Determination of Five-Day Biochemical Oxygen Demand (BOD5) in Surface Water

The five-day biochemical oxygen demand (BOD5) is one of the most widely applied indicators for evaluating the concentration of biodegradable organic matter in surface water. This article presents a comprehensive technical overview of BOD5 determination in surface water, covering the analytical principle, sampling requirements, the dilution and seeding method, dissolved oxygen measurement techniques, quality control procedures, and typical applications in environmental monitoring and regulatory compliance.

Technical Overview

Biochemical oxygen demand (BOD) is defined as the mass of dissolved oxygen consumed by aerobic microorganisms while decomposing organic matter in a water sample under specified incubation conditions. The standard BOD5 test measures the oxygen consumed during five days of incubation at 20 ± 1 °C in complete darkness. Because the microbial oxidation of carbonaceous organic matter is approximately 60–70 percent complete after five days, BOD5 is accepted internationally as a practical and reproducible estimate of the oxygen demand exerted by biodegradable substances in a water body.

For surface water, BOD5 is a core classification parameter in environmental standards worldwide, including GB 3838-2002 (Environmental Quality Standards for Surface Water). The internationally harmonized laboratory procedure is the dilution and seeding method described in ISO 5815-1, which corresponds to the Chinese environmental protection standard HJ 505-2009 and to APHA Standard Methods 5210 B. Where nitrification must be excluded, ISO 5815-2 specifies a companion procedure using a nitrification inhibitor to determine carbonaceous BOD.

The basic principle of the test is as follows: the water sample is diluted with air-saturated dilution water containing essential nutrients, seeded with a microbial culture when necessary, and incubated in airtight bottles. Dissolved oxygen (DO) is measured immediately after dilution and again after five days of incubation. BOD5 is calculated from the oxygen depletion, corrected for the dilution factor and, where applicable, for the oxygen demand of the seed itself. The dilution and seeding method is generally applicable to BOD5 concentrations from approximately 3 mg/L up to 6,000 mg/L by selecting appropriate dilution factors.

Testing Samples

Rivers and Streams

Flowing waters that receive municipal, industrial, or agricultural discharges are the most common surface water matrices for BOD5 testing. Grab or time-composite samples are collected from well-mixed sections at mid-depth, avoiding bank effects, floating debris, and sediment disturbance. Expected BOD5 values typically range from below 2 mg/L in pristine headwaters to more than 20 mg/L in polluted reaches.

Lakes and Reservoirs

In standing water bodies, thermal stratification and algal activity strongly influence oxygen balance. Samples are usually taken from the epilimnion at representative monitoring points. Because algae can release oxygen through photosynthesis, incubation in complete darkness is essential to suppress photosynthetic oxygen production and obtain a valid oxygen depletion value. Samples with heavy algal content may be gently settled before analysis.

Estuarine and Brackish Waters

For estuarine samples, the salinity of the dilution water should be matched to that of the sample, for example by adding a prepared artificial seawater brine, to prevent osmotic shock to the microbial seed. This ensures that oxygen depletion reflects substrate decomposition rather than microbial mortality.

Sampling and Preservation Requirements

• Collect samples in clean glass or polyethylene bottles, filling them completely without entraining air bubbles.
• Minimize aeration and headspace during collection, transfer, and mixing.
• Transport and store samples at 0–4 °C in the dark.
• Begin analysis as soon as practicable, preferably within 6 hours and no later than 24 hours after collection.

Testing Items

The principal deliverable is the BOD5 concentration of the sample, expressed in mg/L. Supporting determinations and control checks that form part of a complete test report include:

• Dissolved oxygen of the freshly prepared diluted sample (D1) and after five days of incubation (D2)
• Dilution water blank depletion, which must not exceed 0.2 mg/L
• Seed control depletion and the corresponding seed correction factor
• Glucose–glutamic acid check sample for method validation
• Pre-incubation screening of sample pH, temperature, residual chlorine, and salinity

Testing Methods

Dilution and Seeding Method

The sample is diluted with nutrient-fortified, air-saturated dilution water so that sufficient dissolved oxygen remains after incubation. For surface waters containing few microorganisms, the dilution water is seeded with a suitable microbial culture. Aliquots are transferred to airtight incubation bottles of typically 250–300 mL capacity, water-sealed, and incubated at 20 ± 1 °C in darkness for five days.

Preparation of Dilution Water

Dilution water is prepared from high-purity water that has been aerated and equilibrated to oxygen saturation at 20 °C (approximately 9.1 mg/L DO in freshwater). Four nutrient stock solutions are added immediately before use: a phosphate buffer maintaining pH near 7.2, magnesium sulfate, calcium chloride, and ferric chloride. Seed sources include settled domestic wastewater aged 24–48 hours, supernatant from a river receiving wastewater, or commercial seed preparations.

Sample Pretreatment

• Neutralize samples with dilute hydrochloric acid or sodium hydroxide if the pH falls outside the range of 6.5 to 7.5.
• Neutralize residual chlorine with sodium sulfite, allowing sufficient contact time before analysis.
• Equilibrate the sample to approximately 20 °C before preparing dilutions.
• Allow samples containing settleable solids to settle quietly, or use the homogeneous portion, depending on the monitoring objective.
• When carbonaceous BOD is required, add a nitrification inhibitor such as allylthiourea in accordance with ISO 5815-2.

Dissolved Oxygen Measurement

Dissolved oxygen may be determined by the iodometric Winkler titration method (ISO 5814, GB 7489, ASTM D888), by membrane-electrode probes, or by optical luminescent dissolved oxygen sensors. Electrochemical and optical sensors must be calibrated against Winkler titration results, and each incubation bottle is measured only once, as the measurement terminates the test for that bottle.

Calculation

For an unseeded dilution, BOD5 = (D1 − D2) / P, where P is the decimal volumetric fraction of the sample in the bottle. When seed is used, BOD5 = [(D1 − D2) − (B1 − B2) × f] / P, in which B1 and B2 are the dissolved oxygen values of the seed control before and after incubation, and f is the ratio of seed volume in the sample bottle to that in the seed control bottle. A result is valid only when the oxygen depletion is at least 2 mg/L and the residual dissolved oxygen after incubation remains above the minimum level, generally 1–2 mg/L, prescribed by the applicable standard.

Quality Assurance and Quality Control

Each analytical batch must include dilution water blanks, replicate dilutions, and a glucose–glutamic acid check standard (150 mg/L of each compound), which should yield a BOD5 of approximately 198 mg/L within established control limits. Dissolved oxygen measurement systems require documented calibration, and competent testing is performed by accredited third-party laboratories or certified in-house facilities operating under an ISO/IEC 17025 quality management system.

Applications

• Classification of surface water quality against GB 3838-2002, which sets BOD5 limits of 3 mg/L for Class I and II waters, 4 mg/L for Class III, 6 mg/L for Class IV, and 10 mg/L for Class V.
• Compliance monitoring of wastewater treatment plant discharges and evaluation of their impact on receiving waters.
• Environmental impact assessments, pollution source identification, and total pollutant load allocation programs.
• Studies of self-purification capacity and oxygen balance in rivers, lakes, and estuaries.
• Biodegradability evaluation using the BOD5-to-COD ratio, which distinguishes readily biodegradable organic pollution from refractory organic matter.
• Baseline monitoring and long-term trend analysis in national and regional water quality networks.

FAQ

Why is the incubation period exactly five days at 20 °C?

The five-day period at 20 °C is a historical convention that provides a reproducible compromise. Complete biochemical oxidation of carbonaceous matter takes roughly twenty days, and the five-day value at 20 °C represents approximately 60–70 percent of that ultimate carbonaceous demand while remaining short enough for routine regulatory monitoring.

What is the difference between BOD5 and COD?

BOD5 measures only the fraction of organic matter that aerobic microorganisms can biologically decompose within five days, whereas chemical oxygen demand (COD) measures the total chemically oxidizable material, including non-biodegradable compounds. A high COD-to-BOD5 ratio indicates a large proportion of refractory or toxic organic substances in the water.

How are dilution ratios selected?

Analysts estimate the expected BOD5, often from a preliminary COD or historical data, and prepare several dilutions bracketing that value. The correct dilution is the one in which oxygen depletion is at least 2 mg/L while at least 1–2 mg/L of dissolved oxygen remains after incubation, ensuring the result falls within the valid measurement window.

What is seeding and when is it required?

Seeding is the addition of a microbial culture to dilution water when the sample itself contains too few organisms, as is common for clean surface waters. The oxygen demand contributed by the seed is measured in a separate seed control and subtracted from the sample result through the seed correction term in the calculation formula.

How long can samples be stored before analysis?

Samples should be kept at 0–4 °C in the dark and analyzed as soon as possible, ideally within 6 hours of collection. Under no circumstances should the holding time exceed 24 hours, because biological activity during storage changes the true BOD5 value.

What validation checks confirm the reliability of a BOD5 result?

Reliability is demonstrated by dilution water blank depletion not exceeding 0.2 mg/L, compliance with the minimum depletion and residual oxygen criteria, acceptable agreement among replicate dilutions, and a glucose–glutamic acid check result falling within the recognized control range around 198 mg/L.