David SmithEnvironmental Express

Technical SpecialistLab Matters

Biochemical Oxygen Demand (BOD) bull 1

Call 8003435319 or 8438816560 bull wwwenvironmentalexpresscom

E N V I R O N M E N T A L E X P R E S S

Copyright Environmental Express Inc Lab Matters 1204

Understanding Biochemical Oxygen Demand (BOD)Introduction ndash 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 BOD

5 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 20degC) 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 Shakespearersquos MacBeth During the scene with the witches and the cauldron he started commenting on how there wasnrsquot 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 ndash 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 ndash A known volume of sample has its initial dissolved oxygen content recorded After a five day incubation period at 20degC 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 ndash 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

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

David SmithEnvironmental Express

Technical SpecialistLab Matters

Biochemical Oxygen Demand (BOD) bull 2Call 8003435319 or 8438816560 bull wwwenvironmentalexpresscom

option for the source water is to purchase it Anything that is labeled ldquosteam distilledrdquo 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 02 mgL Laboratories that are well versed in BOD analysis should be able to routinely produce blanks with a depletion of less than 01 mgL

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 isnrsquot 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 lsquoseed farmrsquo 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 manufacturerrsquos 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 ldquoThe DO uptake attributable to the seed added to each bottle generally should be between 06 and 10 mgLrdquo (SM 5210B 5 d 21st ed)

ldquoThe 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 procedurerdquo (SM 5210B 6 b 21st ed) The requirement for the glucose-glutamic acid (GGA) check is a BOD result of 198 plusmn 305 mgL 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 65 to 75 This implies that any pH outside this range should be adjusted Meanwhile the 21st edition says that samples naturally between 60 and 80 are acceptable but if outside that range they should be adjusted to be between 70 and 72 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 H

2SO

4 and KI to a portion of the sample Add a

few drops of starch indicator solution If the sample turns bluepurple chlorine is present and must be removed

David SmithEnvironmental Express

Technical SpecialistLab Matters

Call 8003435319 or 8438816560 bull wwwenvironmentalexpresscom

Titrate with Na2SO

3 solution to dissipate the color Add a proportional amount of Na

2SO

3 solution to the sample to

be tested Be careful not to overdose with Na2SO

3 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 70 mgL 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 (H

2O

2) 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 measureable 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 ndash 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 13 full with 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 20 mgL oxygen and still have at least 10 mgL oxygen remaining This is known as the 21 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 ndash 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 ldquoA Bugrsquos-Eye-View of the BOD Testrdquo by Perry Brake for a much more in depth discussion of BOD

Biochemical Oxygen Demand (BOD) bull 3

David SmithEnvironmental Express

Technical SpecialistLab Matters

Biochemical Oxygen Demand (BOD) bull 2Call 8003435319 or 8438816560 bull wwwenvironmentalexpresscom

option for the source water is to purchase it Anything that is labeled ldquosteam distilledrdquo 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 02 mgL Laboratories that are well versed in BOD analysis should be able to routinely produce blanks with a depletion of less than 01 mgL

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 isnrsquot 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 lsquoseed farmrsquo 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 manufacturerrsquos 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 ldquoThe DO uptake attributable to the seed added to each bottle generally should be between 06 and 10 mgLrdquo (SM 5210B 5 d 21st ed)

ldquoThe 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 procedurerdquo (SM 5210B 6 b 21st ed) The requirement for the glucose-glutamic acid (GGA) check is a BOD result of 198 plusmn 305 mgL 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 65 to 75 This implies that any pH outside this range should be adjusted Meanwhile the 21st edition says that samples naturally between 60 and 80 are acceptable but if outside that range they should be adjusted to be between 70 and 72 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 H

2SO

4 and KI to a portion of the sample Add a

few drops of starch indicator solution If the sample turns bluepurple chlorine is present and must be removed

David SmithEnvironmental Express

Technical SpecialistLab Matters

Call 8003435319 or 8438816560 bull wwwenvironmentalexpresscom

Titrate with Na2SO

3 solution to dissipate the color Add a proportional amount of Na

2SO

3 solution to the sample to

be tested Be careful not to overdose with Na2SO

3 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 70 mgL 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 (H

2O

2) 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 measureable 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 ndash 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 13 full with 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 20 mgL oxygen and still have at least 10 mgL oxygen remaining This is known as the 21 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 ndash 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 ldquoA Bugrsquos-Eye-View of the BOD Testrdquo by Perry Brake for a much more in depth discussion of BOD

Biochemical Oxygen Demand (BOD) bull 3

David SmithEnvironmental Express

Technical SpecialistLab Matters

Call 8003435319 or 8438816560 bull wwwenvironmentalexpresscom

Titrate with Na2SO

3 solution to dissipate the color Add a proportional amount of Na

2SO

3 solution to the sample to

be tested Be careful not to overdose with Na2SO

3 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 70 mgL 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 (H

2O

2) 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 measureable 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 ndash 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 13 full with 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 20 mgL oxygen and still have at least 10 mgL oxygen remaining This is known as the 21 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 ndash 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 ldquoA Bugrsquos-Eye-View of the BOD Testrdquo by Perry Brake for a much more in depth discussion of BOD

Biochemical Oxygen Demand (BOD) bull 3

David SmithEnvironmental Express

Technical SpecialistLab Matters

Call 8003435319 or 8438816560 bull wwwenvironmentalexpresscom

Method Procedure Note ndash 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 appropriately 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 mLL 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 75 mgL as a minimum oxygen level for the dilution water

3 If using PolySeed measure out the appropriate volume of dilution water (usually 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 plastic or glass BOD bottles and numberlabel 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 (YSI Meter Thermo Meters)

13 Stopper each bottle and add an overcap

14 Incubate the samples for 5 days at 20degC

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

Biochemical Oxygen Demand (BOD) bull 4

David SmithEnvironmental Express

Technical SpecialistLab Matters

Call 8003435319 or 8438816560 bull wwwenvironmentalexpresscom

16 Calculate the BOD value according to the following formula

[(DOinitial

mdash DOfinal

) mdash SCF] 300)

Sample Volume (mL)

The chart below is intended to be a guide in selecting dilution amounts It presumes a SCF value of 08 Each column is for an amount of oxygen depleted and each row is for a volume of sample in the bottle

Chart 1 BOD value in mgL for various sample volumes and depletion amounts

We all like things that make life easier Was this document helpful Or do youhellipdisagree with something

Have something to add Contact me at DavidSenvexpcom to let me know what you think

= BOD

Sample DO Depletion Volume (mL) 20 30 40 50 60 70 80

001 36000 66000 96000 126000 156000 186000 216000

002 18000 33000 48000 63000 78000 93000 108000

005 7200 13200 19200 25200 31200 37200 43200

01 3600 6600 9600 12600 15600 18600 21600

02 1800 3300 4800 6300 7800 9300 10800

05 720 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 72 132 192 252 312 372 432

100 36 66 96 126 156 186 216

150 24 44 64 84 104 124 144

300 12 22 32 42 52 62 72

Assume SCF Value of 08

Biochemical Oxygen Demand (BOD) bull 5