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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Details on test solutions:
Test substance:The test substance is soluble in deionized water in concentrations needed for the test. The stock solution was prepared by dissolution of 5.0 g of the test substance in 1000 mL deionized water on the day of testing. The concentrations 10, 100 and three replicates of 1000 mg·L-1 of the test substance were tested in the preliminary test. Reference substance: The stock solution of the reference substance was prepared by dissolution of 0.1 g of 3,5-dichlorophenol in 200 mL deionized water. The ultrasound for 60 minutes to accelerate the dissolution was used. The resulting solution has a concentration of 500 mg·L-1. The concentration range used was 5 - 20 mg·L-1. It was calculated by geometric progression with factor of 2.0.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated biological sludge containing mixed culture of microorganisms obtained from the second step of sewage treatment plant of Pardubice was used for the testing. The wastewater processed by the sewage treatment plant is predominantly municipal.The activated sludge was collected two days before the day of testing.After the sample collection the sludge was washed with potable water for 30 minutes and subsequently decanted for 30 minutes. This procedure was repeated three times in total. Further the sludge was fed with the 100 mL synthetic sewage feed per 2 L of diluted sludge suspension at permanent aeration till the next day. The next day the sludge was washed with potable water and decanted according to the procedures described for the first day. This procedure was repeated three times in total again.The suspended solids concentration was determined from 10 mL of sludge suspension after the last 30-minutes sedimentation. The sludge was fed with the 100 mL synthetic sewage feed per 2 L of diluted sludge suspension at permanent aeration till day of the test.Before the test the sludge was suspended in water up to concentration about 3000 mg of suspended solids per litre. The pH adjustment to 6.0 was carried out. In that way modified sludge suspension was aerated until the use.
Limit test:
Total exposure duration:
3 h
Test temperature:
20-22 °C
Reference substance (positive control):
3,5-dichlorophenol 97%
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Details on results:
The highest inhibition the respiration rate of the activated sludge caused by the test substance was 3.0 % (see table 4) in the preliminary test. This low level of inhibition of oxygen consumption by the test substance in the preliminary test demonstrated that a definitive test is unnecessary. The result could be used as final one, because preliminary test design includes three replicates required by guidelines. Exact value of EC50could not be calculated, the value of EC50 is given in the form of a range: 3 hour – EC50> 1 000 mg·L-1
Validity criteria fulfilled:
EC50 micro - organism > 1000 mg/l
Executive summary:

Based of the test results, the tested substance does not showed toxic effects on microorganism.

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Based on the available test results, no toxic effect on microorganism were observed. and a value of EC50 > 1000 mg/l was set.