperature adjustmentBring samples and dilution water to desired test
temperature (1C) before making dilutions or transferring to test vessels.
Sample dilution: Use distilled water or water from other appropriate sources free
of organic matter for dilution.
Nutrients, minerals, and buffer : Add sufficient ammonia nitrogen to provide a
COD:N:P ratio of 100:5:1 or a TOC:N:P ratio of 30:5:1.
Add 2 mL each of calcium, magnesium, ferric chloride, and trace mineral solutions
to each liter of diluted sample unless sufficient amounts of these minerals are
present in the original sample.
Phosphorus requirements will be met by the phosphate buffer if it is used (1
mL/50 mg/L COD or ultimate BOD of diluted sample usually is sufficient to
maintain pH between 6.8 and 7.2).
Nitrification inhibition: If nitrification inhibition is desired, add 10 mg 2-
chloro-6-(trichloromethyl) pyridine (TCMP)/L sample in the test vessel.

Seeding: Use sufficient amounts of seed culture to prevent major lags in
the oxygen uptake reaction but not so much that the oxygen uptake of the
seed exceeds about 10% of the oxygen uptake of the seeded sample.
Determine the oxygen uptake of the seeding material as for any other sample.
This is the seed control.

Incubation: Incubate samples at 20C or other suitable temperature 1.0C.

Calculations
To convert instrument readings to oxygen uptake, refer to
manufacturers procedures.
Correct oxygen uptake for seed and dilution by the following equation:
C= *A B(S
A
/S
B
)](1000/N
A
)
where:
C= corrected oxygen uptake of sample, mg/L,
A= measured oxygen uptake in seeded sample, mg,
B= measured oxygen uptake in seed control, mg,
S
A
= volume of seed in Sample A, mL,
S
B
= volume of seed in Sample B, mL, and
N
A
= volume of undiluted sample in Sample A, mL.

Reference
APHA manual for water and waste water
analysis, 4000- 6000