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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was directly weighed into the test flasks. Then the test flasks were filled each with 284 ml tap water.
One test solution with a final volume of 500 ml was tested per concentration in a glasss flask. The flasks were filled according to the scheme given in Table 1 in "any other information on materials and methods including tables".
Samples were prepared in about 15 minute intervals. Thereafter, the pH of the samples was noted, and aeration started using compressed air at a flow-rate of approximately 0.6 l/min.
- Controls: Sewage feed, activated sludge and dilution water with no test substance. Positive control: 3,5-Dichlorophenol at concentrations of 10, 32 and 100 mg/l.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: Groβ-Zimmern, Germany
- Method of cultivation: not reported
- 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 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 2 g dry material per litre, were made up with tap water. To this mixture, 50 ml synthetic sewage feed per litre was added daily starting four days prior to use, and the sludge was kept at room temperature under continuous aeration until use. Immediately before use the dry weight if the sludge was determined to be approximately 2 g/l. The pH of the activated sludge was adjusted to pH 8.0 with 1 n sulphuric acid.
- Initial biomass concentration: not reported
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
not reported
Test temperature:
7.8 - 8.7
Dissolved oxygen:
6.0 - 8.1
not reported
Nominal and measured concentrations:
Nominal: Control, 10, 32, 100, 320 and 1000 mg/l
Details on test conditions:
- Test vessel: Glass flasks
- Type (delete if not applicable): open / closed: not reported
- Material, size, headspace, fill volume: Glass, 1 litre in size, 500 ml fill volume
- Aeration: Yes with compressed air at approximately 0.6 litres/minute
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): n/a
- No. of vessels per abiotic control (replicates): n/a
- Sludge concentration (weight of dry solids per volume): 2g dry material/litre
- Weight of dry solids per volume of reaction mixture per unit of time: not reported
- Nutrients provided for bacteria: Synthetic sewage feed
- Biomass loading rate: not reported

- Source/preparation of dilution water: tap water
- Particulate matter: not reported

- Adjustment of pH: Yes
- Photoperiod: not reported
- Light intensity: not reported
- Details on termination of incubation: For the measurement of 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 10 minutes. During measurement, the samples were continuously stirred on a magnetic stirrer.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Respiration rate measured after 3 hours. pH and oxygen concentrations were determined at the start and the end of the test in all concentrations.

- Spacing factor for test concentrations: approximately 3.2
- Justification for using fewer concentrations than requested by guideline: n/a
- Range finding study: no
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:
inhibition of total respiration
Key result
3 h
Dose descriptor:
other: EC20
Effect conc.:
521 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: not reported
- Adsorption (e.g. of test material to the walls of the test container): none reported
- Blank controls oxygen uptake rate: Oxygen consumption 0.680-0.700 mg O2/L min
- Coefficient of variation of oxygen uptake rate in control replicates: % deviation: 2.9
Results with reference substance (positive control):
- Results with reference substance valid?
- Relevant effect levels: EC50 14 mg/l
Reported statistics and error estimates:
Probit analysis

Table 2: Influence of test substance on oxygen consumption of activated sludge

Flask no. Treatment Concentration (mg/l) Oxygen consumption (mg O2/l min) Inhibition (%) pH value start* pH value end* Oxygen concentration (mg O2/l min) start* Oxygen concentration (mg O2/l min) end*
 1  Control  -  0.680  -  7.9  8.4  6.0  7.2
 10  Control  -  0.700  -  7.8  8.1  6.1  6.2
 mean      0.690          
% deviation      2.9          
 5 Test substance  1000  0.429  37.8  8.3  8.7  6.6  7.2
 6 Test substance  320 0.678   1.7  8.4  8.4  6.0 8.1
 7 Test substance 100   0.627  9.1  8.2  8.4  6.6  7.0
 8 Test substance  32 0.706   -2.3  8.1  8.2  6.0  6.9
 9 Test substance  10 0.723   -4.8  8.0 8.2   6.3  6.3

*start and end of 3-hour aeration

Validity criteria fulfilled:
An ASRI 3hr EC50 value of >1000 mg/l and an EC20 of 521 mg/l have been determined for the effects of the test substance on respiration inhibition of activated sludge.

Description of key information

Toxicity to microorganisms: An ASRI 3-hour EC50 value of >1000 mg/l and an EC20 of 521 mg/l (nominal concentrations). The EC50 is equivalent to >320 mg/L when expressed in terms of the silanol hydrolysis product of the tested substance.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

An ASRI 3 hour EC50 value of >1000 mg/l (highest concentration tested) and an EC20 of 521 mg/l (nominal concentrations) have been determined for the effects of the test substance, N,N',N''-tricyclohexyl-1-methylsilanetriamine on respiration inhibition of activated sludge, in compliance with OECD Test Guideline 209. This indicates that the test substance was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/l. The results may be expressed in terms of concentration of the hydrolysis product of the tested substance, methylsilanetriol, by applying a molecular weight correction: (MW of silanol = 108.17 / MW of parent = 337.62) * >1000 = >320 mg/L. For cyclohexylamine: MW of hydrolysis product / MW of parent * concentration of parent = 3*99.17/337.63 * >1000 = >880 mg/l.