- 1:1 sulfuric acid
- 1 N sulfuric acid (1.5 mL concentrated sulfuric acid / 50 mL distilled water)
5 N sodium hydroxide (10 g NaOH / 50 mL distilled water)
- Dry reagent grade glucose and glutamic acid at 103° C for 1 hour. Cool.
- Weigh 150 mg glucose and 150 mg glutamic acid. Place in a 1 L volumetric flask containing 500 mL distilled water. Mix well and bring to volume.
- Glucose-Glutamic Acid solutions (GGA) can be preserved by:
- Freezing in 20 mL volumetric aliquots - stable for two months; or
- Add 2 pellets of NaOH (to the 1 L solution) and refrigerate at 4° C – stable for 30 days. Allow chilled samples to reach room temperature before using for analysis.
- Seed Material
- Nitrification Inhibitor
- Dechlorination Reagents
- Sodium Thiosulfate – Dissolve 0.1575 g Na2SO3 in distilled water. Dilute to 100 mL. Prepare weekly.
- Sulfuric Acid – Add 1 mL concentrated H2SO4 to 50 mL distilled water or use 1N H2SO4.
- Acetic Acid (1:1) – Mix equal volumes of CH3CO2H and distilled water.
- Potassium Iodide – Mix 10 grams of KI with 100 mL of distilled water.
- Starch solution – Dissolve 2 g of soluble starch in 100 mL of hot distilled water. To preserve the solution, add 0.2 g salicylic acid.
Note: The BOD5 of this solution is 198 mg/L ±30.5 mg/L.
Seed material will vary by facility. The most common source is supernatant from settled raw sewage collected from daily samples. The seed material is best used immediately upon collection, but may be refrigerated for no more than 36 hours.
Reagent grade: 2-chloro-6-(trichloromethyl) pyridine; or
Nitrification Inhibitor can be purchased commercially. [Hach formula 2533]
Samples may be stored in a plastic or glass container and kept at 4° C. See sample storage and preservation table.
The following must be recorded on the data sheet:
- Sample identification (source, name, and date(s) of collection)
- Set up and Read off dates; time Set up was started
- Analyst(s)
- Raw data
- Final results with correct units (reported to nearest mg/L)
- Description of unusual sample characteristics
- Replicates are to be listed in an orderly cluster
- Dilution water blanks should have a 5-day depletion of not more than 0.30 units and not less than -0.15 units.
- Dilution water to be used for analysis should have a pH in the range of 6.50 to 7.50 pH units.
- Samples to be analyzed must have pH adjusted to within the range of 6.50 to 7.50 pH units.
- Individual dilutions must deplete at least 2.00 mg/L DO, and at least 1.00 mg/L DO must remain.
- At least two dilution water blanks, a seed dilution series, and a seeded BOD check sample pair should be prepared for each bottle of dilution water. The frequency of the blank and standard testing may be less as determined by the needs of individual laboratories in order to meet a 10% QC level. Individual laboratories may also determine how often BODs are run, how many dilutions are made, and what type of records are kept.
- Recoveries of seeded BOD check samples should be between ± 2 standard deviations.
- Recoveries of seeded BOD check samples must be between ± 3 standard deviations, or the set of results the check sample accompanies will be declared unreportable. If the results are declared unreportable, "No Data -- Lab Error" must be reported.
- Check sample recovery statistics should be reviewed on a yearly basis and any changes to acceptable ranges should be documented appropriately.
- Results of replicate analyses should yield relative standard deviations (RSD’s) below 15.0 % for the set of replicates.
- Do periodic checks on the DO meter or Winkler testing to assure proper calibration and titration.
- Adhere to sampling protocol and holding times.
- When several sample results meet the range requirements, an average of those results should be reported. The fact that they are an average should be documented.
- Since various methods are correct for setting up the BODs, each laboratory is responsible for documenting the specific technique that was used. Consistency in the technique used is the key factor.
Place the desired volume of water in a suitable container. Add 1 mL each of phosphate buffer, magnesium sulfate, calcium chloride, and ferric chloride solutions per liter of dilution water.
The source of dilution water is not restricted and may be distilled, tap, or receiving-stream water free of biodegradable organics and bioinhibitory substances such as chlorine or heavy metals. Distilled water may contain ammonia or volatile organics; deionized waters often are contaminated with soluble organics leached from the resin bed.
Aerate by shaking, aerating with an aquarium pump for 24 hours, or by drawing a vacuum through the water for 15-20 minutes. If the aerated water has a DO greater than saturation, allow the water to set unstoppered until it is equal to or less than saturation. Store at 20° C for at least 24 hours. The water may be stored for longer periods if dilution water blanks consistently meet quality control limits.
Preparation of Samples for Incubation
- Sample Collection and Storage
Collect the samples in clean, dry containers (glass or plastic) large enough to contain the appropriate amount of sample needed for testing. Samples for BOD analysis may degrade significantly in the period of time between collection and analysis. Maintain the validity of the sample by prompt analysis or by cooling it to near-freezing (4° C). However do not freeze the sample. Allow chilled samples to reach room temperature before analysis.
- Prepare raw data sheets.
- Sample source, sample date, sample types (total or carbonaceous), set up and read off dates, set up time, and analyst’s initials are the preliminary information to record on the raw data sheets.
- Seeded BOD check samples will be analyzed for quality assurance. At least one check sample pair will be analyzed with each bottle of dilution water used.
- Elimination of Residual Chlorine
It is usually recommended that wastewater treatment plants sample the effluent prior to the chlorination process. If the sample has been chlorinated, perform the following procedure.
- To 100 mL of chlorinated sample, add 10 mL of the potassium iodide solution, 10 mL of sulfuric acid solution (or acetic acid solution), and 1 mL of starch solution. The sample will turn blue if chlorine is present.
- If residual chlorine is present, use one of the following methods to remove the chlorine;
- Natural removal -- Expose the sample to sunlight for two to three hours while stirring the sample. The residual chlorine will decrease rapidly due to photodecomposition and agitation.
- Chemical removal -- Using a pipette or an eyedropper, add sodium thiosulfate solution dropwise while stirring the sample prepared in step 1. Count the number of drops needed to remove the blue coloration.
- Calculate the number of drops of sodium thiosulfate needed to eliminate the residual chlorine in a newly measured volume of sample using the following formula:
- Add this number of drops of sodium thiosulfate to the fresh sample and mix well. Use this freshly dechlorinated portion to set up the dilutions.
OR
NOTE: The use of dechlorinated samples for the BOD test is not recommended. Chlorine destroys some BOD, thus lowering the test value. The sodium thiosulfate exerts an oxygen demand and may elevate the BOD. If possible, obtain the sample for BOD analysis at a point before the chlorine is added.
- pH Measurement and Adjustment
- Calibrate the pH apparatus prior to running a set of samples, then perform and record the results of a pH check. (See Use Of pH Meters And Electrodes and Measurement Of pH sections for specifics.)
- Determine the initial pH of the sample, then adjust the sample pH if it is not in the proper range.
- Record the initial pH for the sample in the appropriate space on the raw data sheet.
- Adjust the pH of the sample (if necessary) so that the sample will remain between pH 6.50 and 7.50 during analysis.
- Wear eye protection when delivering acid or alkaline solutions.
- If the pH of the sample is less than 6.5, adjust the pH to 6.5-7.5 with NaOH.
- If the pH of the sample is greater than 7.5, adjust the pH to 6.5-7.5 with H2SO4.
- Use the appropriate acid or alkali solution to adjust the pH of the sample. (Wear appropriate eye protection when delivering acid or alkaline solutions.)
- If the addition of acid solution or the alkaline solution causes too great a change in the sample pH, adjust the sample pH using pH 7.2 phosphate buffer, OR use the following procedure:
- Remove a small aliquot of the sample, then add a drop of acid or alkaline solution to this small portion of sample. Mix thoroughly, then use THIS solution to adjust the pH of the total sample.
- Record the adjusted pH of the sample in the appropriate space on the prepared raw data sheet.
Ensure a well-mixed sample in the BOD bottle by any of the methods below.
- Shaking the sample vigorously.
- Stir the sample manually.
- Place the sample container on a magnetic stirrer (this will also prevent settling while extracting sample portions).
Choosing a Dilution Series for the Individual Samples.
- Use sample appearance, historical sample data, information from the sampling personnel, and/or grab sample data to predict what the BOD value will be for the sample.
- The nature of the wastewater samples changes from day to day. Make sure the dilution ranges chosen take these subtle changes into account.
- Use the chart that correlates sample volume to a range of BOD values as a tool for choosing appropriate dilutions.
- For the BOD results to meet quality control requirements, the oxygen content of the sample dilutions must deplete by at least 2.00 mg/L oxygen during the 5-day incubation time, but at least 1.00 mg/L oxygen must remain.
- Use at least 2 and as many additional dilutions as necessary to insure that the dilution range will encompass the BOD concentration.
- Make sure the individual dilution BOD ranges overlap so there are no gaps between them.
- Some samples may require use of a double dilution technique (i.e.: "as received" and a 1:10 dilution) to cover the necessary range of BODs. When double dilutions are used in pipetting the dilution series that has been chosen, duplicate the % dilution at the crossover point between the two sample strengths. The purpose of this "duplicate" is to demonstrate that the dilution was done properly and should not be used as the duplicate used to demonstrate precision.
- Prepare individual BOD bottles.
- Choose clean, dry, intact individual BOD bottles.
- Bottles must remain stoppered until immediately before sample is pipetted into them.
- Line up the individual BOD bottles on the bench in the same order that the bottles are listed on the raw data sheet(s). Bottles are usually arranged in ascending numerical order for an individual dilution series. Do not use duplicate bottle numbers within the same dilution series. (i.e.: It must be completely clear to the analyst reading off the DO levels at the end of the incubation period which bottle is which.) Use the marking spot on the individual BOD bottle as the place to write additional identification information.
- At least two individual BOD bottles must be prepared as blanks for every stock bottle of dilution water used. Check sample dilutions should be set up in duplicate.
- Deliver sample into individual BOD bottles.
- Wear eye protection when delivering samples.
- Record the sample volume placed in each BOD bottle.
- The following table lists recommended vessels for delivering sample aliquots:
- Mix the sample to be analyzed by placing the sample jar on a magnetic stirplate, placing a clean magnetic stirbar into the jar, and adjusting the stir speed of the magnetic stirplate so the sample mixes completely. Good mixing is usually indicated by a uniform color and an even solids distribution in the sample and is often accompanied by a small "dimple" on the top surface of the sample.
- Use proper procedure when using a mechanical pipette to assure accurate results. (See section on Care And Use Of Mechanical Pipettors for specifics.)
- Select a pipette tip with a tip bore size that is large enough to pipette all the representative solids in the sample, yet is not so large as to cause sample loss when transferring the sample aliquot.
- Be sure that the pipette tip and/or graduated cylinder to be used is clean.
- Take sample aliquots near the vertical midpoint of the liquid level in the sample container.
The goal of the sample set-up is to leave at least 1 mg/L DO in the bottle at the end of the incubation period, yet have the organisms present in the sample use at least 2.0 mg/L DO. As discussed previously, seed may be required with some samples to produce the 2.0 mg/L depletion required.
The following dilution guide may be of some assistance:
|
Volume of Sample added to 300-mL Bottle (mL) |
Minimum BOD (mg/L) |
Maximum BOD (mg/L) |
|
3 |
210 |
560 |
|
6 |
105 |
280 |
|
9 |
70 |
187 |
|
12 |
53 |
140 |
|
15 |
42 |
112 |
|
18 |
35 |
94 |
|
21 |
30 |
80 |
|
24 |
26 |
70 |
|
27 |
24 |
62 |
|
30 |
21 |
56 |
|
45 |
14 |
37 |
|
60 |
11 |
28 |
|
75 |
8 |
22 |
|
150 |
4 |
12 |
Total volumes achieved by combinations of partial volumes from the three vessels below are acceptable.
|
Sample delivery device |
Proper volume range |
|
Variable 1 mL – 5 mL pipette |
Increments of 1.5 - 49.9 mL |
|
50 mL glass, wide-mouth transfer pipette |
Increments of 50.0 mL |
|
250 mL glass graduated cylinder |
Volumes in excess of 200 mL |
Prepare sample dilutions directly in the individual BOD bottles by pipetting sample into the BOD bottle now and adding dilution water later. However, no less than 3 mL should ever be pipetted into an individual BOD bottle. If the sample is so concentrated that a 1% (3 mL) dilution will cause too great of an oxygen depletion, the sample will have to be reduced in strength prior to pipetting. It is convenient and customary to make reduced strength samples as dilutions which are offset by factors of 10 (1/10, 1/100, 1/1000, etc).
- To produce a reduced strength sample for pipetting:
- Measure 100.0 mL of well-mixed sample in a 100 mL glass graduated cylinder which has been rinsed with the sample to be delivered.
- Pour this sample into a clean 1000 mL graduated container and use demineralized, distilled, tap, or BOD dilution water, to rinse the 100 mL glass graduated cylinder at least 3 times. Add rinses to the 1000 mL graduated container that now contains the sample. Fill the large graduated cylinder to the 1000 mL mark with the dilution water chosen.
- Before delivering any of this reduced strength sample, make sure it is well mixed.
- Reduced strength samples must not be reduced by any more than 1/10 in any one dilution step (i.e. use the 1/10 dilution to prepare a 1/100 dilution).
- Record the concentration of any reduced strength sample(s) used in the appropriate column on the raw data sheet.
- Rinse the sample delivery device three times with well-mixed sample, discarding rinses in a waste container.
- Transfer a measured aliquot of sample into each individual BOD bottle.
- If a pipette is used to deliver sample, deliver the exact amount as calculated above.
- If a graduated cylinder is used to deliver sample, use the following procedure:
- Quickly transfer a volume of sample into the graduated cylinder. This volume should approximate the volume calculated above, but does not have to match the calculated volume exactly.
- Determine the exact volume of sample in the graduated cylinder and record this volume (to the appropriate number of significant figures, usually to the nearest mL) in the appropriate space on the prepared raw data sheet
- If more than one type of solution needs to be transferred into the individual BOD bottle (as occurs whenever a sample is seeded), Do Not combine the different solutions until immediately before adding dilution water. (See Seeding Individual BOD Bottles below.)
- Remove the magnetic stirbar from the sample container using the magnetic stirbar retriever.
- Clean the magnetic stirbar by rinsing it with a stream of distilled water.
Be sure to record the type of seed used and concentration of seed in the appropriate spaces on the prepared raw data sheet.
- Deliver Nitrification Inhibitor into individual BOD bottles (if carbonaceous BOD determination is required).
- Nitrification Inhibitor is used in those individual BOD bottles which will be used to determine a carbonaceous BOD (CBOD).
- If the Hach product is being used, use the reagent bottle with the dispensing top. Deliver two aliquots of Nitrification Inhibitor into each individual BOD bottle. Clean the dispensing top on the reagent bottle daily, so the piston on the dispensing top will be less likely to "freeze up".
- If another source of the nitrification inhibitor [2-chloro-6-(trichloromethyl) pyridine (TCMP)] is used, add 3 mg to each 300 mL BOD bottle.
- Use proper procedure when using the DO electrode. (See the manufacturer’s instruction manual for specifics.)
- Select a stir speed on the magnetic stirplate that will ensure that BOD bottle contents are constantly mixed.
- The calibration of the dissolved oxygen electrode must be checked before every set of samples is analyzed. The appropriate items should be recorded in the dissolved oxygen electrode logbook.
Preparing the Dissolved Oxygen Electrode for Measurements.
Prepare BOD blanks by filling clean BOD bottles with dilution water. No other solutions or solids should be added to the BOD bottle. Record final DO at the end of the incubation period.
Primary Standard (Glucose-Glutamic Acid) Preparation
- With Seeded Dilution Water
Add 6 mL (2%) glucose-glutamic acid (at room temperature) solution to a 300 mL BOD bottle.
Record final DO at the end of the incubation period. Use calculations for seeded dilution water. (See Calculations below).
The final calculations of BOD on a glucose-glutamic acid control should be equal to 198 mg/L plus or minus 30.5 mg/L to be considered an acceptable control. Alternatively, using at least 25 tests, determine the mean and standard deviation for your laboratory. If they fall within the above range, the value of the mean plus or minus three standard deviations may be used as the QC parameter for that laboratory.
- With Individual Bottles Seeded
To a seeded BOD bottle add 6 mL (2%) glucose-glutamic acid solution (at room temperature).
Record final DO at the end of the incubation period and use calculations for individually seeded bottles. (See Calculations below).
Samples are seeded to ensure a sufficient population of microorganisms capable of oxidizing the biodegradable organic matter in the sample. The two methods for introducing seed into a sample include seeding the dilution water or seeding of individual BOD bottles. Seeding of individual samples avoids a declining ratio of seed to sample as increasing dilutions are made. However, with proper monitoring, either method is considered correct.
- Seeded Dilution Water
To a known amount of dilution water, add a known amount of seeding material. Note that it will be necessary to remove some of the dilution water before performing this step to be able to set-up the seed control bottles explained below and the blank bottles.
Using the same seeding material, set up a series of seed control bottles using unseeded dilution water (three samples gives a good average). Place varying amounts of seed in dilution bottles.
The ideal situation is to make dilutions of seed such that the largest quantity results in at least 50% DO depletion. A plot of DO depletion in mg/L versus mL seed should present a straight line for which the slope indicates DO depletion per mL of seed. The DO-axis intercept is oxygen depletion caused by the dilution water and should be less than 0.1 mg/L. The slope of this line is the DO uptake of the seeded dilution water and should be between 0.6 and 1.0 mg/L. To determine a sample DO uptake, subtract seed DO uptake from the total DO uptake.
When DO readings are taken at the end of the incubation period, the BOD calculation for seeded dilution water is used. This calculation takes the presence of seed into consideration for all samples. (See Calculations below).
- Seeding Individual BOD Bottles
This seems to be the most common method for seeding BOD samples. It is straightforward and makes the calculations easier.
Set up seed control bottles by placing varying amounts of seed in dilution bottles. (See the information above regarding selecting seed dilutions).
Place a known amount of seed in bottles that will contain samples that require seeding. Proceed with sample set-up.
When DO readings are taken at the end of the incubation period, use the BOD calculation for individually seeded BOD bottles. (See Calculations below).
To samples requiring nitrification inhibition (CBOD), add nitrification inhibitor to the dilution water (if all samples need inhibition); or to the individual samples. Dosage varies by the manufacturer of the inhibitor. The dosage is supposed to be 10 mg/L (or 3 mg per 300 mL BOD bottle) of the active ingredient. Report the results as CBOD5.
- Fill the BOD bottles in the following order
- Fill the blank bottle(s) first.
- Fill the seed series bottles second.
- If the dilution water is to be seeded, it should be seeded at this point.
- Fill the check sample bottles, if appropriate, next.
- Fill the sample bottles last.
- After all samples, seed (where necessary), and nitrification inhibitor (where necessary), have been added to BOD bottles, the dilution water should be carefully added to each of the BOD bottles.
- Siphon the dilution water into the BOD bottles slowly to prevent trapping air bubbles.
- Filling the BOD bottle half way up the ground glass portion is helpful to provide enough water to provide a water seal for a DO probe and the glass stopper that will be used to seal the BOD bottle later
- Take the initial DO reading, stopper, cap, and place in the incubator at 20° C for 5 days.
- After recording the initial DO reading, make sure that some of the water in the neck of the BOD bottle is displaced when the stopper is put into place.
- If there is not water above the stopper at this point, add some to create a water seal and then cap.
- Place the BOD bottles, with the caps on them, into the incubator for 5 days at 20°C.
- Retrieve raw data sheets.
- Remove raw data sheets from the file used for storing data sheets during the incubation period.
- Make sure the read off date on the top of the raw data sheet matches today's date.
- The read off analyst should initial the raw data sheets at this time.
- Prepare the dissolved oxygen electrode for measurements. (See section on Preparing The Dissolved Oxygen Electrode For Measurements.)
- Determine the residual oxygen content of the individual BOD bottles and calculate 5-day BOD (or BOD5) for the sample.
- Use the description of bottle location at the top of the raw data sheet to locate the individual BOD bottles due to be read off.
- Use proper procedure when using the DO electrode. (See the manufacturer’s instruction manual for specifics.)
- The order in which samples shall be read off after calibration is:
- Remove the insulating cap from the individual BOD bottle just before the dissolved oxygen will be read.
- Pour the water seal from the flared neck of the BOD bottle into a waste container before removing the stopper.
- If there is an air pocket present in the BOD bottle, its BOD value may not be valid. Note this on the raw data sheet.
- Record the final dissolved oxygen content of each BOD bottle (to the nearest 0.1 or 0.001 mg/L) in the appropriate space on the raw data sheet. Do this before removing the electrode from the bottle.
- Calculate oxygen depletions and BODs while reading off. (See Calculations below for equations.) This will allow you to identify any problems while the sample is still at hand.
- The 5-day oxygen depletion of the dilution water blanks should not exceed 0.20 mg/L.
- When readings are complete, prepare the DO electrode for overnight storage (See the manufacturer’s instruction manual for specifics.), remove the magnetic stirplate from the refrigerator, and turn off the stirplate.
- Report the data.
- Check for values that deserve notification of plant operations personnel.
Procedure for Analysis of Samples after Incubation
blank, seed, check, samples.
Example: 6 ml sample/300 ml bottle = 0.02
OR
Example: (6 ml sample/300 ml bottle) × 100 = 2%
OR
When Individual Bottles are Seeded
Read the Seed Control Bottles, and use the following equation to calculate mg/L DO depletion caused by each mL of seed added.
Determine how much of the BOD in each sample was caused by the seed by using the following equation:
Then use the following equation to determine BOD5 of each sample.
Goals for a New Analyst Performing BOD Analyses
- Know safety procedures for BOD work
- Know how to fill out raw data sheets
- Understand analysis procedures used to perform BOD analyses
- Need to adjust pH of samples
- How to prepare samples for different types of analysis
- (Total & Carbonaceous)
- Mixing and delivering samples
- How to fill individual BOD bottles
- Incubation time for samples
- Understand equipment used in BOD analyses
- Dissolved oxygen electrode
- How to calculate calibration value
- How to calibrate
- General use and care of electrode
- Glass pipettes / Mechanical pipettes
- Incubators
- Know where to find equipment / supplies
- BOD bottles
- Pipettes and tips
- BOD check samples
- BOD dilution water
- Dilution water chemicals
- Graduated Cylinders
- Use of Nitrification Inhibitor
- Why nitrification inhibitor is used
- When nitrification inhibitor is used
- How nitrification inhibitor is used
- Understand how dilution ranges are selected
- Use of dilution chart
- Know how to establish acceptability of BOD dilution water and the procedure to follow when water must be rejected
- Know how to calculate BOD results
- Know "true" value of BOD check sample
- Understand quality control procedures
- Need for quality control
- Calculation of mean and %RSD
- Calculation of percent recovery
- Know how to clean and prepare glassware
- BOD bottles
- Dilution water bottles
- Know how to prepare dilution water
- Addition of chemicals
- Aeration of dilution water
- Storage of dilution water
- Know how to handle BOD incubator failure situations
- Check the appropriate circuit breaker. Reset the circuit breaker if it is blown.
- Try to reset the incubator by turning control switches OFF, then back ON.
- If the incubator does not respond to the resets, turn all control switches to OFF and proceed with the preservation steps listed below.
- If no other incubator is available, pack the malfunctioning incubator with ice.
- Distribute the containers with ice throughout the interior of the malfunctioning incubator.
- Keep the door of the malfunctioning incubator closed as much as possible.
BOD / CBOD Analysis; the Tips for Techs version
- Prepare dilution water
- Collect sample
- Prepare raw data sheets
- Check residual chlorine
- Check pH
- Prepare blanks
- Prepare check samples (optional)
- Prepare sample(s)
- Add seed (if appropriate)
- Add nitrification inhibitor (if appropriate)
- Calibrate dissolved oxygen electrode (if necessary)
- Add dilution water
- Obtain initial dissolved oxygen readings
- Incubate samples for 5 days
- Calibrate dissolved oxygen electrode (if necessary)
- Obtain final dissolved oxygen readings
- Calculate results
- Review for Quality Control / Quality Assurance compliance