🫧 BOD5 — Biochemical Oxygen Demand
In short
Determination of oxygen consumed by microorganisms for decomposition of organic matter in water over 5 days. The test is performed according to PN-EN ISO 5815-1:2019-12; Measurement range: 0.5–6 000 mg O₂/L (with dilution). The procedure comprises 11 steps (about 5 days 1 h 20 min in total); it is used for: Treated wastewater quality control (water permit compliance), Wastewater treatment plant sizing and operational control, Surface water monitoring (Water Framework Directive).
At a glance
- Standard: PN-EN ISO 5815-1:2019-12
- Category: Physicochemistry
- Procedure steps: 11
- Total time of stages: 5 days 1 h 20 min
- Measurement range: 0.5–6 000 mg O₂/L (with dilution)
- Limit of quantification: 0.5 mg O₂/L
- Incubation temperature: 20 ± 1°C
Overview
Biochemical Oxygen Demand (BOD5) is one of the most important parameters for determining the degree of organic pollution in water and wastewater. It expresses the amount of dissolved oxygen (in mg O₂/L) consumed by aerobic microorganisms for biochemical oxidation of organic substances in a water sample during 5 days of incubation at 20°C in darkness.
BOD5 is a critical parameter in wastewater treatment technology — it is used for treatment plant sizing, monitoring the efficiency of biological processes, and assessing activated sludge loading. In environmental monitoring, it allows evaluation of the impact of wastewater discharges on receiving waters (rivers, lakes). It is a mandatory parameter in water permits and wastewater discharge billing.
Permissible BOD5 values in Poland: drinking water <3 mg O₂/L, treated wastewater discharged to waters <25 mg O₂/L (above 10 000 PE), surface water class I <3 mg O₂/L.
Method principle
The method involves measuring the dissolved oxygen concentration in the sample before and after 5-day incubation at 20±1°C in darkness. The sample is diluted with aerated dilution water (oxygen-saturated, supplemented with mineral salts) at a ratio ensuring a DO decrease of 2–5 mg/L after incubation (a minimum of 2 mg/L dissolved oxygen must remain). Addition of allylthiourea (ATU) inhibits nitrification, eliminating the influence of ammonium nitrogen oxidation on the result.
BOD5 = (DO₀ – DO₅) × dilution factor
where DO₀ — dissolved oxygen at start, DO₅ — after 5 days.
Applications
- Treated wastewater quality control (water permit compliance)
- Wastewater treatment plant sizing and operational control
- Surface water monitoring (Water Framework Directive)
- Drinking water quality testing
- Industrial discharge control
- Biodegradability assessment (BOD5/COD ratio)
Key parameters
| Parameter | Value |
|---|---|
| Measurement range | 0.5–6 000 mg O₂/L (with dilution) |
| Limit of quantification | 0.5 mg O₂/L |
| Incubation temperature | 20 ± 1°C |
| Incubation time | 5 days ± 4 hours |
| Precision (CV) | 5–15% (concentration-dependent) |
| Required DO depletion | min. 2 mg/L, sample must retain min. 2 mg/L DO |
Standard
- Standard number
- PN-EN ISO 5815-1:2019-12
- Title (PL)
- Jakość wody -- Oznaczanie biochemicznego zapotrzebowana tlenu po n dniach (BZTn) -- Część 1: Metoda rozcieńczeń, z dodatkiem materiału zaszczepiającego i allilotiomocznika
- Title (EN)
- Water quality — Determination of biochemical oxygen demand after n days (BODn) — Part 1: Dilution and seeding method with allylthiourea addition
Step-by-step procedure
-
Dilution water preparation
Aerate deionized water for min. 1 hour (to oxygen saturation ~8.5 mg/L at 20°C). Add 1 mL each of phosphate, MgSO₄, CaCl₂, and FeCl₃ solutions per litre of water.
-
ATU addition
Add allylthiourea to a final concentration of 0.5 mg/L (1 mL of 5 g/L solution per 10 L of dilution water) to inhibit nitrification.
-
Seeding
Add inoculum (5–10 mL of raw wastewater per litre of dilution water) or use a prepared seed. Do not seed if the sample contains its own microflora.
-
Dilution selection
Based on the expected BOD5, prepare min. 3 dilutions. E.g. for raw wastewater (BOD ~200 mg/L): 1:50, 1:100, 1:200. For surface water: undiluted or 1:2.
-
Bottle filling
Fill 300 mL Winkler bottles with diluted sample — without air bubbles. Close with ground-glass stopper, apply water seal on top. Minimum 2 bottles per dilution.
-
DO₀ measurement (day 0)
In one bottle from each dilution, measure dissolved oxygen (DO₀). Record the value to 0.1 mg/L accuracy. DO₀ should be >7 mg/L.
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Incubation
Place bottles in the incubator at 20±1°C in darkness (light inhibits photosynthesis → falsified results). Incubate for 5 days ±4 hours.
-
DO₅ measurement (day 5)
After 5 days, measure dissolved oxygen (DO₅) in the remaining bottles. Measure immediately after removal from the incubator — do not mix vigorously.
-
Result validation
Verify: DO depletion ≥2 mg/L, residual DO ≥2 mg/L, blank (dilution water) ΔDO ≤0.2 mg/L. Reject non-compliant results.
-
Calculations
BOD5 = [(DO₀ – DO₅) – (DO₀blank – DO₅blank)] × dilution factor. Result in mg O₂/L. Calculate the mean from selected dilutions.
-
Quality control
GGA standard: BOD5 = 198 ± 30.5 mg O₂/L (certified value). Run with each sample batch.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| BOD incubator (20±1°C) | WTW TS 606-G/2-i, Binder KB 240, POL-EKO ST 5 | 8 000–25 000 PLN |
| Dissolved oxygen meter | WTW Oxi 3310, Hach HQ40d, YSI ProODO | 3 000–12 000 PLN |
| BOD bottles 300 mL | Winkler bottles with ground-glass stopper, DURAN | 30–60 PLN/pc. |
| Aeration system | Aquarium compressor with diffuser, aeration 1 h | 100–500 PLN |
| Automatic pipettes | Eppendorf Research Plus 1–10 mL, Brand Transferpette | 800–2 000 PLN |
| Measuring cylinders | 100, 250, 500, 1000 mL class A | 50–150 PLN/pc. |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Allylthiourea (ATU) | 109-57-9 | Nitrification inhibitor, 5 g/L solution, dose 1 mL/L of dilution water |
| Phosphate buffer solution | — | KH₂PO₄ (8.5 g) + K₂HPO₄ (21.75 g) + Na₂HPO₄·7H₂O (33.4 g) + NH₄Cl (1.7 g) per 1 L |
| MgSO₄ solution | 7487-88-9 | MgSO₄·7H₂O 22.5 g/L |
| CaCl₂ solution | 10043-52-4 | CaCl₂ (anhydrous) 27.5 g/L |
| FeCl₃ solution | 7705-08-0 | FeCl₃·6H₂O 0.25 g/L |
| Inoculum (seed) | — | Raw wastewater from treatment plant or garden soil — source of microorganisms |
| GGA standard | 56-40-6 | Glucose-glutamic acid (150 mg/L glucose + 150 mg/L glutamic acid), BOD5 = 198±30.5 mg/L |
Health and safety (OHS)
- Allylthiourea — toxic, use in fume hood, nitrile gloves required
- Wastewater samples — potentially infectious, wear gloves and face protection
- Iron(III) chloride — irritant, avoid skin contact
- Laboratory coat, gloves, and safety glasses mandatory
- Biological waste must be disposed of according to laboratory protocol
Training and video materials
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 5815-1:2019-12 — “Water quality — Determination of biochemical oxygen demand after n days (BODn) — Part 1: Dilution and seeding method with allylthiourea addition”.
How does this method work?
The method involves measuring the dissolved oxygen concentration in the sample before and after 5-day incubation at 20±1°C in darkness. The sample is diluted with aerated dilution water (oxygen-saturated, supplemented with mineral salts) at a ratio ensuring a DO decrease of 2–5 mg/L after incubation (a minimum of 2 mg/L dissolved oxygen must remain).
What is the measuring range and accuracy?
Measurement range: 0.5–6 000 mg O₂/L (with dilution); Limit of quantification: 0.5 mg O₂/L; Incubation temperature: 20 ± 1°C; Incubation time: 5 days ± 4 hours.
How long does the test take?
The times given for the individual stages add up to approximately 5 days 1 h 20 min. The procedure comprises 11 steps.
What equipment is required?
BOD incubator (20±1°C), Dissolved oxygen meter, BOD bottles 300 mL, Aeration system, Automatic pipettes, Measuring cylinders. Indicative cost of the core instrument (BOD incubator (20±1°C)): 8 000–25 000 PLN.
Where is this test used?
Treated wastewater quality control (water permit compliance); Wastewater treatment plant sizing and operational control; Surface water monitoring (Water Framework Directive); Drinking water quality testing; Industrial discharge control; Biodegradability assessment (BOD5/COD ratio).
What safety precautions apply?
Allylthiourea — toxic, use in fume hood, nitrile gloves required; Wastewater samples — potentially infectious, wear gloves and face protection; Iron(III) chloride — irritant, avoid skin contact; Laboratory coat, gloves, and safety glasses mandatory; Biological waste must be disposed of according to laboratory protocol.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 5815-1.