BIOLOGICAL PROCESS CONTROL EES 4111L

LAB #3 Biochemical Oxygen Demand Lab

INTRODUCTION:

The methods commonly used to determine BOD were developed with the intent of closely simulating conditions found in nature. Essentially, the procedure involves determination of the amount of dissolved oxygen used by bacteria as they consume the organic matter present in a sample being tested. By convention, a five-day test period is used and the samples are incubated in the dark at a temperature of 20C.

The origin of the five-day test period is somewhat obscure. The most commonly held theory (i.e. the legend) is that five days was chosen because where the test was originated (England) the longest time-of- flow to the ocean was about five days. The 20C incubation temperature was chosen as being reasonably representative of field conditions. The samples are incubated in the dark to eliminate the effects of algae that are often present in waters and wastewaters. This is necessary because, when exposed to light, algae will produce dissolved oxygen and, thus, cause the BOD to appear lower than it really is.

LOW BOD SAMPLES:

For samples having a BOD less than about 8 mg/L, the standard three-bottle test may be used. Normally, the best practice is to aerate the sample for a few minutes to assure that it is approximately saturated with dissolved oxygen. This may be accomplished by bubbling air through the sample or by shaking the sample vigorously, admitting air to the container from time to time. Once the sample has been saturated, three BOD bottles are carefully filled. (NOTE - it is very important to pour the sample into the three bottles without causing any additional aeration or deaeration.) The dissolved oxygen is determined in the bottles immediately.

The most common method of measuring D.O. in the field or in wastewater treatment plants is to use a dissolved oxygen probe which measures the activity of oxygen using a permeable membrane and a cathode to generate electric current in the probe which is received by the meter. The results are read on a scale that shows the D.O. concentration in mg/l. Using this technique it is possible to measure the D.O. in all three bottles.

Another dissolved oxygen analysis technique is a wet chemical procedure in which the sample is destroyed. Thus, when this method is used, it is not possible to determine the 'before' and 'after' dissolved oxygen (D.O.) concentration for the same bottle. The other two bottles are incubated for the prescribed period, usually five days, and then subjected to the dissolved oxygen analysis.

The BOD of the sample may be determined as follows for the wet chemical technique:

BOD = IDO - FDO

Where:

BOD = the BOD of the sample in mg/L,

IDO = the D.O. in the bottle analyzed immediately, and

FDO = the average final D.O. in the incubated bottles.

Alternatively, the initial D.O. may be determined with a non-destructive method (i.e. with the D.O. probe) in each of the three bottles. Then, all three may be incubated and subjected to dissolved oxygen analysis again at the end of the prescribed period. If this procedure is used, the BOD is calculated in the same way except as follows:

IDO = average initial D.O. in mg/L, and

FDO = average final D.O. in mg/L.

Several types of reliable dissolved oxygen meters are available for non-destructive testing. Regardless of the dissolved oxygen determination method used, it is good practice to base the results on replicate samples.

HIGHER BOD SAMPLES:

Oxygen is almost insoluble in water, the saturation concentration being only about 9 mg/L at a temperature of 20C. Therefore, the method described above can be used only when the BOD is less than about 8 mg/L. For higher BOD samples, a dilution technique must be used.

The procedure followed when dilution is necessary is similar to that used for low BOD samples, except that the sample must be diluted prior to analysis. The BOD of the original sample is calculated by 'correcting' the test results for the effect of dilution as follows:

BOD = (IDO-FDO)_sample - f(IDO-FDO)_seed

DF

Where:

f = mL seed in sample/mL seed in seed control

DF = the dilution factor = % dilution/100,

The solution used for dilution is generally known as 'dilution water'; however, it is not just plain water. It must be specially prepared to ensure that all essential nutrients and trace elements, except organic carbon, are present. Dilution water "seed" is added to the dilution water to insure that there is a bacterial culture capable of metabolizing the organic material present in the sample(s) to be tested. This is usually accomplished by adding 1 or 2 mL of the seed material to each liter of dilution water. Typically, settled sewage or stream water taken from a point downstream of the wastewater discharge is used for seed. A commercially prepared seed may be used. In this case, the manufacturer states the amount of seed to be used. No seed is required for samples already containing microorganisms such as sewage or unchlorinated activated sludge effluent. In this case "f(IDO-FDO)seed" is simply omitted from the equation on the previous page.

Experience has shown that BOD determinations are not dependable if less than about 2 mg/L of dissolved oxygen is used during the course of the test. Thus, samples exhibiting an oxygen depletion of less than 2 mg/L should not be used in estimating BOD. Also, it is necessary that the final dissolved oxygen concentration be greater than 0.5 mg/L to ensure that aerobic conditions were maintained throughout the test period. (Some analysts say that a minimum of 1.0 mg/L is needed.) Therefore, samples exhibiting a residual dissolved oxygen of less than 0.5 mg/L (or 1.0 mg/L) should not be considered in calculating BOD values. Obviously, one generally does not know the BOD of a sample prior to running the BOD test. Thus, it is necessary to estimate the BOD very accurately prior to running the test, or to run tests using more than one dilution factor. Some suggested dilution factors are shown in Table 1, below

TABLE 1

SUGGESTED DILUTIONS FOR BOD TESTS

PERCENT SAMPLE DILUTION FACTOR RANGE OF VALID

USED BOD VALUES (mg/L)

100 1.0 0 - 7

50 0.5 4 - 14

20 0.2 10 - 35

10 0.1 20 - 70

5 0.05 40 - 140

2 0.02 140 - 350

1 0.01 200 - 700

0.1 0.001 2000 - 7000

0.01 0.0001 20000 - 70000

Generally, several dilutions of each sample are made and tested. By this means, at least one set of valid test results should be obtained. When more than one set of valid test results is obtained, the results of the least dilute valid test may be used. Alternatively an average BOD may be calculated. Analysts disagree as to the best approach.

LABORATORY PROCEDURE:

The first step in determining the BOD of any sample is to select the appropriate dilution factor. Sound engineering judgement plus the use of at least three different dilution factors generally ensure that valid results will be obtained. After selecting the proper dilution factors, you must make the necessary dilutions. To accomplish this, siphon (or carefully pour) about one-half of the required dilution water into a 1000 mL graduated cylinder. Add 2 mL of seed mixture if needed. Then, add the required sample volume (this will vary depending upon the dilution factor selected) into the cylinder and carefully fill the remaining volume with dilution water. If 100 mL or more of sample volume is required, a graduated cylinder may be used in lieu of a pipette. After the final dilution water has been added, use a plunger-type mixer to gently mix the sample and dilution water. It is very important that a good mixture be obtained. For dilution factors of 0.2 or greater, it will be necessary to aerate the diluted sample for approximately 5 minutes to insure adequate initial dissolved oxygen. To make a seed control, dilute the seed mixture with dilution water to a total of 1000mL.

If the initial D.O. can be measured for all duplicates of a dilution using a non-destructive technique (i.e. the D.O. probe) then dilutions can be prepared directly in each BOD bottle. The volume of each bottle is 300 ml and using this the volume of sample to yield the required dilution can be pipetted into the bottle. The bottle is then filled with dilution water, with seed added if it is required.

After the diluted sample has been prepared, carefully pour the contents of the cylinder into three standard BOD bottles. Be sure to fill the bottles completely to the very top. Since each bottle holds 300 mL, you will have more than enough diluted sample to completely fill all three bottles. It is very important that the filling operation be done with care since the validity of the results depends upon the assumption that the contents of each of the three bottles are exactly the same. Each bottle should be stoppered and sealed immediately after filling is completed. The stopper should be inserted so that no air bubbles are trapped in the bottle. This procedure will displace some of the diluted sample and some will remain in the neck of the bottle above the stopper. This serves as a water seal that helps to ensure that air will not leak into or out of the bottle during the test period. The numbers on the bottles should be recorded so they can be identified later. Many of the stoppers are also numbered, so be sure to always record the bottle number.

Determine the dissolved oxygen concentration in one of the three bottles and place plastic caps on the other two and place them in a 20C incubator. The bottles should be inspected occasionally and the water seal replenished as necessary to ensure that no leaks occur. After the prescribed incubation period, these bottles should be removed from the incubator and also subjected to analysis for dissolved oxygen.

Equipment:

300-mL incubator bottles

Air incubator set at 20 C +/- 1 degree

DO meter and probe

Reagents:

Phosphate buffer

Dissolve 8.5 g KH₂PO₄, 21.7 g K₂HPO₄, 33.4 g Na₂HPO₄, and 1.7 g NH₄Cl in deionized water. Adjust pH to 7.2, if necessary, with either 1 N H₂SO4 or NaOH. Dilute to one liter.

Magnesium Sulfate

Dissolve 22.5 g MgSO₄.7H₂O and dilute to one liter.

Calcium Chloride

Dissolve 27.5 g CaCl₂ and dilute to one liter.

Ferric Chloride

Dissolve 0.25 g FeCl₃.6H₂O and dilute to one liter.

Note: To prepare dilution water, add one mL of each of the four solutions listed above to one liter of deionized water. Saturate with DO by drawing a vacuum through the solution.

Glucose-glutamic acid check:

Since the seed must be of certain strength in order for proper test results to be obtained, a check must be performed. Take 0.150 g of each of the dried glucose and glutamic acid and add them to a one-liter volumetric flask. Stir to dissolve and dilute to one liter. Do not prepare this solution until you are ready to begin testing.

BOD Test:

Once the meter is calibrated and the samples are prepared, arrange the proper amount of bottles on a cart. Include three bottles for blanks, three for seed, and three more for the glucose-glutamic acid test. Start with the blanks first. Fill the bottles to capacity with the dilution water and measure the DO by inserting the probe and obtaining a stable reading. This reading should be close to the DO you used for calibration. Refill the bottles with dilution water and seal with a stopper and a cap. Next place the seed water in each of the three bottles. Fill to the top with dilution water. This is a seed control that will be used later for seed corrections. Again take a reading, refill and stopper. For the samples: if a sample has not been chlorinated or is not suspected of having anything toxic present, the sample maybe diluted serially with dilution water and analyzed. For the glucose-glutamic acid check and for any sample that has been chlorinated or is in any way suspect, seeding must be performed. Simply add the seed, the desired volume of sample, and fill to capacity with dilution water. Take a reading, refill with dilution water and stopper. These initial readings are the initial DOs of the samples. The samples are incubated over the course of the next five days +/- two hours at 20 C +/- 1 C. When the incubation period is complete the samples are removed from the incubator and the final DO determined in the same manner as before. The following calculations are then used to obtain the BOD:

Use averaged values for the dilution blanks and seed controls in the following calculations.

Non-seeded sample:

BOD (mg/L) =

Where: D₁ = DO initial

D₂ = DO final

P = volumetric fraction of sample used (Dilution Factor)

Seeded sample:

BOD (mg/L) =

Where: D₁, D₂, and P are as stated above

B₁ = initial DO of seed control

B₂ = final DO of seed control

BOD Data Table:

Date/Time Started:_______ Cal: DO________ Temperature:_______

Date/Time Started:_______ Cal: DO________ Temperature:_______

Sample

Sample I.D.

Vol. Sample (mL)

Vol. Seed (mL)

Dilution Factor

Initial DO

Final DO

mg/L BOD

ASSIGNMENT:

1. Calculate the final BODs for all bottles.

2. Test the blanks, seed controls, and G-G acid test results for outliers. Show all calculations and state if any of the data needs to be thrown out. In addition the dilution water blanks can not deplete more than 0.2 mg/L. Was this criteria met?

3. If a bottle has a final DO of less than 1 mg/L, it is discarded if possible. If a bottle has depleted less than 2 mg/L of dissolved oxygen, it is discarded if possible. For obvious reasons, these criteria are inappropriate for application to the blanks or seed controls. Point out all bottles that you would discard. Perform an outlier check on the remaining sample BOD values. Based on the outlier check, state which values should be discarded. Now average the remaining BOD values and report this as the final BOD in mg/L for each of the samples.

4. Common sense should tell you that the more sample that is present in a BOD dilution the greater the dissolved oxygen depletion should be. When there is something in the sample that may inhibit the test; this may not be the case. A ‘toxic effect’ occurs when a dilution that contains a lower percentage of the sample depletes more dissolved oxygen than dilutions with a higher percentage of the same sample. When a toxic effect occurs all the data for that sample dilution and for all preceding dilutions with higher percentages of that sample become suspect. Are there any samples exhibiting this effect and if so which dilutions are suspect?

5. If a seed of the proper strength was used the value for the G-G acid test, the results would be in the range of 200 +/- 37 mg/L BOD₅. Report the average value for the test. Is it acceptable?

6. For the glucose-glutamic acid and influent wastewater, plot the biochemical oxygen demand exerted with time. Comment on the plot.

REFERNCES:

· Standard Methods for the Examination of Water and Wastewater. 1992. PP 4-98,

4-103 to 4-105, 5-1 to 5-6 referenced 5210-B

· Sawyer, McCarthy and Parkin. Chemistry for Environmental Engineers. Fourth Ed.

McGraw-Hill. 1994. PP 527 to 544.