5210 E. Headspace
Biochemical Oxygen Demand (HBOD) [Proposed]
1. General Discussion
a. Principle: The HBOD test is a variation of a respirometric test
in that it provides direct measurement of oxygen consumption by
microorganisms in a closed-vessel under conditions of constant
temperature and agitation.1 The main difference between the
respirometric HBOD tests is the frequency of analysis: respirometric
tests can provide essentially continuous measurements of oxygen
utilization, while the HBOD test is a batch test. In the HBOD test,
oxygen is continuously resupplied from a large, but finite, supply
in the air in the headspace of a sealed tube through oxygen transfer
from the air into the liquid. Samples must be continuously agitated
in order for oxygen uptake not to be limited by mass transfer of
oxygen from the air into the water.
b. Uses: The HBOD test is useful for many of the same reasons a
respirometer is useful: measuring the oxygen consumption by
microorganisms during biodegradation of solutions containing
specific chemicals or mixed wastes; and assessing the effect of
toxic chemicals, pH, or nutrient additions on oxygen uptake.
c. Relationship to dilution BOD: Because water and wastewater
samples need not be diluted when analyzed in the HBOD test, the
oxygen demand of a sample will be exerted more rapidly than in the
dilution BOD test. In general, a three-day HBOD (HBOD3) is equal to
the 5-day BOD (BOD5).1 Valid correlations between the BOD and HBOD
tests should be established for specific wastewaters at each new
site. Based on respirometric data, a BOD5 is typically exerted after
2 to 3 days for municipal wastewaters.2,3
d. Interferences: The pressure in the tube may change during the
test due to the evolution of gases and the consumption of oxygen,
but there is no evidence that pressure changes affect the
consumption rate of oxygen. Carbon dioxide is not stripped out of
the sample tube, and high concentrations of CO2 have been noted to
slow down or inhibit biological reactions.4 However, dilution tests
suggest that within the recommended limits of the test for oxygen
consumption, CO2 does not interfere with the test.
e. Minimum detectable concentration:
The test is best suited to HBODs slightly larger than the solubility of oxygen, or HBOD>10
mg/L. For HBODs smaller than this, the concentration of dissolved
oxygen in the aqueous sample become important, and must be measured
and included in oxygen depletion calculations.
f. Sampling and Storage:
1) Grab Samples— If analysis is begun within 2 h of sample
collection, cold storage is unnecessary. For longer storage periods,
store samples at 0 to #4oC from the time of collection. Analysis
within 6 h is preferred; do not analyze samples if stored for more
than 24 h, and state storage time if stored between 6 and 24 h.
2) Composite Samples— Keep samples at 0 to #4oC from the time of
collection and during compositing. Use the same criteria for storage
as for grab samples, starting the measurement of holding time from
the end of the compositing period. State storage time and conditions
a. Incubation bottles. Any gas-tight crimp-seal bottles can
potentially be used, but mass transport coefficients must be
measured for new bottles. Currently, the HBOD test has only been
conducted using 28-mL anaerobic test tubes (BellCo) with 25 mm
Teflon-lined caps and aluminum crimp seals.1 Clean bottles with a
detergent, rinse thoroughly, and dry completely before use.
b. Incubator and water bath. All sample tubes must be shaken, on
their side, on a shaker table. Agitation rates typical of shaker
tables are sufficient for domestic wastewater samples. Place shaker
table in an incubator, or using a shaker table with a
temperature-controlled water bath. Exclude light from samples by an
appropriate method, such as placing tubes in a sealed box or
covering tubes (or water bath) with foil.
c. Instrument to measure oxygen in the headspace of a HBOD tube.
There are two currently proven methods to measure oxygen in tube heaspaces: an HBOD probe (Ocean Optics, Inc.) or a gas chromatograph
(many manufacturers). If using the HBOD probe, follow the
manufacturer’s instructinos for calibration. If Using a Gas
Chromatograph (GC) it should be equipped with a thermal conductivity
detector (TCD). Use an appropriate column and temperature to
analyzed for oxygen gas, such as a molecular sieve column and column
temperatures of 30 to 100 oC.1 Follow the manufacturer’s
instructions on operating the GC. Ensure that nitrogen and oxygen
peaks are adequately separated under the selected operating
conditions. Pressure will change inside an HBOD tube during an HBOD
test. A leur-lock gas-tight syringe must be used to maintain a
constant pressure in the syringe; otherwise, laboratory air will be
drawn into the syringe when it is removed from the tube. A 50 or 100
µL syringe is recommended.
d. Barometer, hydrometer, thermometer.
The pressure, relative
humidity and temperature of laboratory air must be know for accurate
calculation of the oxygen in each tube. Completely digital units
measuring all three parameters are commercially available. Make sure
that the absolute pressure is used. (Many weather stations will
adjust pressure to be sea level.)
a. Distilled water: Use only high-quality water distilled from a
block tin or all-glass still (See section 1080). Deionized water may
be used, but filter (through a 0.2 polycarbonate membrane) to remove
excess bacteria if present. The water must contain <0.01 mg/L of
heavy metals, and be free of chlorine, chloramines, caustic
alkalinity, organic material, and acids. When other waters are
required for special-purpose testing, state their source and
b. Potassium hydroxide (KOH) solution, 6 N: (CAUTION: If preparing
your own solution, add KOH to water slowly and use constant mixing
to prevent excessive heat accumulation.) Add 336 g KOH in 700 mL
water and dilute to 1 L. Commercials solutions containing 30 to 50%
KOH (by weight) may also be used.
c. Nitrification Inhibitor. Use pure reagent-grade
2-chloro-6-(trichloro-methyl) pyridine (TCMP) or equivalent. If TCMP
is not pure, adjust required doses accordingly. A final
concentration of 10 mg/L is recommended to inhibit nitrification.
d. Glucose-glutamic acid (GGA) solution. Dry reagent-grade glucose
and glutamic acid powders at 103oC for 1 h. Add 0.3 g of glucose and
0.3 g of glutamic acid to distilled water, and dilute to 1 L for a
final concentration of 600 mg/L. Neutralize to pH 7.0 using 6 N
potassium hydroxide. The solution may be stored for <1 week at 4oC.
e. Sodium sulfite (NasSO3). Use reagent grade sodium sulfite.
f. Cobalt chloride (CoCl2) solution: Cobalt chloride is used as a
catalyst for measuring oxygen transfer rates with sodium sulfite.
Add 1.5 mg CoCl2 to 1 L of distilled water.
a. Laboratory conditions: Record the laboratory air temperature,
pressure, and relative humidity at the time HBOD samples are
b. Sample volume: The proper sample volume for a tube depends on the
expected HBOD. Using Table 1, choose an appropriate liquid volume.
c. Sample preparation:
1) Homogenization— If a sample contains large settleable or
floatable solids, homogenize it with a blender and transfer a
representative portion of the appropriate volume into an HBOD tube.
Skip this step if there is a concern about changing sample
2) pH adjustment— Neutralize samples to pH 7 using H2SO4 or NaOH (if
desired) if the sample will not be diluted >0.5%.
3) Dechlorination— If possible, avoid analyzing samples containing
residual chlorine by collecting samples prior to chlorination
processes. If samples contain residual chlorine, aerate samples as
described below (¶ 5) or let sample stand in the light for 1 to 2 h.
If a chlorine residual is still present, Na2SO3 must be added to
react with the chlorine, although doing this will remove oxygen from
the sample and can injure microbes. The required volume of Na2SO3 is
10 mL 1 + 50 H2SO4, and 10 mL potassium iodide solution (10 g/100
mL) to a portion of the sample. Titrate with a 0.05 M Na2SO3
solution, and after 10 to 20 min check for residual chlorine. If
this dechlorination process is followed, the sample will need to be
seeded as described below.
4) Toxic substances in samples— If samples contain toxic substances,
they will require special treatment beyond the scope of the
development of the present procedure.
5) Initial oxygen concentration— For very low HBODs, it may be
necessary to know the initial DO of the sample. The sample can
either be aerated, or a dissolved oxygen microprobe must be used to
sample the DO in the sample after it has been transferred into a
tube. Samples can be aerated by agitation (using a shaker table or
stir bar) or by using a diffuser connect to either a tank of clean
air or to a filtered air source. Samples should not be aerated for
more than 1 h; aeration will decrease the HBOD of the sample
depending on the strength of the sample and the concentration of
microorganisms in the sample.
6) Sample temperature— All samples must be brought to the desired
incubation temperature (±1oC) before adding samples to the HBOD
d. Dilution: It is not necessary to dilute most samples to conduct a HBOD test. If dilution is desired, for example to examine for
potential toxic effects, to lower the final HBOD to avoid oxygen
transport limitations, or as a result of adding nitrification
inhibitor, dilute samples only with distilled or other water free
from organic and chemical contamination. Dilution water can be added
directly to the HBOD tubes as necessary.
e. Nutrients, minerals and buffer: It is important to remember that
if addition of nutrients, minerals or buffers are necessary to
obtain an estimate of the HBOD, then these a lack of any of these
components will interfere with optimal operation of a biological
wastewater treatment process. The appropriate concentrations of
nutrients should be added to obtain a final ratio of COD:N:P or
100:5:1, or a TOC:N:P ratio of 30:5:1. If minerals or buffering
capacity is suspected to be lacking, add to distilled water and
f. Nitrification inhibition: Many domestic wastewaters at full
strength and river waters will exert an oxygen demand due to
nitrification unless a nitrification inhibitor is added. Prepare a
concentrated solution of the nitrification inhibitor TCMP at a
concentration of 100 mg/L. Dilute wastewater samples in a ratio of
1:10 to obtain a final concentration of 10 mg/L of TCMP in the HBOD
tube. To determine the nitrogenous HBOD (NHBOD), also prepare
samples without any nitrification inhibitor.
High concentrations of microorganisms are necessary to
ensure that an HBOD will be exerted in about three days that is
comparable to a BOD5. See section 5210B.4d1) for seed preparation.
If available, use secondary clarifier overflow prior to
chlorination. In any case, it is important to use sufficient amounts
of seed to prevent major lags in oxygen utilization. When seeded
samples are used, it is necessary to run parallel tests using
full-strength seed to correct the sample HBOD for the seed HBOD.
h. Sample Preparation: Add to each sample tube, in the following
order: (1) any water containing nutrients, minerals, buffers or
nitrification inhibitor (if necessary) ; (2) seed solution
containing microorganisms (if necessary); (3) wastewater sample; (4)
immediately cap bottles, and crimp seal aluminum cap on top with a
i. Seed Preparation: If a seed control is performed, repeat steps
(1) and (2) and (4) in step j above for the same number of bottles
used for each sample.
j. Air blanks: Seal 3 tubes containing one or two mL of distilled
k. Incubation. Incubate all tubes containing samples and seed on
their sides, oriented in a direction that gives the maximum mixing
of the sample. Keep samples in the dark and at 20±1 oC.
l. Oxygen Analysis in Tubes.
After an appropriate incubation time,
take tubes off of shaker table, place them in an upright position
near the oxygen-measuring instrument. If using the HBOD probe,
calibrate the probe in air from the Air Blank tubes. Then, sample
tubes. With this probe oxygen measurements are complete in a few
seconds. If using a GC, using a gas-tight syringe (50 or 100 µL)
with a Luer-lock type adaptor and side port needle, calibrate the GC
first by measuring oxygen in laboratory air until a stable and
repeatable result is obtained (at least 5 injections). Then, measure
oxygen concentrations first in the Air Blank tubes and samples.
Record the areas of both the oxygen and nitrogen peaks. If nitrogen
peaks areas vary by more than >10% for samples versus those for the
air blanks, repeat an injection.
The HBOD of each tube on day n (THBOD) is used to calculate the HBOD
of the water sample (HBODn = WHBODn) after correction for dilution
and seed addition. Depending on the experimental objectives, it is
also possible to calculate the carbonaceous HBOD (CHBOD),
nitrogenous HBOD (NHBOD), and the HBOD of a glucose-glutamic acid
calibration solution (GGHBOD).
Calculate the HBOD for each tube using:
where: THBODn= Headspace BOD on day n of sample in HBOD tube [mg/L];
P0 = Total pressure of laboratory air on day 0 recorded from
p0,w = Vapor pressure of water at temperature of sample on day 0 from
table of water vapor pressures [mmHg];
r0 = Relative humidity of air on day 0 read from relative humidity
An= Oxygen in sample on day n: for HBOD probe, percent of oxygen in
the air in the tube [%]; for GC, peak area [mV-sec ];
A0,n= Oxygen in the day 0-tube analyzed on day n [%] or [mV-sec];
T0= Temperature of air on day 0 [oC].
DO= Saturation dissolved oxygen concentration in water at 760 mmHg
(1 atm) in water-saturated air at temperature T0 from dissolved
oxygen reference table [mg/L];
VT= Total volume of empty HBOD tube [mL];
VL= Volume of liquid wastewater sample put into HBOD tube [mL];
a. Water Sample— No dilution: If the sample was not diluted, the HBOD of the water sample is the same as the HBOD of the tube
calculated using eq. 1, or
b. Sample HBOD with dilution (no seed): If the sample contains any
dilution water, the sample HBOD is
where VL is the total sample liquid volume and Vw is the volume of
the water sample. The term VW/VL is equal to the fraction of the
liquid that this the water sample, or VW/VL= P where P is the
decimal volumetric fraction of sample used. The total liquid volume
must be the sum of the water sample and dilution water volume, or
VL=VW+VD, where VD is the volume of dilution water assumed to have
c. Sample HBOD with Seed: The HBOD of the sample and the seed must
be determined separately. Calculate the HBOD of the seed using
method a) or other methods in this section, as appropriate. The HBOD
of the water sample is then calculated from the HBOD of the seed
(SHBOD) and the HBOD of the tube containing both sample and seed
d. Carbonaceous HBOD (CHBOD): The CHBOD can be measured for a sample
by either adding the nitrification inhibitor TCMP into the water
sample, or into the dilution water. If TCMP is added to the water
sample, calculate the CHBOD as in part a) or if seed is used as in
part c). If TCMP is added into the dilution water, calculate the
CHBOD as in part b), or if seed is used, calculate the HBOD as in
part c) recognizing that TCMP must be added to both the sample and
e. Nitrogenous HBOD (NHBOD): In order to calculate the NHBOD,
samples must be analyzed with and without nitrification inhibitor.
Calculate the total HBOD of just the water sample using either a) or
b) depending on whether dilution of the sample is necessary. Run in
parallel samples containing nitrification inhibitor. The NHBOD is
calculated as the difference between the two results, as
6. Quality Control
In order to test the procedures necessary for the HBOD protocol, it
is necessary to ensure that samples are adequately mixed (aerated)
and that the other procedures are adequately run. In order to show
that samples are well mixed, measure the mass transport coefficient
in tubes for typical mixing conditions. Any time a new mixing speed,
shaker table, or tube-type is used, check to see that the mass
transport coefficient meets the requirements as described below. The
glucose-glutamic acid check is suggested to verify proper operation
of the GC, gas injections, seed (if used) and general laboratory
a. Mass transport coefficient: Add sodium sulfite (Na2SO3) to cobalt
chloride solution at a concentration (>0.5 M Na2SO3) that will
always ensure that sodium sulfite is in excess and oxygen uptake
will be first order with respect to oxygen in the headspace. The
mass of sodium sulfite added is calculated as the sum of the mass of
chemical necessary to react with all oxygen in the air in an empty
tube, and mass in the liquid at a concentration of 0.05 M. The mass
of sodium sulfite Na2SO3 is therefore
where p is the partial pressure of oxygen in air, T the temperature,
Va the volume of air and VL the volume of liquid (Logan and Kohler,
1999). For typical conditions of 0.209 atm of oxygen, 20oC (293 K),
this can be simplified to
The procedure for calculating the mass transport coefficient is as
1) Place the appropriate mass of sodium sulfite (calculated using
eq. 6 or 7) into each of 3 tubes.
2) Add the cobalt chloride solution of volume VL.
3) Immediately cap tubes and place on shaker table. Start timing
from the moment the sample is shaken.
4) Analyze the concentration of oxygen in the tubes at times of 1, 3
and 5 minutes.
5) Calculate mass transport coefficients, kLa [hr-1], as
where A1 and A2 are the oxygen concentrations (peak area or percent
oxygen) in the tube headspaces measured at times t1 and t2. For
example, if laboratory air is analyzed using the HBOD probe to have
a peak area of 20.9%, after sodium sulfite is added, the sample is
capped, and placed on the shaker table, after 1 min analysis of the
air in the tube indicates 18% oxygen, but after 3 min the
concentration is 13.1%. Using eq. 8, kLa=9.6 hr-1. Each run with
three tubes produces two values of kLa.
6) Repeat steps 2 through 6 at least three times. A typical value
for kLa is 8±2 hr-1.
The mass transport coefficient is the maximum rate of oxygen
transport into liquid, and therefore, it represents the maximum rate
that substrate can aerobically be consumed. Extensive BOD testing
(5210 B 5) and some HBOD testing1 indicates that the DO should
remain above 1 and 2 mg/L (respectively) for oxygen not to limit the
rate of BOD exertion. For typical mass transport coefficients, HBODs
as high as 1340 mg/L-d are possible at the start of an HBOD test,
although the maximum daily HBOD declines to 192 mg/L-d at the end of
the test due to oxygen depletion in the sample headspace (Logan and
Kohler, 1999). If greater oxygen transfer rates are needed, the
intensity of mixing can be increased or samples can be diluted.
b. Glucose Glutamic Acid Calibration. Because many full-strength
wastewaters will nitrify, it is important to run a calibration test
with nitrification inhibitor in all samples. A seed solution is
needed to supply concentrated suspension of microorganisms. Prepare
a GGA solution as described above. Add TCMP to both the GGA solution
and the seed at a final TCMP concentration of 10 mg/L. The HBOD of
the GGA solution (GGHBOD) must be determined using separate samples
containing GGA and the seed, and samples containing only seed.
Calculate the HBOD of the seed using method 5a) or 5b). The HBOD of
the GGA water sample is then calculated from the HBOD of the seed
(SHBOD) and the HBOD of the tube containing both sample and GGA
where THBOD is the HBOD of the tube containing GGA and seed filled
to a volume VL and containing a volume of seed, VS, and volume of
GGA, VG, and SHBOD is the HBOD of the seed determined in separate
experiments. Note that VL=VG+VS.
Run at least tubes in triplicate. Three-day GGHBOD’s (211 mg/L) have
been reported to be within the range of BOD5 results (204±10 mg/L).
If values are not within this range, make changes to the protocol
such as using new reagents to ensure high quality, changing the
source of the seed, checking the accuracy of the gas chromatograph,
or the mixing rate of samples on the shaker table.
7. Precision and Bias
a. Precision. There is no standard available to check the accuracy
of the HBOD technique, but it is assumed to compare well with
respirometric techniques. The precision of the test can be examined
using the glucose-glutamic acid calibration technique described
above in section 6b.
b. Control limits. Perform a minimum of 25 glucose-glutamic acid
checks on the GGHBOD3 over a period of several months and calculate
the mean and standard deviation. If the mean of the three-day tests
is outside the range of 204±10 mg/L, re-evaluate the procedure and
make changes accordingly.
Logan, B.E. and D. Kohler. 1999. Oxygen mass transfer coefficients
for different sample containers used in the headspace BOD (HBOD)
Logan, B.E. and R. Patnaik. 1997. A gas chromatographic based
headspace biochemical oxygen demand test. Water Env. Res.,
Young, J.C. and E.R. Baumann. 1976. The electrolytic respirometer—
Factors affection oxygen uptake measurements. Water Res. 10:1031.
Young, J.C. and E.R. Baumann. 1976. The electrolytic respirometer—
Use in water pollution control plant laboratories. Water Res.
Rozich, A.F. and A.F. Gaudy, Jr. 1992. Design and operation of
activated sludge processes using respirometry. Lewis, Chelsea, MI.
Logan, B.E. and G.A. Wagenseller. 1993. The HBOD test: a new method
for determining biochemical oxygen demand. Water Environ. Res.
Table 1. Range of measurable HBODs as a function of headspace and
liquid volumes for a DO change of >1 mg/L and a minimum final DO of
>2 mg/L (VT=28 mL, r=20%, To=20oC, ys=0.209, pw=17.54 mmHg, and
PT=700 mmHg, corresponding to a DO saturation concentration of 9.09
||7 - 50
||12 - 86
||17 - 117
||39 - 236
||51 - 364
||71 - 503