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The test for Biochemical Oxygen Demand is especially important in waste water treatment, food manufacturing, and filtration

facilities where the concentration of oxygen is crucial to the overall process and end products. High concentrations of dissolved oxygen (DO) predict that oxygen uptake by microorganisms is low along with the required break down of nutrient sources in the medium (sample). On the other hand, low DO readings signify high oxygen demand from microorganisms, and can lead to possible sources of contamination depending on the process. Performing the test for Biochemical Oxygen Demand requires a significant time commitment for preparation and analysis. The entire process requires five days, and it is not until the last day where data is collected and evaluated. During this time, samples are initially seeded with microorganisms and supplied with a carbon nutrient source of glucose-glutamic acid. The sample is then introduced to an environment suitable for bacterial growth at reproducible temperatures, nutrient sources, and light within a 20 degree Celsius incubator such that oxygen will be consumed. Quality controls, standards and dilutions are also run to test for accuracy and precision. Determination of the dissolved oxygen within the sample can be determined through Winkler titration methods. The difference in initial DO readings (prior to incubation) and final DO readings (after 5 days of incubation) predicts the BOD of the sample. A suitable detection limit as per environmental QC is 1 mg/L. Click on to the following links for this procedure: Equipment Reagents Sample Preparation Procedure Analysis Calculations References Suggestions for Further Use

Equipment
The following is a list of necessary materials and equipment for starting the procedure: 1. 2. 3. 4. 5. 6. 7. 8. 9. Series of 250-300 ml BOD bottles with ground glass stoppers, and caps Incubator set at 20 degrees Celsius Two large carboys (20 liter capacity depending on sample amount) Series of class A pipets (0.2 ml-10 ml) Aeration device pH meter Six 500 ml ehrlenmeyer flasks Millipore water Phosphate buffer pillows for every 6 liter carboy volume

10. DO meter with membrane electrode 11. Seed 12. 50 ml class A buret 13. Stir plates and stir bars 14. Series of volumetric flasks 15. Series of beakers 16. Deionized water The following is a list of reagents used in this method that are commercially prepared: 1. Concentrated Sulfuric Acid 2. 0.0375 N Sodium Thiosulfate 3. Starch indicator 4. Crystalline D-glucose and glutamic acid 5. Bleach (5.25%) 6. Acetic Acid 7. Potassium Iodide 8. Sodium hydroxide pellets 9. Manganese sulfate 10. Sodium sulfite The remaining reagents are prepared within the laboratory. Caution must be taken since the shelf life of these reagents should not exceed 6 months unless otherwise noted. 1. 0.1 N Sulfuric Acid: Add 2.8 mls concentrated acid to 1 liter distilled water. 2. 0.1 N Sodium Hydroxide: Add 4 grams sodium hydroxide pellets to 1 liter distilled water. 3. Sodium Sulfite solution: Prepare daily. Dissolve 1.575 g sodium sulfite up to one liter deionized water. 4. Manganese Sulfate solution: Dissolve 364 grams manganese sulfate up to one liter deionized water. Slight heating and filtration may be necessary. 5. Glucose-Glutamic solution: Dissolve 75 mg glucose and 75 mg glutamic acid up to 500 ml deionized water. Sufficient stirring is required. 6. Alkali Azide solution: In a 1 liter volumetric, dissolve 500 gram sodium hrdroxide pellets with 150 grams potassium iodide. When dissoliution is complete, add an additional 40 mls distilled water with 10 grams sodium azide. Caution must be taken when handling this solution. 7. 1+1 Acetic Acid: 500 ml pure grade acetic acid is added to 500 ml deionized water with stirring. Caution must be taken since the temperature of the solution will increase rapidly. 8. Potassium Iodide solution: Dissolve 10 grams potassium iodide in 100 ml volumetric flask with distilled water.

The following is a list of reagents used in this method that are commercially prepared: 1. Concentrated Sulfuric Acid 2. 0.0375 N Sodium Thiosulfate 3. Starch indicator 4. Crystalline D-glucose and glutamic acid 5. Bleach (5.25%) 6. Acetic Acid 7. Potassium Iodide 8. Sodium hydroxide pellets 9. Manganese sulfate 10. Sodium sulfite The remaining reagents are prepared within the laboratory. Caution must be taken since the shelf life of these reagents should not exceed 6 months unless otherwise noted. 1. 0.1 N Sulfuric Acid: Add 2.8 mls concentrated acid to 1 liter distilled water. 2. 0.1 N Sodium Hydroxide: Add 4 grams sodium hydroxide pellets to 1 liter distilled water. 3. Sodium Sulfite solution: Prepare daily. Dissolve 1.575 g sodium sulfite up to one liter deionized water. 4. Manganese Sulfate solution: Dissolve 364 grams manganese sulfate up to one liter deionized water. Slight heating and filtration may be necessary. 5. Glucose-Glutamic solution: Dissolve 75 mg glucose and 75 mg glutamic acid up to 500 ml deionized water. Sufficient stirring is required. 6. Alkali Azide solution: In a 1 liter volumetric, dissolve 500 gram sodium hrdroxide pellets with 150 grams potassium iodide. When dissoliution is complete, add an additional 40 mls distilled water with 10 grams sodium azide. Caution must be taken when handling this solution. 7. 1+1 Acetic Acid: 500 ml pure grade acetic acid is added to 500 ml deionized water with stirring. Caution must be taken since the temperature of the solution will increase rapidly. 8. Potassium Iodide solution: Dissolve 10 grams potassium iodide in 100 ml volumetric flask with distilled water.

Procedure
Once reagents have been properly prepared, the sample is ready for analysis. 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. Carboy Set Up Seed Preparation DO Preparation BOD Sample Preparation

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. Once the acid is flushed from the carboys, a small amount of bleach is added to eliminate excess organisms that may serve as sources of interference. The carboys are again flushed with adequate deionized 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 atleast 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.

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 mls 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 effect the results.

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. This method is not contained in the BOD procedure, but rather comes from Standard Methods for the Examination of Water and Wastewater (Method 218B: Azide Modification: 1971) In my experience, we often used a pH meter with an electrode 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 ehrlenmeyer 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.

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 analysed. 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. The blank DO should normally be less than 0.2 mg/L.

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. The following calculations are taken from Standard Methods. A. The BOD of the blanks are calculated by subtracting the final DO from the initial DO: BOD blank=DO1-DO2; B. The BOD of the seeds are calculated by subtracting the final DO from the initial DO and multiplying this number by the dilution factor: BOD seed=(DO1-DO2) * Dilution factor per 300 ml C. The BOD of sample and standards are calculated by subtracting the final DO from the initial DO and multiplying this number by the dilution factor. The final value is determined by substracting out the BOD blank and the seed blank for each delta DO. If the testing procedure was carried out correctly and the dilutions of the sample were made appropriately, the analyst should have obtained BOD values that are within a reasonable percent error and relative percent difference. Generally, values are discarded for a specific sample dilution if the final DO of the sample is < 1.0 mg/L of if delta DO is < 1.0 mg/L. This also stresses the importance of using different dilutions for each sample to key in on the appropraite BOD when little is known about how the sample will react and how high its BOD levels are.

References
The majority of the information for this tutorial came from : Standard Methods for the Examination of Water and Wastewater. 16th Edition: 1985. Method 507, pg 525-532. Standard Methods for the Examination of Water and Wastewater. 13th Edition: 1971. Method 218B, pg 477-481. The laboratory procedure with which I am familiar also makes reference to the United States Environmental Protection Agency Methods for Chemical Analysis of Water and Wastes. March 1979: 1974 editorial revision. Method 405.1.

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