5-2 AGGREGATE ORGANIC CONSTITUENTS (5000)

5210 BIOCHEMICAL OXYGEN DEMAND (BOD)*

5210 A. Introduction

1. General Discussion iron and sulfur compounds, or lack of mixing may affect the
accuracy and precision of BOD measurements. Presently, there
The biochemical oxygen demand (BOD) determination is an is no way to include adjustments or corrections to account for the
empirical test in which standardized laboratory procedures are effect of these factors.
used to determine the relative oxygen requirements of wastewa- Oxidation of reduced forms of nitrogen, such as ammonia and
ters, effluents, and polluted waters. The test has its widest ap- organic nitrogen, can be mediated by microorganisms and exert
plication in measuring waste loadings to treatment plants and in nitrogenous demand. Nitrogenous demand historically has been
evaluating the BOD-removal efficiency of such treatment sys- considered an interference in the determination of BOD, and the
tems. The test measures the molecular oxygen utilized during a inclusion of ammonia in the dilution water contributes an exter-
specified incubation period for the biochemical degradation of nal source of nitrogenous demand. The interference from nitrog-
organic material (carbonaceous demand) and the oxygen used to
enous demand can now be prevented by an inhibitory chemical.1
oxidize inorganic material such as sulfides and ferrous iron. It
If an inhibiting chemical is not used, the oxygen demand mea-
also may measure the amount of oxygen used to oxidize reduced
sured is the sum of carbonaceous and nitrogenous demands.
forms of nitrogen (nitrogenous demand) unless their oxidation is
prevented by an inhibitor. The seeding and dilution procedures Measurements that include nitrogenous demand generally are not
provide an estimate of the BOD at pH 6.5 to 7.5. useful for assessing the oxygen demand associated with organic
Measurements of oxygen consumed in a 5-d test period (5-d material. Nitrogenous demand can be estimated directly from am-
BOD or BOD5, 5210B), oxygen consumed after 60 to 90 d of monia nitrogen (Section 4500-NH3); and carbonaceous demand can
incubation (ultimate BOD or UBOD, 5210C), and continuous be estimated by subtracting the theoretical equivalent of the nitrite
oxygen uptake (respirometric method, 5210D) are described and nitrate produced in uninhibited test results. However, this
here. Many other variations of oxygen demand measurements method is cumbersome and is subject to considerable error. Chem-
exist, including using shorter and longer incubation periods and ical inhibition of nitrogenous demand provides a more direct and
tests to determine rates of oxygen uptake. Alternative seeding, more reliable measure of carbonaceous demand.
dilution, and incubation conditions can be chosen to mimic The extent of oxidation of nitrogenous compounds during the
receiving-water conditions, thereby providing an estimate of the 5-d incubation period depends on the concentration and type of
environmental effects of wastewaters and effluents. microorganisms capable of carrying out this oxidation. Such
The UBOD measures the oxygen required for the total degra- organisms usually are not present in raw or settled primary
dation of organic material (ultimate carbonaceous demand) sewage in sufficient numbers to oxidize sufficient quantities of
and/or the oxygen to oxidize reduced nitrogen compounds (ul- reduced nitrogen forms in the 5-d BOD test. Many biological
timate nitrogenous demand). UBOD values and appropriate ki- treatment plant effluents contain sufficient numbers of nitrifying
netic descriptions are needed in water quality modeling studies organisms to cause nitrification in BOD tests. Because oxidation
such as UBOD:BOD5 ratios for relating stream assimilative of nitrogenous compounds can occur in such samples, inhibition
capacity to regulatory requirements; definition of river, estuary, of nitrification as directed in 5210B.5e) is recommended for
or lake deoxygenation kinetics; and instream ultimate carbona-
samples of secondary effluent, for samples seeded with second-
ceous BOD (UCBOD) values for model calibration.
ary effluent, and for samples of polluted waters.
2. Carbonaceous Versus Nitrogenous BOD
3. Reference
A number of factors, for example, soluble versus particulate
organics, settleable and floatable solids, oxidation of reduced
1. YOUNG, J.C. 1973. Chemical methods for nitrification control. J.
Water Pollut. Control Fed. 45:637.
* Approved by Standard Methods Committee, 2001.
Joint Task Group: James C. Young (chair), George T. Bowman, Sabry M.
Kamhawy, Terry G. Mills, Marlene Patillo, Ray C. Whittemore.

5210 B. 5-Day BOD Test

1. General Discussion it at the specified temperature for 5 d. Dissolved oxygen is

measured initially and after incubation, and the BOD is com-
The method consists of filling with diluted and seeded sample, puted from the difference between initial and final DO. Because
to overflowing, an airtight bottle of specified size and incubating the initial DO is determined shortly after the dilution is made, all
BIOCHEMICAL OXYGEN DEMAND (BOD) (5210)/5-Day BOD Test 5-3

oxygen uptake occurring after this measurement is included in i. Ammonium chloride solution: Dissolve 1.15 g NH4Cl in
the BOD measurement. about 500 mL distilled water, adjust pH to 7.2 with NaOH
For sampling and storage procedures, see 4a below. solution, and dilute to 1 L. Solution contains 0.3 mg N/mL.
j. Source water for preparing BOD dilution water: Use de-
2. Apparatus mineralized, distilled, tap, or natural water for making sample
dilutions (see 4c).
a. 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, 4. Preparatory Procedures
rinse thoroughly, and drain before use.
b. Air incubator or water bath, thermostatically controlled at a. Sampling and storage: Samples for BOD analysis may
20 1C. Exclude all light to prevent possibility of photosyn- degrade significantly during storage between collection and anal-
thetic production of DO. ysis, resulting in low BOD values.
1) Grab samplesIf analysis is begun within 2 h of collec-
3. Reagents tion, cold storage is unnecessary. If analysis is not started within
2 h of sample collection, keep sample at or below 4C from the
Prepare reagents in advance but discard if there is any sign of time of collection. Begin analysis within 6 h of collection; when
precipitation or biological growth in the stock bottles. Commer- this is not possible because the sampling site is distant from the
cial equivalents of these reagents are acceptable and different laboratory, store at or below 4C and report length and temper-
stock concentrations may be used if doses are adjusted propor- ature of storage with the results. In no case start analysis more
tionally. Use reagent grade or better for all chemicals and use than 24 h after grab sample collection. When samples are to be
distilled or equivalent water, preferably sterilized, for making all used for regulatory purposes make every effort to deliver sam-
solutions. ples for analysis within 6 h of collection.
a. Phosphate buffer solution: Dissolve 8.5 g KH2PO4, 21.75 g 2) Composite samplesKeep samples at or below 4C during
K2HPO4, 33.4 g Na2HPO47H2O, and 1.7 g NH4Cl in about 500 compositing. Limit compositing period to 24 h. Use the same
mL distilled water and dilute to 1 L. The pH should be 7.2 criteria as for storage of grab samples, starting the measurement
without further adjustment. Alternatively, dissolve 42.5 g of holding time from end of compositing period. State storage
KH2PO4 and 1.7 g NH4Cl in about 700 mL distilled water. time and conditions as part of the results.
Adjust pH to 7.2 with 30% NaOH and dilute to 1 L. b. Sample preparation and pretreatment:
b. Magnesium sulfate solution: Dissolve 22.5 g MgSO47H2O 1) All samples Check pH; if it is not between 6.0 and 8.0,
in distilled water and dilute to 1 L. adjust sample temperature to 20 3C, then adjust pH to 7.0 to
c. Calcium chloride solution: Dissolve 27.5 g CaCl2 in dis- 7.2 using a solution of sulfuric acid (H2SO4) or sodium hydrox-
tilled water and dilute to 1 L. ide (NaOH) of such strength that the quantity of reagent does not
d. Ferric chloride solution: Dissolve 0.25 g FeCl36H2O in dilute the sample by more than 0.5%. Exceptions may be justi-
distilled water and dilute to 1 L. fied with natural waters when the BOD is to be measured at
e. Acid and alkali solutions, 1N, for neutralization of caustic or in-situ pH values. The pH of dilution water should not be
acidic waste samples. affected by the lowest sample dilution. Always seed samples that
1) AcidSlowly and while stirring, add 28 mL conc sulfuric have been pH adjusted.
acid to distilled water. Dilute to 1 L. 2) Samples containing residual chlorine compoundsIf pos-
2) AlkaliDissolve 40 g sodium hydroxide in distilled water. sible, avoid samples containing residual chlorine by sampling
Dilute to 1 L. ahead of chlorination processes. If residual chlorine is present,
f. Sodium sulfite solution: Dissolve 1.575 g Na2SO3 in 1000 dechlorinate sample. In some samples chlorine will dissipate
mL distilled water. This solution is not stable; prepare daily. within 1 to 2 h of standing in the light. This dissipation often
g. Nitrification inhibitor: occurs during sample transport and handling. For samples in
1) 2-chloro-6-(trichloromethyl) pyridineUse pure TCMP or which chlorine residual does not dissipate in a reasonably short
commercial preparations*. time, destroy chlorine residual by adding Na2SO3 solution. De-
2) Allylthiourea (ATU) solutionDissolve 2.0 g allylthio- termine required volume of Na2SO3 solution on a 100- to
urea (C4H8N2S) in about 500 mL water and dilute to 1 L. Store 1000-mL portion of neutralized sample by adding 10 mL 1 1
at 4C. The solution is stable for not more than 2 weeks. acetic acid or 1 50 H2SO4, 10 mL potassium iodide (KI)
h. Glucose-glutamic acid solution: Dry reagent-grade glucose solution (10 g /100 mL) per 1000 mL sample and titrating with
and reagent-grade glutamic acid at 103C for 1 h. Add 150 mg Na2SO3 solution to the starch-iodine end point for residual. Add
glucose and 150 mg glutamic acid to distilled water and dilute to to neutralized sample the proportional volume of Na2SO3 solu-
1 L. Prepare fresh immediately before use unless solution is tion determined by the above test, mix, and after 10 to 20 min
maintained in a sterile condition. Store all glucose-glutamic acid check sample for residual chlorine. (NOTE: Excess Na2SO3 exerts
mixtures at 4C or lower. Commercial preparations may be used an oxygen demand and reacts slowly with certain organic chlo-
but concentrations may vary. ramine compounds that may be present in chlorinated samples.)
Do not test chlorinated/dechlorinated samples without seeding.
3) Samples containing other toxic substancesCertain indus-
* Nitrification Inhibitor Formula 2533 (2% TCMP on sodium sulfate), Hach Co., trial wastes, for example, plating wastes, contain toxic metals.
Loveland, CO, or equivalent. Such samples often require special study and treatment.
5-4 AGGREGATE ORGANIC CONSTITUENTS (5000)

4) Samples supersaturated with DOSamples containing DO Do not filter seed sources; filtering removes the seed microor-
concentration above saturation at 20C may be encountered in ganisms.
cold waters or in water where photosynthesis occurs. To prevent When acclimated seed sources are not available, develop an
loss of oxygen during incubation of such samples, reduce DO to acclimated seed in the laboratory by continuously aerating a
saturation by bringing sample to about 20 3C in partially sample of settled domestic wastewater and adding small daily
filled bottle while agitating by vigorous shaking or by aerating increments of sample from the waste in question. Use a soil
with clean, filtered compressed air. suspension, activated sludge, or a commercial seed preparation
5) Samples containing hydrogen peroxideHydrogen perox- to obtain the initial microbial population. Determine the exis-
ide remaining in samples from some industrial bleaching pro- tence of a satisfactory population by testing the performance of
cesses such as those used at paper mills and textile plants can the seed in BOD tests on the sample. BOD values that increase
cause supersaturated oxygen levels in samples collected for with time of adaptation to a steady high value indicate successful
BOD testing. Mix such samples vigorously in open containers seed acclimation.
for sufficient time to allow the hydrogen peroxide to dissipate
before setting up BOD tests. Check adequacy of peroxide re- 5. Testing Procedure
moval by observing dissolved oxygen concentrations over time
during mixing or by using peroxide-specific test strips. Mixing a. Preparation of dilution water: Transfer desired working
times can vary from 1 to 2 h depending on the amount of volume of source water ( 4c) to a suitably sized bottle (glass is
hydrogen peroxide present. The peroxide reaction can be con- preferred). Check to ensure that the dissolved oxygen concentra-
sidered complete when the DO no longer increases during a tion is at least 7.5 mg/L before using water for BOD tests. If not,
30-min period without mixing. add DO by shaking bottle or by aerating with organic-free
c. Selection and storage of source water for BOD sample filtered air. Alternatively, store the water in cotton-plugged bot-
dilution: Obtain water from suitable source distilled, tap, or tles long enough for the DO concentration to approach satura-
receiving water. Make sure the water is free of heavy metals, tion. Add 1 mL each of phosphate buffer, MgSO4, CaCl2, and
specifically copper, and toxic substances, such as chlorine, that FeCl3 solution/L to prepared source water ( 4c). Mix thoroughly
can interfere with BOD measurements. Protect source water and bring temperature to 20 3C. Prepare dilution water
quality by using clean glassware, tubing, and bottles. Deionized immediately before use unless dilution water blanks ( 6c) show
water often contains sufficient amounts of organics and micro- that the water is acceptable after longer storage times. If the
organisms to cause failure of the dilution water quality control dilution water blanks show a DO depletion greater than 0.20
check ( 6c). Source water may be stored before use as long as mg/L, obtain a satisfactory water by improving purification or
the prepared dilution water ( 5a) meets quality control criteria use water from another source. Do not add oxidizing agents or
in the dilution water blank ( 6c). Such storage may improve the expose dilution water to ultraviolet light in attempts to bring the
quality of some source waters but may allow biological growth dilution blank into range.
to cause deterioration in others. Storage of prepared dilution b. Sample temperature adjustment: Bring samples to 20
water ( 5g) for more than 24 h after adding nutrients, minerals, 3C before making dilutions.
and buffer is not recommended unless dilution water blanks c. Preparation of dilutions: Using the dilution water prepared
consistently meet quality control limits. Discard stored source as in 5a, make at least three dilutions of prepared sample
water if the dilution water blank shows more than 0.20 mg/L DO estimated to produce a residual DO of at least 1.0 mg/L and a DO
depletion in 5 d (see 6c). uptake of at least 2.0 mg/L after a 5-d incubation. Five dilutions
d. Preparation of seed suspension: It is necessary to have are recommended if experience with a particular sample does not
present in each BOD bottle a population of microorganisms produce at least three bottles having acceptable minimum DO
capable of oxidizing the biodegradable organic matter in the depletions and residual limits ( 6a). A more rapid analysis, such
sample. Domestic wastewater, unchlorinated or otherwise undis- as COD (Section 5220), may be correlated approximately with
infected effluents from biological wastewater treatment plants, BOD and serve as a guide in selecting dilutions. In the absence
and surface waters receiving wastewater discharges usually con- of prior knowledge, use the following percentages of wastewater
tain satisfactory microbial populations. Some samples (for ex- when preparing dilutions: 0.01 to 1.0% for strong industrial
ample, some untreated industrial wastes, disinfected wastes, wastes, 1 to 5% for raw and settled wastewater, 5 to 25% for
high-temperature wastes, wastes having pH values less than 6 or biologically treated effluent, and 25 to 100% for polluted river
greater than 8, or wastes stored more than 6 h after collection) do waters. The number of bottles to be prepared for each dilution
not contain a sufficient microbial population. Seed such samples depends on the DO technique and the number of replicates
by adding a population of suitable microorganisms. The pre- desired. Prepare dilutions in volumetric containers (Class A glass
ferred seed is obtained from a biological treatment system pro- or equivalent) and then transfer to BOD bottles or prepare
cessing the waste. In this case, use supernatant from settled directly in BOD bottles. Either dilution method can be combined
domestic wastewater, effluent from primary clarifiers, diluted with any DO measurement technique.
mixed liquor from an aeration basin, undisinfected effluent, or 1) Dilutions prepared in volumetric containersUsing a
receiving water from below the point of discharge. When efflu- wide-tipped pipet, add desired amount of prepared sample to
ent or mixed liquor from a biological treatment process is used individual volumetric cylinders or flasks. Mix the sample well
as a seed source, inhibition of nitrification is recommended. Do immediately before pipetting to avoid loss of solids by settling.
not use seed from effluents that have been disinfected by chlo- For dilutions greater than 1:100 make a primary dilution before
rine or other means. Commercial seed sources may be used but making final dilution in the bottle. Fill cylinders or flasks at least
are more likely to be unadapted to the wastewater constituents. two-thirds full of dilution water without entraining air. Add
BIOCHEMICAL OXYGEN DEMAND (BOD) (5210)/5-Day BOD Test 5-5

appropriate amounts of seed suspension ( 5d) and nitrification Some commercial TCMP formulations are not 100% TCMP;
inhibitor ( 5e). Dilute to final level with dilution water ( 5a). adjust dosage appropriately.
Mix well but avoid entraining air. Siphon mixed dilution into a 2) Nitrification inhibition using allylthiourea (ATU)Add 1
suitable number of BOD bottles, taking care not to let solids mL ATU solution ( 3g)/L diluted sample or 0.3 mL/300mL test
settle in the cylinder or flask during transfer. bottle or sample dilution vessel. Do not add ATU to BOD bottles
2) Dilutions prepared directly in BOD bottlesUsing a wide- until they are at least two-thirds filled with diluted sample.
tip volumetric pipet, add the desired sample volume to individual f. Sealing of bottles: Complete filling of each bottle by adding
BOD bottles. Fill each BOD bottle approximately two-thirds full enough dilution water that insertion of the stopper leaves no
with dilution water. Add appropriate amounts of seed suspension bubbles in the bottle. Mix the sample by turning the bottle
( 5d) and nitrification inhibitor ( 5e) to the individual BOD manually several times unless a DO probe having a stirrer is used
bottles. When a bottle contains more than 67% of the sample immediately to measure initial DO concentration. As a precau-
after dilution, nutrients may be limited in the diluted sample and tion against drawing air into the dilution bottle during incuba-
subsequently reduce biological activity. In such samples, add the tion, use a water seal. Obtain satisfactory water seals by invert-
nutrient, mineral, and buffer solutions (s 3a e) directly to ing bottles in a water bath or by adding water to the flared mouth
diluted sample at a rate of 1 mL/ L (0.30 mL/300-mL bottle) or of special BOD bottles. Place a paper or plastic cup or foil cap
use commercially prepared solutions designed to dose the ap- over flared mouth of bottle to reduce evaporation of the water
propriate bottle size. seal during incubation.
d. Addition of seed suspension: If seeding is used, add seed g. Determination of initial DO: Use the azide modification of
suspensions to the dilution vessels or to individual BOD bottles the iodometric method (Section 4500-O.C) or the membrane
before final dilution as described in 5c. Do not add seed electrode method (Section 4500-O.G) to determine initial DO on
directly to wastewater samples if they contain materials that are all sample dilutions, dilution water blanks, and, where appropri-
toxic before dilution. Generally, 1 to 3 mL of settled raw waste- ate, seed controls. Replace any displaced contents with sufficient
water or primary effluent or 1 to 2 mL of a 1:10 dilution of mixed diluted sample or dilution water to fill the bottle, stopper all
liquor/300-mL bottle will provide a suitable amount of micro- bottles tightly, and water seal before beginning incubation. After
organisms. Do not filter seed suspension before use. Agitate the preparing dilution, measure initial DO within 30 min. If the
membrane electrode method is used, take care to eliminate drift
seed suspension during transfer to ensure that the same quantity
in calibration between initial and final DO readings. If the azide
of microorganisms is added to each BOD bottle. Always record
modification of the titrimetric iodometric method is used, pre-
the exact volume of seed suspension added to each bottle. The
pare an extra bottle for initial DO determination for each sample
DO uptake attributable to the seed added to each bottle generally
dilution.
should be between 0.6 and 1.0 mg/L, but the amount of seed
h. Sample incubation: Incubate at 20C 1C the stoppered
added should be adjusted from this range to that required to
and sealed BOD bottles containing desired dilutions ( 5a), seed
provide glucose-glutamic acid (GGA) check results of 198
controls ( 6d), dilution water blanks ( 6c), and glucose-glu-
30.5 mg/L. For example, if 1 mL of seed suspension is required
tamic acid checks ( 6b). Exclude light to avoid growth of algae
to achieve 198 30.5 mg/L BOD in the glucose-glutamic acid in the bottles during incubation.
check, then use 1 mL in each BOD bottle receiving the test i. Determination of final DO: After 5 d 6 h of incubation,
wastewater. determine DO in all sample dilutions, and in all blanks and
e. Addition of nitrification inhibitor: Samples that may require checks as in 6b d, using the azide modification of the titri-
nitrification inhibition1 include, but are not limited to, biologi- metric method or the membrane electrode method.
cally treated effluents, samples seeded with biologically treated
effluents, and river waters. Note the use of nitrogen inhibition 6. Quality Control Checks
and the chemical used when reporting results. (NOTE: TCMP is
the preferred nitrification inhibitor but requires handling and a. Minimum residual DO and minimum DO depletion: Only
transfer in a solid form. Allylthiourea is not always effective in bottles, including seed controls, giving a minimum DO depletion
inhibiting nitrification within the 5-d incubation period and con- of 2.0 mg/L and a residual DO of at least 1.0 mg/L after 5 d of
centrations above 2 mg/L may cause increases in carbonaceous incubation are considered to produce valid data, because at least
BOD measurements. ATU concentrations above 2 mg/L also can 2.0 mg oxygen/uptake L is required to give a meaningful mea-
adversely affect the azide modification of the iodometric meth- sure of oxygen uptake and at least 1.0 mg/L must remain
od). Seed all samples to which nitrification inhibitor has been throughout the test to ensure that insufficient DO does not affect
added. The amount of seed should be consistent with that re- the rate of oxidation of waste constituents. Exceptions occur for
quired to achieve GGA test results in the range of 198 30.5 reporting purposes only when the depletions for tests using
mg/L ( 6b). undiluted samples in all bottles fall below 2.0 mg/L and when the
1) Nitrification inhibition using 2-chloro-6-(trichloromethyl) residual DO in all dilutions is less than 1.0 mg/L (see 7). When
pyridine (TCMP)Add 10 mg TCMP/L to diluted sample or 3 using membrane electrodes for measuring DO, make frequent
mg TCMP to each 300-mL bottle or sample dilution vessel, or calibration checks to ensure accurate DO readings (see 4500-
proportional amounts to other sized bottles, after initial sample O.C).
dilution but before final filling of the bottles with dilution water. b. Glucose-glutamic acid check: The glucose-glutamic acid
Do not add TCMP to BOD bottles before they are at least check is the primary basis for establishing accuracy and preci-
two-thirds filled with diluted sample. (NOTE: TCMP dissolves sion of the BOD test and is the principal measure of seed quality
slowly and can float on top of the sample if not mixed well). and set-up procedure. Together with each batch of samples,
5-6 AGGREGATE ORGANIC CONSTITUENTS (5000)

check seed effectiveness and analytical technique by using pro- (D 1 D 2) (S)V s

cedures in 5 to make BOD measurements on an equal weight BOD5, mg/L
P
mixture of glucose and glutamic acid as follows: Add sufficient
amounts of standard glucose-glutamic acid solution ( 3h) to
where:
give 3.0 mg glucose/L and 3.0 mg glutamic acid /L in each of D1 DO of diluted sample immediately after preparation, mg/L,
three test bottles (20 mL GGA solution/L seeded dilution water, D2 DO of diluted sample after 5 d incubation at 20C, mg/L,
or 6.0 mL/300-mL bottle). Commercial solutions may contain S oxygen uptake of seed, DO/mL seed suspension added
other glucose-glutamic acid concentrations; adjust doses accord- per bottle ( 6d) (S 0 if samples are not seeded),
ingly. Add nitrification inhibitor if seed is obtained from a source Vs volume of seed in the respective test bottle, mL, and
that is nitrifying. Evaluate data as described in 8, Precision and P decimal volumetric fraction of sample used; 1/P dilution
Bias. The resulting average BOD for the three bottles, after factor.
correction for dilution and seeding, must fall into the range of
198 30.5 mg/L. If the average value falls outside this range, 2) If DO depletion is less than 2.0 mg/L and sample concen-
evaluate the cause and make appropriate corrections. Consis- tration is 100% (no dilution except for seed, nutrient, mineral,
and buffer solutions), actual seed-corrected, DO depletion may
tently high values can indicate the use of too much seed suspen-
be reported as the BOD even if it is less than 2.0 mg/L.
sion, contaminated dilution water, or the occurrence of nitrifica-
3) When all dilutions result in a residual DO 1.0, select the
tion; consistently low values can indicate poor seed quality or
bottle having the lowest DO concentration (greatest dilution) and
quantity or the presence of a toxic material. If low values persist,
report:
prepare a new mixture of glucose and glutamic acid and check
the sources of dilution water and source of seed.
c. Dilution water quality check: With each batch of samples (D 1 D 2) (S)V s
BOD, mg/L
incubate one or more bottles of dilution water that contains P
nutrient, mineral, and buffer solutions but no seed or nitrification
inhibitor. This dilution water blank serves as a check on quality In the above calculations, do not make corrections for DO
of unseeded dilution water and cleanliness of incubation bottles. uptake by the dilution water blank during incubation. This cor-
Determine initial and final DO as in s 5e and g. The DO uptake rection is unnecessary if dilution water meets the blank criteria
in 5 d must not be more than 0.20 mg/L and preferably not more stipulated in 6c. If the dilution water does not meet these
than 0.10 mg/L, before making seed corrections. If the dilution criteria, proper corrections are difficult; do not record results or,
water blank exceeds 0.20 mg/L, discard all data for tests using as a minimum, mark them as not meeting quality control criteria.
this dilution water or clearly identify such samples in data b. Reporting: Average the test results for all qualified bottles
records. within each dilution series. Report the result as BOD5 if nitrifi-
d. Seed control: Determine BOD of the seed suspension as for cation is not inhibited. Report results as CBOD5 if nitrification is
any other sample. This is the seed control. Ideally, make three inhibited. Samples showing large differences between the com-
dilutions of seed such that the smallest quantity gives at least 2.0 puted BOD for different dilutions, for example, greater than
mg/L DO depletion and the largest quantity results in at least 1.0 30%, may indicate the presence of a toxic substance or analytical
mg/L DO residual after 5 d of incubation. Determine the DO problems. When the effect becomes repetitive, investigate to
uptake per milliliter of seed added to each bottle using either the identify the cause. Identify results in the test reports when any of
slope method or the ratio method. For the slope method, plot DO the following quality control parameters is not met:
depletion in milligrams per liter versus milliliters of seed for all Dilution water blank exceeds 0.20 mg/L ( 6c),
seed control bottles having a 2.0 mg/L depletion and 1.0 mini- Glucose-glutamic acid check falls outside acceptable limits
mum residual DO. The plot should present a straight line for ( 6b),
which the slope indicates DO depletion per milliliter of seed. The Test replicates show more than 30% difference between high
DO-axis intercept is oxygen depletion caused by the dilution and low values,
water and should be less than 0.20 mg/L (see 6c). For the ratio Seed control samples do not meet the above criteria in all
dilutions ( 6d), or
method, divide the DO depletion by the volume of seed in
Minimum DO is less than 1.0 mg/L [ 7a3)].
milliliters for each seed control bottle having a 2.0 mg/L deple-
tion and greater than 1.0 mg/L minimum residual DO and
8. Precision and Bias
average the results. Seed dilutions showing widely varying de-
pletions per milliliter of seed ( 30%) suggest the presence of
There is no measurement for establishing bias of the BOD
toxic substances or large particulates in the seed suspension. In
procedure. The glucose-glutamic acid check prescribed in 6b is
this case, check or change the seed source.
intended to be a reference point for evaluation of dilution water
quality, seed effectiveness, and analytical technique. Single-
laboratory tests using a 300-mg/L mixed glucose-glutamic acid
7. Data Analysis and Reporting
solution provided the following results:
a. Calculations: Number of months: 14
1) For each test bottle having 2.0 mg/L minimum DO deple- Number of triplicates: 421
tion and at least 1.0 mg/L residual DO, calculate BOD as Average monthly recovery: 204 mg/L
Average monthly standard deviation: 10.4 mg/L
follows:
BIOCHEMICAL OXYGEN DEMAND (BOD) (5210)/Ultimate BOD Test 5-7

In a series of interlaboratory studies,2 each involving 2 to 112 The lower detection limit for unseeded samples that require
laboratories (and as many analysts and seed sources), 5-d BOD dilution (S 0; P 1.0) is 2 mg/L multiplied by the dilution
measurements were made on synthetic water samples containing factor as established by the requirement for a minimum DO
a 1:1 mixture of glucose and glutamic acid in the total concen- depletion of 2 mg/L.
tration range of 3.3 to 231 mg/L. The regression equations for The lower limit for seeded samples that require dilution
mean value, X, and standard deviation, S, from these studies (S 0; P 1.0) is approximately 1 mg/L as established by the
were: minimum depletion of 2.0 mg/L minus the maximum seed cor-
rection, which should be less than about 1 mg/L.
X 0.658 (added concentration, mg/L) 0.280 mg/L The lower limit for unseeded samples that require no dilution
(S 0; P 1.0) is equal to the detection limit of the DO
S 0.100 (added concentration, mg/L) 0.547 mg/L measurement method (0.1 mg/L).
The lower detection limit for seeded samples that require no
For the 300-mg/L mixed primary standard, the average 5-d dilution (S 0; P 1.0) is 0 mg/L, as established by the
BOD would be 198 mg/L with a standard deviation of 30.5 difference between the sample DO depletion and the seed cor-
mg/L. When nitrification inhibitors are used, GGA test results rection.
falling outside the 198 30.5 control limit quite often indicate
use of incorrect amounts of seed. Adjust amount of seed added 9. References
to the GGA test to achieve results falling within this range.
a. Control limits: Because of many factors affecting BOD tests 1. YOUNG, J.C. 1973. Chemical methods for nitrification control. J.
in multilaboratory studies and the resulting extreme variability in Water Pollut. Control Fed. 45:637.
test results, one standard deviation, as determined by interlabo- 2. U.S. ENVIRONMENTAL PROTECTION AGENCY, OFFICE OF RESEARCH AND
ratory tests, is recommended as a control limit for individual DEVELOPMENT. 1986. Method-by-Method Statistics from Water Pol-
laboratories. Alternatively, each laboratory may establish its lution (WP) Laboratory Performance Evaluation Studies. Quality
control limits by performing a minimum of 25 glucose-glutamic Assurance Branch, Environmental Monitoring and Support Lab.,
Cincinnati, Ohio.
acid checks ( 6b) over a period of several weeks or months and
calculating the mean and standard deviation. Use the mean 3 10. Bibliography
standard deviations as the control limit for future glucose-glu-
tamic acid checks. Compare calculated control limits to the THERIAULT, E.J., P.D. MCNAMEE & C.T. BUTTERFIELD. 1931. Selection of
single-laboratory tests presented above and to interlaboratory dilution water for use in oxygen demand tests. Pub. Health Rep.
results. If the glucose-glutamic acid test results are outside the 46:1084.
range of 198 30.5, re-evaluate the control limits and investi- LEA, W.L. & M.S. NICHOLS. 1937. Influence of phosphorus and nitrogen
gate source of the problem. If measured BOD for a glucose- on biochemical oxygen demand. Sewage Works J. 9:34.
glutamic acid check is outside the accepted control limit range, RUCHHOFT, C.C. 1941. Report on the cooperative study of dilution waters
reject tests made with that seed and dilution water or identify made for the Standard Methods Committee of the Federation of
such tests clearly in all data records and reports. Sewage Works Associations. Sewage Works J. 13:669.
MOHLMAN, F.W., E. HURWITZ, G.R. BARNETT & H.K. RAMER. 1950.
b. Working range and detection limit: The working range is
Experience with modified methods for BOD. Sewage Ind. Wastes
equal to the difference between the maximum initial DO (7 to 9 22:31.
mg/L) and minimum DO residual of 1 mg/L corrected for seed, YOUNG, J.C., G.N. MCDERMOTT & D. JENKINS . 1981. Alterations in the
and multiplied by the dilution factor. BOD procedure for the 15th edition of Standard Methods for the
Detection limits are established by the minimum DO depletion Examination of Water and Wastewater. J. Water Pollut. Control
and minimum DO residuals as follows: Fed. 53:1253.

5210 C. Ultimate BOD Test

1. General Discussion test. From the DO versus time series, UBOD is calculated by an
appropriate statistical technique. For improved accuracy, run
The ultimate BOD test is an extension of the 5-d dilution BOD tests in triplicate.
test as described in 5210B but with a number of specific test Bottle size and incubation time are flexible to accommodate
requirements and differences in application. The user should be individual sample characteristics and laboratory limitations. In-
familar with the 5210B procedure before conducting tests for cubation temperature, however, is 20C. Most effluents and
UBOD. some naturally occurring surface waters contain materials with
a. Principle: The method consists of placing a single sample oxygen demands exceeding the DO available in air-saturated
dilution in full, airtight bottles and incubating under specified water. Therefore, it is necessary either to dilute the sample or to
conditions for an extended period depending on wastewater, monitor DO frequently to ensure that low DO or anaerobic
effluent, river, or estuary quality.1 Dissolved oxygen (DO) is conditions do not occur. When DO concentrations approach 2
measured (with probes) initially and intermittently during the mg/L, the sample should be reaerated.
5-8 AGGREGATE ORGANIC CONSTITUENTS (5000)

Because bacterial growth requires nutrients such as nitrogen, b. Reservoir bottle: 4-L or larger glass bottle. Close with
phosphorus, and trace metals, the necessary amounts may be screw plastic cap or non-rubber plug.
added to the dilution water together with buffer to ensure that pH c. Incubator or water bath, thermostatically controlled at 20
remains in a range suitable for bacterial growth and seed to 1C. Exclude all light to prevent the possibility of photosynthetic
provide an adequate bacterial population. However, if the result production of DO.
is being used to estimate the rate of oxidation of naturally d. Oxygen-sensitive membrane electrode: See Section 4500-
occurring surface waters, addition of nutrients and seed probably O.G.2.
accelerates the decay rate and produces misleading results. If
only UBOD is desired, it may be advantageous to add supple- 3. Procedure
mental nutrients that accelerate decay and reduce the test dura-
tion. When nutrients are used, they also should be used in the a. River water samples: Preferably fill large BOD bottle (2
dilution water blank. Because of the wide range of water and L, or alternatively 6 or more 300-mL BOD bottles) with sample
wastewater characteristics and varied applications of UBOD at 20C. Add no nutrients, seed, or nitrification inhibitor if
data, no specific nutrient or buffer formulations are included. in-bottle decay rates will be used to estimate in-stream rates. Do
The extent of oxidation of nitrogenous compounds during the not dilute sample unless it is known by pretesting or by experi-
prescribed incubation period depends on the presence of micro- ence to have a high ultimate BOD (20 mg/L).
organisms capable of carrying out this oxidation. Such organ- Measure DO in each bottle, stopper, and make an airtight seal.
isms may not be present in wastewaters in sufficient numbers to Incubate at 20C in the dark.
oxidize significant quantities of reduced nitrogen. This situation Measure DO in each bottle at intervals of at least 2 to 5 d over
may be reversed in naturally occurring surface waters. Erratic a period of 30 to 60 d (minimum of 6 to 8 readings) or longer
results may be obtained when a nitrification inhibitor is used;2 under special circumstances. To avoid oxygen depletion in sam-
therefore, the specified method precludes use of a nitrogen ples containing NH3-N, measure DO more frequently until ni-
inhibitor unless prior experimental evidence on the particular trification has taken place. If DO falls to about 2 mg/L, reaerate
sample suggests that it is acceptable.* Monitor NO2-N and as directed below. Replace sample lost by the cap and DO probe
NO3-N to compute the oxygen equivalency of the nitrification displacement by adding 1 to 2 mL sample from the reservoir
reaction. When these values are subtracted from the DO vs. time bottle.
series, the carbonaceous BOD time series can be constructed.3 When DO approaches 2 mg/L, reaerate. Pour a small amount
b. Sampling and storage: See Section 5210B.4a. of sample into a clean vessel and reaerate the remainder directly
in the bottle by vigorous shaking or bubbling with purified air
2. Apparatus (medical grade). Refill bottle from the storage reservoir and
measure DO. This concentration becomes the initial DO for the
a. Incubation bottles: Glass bottles with ground-glass stop- next measurement. If using 300-mL BOD bottles, pour all of the
pers, 2-L (or larger) capacity. Glass serum bottles of 4- to 10-L sample from the several bottles used into a clean vessel, reaerate,
capacity are available. Alternatively use nonground-glass bottles and refill the small bottles.
with nonbiodegradable plastic caps as a plug insert. Do not reuse Analyze for nitrate plus nitrite nitrogen (NO3-N NO2-N)
the plugs because discoloration occurs with continued use. Re- (see Sections 4500-NO2 and 4500-NO3) on Days 0, 5, 10, 15,
place plugs every 7 to 14 d. Do not use rubber stoppers that may 20, and 30. Alternatively, determine NO2-N and NO3-N each
exert an oxygen demand. Clean bottles with a detergent and time DO is determined, thereby producing corresponding BOD
wash with dilute HCl (3N) to remove surface films and precip- and nitrogen determinations. If the ultimate demand occurs at a
itated inorganic salts; rinse thoroughly with DI water before use. time greater than 30 d, make additional analyses at 30-d inter-
Cover top of bottles with paper after rinsing to prevent dust from vals. Remove 10 to 20 mL from the bottle for these analyses.
collecting. To prevent drawing air into the sample bottle during Refill bottle as necessary from the reservoir bottle. Preserve
incubation, use a water seal. If the bottle does not have a flared NO2-N NO3-N subsample with H2SO4 to pH 2 and
mouth, construct a water seal by making a watertight dam around refrigerate. If the purpose of the UBOD test is to assess the
the stopper (or plug) and fill with water from the reservoir as UBOD and not to provide data for rate calculations, measure
necessary. Cover dam with clean aluminum foil to retard evap- nitrate nitrogen concentration only at Day 0 and on the last day
oration. If a 2-L BOD bottle is used, fill reservoir with sample of the test (kinetic rate estimates are not useful when the nitri-
and cover with a polyethylene cap before incubation. fication reaction is not followed).
Place a clean magnetic stirring bar in each bottle to mix Calculate oxygen consumption during each time interval and
contents before making DO measurement or taking a subsample. make appropriate corrections for nitrogenous oxygen demand.
Do not remove the magnets until the test is complete. Correct by using 3.43 the NH3-N to NO2-N conversion plus
Alternatively use a series of 300-mL BOD bottles as described 1.14 the NO2-N to NO3-N conversion to reflect the stoi-
in 5210B, if larger bottles are not available or incubation space chiometry of the oxidation of NH4 to NO2 or NO3.
is limited. 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 1 mg DO/L
* Some analysts have reported satisfactory results with 2-chloro-6-(trichloro- in a 30- to 90-d period. If DO uptake of the dilution water is greater
methyl) pyridine (Nitrification Inhibitor, Formula 2533, Hach Co., Loveland, CO, than 0.5 mg/L for a 20-d period, or 1 mg/L for a 90-d period, report
or equivalent).
Wheaton 2-L BOD bottle No. 227580, 1000 North Tenth St., Millville, NJ, or the magnitude of the correction and try to obtain higher-quality
equivalent. dilution water for use with subsequent UBOD tests.
BIOCHEMICAL OXYGEN DEMAND (BOD) (5210)/Ultimate BOD Test 5-9

TABLE 5210:I. UBOD RESULTS FOR WASTEWATER SAMPLE

(1) (2) (3) (4) (5) (6)
Accumulated
Average Average DO Consumed Average
DO* Blank DO by Sample NO3-N NBOD CBOD
Day mg/L mg/L mg/L mg/L mg/L mg/L

0 8.1 0 0.0 0 0
3 5.6 2.5 0 2.5
5 3.5/8.0 4.6 0.0 0 4.6
7 6.2 6.4 0.23 6.2
10 3.2/8.2 9.4 0.10 0.46 8.9
15 4.3 13.3 0.58 12.7
18 2.7/8.1 14.9 0.15 0.69 14.2
20 6.6 16.4 0.80 15.6
25 5.4 17.6 0.20 0.92 16.7
30 2.6/8.2 20.4 0.92 19.5
40 5.3 23.3 0.20 0.92 22.4
50 3.1/8.0 25.5 0.92 24.6
60 4.5 29.0 0.92 28.1
70 3.3/8.1 30.2 0.92 29.3
90 5.4 32.9 0.20 0.92 32.0
* Two readings indicate concentrations before and after reaeration.
None was used.
Column (1) blank correction (none needed in the example).
Column (4) 4.57 (linear interpolation between values).
[Column (3)Column (5)] dilution factor.

Ultimate CBOD 34.5 mg/L; CBOD decay rate 0.03/d (calculated with first-order equation from 5210C.4).

When the weekly DO consumption drops below 1 to 2% of the

total accumulative consumption, calculate the ultimate BOD The data in Table 5210:I were analyzed with a nonlinear
using a nonlinear regression method. regression technique applied to the above first-order model.4
b. Wastewater treatment plant samples: Use high-quality re- However, a first-order kinetic model may not always be the best
agent water (see Section 1080) for dilution water. Add no nitri- choice. Significantly better statistical fits usually are obtained
fication inhibitors if decay rates are desired. If seed and nutrients with alternative kinetic models including sum of two first-order
are necessary, add the same amounts of each to the dilution water and logistic function models.1,3 8
blank. Use minimal sample dilution. As a rule of thumb, the
ultimate BOD of the diluted sample should be in the range of 20 5. Precision and Bias
to 30 mg/L. Dilution to this level probably will require two or
three sample reaerations during the incubation period to avoid The precision of the ultimate BOD test was assessed with a
having dissolved oxygen concentrations fall below 2 mg/L. series of replicate tests in a single laboratory. Interlaboratory
Use 2-L or larger BOD bottles (alternatively, multiple 300-mL studies have not been conducted.
BOD bottles) for each dilution. Add desired volume of sample to
each bottle and fill with dilution water. UBOD
Fill a BOD bottle with dilution water to serve as a dilution water Reference Replicate No. mg/L Precision Summary*
blank. Treat blank the same as all samples. Follow procedure given 2 1 154 151 mg/L
in 3a and incubate for at least as long as UBOD test. 2 154
3 145 CV 3.5%
4. Calculations 5 1 10.3
2 11.1
An example of results obtained for a wastewater sample, 3 9.6 10.0 mg/L
undiluted, without seed and nutrients, is given in Table 5210:I. 4 9.9
5 9.8 CV 5.8%
UBOD can be estimated by using a first-order model described
6 9.6
as follows: 6 1 12.8 12.4 mg/L
kt 2 12.6
BOD t UBOD (1 e )
3 12.6 CV 4.4%
where: 4 11.6
BODt oxygen uptake measured at time t, mg/L, and * mean,
k first-order oxygen uptake rate. CV coefficient of variation.
5-10 AGGREGATE ORGANIC CONSTITUENTS (5000)

Bias was assessed by determining the BOD of a known ceous and Nitrogenous BOD in Long-Term BOD Measurements.
concentration of glucose (150 mg/L) and glutamic acid (150 Tech. Bull. No. 461, New York, N.Y.
mg/L). This solution has a UBOD of 321 mg/L to 308 mg/L, 3. NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM
depending on extent of nitrification. The results of the study IMPROVEMENT, INC. 1982. A Review of Ultimate BOD and Its Kinetic
conducted in triplicate were:1 Formulation for Pulp and Paper Mill Effluents. Tech. Bull. No. 382,
New York, N.Y.
4. BARNWELL, T. 1980. Least Squares Estimates of BOD Parameters. J.
Estimated* Theoretical Environ. Eng. Div., Proc. Amer. Soc. Civil Eng. 107(EE6):1197.
UBOD BOD Percent 5. NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM
mg/L mg/L Difference IMPROVEMENT, INC. 1982. A Proposal to Examine the Effect of Mixing
on Long Term BOD Test. NE82-01, New York, N.Y.
276 308/321 10/14
310 308/321 1/3 6. NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM
303 308/321 2/6 IMPROVEMENT, INC. 1982. A Study of the Selection, Calibration, and
Verification of Mathematical Water Quality Models. Tech. Bull. No.
* By statistical model.
367, New York, N.Y.
6. References 7. NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM
IMPROVEMENT, INC. 1987. Users Manual for Parameter Estimation for
1. MARTONE, C.H. 1976. Studies Related to the Determination of Bio- First Order Ultimate BOD Decay, BODFO. Tech. Bull. No. 529,
degradability and Long Term BOD. M.S. thesis, Dep. Civil Engi- New York, N.Y.
neering, Tufts Univ., Medford, Mass. 8. CHU, W.S. & E.W. STRECKER. 1972. Parameter Identification In Water
2. NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM System Model. Dep. Civil Engineering, Univ. Washington, Seattle.
IMPROVEMENT, INC. 1986. A Review of the Separation of Carbona-

5210 D. Respirometric Method

1. General Discussion consumption while maintaining a constant volume. Volumetric

respirometers measure oxygen uptake in incremental changes in
a. Principle: Respirometric methods provide direct measure- gas volume while maintaining a constant pressure at the time of
ment of the oxygen consumed by microorganisms from an air or reading. Electrolytic respirometers monitor the amount of oxy-
oxygen-enriched environment in a closed vessel under condi- gen produced by electrolysis of water to maintain a constant
tions of constant temperature and agitation. oxygen pressure within the reaction vessel. Direct-input respi-
b. Uses: Respirometry measures oxygen uptake more or less rometers deliver oxygen to the sample from a pure oxygen
continuously over time. Respirometric methods are useful for supply through metering on demand as detected by minute
assessing: biodegradation of specific chemicals; treatability of pressure differences. Most respirometers have been instrumented
organic industrial wastes; the effect of known amounts of toxic to permit data collection and processing by computer. Reaction-
compounds on the oxygen-uptake reaction of a test wastewater vessel contents are mixed by using a magnetic or mechanical
or organic chemical; the concentration at which a pollutant or a stirring device or by bubbling the gaseous phase within the
wastewater measurably inhibits biological degradation; the ef- reaction vessel through the liquid phase. All respirometers re-
fect of various treatments such as disinfection, nutrient addition, move carbon dioxide produced during biological growth by
and pH adjustment on oxidation rates; the oxygen requirement suspending a concentrated adsorbent (granular or solution)
for essentially complete oxidation of biologically oxidizable within the closed reaction chamber or by recirculating the gas
matter; the need for using adapted seed in other biochemical phase through an external scrubber.
oxygen-uptake measurements, such as the dilution BOD test; and d. Interferences: Evolution of gases other than CO2 may
stability of sludges. introduce errors in pressure or volume measurements; this is
Respirometric data typically will be used comparatively, that uncommon in the presence of dissolved oxygen. Incomplete CO2
is, in a direct comparison between oxygen uptakes from two test absorption will introduce errors if appropriate amounts and con-
samples or from a test sample and a control. Because of inherent centrations of alkaline absorbent are not used. Temperature
differences among uses, among seed cultures, among applica- fluctuations or inadequate mixing will introduce error. Fluctua-
tions of results, and among instruments, a single procedure for tions in barometric pressure can cause errors with some respi-
respirometric tests applicable to all cases cannot be defined. rometers. Become familiar with the limits of the instrument to be
Therefore, only basic recommendations and guidelines for over- used.
all test setup and procedure are given. Follow manufacturers e. Minimum detectable concentration: Most commercial re-
instructions for operating details for specific commercial instru- spirometers can detect oxygen demand in increments as small as
ments. 0.1 mg but test precision depends on the total amount of oxygen
c. Types of respirometers: Four principal types of commercial consumed at the time of reading, the precision of pressure or
respirometers are available. Manometric respirometers relate volume measurement, and the effect of temperature and baro-
oxygen uptake to the change in pressure caused by oxygen metric pressure changes. Upper limits of oxygen uptake rate are
BIOCHEMICAL OXYGEN DEMAND (BOD) (5210)/Respirometric Method 5-11

determined by the ability to transfer oxygen into the solution 3. Reagents

from the gas phase, which typically is related to mixing intensity.
Transfer limits typically range from less than 10 mg O2/L/h for Formulations of reagent solutions are given for 1-L volumes,
low-intensity mixing to above 100 mg O2/L/h for high-intensity but smaller or larger volumes may be prepared according to
mixing. need. Discard any reagent showing signs of biological growth or
f. Relationship to dilution BOD: Variations in waste compo- chemical precipitation. Stock solutions can be sterilized by au-
sition, substrate concentration, mixing, and oxygen concentra- toclaving to provide longer shelf life.
tions from one wastewater source to another generally preclude a. Distilled water: Use only high-quality water distilled from
use of a general relationship between oxygen uptake by respi- a block tin or all-glass still (see Section 1080). Deionized water
rometers and the 5-d, 20C, BOD (see 5210B, above). Reason- may be used but often contains high bacterial counts. The water
ably accurate correlations may be possible for a specific waste- must contain less than 0.01 mg heavy metals/L and be free of
water. The incubation period for respirometric measurements
chlorine, chloramines, caustic alkalinity, organic material, or
need not be 5 d because equally valid correlations can be made
acids. Make all reagents with this water. When other waters are
between the 5-d dilution BOD and respirometric oxygen uptake
required for special-purpose testing, state clearly their source and
at any time after 2 d.1,2 The point of common dilution and
respirometric BOD seems to occur at about 2 to 3 d incubation quality characteristics.
for municipal wastewaters. Correlations between respirometric b. Phosphate buffer solution, 1.5N: Dissolve 207 g sodium
measurements and 5-d BOD for industrial wastes and specific dihydrogen phosphate, NaH2PO4 H2O, in water. Neutralize to
chemicals are less certain. Respirometric measurements also can pH 7.2 with 6N KOH ( 3g below) and dilute to 1 L.
provide an indication of the ultimate biochemical oxygen de- c. Ammonium chloride solution, 0.71N: Dissolve 38.2 g am-
mand (UBOD) (see Section 5210C). In many cases, it is reason- monium chloride, NH4Cl, in water. Neutralize to pH 7.0 with
able to consider that the 28- to 30-d oxygen uptake is essentially KOH. Dilute to 1.0 L; 1 mL 10 mg N.
equal to the UBOD.3 d. Calcium chloride solution, 0.25N: Dissolve 27.7 g CaCl2 in
More commonly, respirometers are used as a diagnostic tool. water and dilute to 1 L; 1 mL 10 mg Ca.
The continuous readout of oxygen consumption in respirometric e. Magnesium sulfate solution, 0.41N: Dissolve 101 g
measurements indicates lag, toxicity, or any abnormalities in the MgSO4 7H2O in water and dilute to 1 L; 1 mL 10 mg Mg.
biodegradation reaction. The change in the normal shape of an f. Ferric chloride solution, 0.018N: Dissolve 4.84 g
oxygen-uptake curve in the first few hours may help to identify FeCl3 6H2O in water and dilute to 1 L; 1 mL 1.0 mg Fe.
the effect of toxic or unusual wastes entering a treatment plant in g. Potassium hydroxide solution, 6N: Dissolve 336 g KOH in
time to make operating corrections. about 700 mL water and dilute to 1 L. CAUTION: Add KOH to
g. Relationship to other test methods and protocols: This water slowly and use constant mixing to prevent excessive heat
method supports most of the protocols and guidelines established buildup. Alternately, use commercial solutions containing 30 to
by the European Organization for Economic Co-operation and 50% KOH by weight.
Development3 (OECD) that require measurement of oxygen h. Acid solutions, 1N: Add 28 mL conc H2SO4 or 83 mL conc
uptake.
HCl to about 700 mL water. Dilute to 1 L.
h. Sampling and storage:
i. Alkali solution, 1N: Add 40 g NaOH to 700 mL water.
1) Grab samplesIf analysis is begun within 2 h of sample
Dilute to 1 L.
collection, cold storage is unnecessary. Otherwise, keep sample
at or below 4C from the time of collection. Begin analysis j. Nitrification inhibitor: Reagent-grade 2-chloro-6-(trichlo-
within 6 h of collection; when this is not possible, store at or romethyl) pyridine (TCMP) or equivalent.3*
below 4C and report length and temperature of storage. Never k. Glucose-glutamic acid solution: Dry reagent-grade glucose
start analysis more than 24 h after grab sample collection. and reagent-grade glutamic acid at 103C for 1 h. Add 15.0 g
2) Composite samplesKeep samples at or below 4C during glucose and 15.0 g glutamic acid to distilled water and dilute to
compositing. Limit compositing period to 24 h. Use the same 1 L. Neutralize to pH 7.0 using 6N potassium hydroxide ( 3g).
criteria as for storage of grab samples, starting the measurement This solution may be stored for up to 1 week at 4C.
of holding time from the end of the compositing period. State l. Electrolyte solution (for electrolytic respirometers): Use
storage time and conditions with results. manufacturers recommended solution.
m. Sodium sulfite solution, 0.025N: Dissolve 1.575 g Na2SO3
in about 800 mL water. Dilute to 1 L. This solution is not stable;
2. Apparatus prepare daily or as needed.
n. Trace element solution: Dissolve 40 mg MnSO4 4H2O, 57
a. Respirometer system: Use commercial apparatus and check mg H3BO3, 43 mg ZnSO4 7H2O, 35 mg (NH4)6 Mo7O24, and
manufacturers instructions for specific system requirements, 100 mg Fe-chelate (FeCl3-EDTA) in about 800 mL water. Dilute
reaction vessel type and volume, and instrument operating char- to 1 L. Sterilize at 120C and 200 kPa (2 atm) pressure for 20
acteristics. min.
b. Incubator or water bath: Use a constant-temperature room,
incubator chamber, or water bath to control temperature to
1C. Exclude all light to prevent oxygen formation by algae in
* Formula 2533, Hach Chemical Co., Loveland, CO, or equivalent. NOTE: Some
the sample. Use red, actinic-coated bottles for analysis outside of commercial formulations are not pure TCMP. Check with supplier to verify
a darkened incubator. compound purity and adjust dosages accordingly.
5-12 AGGREGATE ORGANIC CONSTITUENTS (5000)

o. Yeast extract solution:3 Add 15 mg laboratory- or pharma- pure oxygen may be added to respirometer vessels to increase
ceutical-grade brewers yeast extract to 100 mL water. Make this oxygen levels above ambient.
solution fresh immediately before each test in which it is used. 6) Temperature adjustmentBring samples and dilution water
p. Nutrient solution:3 Add 2.5 mL phosphate buffer solution to desired test temperature (1C) before making dilutions or
(3b), 0.65 mL ammonium chloride solution (3c), 1.0 mL calcium transferring to test vessels.
chloride solution (3d), 0.22 mL magnesium sulfate solution (3e), e. Sample dilution: Use distilled water or water from other
0.1 mL ferric chloride solution (3f), 1 mL trace element solution appropriate sources free of organic matter. In some cases, re-
(3n), and 1 mL yeast extract solution (3o) to about 900 mL ceiving stream water may be used for dilution. Add desired
water. Dilute to 1 L. This nutrient solution and those of s n and sample volume to test vessels using a wide-tip volumetric pipet
o above are specifically formulated for use with the OECD or other suitable volumetric glassware. Add dilution water to
method.3 (NOTE: A 10:1 concentrated nutrient solution can be bring sample to about 80% of desired final volume. Add appro-
made and diluted accordingly.) priate amounts of nutrients, minerals, buffer, nitrification inhib-
itor if desired, and seed culture as described in s 4f and h below.
Dilute sample to desired final volume. The number of test vessels
4. Procedure to prepare for each dilution depends on test objectives and
number of replicates desired.
a. Instrument operation: Follow respirometer manufacturers f. Nutrients, minerals, and buffer: Add sufficient ammonia
instructions for assembly, testing, calibration, and operation of nitrogen to provide a COD:N:P ratio of 100:5:1 or a TOC:N:P
the instrument. NOTE: The manufacturers stated maximum and ratio of 30:5:1. Add 2 mL each of calcium, magnesium, ferric
minimum limits of measurement are not always the same as the chloride, and trace mineral solutions to each liter of diluted
instrument output limits. Make sure that test conditions are sample unless sufficient amounts of these minerals are present in
within the limits of measurement. the original sample. Phosphorus requirements will be met by the
b. Sample volume: Sample volume or concentration of organic phosphate buffer if it is used (1 mL/50 mg/L COD or ultimate
chemicals to be added to test vessels is a function of expected BOD of diluted sample usually is sufficient to maintain pH
oxygen uptake characteristics and oxygen transfer capability of between 6.8 and 7.2). Be cautious in adding phosphate buffer to
the instrument. Small volumes or low concentrations may be samples containing metal salts because metal phosphates may
required for high-strength wastes. Large volumes may be re- precipitate and show less toxic or beneficial effect than when
quired for low-strength wastes to improve accuracy. phosphate is not present. For OECD-compatible tests, substitute
c. Data recording interval: Set instrument to give data read- the nutrient, mineral, and buffer amounts listed in 3p for the
ings at suitable intervals. Intervals of 15 min to 6 h typically are above nutrient/mineral/buffer quantities.
used. g. Nitrification inhibition: If nitrification inhibition is desired,
d. Sample preparation: add 10 mg 2-chloro-6-(trichloromethyl) pyridine (TCMP)/L
1) HomogenizationIf sample contains large settleable or sample in the test vessel. Samples that may nitrify readily in-
floatable solids, homogenize it with a blender and transfer rep- clude biologically treated effluents, samples seeded with biolog-
resentative test portions while all solids are in suspension. If ically treated effluents, and river waters.4
there is a concern for changing sample characteristics, skip this h. Seeding: See 5210B.4d for seed preparation. Use sufficient
step. amounts of seed culture to prevent major lags in the oxygen
2) pH adjustmentNeutralize samples to pH 7.0 with H2SO4 uptake reaction but not so much that the oxygen uptake of the
or NaOH of such strength (s 3h and i) that reagent quantity does seed exceeds about 10% of the oxygen uptake of the seeded
not dilute the sample more than 0.5%. sample.
3) DechlorinationAvoid analyzing samples containing re- Determine the oxygen uptake of the seeding material as for
sidual chlorine by collecting the samples ahead of chlorination any other sample. This is the seed control. Typically, the seed
processes. If residual chlorine is present, aerate as described in volume in the seed control should be 10 times the volume used
5) below or let stand in light for 1 to 2 h. If a chlorine residual in seeded samples.
persists, add Na2SO3 solution. Determine required volume of i. Incubation: Incubate samples at 20C or other suitable
Na2SO3 solution by adding 10 mL 1 1 acetic acid or 1 50 temperature 1.0C. Take care that the stirring device does not
H2SO4 and 10 mL potassium iodide solution (10 g/100 mL) to a increase the temperature of the sample.
portion of the sample. Titrate with 0.025N Na2SO3 solution to
the starch-iodine end point (see Section 4500-Cl.B). Add to the 5. Calculations
neutralized sample a proportional volume of Na2SO3 solution
determined above, mix, and after 10 to 20 min check for residual To convert instrument readings to oxygen uptake, refer to
chlorine. Re-seed the sample (see 4h below). manufacturers procedures.
4) Samples containing toxic substancesCertain industrial Correct oxygen uptake for seed and dilution by the following
wastes contain toxic metals or organic compounds. These often equation:
require special study and treatment.3
5) Initial oxygen concentrationIf samples contain dissolved C [A B(S A/S B)](1000/N A)
oxygen concentrations above or below the desired concentration,
agitate or aerate with clean and filtered compressed air for about where:
1 h immediately before testing. Minimum and maximum actual C corrected oxygen uptake of sample, mg/L,
DO concentrations will vary with test objectives. In some cases, A measured oxygen uptake in seeded sample, mg,
BIOCHEMICAL OXYGEN DEMAND (BOD) (5210)/Respirometric Method 5-13

B measured oxygen uptake in seed control, mg, ficient of variation, Cv, is approximately 5% for samples having
SA volume of seed in Sample A, mL, total oxygen uptakes of 50 to 100 mg/L and 3% for more
SB volume of seed in Sample B, mL, and concentrated samples.1,2 Individual instruments have different
NA volume of undiluted sample in Sample A, mL. readability limits that can affect precision. The minimum re-
sponse or sensitivity of most commercial respirometers ranges
6. Quality Control from 0.05 to 1 mg oxygen. Check manufacturers specifications
for sensitivity of the instrument at hand.
Periodically use the following procedure to check distilled b. Control limits: To establish laboratory control limits, per-
water quality, instrument quality, instrument function, and ana- form a minimum of 25 glucose-glutamic acid checks over a
lytical technique by making oxygen uptake measurements using period of several weeks or months and calculate mean and
a mixture of glucose and glutamic acid as a standard check standard deviation. If measured oxygen uptake in 5 d at 20C is
solution. outside the 260 30 mg/L range, re-evaluate procedure to
Adjust water for sample formulation to test temperature and identify source of error. For other samples, use the mean 3
saturate with DO by aerating with clean, organic-free filtered air. standard deviations as the control limit.
Protect water quality by using clean glassware, tubing, and c. Working range and detection limits: The working range and
bottles. detection limits are established by the limits of each commercial
Prepare a test solution by adding 10 mL glucose-glutamic acid instrument. Refer to manufacturers specifications.
solution (3k); 6 mL phosphate buffer (3b); 2 mL each of ammo-
nium chloride (3c), magnesium sulfate (3e), calcium chloride 8. References
(3d), ferric chloride (3f), and trace element solution (3n) to
approximately 800 mL water. Add 10 mg nitrification inhibitor 1. YOUNG, J.C. & E.R. BAUMANN. 1976. The electrolytic respirometer
(TCMP)/L. Add sufficient seed from a suitable source as de- Factors affecting oxygen uptake measurements. Water Res. 10:1031.
2. YOUNG, J.C. & E.R. BAUMANN. 1976. The electrolytic respirometer
scribed in 4h to give a lag time less than 6 h (usually 25 mL
Use in water pollution control plant laboratories. Water Res. 10:1141.
supernatant from settled primary effluent/L test solution is suf- 3. ORGANIZATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT. 1981.
ficient). Dilute to 1 L. Adjust temperature to 20 1C. Method 5.2, Annex V, Part C in OECD Guidelines for Testing of
Prepare a seed blank by diluting 500 mL or more of the seed Chemicals. OECD, Paris, France.
solution to 800 mL with distilled water. Add the same amount of 4. YOUNG, J.C. 1973. Chemical methods for nitrification control. J.
buffer, nutrients, and TCMP as in the test solution, and dilute to Water Poll. Control Fed. 45:637.
1 L. Adjust temperature to 20 1C.
Place test solution and seed blank solution in separate reaction 9. Bibliography
vessels of respirometer and incubate for 5 d at 20C. Run at least
three replicates of each. The seed-corrected oxygen uptake after HEUKELEKIAN, H. 1947. Use of direct method of oxygen utilization in
5 d incubation should be 260 30 mg/L. If the value of the waste treatment studies. Sew. Works J. 19:375.
check is outside this range, repeat the test using a fresh seed CALDWELL, D.H. & W.F. LANGELIER. 1948. Manometric measurement of
culture and seek the cause of the problem. the biochemical oxygen demand of sewage. Sew. Works J. 20:202.
GELLMAN, I. & H. HEUKELEKIAN. 1951. Studies of biochemical oxidation
7. Precision and Bias by direct methods. Sew. Ind. Wastes. 23:1267.
JENKINS, D. 1960. The use of manometric methods in the study of sewage
and trade wastes. In Waste Treatment, p. 99. Pergamon Press,
a. Precision: No standard is available to check the accuracy of
Oxford, U.K.
respirometric oxygen uptake measurements. To obtain labora- MONTGOMERY, H.A.C. 1967. The determination of biochemical oxygen
tory precision data, use a glucose-glutamic acid mixture ( 6 demand by respirometric methods. Water Res. 1:631.
above) having a known theoretical maximum oxygen uptake CEDENA, F., A. DROHOBYCZAR, M.I. BEACH & D. BARNES. 1988. A novel
value. Tests with this and similar organic compound mixtures approach to simplified respirometric oxygen demand determina-
have shown that the standard deviation, expressed as the coef- tions. Proc. 43rd Ind. Waste Conf., Purdue Univ., Lafayette, Ind.