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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2015-09-30 to 2015-09-30
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
Version / remarks:
(July 2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(May 2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: PCSPP 850.3300: Modified Activated Sludge, Respiration Inhibition Test (January 2012)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
One test solution with a final volume of 300 mL was tested per treatment in a glass flask. 9.6 mL synthetic sewage and an adequate volume of the stock solution of the N-allylthiourea adequate volumes of the stock solution of reference item were filled up with water (with deionised water) to 150 mL before the start of the test. At the test item treatments the 300 mg test item was measured directly into the empty test containers the 150 mL synthetic sewage-water mixture was filled into these test containers just before the inoculation. At the start of the test 150 mL activated sludge inoculum with a sludge concentration of 3 g/L (on dry weight basis) was added to the test containers, first to the blank controls (CBA, thereafter CBB CBC and CBD the “start” CB group), then in appropriate time intervals to the nitrification controls, the test solutions of the reference item and the test item and finally to the “end” CB group (CBE, CBF, CBG and CBH). In time intervals of approximately 12 minutes (an arbitrary but convenient interval) a maximum of four test vessels were started.
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). At the concentration calculation the dilution resulted by the fed synthetic sewage was taken into consideration. The activated sludge was not used on the day of the collection but continuously aerated (0.5 L/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 just before use: 8.01. pH adjustment of the inoculum was considered as not necessary.
- Initial biomass concentration: 3 g/L (on dry weight basis)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
19.8 - 22.0 °C
pH:
The pH of the activated sludge inoculum was checked just before use: 8.01. pH adjustment of the inoculum was considered as not necessary.
Nominal and measured concentrations:
Nominal: 100 mg/L
No measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Type: closed
- Material, fill volume: Glass flasks with a final volume of 300 mL
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): Five
- No. of vessels per control (replicates): Eight blanc controls (containing water, synthetic sewage and inoculum, but without addition of the test or reference item), three reference controls (3,5-Dichlorophenol at the nominal concentrations of 2,7 and 24.5 mg/L) and three nitrification controls (same as the blank controls, however containing 11.6 mg/L N-allylthiourea)

TEST MEDIUM / WATER PARAMETERS
- in accordance to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: not considered necessary

EFFECT PARAMETERS MEASURED: Respiration rate after 3 hours

TEST CONCENTRATIONS
- Range finding study: In a preliminary range-finding study concentrations of 10, 100 and 100 mg/L were tested (with three replicates at the highest test item concentration, with two replicates at the lower concentrations). Blank controls, nitrification controls and a abiotic control group were inculded in the test.
- Results used to determine the conditions for the definitive study: Based on the preliminary information about the test item caused effect on the activated sludge inoculum, in the main test the test Oligomeric Silane Adduct was investigated at the 1000 mg/L test item concentration as limit concentration, only, without pH adjustment. In the preliminary tests the nitrification respiration was insignificant and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification is occurred. Therefore the main test was performed in one set of test vessels, without N-allylthiourea (ATU) addition. In the main test (for informative reason) a nitrification control group (containing N-allylthiourea) was investigated with three parallels. The abiotic control group in the preliminary experiments did not show oxygen uptake therefore in the main test abiotic control group was not included.
Reference substance (positive control):
yes
Remarks:
(3,5-Dichlorophenol)
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
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:
respiration rate
Details on results:
The observed-calculated oxygen consumption rates were in the range of the blank controls, significant inhibitory effect (the observed slight in average 2.89 % inhibition was within the biological variability of the applied test system) of the test item at the examined limit concentration of 1000 mg/L was not observed.
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 15.2 mg/L, (95 % confidence limits: 12.5-18.5 mg/L).
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 test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. 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. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item Oligomeric Silane Adduct was investigated at the concentration of 1000 mg/L as a limit concentration, only. 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.

Description of key information

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.

Key value for chemical safety assessment

Additional information

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. 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. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item was investigated at the concentration of 1000 mg/L as a limit concentration, only. 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.

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