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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 5, 2015 to Sept 10, 2015
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:
2010
Deviations:
yes
Remarks:
one deviation noted; considered uncritical
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008
Deviations:
yes
Remarks:
one deviation noted; considered uncritical
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 6F11027000
- Expiration date of the lot/batch: 17-Nov-2016
- Purity: 96%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Due to the poor solubility of the test item, the test item was added directly into the test vessel using pipettes.

Tap water was used as the dilution water along with a nutrient solution.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Collected from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant D-67435 NW-Lachen-Speyerdorf
- Method of cultivation: Field-collected
- Preparation of inoculum for exposure: sludge was filtrated, washed with tap water and re- suspended in tap water.
- Pretreatment: The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L (the nutrient solution was thawed and the sludge was fed with 50 mL/L sludge)
- Initial biomass concentration: 2.52 g suspended solids/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
22.6 – 23.4°C
pH:
7.6-8.0
Nominal and measured concentrations:
1, 100, 1000 mg/L; Nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 2000 mL Schott flasks (250-mL narrow-neck glass bottles with flat bottoms used as measuring flasks)
- Type: closed
- Material, size, headspace, fill volume: a large headspace
- No. of vessels per concentration (replicates): 5 replicates for 1000 mg/L test item concentration; 1 replicate for 100 mg/L test item concentration; 1 replicate for 10 mg/L test item concentration; 1 replicate for 1 mg/L test item concentration
- No. of vessels per control: 6 replicates
- No. of vessels per positive control: 4 replicates
- Sludge concentration: 1.26 g suspended solids/L
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenole
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:
other:
Remarks:
Calculated % Inhibition values are presented based on the O2 consumption measured (which is a measure for the viability of the bacteria). However it is not explicitly stated if this represents (total?) respiration.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
Calculated % Inhibition values are presented based on the O2 consumption measured (which is a measure for the viability of the bacteria). However it is not explicitly stated if this represents (total?) respiration.
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
Calculated % Inhibition values are presented based on the O2 consumption measured (which is a measure for the viability of the bacteria). However it is not explicitly stated if this represents (total?) respiration.
Results with reference substance (positive control):
Four concentrations were tested; an EC50 of 8.9 mg/L (95% confidence interval: 7.3 – 11 mg/L) was determined, which lies within the range of 2 – 25 mg/L.
Reported statistics and error estimates:
For the treatments with the test item concentration 1000 mg/L, it was tested whether the differences between treatment and control were significant. Equality of variance was tested (F-value), followed by a t-test.
The difference between treatment 1000 mg/L and the control was considered as not significant as the calculated t-value lay below the significance level. Therefore, the concentration 1000 mg/L is stated as NOEC.

Summary of validation criteria:

- The coefficient of variation of oxygen uptake rate in control replicates (8.0%) did not exceed 30% at the end of the test

- The blank controls oxygen uptake rate (41.50 mg oxygen per gram sludge in 1 hour) was less than 20 mg oxygen per gram of activated sludge (dry weight of suspended solids) in 1 hour

 

The following deviations from the study plan were reported:

- The temperature in the Test was in a range of 22.6 – 23.4°C instead of 20 ± 2°C.This deviation was stated as uncritical, as normal respiration activity of the control could be observed.

- The test item was stored from the date of receipt to last usage for the study at 20 ± 5°C except of 10 days below 15°C and short-term for 25.8°C. This is considered as uncritical concerning the outcome of the study.

- The concentration in the lowest concentration was 1.7 mg/L instead of 1 mg/L because direct addition of 1 mg/L (corresponding to 0.56 µL / 500 mL Test solution) is not possible.                                            

Validity criteria fulfilled:
yes
Conclusions:
The test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal con-centration. As the test item is volatile, the experiment was performed in a closed system without aeration. Under the test conditions, the 3-hr EC10 and 3-hr EC50 were determined to be > 1000 mg/L (nominal).
Executive summary:

The effect of 1,3,5-triisopropylbenzene was tested under static conditions in a 3-hour activated sludge respiration inhibition study with 4 concentrations according to OECD TG 209. Based on nominal concentrations, the 3-h NOEC was determined to be 1000 mg/L.

Description of key information

The effect of 1,3,5-triisopropylbenzene was tested under static conditions in a 3-hour activated sludge respiration inhibition study with 4 concentrations according to OECD TG 209. Based on nominal concentrations, the 3-h NOEC was determined 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