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OECD 209: Activated Sludge Respiration Inhibition Test

OECD 209 is designed to assess the effects of a substance on microorganisms from activated sludge of wastewater treatment plants by measuring their respiration rate (carbon and/or ammonium oxidation) as oxygen consumption.

The test results may also serve as an indicator of suitable non-inhibitory concentrations of test substances to be used in biodegradability tests. 

OECD 209 Key Summary

The test allows the determination of ECx and/or NOEC values of the test substance. The inhibition of three different oxygen uptakes may be determined, i.e., total, heterotrophic only, and that due to nitrification in the absence and presence of N-allylthiourea, a specific nitrification inhibitor. For obtaining both NOEC and ECx, six controls and five treatment concentrations in a geometric series with five replicates are recommended. 

In each test vessel, test mixtures containing water, synthetic sewage feed and the test substance are incubated at the pH of 7.5 ±0.5 and the temperature within 20 ± 2°C under forced aeration to keep the dissolved oxygen concentration above 60-70% saturation. The oxygen consumption is measured after 3 hours of exposure and additional measurements at 30 minutes of exposure can be performed in the case that the test substance is rapidly degraded.

▶ Materials that can be tested:
     ➢ Materials that are normally not volatile;
▶ Test substance dosage: the test should be performed using at least three concentrations of the test substance, for example, 10 mg/L, 100 mg/L and 1000 mg/L with a blank control (based on the preliminary test). Depending on the purpose of the test, it is also possible to simply determine the toxicity of a limit concentration, e.g. 1000 mg/L. If no statistically significant toxic effect occurs at this concentration, further testing at higher or lower concentrations is not necessary;
▶ Reference substance: 3,5-dichlorophenol or copper (II) sulphate pentahydrate for inhibiting total respiration, and N-methylaniline for inhibiting nitrification;
▶ Inoculum information:
     ➢ Activated sludge collected from a wastewater treatment plant which treats predominantly domestic sewage;
     ➢ May be washed if the endogenous respiration rate is low;
     ➢ Normally used within one day of collection;
▶ Inoculum dosage: 1.5 g/L of total suspended solids;
▶ Reactor: 1000 mL reactors to contain 500 mL of reaction mixture; BOD bottles;
▶ Number of reactors: 3 for each group;
▶ Testing conditions: 
     ➢ Temperature: 20 ± 2°C;
     ➢ pH: 7.5 ± 0.2;
▶ General procedures: prepare test mixtures, reference mixtures and the blank controls, and aerate them with mixing for normally 3 hours. Then the samples are filled into closed cells (e.g., BOD bottles), where the dissolved oxygen decrease is monitored to determine the respiration rates, and the inhibitory effects of the test/reference substances on the respiration rates;
▶ Incubation/exposure duration: normally 3 hours;
▶ Measurement: 
     ➢ Dissolved oxygen (DO) consumption when DO is between 2 and 7 mg/L (measurement normally lasts for 5-10 minutes, or until DO falls below 2 mg/L);
     ➢ Normally, the measurement of total oxygen uptake inhibition should be adequate. When necessary, the inhibition of three different oxygen uptakes may be determined, total, heterotrophic only and that due to nitrification;
▶ Test validity criteria: 
     ➢ Blank controls have over 20 mg oxygen per one gram of activated sludge in an hour;
     ➢ The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test;
     ➢ The EC50 of 3,5-DCP should lie in the expected range as found in a previous ring test.

OECD 209 Requirement and Applicability

Different testing methods are applicable for materials with different properties. Below is a summary of the applicability of OECD 209. Please check our Method Selection Guide to select the most appropriate method for your materials. You can also find the applicability for many other methods on Aropha Resource Center

TestAnalytical methodSample info required *Poorly solubleVolatileAdsorbing
OECD 209Dissolved oxygen consumptionN/A++/-+

*"Sample info required" is the information needed to calculate the biodegradation percentages. This must be available for a selected method.
*"Organic carbon content" is the ratio of the organic carbon weight to the weight of the sample. It can be calculated by the sample formula (e.g., acetic acid C2H4O2, carbon content is 12*2/(12*2+1*4+16*2)=40%). Try our Online C% Calculator. If the formula is unknown, we can send the sample out to a third party lab for you for analysis (normally $110 with a 10-day turnaround time).

Aropha Resource Center

To learn more about different types of biodegradation tests, their applicability, biodegradation mechanisms, and many other information such as case studies, publications, and blogs, please check our Aropha Resource Center

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