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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-09-04 to 2009-09-04
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
Analytical monitoring:
Details on test solutions:
- Method: 54 µl of the test substance, corresponding to 50 mg, was pipetted below the surface of the
Milli-RO-water and stirred briefly to mix (5 minutes). Subsequently, synthetic sewage feed and sludge were added (final volume 500 ml) and the test was initiated. A loading rate of 100 mg/l was tested in duplicate. Optimal contact between the test substance and test medium was ensured by applying continuous aeration and stirring during the exposure period.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Activated sludge from municipal sewage treatment plant: ‘Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

- Preparation of inoculum for exposure: The sludge was coarsely sieved to remove large particles. The sludge was then washed by centrifuging at 1000 g for 10 minutes. The supernatant was decanted and replaced with ISO-medium. The washing step was performed three times. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.8 g/l of sludge, as used for the test). The pH was 7.8 on the day of testing. The batch of sludge was used within two days after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of each day. The sludge was kept aerated at test temperature until use.

- Initial biomass concentration: 3.8 g/l of sludge, as used for the test
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
18.5 to 19.0 °C
Details on test conditions:
- Test vessel: All glass, approximately 300 ml oxygen bottles and 1 litre test bottles.

- Aeration: Strong aeration with clean, oil-free air.

- No. of vessels per concentration (replicates): Duplicate

- No. of vessels per control (replicates): Two controls without test substance were tested in each test series, one at the start and one at the end.

- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp.)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After the 3-hour contact time, a well mixed sample of the contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. Oxygen meter: WTW inolab Oxi 730 supplied with a WTW CellOx 325 oxygen electrode, electrolyte type ELY/G.

- Adjustment of pH: The pH of synthetic sewage feed was adjusted with 1 M NaOH (Merck) to 7.4.

- Spacing factor for test concentrations: Performance of a limit concentration at 100 mg/l is acceptable, because 100 mg/l is far above the water solubility of Tetraethyl orthosilicate and thus exceeds the expected concentration in a Sewage Treatment Plant.
Reference substance (positive control):
3, 5-dichlorophenol
Key result
3 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Results with reference substance (positive control):
The 3-hour EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/l
Validity criteria fulfilled:
An activated sludge respiration inhibition EC50 value of >100 mg/L was determined for the substance in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

Description of key information

Toxicity to microorganisms: ASRI 3-hour EC50 >100 mg/L (OECD 209).

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

An activated sludge respiration inhibition EC50 value of >100 mg/L was determined in a reliable study conducted according to an appropriate test protocol (OECD 209) and in compliance with GLP.

The key study was chosen because it used a standard method and was carried out most recently.

In a supporting study conducted for the substance, an activated sludge respiration inhibition 5 hour EC10 value of >2 ml/L (equivalent to >1.9 g/L based on a relative density of 0.934, Pseudomonas putida) based on oxygen consumption/respiration inhibition was determined for the substance using a relevant test method and in compliance with GLP (Degussa 1993). The result is considered to be reliable but is not used as key study because (i) the study is a 5 hour study instead of the recommended 16 hour growth inhibition type test recommended in ISO 10712 Test Method (ii) the study uses an emulsifier - nonylphenol ethoxylate to improve solubility of the test substance and (iii) no reference substance was used during the study.

Under the conditions of the key test, tetraethyl orthosilicate was not toxic to wastewater (activated sludge) bacteria up to and including a loading rate of 100 mg/L.

The EC50 exceeded a loading rate of 100 mg/L.