OECD 209 Activated Sludge, Respiratory Inhibition Test (Carbon and Ammonium Oxidation)

The “OECD 209 Activated sludge, respiration inhibition test (carbon and ammonium oxidation)” standard, published under the OECD guidelines on responsible business conduct for multinational companies (OECD guidelines) , is designed to assess the effects of a substance on microorganisms obtained from activated sludge in wastewater treatment plants by measuring their respiration rates (carbon or ammonium oxidation) as oxygen consumption. Test results can also be used as an indicator of appropriate non-inhibitory concentrations of test substances to be used in biodegradability tests.

This test method allows the determination of the effective concentration and the observed no-effect concentration of the test substance. Three different types of oxygen uptake inhibition can be determined: total, heterotrophic only, and nitrification-induced inhibition in the presence and absence of N-allylthiourea, a specific nitrification inhibitor.

To obtain both the observed no-effect concentration and the effective concentration, a geometric series of five replicates of six control and five treatment concentrations is recommended. In each test vessel, test mixtures containing water, synthetic sewage feed, and the test substance are incubated at pH 7,5 plus/minus 0,5 and at 20°C plus/minus 2°C under forced aeration to maintain the dissolved oxygen concentration above 60-70 percent saturation. Oxygen consumption is measured after 3 hours of exposure, and if the test substance degrades rapidly, additional measurements may be made after 30 minutes of exposure.

The OECD 209 standard is a standard method developed by the Organisation for Economic Co-operation and Development (OECD) to assess the potential toxicity of chemicals to microorganisms in wastewater treatment systems. This test is critical for assessing the environmental impacts of chemicals, particularly in the context of wastewater treatment plants where activated sludge plays a vital role in the degradation of organic matter.

The primary objective of the OECD 209 test method is to assess the effects of a substance on microorganisms present in activated sludge, a key component of aerobic wastewater treatment processes. The respiration rate of these microorganisms, specifically their oxygen consumption during carbon and ammonium oxidation, is measured to determine whether a substance inhibits microbial activity. This information is crucial for:

• Environmental risk assessments: Testing helps evaluate the potential toxicity of chemicals on microbial communities necessary for the degradation of organic pollutants in wastewater.
• Biodegradability testing: Results may indicate appropriate, non-inhibitory concentrations of a test substance for use in subsequent biodegradability studies.
• Regulatory compliance: Testing provides data to support regulatory decisions regarding the safety of chemicals and ensuring they do not disrupt wastewater treatment processes.

This test method measures key endpoints used to quantify the toxicity of a substance, such as the effective concentration (e.g., EC50, the concentration that causes 50 percent inhibition) and the no-observed-effect concentration.

The OECD 209 test involves exposing activated sludge microorganisms to various concentrations of a test substance under controlled conditions. The methodology is designed to ensure reproducibility and reliability while adhering to stringent validation criteria.

The basic steps of the testing method are as follows:

• Test organisms: Activated sludge, typically collected from the aeration tank of a well-operated wastewater treatment plant that primarily treats municipal wastewater, is used in the test. The sludge contains a diverse microbial community responsible for organic matter degradation and nitrification. To ensure consistency, sludge can be washed if the endogenous respiration rate is low and is typically used within one day of collection.
• Test setup:
  • Test mixtures: The test involves incubating mixtures of activated sludge, synthetic wastewater feed (which mimics wastewater nutrients), and test substance in 1000 mL reactors containing 500 mL of the reaction mixture. A blank control (without test substance) and a reference substance (e.g., 3,5-dichlorophenol or copper(II) sulfate pentahydrate) are added to verify the sensitivity of the test system.
  • Concentrations: The test usually includes at least three concentrations of the test substance in a geometric series (e.g., 10 mg/L, 100 mg/L, and 1000 mg/L), and for dose-response testing, six controls and five replicates per treatment. In limit testing, a single concentration (e.g., 1000 mg/L) may be used to determine whether significant toxicity occurs.
  • Conditions: Testing is conducted at a pH of 7,5 plus/minus 0,5 and a temperature of 20 degrees Celsius plus/minus 2 degrees Celsius, with forced ventilation to maintain dissolved oxygen levels above 60-70 percent saturation. Oxygen consumption is measured after a 3-hour exposure period; 30-minute measurements are optional for rapidly degrading substances.
• Measurement: Respiratory rate is determined by measuring dissolved oxygen consumption in the range of 2–7 mg/L using an oxygen electrode in a closed cell. The test can assess three types of oxygen uptake:
  • Total respiration: Total oxygen consumption of the sludge.
  • Heterotrophic respiration: The oxygen used for carbon oxidation is measured in the presence of N-allylthiourea, a nitrification inhibitor.
  • Nitrifying respiration: The oxygen used for ammonium oxidation is calculated as the difference between total and heterotrophic respiration.

The respiration rates of the test substance and the reference substance are compared with blank controls and expressed as percent inhibition. From these data, the effective concentration and the observed no-effect concentration are calculated.

• Validity criteria: To ensure the reliability of the test, the following criteria must be met:
  • Blank controls should show an oxygen uptake rate of at least 20 mg per gram of activated sludge per hour.
  • The coefficient of variation of oxygen uptake in control repetitions should not exceed 30 percent.
  • The EC50 value of the reference substance (e.g. 3,5-dichlorophenol) should be within the expected range from previous ring tests.

The OECD 209 test is applicable to non-volatile substances, including poorly soluble and absorbent substances. It is widely used in regulatory frameworks such as European Union Commission Regulation (EC) No 440/2008 and is compatible with standards such as ISO 8192.

However, this method has some limitations:

• Short exposure time: The standard exposure time of 3 hours (or 30 minutes for rapidly degrading substances) may not capture long-term effects. A modified version with a 27-hour incubation time has been proposed to increase sensitivity.
• Complex mixtures: Testing may be less effective for complex mixtures or rapidly degrading substances and may require additional assessments or measurements.
• Specificity: While the test distinguishes between carbon and ammonium oxidation, it may not fully capture the ecological impact of substances on other microbial processes.

The OECD 209 test is a cornerstone of ecotoxicological testing, providing a rapid and cost-effective screening method for assessing the potential hazards of chemicals in wastewater treatment systems. Its results are critical for industries including chemical manufacturing, pesticides, and dyes to ensure their products do not disrupt microbial activity in treatment plants. The test's compliance with international standards and its adoption by testing laboratories worldwide underscore its importance in environmental safety assessments.

In conclusion, the OECD 209 standard provides a robust framework for assessing the toxicity of substances to activated sludge microorganisms. By measuring respiratory inhibition, it provides valuable data for environmental risk assessments and regulatory decision-making. Its standardized methodology and rigorous validation criteria ensure reliable results, while ongoing improvements, such as extended incubation periods, aim to increase its applicability. For researchers, regulators, and industry, the OECD 209 test remains an important tool for safeguarding wastewater treatment processes and protecting environmental health.

Our organization, which has been operating for many years and meticulously follows global developments in science and technology, conducts testing, measurement, and analysis in a wide range of areas for businesses in every sector with a skilled team and extensive infrastructure. In this context, we also provide testing services in accordance with the OECD 209 Activated sludge, respiratory inhibition test (carbon and ammonium oxidation) standard.