Understanding Biochemical Oxygen Demand (BOD)


BOD, like COD, is not one definable particle. You cannot count BOD molecules. BOD is the amount of oxygen consumed by decomposition of the sample during the incubation period. The intent is to measure what affect the sample will have on oxygen available to living organisms in the waters into which the waste is discharged. If the BOD of a waste is high enough, the microbial population will quickly deoxygenate the water and render it unsuitable for other forms of marine life. This can cause dead zones in a river or other body of water. There is an additional subset of BOD that is required in certain areas. This is referred to as carbonaceous BOD or CBOD. This measures along the same basic principle as BOD, except that an inhibitor is added to exclude the oxygen consumption by nitrogen fixing bacteria. Sometimes BOD will be referred to as BOD5 or five-day BOD. This number is related to the incubation period required for the standard analysis. Other versions of the test are possible and are distinguished by the proper numerical qualifier which is equal to the number of days of incubation. This can go all the way up to ultimate BOD; incubating the sample and reading the depletion until nothing else happens. According to BOD legend, the source of the particular requirements for BOD incubation (5 days at 20°C) arise from the average temperature and flow time of the Thames river from London to the sea. An alternative theory, proposed by yours truly, is that the original BOD chemist, or possibly alchemist, went to a production of Shakespeare’s MacBeth. During the scene with the witches and the cauldron he started commenting on how there wasn’t any QC for the method the witches were following. His fellow viewers got annoyed and put the poor guy in the hospital where he remained for five days. Once he got back to his lab, instead of redoing his oxygen demand experiment with overnight values he wrote 5 days into his procedure and left it as is. You decide which version is more likely.

Approved methods

While there are a few different methods approved for BOD, one of them is used overwhelmingly by the analytical community, Standard Methods 5210B.

Method summary

A known volume of sample has its initial dissolved oxygen content recorded. After a five-day incubation period at 20°C, the sample is removed from the incubator and the final dissolved oxygen content is taken. The BOD value is calculated from the depletion and the amount of sample used.

What you should know

The actual procedure is so much more involved than the summary given above. The biggest challenge in the BOD test is that of time. The holding time for a BOD sample is 48 hours from collection. The test itself requires a five-day incubation period. Doing the math shows that by the time you find out if your test is valid, it is too late to do anything about potential problems. Therefore, you must give the utmost care to each step of your procedure to avoid the possibility of having invalid data.

Environmental Express Disposable BOD Bottle

Source water can be the trickiest part of beginning your BOD analysis. Your water will eventually be conditioned by the addition of certain trace nutrients so that the bacteria population can survive. Before this happens, you need water that is free from any and all contaminants that could contribute to the oxygen demand of the samples. It also needs to be free of certain metal contaminants that could inhibit the microbial growth. Simple distillation is not always suitable because volatile components and residue from certain metals can distill over with the water. Many labs have good results from a deionized water system. Special care must be paid to the maintenance of the tanks with these systems as organics can leach from the resin beds. Another option for the source water is to purchase it. Anything that is labeled “steam distilled” has gone through a specific process that further cleanses it from unwanted impurities. The ultimate determination of suitability of the water is if the oxygen depletion after a 5-day incubation is less than 0.2 mg/L. Laboratories that are well versed in BOD analysis should be able to routinely produce blanks with a depletion of less than 0.1 mg/L.

For BOD to operate properly, there must be a sufficient population of healthy bacteria in the bottle. Maintaining that bacterial population can be difficult for the average analyst, especially one in a commercial lab. Wastewater treatment plants will typically have a ready supply of bacteria, but it isn’t always in the places one would expect to find it. The influent typically has a very high oxygen demand, but not necessarily a good population of bacteria. It tends to be variable with the fluctuations depending on time of day and weather. Also, influents run the risk of being toxic in nature. The best source of seed material is found in the plant that treats the waste. Depending on the process and operating conditions in the plant, effluent from the primary or secondary treatment process will contain sufficient numbers of bacteria to inoculate the sample. Be sure to pull from a spot in the process stream ahead of the disinfection stage. If you do not work at a wastewater plant, it may be possible to obtain a suitable amount of the effluent and keep a ‘seed farm’ in your lab. You will need to pay special attention to the feeding and aeration of the stock, as well as its performance over time. If a natural source of seed is unavailable to you will need to use a freeze-dried seed. These seed materials will need to be rehydrated prior to use. Follow the manufacturer’s instructions to prepare the seed. Generally, the volume of water used to rehydrate the seed can be increased if the seed concentration needs to be lowered or decreased if the seed concentration needs to rise. Always do a trial run before using any new seed source or new seed lot. This will inform you of the strength of that seed and will allow you to use the proper volume. “The DO uptake attributable to the seed added to each bottle generally should be between 0.6 and 1.0 mg/L,” (SM 5210B 5. d. 21st ed.)

"The glucose-glutamic acid check is the primary basis for establishing accuracy and precision of the BOD test and is the principal measure of seed quality and set-up procedure." (SM 5210B 6. b. 21st ed.) The requirement for the glucose-glutamic acid (GGA) check is a BOD result of 198 ± 30.5 mg/L. Depending upon the version used you may have different requirements for arriving at the final value. The 21st edition requires 3 bottles being set up with GGA. The results for all three bottles are averaged together and the final average is what must be within the acceptance range. If results consistently fall outside of the acceptable limits, you will need to evaluate possible sources of error. The two most common are the source water and the seed material. If the water is the problem, you will almost always see failing water blanks associated with the failing GGA. If the problem is from the seed, you may still see acceptable seed checks and not have passing GGA. If the results are consistently low add larger volumes of seed to the samples, likewise if the results are consistently high reduce the amount of seed added.

All samples for BOD analysis must be checked for certain conditions to ensure they are suitable for the bacteria to perform properly. All samples must fall within a certain pH range to provide proper growth conditions. This pH requirement differs according to which edition is being cited. For example, the 18th edition says to neutralize samples to a pH of 6.5 to 7.5. This implies that any pH outside this range should be adjusted. Meanwhile, the 21st edition says that samples naturally between 6.0 and 8.0 are acceptable but if outside that range they should be adjusted to be between 7.0 and 7.2. The 19th and 20th editions have their own slight modifications on the acceptable range and the required adjustment range. The presence of chlorine will be detrimental to the health of the bacteria in the sample. Therefore, all samples should also be checked for the presence of residual chlorine compounds. This is done by adding a small amount (1% of the sample volume) of H2SO4 and KI to a portion of the sample. Add a few drops of starch indicator solution. If the sample turns blue/purple, chlorine is present and must be removed.

Titrate with Na2SO3 solution to dissipate the color. Add a proportional amount of Na2SO3 solution to the sample to be tested. Be careful not to overdose with Na2SO3 as this solution has an oxygen demand of its own. Samples that were collected at colder temperatures may be supersaturated with DO. To overcome this, you should simply warm the sample to approximately 20 C and shake the sample vigorously. Along the same lines are samples that do not contain sufficient initial DO. The method does not give a minimum initial value, but 7.0 mg/L oxygen is a good baseline for sample set up. This will give you enough initial DO to be able to satisfy the rules for acceptable depletion. The 21st edition introduced the requirement to check for hydrogen peroxide (H2O2) in samples. This compound will readily degrade to oxygen gas and water. Peroxide can be detected directly via peroxide specific test strips or by taking two DO measurements 30 minutes apart. If the DO increases by a measurable amount in the interval there is peroxide present in the sample. Treatment consists of vigorously mixing or stirring in an open container.

Measurement of the DO can be accomplished in a few different ways. The titration method is rapidly decreasing in popularity. While it is very accurate it is somewhat difficult to perform, especially in the field. It also makes use of azide reagents, which are very dangerous to handle and use. Electronic probes of one form or another are used by virtually everyone in measuring DO. The most common is the membrane electrode. The operating principle is that oxygen diffuses across the membrane and generates a current in the electrode. The amount of current is proportional to the concentration of oxygen. Because the measurement actually consumes small amounts of oxygen a stirrer is required to constantly bring a fresh supply of the sample across the membrane until stabilization. The other type of probe is based on optical luminescence technology. The probe uses an LED to cause luminescence in the water. Oxygen will cause the luminescence to be quenched at a rate proportional to the concentration of the oxygen. This type of probe has several advantages over the membrane electrode – it requires no electrolyte that can degrade, it does not have an electrode that can corrode, there is no membrane to foul up, it does not require a continuous flow, and it has a much wider linear range. Keep in mind that although the luminescence probe does not require a stirrer the method requires a probe with a stirrer. Having the stirrer also decreases the time to achieve a stable reading. Both types of probes require calibration prior to use. Typically, this is done via the water saturated air method. This entails filling a BOD bottle approximately 1/3 full of DI water, making sure that the water level is below the reach of the probe. Place a stopper in the mouth of the bottle and shake vigorously for one minute. Remove the stopper and the bottle is ready for use in calibration.

To get valid results from your samples it is critical to choose appropriate dilutions. Standard Methods 21st edition says to set up 3 different dilutions for a well-known sample and as many as 5 dilutions for a sample of unknown behavior. The goal is to have at least one sample deplete by more than 2.0 mg/L oxygen and still have at least 1.0 mg/L oxygen remaining. This is known as the 2:1 rule. If more than one dilution bottle has acceptable depletion, the final results for each bottle are averaged together to give one reported result.

Note – BOD is a very complex test and cannot be fully explained in this space. All of the items discussed in this section have been simplified in one way or another for space constraints. Please see “A Bug’s-Eye-View of the BOD Test” by Perry Brake for a much more in-depth discussion of BOD.

Method procedure

Note – This is not intended to be a standalone method and does not address all safety or quality control aspects that may be required. Please consult your local regulations to comply with all requirements.

1. Collect your sample in an appropriate sized plastic container, wide mouth or narrow mouth

2. Add calcium chloride, ferric chloride, magnesium sulfate, and phosphate buffer(or set of all four) to your source water at the rate of 1 mL/L for each reagent. Swirl gently to mix if your water has been allowed to equilibrate for a sufficient time at the proper temperature. Mix vigorously if additional aeration is required. SM 21st edition requires 7.5 mg/L as a minimum oxygen level for the dilution water.

3. If using PolySeed, measure out the appropriate volume of dilution water (usuplastially 500 mL) and add the contents of one capsule. Stir and aerate for one hour before using. After rehydration is complete allow the bran to settle and decant the liquid suspension for use. Steps 4-6 can usually be accomplished during the rehydration period.

4. Check the pH of each sample using a pH meter. If outside the correct range, adjust the pH of an appropriate amount of sample to an acceptable pH value using dilute sodium hydroxide or sulfuric acid.

5. Check for the presence of chlorine with potassium iodide and starch indicator If chlorine is present, neutralize with sodium sulfite solution.

6. Determine the number of dilutions and the amount of sample to be added to each one.

7. Get out the appropriate number of glass or plastic BOD bottles and number/label them as necessary.

8. Add the appropriate amount of sample (pH adjusted, and chlorine neutralized if necessary) to each bottle.

9. Fill each bottle at least two thirds full with dilution water. Do not fill completely as you will need to leave room to add seed. Standard Methods specifically requires the bottle have a minimum volume present before seed addition.

10. Add GGA to the correct bottles and the appropriate amounts of seed to the seed control bottles, GGA bottles, and samples.

11. Top the bottle off with dilution water. Make sure the level of the liquid rises up into the neck of the bottle. This will help ensure a proper water seal during incubation.

12. Take an initial DO reading according to the requirements at your facility with a DO meter.

13. Stopper each bottle and add an overcap.

14. Incubate the samples for 5 days at 20 C.

15. Take the final DO reading with the same method as the initial.

16. Calculate the BOD value according to the following formula:
BOD calculation formula
The chart below is intended to be a guide in selecting dilution amounts. It presumes a SCF value of 0.8. Each column is for an amount of oxygen depleted and each row is for a volume of sample in the bottle.

Table scrolls horizontally

Table 1: BOD value in mg/L for various sample volumes and depletion amounts
DO Depletion
Sample Volume (ml) 2.0 3.0 4.0 5.0 6.0 7.0 8.0
0.01 3600 66000 96000 126000 156000 186000 216000
0.02 18000 33000 48000 63000 78000 93000 108000
0.05 7200 13200 19200 25200 31200 37200 43200
0.1 3600 6600 9600 12600 15600 18600 21600
0.2 1800 3300 4800 6300 7800 9300 10800
0.5 720 1320 1920 2520 3120 3720 4320
1 360 660 960 1260 1560 1860 2160
2 180 330 480 630 780 930 1080
5 72 132 192 252 312 372 432
10 36 66 96 126 156 186 216
20 18 33 48 63 78 93 108
50 7.2 13.2 19.2 25.2 31.2 37.2 43.2
100 3.6 6.6 9.6 12.6 15.6 18.6 21.6
150 2.4 4.4 6.4 8.4 10.4 12.4 14.4
300 1.2 2.2 3.2 4.2 5.2 6.2 7.2
Assume SCF Value of 0.8