ISO 9408:1999

Overview

ISO 9408:1999 specifies a laboratory method to evaluate the ultimate aerobic biodegradability of organic compounds (including wastewaters) in aqueous media by measuring oxygen demand in a closed respirometer. The test follows aerobic microbial mineralization (to CO2, H2O and salts) and expresses biodegradation as the oxygen consumed relative to the compound’s theoretical oxygen demand (ThOD) or chemical oxygen demand (COD). Measurements are typically followed over 28 days under controlled temperature and lighting.

Key topics and technical requirements

• Test principle: Static aqueous test with the test substance as the sole carbon source; oxygen uptake in a closed respirometer is measured while CO2 is absorbed by a suitable trap.
• Exposure conditions: Incubation in the dark or diffused light at 20–25 °C (±1 °C); agitation and oxygen/CO2-controlled vessels required.
• Substance applicability: Applies to water‑soluble compounds, poorly soluble substances with dispersion measures, volatile compounds (with adapted apparatus), and substances that do not react with the CO2 absorbent. Test concentrations are typically set to provide ≈100 mg/L test compound or at least 100 mg/L ThOD.
• Inoculum: Mixed microbial inoculum (commonly activated sludge) is used. Inoculum volume, activity and quality criteria are specified to ensure sufficient biodegradative activity while limiting background organic carbon contribution.
• Measurements & endpoints:
  • Oxygen consumption tracked automatically or manually over the test period.
  • Results reported as percent biodegradation versus ThOD or COD; optional dissolved organic carbon (DOC) removal may be measured for additional information.
  • Validity criteria and control treatments (reference compound, inhibition check, blanks) are defined.
• Reagents & apparatus: Analytical-grade reagents, specified test medium and trace supplements, CO2 absorbent (e.g., KOH, soda lime), and a closed respirometer with oxygen‑impermeable tubing and temperature control.
• Safety note: Activated sludge and sewage may contain pathogens; toxic test compounds require appropriate handling.

Applications and users

ISO 9408:1999 is used by:

• Environmental testing laboratories performing biodegradation screening and regulatory submissions
• Chemical manufacturers assessing environmental fate of organic compounds
• Wastewater treatment researchers evaluating treatability and biodegradability
• Regulatory and risk assessment bodies requiring standardized biodegradability data

Typical applications include screening for ultimate biodegradability, comparing biodegradation kinetics, and supporting environmental risk assessments and wastewater treatment design.

Related standards

Standards referenced for complementary methods and checks include:

• ISO 10634 (poorly water‑soluble substances handling)
• ISO 8192 (toxicity/inhibition testing)
• ISO 15462 (alternative biodegradation methods)
• ISO 6060 (COD determination)
• ISO 9888 / ISO 9887 (pre‑exposure / related biodegradation tests)
• ISO 11923 (suspended solids determination)

Annexes in ISO 9408 provide calculation examples, nitrification corrections, biodegradation curves and respirometer guidance.