biological oxygen demand test by apha

8/11/2019 Biological Oxygen Demand Test by APHA

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BIOCHEMICAL OXYEGEN DEMAND(BOD)

GAYATHRI S MOHAN

CPET 712


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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) unlesstheir 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.


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IntroductionInterference: 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 UBODmeasures 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 (CBOD5) when

inhibiting the nitrogenous oxygen demand. When nitrification is not inhibited,

report results as BOD5


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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, andtrace 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, orreceiving-stream water free of biodegradable organics and bioinhibitory

substances such as chlorine or heavy metals.


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5-Day BOD TestPrinciple

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.


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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.


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Reagentsa. 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


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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.


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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


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Procedure

d. Seeding

Seed source: Some samples do not contain a sufficient microbial population (forexample, some untreated industrial wastes, disinfected wastes, high-temperaturewastes, or wastes with extreme pH values). For such wastes seed the dilutionwater or sample by adding a population of microorganisms.

The preferred seed is effluent or mixed liquor from a biological treatment systemprocessing the waste.

Where such seed is not available, use supernatant from domestic wastewater aftersettling 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 themicroorganisms. In such cases develop an adapted seed in the laboratory bycontinuously aerating a sample of settled domestic wastewater and adding smalldaily increments of waste.


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ProcedureSeed 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


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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

Na2SO

3.

Determine required volume of Na2SO3 solution on a 100- to1000-mL portion of

neutralized sample by adding 10mL of 1 + 1 acetic acid or 1 + 50 H2SO4, 10 mL

potassium iodide (KI) solution (10 g/100 mL) per 1000 mL portion, and titrating

with Na2SO3solution 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 addsufficient amounts to the dilution water to make a final concentration. Note the

use of nitrogen inhibition in reporting results.


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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 atleast 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.


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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 BODbottles

Add appropriate amounts of seed material either to the individual BOD bottles orto 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.


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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 preparingdilution 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 ofunseeded 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/Land preferably not more than 0.1 mg/L

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

Determination of final DO: After 5 d incubation determine DO in sample dilutions,

Blanks.


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Methods to find DOTwo 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 ofmolecular oxygen across a membrane

The choice of procedure depends on the interferences present, the

accuracy desired, and, in some cases, convenience or expedience.


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Iodometric MethodsPrinciple

It is based on the addition of divalent manganese solution, followed by strongalkali, 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 (I2)equivalent to theoriginal DO content

The released I2 can then be titrated with standard solution of sodium thiosulfate

(Na2S2O3) 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).


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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.


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Azide ModificationReagents

a. Manganous sulfate solutionb. 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 H2SO4+ 20.00 mL standard bi-iodate solution

Dilute to 200 mL

Titrate the liberated iodine against Na2S2O3Starch indicator, pale straw color end point


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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 H2SO4

Restopper and mix by inverting several times until dissolution is complete.

Titrate a volume corresponding to 200mL original sample after correction forsample loss by displacement with reagents. Thus, for a total of 2mL (1 mL each) ofMnSO4 and alkali-iodide-azide reagents in a 300-mL bottle, titrate

200 300/(300 2) = 201 mL.

b. Titrate with 0.025M Na2S2O3solution to a pale straw color. Add a few drops ofstarch solution and continue titration to first disappearance of blue color.


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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


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

0.70 mL conc H2SO4+ 1 mL KMnO4solution + 1 mL KF solution

Stopper and mix by inverting

Remove permanganate color completely by adding 0.5 to 1.0 mL K2C2O4solution

Mix well and allow to stand in the dark to facilitate

the reaction.

1 ml of MnSO4+ 1 mL alkali-iodide-azide reagent

Stopper, mix, and let precipitate settle for short time

Acidify with 2 ml H2SO4

take 200 300/(300 7.7) = 205 mL for titration


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Alum Flocculation Modification

The interference due to solidsmay 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. Starchg. Standard sodium thiosulfate titrant

All the reagents required for the azide modification


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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.

C S lf t S lf i A id


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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

ProcedureAdd 10 mL CuSO4-NH2SO2OH inhibitor to a 1-L glass-stoppered bottle.

Insert bottle in a special sampler designed so that bottle fills from a tube near bottomand 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


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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 asthey 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.


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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 asample 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


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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 t0)

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


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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:

D1= DO of diluted sample immediately after preparation, mg/L,

D2= DO of diluted sample after 5 d incubation at 20C, mg/L,

P= decimal volumetric fraction of sample used


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CalculationBOD (mg/L)= [(D1D2)f(B1B2)]/P

where:

D1= DO of diluted sample immediately after preparation, mg/L,D2= DO of diluted sample after 5 d incubation at 20C, mg/L,

P= decimal volumetric fraction of sample used,

B1= DO of seed control before incubation, mg/L,

B2= 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)


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Ultimate BOD Test

An extension of 5-day BOD test.

Principle

Placing a single sample dilution in full, airtight bottles and incubating underspecified conditions for an extended period depending on wastewatereffluent, river, or estuary quality.

Dissolved oxygen (DO) is measured (with probes) initially and intermittentlyduring 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 naturallyoccurring surface waters, addition of nutrients and seed probably acceleratesthe decay rate and produces misleading results.

If only UBOD is desired, it may be advantageous to add supplemental nutrientsthat accelerate decay and reduce the test duration. When nutrients are used,they also should be used in the dilution water blank.


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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 ornon-rubber plug.

Incubator or water bath, thermostatically controlled at 20 1C. Exclude

all light to prevent the possibility of photosynthetic production of DO.


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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 specialcircumstances.

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 theremainder 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 smallbottles.


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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 theultimate demand occurs at a time greater than 30 d, make additionalanalyses 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 atDay 0 and on the last day of the test.

When using a dilution water blank, subtract DO uptake of the blank fromthe total DO consumed.

High-quality reagent water without nutrients typically will consume amaximum 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-dperiod, report the magnitude of the correction and try to obtain higher-quality dilution water for use with subsequent UBOD tests.


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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.


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Calculations

UBOD can be estimated by using a first-order model described

as follows:

BODt= UBOD(1 e-Kt)

where:

BODt= oxygen uptake measured at time t, mg/L, and

k= first-order oxygen uptake rate.


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Respirometric MethodRespirometric 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 ofbiologically 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 sampleand a control.


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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 changesin 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 vesselthrough 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.


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Interferences Evolution of gases other than CO2may introduce errors in pressure or volume

measurements; this is uncommon in the presence of dissolved oxygen. Incomplete CO2absorption 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 smallas 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.


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Apparatus

Respirometer system

Incubator or water bath

Reagentsa. Distilled water

b. Phosphate buffer solution

c. Ammonium chloride solution

d. Calcium chloride solutione. Magnesium sulfate solution,

f. Ferric chloride solution


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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


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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 H2SO4or 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 Na2SO3solution.


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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 freeof 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).


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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.


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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(SA/SB)](1000/NA)

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,

SA= volume of seed in Sample A, mL,

SB = volume of seed in Sample B, mL, andNA= volume of undiluted sample in Sample A, mL.


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Reference

APHA manual for water and waste water

analysis, 4000- 6000


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biological oxygen demand test by apha

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