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BIOCHEMICAL OXYGEN DEMAND

Training-Workshop in Wastewater Analysis RLC Laboratory, Robinsons Place Manila, Ermita , Manila September 17 21, 2012

by Diana C. Galicia

Measurement of Organic Content


Over the years, a number of different tests have been developed to determine the organic content of wastewaters. Generally, the tests may be divided into two:
1.

2.

those used to measure gross concentrations of organic matter greater than about 1 mg/L those used to measure trace concentration in the range of 10-6 to 10-3 g/L.
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Measurement of Organic Content


Laboratory methods commonly used today to measure gross amounts of organic matter greater than 1 mg/L in wastewater include: (1) biochemical oxygen demand (BOD); (2) chemical oxygen demand (COD); and (3) total organic carbon.

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Biochemical Oxygen Demand

The 5-day BOD (BOD5) is the most widely used parameter of organic pollution applied to both the wastewater and surface water. This determination involves the determination of the dissolved oxygen used by the microorganisms in the biochemical oxidation of organic matter.

Organic Matter (CaHbOc) + nO2 nCO2 + nH2O

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Importance of BOD test


Determination the quality of natural waters. Measuring the effect of oxidation of wastes on streams, Assessing the suitability of water for fish and other organisms Monitoring progress of self-purification of various body of waters In aerobic sewage treatment units the minimum objectionable odor potential, maximum treatment efficiency and stabilization of wastewater are dependent on maintenance of adequate dissolved oxygen. Frequent dissolved oxygen measurement is essential for adequate process control.
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Dissolved Oxygen

Dissolved oxygen is essential for the survival of aquatic plant and animal life. Generally, 5 mg/L of dissolved oxygen content is borderline concentration if considering an extended time period. For adequate game fish population, the dissolved oxygen content should be in the 8 to 15 mg/L range.

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Dissolve oxygen concentration varies with water depth, sludge deposits temperature, clarity and flow rate. Thus, a single water sample rarely is representative of the overall condition of a body of water.

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Biochemical Oxygen Demand


Despite the widespread used of the BOD Test it has a number of limitations. It is hoped that, through the continued efforts of workers in the field one of the other measures of organic content, or perhaps a new measure, will ultimately be used in its place.

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Limitations in the BOD Test


A high concentration of active, acclimated seed bacteria is required. Pretreatment is needed when dealing with toxic wastes, and the effects of nitrifying organisms must be reduced; Only the biodegradable organics are measured; The test does not have stoichiometric validity after the soluble organic matter present in solution has been used ; and An arbitrary, long period of time is required to obtain results.
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Why, then, if the test suffers from serious limitations, is still widely used?

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The reason is that BOD test results are now used to


1.

2.

3.

4.

determine the approximate quantity of oxygen that will be required to biologically stabilize the organic matter present, determine the size of wastewater treatment facilities, measure the efficiency of some treatment processes, and determine compliance with wastewater discharge permits.
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BOD ANALYSIS

Nitrification in BOD Test Non-carbonaceous matter, such as, ammonia is produced during the hydrolysis of proteins. Autotrophic bacteria are capable of oxidizing ammonia to nitrite and subsequently to nitrate the generalized reactions are:

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BOD ANALYSIS
Nitrification in BOD Test nitrite-forming bacteria (a) NH3 + 3/2 O2 HNO2 + H2O

(b) HNO2 + O2 NH3 + 2O2

nitrate-forming bacteria

HNO3

HNO3 + H2O

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BOD ANALYSIS

Nitrification in BOD Test The oxygen demand associated with the oxidation of ammonia to nitrate is called the nitrogenous biochemical oxygen demand (NBOD). It normally takes 6 to 10 days for a significant number of nitrifying bacteria to exert measurable oxygen demand because their reproductive rate is slow
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BOD ANALYSIS

Carbonaceous BOD The interference caused by the nitrifying bacteria can be eliminated by pretreatment of the sample or by the use of inhibitory agents. The results of the suppressed BOD test is reported as CBOD (carbonaceous biochemical oxygen demand).
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BOD ANALYSIS
I.

II.
III. IV. V.

Materials and Equipment Reagents Preparatory Procedure Testing Procedure Chemical Reaction

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I. Materials and Equipment


1.

Incubation Bottles, 300ml

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Incubator
Thermostatically controlled at 20oC+1oC. Exclude all light to prevent possibility of photosynthetic production of DO.

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II. REAGENTS
1.

Nutrients
a. b. c. d.

Phosphate buffer Magnesium sulfate solution Calcium chloride solution Ferric chloride solution

2.

Acid and alkali solutions


- for neutralization of basic or acidic waste samples

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II. REAGENTS
3. 4.
a. b.

Sodium sulfite solution Nitrification inhibitor


2-chloro-6-(trichloromethyl)pyridine Allylthiourea (ATU) solution

5.
a. b.

Determination of Dissolved Oxygen


Manganous sulfate solution Alkali-iodide azide reagent
CAUTION! Do not acidify this solution because toxic hydrazoic acid fumes may be produced.
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II. REAGENTS
c.

d.
e. f.

Sulfuric acid, concentrated Starch Standard sodium thiosulfate titrant Standard potassium bi-iodate solution or potassium iodide

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II. REAGENTS
6.

QC Standard Solution
a.

Glucose-Glutamic acid solution, 198+30.5 mg O2/L This solution should be prepared fresh immediately before use unless the solution is maintained in a sterile condition, the mixture can be stored at temperature 4oC or lower
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III. PREPARATORY PROCEDURE


1.

Sampling and Storage Samples for BOD analysis may degrade significantly during storage between collection and analysis, resulting in low BOD values.
a.

Grab sampling if analysis is begun within 2h of collection cold storage is unnecessary. If analysis is not started within 2h of sample collection, keep sample at or below 4oC from the time of collection.
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III. PREPARATORY PROCEDURE


1. Sampling and Storage
b.

Composite sampling

Keep samples at or below 4oC during composting. Limit composting period to 24h. Use the same criteria for storage of grab samples, starting measurement of holding time from end of composting period.
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III. PREPARATORY PROCEDURE


2.

Sample Preparation and Pretreatment


a.

All samples

Check pH. If it is not between 6.0 to 8.0, adjust sample temperature to 20 + 3C, then adjust pH to 7.0 to 7.2 using solution of sulfuric acid or sodium hydroxide. Always seed samples that have been pH adjusted.
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III. PREPARATORY PROCEDURE


2.
b.

Sample Preparation and Pretreatment


Samples containing residual chlorine compounds.
In some samples, chlorine dissipates within 1 to 2 h of standing in light which occurs during sample transport and handling. For samples in which chlorine residual does not dissipate in reasonably short time, dechlorinate sample by adding Na2SO3 Solution
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III. PREPARATORY PROCEDURE


2.
c.

Sample Preparation and Pretreatment


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

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III. PREPARATORY PROCEDURE


2.

Sample Preparation and Pretreatment


d.

Samples supersaturated with DO Samples containing DO concentration above saturation at 20C (>9 mg O2/L) may be encountered in cold water or in water where photosynthesis occurs. To prevent loss of oxygen during incubation of such samples reduce DO to saturation by bringing sample to about 20 + 3C in partially filled bottle while agitating by vigorous shaking or by aerating with clean, filtered compressed air.
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III. PREPARATORY PROCEDURE


2.
e.

Sample Preparation and Pretreatment


Samples containing hydrogen peroxide.
Mix samples vigorously in open containers for sufficient time to allow the hydrogen peroxide to dissipate before setting up BOD Tests. Mixing time may vary from 1 to 2 hours depending on the amount of hydrogen peroxide present, the peroxide reaction can be considered complete when the DO no longer increases during 30-min period without mixing.
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III. PREPARATORY PROCEDURE


3.

Selection and storage of source water for BOD sample dilution > Distilled > Tap water > Deionized water

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Selection and storage of source water for BOD sample dilution


Make sure the water is free from heavy metals, specifically copper, and toxic substances, such as chlorine, that can interfere with BOD measurements. Deionized water often contains sufficient amounts of organics and microorganisms to cause failure of the dilution water quality control check. Discard stored source water if the dilution water blank shows more than 0.20 mg/L DO depletion in 5 days.
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III. PREPARATORY PROCEDURE


4.

Preparation of seed suspension It is necessary to have present in each BOD bottle a population of microorganisms capable or oxidizing the biodegradable organic matter in the sample. Seed Sources

Domestic wastewater Unchlorinated effluents from biological wastewater treatment plants Surface water receiving wastewater discharges. Commercial seed.
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V. Testing Procedure
A. Preparation of Dilution water
1.

Transfer desired working volume of source water in a Damajuana. Ensure DO concentration is at least 7.5 mg/L before using water for BOD tests.

Volume of desired = total no. of BOD btls. x 300 ml x 1L/1000 ml dilution water (values round off to whole no. as liters)
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V. Testing Procedure
A. Preparation of Dilution water
2.

3.

Add 1 ml each of the nutrients per liter of the source water. Bring temperature to 20 + 3C and aerate for 1 hour.
The DO depletion of dilution water blank should be lower than 0.2 mg/L. if not, obtain satisfactory water by improving purification or use water from another source.
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V. Testing Procedure
B. Sample temperature adjustment
Bring samples to 20 + 3C before making dilutions.

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V. Testing Procedure
c. Preparation of dilutions
1.

2.

Using the prepared dilution water make three dilutions of prepared sample. Compute for the volume of sample from the computed value of COD.

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V. Testing Procedure
c. Preparation of dilutions
Dilution Techniques
Basis for seeded sample dilution: 1. Compute for COD results for samples 2. Min. sample dilution = 300 x 5 COD 3. Max. sample dilution = 1500 0.4 x COD 4. Select tolerance volume at three sample dilution
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V. Testing Procedure
c. Preparation of dilutions
In the absence of prior knowledge of COD values, use the following percentages of wastewater when preparing dilution:
Percent dilution 0.1 1.0 % Type of sample Strong industrial waste

15%
5 25 % 25 100 %

Raw and settled wastewater


Biologically treated Polluted river waters
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V. Testing Procedure
d.

Addition of seed suspension


1.

2.

3.

4.

To make seed solution, place the entire contents of one Polyseed capsule into 500 ml of dilution water Aerate and stir the Polyseed solution for 1 h. Decant the supernatant carefully so as not to allow any bran in the solution. Pour the decanted Polyseed solution in a clean 500 ml beaker with a sterile stir bar, place on magnetic stirrer and gently stir for the remainder of the test.

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V. Testing Procedure
d.

Addition of nitrification inhibitor


1.

2.

Samples that may require nitrification inhibition include but are not limited to , biologically treated effluents, samples seeded with biologically treated effluents and river waters. Seed all samples to which nitrification inhibitor has been added.

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V. Testing Procedure
e.

Sealing of bottles

Complete filling of each bottle by adding enough dilution water that insertion of the stopper leaves no bubbles in the bottle. As a precaution against drawing air into the dilution bottle during incubation, use water seal. Obtain satisfactory water seals by adding water to the flared mouth of BOD bottles. Place a foil cap over flare mouth of bottle to reduce evaporation of the water seal during incubation
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V. Testing Procedure
f.

Determination of initial DO

Use the Azide modification of the iodometric method to determine the initial DO on all sample dilutions, dilution water blanks, GGA checks and seed controls

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V. Testing Procedure
f.

Determination of initial DO
1.

2.

Add 1 ml of MnSO4 solution directly to the BOD bottle Then add 1 ml of alkali-iodide-azide solution.

If pipettes are dipped into sample, rinse them before returning them to reagent bottles. Alternatively, hold pipette tips just above the liquid surface when adding reagents.
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V. Testing Procedure
f.

Determination of initial DO
3.

4.

Stopper carefully to exclude air bubbles and mix by inverting bottle for 15 times. When precipitate has settled sufficiently (to approximately half the bottle volume) to leave clear supernate above the manganese hydroxide floc, add 1.0 ml of concentrated H2SO4.
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V. Testing Procedure
f.

Determination of initial DO Re-stopper and mix by inverting bottle 15 times or until dissolution is complete. Measure 99 ml (corrected volume after sample loss by reagent) of the sample and discard.

5.

6.

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V. Testing Procedure
f.

Determination of initial DO Thus, for a total of 2 ml (1ml each) of MnSO4 and AAS reagents in a 300-ml bottle, titrate the remaining 201 ml with standard Na2S2O3 titrant to pail straw color. 200 x 300 = 201 ml (300 - 2 ) Add a few drops of starch solution and continue to first disappearance of blue color.
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5.

5.

V. Testing Procedure
g.

Sample dilution

Incubate at 20 + 1C the stoppered and sealed BOD bottles of samples, seed controls, dilution water blanks and glucose-glutamic acid check. Exclude light to avoid growth of algae in the bottles during incubation.
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V. Testing Procedure
h.

Determination of final DO

After 5 days + 6 h of incubation, determine DO in all sample dilutions, and in all blanks and checks using the azide modification of the titrimetric method.

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V. Chemical Reaction

In the analysis, Mn2+ (manganous ion) reacts with the dissolved oxygen present in the alkaline solution to for a Mn4+ oxide hydroxide floc.

2Mn2+ + O2 + 4OH 2MnO(OH)2

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V. Chemical Reaction

In the analysis, Mn2+ (manganous ion) reacts with the dissolved oxygen present in the alkaline solution to for a Mn4+ oxide hydroxide floc.

2+ 2Mn

+ O2 + 4OH 2MnO(OH)2
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V. Chemical Reaction

In the analysis, Mn2+ (manganous ion) reacts with the dissolved oxygen present in the alkaline solution to for a Mn4+ oxide hydroxide floc.

2+ 2Mn

+ O2 + 4OH 2MnO(OH)2
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V. Chemical Reaction

In the analysis, Mn2+ (manganous ion) reacts with the dissolved oxygen present in the alkaline solution to for a Mn4+ oxide hydroxide floc.

2Mn2+ + O2 + 4OH 2MnO(OH)2

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V. Chemical Reaction

Azide is added at this time to suppress interference from any nitrate present which would react with the iodide. The solution is then acidified and the manganese floc is reduced by iodide to produce Mn2+ and free iodine as I3(I2 + I in solution).
MnO(OH)2 + 6I + 6H+ Mn2+ + 2I3 + 3H2O
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V. Chemical Reaction

Azide is added at this time to suppress interference from any nitrate present which would react with the iodide. The solution is then acidified and the manganese floc is reduced by iodide to produce Mn2+ and free iodine as I3(I2 + I in solution).

2+ MnO(OH)2 + 6I + 6H+ Mn

+ 2I3 + 3H O
2
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V. Chemical Reaction

The iodine gives the clear supernate a brown color. Phenylasine oxide (PAO) or thiosulfate is then used to titrate the iodine to a colorless endpoint.

Starch indicator can be added to enhance the determination of the end point by producing a color change from dark blue to colorless. The dissolved oxygen of the sample is then calculated from the quantity of titrant used.
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References

SMEWW 21st Edition. 2005. APHA, AWWA, WEF

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END
Thank you.