Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-05-11 to 1999-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 04 April 1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
Official Journal of the European Communities No. L 133/118-22, 30 May 1988
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
DHA

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Sewage plant Groß-Zimmern, Germany
- Method of cultivation: 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. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per liter, were made up with tap water. To this mixture, 50 mL synthetic sewage feed per liter was added daily, starting three days prior to use, and the sludge was kept at room temperature under continuous aeration until use.
- Preparation of inoculum for exposure: Immediately before use, the dry weight of the activated sludge was determined again, and adjusted to 4 g/L with tap water. The pH of the activated sludge was determined to be 7.8.
- Initial biomass concentration: 4 g/L

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
21 - 22 °C during incubation period
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume
- Type (delete if not applicable): open
- Fill volume: 500 mL
- Aeration: with compressed air (approximately 0.6 litre per minute)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 4 g/L in activated sludge; 1.6 g/L in final test medium

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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 continuosly stirred on a magnetic stirrer. The oxygen consumption (in mg O2/L per min) was determined from the most linear part of the respiration curve.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 10, 32, 100, 320, 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3 ,5-Dichlorophenol

Results and discussion

Effect concentrations
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
Results with reference substance (positive control):
The 3-hour EC 50 of the positive control 3,5-Dichlorophenol, which was tested in the same way as the test substance, was found to be 7 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge.

Any other information on results incl. tables

Toxicity of Test Substance:

In comparison to the inoculum controls the respiration rates of the activated sludge were not inhibited (by -0.6 % to -7.5 %) up to the highest tested concentration of nominal 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested.

3-hour EC 20 and EC 50 of Test Substance:

From the determined 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 no inhibition of the respiration rate was noted after three hours contact time. Nevertheless, the 3-hour EC 20 and EC 50 are clearly higher than 1000 mg/L under the present test conditions. Considering the fact that the maximum inhibition was below 10 %, it is concluded that the EC10 is also above the limit concentration of 1000 mg/L.

Toxicity of 3,5-Dichlorophenol:

The 3-hour EC 50 of the positive control 3,5-Dichlorophenol, which was tested in the same way as the test substance, was found to be 7 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
conditions for the validity of OECD 209 are fulfilled
Conclusions:
In comparison to the inoculum controls the respiration rates of the activated sludge were not inhibited (by -0.6 % to -7.5 %) by the test item up to the highest tested concentration of nominal 1000 mg/L.
Executive summary:

The influence of the test substance on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions. 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 substance after an incubation period of 3 hours. The test item was tested at concentrations of 10, 32, 100, 320 and 1000 mg/L. The positive control, 3,5 -dichlorophenol, was tested at concentrations of 10, 32, and 100 mg/L. Two inoculum controls were included.

In comparison to the inoculum controls the respiration rates of the activated sludge were not inhibited (by -0.6 % to -7.5 %) by the test item up to the highest tested concentration of nominal 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. From the determined 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 no inhibition of the respiration rate was noted after three hours contact time. Considering the fact that the maximum inhibition was below 10 %, it is concluded that the EC10 is also above the limit concentration of 1000 mg/L. The 3-hour EC 10/20 and EC 50 are clearly higher than 1000 mg/L under the present test conditions. The 3-hour EC 50 of the positive control 3,5-Dichlorophenol, which was tested in the same way as the test substance, was found to be 7 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge.