Determination of Biochemical Oxygen Demand

Biochemical Oxygen Demand (BOD₅) Determination: A Comprehensive Guide

1. Introduction to BOD₅

Biochemical Oxygen Demand (BOD) is a critical water quality parameter that quantifies the amount of dissolved oxygen (DO) consumed by aerobic microorganisms during the decomposition of organic matter in a water sample. The standard test, BOD₅, measures this oxygen depletion over a five-day incubation period at 20°C. High BOD values indicate high organic pollution; low BOD means the water is cleaner for aquatic life.

Caption: BOD bottles in an incubator, illustrating the test setup.

Principle:

Microorganisms naturally present in water utilize organic matter as food. In an aerobic environment, they consume dissolved oxygen to break down this organic material. The BOD₅ test directly measures this oxygen consumption, providing an indication of the biodegradable organic pollution load in a water sample.

2. Essentials Required for BOD₅ Determination

A. Equipment:

• BOD Bottles: 300 mL glass bottles with stoppers, ensuring an airtight seal.
• Dissolved Oxygen (DO) Meter: Calibrated DO probe with temperature compensation (or equipment for Winkler Titration Method).
• Incubator: Capable of maintaining a constant temperature of 20 ± 1°C.
• Measuring Cylinders/Volumetric Flasks: Various sizes for preparing solutions and dilutions.
• Pipettes: Accurate volumetric pipettes (e.g., 1 mL, 5 mL, 10 mL, 25 mL) for precise sample and reagent addition.
• Burettes: For titrimetric methods (if using Winkler).
• Magnetic Stirrer and Stir Bars.
• pH Meter: For checking and adjusting sample pH.
• Aeration Device: Air pump with diffuser stone (for saturating dilution water).

B. Reagents and Solutions:

All reagents should be analytical grade. Use deionized or distilled water for preparing solutions.

• Dilution Water: High-purity water (distilled or deionized, free of toxic substances and organic matter).
• Phosphate Buffer Solution: (e.g., containing KH₂PO₄, K₂HPO₄, Na₂HPO₄·7H₂O, NH₄Cl).
• Magnesium Sulfate Solution (MgSO₄. 7H₂O).
• Calcium Chloride Solution (CaCl₂).
• Ferric Chloride Solution (FeCl₃. 6H₂O).
• Acid/Base Solutions: Dilute H₂SO₄ (1:50) and NaOH (1 M) for pH adjustment.
• Sodium Sulfite Solution (Na₂SO₃): For dechlorination (if required).
• Nitrification Inhibitor (e.g., TCMP): If carbonaceous BOD (cBOD) is to be determined.
• Seed Material: Unfiltered domestic sewage effluent (settled for a few hours) or a commercially available seed culture.
• Glucose-Glutamic Acid (GGA) Standard Solution: For quality control (150 mg/L glucose + 150 mg/L glutamic acid).
• For Winkler Titration (if used): Manganous Sulfate Solution, Alkali-Iodide-Azide Reagent, Concentrated H₂SO₄, Starch Indicator Solution, Sodium Thiosulfate Standard Solution.

3. Step-by-Step Procedure (BOD₅ Dilution Method)

A. Preparation Phase:

1. Prepare Dilution Water:
   • Add 1 mL each of phosphate buffer, MgSO₄, CaCl₂, and FeCl₃ solutions per liter of high-purity water.
   • Aerate this dilution water vigorously for at least 12 hours (preferably 24 hours) at 20°C to ensure it's saturated with DO.
2. Sample Pretreatment:
   • Temperature: Bring sample temperature to 20°C.
   • pH Adjustment: Check the pH of the sample. If it's outside 6.5-7.5, adjust it using dilute H₂SO₄ or 1 M NaOH.
   • Dechlorination: If the sample contains residual chlorine (e.g., chlorinated effluent), add sodium sulfite solution dropwise until chlorine is no longer detectable (test with starch-iodide paper) or the DPD method. Do not add excess.
   • Homogenization: For samples with suspended solids, gently mix to ensure homogeneity.
3. Prepare Seed (if required):
   • Allow domestic sewage to settle for approximately 2 hours. Use the supernatant as seed.
   • For industrial samples or highly treated effluents, seeding is usually necessary to provide active microorganisms.

B. Bottle Setup and Initial DO Measurement:

4. Prepare Dilution Water Blank:
   • Fill two 300 mL BOD bottles completely with only the prepared dilution water.
5. Prepare Seed Blank (if seeding):
   • Prepare two 300 mL BOD bottles with a small, known volume of seed material (e.g., 5-10 mL) and fill the rest with dilution water. These blanks will account for the oxygen demand of the seed itself.
6. Prepare GGA Standard:
   • Prepare one (or two) 300 mL BOD bottles with the Glucose-Glutamic Acid (GGA) standard solution, diluted as appropriate, and seeded if your samples are seeded. Fill the rest with dilution water.
7. Prepare Sample Dilutions:
   • Based on expected BOD values (e.g., from COD estimation), select at least three different dilutions for your sample. A typical range might be 1%, 3%, 5%, or 10%.
   • Accurately pipette the measured volume of the sample into a 300 mL BOD bottle.
   • Fill the bottle completely with the prepared dilution water (and seed, if required), ensuring no air bubbles are trapped. Prepare at least two replicate bottles for each dilution.
8. Initial Dissolved Oxygen (DO₁) Measurement:
   • Immediately after preparation (Step 4-7), measure the initial DO (DO₁) for one bottle from each set (one dilution water blank, one seed blank, one GGA, and one of each sample dilution).
   • Use a calibrated DO meter by carefully inserting the probe into the bottle without trapping air. Record the value in mg/L.

C. Incubation and Final DO Measurement:

9. Incubation:
   • Seal the remaining BOD bottles (one from each dilution water blank, seed blank, GGA, and sample dilution) with their stoppers, ensuring an airtight seal (e.g., with a water seal or parafilm).
   • Place all sealed bottles in a dark incubator maintained at 20 ± 1°C for exactly 5 days (± 3 hours).
10. Final Dissolved Oxygen (DO₂) Measurement:
    • After 5 days, remove the bottles from the incubator.
    • Measure the final DO (DO₂) for each incubated bottle using the same calibrated DO meter. Record the value in mg/L.

4. Calculations

A. Dilution Factor (DF):

DF = Total Volume of BOD Bottle (mL) / Volume of Sample (mL)

For a 300 mL bottle:

DF = 300 mL / Volume of Sample (mL)

B. Seed Correction (C) - If using seeded samples:

Calculate the oxygen consumed by the seed material from the seed blank:

Seed Depletion Rate (mg/L/mL) = (DO_{Seed Blank Initial} - DO_{Seed Blank Final}) / Volume of Seed in Blank (mL)

C (mg/L) = Seed Depletion Rate × Volume of Seed in Sample (mL)

We can write:
C (mg/L) = [(DO_{Seed Blank Initial} - DO_{Seed Blank Final}) / Volume of Seed in Blank (mL)] × Volume of Seed in Sample (mL)

C. BOD₅ Calculation:

1. For Unseeded Samples:

BOD₅ (mg/L) = (DO₁ - DO₂) × DF

Where:

• DO₁: Initial DO of diluted sample (mg/L)
• DO₂: Final DO of diluted sample (mg/L)
• DF: Dilution Factor

2. For Seeded Samples:

BOD₅ (mg/L) = [(DO₁ - DO₂) - C] × DF

Where:

• DO₁: Initial DO of diluted sample (mg/L)
• DO₂: Final DO of diluted sample (mg/L)
• C: Seed Correction (mg/L)
• DF: Dilution Factor

Typical Results Interpretation
BOD Value (mg/L)	Water Quality	Notes
0–2	Very Clean	Natural springs, well-maintained rivers
3–5	Moderately Polluted	Treated domestic sewage
6–12	Poor Quality	Untreated sewage/industrial discharge
>12	Heavily Polluted	Oxygen depletion, hazardous to life

5. Quality Assurance / Quality Control (QA/QC)

These checks are CRITICAL for valid and reportable BOD₅ results.

A. Dilution Water Blank:

• Acceptable Range: DO depletion must be < 0.2 mg/L over 5 days.
• Action: If higher, the dilution water is contaminated or poorly prepared; remake it.

B. Glucose-Glutamic Acid (GGA) Standard:

• Acceptable Range: Measured BOD₅ should be 198 ± 30.5 mg/L (as per APHA Standard Methods).
• Action: If outside this range, the seed activity is compromised, or there is a procedural error. The entire batch of BOD tests is invalid.

C. Sample Depletion Criteria:

• DO Depletion: Must be at least 2.0 mg/L.
• Residual DO (DO₂): Must be at least 1.0 mg/L.
• Action: If these criteria are not met, the dilution chosen was inappropriate (either too dilute or not dilute enough). The sample must be re-analyzed with different dilutions. Select the results from dilutions that meet both criteria.

D. Reporting:

• If multiple dilutions meet the criteria, calculate BOD₅ for each valid dilution and report the average of those values.
• Always mention the incubation time and temperature (e.g., BOD₅ at 20°C).

6. Troubleshooting Common Issues

• No DO depletion: Sample might be sterile or toxic. Try adding more seed or diluting further.
• Complete DO depletion (DO₂ = 0): Sample was too concentrated; use a higher dilution.
• Inconsistent replicates: Indicates poor mixing, air bubbles, or equipment malfunction.
• GGA outside range: Seed quality, temperature fluctuation, or contaminated reagents are likely culprits.

Remember: Precision in measurements, strict adherence to temperature, and meticulous attention to detail in sealing bottles are paramount for accurate BOD₅ results.

NOTE: This is a requested topic.