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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
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 (Carbon and Ammonium Oxidation))
Deviations:
yes
Remarks:
In some test vessels temperatures near 17°C were observed, however the slightly lower temperature has no impact on the respiration rate of the activated sludge
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
In some test vessels temperatures near 17°C were observed, however the slightly lower temperature has no impact on the respiration rate of the activated sludge
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
yes
Remarks:
In some test vessels temperatures near 17°C were observed, however the slightly lower temperature has no impact on the respiration rate of the activated sludge
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was directly dosed into each test flask
- Controls: Tap water, synthetic sewage and inoculum
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: municipal sewage treatment plant (Bensheim, Germany)
- Preparation of inoculum for exposure: The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (30 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at room temperature overnight.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in tap water to yield a concentration equivalent to 3.0 g/L on dry weight basis. This level gave a concentration of 1.2 g/L suspended solids in the test medium.
Total exposure duration:
3 h
Test temperature:
17 to 21°C
pH:
7.5 to 7.7 at the start and 8.3 to 8.5 at the end of the incubation period in all test concentrations and controls
Nominal and measured concentrations:
Nominal: 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume
- Aeration: With compressed air (1.026 litre per minute)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): concentrations of 3.2, 10 and 32 mg/L (one replicate each)
- Sludge concentration (weight of dry solids per volume): 1.2 g/L suspended solids in the test medium

Preparation of Test Flasks:
Every solution with a final volume of 500 mL was tested per treatment in a glass flask. Samples were prepared in 15-minute intervals. 16 mL synthetic sewage and an adequate amount of the test item or an adequate volume of the reference item stock solution were filled up with tap water to 300 mL before the start of the test. At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 3.0 g/L suspended solids was added, first to control 1 and 2, then in time intervals of 15 minutes to the reference item, to control 3 and 4, then to the test item and finally to additional controls. During 3 hour aeration period the flasks were stirred on a magnetic stirrer to maintain sludge flocs in suspension.

EFFECT PARAMETERS MEASURED: For the measurement of the respiration rate a well mixed sample of test medium from each flask was poured into a Karlsruher flask after exactly 3 hours incubation time and was not further aerated during measurement. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2/L/minute) was determined over periods up to 10 minutes in the range between approximately 9.4 – 2.3 mg O2/L.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The test substance had no effects on the respiration of activated sludge microorganisms. The mean respiration rate of activated sludge treated with the test item at a concentration of 100 mg/L differed from the respiration rate in the control by less than 10%. In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited by 3.5%.
Results with reference substance (positive control):
- Relevant effect levels: 3-hour EC50: 2.6 mg/L.
- Other: In comparison to the controls the respiration rate of the activated sludge was moderately inhibited by 54.2% at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg/L, the respiration rate was inhibited by 68.8% and 85.4%, respectively.
Reported statistics and error estimates:
The EC50 including the 95% confidence limits of the reference item was calculated by Probit analysis. For the determination of the NOEC of the test item the Student t-test for homogenous variances with Bonferroni-Holm Adjustment (α=0.05) was used.
Validity criteria fulfilled:
yes
Conclusions:
The test substance at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge.
3h NOEC: >100 mg/L
Executive summary:

The influence of the test substance on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions following OECD guideline 209. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of one concentration of the test item (100 mg) after an incubation period of 3 hours.

This limit test was performed in compliance with the test guidelines to demonstrate that the test substance had no toxic effect on activated sludge up to at least this concentration. The test substance had no effects on the respiration of activated sludge microorganisms. The mean respiration rate of activated sludge treated with the test item at a concentration of 100 mg/L differed from the respiration rate in the control by less than 10%. In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited by 3.5%. Therefore, the test substance at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge. The NOEC is established to be above 100 mg/L (Student t-test with Bonferroni-Holm-adjustment). The 3-hour EC50 for the positive control 3,5-dichlorophenol, which was tested in the same way as the test item, was found to be 2.6 mg/L and is within the range of 2 - 25 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge.

The test substance at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge. The NOEC is established to be above 100 mg/L (Student t-test with Bonferroni-Holm-adjustment).

Description of key information

3-hour NOEC (activated sludge, domestic): >100 mg/L; OECD 209; Reliability = 1

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

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