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Lab 15: Biochemical Oxygen Demand

The typical wastewater treatment plant depends on microorganisms in the wastewater to decompose waste. These microorganisms are primarily aerobic, so they use up oxygen as they break down organic matter. The Biochemical Oxygen Demand, or BOD, is the amount of dissolved oxygen which is used up by these microorganisms and is roughly equivalent to the amount of "food" (organic matter) found in the wastewater. The more "food" that is present in the water, the more DO will be used up by the bacteria and the greater the BOD reading will be. A major disadvantage of the BOD test is that results are not available for 5 days. Nevertheless, BOD tests are an integral part of the wastewater operator's repertoire. Wastewater treatment plants use BOD as an estimate of the waste load in the influent water. They can also test BOD of the effluent to determine the plant's efficiency, to control plant processes, and to determine the effects of discharges on receiving waters.


Incubation bottles. (A typical incubation bottle is shown above. Incubation bottles should be made of glass and should have a volume of 60 mL or greater. Preferred bottles have a 300 mL capacity, a ground-glass stopper, and a flared mouth.)

Air incubator or water bath. (The incubator must be capable of maintaining a temperature of 20 + 1C and must keep the bottles in complete darkness.) Oxygen-sensitive membrane electrode, with appropriate meter


Phosphate buffer solution Magnesium sulfate solution Calcium chloride solution Ferric chloride solution Acid and alkali solutions, 1 N Sodium sulfite solution Nitrification inhibitor Glucose-glutamic acid solution Ammonium chloride solution Distilled water Seed source. (In some cases, it is necessary to add a population of microorganisms to the sample water. If so, a small amount of wastewater effluent or mixed liquor can be used as the seed source. Alternatively, supernatant from domestic wastewater, settled at room temperature for between 1 hour and 36 hours can be used.)

Laboratory Procedure
. 1. Clean the incubation bottles. Clean the bottles with a detergent, rinse thoroughly, and drain. Invert bottles in a water bath to prevent contamination between cleaning and use. 2. Determine the sample size(s). Most samples of wastewater will require more oxygen during the incubation period than is found in the BOD bottle, so the samples must be diluted. At the proper

dilution, the residual DO after five days will be at least 1 mg/L and the DO uptake will be at least 2 mg/L. If your treatment plant has influent and effluent BODs which do not vary greatly over time, then you can use the dilutions which have been used in the past at that plant. At other plants, however, the BOD fluctuates widely. In this case, it is usually best to produce at least five different dilutions of the sample water. The range of dilutions should be approximately:
Source Strong industrial wastes Raw and settle wastewater Polluted river waters Dilute the Sample Water to the Following Concentration: 0.1 to 1.0% 1 to 5% 100%

Biologically treated effluent 5 to 25%

If you have an estimate of the BOD likely to be found in your sample water, you can use the two equations below to determine the maximum and minimum amount of sample water to be used in your dilutions.

For example, suppose that the estimated BOD of an influent sample is 400 mg/L. The D.O. of saturated dilution water is approximately 8.0 mg/L. If you want the D.O. depletion to be at least 2.0 mg/L after 5 days of incubation and the residual D.O. to be at least 1.0 mg/L, then you would calculate the dilutions as follows. First, to calculate the least concentrated dilution, use the minimum allowable depletion:

This means that you will use 1.5 mL of sample water in a 300 mL sample bottle, filling the bottle the rest of the way with dilution water. Next, to calculate the most concentrated dilution, use the maximum allowable depletion. The maximum allowable depletion can be calculated as follows:
Max. allowable depletion = (Saturated D.O. concentration) - (Min. residual concentration) Max. allowable depletion = 8.0 mg/L - 1.0 mg/L Max. allowable depletion = 7.0 mg/L

Then the amount of sample water used in the dilution is calculated as follows:

From the calculations above, we can estimate that we should use a 1.5 mL sample and a 5.25 mL sample. Since the BOD value we used was just an approximation, we should probably use slightly lower and slightly higher sample sizes, as well as a few sample sizes in between. We might choose to use samples of 1 mL, 3 mL, 4 mL, 5 mL, and 6 mL. Once you have determined the amount of sample water which will go in each bottle, record the amount in the Data section. Be sure to label each bottle. 3. Prepare the dilution water in each incubation bottle. a. Place the desired volume of distilled water in an incubation bottle. Use the formula below to calculate the amount of water to place in the BOD bottle:
(Vol. of dilution water) = (Vol. of BOD bottle) - (4 mL) - (Vol. of seed water) - (Vol. of sample water)

For example, if 5 mL of sample water is going to be added to a 300 mL BOD bottle and the sample is not going to be seeded, then the amount of dilution water would be:

(Vol. of dilution water) = 300 mL - 4 mL - 0 mL - 5 mL (Vol. of dilution water) = 291 mL b. Add 1 mL each of phosphate buffer solution, magnesium sulfate solution, calcium chloride solution, and ferric chloride solution to the water. c. Bring the water to a temperature of 20 + 3C. d. Saturate the water with dissolved oxygen by shaking in a partially filled bottle or by aerating with organic-free filtered air. e. If the pH of the sample water is altered in the next step, or if the water has been chlorinated, then the dilution water solution should be seeded. If the water is seeded, record information about the seed source and seed amount in the Data section. 4. Prepare the sample water. a. Collect the sample water. Samples can be either grab or composite, with the type of sample taken depending on monitoring requirements, plant operations, and storage facilities at the plant. Regardless of the type of sample, the sample should be taken at a point where the water is well-mixed and representative of the plant's flow. The BOD test should be begun as soon as possible after sample collection. If samples must be stored, they should be kept at a temperature of 4C or below and should not be allowed to freeze. Samples may be stored for no more than 48 hours before beginning the BOD test. b. Check the pH of the sample water. Record in the Data section. c. If the pH is greater than 8.5 or less than 6.0, then small amounts of acid or alkali solution should be added to bring the pH back into the desired range. Do not dilute the solution with more than 0.5% acid or alkali. Record the final pH in the Data section. d. If the sample water contains chlorine, add sodium sulfite. If sodium sulfite is added, record the amount in the Data section. e. Ensure that the sample water is not supersaturated.

Test the DO content of the sample water. If the water contains more than 9 mg/L of DO at 20C, then it is supersaturated and will lose some oxygen to the air. This problem may occur when testing cold water or water in which photosynthesis occurs. If the water is supersaturated, place the sample in a partially filled bottle and agitate it by vigorous shaking or by aerating with clean, filtered compressed air. Continue until the DO content has dropped below 9 mg/L. e. Bring the sample water to 20 + 1C. f. If the sample water contains biologically treated effluent or river water or was seeded with biology treated effluent, then 3 mg of nitrification inhibitor should be added to each 300 mL BOD bottle. Record the addition in the Data section. 5. Using the sample sizes determined in step 2, prepare each sample bottle. Using a wide-mouth volumetric pipet, add the desired sample volume to individual BOD bottles of known capacity. Fill the bottles with enough dilution water so that the insertion of the stopper displaces all air, leaving no bubbles. Water-seal the bottle. If the dilution is greater than 1:100, you should first make a primary dilution in a graduated cylinder then make a second dilution in the bottle. 6. Produce a dilution water blank containing unseeded dilution water. Fill a BOD bottle completely with unseeded dilution water. This bottle will serve as a rough check of the quality of the dilution water and the cleanliness of the bottles. If the DO uptake of this bottle is more than 0.2 mg/L, then an alternative source of dilution water or an alternative method of cleaning the bottles should be used and the entire procedure should be repeated. 7. If the water was seeded in step 3, then prepare a seed control bottle. Use the seed source water instead of the sample water to prepare a seed control bottle. 8. Determine the initial DO content of each sample bottle using the procedure outlined in Lab 13. Record the readings in the Data section. 9. Incubate each bottle at 20C + 1C for 5 days.

10. After five days of incubation, determine the final DO content of each sample bottle. Record the readings in the Data section. 11. For each bottle which meets the 2.0 mg/L DO depletion and 1.0 mg/L residual DO requirements, calculate the BOD5 as follows: a. First, calculate, the fraction of sample found in each bottle using the equation shown below. Record this value in the Data section.

b. Next, if bottles were seeded, calculate the "f" value of each bottle using the equation shown below. Record this value in the Data section.

c. Finally, calculate the BOD5 of each sample using the following formula. Record this value in the Data section.

Where: D1 = initial DO of sample, mg/L D2 = final DO of sample, mg/L B1 = initial DO of seed control, mg/L B2 = final DO of seed control, mg/L f = the value calculated in step b. P = fraction of sample as calculated in step a.

Example problem: A BOD test was done on a 5 mL sample. The initial DO of the sample and dilution water was 7.82 mg/L. The DO of the sample after 5 days of incubation was 4.17 mg/L. What was the BOD of the sample?

Initial DO: 7.82 mg/L Final DO: 4.17 mg/L DO Depletion: 3.65 mg/L The BOD bottle used is 300 mL. Therefore the sample fraction was: 5 mL divided by 300 mL = 0.0167 mL

Source Volume of incubation bottle (mL) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Dilution water blank Seed control 0 0 Volume of Initial sample DO added (mL) (mg/L) Final DO (mg/L) Fraction of sample f BOD5

The tables below should be filled out if the pH of the sample is adjusted, if the water is seeded, or if sodium sulfite or nitrification inhibitor are added to the water. pH adjustment

Initial pH Final pH Type of alkali or acid added Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

Seed source Amount of seed added (mL) Seed BOD Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

Other treatments
Sodium sulfite added (mL) Nitrification inhibitor added (mL) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5