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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 May 2018 to 18 May 2018
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
Qualifier:
according to
Guideline:
other: The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 209 Activated Sludge, Respiration Inhibition Test [M].
Version / remarks:
The editorial board of “The Guidelines for the Testing of Chemicals” of Chemical Registration Center of the Ministry of Environmental Protection. Second Edition Beijing: China Environmental Press. 2013, page 73-85.
Deviations:
no
Qualifier:
according to
Guideline:
other: Department of Science and Technology Standards of State Environmental Protection Administration. HJ/T 153-2004 Guidelines for Testing of Chemicals [S].
Version / remarks:
Beijing: China Standard Press. 2004.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Total respiration inhibition test
- To prepare the test solution 16 mL synthetic sewage and 500.1-504.7 mg of test material were added to 234 mL of test water, this was added to 250 mL activated sludge inoculum with concentration of 3 g/L to get the test mixture with total volume of 500 mL.
- The blank control was prepared in the same way without the addition of the test material.

Heterotrophic respiration inhibition test
- To prepare the test solution 16 mL synthetic sewage and 499.9-504.2 mg of test material were added to 230 mL of test water and 4.000 mL of ATU stock solution (1.45 g/L), this was added to 250 mL activated sludge inoculum with concentration of 3 g/L to get the test mixture with total volume of 500 mL.
- The blank control was prepared in the same way without the addition of the test material.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: The activated sludge derived from sewage treatment plant in the east of Suzhou city sewage (a conventional treatment plant for domestic waste) was selected as inoculum.
- Collection and pre-treatment: the freshly sampled activated sludge was collected, kept aerobic during transport. On the day of sampling, the activated sludge was filtered through a fine filter sieve to remove the coarse particles, and then washed using chlorine-free tap water. After settling for about 10 minutes, the supernatant was discarded. The sludge was re-suspended in chlorine-free tap water and the above operation was repeated 3 times.
- 10 mL of washed activated sludge was sampled (4 replicates) and dried at 105 °C for 2 hours to determine the concentration of suspended solids. The concentration of the sludge was determined to be 3.8 g suspended solid (SS)/L. The suspended sludge was prepared with chlorine-free tap water to yield a concentration of 3 g SS/L. The sludge was continuously aerated at the test temperature and not used on the day of collection. The sludge was fed daily with synthetic sewage feed (not more than 50 mL synthetic sewage feed/ L activated sludge) for 2 days. The pH of the activated sludge was 6.08 and adjusted to 7.22 with sodium hydrogen carbonate solution before use.
- The heterotrophic and nitrification respiration rate of activated sludge was determined on the day of collection and on the day before use. The results showed that the endogenous heterotrophic respiration rate was 58 % and 63 % of total respiration rate respectively on the day of collection and on the day before use. The nitrification respiration rate was 42 % and 37 % of total respiration rate respectively on the day of collection and on the day before use. It was considered that no significant change in its activity, assessed by its endogenous heterotrophic and nitrification respiration rate, had occurred. The activated sludge could be used for the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20.5 - 21.6 °C
Dissolved oxygen:
Day of Collection Total respiration: 4.08 - 7.86 mg/L
Day of Collection Heterotrophic respiration: 5.61 - 8.04 mg/L
Before Using Total respiration: 2.61 - 7.86 mg/L
Before Using Heterotrophic respiration: 4.41 - 7.79 mg/L
Nominal and measured concentrations:
1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: BOD bottles
- Aeration: Compressed air was passed through an appropriate filter to remove dust and oil. Forced aeration of 0.5-1 L/min was used to keep the dissolved oxygen concentration above 60-70 % saturation. Aerated period: 3 hours. The aeration began with the initial contact of the activated sludge with the other constituents of the final mixture. The test mixture was prepared first, but without inoculum addition. The activated sludge was added to the test vessels at the start of each batch of aeration. The procedures were repeated in time intervals of 3 - 15 min. The activated sludge was added to several (e.g. 2 - 3) test vessels simultaneously, and the aeration began. The controls were aerated at the start, middle and end of test.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Nutrients provided for bacteria: 2 L of synthetic sewage feed needed in the test was prepared: Peptone 32.023 g, Meat extract 22.021 g, Urea 6.003 g, NaCl 1.404 g, CaCl2·2H2O 0.803 g, MgSO4·7H2O 0.401 g and K2HPO4·3H2O 7.405 g were dissolved in purified water and made up to 2 L. The pH of the solution was 6.95, adjusted to be 7.03 with NaOH solution. The prepared medium was not used immediately; it was stored in the dark at 0-4 °C for no longer than one week.
- Nitrification inhibitor used: All the test vessels contained the specific inhibition of nitrification at the concentration of 11.6 mg/L in the final test solution. 0.363 g ATU was weighed, dissolved and made up to 250 mL with purified water to obtain the stock solution with concentration of 1.45 g/L.

TEST MEDIUM / WATER PARAMETERS
- Chlorine-free tap water (also named as treated water in the lab)

EFFECT PARAMETERS MEASURED
- After the aeration period, the test mixture from the first batch of aeration vessels was transferred to the BOD bottles equipped with a magnetic stirrer and the concentration of dissolved oxygen was continuously measured for a period of 10 min. After a time interval of 3 - 15 min, another batch of the aeration test mixture was measured. The temperature of the testing area was monitored at the start and end of test.

TEST CONCENTRATIONS
- Preliminary Test (Non-GLP): The preliminary test was conducted prior to the definitive test to help select the concentrations for the definitive study. The concentration of test material was 10, 33, 100, 330 and 1000 mg/L, and the dissolved oxygen was determined after aeration for 3 hours. The test results showed when the test material concentration was 10, 33, 100, 330 and 1000 mg/L, the total respiration inhibition percentage of activated sludge was 1.0 %, 2.2 %, -0.4 %, -0.2 %, and -9.1 %; the heterotrophic respiration inhibition percentage was 4.7 %, -6.1 %, -8.7 %, -7.6 % and -9.6 %; the nitrification respiration inhibition percentage was -5.6 %, 17.2 %, 14.7 %, 13.2 % and -8.2 %. A test solution (1000 mg/L) was prepared in the preliminary test. The pH value of the solution was 7.56 without adding inoculums and in the range of 7.5 ± 0.5. Therefore, the pH of test solution was not determined before adding inoculums in definitive test.
- According to the results of preliminary test, test material showed no toxic effect on microorganisms of activated sludge at the concentration of 1000 mg/L. The definitive test was therefore conducted as a limit test.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
and nitrification respiration rate
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
Remarks:
and nitrification respiration rate
Details on results:
VALIDITY OF THE TEST
- The blank controls (FTB) oxygen uptake rate of total respiration was 23.3 mg/ (h·g SS) (not less than 20 mg oxygen one gram of activated sludge (dry weight of suspended solid) in an hour).
- The coefficient of variation of total respiration and heterotrophic respiration oxygen uptake rate in control replicates was 2.7 % and 2.9 % respectively (not more than 30 %) at the end of the definitive test.
- The EC50 of reference material to total respiration of activated sludge was 4.5 mg/L, which lies in the range of 2-25 mg/L. The EC50 of reference material to heterotrophic respiration of activated sludge was 11.7 mg/L, which lies in the range of 5-40 mg/L. The EC50 of reference material to nitrification respiration of activated sludge was 1.2 mg/L, which lies in the range of 0.1-10 mg/L.
Thus, the test satisfied all the criteria of test validity.

ECx AND NOEC
- Compared to blank control, the test material had no significant effect on the total, heterotrophic and nitrification respiration rate of activated sludge at the concentration of 1000 mg/L. The 3-hour NOEC of the test material to total, heterotrophic and nitrification respiration was 1000 mg/L. Therefore, the 3-hour EC50 of test material to total, heterotrophic and nitrification respiration was found to be > 1000 mg/L.
Results with reference substance (positive control):
- 125.0 mg of reference item was weighed and dissolved in purified water. Ultrasonication was used to accelerate the dissolution. The solution was made up to 250 mL with purified water to obtain the stock solution with concentration of 500 mg/L. The pH of the solution was measured to be 6.24 and adjusted to 7.36 with NaOH solution. 5.000 mL of the stock solution was taken and made up to 500 mL with purified water to obtain the reference item solution with concentration of 5 mg/L.
- The concentration of reference item was 0.03, 0.1, 0.3, 1, 3, 9, 27 and 81 mg/L. Two replicates were set each concentration and one replicate contained the specific inhibition of nitrification at the concentration of 11.6 mg/L in the final test solution.
- The EC50 of reference item to total respiration of activated sludge was 4.5 mg/L and its 95 % confidence limit was 3.7-5.4 mg/L. The EC50 of reference item to heterotrophic respiration of activated sludge was 11.7 mg/L and its 95 % confidence limit was 7.6-17.9 mg/L. The EC50 of reference item to nitrification respiration of activated sludge was 1.2 mg/L and its 95 % confidence limit was 0.8-1.7 mg/L.
Reported statistics and error estimates:
The probit analysis method was used to calculate the EC50 of reference item to total respiration and its 95 % confidence limit.
The F test and T test was used to calculate NOEC.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 3-hour NOEC of the test material to total, heterotrophic and nitrification respiration was 1000 mg/L. Therefore, the 3-hour EC50 of the test material to total, heterotrophic and nitrification respiration was found to be > 1000 mg/L.
Executive summary:

The toxicity of the test material to microorganisms was investigated in accordance with the standardised guideline OECD 209 and other Chinese guidelines, under GLP conditions.

The objective of the study was to evaluate toxic effects of the test material on activated sludge microorganisms. According to the results of preliminary test, a limit test was conducted at the nominal concentration of 1000 mg/L as the definitive test using five replicates and 6 replicates of the blank control were set in parallel. The reference material used was 3,5-dichlorophenol to check the sensitivity of activated sludge, which included total respiration inhibition test and heterotrophic respiration inhibition test. The concentration of reference material was 0.03, 0.1, 0.3, 1, 3, 9, 27 and 81 mg/L, two replicates were set each concentration and one replicate contained the specific inhibition of nitrification at the concentration of 11.6 mg/L in the final test solution.

After aeration for 3 hours, the concentration of dissolved oxygen was continuously measured for a period of 10 min. The no-observed effect concentration (NOEC) of the test material on the respiration inhibition of activated sludge were calculated.

The EC50 of reference material to total respiration of activated sludge was 4.5 mg/L and its 95 % confidence limit was 3.7-5.4 mg/L. The EC50 of reference material to heterotrophic respiration of activated sludge was 11.7 mg/L and its 95% confidence limit was 7.6-17.9 mg/L. The EC50 of reference material to nitrification respiration of activated sludge was 1.2 mg/L and its 95 % confidence limit was 0.8-1.7 mg/L.

Compared to blank control, the test material had no significant effect on the total, heterotrophic and nitrification respiration rate of activated sludge at the concentration of 1000 mg/L. The NOEC of test material to total, heterotrophic and nitrification respiration was 1000 mg/L.

Under the conditions of this study, the 3-hour NOEC of the test material to total, heterotrophic and nitrification respiration was 1000 mg/L. Therefore, the 3-hour EC50 of the test material to total, heterotrophic and nitrification respiration was found to be > 1000 mg/L.

Description of key information

Under the conditions of this study, the 3-hour NOEC of the test material to total, heterotrophic and nitrification respiration was 1000 mg/L. Therefore, the 3-hour EC50 of the test material to total, heterotrophic and nitrification respiration was found to be > 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The toxicity of the test material to microorganisms was investigated in accordance with the standardised guideline OECD 209 and other Chinese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to evaluate toxic effects of the test material on activated sludge microorganisms. According to the results of preliminary test, a limit test was conducted at the nominal concentration of 1000 mg/L as the definitive test using five replicates and 6 replicates of the blank control were set in parallel. The reference material used was 3,5-dichlorophenol to check the sensitivity of activated sludge, which included total respiration inhibition test and heterotrophic respiration inhibition test. The concentration of reference material was 0.03, 0.1, 0.3, 1, 3, 9, 27 and 81 mg/L, two replicates were set each concentration and one replicate contained the specific inhibition of nitrification at the concentration of 11.6 mg/L in the final test solution.

After aeration for 3 hours, the concentration of dissolved oxygen was continuously measured for a period of 10 min. The no-observed effect concentration (NOEC) of the test material on the respiration inhibition of activated sludge were calculated.

The EC50 of reference material to total respiration of activated sludge was 4.5 mg/L and its 95 % confidence limit was 3.7-5.4 mg/L. The EC50 of reference material to heterotrophic respiration of activated sludge was 11.7 mg/L and its 95% confidence limit was 7.6-17.9 mg/L. The EC50 of reference material to nitrification respiration of activated sludge was 1.2 mg/L and its 95 % confidence limit was 0.8-1.7 mg/L.

Compared to blank control, the test material had no significant effect on the total, heterotrophic and nitrification respiration rate of activated sludge at the concentration of 1000 mg/L. The NOEC of test material to total, heterotrophic and nitrification respiration was 1000 mg/L.

Under the conditions of this study, the 3-hour NOEC of the test material to total, heterotrophic and nitrification respiration was 1000 mg/L. Therefore, the 3-hour EC50 of the test material to total, heterotrophic and nitrification respiration was found to be > 1000 mg/L.