Standard Dilution Method for BOD Measurement – A Practical Guide

The five‑day biochemical oxygen demand (BOD₅) is a key indicator of organic pollution in water. The standard dilution (or dilution‑and‑seeding) method is the classic and widely accepted procedure for determining BOD₅. It involves incubating a diluted water sample at 20 °C in the dark for five days and measuring the dissolved oxygen (DO) consumed by microorganisms. Because most samples contain more organic matter than the available oxygen in the bottle, proper dilution is essential.

A water sample is diluted with oxygen‑saturated dilution water (sometimes seeded with microorganisms). The initial DO (D₁) and the DO after five days (D₂) are measured. The difference, multiplied by the dilution factor, gives the BOD₅ value. For valid results, the residual DO after five days must be ≥1 mg/L and the consumed DO ≥2 mg/L.

Reagents and Dilution Water

Dilution water is prepared from deionised water. Phosphate buffer, magnesium sulfate, calcium chloride, and ferric chloride are added to provide essential nutrients. The water is aerated to achieve DO ≥8 mg/L and adjusted to pH 7.2. If the sample lacks sufficient microorganisms (e.g., industrial wastewater), a seeding suspension (e.g., settled activated sludge supernatant) is added to the dilution water. To suppress nitrification, a nitrification inhibitor (allylthiourea) may be added.

Determining Dilution Factors

The correct dilution is critical. It is usually estimated from chemical oxygen demand (COD). A common rule: dilution factor ≈ COD ÷ (4–5). For safety, three different dilutions are prepared. For high‑strength wastewater, factors of 100–1000 may be needed; for clean river water, dilution may be unnecessary.

Procedure

Prepare the dilution series by mixing the sample with dilution water in graduated cylinders. Carefully fill 300 mL BOD bottles to overflowing to eliminate air bubbles, then seal with a glass stopper and water‑seal the cap. Prepare blank bottles containing only seeded dilution water. Measure initial DO (using a DO meter or Winkler titration) immediately after filling. Incubate all bottles at 20 °C ±1 °C in the dark for five days. After incubation, measure the final DO.