The Perfect BOD Procedure

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Scope and Application

The Biochemical Oxygen Demand (BOD) test measures the oxygen required for the biochemical degradation of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic material such as sulfides and ferrous ions. The test is used to evaluate waste loadings and BOD-removal efficiency of the treatment process, and for compliance reporting purposes.

Summary of Method

A bottle is filled airtight with sample, and incubated at a specific temperature for five days. Dissolved Oxygen (DO) is measured initially and after incubation. The BOD is computed from the difference between initial and final DO.



Caustic alkalinity, mineral acid, free chlorine, heavy metals, ammonia and sunlight can interfere with analysis. A nitrification agent is added to inhibit ammonia interference in the BOD test. Exclude light during incubation to prevent the possibility of photosynthetic production of dissolved oxygen. Maintain temperature between 19-21 oC during incubation and analysis. Keep all glassware thoroughly clean to prevent organic interference. Tygon tubing can contaminate dilution water. Flush carboy tubing before each use to minimize contamination.


Avoid unnecessary exposure to the sample and ensure prompt removal from skin, eyes and clothing. Follow the guidelines of the Chemical Hygiene Plan and manufacturers' recommendations for handling all standards and reagents.

Equipment and Supplies

  • Plastic BOD Bottles with acrylic stopper, 300 mL – 2 or 3 per sample
  • Plastic cap to cover bottle during incubation
  • Incubator, 20 oC +/- 1 oC
  • Measuring pipettes
  • 12 or 13 L Bottle with outlet (for BOD dilution water), 1 per day
  • Tubing, PVC, 3/8ID X 1/16 wall X 1/2 OD, for BOD dilution water bottle (DO not use Tygon tubing)
  • Graduated cylinders, 100 mL, 50 mL
  • Personal Pump with tubing for aerating final effluent, secondary samples and seed
  • LDO probe and meter, Hach Company
  • Erlenmeyer Flasks, 2000 mL, 1000 mL, 500 mL
  • Volumetric pipets, 3 mL
  • Thermometer

Reagents and Standards

For instructions on laboratory preparation of these standards, see reference 16.1. Hach reagents are individually packaged for maximum stability, discard after the expiration date on the package.

  • Nitrification Inhibitor- (2-chloro-6(trichloromethyl) pyridine (CTCMP), Hach Company, Stable indefinitely, product number 25335.
  • BOD Nutrient Buffer Pillows, Hach Company, comes in 5 volumes
  • 19L, 25/pk, product number 1486309.
  • 6 L, 50/pk, product number 1486266.
  • 4 L, 50/pk, product number 2436466
  • 3 L, 50/pk, product number 1486166
  • 300 mL, 50/pk, product number 1416066
  • Acid & Alkali Solutions- Stable indefinitely.
    • Acid: Sulfuric Acid, Concentrated
    • Alkali: 10 N NaOH
  • Glucose-Glutamic Acid Solution – Hach Company, 300 mg/L pk/16 10 mL voluette ampoules, product number 14986510
  • BOD Dilution Water-
  1. Clean carboy thoroughly before use with phosphate free soap soak and acid rinse. Rinse thoroughly with deionized water.
  2. Fill carboy with deionized water. To verify cleaning, check pH of water in the carboy and deionized source water. If the pH is not the same between the two within 0.50 pH units, rinse carboy with deionized water again and recheck pH. Record pH in logbook.
  3. Add enough of the BOD dilution water pillows to equal exceed the volume of the water that it is being added to. For example if one had a 13L carboy it would be necessary to add 2 6L pillows and 1 3 L pillow to the carboy.
  4. Swirl the water to dissolve the slurry before use.
  5. The temperature should be 19-21 oC.
  6. The pH should be 6.5 – 7.5
  7. Also check dissolved oxygen level. Dissolved oxygen should be near the oxygen solubility value as compared to the sample temperature. If the DO of the dilution water is greater or less than 100 % of the oxygen solubility value by 2% or more shake the carboy (with the cap on) until the oxygen solubility value is within bounds.
  8. Do not store the dilution water once prepared for longer than it takes to prepare the BODs for analysis. As soon as the BODs are prepared rinse the container out with copious amounts of deionized water. Prior to the addition of nutrients it is advisable to allow the water a “settling time” in the incubator of anywhere from 1 day to 1 week. Never allow deionized water with nutrient sit in the incubator for longer than half a day (long enough to do the test).
  9. Do not aerate dilution water immediately prior to use. If the dilution water must be aerated just prior to use verify that the saturation of the water is with 2% of 100% of the expected value.
  • Sodium Sulfite Solution, approximately 0.025 N-Dissolve 1.575 g Na2SO3 in 1.9 L deionized water. Unstable. Prepare fresh daily.
  • Potassium Iodide, crystals, Stable indefinitely.
  • Starch Solution, 1 % Used for chlorine test. Chempure Cat. No. RS-565-500. Stable indefinitely.
  • Polyseed capsules- Hach Company
  1. Use 400 mL of dilution water to dissolve Polyseed capsule.
  2. If possible get seed from a WWTP. Settled influent works best although primary effluent is also often quite good. Lyophilized seed is never as good as seed found in the environment.

Sample Collection Preservation and Storage

  1. Samples for BOD are collected as 24-hour composites from whichever points the regulatory body requires and whatever frequency that they require. It is common to be regulated on influent and effluent 3-5 days per week.
  2. Samples that cannot be analyzed immediately are refrigerated at 4 oC until time of analysis.
  3. Recommended holding time is 6 hours. Maximum holding time for regulatory samples is 48 hours. Warm samples to 19-21 oC before making dilutions for analysis, using dial thermometer.

Quality Control

  1. An initial demonstration of laboratory capability and ongoing analyses of laboratory prepared water are requirements of the LDO reference method to demonstrate accuracy and precision. A record of this information is available in the laboratory.
  2. Ongoing precision and accuracy will be verified daily by calibration verification and ongoing precision and recovery (See Section 6.2 of Reference Method 16.4).
  3. Blanks-Run three (3) dilution water blanks with each set of samples. Dilution water at 20 oC contains approximately 7 -9 mg/L D.O.
  4. Standards-A glucose-glutamic acid standard check is run with each set to monitor the performance of all systems including the meter. Two 300-mL BOD bottles are prepared using 3 mL of Glucose-Glutamic acid solution and sufficient seed to get a seed depletion of 0.5-1.0 mg/L. If the GGA is not at 198 on average increase or decrease the seed amount used until it is on average 198 mg/L. Break the GGA ampoule, pour into a dry clean 50 mL beaker then pipette 3 mL directly from Glucose-Glutamic acid solution stock using a volumetric pipette.
  5. If check standard is outside the in-house range, reject any BOD determinations and seek the cause of the problem. The standard deviation of the range should be 10-15. Make sure that the GGA value is 198 mg/L on average.
  6. Laboratory Duplicates-Duplicates are run daily on three samples (approximately 10% of the samples analyzed). The range and UCL of the duplicate results will be used to measure method precision.
  7. The range and the UCL are determined using the formulas in 12.1.3. The UCL value will be updated when a minimum of n=20 data points have been collected. The control data form and control chart containing updated limits for this method are maintained in the BOD QC folder.
  8. Incubator Temperature-Verify daily that temperature is 19-21 oC. Record reading on temperature on door of each incubator. Check temperature on all incubator shelves monthly to verify constant temperature throughout incubator. Store dilution water for not more than 4 houre in incubator until just before use.

Calibration and Standardization

  1. Standards
    1. Dilution Water- depletion of less than 0.2.
    2. Glucose-Glutamic Acid Check-Determine the five (5) day, 20 oC, BOD of a 1% dilution of the Hach Company glucose-glutamic acid standard solution following Analytical Procedure.
  2. Calibration
    1. Sensor will be calibrated daily and when a cap is replaced. The manufacturer recommends air calibration, since it is most accurate.
    2. Add approximately ¼ inch of reagent water to a clean BOD bottle and stopper.
    3. Shake vigorously for ~ 30 seconds.
    4. Allow 30 minutes for the BOD bottle and its contents to equilibrate to room temperature.
    5. The stopper may now be removed from the BOD bottle and the probe inserted for calibration purposes.
    6. Turn on meter by pressing the on button on the keypad.
    7. Press the Blue, Left key under Calibrate on the display. Screen will display “Dry the probe. Place it in water-saturated air and press Read”.
    8. Dry the probe and place in bottle.
    9. Press the Green, Right key under Read on the display.
    10. Wait for the display to stabilize.
    11. Press the Green, Right key under Store to accept the calibration. The display will return to measurement mode. An OK in the upper left corner indicates the calibration was successful.
    12. Record date, time, calibration data and temperature.


Carbonaceous BOD Analysis Sample Pretreatment

  1. pH Adjustment-Warm samples to 19-21 oC in sink filled with warm water. Use dial thermometer to get general idea of sample temperature. Verify the pH of the samples by taking a new pH of the composite or observing individual readings on the pH lab sheet. If the pH is not 6.0 to 8.5, neutralize with concentrated sulfuric acid or 10 N NaOH. Do not dilute samples more than 0.5%. Seed any samples that are pH-adjusted.
  2. Aerate Effluent and Secondary plant samples for a minimum of 5 minutes to remove super saturation.
  3. Dechlorination-Test final effluent samples for chlorine by the following procedure when the effluent sample chlorine residual exceeds the method detection limit:
    1. Measure 100 mL of well-mixed sample into a 250 mL Erlenmeyer flask.
    2. Add a few crystals of potassium iodide (KI) to the flask and dissolve the crystals.
    3. Add 1 mL of concentrated sulfuric acid and mix well. Finally, add five drops of starch. If a blue color is produced, chlorine is absent. If a purple/red color is produced, chlorine is present. Regardless, the sample must be seeded.
    4. If a purple/red color is produced, titrate the sample with 0.025 N sodium sulfite (Na2SO3) to the endpoint between the last trace of blue color and a colorless solution. Make the titration very slowly, counting the number of drops of 0.025 N sodium sulfite used.
    5. Dechlorinate sample for BOD test using the following procedure:
    6. Measure another 100-mL portion of the well-mixed composite sample into a clean 250-mL Erlenmeyer flask.
    7. Add the number of drops of 0.025 N sodium sulfite (n) determined necessary for dechlorination in section and mix well.
    8. Seeding of Samples
    9. If you use Polyseed, prepare it according to the manufacturers directions.
    10. It is preferable to use plant influent or primary effluent for seed, if this is possible.
    11. Evaluate at least 2 dilutions of the seed so that it will be possible to determine how much of the seed is needed to obtain a depletion of 0.6-1.0 mg/L on subsequent runs.
    12. The formula for deciding how to seed is to take the volume used to make the seed controls and multiply that value by 0.8 then divide that number by the original ~ mg/L of the seed’s depletion: For example if we had 3 mg/L of depletion when we placed 9 mL of seed in dilution water, we would expect the same seed to have ~ 0.8 mg/L if we used 2.4 mL of seed instead. So we should use 2.4 mL of seed to seed our GGA and seedable samples in this example.
    13. Seed any sample that has been chlorinated, pH adjusted, or disinfected.
    14. Add a small amount of dilution water to two seed control bottles. Add the quantity of seed to the control bottles that will allow them to deplete by approximately half. These bottles measure the actual oxygen depletion of the seed and serve as guide for how much seed to use in subsequent analyses

BOD Analysis of Samples With and Without Seeding, Influent, Stormwater and Effluent Samples

  1. Prepare three blanks by filling three bottles completely with dilution water that has been checked for temperature, pH and dissolved oxygen. Insert stopper in the bottle and ensure that no air is trapped in the bottle.
  2. Using a graduated cylinder, prepare dilutions (3-5 for each sample) to meet approximately 4.0 mg/L of Dissolved Oxygen. Typical dilutions are listed in TABLE 1. Dilutions should be adjusted according to trends in BOD values in order to meet criteria. Effluent and secondary samples are always seeded.Note: Each sample bottle must contain at least 5% (15 mL) dilution water, if this is not practical use DI water for dilution and a single 300 mL BOD buffer pillow per bottle.
  3. Read the DO and temperature immediately after preparing dilutions for samples, duplicates and blanks and record on the forms listed in Data Analysis and Calculations. Sample temperature should be 17-23 oC.
  4. Samples are incubated in the dark at 19-21 oC for five (5) days +/- 3 hours. Place all seeded sample bottles on the same shelf during incubation.
  5. Read the D.O. reading after incubation and record on the forms in Data Analysis and Calculations. NOTE: Verify sample temperature is 19-21 C before reading final DO.
  6. Add 10-mg (or 2 measures from the dispenser) of the nitrification inhibitor to each bottle before incubation. Ensure that no air bubble is present in the BOD bottle
  7. Instrument Readings-Set the instrument calibration as listed in Calibration.
  8. Sample dilutions must meet the criteria of a residual DO of at least 1 mg/L and a depletion of at least 2 mg/L. If both dilutions meet these criteria, the results can be averaged.
  9. Place the LDO sensor in the sample.
  10. Press the Green/Right key under Read on the display.
  11. Wait for the display to stabilize. The result will be automatically stored in the data log.
  12. Repeat for each measurement.
  13. Clean all glassware, aerators and carboys after use following the glassware and plastic ware cleaning standard operating procedure.

Data Analysis and Calculations

  1. Calculate according to standard calculations.
  2. Precision and Accuracy-Equations used to calculate precision and accuracy are listed on the control data sheets located in the BOD folder.
    1. Reporting
    2. Record all bottle numbers, dilutions, pH, temperature, date and time of sampling and initial DO (mg/L) on the BOD data sheet. Once the final values have been obtained, record the final DO (mg/L) readings on the same data sheet.
    3. Do not use a meter reading less than 0.2 mg/L. Calculate the sample concentration using the formulas above and record results (mg/L) for the seeded samples.
    4. Sample concentration results are transferred from the BOD Data sheet to the computer for reporting on the monthly report and industrial reports.

Method Performance

Method Detection Limit (MDL)-This method is accurate for BOD in the concentration range of 1 mg to 3000 mg/L based on the data contained in the reference method 14.1.


  1. Environmental Protection Agency, Methods for the Chemical Analysis of Water and Wastes, Method 405.1, June 1974, Revised, March 1983
  2. APHA, AWWPCF, Standard Methods for the Examination of Water and Wastewater, Method 5210 B, p.p. 5-2 - 5-6, 20th Edition, ,1998.
  3. Hach Company, HQ Series portable Meters User Manual, June 2006 Edition 4.
  4. HACH Method 10360 – Luminescence Measurement of Dissolved Oxygen (LDO) in Water and Wastewater
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