BIOCHEMICAL OXYEGEN DEMAND (BOD)

GAYATHRI S MOHANCPET 712

BIOCHEMICAL OXYGEN DEMAND

• The biochemical oxygen demand (BOD) determination is an empirical test in which standardized laboratory procedures are used to determine the relative oxygen requirements of wastewaters, effluents, and polluted waters.

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

The test measures the

Oxygen utilized during a specified incubation period for the biochemical degradation of organic material (carbonaceous demand)

Oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless their oxidation is prevented by an inhibitor.

Oxygen used to oxidize inorganic material such as sulfides and ferrous iron. The seeding and dilution procedures provide an estimate of the BOD at pH 6.5 to

7.5.

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

UCBOD + UNBOD UBOD

Report results as carbonaceous biochemical oxygen demand (CBOD5) when inhibiting the nitrogenous oxygen demand. When nitrification is not inhibited, report results as BOD5

Dilution Requirements

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

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

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

5-Day BOD Test Principle The method consists of filling with sample, to overflowing, an airtight bottle of the

specified size and incubating it at the specified temperature for 5 d. Dissolved oxygen is measured initially and after incubation, and the BOD is computed from the difference between initial and final DO.

Sampling and storage1) Grab samples—Store the sample at or below 4°C 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 samples—Keep samples at or below 4°C during compositing. Limitcompositing period to 24 h.

Apparatus

Incubation bottles Use glass bottles having 60 mL or greater capacity (300-mL bottles having a ground-

glass stopper and a flared mouth are preferred). Clean bottles with a detergent, rinse thoroughly, and drain before use. Use a water seal to prevent the drawing of air in to the bottle. Place a paper or plastic cup or foil cap over flared mouth of bottle to reduce

evaporation of the water seal during incubation.

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

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

• a

Procedurea. 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 ± 3°C. • Saturate with DO by shaking in a partially filled bottle or by aerating with

organic-free filtered air. Alternatively, store in cotton-plugged bottles long enough for water to become saturated with DO.

b. Dilution water storage• Source water may be stored before use as long as the prepared dilution water

meets quality control criteria in the dilution water blank. • Discard stored source water if dilution water blank shows more than 0.2 mg/L DO

depletion in 5 d.

Procedure

c. Glucose-glutamic acid check To check the dilution water quality. Periodically check dilution water quality, seed effectiveness, and analytical

technique by making BOD measurements on a mixture of 150 mg glucose/L and 150 mg glutamic acid/L as a ‘‘standard’’ check solution.

Glucose has an exceptionally high and variable oxidation rate but when it is used with glutamic acid, the oxidation rate is stabilized and is similar to that obtained with many municipal wastes.

Determine the 5-d 20°C BOD of a 2% dilution of the glucose-glutamic acid standard check solution

Procedure

d. Seeding• Seed source: Some samples do not contain a sufficient microbial population (for example,

some untreated industrial wastes, disinfected wastes, high-temperature wastes, or wastes with extreme pH values). For such wastes seed the dilution water or sample by adding a population of microorganisms.

• The preferred seed is effluent or mixed liquor from a biological treatment system processing the waste.

• Where such seed is not available, use supernatant from domestic wastewater after settling at room temperature for at least 1 h but no longer than 36 h.

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

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

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 <6.0)—Neutralize samples to pH 6.5 to 7.5 with a solution of sulfuric acid (H2SO4) or sodium hydroxide (NaOH) of such strength that the quantity of reagent does not dilute the sample by more than 0.5%.

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

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

Sample treatment• In some samples chlorine will dissipate within 1 to 2 h of standing in the light. If

residual chlorine is present, dechlorinate sample and seed the dilution water using Na2SO3.

• 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 Na2SO3 solution to the starch-iodine end point for residual.

• Samples containing other toxic substances—Certain industrial wastes, for example,plating wastes, contain toxic metals. Such samples often require special study and

treatment.• Sample temperature adjustment—Bring samples to 20 ± 1°C before making

dilutions.• Nitrification inhibition—If nitrification inhibition is desired add 3 mg of 2-chloro-6-

(trichloro methyl) pyridine (TCMP) to each 300-mL bottle before capping or add sufficient amounts to the dilution water to make a final concentration. Note the use of nitrogen inhibition in reporting results.

Dilution technique

• Make several dilutions of sample that will result in a residual DO of atleast 1 mg/L and a DO uptake of at least 2 mg/L after a 5-d incubation.

• Five dilutions are recommended unless experience with a particular sample shows that use of a smaller number of dilutions produces at least two bottles giving acceptable minimum DO depletion and residual limits.

• In the absence of prior knowledge, use the following dilutions: 0.0 to 1.0% for strong industrial wastes, 1 to 5% for raw and settled wastewater, 5 to25% for biologically treated effluent, and 25 to 100% for polluted river waters.

• When using graduated cylinders or volumetric flasks to prepare dilutions, and when seeding is necessary, add seed either directly to dilution water or to

individual cylinders or flasks before dilution.

Dilution technique• Seeding of individual cylinders or flasks avoids a declining ratio of seed to sample

as increasing dilutions are made. • When dilutions are prepared directly in BOD bottles and when seeding is

necessary, add seed directly to dilution water or directly to the BOD bottles.

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

↓• Add appropriate amounts of seed material either to the individual BOD bottles or

to the dilution water↓

• Fill bottles with enough dilution water, seeded if necessary ↓

• Determine initial DO on one bottle ↓

• Stopper tightly, water-seal, and incubate for 5 d at 20°C.

Determination of initial DO• If the sample contains materials that react rapidly with DO, determine initial DO

immediately after filling BOD bottle with diluted sample.

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

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

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

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

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

Methods to find 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 of molecular oxygen across a membrane

• The choice of procedure depends on the interferences present, the accuracy desired, and, in some cases, convenience or expedience.

Iodometric MethodsPrinciple• It is based on the addition of divalent manganese solution, followed by strong

alkali, to the sample in a glass-stoppered bottle. • DO rapidly oxidizes an equivalent amount of the dispersed divalent manganous

hydroxide precipitate to oxides of higher valence state.• In the presence of iodide ions (I -) in an acidic solution, the oxidized manganese

reverts to the divalent state, with the liberation of iodine (I2)equivalent to the original 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).

Modifications of Iodometric method

• azide modification-removes interference caused by nitrite

• the permanganate modification-removes the interference of ferrous iron

• the alum flocculation modification- suspended solids interference

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

• The choice is based on the effect of interferences, particularly oxidizing or reducing materials that may be present in the sample.

Azide ModificationReagentsa. Manganous sulfate solutionb. Alkali-iodide-azide reagentc. Sulfuric acidd. Starche. Standard sodium thiosulfate titrantf. Standard potassium bi-iodate solution

Standardization—Dissolve 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 Na2S2O3

Starch indicator, pale straw color end point

Procedure

a. To the sample collected in a 250- to 300-mL bottle ↓ 1 ml of MnSO4 ↓

1 mL alkali-iodide-azide reagent

• Stopper carefully to exclude air bubbles and mix by inverting bottle a few times.• When precipitate has settled sufficiently add 1.0 mL conc H2SO4

• Restopper and mix by inverting several times until dissolution is complete.• Titrate a volume corresponding to 200mL original sample after correction for sample

loss by displacement with reagents. Thus, for a total of 2mL (1 mL each) of MnSO4 and alkali-iodide-azide reagents in a 300-mL bottle, titrate

200 × 300/(300 −2) = 201 mL.

b. Titrate with 0.025M Na2S2O3 solution to a pale straw color. Add a few drops of starch solution and continue titration to first disappearance of blue color.

Permanganate Modification

only on samples containing ferrous iron ineffective for oxidation of sulfite, thiosulfate, polythionate, or the organic matter

in wastewater.

Reagents

a. Potassium permanganate solutionb. Potassium oxalate solutionc. Potassium fluoride solutionAll the reagents required for the azide modification

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

↓ 0.70 mL conc H2SO4 + 1 mL KMnO4 solution + 1 mL KF solution

↓ Stopper and mix by inverting

Remove permanganate color completely by adding 0.5 to 1.0 mL K2C2O4 solution

↓ 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

Alum Flocculation Modification

• The interference due to solids may be removed by alum flocculation.

Reagentsa. Alum solutionb. Ammonium hydroxidec. Manganous sulfate solutiond. Alkali-iodide-azide reagente. Sulfuric acidf. Starchg. Standard sodium thiosulfate titrantAll the reagents required for the azide modification

Procedure

• To a sample collected in a 250- to 300-mL bottle ↓

10 mL alum solution + 1 to 2 mL conc NH4OH↓

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

• Continue sample treatment as same of azide modification.

Copper Sulfate-Sulfamic Acid Flocculation

Modification• Used for biological flocs such as activated sludge mixtures, which have high oxygen utilization rates.

Reagentsa. Copper sulfate-sulfamic acid inhibitor solutionb. 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 bottom and

overflows only 25 to50% of bottle capacity. ↓Collect sample, stopper, and mix by inverting

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

DO bottle

Continue as per azide modification

Membrane Electrode Method

• Being completely submersible, membrane electrodes are suited for analysis in situ. Principle• Oxygen-sensitive membrane electrodes of the polarographic or galvanic type are

composed of two solid metal electrodes in contact with supporting electrolyte separated from the test solution by a selective membrane.

• In polarographic, electrode reaction is spontaneous• In galvanic types external source of applied voltage is needed to polarize the

indicator electrode.• Membranes like Polyethylene and fluorocarbon membranes are normally used as

they are permeable to molecular oxygen and are relatively rugged.• In the device ‘‘diffusion current’’ is linearly proportional to the concentration of

molecular oxygen.• The current can be converted to concentration units by calibration.

Apparatus

• Oxygen-sensitive membrane electrode, polarographic or galvanic, with appropriate meter.

Procedurea. Calibration :calibrate membrane electrodes by reading against air or asample of known DO concentration (determined by iodometric method) as well as in a

sample with zero DO.

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

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

Calculation

• Membrane electrodes exhibit a relatively high temperature coefficient largely due to changes in the membrane permeability

• The effect of temperature on the electrode sensitivity, φ (microamperes per milligram per liter)

log φ= 0.43 mt+ bwhere: 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 (φ and t)= log φ= log φ0+ 0.43 m(t −t 0)

Temperature correction can be found from the nomographic charts

Electrode sensitivity varies with salt concentration according to the following relationship:

log φ s= 0.43 mSCs+ log φ 0

Calculation

• For each test bottle meeting the 2.0-mg/L minimum DO depletion and the 1.0-mg/L residual DO, calculate BOD5 as follows:

• When dilution water is not seeded:

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

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

P= decimal volumetric fraction of sample used

CalculationBOD (mg/L)= [(D1 –D2 ) –f(B1 –B2 )]/P

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

D2= DO of diluted sample after 5 d incubation at 20°C, 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)

Ultimate BOD Test

• An extension of 5-day BOD test.Principle• Placing a single sample dilution in full, airtight bottles and incubating under

specified conditions for an extended period depending on wastewater effluent, river, or estuary quality.

• Dissolved oxygen (DO) is measured (with probes) initially and intermittently during the test. From the DO versus time series, UBOD is calculated.

• Temp: 20°C• When DO concentrations approach 2 mg/L, the sample should be reaerated. • If the result is being used to estimate the rate of oxidation of naturally occurring

surface waters, addition of nutrients and seed probably accelerates the decay rate and produces misleading results.

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

Apparatus

Incubation bottles: Glass bottles with ground-glass stoppers 2-L (or larger) capacity clean bottles with a detergent and wash with dilute HCl (3N) to remove surface films and precipitated inorganic salts; rinse thoroughly with DI water before use.

• To prevent drawing air into the sample bottle during incubation, use a water seal.

• Place a clean magnetic stirring bar in each bottle to mix contents before making DO

• measurement or taking a subsample. Reservoir bottle:4-L or larger glass bottle. Close with screw plastic cap or

non-rubber plug. Incubator or water bath, thermostatically controlled at 20 ± 1°C. Exclude

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

Procedure: River water samples• Fill large BOD bottle with sample at 20°C. 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 20°C in the dark.

• Measure DO in each bottle at intervals of at least 2 to 5 d over a period of 30 to 60 d(minimum of 6 to 8 readings) or longer under special circumstances.

• To avoid oxygen depletion in samples containing NH3-N, measure DO more frequently until nitrification has taken place. If DO falls to about 2 mg/L, reaerate .

• Pour a small amount of sample into a clean vessel and reaerate the remainder directly in the bottle by vigorous shaking or bubbling with purified air (medical grade). Refill bottle from the storage reservoir and measure DO. This concentration becomes the initial DO for the next measurement. If using 300-mL BOD bottles, pour all of the sample from the several bottles used into a clean vessel, reaerate, and refill the small bottles.

• Analyze for nitrate plus nitrite nitrogen on Days 0, 5, 10, 15, 20, and 30. Alternatively, determine NO2–-N andNO3–-N each time DO is determined, thereby producing corresponding BOD and nitrogen determinations. If the ultimate demand occurs at a time greater than 30 d, make additional analyses at 30-d intervals.

• If the purpose of the UBOD test is to assess the UBOD and not to provide data for rate calculations, measure nitrate nitrogen concentration only at Day 0 and on the last day of the test.

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

• High-quality reagent water without nutrients typically will consume a maximum of 1mg DO/L in a 30- to 90-d period. If DO uptake of the dilution water is greater than 0.5 mg/L for a 20-d period, or 1 mg/L for a 90-d period, report the magnitude of the correction and try to obtain higher-quality dilution water for use with subsequent UBOD tests.

Wastewater treatment plant samples

• Use high-quality reagent water for dilution water.

• Add no nitrification inhibitors if decay rates are desired.

• If seed and nutrients are necessary, add the same amounts of each to the dilution water blank the ultimate BOD of the diluted sample should be in the range of 20 to 30 mg/L.

• Dilution to this level probably will require two or three sample reaerations during the incubation period to avoid having dissolved oxygen concentrations fall below 2 mg/L.

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

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

• Treat blank the same as all samples.

Calculations

• UBOD can be estimated by using a first-order model described as follows:

BODt= UBOD(1 −e -Kt)

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

k= first-order oxygen uptake rate.

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 chemicalstreatability of organic industrial wastesthe effect of known amounts of toxic compounds on the

oxygen-uptake reaction of a test wastewater or organic chemicalthe concentration at which a pollutant or a wastewater

measurably inhibits biological degradationthe effect of various treatments such as disinfection, nutrient

addition, and pH adjustment on oxidation ratesthe oxygen requirement for essentially complete oxidation of

biologically oxidizable matterthe need for using adapted seed in other biochemical oxygen

uptake measurements, such as the dilution BOD test• Respirometric data typically will be used comparatively, that is, in a direct

comparison between oxygen uptakes from two test samples or from a test sample and a control.

Types of Respirometers• Manometric respirometers relate oxygen uptake to the change in pressure

caused by oxygen consumption while maintaining a constant volume. • Volumetric respirometers measure oxygen uptake in incremental changes

in gas volume while maintaining a constant pressure at the time of reading.

• Electrolytic respirometers monitor the amount of oxygen produced by electrolysis of water to maintain a constant oxygen pressure within the reaction vessel.

• Direct-input respirometers deliver oxygen to the sample from a pure oxygen supply through metering on demand as detected by minute pressure differences.

• Reaction-vessel contents are mixed by using a magnetic or mechanical stirring device or by bubbling the gaseous phase within the reaction vessel through the liquid phase.

• All respirometers remove carbon dioxide produced during biological growth by suspending a concentrated adsorbent (granular or solution) within the closed reaction chamber or by recirculating the gas phase through an external scrubber.

Interferences• Evolution of gases other than CO2 may introduce errors in pressure or volume

measurements; this is uncommon in the presence of dissolved oxygen.• Incomplete CO2 absorption will introduce errors if appropriate amounts and

concentrations of alkaline absorbent are not used. • Temperature fluctuations or inadequate mixing. • Fluctuations in barometric pressure• Most commercial respirometers can detect oxygen demand in increments as small

as 0.1 mg.• Upper limits of oxygen uptake rate are determined by the ability to transfer oxygen

into the solution from the gas phase, which typically is related to mixing intensity.• The continuous readout of oxygen consumption in respirometric measurements

indicates lag, toxicity, or any abnormalities in the biodegradation reaction. The change in the normal shape of an oxygen-uptake curve in the first few hours may help to identify the effect of toxic or unusual wastes entering a treatment plant in time to make operating corrections.

Apparatus

• Respirometer system• Incubator or water bath

Reagentsa. Distilled waterb. Phosphate buffer solutionc. Ammonium chloride solutiond. Calcium chloride solutione. Magnesium sulfate solution,f. Ferric chloride solution

Reagents

• Potassium hydroxide solution,6N• Acid solutions,1N• Alkali solution,1N• Nitrification inhibitor: Reagent-grade 2-chloro-6

(trichloromethyl) pyridine (TCMP) or equivalent.• Glucose-glutamic acid solution• Electrolyte solution• Sodium sulfite• Trace element solution• Yeast extract solution• Nutrient solution

Procedure• Instrument operation: Follow respirometer manufacturer’s 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• Homogenization—If 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 adjustment—Neutralize samples to pH 7.0with H2SO4 or NaOH of such strength that reagent that reagent quantity does not dilute the sample more than 0.5%.

• Dechlorination—Avoid 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 Na2SO3 solution.

Procedure• Initial oxygen concentration—If 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 adjustment—Bring samples and dilution water to desired test temperature (±1°C) before making dilutions or transferring to test vessels.

• Sample dilution: Use distilled water or water from other appropriate sources free of organic matter for dilution.

• Nutrients, minerals, and buffer : Add sufficient ammonia nitrogen to provide a COD:N:P ratio of 100:5:1 or a TOC:N:P ratio of 30:5:1.

• Add 2 mL each of calcium, magnesium, ferric chloride, and trace mineral solutions to each liter of diluted sample unless sufficient amounts of these minerals are present in the original sample.

• Phosphorus requirements will be met by the phosphate buffer if it is used (1 mL/50 mg/L COD or ultimate BOD of diluted sample usually is sufficient to maintain pH between 6.8 and 7.2).

• Nitrification inhibition: If nitrification inhibition is desired, add 10 mg 2-chloro-6-(trichloromethyl) pyridine (TCMP)/L sample in the test vessel.

• Seeding: Use sufficient amounts of seed culture to prevent major lags in the oxygen uptake reaction but not so much that the oxygen uptake of the

seed exceeds about 10% of the oxygen uptake of the seeded sample.Determine the oxygen uptake of the seeding material as for any other sample.

This is the seed control.

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

Calculations• To convert instrument readings to oxygen uptake, refer to

manufacturer’s 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, and

NA= volume of undiluted sample in Sample A, mL.

Reference

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