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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-16 - 2011-08-18
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
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- For each experiment, a stock solution of an appropriate concentration in deionised water was prepared. The stock solution then was used to prepare the treatments.
- A stock solution in deionised water containing 500 mg/L (nominal) was freshly prepared for each experiment.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
ACTIVATED SLUDGE
- Laboratory culture: no
- Source: activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant D-67435 NW-Lachen-Speyerdorf
- Method of cultivation:
* upon arrival the sludge was filtrated, washed with tap water and re-suspended in tap water.
* The activated sludge was aerated until usage in the test and fed daily with 50 ml /L synthetic sewage feed.
- Initial suspended solids concentration in the test was 1.34 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
yes, for measurement of oxygen uptake rate
Hardness:
Not reported.
Test temperature:
• Temperature in the pre-test was in a range of 20.7 – 24.2 °C instead of 20 ± 2 °C.
• Temperature in the main study was in a range of 22.1 – 23.0 °C instead of 20 ± 2 °C.
These deviations were stated as uncritical, as normal respiration activity of the control could be observed
pH:
- Control: at start 7.7 - 7.8 and at the end 7.7.
- Test item: at start 7.2 and at end 7.6
Dissolved oxygen:
Not applicable.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal concentrations: blank, 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: At first, in the pre test, glass beakers were used as test vessels. Because of extreme foam formation, aeration was stopped and the test solutions were transferred into Schott flasks.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: fill volume of 2L, no other details reported
- Aeration: no, but vigorous stirring
- No. of vessels per concentration (replicates):
* one replicate/treatment (positive control all experiments, test item pre-test lower concentrations)
* five replicates/treatment (test item pre-test highest concentration and main study all concentrations)
- No. of vessels per control (replicates): two replicates before and two after measuring positive control and test item, respectively

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dionised water
- Culture medium different from test medium: no
- The activated sludge was aerated until usage in the test and fed daily with 50 ml /L synthetic sewage feed. The dry matter was determined as 2.68 g suspended solids/L, giving a concentration of 1.34 g suspended solids/L in the test

OTHER TEST CONDITIONS
- Adjustment of pH: yes, prior to start of exposure
- Photoperiod: not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate, measured after 3 hours in narrow neck glass bottles with flat bottoms

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes, pre-test was performed
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
970 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 1900-4600 mg/L for on test material
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
As inhibition > 20% was observed, a study plan amendment was issued and a main study was performed. The inhibition which was caused by the test item C12-C18 AAAO did not rise above 68% at the highest concentration of 8962 mg/L corresponding to 3200 mg/L active ingredient. The correlation in the inhibition values in the higher concentrated treatments was good. But in the lower treatments, especially in treatment 279 mg/L, the inhibition values were partly unexpectedly high and scattering. It might be possible that in spite of the closed system, foam formation in treatment 894 mg/L results in a loss of test item by adsorption on the inner surface of the glass flasks. In the lower concentrated treatment 280 mg/L, foam formation was only marginal.
The extrapolated value for the EC10 lies below the statistically determined NOEC. Based on statistical evaluation, the mean inhibition in the lowest concentrated treatment 89 mg/L can be considered as not significant.
The result of the test can be considered valid.
Results with reference substance (positive control):
- 3-h EC50 = 11 mg/L, which is within the normal range of 2 to 25 mg/L.
- The results with the reference substance indicate that the sludge in this test is responding normally and confirm the viability of the sludge organisms.
Reported statistics and error estimates:
t-Test was used to determine NOEC. For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-logarithmic diagram (see chapter 7.6.5, page 23). EC10 and EC50 were determined from the x values of the regression line at y = 10% and y = 50%.
Validity criteria fulfilled:
yes
Conclusions:
Two valid experiments were performed.
In the pre-test, the test item was tested using four concentrations ranging from 2793.3 to 2.8 mg/L test item, corresponding to 1000 to 1 mg/L active ingredient. Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.68 g suspended solids/L, giving a concentration of 1.34 g suspended solids/L in the test.
3.5-Dichloro-phenole was used as positive control. Four concentrations were tested; an EC50 of 8.8 mg/L (95% confidence interval: < 22 mg/L) was determined, which lies within the range of 2 – 25 mg/L.
As inhibition > 20% was observed, a study plan amendment was issued and a main study was performed.
Because of extreme foam formation and loss of test item in the pre-test, the main study was performed in a closed system without aeration. In order to ensure sufficient oxygen supply, a large headspace was provided and the mixtures were stirred vigorously.
The main study was performed using five concentrations, ranging from 8939 to 89 mg/L test item corresponding to 3200 to 32 mg/L active ingredient. The dry matter of the activated sludge was determined as 2.28 g suspended solids/L, giving a concentration of 1.14 g suspended solids/L in the test. The EC50 of the positive control was determined as 11 mg/L (95% confidence interval: 6.9 – 16 mg/L), which lies within the range of 2 –25 mg/L.
The following results for the test item C12-C18 AAAO were determined:
3h NOEC = 89 mg/L
3h EC10 = 46 mg/L test item (corresponding to 16 mg/L active ingredient)
3h EC50 = 2700 mg/L (corresponding to 970 mg/L active ingredient)
The results of this study can be considered reliable.

Description of key information

An activated sludge inhibition test (Muckle, 2012) carried out according to OECD guideline 209 with activated sludge resulted in a 3h-EC50 of 970 mg a.i./L and a 3h-NOEC of 32 mg a.i./L. The study is given a Klimisch score of 1, was conducted under GLP and is considered as the key study for endpoint coverage.

Key value for chemical safety assessment

EC50 for microorganisms:
970 mg/L
EC10 or NOEC for microorganisms:
32 mg/L

Additional information