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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-01-14 to 2016-01-15
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
Specific details on test material used for the study:
Batch No.: 150209
Purity: 97.7 wt%
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Source: Aeration tank of Liede Sewage Treatment Plant of Guangzhou
- Treatment: The sludge was removed any coarse particles and impurities. As the activated sludge was not used on day of collection, 50mL of synthetic sewage feed per litre of activated sludge was fed daily for another two days and aerated under the test temperature of 200 °C ± 20 °C. The dry weight of the suspended sludge was determined as 5.5 g/L before testing. When testing, 10910 mL of this activated sludge was diluted with 9090 mL of the deionized water, then the sludge suspension with the concentration of 3.0 g/L was obtained, with the pH value of 7.55. Keep the sludge suspension aerating at test temperature until required.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Test temperature:
18.0 °C -20.3 °C (the activated sludge sensitivity test)
18.0 °C -20.6 °C (the test substance toxicity test)
pH:
7.00-7.01
Dissolved oxygen:
less than 2.0 mg/L
Nominal and measured concentrations:
Nominal: 10, 100, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL vessel which has a ground glass stopper and an outlet pipe for the aeration of the test mixtures
- Aeration: Aerating and agitating
- Activated sludge final concentration: 1.5 g/L
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): two replicates
- No. of vessels per abiotic control (replicates): three replicates
- Nitrification inhibitor used: N-allylthiourea
- Biomass loading rate: MLSS = 3.0 g/LTEST SYSTEM

Test mixtures:
FT: containing test substance, inoculum, synthetic sewage feed and deionized water
FH: containing test substance, inoculum, synthetic sewage feed, ATU and deionized water
F1-3: containing test substance, synthetic sewage feed and deionized water
Blank control:
FTB: containing inoculum, synthetic sewage feed and deionized water
FHB: containing inoculum, synthetic sewage feed, ATU and deionized water
Reference mixtures:
FR: containing reference substance, inoculum, synthetic sewage feed and deionized water
FHR: containing reference substance, inoculum, synthetic sewage feed, ATU and deionized water
Abiotic control:
FA: containing test substance, synthetic sewage feed and deionized water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water containing less than 1 mg/L DOC was employed

Test for the sensitivity of the activated sludge
A certain volumes of the reference substance stock solution, synthetic sewage feed, ATU stock solution and deionized water were added into each test vessel respectively.
At the beginning of the test (0 min), 250 mL of activated sludge suspensions with the concentration of 3.0 g/L were added into FTB1, FR1, FR2, FR3, FR4 and FTB2, respectively, with a final volume of 500 mL, aerating and agitating. At the moment of 15 min, 250 mL of activated sludge suspensions were added into FHB1, FHR1, FHR2, FHR3, FHR4 and FHB2, respectively, with a final volume of 500 mL, aerating and agitating.
After the exposure times of three hours, the dissolved oxygen concentrations of FTB1, FR1, FR2, FR3, FR4 and FTB2 were measured automatically with an appropriate interval for 8 minutes, and those of the residual test groups were measured in tum. Also the test temperatures were recorded.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3, 5-dichlorophenol
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
316.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Details on results:
The activated sludge sensitivity test:
The average oxygen uptake rates of the blank controls (FTB) was 32.0 mg / (g. h), the coefficient of variation of the oxygen uptake rates in the blank controls (FTR FHB) were 19% - 2.6%, which met the requirement of the test.
The percentage inhibition of the reference substance for the total oxygen uptake with the exposure concentrations of 1.0 mg/L, 3.0 mg/L, 9.0 mg/L, 27.0 mg/L were 3.7%, 12.9%, 68.5% and 93.0%, respectively, 3h-EC50 was 5.7 mg/L, with 95% confidence limits of 5.4 mg/L - 6.0 mg/L.
The percentage inhibition of heterotrophic respiration were 12.9%, 20.4%, 63.2% and 91.8%, respectively, 3h-EC50 was 6.4mg/L, with 95% confidence limits of 6.1 mg/L - 6.8 mg/L.
The percentage inhibition of nitrification were -150%, -112%, 100% and 100%, respectively with 3h-EC50 of 5.2 mg/L.

The test substance toxicity test:
The average oxygen uptake rate of abiotic control was 1.2 mg/(L.h), which was 3.1% of the oxygen uptake rate of the blank control, it indicated that there was no significant abiotic oxygen consumption.
The average oxygen uptake rates of the blank controls (FTB) was 26.4 mg / (g. h), the coefficient of variation of the oxygen uptake rates in the blank controls (FR, FHB) were 5.4% - 81%, which met the validity of the test.
The percentage inhibition of the test substance for total oxygen uptake with the exposure concentrations of 10 mg/L, 100 mg/L and 1000 mg/L (three replicates) were -16.4%, -7.0% and 40.6%, 45.7%, 44.0% (with a mean of 43.4%), respectively. The percentage inhibition of heterotrophic respiration were -8.5%, 11.8% and 28.1 %, 30.9%, 28.8% (with a mean of 29.3%), respectively. The EC50 of total oxygen uptake and heterotrophic respiration were greater than 1000 mg/L.
The percentage inhibition of nitrification were -48.1% (FN1), -82.9% (FN2) and 100% (FN3-5), respectively with the EC50 of 316.2 mg/L. The ratio of RN and RT in the blank control was 19.9%, which showed that there was nitrification occurred in the test. As there was no significant inhibition on the activated sludge in the total oxygen uptake and heterotrophic respiration under the test substance concentration of 1000 mg/L, therefore the definitive test of the test substance for inhibition of nitrification rate was not be carried out.
Results with reference substance (positive control):
The activated sludge sensitivity test:
The percentage inhibition of the reference substance for the total oxygen uptake with the exposure concentrations of 1.0 mg/L, 3.0 mg/L, 9.0 mg/L, 27.0 mg/L were 3.7%, 12.9%, 68.5% and 93.0%, respectively, 3h-EC50 was 5.7 mg/L, with 95% confidence limits of 5.4 mg/L - 6.0 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
EC50 of the test substance for total oxygen uptake and heterotrophic respiration were higher than 1000 mg/L in the presence of this test conditions. EC50 of the test substance for nitrification was 316.2 mg/L.
Executive summary:

The study was performed to determine the ECx value of the respiration Inhibition of the test substance on activated sludge according to OECD Guideline 209.

The dissolved oxygen (DO) of the blank controls and the test mixtures in the presence of different concentrations were determined after the exposure time of 30 minutes, which were withdrawn to calculate the oxygen uptake rates and the percentage inhibition.

 

The average oxygen uptake rate of abiotic control was 1.2 mg / (L. h), which was 3.1% of the oxygen uptake rate of the blank control, it indicated that there was no significant abiotic oxygen consumption.

In the test substance toxicity test , the average oxygen uptake rates of the blank controls (FTB) was 26.4 mg / (g. h), the coefficient of variation of the oxygen uptake rates in the blank controls (FR, FHB) were 5.4% - 81%, which met the validity of the test. The percentage inhibition of the test substance for total oxygen uptake with the exposure concentrations of 10 mg/L, 100 mg/L and 1000 mg/L (three replicates) were -16.4%, -7.0% and 40.6%, 45.7%, 44.0% (with a mean of 43.4%), respectively. The percentage inhibition of heterotrophic respiration were -8.5%, 11.8% and 28.1 %, 30.9%, 28.8% (with a mean of 29.3%), respectively. The EC50 of total oxygen uptake and heterotrophic respiration were greater than 1000 mg/L. The percentage inhibition of nitrification were -48.1% (FN1), -82.9% (FN2) and 100% (FN3-5), respectively with the EC50 of 316.2 mg/L. The ratio of RN and RT in the blank control was 19.9%, which showed that there was nitrification occurred in the test. As there was no significant inhibition on the activated sludge in the total oxygen uptake and heterotrophic respiration under the test substance concentration of 1000 mg/L, therefore the definitive test of the test substance for inhibition of nitrification rate was not be carried out.

 

EC50 of the test substance for total oxygen uptake and heterotrophic respiration were higher than 1000 mg/L in the presence of this test conditions. EC50 of the test substance for nitrification was 316.2 mg/L.

Description of key information

EC50 of the test substance for total oxygen uptake and heterotrophic respiration were higher than 1000 mg/L in the presence of this test conditions. EC50 of the test substance for nitrification was 316.2 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The study was performed to determine the ECx value of the respiration Inhibition of the test substance on activated sludge according to OECD Guideline 209.

The dissolved oxygen (DO) of the blank controls and the test mixtures in the presence of different concentrations were determined after the exposure time of 30 minutes, which were withdrawn to calculate the oxygen uptake rates and the percentage inhibition.

 

The average oxygen uptake rate of abiotic control was 1.2 mg / (L. h), which was 3.1% of the oxygen uptake rate of the blank control, it indicated that there was no significant abiotic oxygen consumption..

In the test substance toxicity test , the average oxygen uptake rates of the blank controls (FTB) was 26.4 mg / (g. h), the coefficient of variation of the oxygen uptake rates in the blank controls (FR, FHB) were 5.4% - 81%, which met the validity of the test. The percentage inhibition of the test substance for total oxygen uptake with the exposure concentrations of 10 mg/L, 100 mg/L and 1000 mg/L (three replicates) were -16.4%, -7.0% and 40.6%, 45.7%, 44.0% (with a mean of 43.4%), respectively. The percentage inhibition of heterotrophic respiration were -8.5%, 11.8% and 28.1 %, 30.9%, 28.8% (with a mean of 29.3%), respectively. The EC50 of total oxygen uptake and heterotrophic respiration were greater than 1000 mg/L. The percentage inhibition of nitrification were -48.1% (FN1), -82.9% (FN2) and 100% (FN3-5), respectively with the EC50 of 316.2 mg/L. The ratio of RN and RT in the blank control was 19.9%, which showed that there was nitrification occurred in the test. As there was no significant inhibition on the activated sludge in the total oxygen uptake and heterotrophic respiration under the test substance concentration of 1000 mg/L, therefore the definitive test of the test substance for inhibition of nitrification rate was not be carried out.

 

EC50 of the test substance for total oxygen uptake and heterotrophic respiration were higher than 1000 mg/L in the presence of this test conditions. EC50 of the test substance for nitrification was 316.2 mg/L.

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