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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2015 - June 2015
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
Qualifier:
according to guideline
Guideline:
other: MEP, P.R. China, the Guidelines for the Testing of Chemicals, Effects on Biotic Systems (the Second Edition), No. 209 “Activated Sludge, Respiration Inhibition Test”. (2013.8
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TEST MATERIAL
- batch No.of test material: 3706OB
- Expiration date of the lot/batch: March 2019
- Purity: >99%
- Appearance: white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature at 15-25°C.




Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: aeration tank of Shenyang North Sewage Treatment Plant
- 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. 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 1.5h). 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.
- 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 (19.8~21.3°C) overnight prior to use in the test.
- Initial biomass concentration: 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:
yes
Total exposure duration:
3 h
Hardness:
Not required
Test temperature:
20±2°C in a temperature controlled room, actual recorded temperature was 19.8~21.6°C.
Dissolved oxygen:
Not required
Salinity:
Not required
Conductivity:
Not required
Nominal and measured concentrations:
According to the results of the range-finding test, the definitive test was conducted with 6 concentrations of 0.100 mg/L, 0.496 mg/L, 2.46 mg/L, 12.2 mg/L, 60.5 mg/L and 300 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- No. of vessels per concentration (replicates): 5 replicates
- No. of vessels per control (replicates): 6 replicates without ATU and with ATU
- Sludge concentration (weight of dry solids per volume):
- Weight of dry solids per volume of reaction mixture per unit of time:
- Nutrients provided for bacteria:
- Nitrification inhibitor used : N-allylthiourea, the final concentration was 11.6 mg ATU/L in the test solutions
- Biomass loading rate:


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

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:
- Details on termination of incubation:
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using fewer concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP), the nominal concentrations were 0.10, 0.30, 0.91, 2.7, 8.3 and 25 mg/L, one replicate at each test concentration.
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
346.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
506.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
154.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Details on results:
Based on the results of test item test, the EC50 (3 hours) of test item for three different respirations are as follows:
- EC50 (3h) Total = 346.1 mg/L with a 95% confidence limits of (214.5 - 676.6mg/L)
- EC50 (3h) Heterotrophic = 50.6.3 mg/L with a 95% confidence limits of (311.0 -1061.6 mg/L)
- EC50 (3h) Nitrification = 154.6 mg/L with a 95% confidence limits of (60.7 - 826.9 mg/L)

Results with reference substance (positive control):
The validity criteria for the reference item EC50 values were satisfied:
EC50 Total = 7.4 mg/L
EC50 Heterotrophic = 10.6 mg/L
EC50 Nitrification = 3.5 mg/L

The specific oxygen uptake rate of the blank controls (without the test item or reference item) was 25.3 mg/(g·h) oxygen. The coefficient of variation of oxygen uptake rate in control replicates was 8.2% at the end of definitive test. The validity criteria have therefore been satisfied.

Validity criteria:

- The specific oxygen uptake rate of the blank controls (without the test item or reference item) was 25.3 mg/(g·h) oxygen ( > 20 mg oxygen per one gram of activated sludge in an hour). The coefficient of variation of oxygen uptake rate in control replicates was 8.2% at the end of definitive test (> 30%.).

- The validity criteria for the reference item EC50 values were satisfied.

EC50 (mg/L) Total = 7.4 mg/L

EC50 (mg/L) Heterotrophic = 10.6 mg/L

EC50 (mg/L) Nitrification = 3.5 mg/L

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of test item test, the EC50 (3 hours) of test item are as follows:
- EC50 (mg/L) Total = 346.1 mg/L with a 95% confidence limits of (214.5 - 676.6mg/L)
- EC50 (mg/L) Heterotrophic = 50.6.3 mg/L with a 95% confidence limits of (311.0 -1061.6 mg/L)
- EC50 (mg/L) Nitrification = 154.6 mg/L with a 95% confidence limits of (60.7 - 826.9 mg/L)

Executive summary:

The study was conducted to determine the inhibition effects of XA 31 micro-organisms from activated sludge by measuring their respiration rate (including total, heterotrophic only and that due to nitrification) following an exposure time of 3 hours. The study was performed according to OECD Guideline 209 (2013) and was compliant with the GLP.

Based on the results of previous preliminary tests, the definitive test was conducted with 6 concentrations of 0.100 mg/L, 0.496 mg/L, 2.46 mg/L, 12.2 mg/L, 60.5 mg/L and 300 mg/L (5 replicates for each concentration). Two sets of test item mixtures at each concentration were prepared, one set of test mixture was used to determine inhibition effects of total oxygen uptake, another set contained 11.6 mg ATU/L was used to determine inhibition effects of heterotrophic respiration only, the differences between heterotrophic and the corresponding total respiration rates represent nitrification. Inoculums blank controls without ATU and containing ATU were performed with 6 replicates at the beginning, medium and end of the exposure period. The rate of respiration of each incubation mixture was determined after 3 hours contact time.

After 3 hours contact time at the highest tested concentration of 300 mg/L, the percentage inhibition were 51.3% for the total respiration, 43.6% for the inhibition of heterotrophic respiration and 64.7% for the inhibition of respiration due to nitrification.

Therefore based on the results of test item test, the EC50 (3 hours) of test item for three different respirations are as follows:

- EC50 (3h) Total = 346.1 mg/L with a 95% confidence limits of (214.5 - 676.6mg/L)

- EC50 (3h) Heterotrophic = 50.6.3 mg/L with a 95% confidence limits of (311.0 -1061.6 mg/L)

- EC50 (3h) Nitrification = 154.6 mg/L with a 95% confidence limits of (60.7 - 826.9 mg/L)

 The validity criteria of the OECD guideline 209 were fulfilled, therefore this study is considered as reliable without restrictions.

Description of key information

 One reliable study is available for the Reaction mass of AminoPhosphonium salt and BisphenolAF (Xa 31) for this endpoint.

The study was conducted to determine the inhibition effects of XA 31 micro-organisms from activated sludge by measuring their respiration rate (including total, heterotrophic only and that due to nitrification) following an exposure time of 3 hours. The study was performed according to OECD Guideline 209 (2013) and was compliant with the GLP.

Based on the results of previous preliminary tests, the definitive test was conducted with 6 concentrations of 0.100 mg/L, 0.496 mg/L, 2.46 mg/L, 12.2 mg/L, 60.5 mg/L and 300 mg/L (5 replicates for each concentration). Two sets of test item mixtures at each concentration were prepared, one set of test mixture was used to determine inhibition effects of total oxygen uptake, another set contained 11.6 mg ATU/L was used to determine inhibition effects of heterotrophic respiration only, the differences between heterotrophic and the corresponding total respiration rates represent nitrification. Inoculums blank controls without ATU and containing ATU were performed with 6 replicates at the beginning, medium and end of the exposure period. The rate of respiration of each incubation mixture was determined after 3 hours contact time.

After 3 hours contact time at the highest tested concentration of 300 mg/L, the percentage inhibition were 51.3% for the total respiration, 43.6% for the inhibition of heterotrophic respiration and 64.7% for the inhibition of respiration due to nitrification.

Therefore based on the results of test item test, the EC50 (3 hours) of test item for three different respirations are as follows:

- EC50 (3h) Total = 346.1 mg/L with a 95% confidence limits of (214.5 - 676.6mg/L)

- EC50 (3h) Heterotrophic = 50.6.3 mg/L with a 95% confidence limits of (311.0 -1061.6 mg/L)

- EC50 (3h) Nitrification = 154.6 mg/L with a 95% confidence limits of (60.7 - 826.9 mg/L)

However, as after 3 hours contact time at the highest tested concentration of 300 mg/L the percentage inhibition reached 51.3% for the total respiration, 43.6% for the inhibition of heterotrophic respiration and 64.7% for the inhibition of respiration due to nitrification, the final EC50 (3h) Total and Heterotrophic > 300 mg/L was preferred.

Key value for chemical safety assessment

EC50 for microorganisms:
300 mg/L

Additional information

the Key values are as follows ( not key data as < or > sign not allowed)

EC50 (3h) Total > 300 mg/L

EC50 (3h) Heterotrophic > 300 mg/L