1. The CBOD₅ Test

The CBOD tests have the widest application in measuring waste loadings to treatment plants and in evaluating the CBOD-removal efficiency of such treatment systems. The test measures the molecular oxygen utilized during a specified incubation period for the biochemical degradation of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic material such as sulfides and ferrous iron. It also may measure the amount of oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless their oxidation is prevented by an inhibitor. The seeding and dilution procedures provide an estimate of the CBOD at pH 6.5 to 7.5.

There are two recognized EPA methods for the measurement of CBOD:

• Standard Methods for the Examination of Water and Wastewater, Method 5210B^[1]
• In-Situ Inc. Method 1004-8-2009 Carbonaceous Biochemical Oxygen Demand (CBOD) Measurement by Optical Probe.^[2]

2. Dissolved Oxygen Probes: Membrane and Luminescence

Since the publication of a simple, accurate and direct dissolved oxygen analytical procedure by Winkler,^[3] the analysis of dissolved oxygen levels for water has been key to the determination of surface water purity and ecological wellness. The Winkler method is still one of only two analytical techniques used to calibrate oxygen electrode meters; the other procedure is based on oxygen solubility at saturation as per Henry's law. Though many researchers have refined the Winkler analysis to dissolved oxygen levels in the low PPB range, the method does not lend itself to automation.

The development of an analytical instrument that utilizes the reduction-oxidation (redox) chemistry of oxygen in the presence of dissimilar metal electrodes was introduced during the 1950s.^[4] This redox electrode utilized an oxygen-permeable membrane to allow the diffusion of the gas into an electrochemical cell and its concentration determined by polarographic or galvanic electrodes. This analytical method is sensitive and accurate down to levels of ± 0.1 mg/l dissolved oxygen. Calibration of the redox electrode of this membrane electrode still requires the use of the Henry’s law table or the Winkler test for dissolved oxygen.

During the last two decades, a new form of electrode was developed based on the luminescence emission of a photo-active chemical compound and the quenching of that emission by oxygen. This quenching photophysics mechanism is described by the Stern–Volmer equation for dissolved oxygen in a solution:^[5]

[math]\displaystyle{ I_0/I~=~1~+~K_{SV}~[\ce{O2}] }[/math]

• [math]\displaystyle{ I }[/math]: Luminescence in the presence of oxygen
• [math]\displaystyle{ I_0 }[/math]: Luminescence in the absence of oxygen
• [math]\displaystyle{ K_{SV} }[/math]: Stern-Volmer constant for oxygen quenching
• [math]\ce{ [O2] }[/math]: Dissolved oxygen concentration

The determination of oxygen concentration by luminescence quenching has a linear response over a broad range of oxygen concentrations and has excellent accuracy and reproducibility.^[6] There are two recognized EPA methods for the measurement of dissolved oxygen for CBOD:

• Standard Methods for the Examination of Water and Wastewater, Method 4500 O^[7]
• In-Situ Inc. Method 1002-8-2009 Dissolved Oxygen Measurement by Optical Probe.^[8]

3. Method Summary

Bring the sample to ambient room temperature. If pH of sample is <6.5 or >7.5 neutralize the sample to approximately a pH of 7.0 using either sulfuric acid or sodium hydroxide. Aliquots of the neutralized sample are transferred to 300 mL CBOD bottles. These CBOD samples must be at concentrations that will deplete by at least 2 mg/L dissolved oxygen (DO) and have at least 1 mg/L DO left after five days of incubation. Therefore, make enough dilutions (minimum of 3) of the prepared sample to bracket the predicted CBOD.

The minimum aliquot volume transferred to a 300 mL CBOD bottle will be 3 mL as set by Standard Methods. If a smaller volume is needed to meet the DO depletion requirements, then you must make dilutions to the sample. Add approximately 0.1 g of Nitrification Inhibitor (2-chloro-6-(trichloro-methyl) pyridine) to each 300mL CBOD bottle before adding CBOD dilution water. If the sample is being prepared as a seeded sample, add enough prepared seed to the sample to achieve acceptable dissolved oxygen depletion. Add CBOD Dilution water to each CBOD sample bottle so as to completely fill the bottle with no air spaces or bubbles when the stopper is placed in the bottle.

Place the dissolved oxygen probe in the bottle and allow the dissolved oxygen meter to come to equilibrium. Allow the meter to come to equilibrium prior to accepting dissolved oxygen value. Record the DO of the sample, stopper the bottle, add DI water to the water seal if needed, cap the water seal, and incubate for 5 days at 20 °C ± 1 °C. Exclude light to avoid growth of algae in the bottles during incubation.

Upon completion of the 5-day incubation± 6 hours, record the DO of the depleted samples with a calibrated DO meter. Allow the meter to come to equilibrium prior to accepting dissolved oxygen value. Calculate the CBODs from the formula below. Only bottles, including seed controls, giving a minimum DO depletion of 2.0 mg/L and a residual DO of at least 1.0 mg/L after 5 days of incubation are considered to produce valid data, because at least 2.0 mg oxygen uptake per L is required to give a meaningful measure of oxygen uptake and at least 1.0 mg/L must remain throughout the test to ensure that insufficient DO does not affect the rate of oxidation of waste constituents.

4. Bacterial Seed CBOD Correction

Seed CBOD Uptake: Typically a 10, 20, and 30 mL sample of seed added to 3 separate CBOD bottles with approximately 0.1 g Nitrification Inhibitor and diluted with CBOD dilution water. Run these QC samples with each batch of seeded CBOD. Calculate the DO uptake per mL of seed added to each bottle using either the slope method or the ratio method.

For the slope method, plot DO depletion in milligrams per liter versus mLs of seed for all seed control bottles having a 2.0 mg/L depletion and 1.0 minimum residual DO. The plot 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 0.20 mg/L.

For the ratio method, divide the DO depletion by the volume of seed in mLs for each seed control bottle having a 2.0 mg/L depletion and greater than 1.0 mg/L minimum residual DO and average the results.

5. CBOD Seed

The CBOD test is method defined. Factors such as bacterial seed viability, anoxic stress during the 5 days, and nitrogenous inhibition efficacy will produce method variability between duplicates, analysts and laboratories. Clear quality assurance and quality control limits must be developed to produce valid results.

6. Sample Toxicity

Wastewater by definition may contain pollutants that inhibit bacterial seed metabolisms or are toxic to the seed. In these cases, all samples should be seeded with a known amount of viable bacteria for the MBOD analysis. Toxicity or inhibition is observed in CBOD analysis when the calculated CBOD increases with progressive dilutions of the sample.

7. Appropriate Microbial Population

Selection of a viable microbial population for the CBOD analysis is key in obtaining valid results. The bacterial population needs both carbonaceous and nitrogenous strains present. Sources of viable bacterial seed can be primary clarifier effluent, non-disinfected secondary clarifier effluent or a commercial seed preparation. Each source should have clear quality assurance and quality control requirements set by the glucose-glutamic acid check sample.

8. Glucose-Glutamic Acid Check Sample

Transfer a known amount of glucose-glutamic acid solution to a CBOD bottle and add sufficient seed to achieve acceptable dissolved oxygen depletion. Fill CBOD bottle with CBOD dilution water and nitrification inhibitor. Determine the 5 Day CBOD. Passing results will have a CBOD of 198 (+ 30.5) mg/L. Run these check samples with each batch of CBOD samples. It is important to realize that glucose-glutamic acid is not intended to be an accuracy check in the test. Its sole purpose is to demonstrate that the seed is viable and metabolizing in the proper range of activity under the conditions of the test.

9. Regulatory Use

In order to reduce a wastewater plants BOD₅ values to meet regulatory compliance requirements, some plant operators try to suppress nitrification when they are not required to meet ammonia limits. This practice usually results in increased effluent toxicity and oxygen demand on the receiving waters. Therefore, to eliminate this situation and because the BOD₅ test is not reflective of effluent quality under nitrifying conditions, the wastewater plant should:

1. Perform parallel CBOD₅ and BOD₅ tests to indicate whether there is a problem with BOD₅ compliance due to nitrification in the BOD₅ test results and that the CBOD₅ is not directly correlated with the BOD₅ test results, and
2. Baseline wastewater plant influent and effluent ammonia, nitrite and nitrate data (same frequency and duration as the parallel CBOD₅ and BOD₅ data) have been provided to perform mass balances for nitrification inhibition.

The results of these analysis can show that CBOD₅ should be utilized for regulatory compliance with wastewater discharge requirements.