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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 14 - 20, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted April 04, 1984
Deviations:
no
Remarks:
(the temperature limit of 22°C was overstepped for a short time)
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
Commission Directive 88/302/EEC: "Activated Sludge Respiration Inhibition Test", Official Journal of the European Communities No. L 133/118-122, dated May 30, 1988
Deviations:
no
Remarks:
(the temperature limit of 22°C was overstepped for a short time)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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.
- Controls:
- 3,5-Dichlorophenol was used as a reference item (positive control): 0.5 g of 3,5-Dichlorophenol was dissolved according to the OECD Guideline No. 209 in 10 mL 1 mol/L NaOH and diluted to about 30 mL with deionised water. Excess of NaOH was neutralized with 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one litre with deionised water. The final pH was measured to be 8.0 and the final concentration amounted to 500 mg/L.
- negative control
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: sewage plant Darmstadt, Germany
- Preparation of inoculum for exposure: 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 procedure was repeated twice. An aliquot of the final sludge suspension 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 per litre was added and the sludge was kept at room temperature under continuous aeration for one day 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 6.9
Test type:
static
Water media type:
freshwater
Remarks:
aerobic
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
18 - 22.6 °C
pH:
7 - 8
Dissolved oxygen:
8.1 - 7.2 mg/L
Nominal and measured concentrations:
1000, 320, 100, 32, 10 mg/L nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks and Karlsruher flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 1 litre volume and 250 mL
- Aeration: continuous, with compressed air, no aeration at 3h incubation
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 2 g/L
- Nutrients provided for bacteria: 50 mL synthetic sewage feed

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Particulate matter:

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod/ Light intensity: controlled environment room


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Measurement of Respiration Rate: For the measurement of the respiration rate a well-mixed sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with an oxygen electrode, and was recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve, and were determined in the range between approximately 8.5 - 3.4 mg O2/L.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (3.2, 10 and 32 mg/L)
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Based on measured inhibition rates, the 3-hour EC20/80 and EC50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 20 % inhibition was noted after three hours incubation. Nevertheless, the 3-hour EC20/80 and EC50 are clearly higher than 1000 mg/L under the present test conditions.
Inoculum control: The respiration rates of the two controls did not differ by more than 15 % (actual: 5.7%).
Dissolved Oxygen: The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.1 mg O2/L and thus >6.5 mg O2/L
Results with reference substance (positive control):
The 3-hour EC50 for the positive control 3,5-Dichlorophenol, which was tested in the same way as the test item, was found to be 11.6 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge. The 95 % confidence limits could not be determined due to mathematical reasons.
Reported statistics and error estimates:
The 3-hour EC50 and their 95 %-confidence limits of the reference item were calculated by Probit analysis. The 3-hour EC20 and EC50 of the test item could not be calculated due to the low toxicity of the test item to micro organisms under the tested conditions.
Validity criteria fulfilled:
yes
Conclusions:
In comparison to the inoculum controls the respiration rate of the activated sludge was slightly inhibited between 5.9 % and 8.8 % in a concentration range from 10 mg/L up to 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. Based on measured inhibition rates, no 3-hour EC20/80 and EC50 could be quantified because up to the highest nominal test concentration of 1000 mg/L less than 10 % inhibition was noted after three hours incubation. The 3-hour EC20 and EC50 are clearly higher than 1000 mg/L under the present test conditions.
Executive summary:

Study design 

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions. Therefore, 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 of the test item (10, 32, 100, 320 and 1000 mg/L) 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 slightly inhibited between 5.9 % and 8.8 % in a

concentration range from 10 mg/L up to 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. 
Based on measured inhibition rates, the 3-hour EC 20 and EC 50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 20 % inhibition was noted after three hours incubation. Nevertheless, the 3-hour EC 20 and EC 50 are clearly higher than 1000 mg/L under the present test conditions. 
The 3-hour EC 50 for the positive control 3,5 -dichlorophenol, which was tested in the same way as the test item, was found to be 11.6 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge.

Conclusion

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

Description of key information

Toxicity to microorganisms EC50(3h) > 1000 mg/L nominal for domestic, activated sludge respiration inhibition (static, open, OECD 209, GLP)

Key value for chemical safety assessment

Additional information

Study design 

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions. Therefore, 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 of the test item (10, 32, 100, 320 and 1000 mg/L) 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 slightly inhibited between 5.9 % and 8.8 % in a

concentration range from 10 mg/L up to 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. 
Based on measured inhibition rates, the 3-hour EC 20 and EC 50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 20 % inhibition was noted after three hours incubation. Nevertheless, the 3-hour EC 20 and EC 50 are clearly higher than 1000 mg/L under the present test conditions. 
The 3-hour EC 50 for the positive control 3,5 -dichlorophenol, which was tested in the same way as the test item, was found to be 11.6 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge.

Conclusion

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