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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 guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Physical state: Clear colourless liquid
- Analytical purity: 97.8%
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 01 January 2004
- Storage condition of test material: at room temperature in the dark
- Other: specific gravity: > 0.94 g/cm3
Analytical monitoring:
Details on test solutions:
A stock solution of 0.5 g/L was prepared by adding 502.0 mg test substance to 1000 mL of Milli-RO water (tap water purified by reverse osmosis). The stock solution was a clear colourless solution with a pH of 9.9. The stock solution was used to prepare a test concentration of 100 mg/L. 500 mL test solutions contained 16 mL synthetic sewage feed and an adequate amount of stock solution (made up to 300 mL with Milli-RO water) + 200 mL activated sludge.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Municipal sewage treatment plant 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands
- Pretreatment: coarsely sieved, washed and diluted with tap water
- Amound of suspended solids: 3.0 g/L
- A batch of sludge was used on the subsequent day and therefore 50 mL of synthetic sewage feed was added per L activated sludge at the end of the day and the mixture was kept aerated at room temperature.
- Initial biomass concentration: 1.2 g/L
Test type:
Water media type:
Limit test:
Total exposure duration:
0.5 h
Post exposure observation period:
ca. 10 min (for measurement of oxygen consumption)
No data
Test temperature:
19.3 - 20.0 °C.
6.8 (control) / 7.3-7.4 (100 mg/L)
Dissolved oxygen:
Measured at the start of oxygen consumption measurement.
8.3 mg/L (control) / 8.2-8.6 (100 mg/L)
Not applicable
Nominal and measured concentrations:
Nominal concentrations: Control + 100 mg/L
Concentrations not measured, but test substance can be considered stable during test period.
Details on test conditions:
- Test vessel: 1-L glass bottles (fill-volume 500 mL) for 30 min aeration and 300-mL glass oxygen bottles (completely filled) for 10-min measurements of oxygen consumption
- Closed static system, continuously aerated with clean, oil-free air during 30 min
- Initial loading: 1.2 mg/L (suspended solids)
- Number of replicates tested: 2

- Dilution water: Milli-RO/Milli-Q
- Synthetic sewage feed: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4, dissolved in 1 L Milli-Q water and filtered. 16 mL added in 500 mL test solution.

- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Respiration inhibition (after 30 min contact time 10 min measurement of oxygen consumption using an oxygen electrode connected to a recorder).

- Control + 100 mg/L (limit test)
- No range finding study performed
Reference substance (positive control):
Key result
30 min
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
No inhibition of the respiration rate of the sludge was recorded at 100 mg substance/L. In the duplicate measurement very slight inhibition (11%) of respiration rate of the sludge was recorded at 100 mg substance/L. Therefore, no further testing was needed.
Rspiration rate of control was 36 mg O2/L/h, which is acceptable.
Variation between controls was within 15% of each other, which is also acceptable.

Acceptability of the test: The mean respiration rate of control 1 and 2 was 36 mg O2/l/hr. The difference between the controls was within 15% of each other (6%).
The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L (7 mg/L, regression line: Y = 63.89X-2.11, Y = % inhitibion and X = log concentration (mg/L)).
Since all criteria for acceptability of the test were met, this study was considered to be valid.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L (7 mg/L).
Reported statistics and error estimates:
No statistics used.
Validity criteria fulfilled:
In this study the effect of the test substance on respiration rate of domestic sewage sludge was investigated at a single concentration of 100 mg/L. Contact time was 30 min, followed by a 10-min measurement of oxygen consumption. In one of the duplicate testing vessels, no inhibition was observed compared to the control, whereas in the other vessel 11% inhibition was observed. The EC50 for the test substance exceeded 100 mg/L. The test results can be considered valid and reliable (Klimisch 1 study).

Description of key information

The Klimisch 1 activated sludge respiration inhibition study of Desmares-Koopmans (2003) performed at NOTOX according to OECD Guideline 209 and EU Method C.11 was considered as the key study. The EC50 for the test substance after 30 minutes of exposure exceeded 100 mg/L and can be used for PNEC derivation.

Key value for chemical safety assessment

Additional information

A single study was available on toxicity to microorganisms (Desmares-Koopmans, 2003). This study is an activated sludge respiration inhibition test performed at NOTOX in which the effect of the substance on respiration was investigated through 10-min measurements of oxygen consumption after a 30-min contact period. A control and a single concentration of 100 mg/L were tested in duplicate. In one of the duplicates at 100 mg/L no inhibition of the respiration rate of the sludge was recorded, whereas in the other a very slight inhibition (11%) was observed compared to the control. Therefore, no further testing was needed. The EC50 is > 100 mg/L. The results of this test are valid and reliable (Klimisch 1).