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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-03-03 to 2003-07-16
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
: There were no circumstances that may have affected the quality or integrity of the data.
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
: There were no circumstances that may have affected the quality or integrity of the data.
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): T002615
- Substance type: not applicable
- Physical state: solid white
- Analytical purity: > 98%
- Purity test date: no data
- Lot/batch No.: 00403487
- Expiration date of the lot/batch: December 2003 (retest date)

- Stability under test conditions: Determined in RCC study No. 847007
- Storage condition of test material: at room temperature at about 20 °C, away from direct sunlight
- Solubility in water: Determined in RCC study No. 846983
Analytical monitoring:
Details on sampling:
Not applicable.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: No stock solutions were prepared due to the low solubiltiy. Therefore, the test items were directly weighed in the test flasks. Then 284 mL of tap water was added to each flask. Mixed by ultrasonic treatment for 10 minutes and intense stirring for 24h at room temperature in the dark to dissolve a maximum amount. Then 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added.
- Controls: two controls, identical but without inoculum.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Yes, down to the lowest test concentration of 10 mg/L part of the test item was suspended in the test media. Thus, all test concentrations were clearly above the water solubility limit of T002615 under the present test conditions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater.
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 2.5 g dry material per liter. During holding, the sludge was fed daily for three days with 50 mL synthetic wastewater per liter and kept at room temperature under continuous aeration until use. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used for the test.
- Initial biomass concentration: 3.9 g/L of sludge

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
Control: 7.2-8.2
Test substance: 7.2-8.2
Positive control: 7.2-8.2
Dissolved oxygen:
Control: 8.0-8.6 mg O2/L
Test substance: 7.9-8.7 mg O2/L
Positive control: 8.0-8.6 mg O2/L
not applicable
Nominal and measured concentrations:
Nominal: 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
- Test vessel: 1000mL glass flasks
- Material, size, headspace, fill volume: 200 mL
- Aeration: During the incubation period of exactly 3 hours all test media and the controls were continuously aerated with compressed air at a flow of approximately one liter per minute. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before the measurements of the respiration rates the dissolved oxygen concentrations were at least 7.9 mg/L.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 3

- Source/preparation of dilution water: tap water

- Adjustment of pH: no
- Photoperiod: no data
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after exactly three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW OXI 539 meter, Wissenschaftlich-Technische Werkstaetten WTW, Weilheiml Germany), and was continuously recorded for about ten to fifteen minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2L-1minute-1) was determined from the linear part of the respiration curve in the range of at least 6.5 -2.5 mg O2/L. In the case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least 10 minutes.

- Spacing factor for test concentrations: x3.2
- Range finding study: yes
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: results from the range finding study and recommended from the testing guidelines
Reference substance (positive control):
Key result
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
no data
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The 3-h EC50 of the reference test substance 3.5-dichlorophenol (positive control) was calculated to be 14 mg/L (the 95% confidence limits were not calculable). The 3-h EC50 is within the guideline recomended range of 5-30 mg/L, confirming suitability of the activated sludge used.
Reported statistics and error estimates:
The 3-h EC50, EC20 and EC80 values for the test item and their 95% confidence limits could not be calculated due to the absence of a toxic effect.
The 3-h EC50 value of the reference item 3,5-dichlorophenol was calculated by Probit Analysis.
Validity criteria fulfilled:
the oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 12% (guideline recommended maximum variation : 15%)
An activated sludge respiration inhibition test (according to OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h NOEC and a 3-h EC50 > than the water solubility limit of T002615 under the present test conditions or higher than 1000 mg/L. The results of the study can be considered reliable without restriction.

Description of key information

The study of Seyfried (2003), investigating the toxicity of T002615 to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h ELR50 was > 1000 mg/L.

Key value for chemical safety assessment

Additional information