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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-06-19 to 2019-06-20
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))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 707001
Purity: 98.5%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test item was directly weighed into the test vessel and distilled water was added.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of test system: collected from the aeration tank of Shenyang North Sewage Treatment Plant 1 day before the test was started and feed the sludge daily with synthetic sewage feed.

- Treatment of activated sludge:
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 for5 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. Re-suspend the sludge pellet in chlorine-free tap water, and the concentration of the re-suspended sludge was determined by drying method (10 mL suspended sludge with 3 replicate was dried at 105°C for 1hour and 50 minutes). From the measured results, the sludge was diluted further in chlorine-free tap water to obtain the required sludge solids concentration of 3 g/L.

- Cultivation and preparation of activated sludge:
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 (19.8~20.9°C) 1 day prior to use in the test.
Before the activated sludge was used as the inoculums, the concentration of the sludge was determined again by drying method, and the concentration was adjusted to be 3.0 g/L.



Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.4 - 21.6 °C
pH:
7.26 - 7.99
Dissolved oxygen:
1.47 - 9.42 mg/L
Nominal and measured concentrations:
Nominal: 10, 100, 300, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Aeration: clean oil-free air, flow rate 0.5~ 1 L/min
- Inoculums blank controls: 4 replicates
- Test concentrations: 10, 100, 300 mg/L were tested with 1 replicate, 1000 mg/L was tested with triplicate
-Abiotic control: 1 replicate at concentration of 1000 mg/L
- Sludge concentration (weight of dry solids per volume): 0.75 g/L
- Synthetic sewage feed: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2•2H2O, 0.2 g MgSO4•7H2O, 2.8 g K2HPO4 were dissolved with distilled water and maken up to 1L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled water

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED
The rate of respiration of each incubation mixture was determined after 3 hours contact time.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 100, 300, 1000 mg/L
- Results used to determine the conditions for the definitive study: Due to the absence of inhibition of oxygen consumption by the test item in a preliminary test a definitive test is unnecessary
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP)
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:
The specific oxygen uptake rate of the blank controls (without the test item or reference item) was 20.7 mg/(h•g SS) oxygen. The coefficient of variation of oxygen uptake rate in control replicates was 1.4% at the end of preliminary test. The validity criteria have therefore been satisfied.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3h-EC50: 6.0 mg/L, 95% confidence limits: 3.9-9.2 mg/L
Validity criteria fulfilled:
yes
Conclusions:
Based on the results, the EC 50 (3 hours) of test item for total respirations was > 1000 mg/L.
Executive summary:

The study was conducted to determine the inhibition effects of test item on micro-organisms from activated sludge (largely bacteria) by measuring their respiration rate according to OECD 209. The contact time for exposure was 3 hours.

The preliminary test was conducted with 4 concentrations of 10, 100, 300 and 1000 mg/L. The rate of respiration of each incubation mixture was determined after 3 hours contact time.

Due to the absence of inhibition of oxygen consumption by the test item in a preliminary test a definitive test is unnecessary.

Based on the results, the EC 50 (3 hours) of test item for total respirations was > 1000 mg/L.

Description of key information

The study was conducted to determine the inhibition effects of test item on micro-organisms from activated sludge (largely bacteria) by measuring their respiration rate according to OECD 209. The contact time for exposure was 3 hours.

The EC 50 (3 hours) of test item for total respirations was > 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

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