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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 12 - 15, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Due to the low solubility of the test item in test water, adequate amounts of the test item were directly weighed into each test flask. One test solution with a final volume of 500 mL was tested per treatment in a glass flask (>500 mL). Samples were prepared in about 15-minute intervals.
The following test item concentrations have been used: 1000, 320, 100, 32, and 10 mg/L
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Species: activated sludge, micro organisms from a domestic waste water treatment plant.
Origin: supplied by the sewage plant Groß Zimmern, Germany

Conditioning:
The sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. The latter pro¬cedure was repeated twice. An aliquot of the fmal sludge sus-pension was weighed, dried and the ratio of wet sludge (g) to its dry weight (g) determined. The washed sludge suspension was made up with tap water and 50 mL synthetic sewage feed (see 6.6) per litre was added and the sludge was kept at room temperature under continuous aeration for two days until use. Immediately before use, the dry weight of the activated sludge was determined, and diluted to 2 g/L with tap water. The pH of the activated sludge was 7.1.

Test type:
other: aerobic
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19-21 °C
pH:
7-8.5
Dissolved oxygen:
7 - 8 mg/L
Nominal and measured concentrations:
1000, 320, 100, 32, and 10 mg/L
Reference substance (positive control):
yes
Remarks:
DCP
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 496.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 269.6 - 925.7
Details on results:
In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited by 26.9 and 16.9% at the two highest nominal concentrations of 1000 and 320 mg/L, respectively. At the next higher nominal concentration of 100 mg/L, the respiration rate was inhibited by 10.3 %. The low test concentrations of 32 and 10 mg/L were in the range of control (maximum: 0.3 % respiration inhibition). Test item concentrations exceeding 1000 mg/L were not tested.

The 3-hour EC 20 was calculated by linear regression analysis using the inhibition rates of the 1000, 320, 100, 32 and 10 mg/L test concentrations, and was calculated to be 496.5 mg/L nominal with a 95 % confidence limit of 269.6 to 925.7 mg/L. The 3-hour EC 50 was calculated by linear regression analysis using the inhibition rates of the 1000, 320, 100, 32 and 10 mg/L test concentrations, and was calculated to be clearly above 1000 mg/L.
Results with reference substance (positive control):
EC50 = 5.3 mg/L (acceptance range: 5-30 mg/L)
Validity criteria fulfilled:
yes
Conclusions:
The 3-hour EC20 was calculated to be at 496.5 mg/L and the 3-hour EC50 was clearly above 1000 mg/L.
Executive summary:

Purpose
The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions.

Study design

The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations (10, 32, 100, 320 and 1000 mg/L) of the test item after an incubation period of 3 hours. This study was conducted according to Good Laboratory Practice (GLP) and the method applied followed the OECD Guideline for Testing of Chemicals, Section 2, No.209: "Activated Sludge, Respiration Inhibition Test", adopted April 04, 1984.

Results

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited by 26.9 and 16.9% at the two highest nominal concentrations of 1000 and 320 mg/L, respectively. At the next higher nominal concentration of 100 mg/L, the respiration rate was inhibited by 10.3 %. The low test concentrations of 32 and 10 mg/L were in the range of control (maximum: 0.3 % respiration inhibition). Test item concentrations exceeding 1000 mg/L were not tested.

The 3 -hour EC20 was calculated by linear regression analysis using the inhibition rates of the 1000, 320, 100, 32 and 10 mg/L test concentrations, and was calculated to be 496.5 mg/L nominal with a 95 % confidence limit of 269.6 to 925.7 mg/L. The 3 -hour EC 50 was calculated by linear regression analysis using the inhibition rates of the 1000, 320, 100, 32 and 10 mg/L test concentrations, and was calculated to be clearly above 1000 mg/L.

The 3 -hour EC50 for the positive control 3,3 -Dichlorophenol, which was tested in the same way as the test item, was found to be 5.3 mg/L which is within the acceptance range recommended by the guidelines thus confirming the suiteability of the activated sludge.

Conclusion

The 3 -hour EC20 and EC50 of the test material are clearly higher than 1000 mg/L under the present test conditions.

Description of key information

The 3-hour EC20 was calculated to be at 496.5 mg/L and the  3-hour  EC50  was clearly above 1000 mg/L.

Key value for chemical safety assessment

Additional information