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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-05-31 to 2018-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Purity: > 99%
Batch No.: 102Z4
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Preparation of test item mixture: the test item was directly weighed into the test vessel, synthetic sewage feed, activated sludge suspension and distilled water was added.
- Concentration of test item: 10, 100, 300 and 1000 mg/L, a abiotic control at concentration of 1000 mg/L
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: a treatment plant receiving predominantly domestic sewage
- Cultivation and Preparation of inoculum for exposure: The activated sludge was fed with the synthetic sewage feed (50 mL synthetic sewage feed/L activated sludge) and continuously aerated at the test temperature (19.9~21.7 °C) 1 day prior to use in the test.
- Pretreatment: On the day of activated sludge collection, remove coarse particles by settling for 15 minutes, and decanting the upper layer of finer solids. The sludge was washed as followed. The sludge was first centrifuged for 5 minutes at ca. 2500 rpm to produce a clear supernatant and pellet of sewage solids. The supernatant liquid was discarded and the sludge was re-suspended in chlorine-free tap water, with shaking, and the wash-water was removed by re-centrifuging and discarding again.
- Initial biomass concentration: 3.0 g/L
Test type:
static
Water media type:
other: distilled water
Total exposure duration:
3 h
Test temperature:
20.2 ~ 21.6 °C
pH:
6.72-7.02
Dissolved oxygen:
forced aeration (0.5 to 1 L/min) to keep the dissolved oxygen concentration above 60 – 70% saturation
Nominal and measured concentrations:
Nominal: 10, 100, 300 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Aeration: clean oil-free air, flow rate 0.5 ~ 1 L/min
- No. of vessels per concentration (replicates): 10, 100, 300 mg/L were tested with 1 replicate, 1000 mg/L was tested with triplicate
- No. of vessels per control (replicates): 4
- No. of vessels per abiotic control (replicates): 1
- Concentration of activated sludge (suspended solids): 1.5 g/L
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:
inhibition of total respiration
Details on results:
- Blank controls oxygen uptake rate: 21.3 mg/(h·g SS) oxygen
- Coefficient of variation of oxygen uptake rate in control replicates: 2.0%
Results with reference substance (positive control):
EC50 (3 hours): 3.5 mg/L, 95% confidence limits: 2.2~5.6 mg/L
The validity criteria for the reference item EC50 values were satisfied.
Validity criteria fulfilled:
yes
Conclusions:
The EC50 (3 hours) of test item for inhibition of total respiration was more than 1000 mg/L.
Executive summary:

The study was conducted to determine the inhibition effects of the test substance on micro-organisms from activated sludge (largely bacteria) by measuring their respiration rate according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)).

The contact time for exposure was 3 hours.The test was conducted with 4 concentrations of 10, 100, 300 and 1000 mg/L. 10, 100, 300 mg/L were tested with 1 replicate, 1000 mg/L was tested with triplicate and an abiotic control at concentration of 1000 mg/L was performed. Inoculums blank controls were performed with 4 replicates. The rate of respiration of each incubation mixture was determined after 3 hours contact times. 3,5-dichlorophenol was used as a reference item in order to check the sensitivity of the activated sludge used as microbial inoculum on the day of exposure.The reference item was tested at 0.5, 1.3, 3.5, 9.4 and 25 mg/L with 1 replicate.

The specific oxygen uptake rate of the blank controls (without the test item or reference item) was 21.3 mg/(h·g SS) oxygen. The coefficient of variation of oxygen uptake rate in control replicates was 2.0% at the end of definitive test. The EC50 (3 hours) of 3,5-dichlorophenol was 3.5 mg/L, 95% confidence limits were 2.2~5.6 mg/L.

Based on the results of test item test, the EC50 (3 hours) for inhibition of total respiration was more than 1000 mg/L.

Description of key information

The EC50 (3 hours) of test item for inhibition of total respiration was more than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The study was conducted to determine the inhibition effects of the test substanceon micro-organisms from activated sludge (largely bacteria) by measuring their respiration rate according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)).

The contact time for exposure was 3 hours.The test was conducted with 4 concentrations of 10, 100, 300 and 1000 mg/L. 10, 100, 300 mg/L were tested with 1 replicate, 1000 mg/L was tested with triplicate and an abiotic control at concentration of 1000 mg/L was performed. Inoculums blank controls were performed with 4 replicates. The rate of respiration of each incubation mixture was determined after 3 hours contact times.3,5-dichlorophenol was used as a reference item in order to check the sensitivity of the activated sludge used as microbial inoculum on the day of exposure.The reference item was tested at 0.5, 1.3, 3.5, 9.4 and 25 mg/L with 1 replicate.

The specific oxygen uptake rate of the blank controls (without the test item or reference item) was 21.3 mg/(h·g SS) oxygen. The coefficient of variation of oxygen uptake rate in control replicates was 2.0% at the end of definitive test.The EC50 (3 hours) of 3,5-dichlorophenol was 3.5 mg/L, 95% confidence limits were 2.2~5.6 mg/L.

Based on the results of test item test, the EC50 (3 hours) for inhibition of total respiration was more than 1000 mg/L.