OECD 209: Activated Sludge Respiration Inhibition Test

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.

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