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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-04-17 to 2019-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of the test item was prepared at a concentration of 10 g/L. Bottles were prepared with deionized water, synthetic sewage feed and the test was performed at 3 different concentrations of the test item (1000 mg/L, 100 mg/L and 10 mg/L). pH of the solutions was checked. The mud was added before starting the test.
- Controls: Blanks were used which contained deionized water and an equal volume of activated sludge and synthetic medium (without reference substance and test item).

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: San Rocco near Monza
- Method of cultivation: Activated sludge was collected and used on day of collection.
- Preparation of inoculum for exposure: No coarse particles were present so settling was not necessary. Then the mud samples were washed and analyzed to prepare the inoculum before their use. The sludge was continuously aerated at the test temperature.
- Initial biomass concentration: The suspended solids in the mud solution was 3 g/L to have a final mixture concentration of 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
7.5 ± 0.5
Nominal and measured concentrations:
Nominal: 1000 mg/L, 100 mg/L and 10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass bottles
- Material, size, headspace, fill volume: Glass
- Aeration: The vessels were aerated to keep the dissolved oxygen concentration above 60-70% saturation and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): The suspended solids in the mud solution was 3 g/L to have a final mixture concentration of 1.5 g/L.
- Nutrients provided for bacteria: Synthetic sewage feed
- Nitrification inhibitor used (delete if not applicable): None
OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the reference substance stock solution was adjusted to pH 7.45 with NaOH (1 M).
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Respiration rate
TEST CONCENTRATIONS
- Range finding study : Only range finding study was conducted since results were already sufficient.
- Test concentrations: 1000 mg/L, 100 mg/L and 10 mg/L
- Results used to determine the conditions for the definitive study: No definitive study necessary
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Inhibition of oxygen uptake
Details on results:
- Blank controls oxygen uptake rate: 33.62 mg oxygen per one gram of activated sludge in an hour.
- Coefficient of variation of oxygen uptake rate in control replicates: 9.17%
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 of 3,5 – Dichlorophenol: 5.71 mg/L for total respiration

Table 1: Mean percent inhibition total respiration rate of the test item

Concentration

% of inhibition total respiration rate Test item

“3-Methoxy-3-methylbutyl acetate (MMB-AC)”

10 mg/L

0.46

100 mg/L

1.32

1000 mg/L

4.43

 

Table 2: Mean percent inhibition total respiration rate of the reference substance.

Concentration

% of inhibition total respiration rate

Reference substance “3,5 - Dichlorophenol”

1 mg/L

19.37

10 mg/L

61.60

50 mg/L

85.19

 

Validity criteria fulfilled:
yes
Conclusions:
The activated sludge respiration inhibition test was conducted in compliance with GLP and in accordance with OECD 209. All validity criteria were met and no deviations were reported. The EC50 value of the test item "3-Methoxy-3-methylbutyl acetate (MMB-AC)” after 3 hours was determined to be greater than 1000 mg/L.

Description of key information

The activated sludge respiration inhibition test was conducted in compliance with GLP and in accordance with OECD 209. All validity criteria were met and no deviations were reported. The EC50 value of the test item "3-Methoxy-3-methylbutyl acetate (MMB-AC)” after 3 hours was determined to be greater than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information