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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
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Details on test solutions:
Test item stock solution (TISS): 10.0 mg/mL (5.0 g test item in 500 mL – diluent: ultrapure water)
The test mixtures were prepared at intervals of 10/15 minutes. Each mixture contained an amount of 1.5 g SS (suspended solid).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The inoculum will be a sample of activated sludge from the aeration tank of a domestic sewage treatment plant (e.g. Pian della Genna - Perugia, Italy). The activated sludge will be mixed for about 15 seconds. Washing the sludge will be necessary in order to remove inhibitory substances eventually used in the plan. The sludge will be suspended in chlorine-free tap water to yield a concentration of 3 g suspended solid/L. The sludge will be fed with the synthetic sewage (50 mL/L activated sludge) and will be aerated (e.g. 2 L/minute) at the test temperature then used within two additional days from the day of collection.
Test type:
Limit test:
Total exposure duration:
3 h
Test temperature:
20.8 – 22.0° C
pH of mineral medium was in the range of 7.5 ± 0.5 (actual value: 7.39).
Dissolved oxygen:
dissolved oxygen concentration above 60 – 70% saturation
Nominal and measured concentrations:
Test concentrations: 10, 100, 1000 mg/L (nominal)
Reference substance (positive control):
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Details on results:
No statistically significant inhibition of the respiration rate (Williams multiple sequential t-test) was observed in the groups treated with test item.
The % total inhibition evaluated in the test item treatments were respectively 14.10%, 0.60% and -65.14%.
Results with reference substance (positive control):
The 3-hours 50% effect concentration (EC50) of the reference substance 3,5 DCP on the total respiration rate, was calculated to be 6.18 mg/L.
Reported statistics and error estimates:
The statistical significance of the effects of the test item compared to controls were investigated using the Williams Multiple Sequential t-test Procedure.
To evaluate the effects of the reference item, a Williams Multiple Sequential t-test Procedure was performed for significance and a non-linear regression (3-param. Normal CDF) was used to evaluate ECx values. The software ToxRat Pro Version 3.2.1 was used for the statistics.
Validity criteria fulfilled:
No inhibition of the total respiration rate was observed in the test item treated groups. It can be concluded that the 3h-EC50 of TEA Trioleate is >1000 mg/L.
Executive summary:

The effects of TEA Trioleate on the respiration rate of activated sludge was assessed following the OECD No. 209.

The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The absence of inhibition of oxygen consumption by the test item in a preliminary trial, demonstrate that a definitive test is unnecessary, so 3 replicates at the highest tested concentration (1000 mg/L) were used in this study. The preliminary trials showed no abiotic respiration of the test item.

No inhibition of the total respiration rate was observed in the test item treated groups. The % inhibition evaluated in the T1, T2 and T3 were respectively 14.10%, 0.60% and -65.14%. Therefore, it can be concluded that the 3h-EC50 of the test item Trioleato di Tea is >1000 mg/L.

Description of key information

No inhibition of the total respiration rate was observed in the test item treated groups. It can be concluded that the 3h-EC50 of TEA Trioleate is >1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information