The 5-Day BOD₅ Test: Overview and Exam Essentials

Biochemical Oxygen Demand (BOD₅) is a fundamental test in wastewater treatment, measuring how much dissolved oxygen (DO) aerobic bacteria consume when breaking down organic matter. The standard 5-day BOD₅ test incubates a sealed sample for 5 days at 20°C (±1°C) in the dark (to prevent algae from producing oxygen via photosynthesis). This test is crucial for assessing water quality and wastewater treatment efficiency. It’s also a common topic on Level 1 and Level 2 wastewater treatment operator exams, where specific conditions and numbers (like incubation time, temperature, and DO levels) often appear as exam questions.

What is the 5-Day BOD₅ Test?

The five-day Biochemical Oxygen Demand test (BOD₅) measures the amount of oxygen microorganisms require to digest organic material in a water sample. In simple terms, it gauges how much oxygen is demanded as bacteria feed on waste in the water. The test is run over a period of 5 days at a controlled temperature of 20°C (±1°C). By comparing the dissolved oxygen in the sample before and after incubation, we determine how much oxygen was consumed. A high BOD indicates a lot of organic pollution (since microbes used up more oxygen), while a low BOD means the water is cleaner. BOD₅ is an important indicator used by treatment plants and often shows up in exam questions to assess your understanding of wastewater organic load and treatment effectiveness.

Basic BOD₅ Test Steps

To perform a BOD₅ test, you’ll follow a series of steps. Here’s an overview of the basic procedure:

1. Measure Initial DO: Before incubation, measure the sample’s initial dissolved oxygen using a DO meter. Record this starting DO level.
2. Incubate for 5 Days: Seal the sample in a 300 mL BOD bottle (with any needed dilution water and a stopper) and place it in a dark incubator at 20°C (±1°C). The bottle stays in the incubator for 5 days (allowable variance of about ±3 hours) in complete darkness so that no algae can produce oxygen. During this period, naturally occurring (or added seed) bacteria consume the organic matter, using up oxygen.
3. Measure Final DO: After the five-day incubation, measure the dissolved oxygen in the bottle again. This final DO reading will be lower than the initial if oxygen was consumed.
4. Calculate BOD₅: Calculate the BOD₅ value using the difference between initial and final DO. The formula (for a 300 mL bottle) is:

BOD₅ (mg/L) = (Initial DO – Final DO) × (300 mL bottle volume) / (mL of sample used).

In practice, you dilute the sample so that this DO difference is measurable. For example, if you used a 10 mL wastewater sample in a 300 mL bottle, you’d multiply the DO drop by 300/10 (which is 30) to get the BOD₅ in mg/L.

Record the result, typically in mg/L, as the five-day BOD of the sample.

Each of these steps is essential. Operator certification exams (Level 1 and 2) may ask about the order of steps, the incubation conditions (time and temperature), or how to perform the BOD calculation, so be sure to understand each part.

Sample Collection and Preservation

Proper sample handling is critical for accurate BOD₅ results. Here are key points on sample collection:

• Temperature Control: Keep BOD samples at or below 4°C from the moment of collection. Chilling the samples (on ice or refrigeration) slows down biological activity so the oxygen demand doesn’t change before testing.
• Holding Time: BOD₅ tests should be started as soon as possible. Standard guidelines (Hach, 2002) recommend that samples be tested within 48 hours of collection. (Importantly, that 48-hour clock starts when the first sample was taken, not at the end of a sampling day.)
• Avoid Contamination: Use clean containers and avoid agitating the sample too much (to prevent adding extra oxygen). Keep samples sealed until you’re ready to set up the BOD test.

By following these practices, you ensure the BOD₅ test reflects the water’s original condition. Remember, exam questions might ask about sample storage temperature or maximum holding times (and 4°C and 48 hours are values you should know!).

BOD₅ Bottle Preparation

BOD tests use specialized bottles and dilution techniques to get reliable results:

• Standard BOD Bottles: Use 300 mL BOD bottles for the test. These bottles have a narrow neck and a special ground-glass stopper. Fill each bottle so that liquid completely fills the bottle and neck, then stopper it tightly. This prevents any air bubbles from being trapped. (Air bubbles would contain oxygen and could invalidate your test by adding oxygen during incubation.)
• Dilution Water: Most wastewater samples are too concentrated (oxygen demand too high) to test directly. You’ll add dilution water to the bottle along with a measured volume of your sample. Fill the bottle to the brim with this oxygen-saturated dilution water after adding the sample. The dilution water is formulated with nutrients and pH buffers to keep bacteria happy during the 5 days.
• Prevent Oxygen Exchange: BOD bottles are designed to minimize oxygen exchange with the environment. The flared lip and the water seal created by the filled neck ensure that the only oxygen used is what was initially in the bottle. This way, the drop in DO truly represents what the sample’s bacteria consumed.
• Initial DO Check: After filling and sealing the bottle (with sample + dilution water), measure the initial DO. It must be ≤ 9 mg/L at the start (Hach, 2002). If the initial DO is higher (supersaturated), you might need to gently aerate or adjust the sample. Having more than 9 mg/L initially can throw off the test or leave too much oxygen, making it hard to see a 2 mg/L drop. Exams will expect you to know that 9 mg/L is the typical maximum starting DO for a valid BOD₅ test.

Key Terms in BOD Testing

Understanding a few key terms will help clarify the BOD₅ procedure (and these often appear in exam questions):

• Dilution Water: This is specially prepared water containing nutrients and buffers that support the microorganisms during the test. It’s used to dilute the wastewater sample so the oxygen consumption falls within a measurable range. Dilution is important because you need some oxygen left after 5 days (at least 1 mg/L DO remaining). For example, a common test setup might use 10 mL of wastewater mixed with 290 mL of dilution water in a 300 mL bottle. This ensures the bottle isn’t oxygen-depleted before the test ends.
• BOD Blank: A blank is a control bottle that contains only dilution water (no sample). It’s incubated alongside your samples to measure any background oxygen demand coming from the water, nutrients, or even the bottle itself. A proper BOD blank should have very little DO depletion (ideally ≤ 0.2 mg/L drop in 5 days). If the blank depletes more than about 0.2 mg/L (Hach, 2002), it indicates a problem with the dilution water quality or contamination. In practice, you subtract the blank’s DO drop from your sample’s drop to get a corrected BOD₅, if needed.
• Seed: A seed is a source of additional bacteria added to a BOD test. Some samples (like industrial waste or very clean water) don’t have enough microbes on their own to consume organic matter. In such cases, we "seed" the BOD test by adding a small volume of mixed liquor from a treatment plant or a commercial seed culture. This ensures there are enough bacteria to carry out the test. When seeding is used, you have to account for the seed’s contribution (often by doing a seeded blank) and ensure that the sample’s DO drops by at least 2 mg/L (to confirm the seed is active and the test worked correctly). The 2 mg/L minimum drop (Hach, 2002) is also part of validating any BOD test.

Verifying BOD₅ Test Results (the 9-2-1 Rule)

After five days and final readings, you need to verify that the test results are valid. In BOD₅ testing, there’s a simple “9-2-1” rule to check this:

• Initial DO ≤ 9 mg/L: The dissolved oxygen in the bottle before incubation should be no more than 9 mg/L.
• Final DO ≥ 1 mg/L: The dissolved oxygen after the 5-day incubation should be at least 1 mg/L (i.e. not completely depleted).
• DO Depletion ≥ 2 mg/L: The drop in DO (initial minus final) should be 2 mg/L or more.

These criteria ensure your BOD₅ test had the right conditions: enough oxygen was consumed to be measurable, but not so much that the bottle ran out of oxygen entirely. If a sample doesn’t meet all three conditions (for example, if the final DO was 0 mg/L or the drop was less than 2 mg/L), the result isn’t considered valid. You would need to dilute the sample more (or add seed) and run the test again.

Exam tip: Memorize the numbers 9-2-1 – many exam questions will check that you know these standard BOD₅ validity criteria!

BOD₅ and Wastewater Operator Exams

The 5-day BOD test is a must-know topic for wastewater treatment operator certification, especially at Level 1 and Level 2 exams. Regulators and exam committees love to ask about the specifics of BOD₅ because it tests your understanding of lab procedures and environmental science basics. Here are a few exam-focused pointers:

• Be clear on key values: 5 days, 20°C incubation, sample storage at 4°C, 48-hour holding time, 300 mL bottle, initial DO no more than 9 mg/L, at least 2 mg/L drop, at least 1 mg/L remaining, and blank depletion ≤ 0.2 mg/L. These numbers often appear in exam questions (sometimes as direct questions, other times hidden in a scenario you have to analyze).
• Know the purpose of each step and component: For instance, an exam question might ask, “Why is the BOD test incubated in the dark?” (Answer: to prevent algae from producing oxygen and skewing the results) or “What is the purpose of a BOD blank?”.
• Understand how to troubleshoot: If given a scenario where the BOD test didn’t meet the 9-2-1 criteria, you should know what to do (e.g., “DO depletion was only 1.5 mg/L – this indicates not enough oxygen was consumed, possibly due to lack of bacteria or too low sample volume. The solution could be to add seed or use less dilution next time.”).

By mastering the BOD₅ test procedure and its criteria, you’ll be well-prepared for the lab section of the exam and for real-world wastewater monitoring. All the bolded numbers and facts in this article are common exam points, so make sure you can recall them easily!

Need more help preparing for the exam? Check out our Level 1 Wastewater Treatment Study Bundle for a complete exam prep guide, including study materials, practice questions, and flash cards. It’s designed to cover the essential 80/20 of what you need to know to pass your Level 1 (and build a foundation for Level 2) wastewater operator exams. Good luck with your studies, and remember – understanding core tests like BOD₅ is key to both operating a plant effectively and acing your certification exams!