Biological Oxygen Demand (BOD): A Complete Laboratory Guide
Biological Oxygen Demand (BOD) is one of the most widely used parameters for evaluating organic pollution in water and wastewater. It reflects the amount of oxygen required by microorganisms to biologically decompose organic matter under aerobic conditions. This blog presents a clear, original, and laboratory-oriented explanation of the BOD test procedure, including reagent preparation, dilution techniques, incubation, and calculation
Purpose of BOD Analysis
The purpose of this procedure is to describe the laboratory method for measuring Biological Oxygen Demand (BOD) in water and wastewater samples.
Scope
This method is applicable to environmental laboratories involved in the analysis of:
- Surface water
- Groundwater
- Treated and untreated wastewater
- Industrial effluents
where BOD determination is required for monitoring, treatment efficiency, or regulatory compliance.
Roles and Responsibilities
- Laboratory Chemist: Performs sample preparation, dilution, incubation, DO measurement, and calculation of BOD.
- Technical Manager: Reviews analytical activities and validates results.
- Quality Manager: Ensures SOP implementation and adherence to quality requirements.
Principle of the BOD Test
Biological Oxygen Demand is defined as the quantity of dissolved oxygen consumed by microorganisms while stabilizing biodegradable organic matter present in a water or wastewater sample under aerobic conditions. The reduction in dissolved oxygen over a fixed incubation period reflects the BOD of the sample.
Instruments and Equipment
- BOD bottles (300 mL capacity)
- BOD incubator maintained at 27 ± 1°C
- Measuring cylinders and volumetric flasks
- DO titration setup (as per Winkler method)
Reagents and Their Preparation
1. Phosphate Buffer Solution
Reagents required:
- Potassium dihydrogen phosphate (KH₂PO₄): 8.5 g
- Dipotassium hydrogen phosphate (K₂HPO₄): 21.75 g
- Disodium hydrogen phosphate heptahydrate (Na₂HPO₄·7H₂O): 33.4 g
- Ammonium chloride (NH₄Cl): 1.7 g
- Distilled water: up to 1000 mL
Preparation: Dissolve all salts in distilled water, make up to 1000 mL, and adjust the pH to 7.2.
2. Magnesium Sulphate Solution
Reagents required:
- Magnesium sulphate heptahydrate (MgSO₄·7H₂O): 82.5 g
- Distilled water: up to 1000 mL
Preparation: Dissolve MgSO₄·7H₂O in distilled water and dilute to 1000 mL.
3. Calcium Chloride Solution
Reagents required:
- Calcium chloride (CaCl₂): 27.5 g
- Distilled water: up to 1000 mL
Preparation: Dissolve CaCl₂ in distilled water and dilute to 1000 mL.
4. Ferric Chloride Solution
Reagents required:
- Ferric chloride hexahydrate (FeCl₃·6H₂O): 0.25 g
- Distilled water: up to 1000 mL
Preparation: Dissolve FeCl₃·6H₂O in distilled water and dilute to 1000 mL.
5. Sodium Thiosulfate Solution (0.025 N)
Reagents required:
- Sodium thiosulfate (Na₂S₂O₃): 6.205 g
- Distilled water: up to 1000 mL
Preparation: Dissolve sodium thiosulfate in distilled water and dilute to 1000 mL.
Test Method
Preparation of Dilution Water
- Aerate the required quantity of distilled water by bubbling compressed air for 1–2 days to achieve DO saturation.
- Add 1 mL each of phosphate buffer, magnesium sulphate, calcium chloride, and ferric chloride solutions per liter of dilution water.
- Mix thoroughly.
- For samples lacking sufficient microbial population, add seed—generally 2 mL of settled sewage per liter of dilution water.
Sample Preparation and Pretreatment
- Adjust sample pH to approximately 7.0 if highly acidic or alkaline.
- Ensure the sample is free from residual chlorine. If chlorine is present, remove it using sodium thiosulfate.
Removal of Residual Chlorine
- Take 50 mL of sample and acidify with 10 mL of 1+1 acetic acid.
- Add approximately 1 g potassium iodide (KI).
- Titrate with sodium thiosulfate using starch as an indicator.
- Calculate the amount of sodium thiosulfate required per milliliter of sample and treat the BOD sample accordingly.
- If the sample has unusually high DO (above 9 mg/L), reduce it by gentle aeration or agitation.
Sample Dilution
Prepare multiple dilutions to achieve:
- At least 2 mg/L DO depletion
- Residual DO not less than 1 mg/L after incubation
- Approximately 50% DO depletion
Dilution is prepared by siphoning seeded dilution water, adding the required volume of sample, and making up to volume with dilution water.
Suggested Dilutions and BOD Ranges
| % Dilution | Expected BOD (mg/L) |
|---|---|
| 0.01 | 20,000 – 70,000 |
| 0.02 | 10,000 – 35,000 |
| 0.05 | 4,000 – 14,000 |
| 0.1 | 2,000 – 7,000 |
| 0.2 | 1,000 – 3,500 |
| 0.5 | 400 – 1,900 |
| 1 | 200 – 700 |
| 2 | 100 – 350 |
| 5 | 40 – 140 |
| 10 | 20 – 70 |
| 20 | 10 – 35 |
| 50 | Up to 14 |
| 100 | 1 – 7 |
Incubation and DO Measurement
- Fill labeled BOD bottles with prepared dilutions and stopper immediately.
- Measure initial DO (D₀) in one bottle.
- Incubate three bottles at 27°C for 3 days with a proper water seal.
- Prepare blank bottles using dilution water only.
- Determine DO for samples and blanks on day 0 and after 3 days using the Winkler method.
Calculation of BOD
Let:
- D₀ = DO of sample on day 0 (mg/L)
- D₁ = DO of sample after 3 days (mg/L)
- C₀ = DO of blank on day 0 (mg/L)
- C₁ = DO of blank after 3 days (mg/L)
BOD (mg/L) = {(D₀ - D₁) - (C₀ - C₁)}/{Decimal fraction of sample used}}
If the sample is seeded, determine the BOD contribution of the seed separately and apply the appropriate correction.
Final Remarks
BOD testing provides critical insight into the biodegradable organic load of water and wastewater. Accurate dilution, proper incubation, and careful DO measurement are essential for reliable results. When followed correctly, this method remains a cornerstone of environmental water quality assessment.
🌱 Healthy microbes tell the true story of water quality.
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