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AGR-WATE71-02 · Lesson 5 · 13.5 clock hours · lab

Dissolved Oxygen, BOD, and the Oxygen Demand Concept

Objective. Students will measure dissolved oxygen using a DO meter and Winkler titration, set up a 5-day BOD test, and explain why BOD is a critical parameter in wastewater treatment.

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This lesson, its worked examples and its practice questions has not yet been verified by a licensed professional in the field or by a certification or approval body. It is a study draft and may contain errors. Do not use it as instructions for work on a job site or on live equipment. Always follow your instructor, your supervising professional, and current official references and regulations.

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Why Dissolved Oxygen Matters

Dissolved oxygen (DO) is the amount of oxygen gas (O₂) dissolved in water, measured in milligrams per liter (mg/L). Aquatic organisms depend on DO to survive. In wastewater treatment, operators track DO because microorganisms in aerobic treatment processes consume oxygen to break down organic waste. Without enough DO, those organisms die off and treatment fails.

Clean, cold surface water can hold roughly 9–12 mg/L of DO under typical conditions. Wastewater, which carries organic matter, nutrients, and biological solids, often enters a treatment plant with very low DO—sometimes near zero. Restoring and maintaining adequate DO is a core operational goal.

Oxygen Solubility: Temperature and Altitude

Oxygen solubility in water decreases as temperature rises. Warm water simply cannot hold as much dissolved gas as cold water. At sea level, water near 0 °C can hold about 14 mg/L, while water at 30 °C holds only about 7.5 mg/L. This inverse relationship is why summer months stress treatment systems: warmer water holds less DO while biological activity speeds up and demands more.

Altitude also reduces DO saturation. Higher elevations have lower atmospheric pressure, so less oxygen is available to dissolve into water. Operators at high-elevation plants must account for this when evaluating aeration efficiency and setting operational targets.

Sources of DO Depletion in Wastewater

Several factors consume DO in wastewater:

Understanding these demands helps operators size aeration equipment, manage sludge wasting, and protect effluent quality.

What Is BOD?

Biochemical Oxygen Demand (BOD) measures the amount of oxygen that microorganisms need to biologically degrade organic matter in a water sample under controlled conditions. It is expressed in mg/L. A high BOD means a large amount of degradable organic matter is present; a low BOD indicates cleaner water.

The standard test is the BOD₅: a sample is incubated in the dark at 20 °C for exactly five days. The DO is measured before incubation begins and again after five days. The difference, adjusted for dilution, equals the BOD₅.

BOD₅ is a critical regulatory parameter. Wastewater treatment plants are typically required to reduce BOD to meet their National Pollutant Discharge Elimination System (NPDES) permit limits before discharge. Exceeding those limits can harm receiving streams and result in regulatory penalties.

Measuring DO: Two Methods

DO Meter (Electrochemical probe): The probe contains a membrane that allows oxygen to diffuse to a sensor. The instrument converts the sensor signal to a mg/L readout. Before use, the probe must be calibrated—usually using air-saturated water or water-saturated air at a known temperature and barometric pressure. A well-calibrated meter offers quick, repeatable readings.

Winkler Titration: This wet-chemistry method is the reference technique listed in Standard Methods for the Examination of Water and Wastewater. Manganese sulfate is added first, fixing the oxygen as a manganese compound. Alkaline iodide solution is then added; the fixed oxygen oxidizes iodide to iodine. Sulfuric acid acidifies the sample, and the liberated iodine is titrated with sodium thiosulfate using starch as an indicator. The volume of thiosulfate used corresponds directly to the DO concentration. Winkler titration is more time-intensive but is used to verify meter accuracy.

Setting Up the BOD₅ Test

1. Prepare dilution water: aerated, buffered, nutrient-supplemented reagent water that supplies microorganisms with what they need besides organic food.

2. Fill BOD bottles completely with diluted wastewater sample—no air bubbles.

3. Measure and record initial DO immediately.

4. Seal bottles with glass stoppers and water seals to exclude air.

5. Place in an incubator at 20 °C (±1 °C) in the dark for five days.

6. After exactly five days, measure final DO and calculate BOD₅.

BOD₅ Calculation

BOD₅ (mg/L) = (DO_initial − DO_final) × Dilution Factor

The dilution factor accounts for how much the sample was diluted with dilution water before filling the bottles.

Safety in the Lab

Key terms8

Dissolved Oxygen (DO)The concentration of oxygen gas dissolved in water, measured in mg/L; essential for aerobic biological treatment and aquatic life.
BOD₅ (5-Day Biochemical Oxygen Demand)The amount of oxygen, in mg/L, consumed by microorganisms decomposing organic matter in a water sample incubated at 20 °C for five days in the dark.
Winkler TitrationA wet-chemistry reference method for measuring DO that uses manganese sulfate, alkaline iodide, sulfuric acid, and sodium thiosulfate in a sequential series of reactions.
Oxygen SaturationThe maximum concentration of DO that water can hold at a given temperature and atmospheric pressure; decreases as temperature or altitude increases.
Dilution FactorThe ratio of total bottle volume to sample volume used in a BOD test; applied to the measured DO difference to calculate the BOD of the undiluted sample.
NPDES PermitNational Pollutant Discharge Elimination System permit; the legal authorization that sets effluent limits, including BOD, for discharges from wastewater treatment plants to surface waters.
NitrificationThe biological conversion of ammonia to nitrate by specialized bacteria; a process that exerts a significant oxygen demand in wastewater and receiving waters.
Standard MethodsShort name for Standard Methods for the Examination of Water and Wastewater, the authoritative reference for laboratory procedures used by water and wastewater operators.

Worked examples4

1. A BOD bottle has a total volume of 300 mL. A student added 15 mL of wastewater and filled the rest with dilution water. Initial DO = 8.6 mg/L; final DO after 5 days = 3.1 mg/L. Calculate BOD₅.
  1. Step 1 – Find the dilution factor: Dilution Factor = Total volume ÷ Sample volume = 300 mL ÷ 15 mL = 20.
  2. Step 2 – Find the DO difference: DO_initial − DO_final = 8.6 mg/L − 3.1 mg/L = 5.5 mg/L.
  3. Step 3 – Apply the BOD formula: BOD₅ = DO difference × Dilution Factor = 5.5 mg/L × 20 = 110 mg/L.
Answer: BOD₅ = 110 mg/L
2. A student calibrates a DO probe and then measures DO in a sample, getting 7.8 mg/L. The Winkler titration on the same sample required 7.3 mL of 0.025 M sodium thiosulfate. Using the simplified relationship that 1 mL of 0.025 M thiosulfate corresponds to 0.2 mg of O₂ in a 200 mL sample (a standard Winkler scaling), calculate the Winkler DO and determine whether the meter passes the ±0.5 mg/L check.
  1. Step 1 – Calculate DO from Winkler: DO (mg/L) = (mL thiosulfate × 0.2 mg O₂ per mL) ÷ 0.200 L sample = (7.3 × 0.2) ÷ 0.200 = 1.46 ÷ 0.200 = 7.3 mg/L.
  2. Step 2 – Find the difference between meter and Winkler: |7.8 − 7.3| = 0.5 mg/L.
  3. Step 3 – Compare to acceptance criterion: The criterion is within ±0.5 mg/L. The difference equals exactly 0.5 mg/L, which meets the criterion.
Answer: Winkler DO = 7.3 mg/L; difference = 0.5 mg/L; meter reading passes the ±0.5 mg/L check.
3. A wastewater plant treats an influent with a BOD₅ of 240 mg/L. The effluent BOD₅ is measured at 18 mg/L. What is the percent BOD removal?
  1. Step 1 – Calculate BOD removed: 240 mg/L − 18 mg/L = 222 mg/L removed.
  2. Step 2 – Calculate percent removal: (222 ÷ 240) × 100 = 92.5%.
Answer: BOD removal = 92.5%
4. On an instructor-provided dataset: Initial DO = 9.0 mg/L; Final DO = 2.4 mg/L; sample volume = 30 mL; bottle volume = 300 mL. Verify this BOD₅ calculation to within 5%.
  1. Step 1 – Dilution factor: 300 ÷ 30 = 10.
  2. Step 2 – DO difference: 9.0 − 2.4 = 6.6 mg/L.
  3. Step 3 – BOD₅: 6.6 × 10 = 66 mg/L.
  4. Step 4 – 5% tolerance check: 5% of 66 = 3.3 mg/L, so an acceptable student answer falls between 62.7 and 69.3 mg/L.
Answer: BOD₅ = 66 mg/L (acceptable range 62.7–69.3 mg/L)

The math in this lesson1

Learners compute a 300 ÷ 15 = 20 dilution factor and BOD₅ = 5.5 × 20 = 110 mg/L.
6.RP.3
In class: Lecture: oxygen solubility vs. temperature and altitude, sources of DO depletion in wastewater, and the significance of BOD5 in evaluating treatment plant effluent quality; students complete a fill-in notes guide. · DO measurement lab: students calibrate a DO probe using air-saturated water, then measure DO in at least four samples at different temperatures, comparing meter readings with Winkler titration results on the same samples. · BOD5 setup: students prepare diluted wastewater samples, measure initial DO, seal BOD bottles, and incubate at the standard temperature; instructor demonstrates the final-day measurement and BOD calculation using a completed example dataset.
Materials: DO meter and probe, Winkler titration reagents (manganese sulfate, alkaline iodide, sulfuric acid, sodium thiosulfate), BOD bottles, incubator set to standard temperature, dilution water, wastewater samples, fill-in notes guide, Standard Methods, safety glasses, gloves, lab aprons.
Check: DO meter reading within ±0.5 mg/L of Winkler titration on same sample; BOD5 calculation correct to within 5% on instructor-provided dataset; lab log entry shows all required data fields completed (all three required to pass).

Practice check8

Answer each question, then check it. Nothing is recorded; this is your own practice.

Water/Wastewater Treatment Processes
1. Which condition produces the LOWEST dissolved oxygen saturation in water?
Laboratory Procedures and Sampling
2. In a BOD₅ test, the sample is incubated at which standard temperature?
Mathematics and Calculations
3. A BOD bottle (300 mL total) is filled with 10 mL of wastewater and 290 mL of dilution water. Initial DO = 9.2 mg/L; final DO = 4.2 mg/L. What is the BOD₅?
Laboratory Procedures and Sampling
4. What is the primary purpose of calibrating a DO probe using air-saturated water before taking measurements?
Laboratory Procedures and Sampling
5. Which sequence of reagents is correct for the Winkler titration method for dissolved oxygen?
Mathematics and Calculations
6. A wastewater plant has an influent BOD₅ of 200 mg/L and an effluent BOD₅ of 20 mg/L. What is the percent BOD removal?
Environmental Regulations and Permit Compliance
7. Why is a high BOD in a wastewater treatment plant effluent a concern for the receiving stream?
Safety and Emergency Response
8. Which personal protective equipment (PPE) is REQUIRED when handling sulfuric acid during the Winkler titration?
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