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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2011-08-18 to 2011-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted July 22, 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
April 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Based on the results of a performed preliminary range-finding test, the test item was investigated at the concentration level of 1000 mg/L.
Based on the solubility properties of the test item the adequate amounts of the test item were weighted in a small inert plastic dish (“plate”: to allow the contact between the inoculum and the whole test item amount) and this plastic dish containing the adequate test item amount was thrown into each test flask. Thereafter a pH measurement was carried out and finally the test item solutions were inoculated.
Because of the test item direct addition an analytical estimation of the test substance concentration in the test vessels was considered not necessary, and the subsequent calculations referred to the nominal test item concentration.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Preparation of inoculum for exposure: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre (on dry weight basis).
The activated sludge was not used on the day of the collection but continuously aerated (2L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation and before use. The pH of the activated sludge inoculum after preparation was 7.47, before use: 7.61. The pH adjustment before use was considered not necessary.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
The temperature in the test mixtures during the measurements in average: 21.4 °C, the measured minimum: 20.8 °C, maximum: 22.0 °C.
pH:
This test was performed without pH adjustment.
Start: 7.75 - 7.97
End: 8.05 - 8.15
Nominal and measured concentrations:
1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles of approximately 300 mL volume
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): five
- No. of vessels per control (replicates): six
- No. of vessels per reference control (replicates): three concentrations with three replicates
- No. of vessels per nitrification control (replicates): three

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: the test was performed without pH adjustment

TEST CONCENTRATIONS
- Range finding study:
Pre-experiments were performed to determine the solubility of the test item in the used aqueous system, to investigate the test item effect on the pH within the test system and to select suitable methods for the preparation of a stock solution and for the dosage of the test item in the test medium.
In the preliminary range-finding test one test container from the concentration of 10 mg/L, one container from the concentration of 100 mg/L, triplicates at the highest test item concentration (1000 mg/L), blank, abiotic (three abiotic controls with the highest concentration of the test substance: 1000 mg/L) and reference controls were included.
In the preliminary experiment one series of the test containers was investigated without pH adjustment and one series with adjusted pH (to the range of pH 6 - 8). (In the preliminary experiment two test item stock solutions were prepared with a concentration of 30 mg/mL (clear solutions) one with adjusted pH and one without pH adjustment.)
The nitrification potential of sludge was examined with additional control mixture that contained N-allylthiourea at 11.6 mg/L.
In the preliminary range-finding test the test item did not show any inhibitory effect up to and including the concentration level of 1000 mg/L when pH adjustment was performed and slight inhibition (~22 %) was noticed at the concentration of 1000 mg/L, without pH adjustment and therefore pH relevant effects cannot be excluded.
The abiotic control group did not show significant oxygen uptake (some oxygen uptake was detected in the range of the biological variability of the test system), therefore in the main test the abiotic control was not repeated.
In the preliminary test the nitrification respiration was insignificant and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification is occurring. Therefore the main test was performed in one set of test vessels, without ATU addition.
In the main test (for informative reason) a nitrification control group (containing N-allylthiourea) was investigated with three parallels.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
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:
respiration rate
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:
respiration rate
Details on results:
Inhibition of Respiration Rate:
An additional nitrification control was examined in the main test with three parallels to check the possible nitrification potential of the applied activated sludge batch. With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 73.53 mg/Lh, the heterotrophic respiration (RH) was 71.36 mg/Lh, the nitrification respiration (RN): 2.18 mg/Lh was calculated according to the following equation: RN= RT-RH.
The obtained 2.18 mg/Lh were considered as not significant difference within a biological variability range of the applied test system, that lower than the 5 % of RT (3.68 mg/Lh) in blank controls.
According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 13.72 mg/L, (95 % confidence limits: 11.39 - 16.54).
- Other: The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. Each concentration of the 3,5-Dichlorophenol reference item was tested in three parallels. In comparison to the blank controls the specific respiration rate of the activated sludge was inhibited by 7.26 % at the lowest concentration of 2 mg/L, at the nominal concentrations of 7 and 24.5 mg/L, the respiration rate was inhibited by 13.40 % and 78.66 %, respectively.
Reported statistics and error estimates:
The EC50 value of the reference item and its confidence limits were calculated using Probit analysis. The analysis was done using the TOXSTAT statistical software program.
Since one concentration (as a limit concentration) of the test item was tested, this concentration level of the test item was compared to the blank control using the 2 Sample t-Test (α=0.05).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.
Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the 1,1'-(1,1,2,2-tetramethylethylene)dibenzene on the activity of activated sludge by measuring the respiration rate under defined conditions according to OECD 209, Regulation (EC) No. 440/2008 Method C.11, EPA OPPTS 850.6800. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The test item was investigated at one (limit) concentration level of 1000 mg/L. The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect of the test item was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined to be higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The purpose of the 3-hour test was to evaluate the influence of the 1,1'-(1,1,2,2-tetramethylethylene)dibenzene on the activity of activated sludge by measuring the respiration rate under defined conditions according to OECD 209, Regulation (EC) No. 440/2008 Method C.11, EPA OPPTS 850.6800. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The test item was investigated at one (limit) concentration level of 1000 mg/L. The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect of the test item was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.