Biochemical Oxygen Demand

Biochemical Oxygen Demand Procedure for BOD Testing The BOD procedure including the DO analysis is actually quite lengthy, so time maintenance is important. This portion can be carried out in four separate steps including carboy setup, adjustment of DO, preparation of seed inoculum and BOD sample preparation. It is best to set up the carboy as soon as possible to ensure proper results. In addition, the BOD sample preparation is the last step of the procedure.

1. Carboy Set Up The BOD procedure calls for two carboys to be used each time the procedure is carried out. The carboys will contain the water necessary for the procedure, and two are used instead of one to serve as a source of comparison amongst carboys. Also, it is a good idea to have two sources of water in case something goes terribly wrong with one of the carboys; there will be some sort of back up. To ensure that the two carboys involved in the procedure are free of contaminants, the carboys must first be rinsed with acid and water. Sufficient water should be rinsed to eliminate all traces of acid. The carboys are again flushed with adequate de-ionized water until the presence of bleach is eliminated. The carboys are then filled with pure water that has been finely filtered. Millipore water seems to work best in this procedure. The carboys should be filled up to supply at least six liters for the first sample, and three liters for each successive sample. The six liters for the first sample are necessary because quality controls will also be included as part of the run. Once filled, one phosphate buffer pillow is added to the carboy per six liters water. These pillows are commercially available, and save the analyst time from preparing the reagents. After addition, the water solutions must be aerated until the point of saturation. Commonly, many laboratories will prepare this water the day before the procedure, but five hours prior to analysis appears to be sufficient. After aeration, the water is ready to be used to fill the BOD bottles, and must be capped until then.

2. Seed Preparation The seed solution must be set up some time before the BOD bottle are filled with water. This is done by adding 0.045 grams of a polyseed inoculum (or sometimes a BOD sample can be used as the inoculum) to 250 ml distilled water. The seed will not dissolve but it is important that the seed is stirred continously at moderate speed for about two hours. At approxiamtley 30 minutes prior to use, the seed solution is allowed to settle undisturbed. Caution must be taken not to disturb the seed particles because the liquid portion of the solution is used in the procedure. Remnants of actual seed within the prepared BOD bottles could greatly affect the results.

3. DO Preparation The DO (dissolved oxygen) of the prepared carboy water must be determined to serve as a reference to all other sample and standard readings. This is most often accomplished by performing a Winkler titration on the carboy water, and adjusting the DO meter to this reading. A pH meter with an electrode can also be used, that could also determine the DO of the solution as our DO meter. The Winkler titration is carried out by first withdrawing three samples of water from each carboy. Caution must be taken that no air bubbles become trapped in the bottle. As a result, it is best to withdraw the water slowly while the bottles are tilted slightly. Once filled, one bottle from each carboy is set aside until later. The remaining four bottles are used in the actual titration. 2 mls manganese sulfate is added into each bottle under the surface of the water, followed by 2 mls alkali azide solution. The bottles are stoppered and shaken until a brown floc appears. The bottles are allowed to settle until the floc is halfway settled. The bottles are then shaken again and allowed to settle. Once settled, 2 mls concentrated sulfuric acid is added down the neck of each bottle and the bottles are shaken again. At this point, the floc will disappear and the solutions should be amber in color. Now, the solutions are ready for titration.

The solutions are first transfered to 500 ml Erlenmeyer flasks. Each solution is titrated with 0.0375 N sodium thiosulfate until the solution turns a pale yellow. (Standard Methods suggests using 0.025 N sodium thiosulfate for a different volume of sample.) At this point, a few drops of starch indicator are added to turn the solution a dark blue. Titration continues until the solution turns clear. The volume of titrant used in mls directly corresponds to the DO of the sample. The average DO reading from each carboy is calculated and recorded. If the DO readings from each carboy set are not relatively close to each other (within 0.1 mls) the process must be repeated until consistent DO readings are obtained. The DO readings should normally fall between 6.0 and 9.0 mg/L. If values are greater than 9.0 mg/L, the carboy water must be aerated again to reduce the DO.

Once the DO readings are calculated, the two bottles that were withdrawn at the beginning are now used. The carboy bottle from which the samples will be made up from is used to adjust the DO meter (in our case, the pH meter). Once adjusted, the DO of the other bottle is measured to determine whether the meter is reading correctly. If the meter reading comes within 0.1 of the Winkler reading, the calibration is complete. If not, the entire Winkler procedure must be completed for both carboys.

4. BOD Sample Preparation Finally, the last step in the BOD procedure involves inoculating the sample with various dilutions along with standards and blanks for quality control. The following quality control must always accompany each BOD run: - 2 carboy water blanks - 4 standards - 2 seeded blanks - optional control sample Usually, many laboratories will include one set of QC for every ten samples. Additional QC is necessary for more than 10 samples.

Preparation: 1. Water blanks - carboy water is withdrawn to the rim of the bottles. 2. Standards - 4, 6, 6, and 8 mls of standard solution are added to separate bottles. An additional 2 mls of seed solution is added to each bottle and the bottles are then filled to the rim with carboy water. 3. Seeded blanks - 2mls of seed solution are added to each of 2 BOD bottles. The bottles are filled to the rim with carboy water. 4. Samples- 4-5 bottles are usually necessary for each sample. Some samples may have to be diluted in order for the DO range to be detected by the meter. Observation of the sample will usually give an indication to its dilution. Clean samples usually require small dilutions whereas wastewater samples will need high dilutions due to their high BOD values. Once reasonable dilutions have been determined, the specific volume of sample is added to each bottle along with 2 mls polyseed. The bottles are filled to the rim with carboy water.

Once all the bottles have been filled, the initial DO's of each solution is determined on the meter and recorded. Once recorded, the bottles are capped with ground glass stoppers to avoid excess bubbles and capped. The bottles are placed in an incubator at approxiamtely 20 degrees celsius where they will remian for five days.

Analysis After five days of incubation, the samples are ready to be analyzed. The samples are removed from the incubator and allowed to equilibrate to room temperature. In the meantime, the analyst should calibrate the meter again with the carboy water as discussed in the DO Sample Preparation section. It may be desired to use fresh carboy water for the calibration as discussed in the Carboy Set Up page.

Once the meter is calibrated, the samples are read starting with the blanks and ending with the actual samples. The final DO of each solution is recorded and the initial and final readings will be used to calculate the BOD. The best results come about when the initial and final DO values for the blanks are similar indicating the absence of organisms and reliable equipment.

BOD Calculations

The calculations for BOD take into account the unseeded blanks and the seeded solutions. These values must be subtracted out in order to obtain reasonable BOD results. 1. The BOD of the blanks are calculated by subtracting the final DO from the initial DO: BOD blank=DO1-DO2; = 7.4 – 7.24 = 0.16 mg/L

By: Varundeep Singh MFTech, NIFT, New Delhi