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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
Study period:
March 2 - March 9, 2012
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:
but the study integrity was not adversely affected by the deviations. The deviations are mentioned in their corresponding sections.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(2008)
Deviations:
yes
Remarks:
but the study integrity was not adversely affected by the deviations. The deviations are mentioned in their corresponding sections.
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
(2007)
Deviations:
yes
Remarks:
but the study integrity was not adversely affected by the deviations. The deviations are mentioned in their corresponding sections.
GLP compliance:
yes
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
- The test concentrations were expressed as solid content
- A stock solution of 10 g/l expressed as solid content was prepared by adding 25.84 g test substance to 1000 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Magnetic stirring for 10 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear colourless, slightly foaming, stock solution corresponding to the test concentration were then added to the test media.
- Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of the sludge: The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (2.74 g/l of sludge, as used for the test). The pH was 7.8 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration:
1.5 g/L of suspended solids in the final text mixture (combined limit/range-finding test)
1.4 g/L of suspended solids in the final text mixture (final test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
with aeration and stirring
Post exposure observation period:
After the 3-hour contact time the oxygen consumption was recorded for a period of 8-11 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations and controls.
Test temperature:
The temperatures measured in all vessels ranged between 18.0 and 22.0°C, except in 1 vessel used in the final test, T5a (1000 mg/l, vessel A), in which the temperature was 22.5°C. As a temperature deviation of 0.5°C was considered to have no effect on the final test results, the study integrity was not adversely affected by this deviation.
pH:
- Combined limit/range-finding test: 7.6-8.7
- Final test: 7.3-8.4
Dissolved oxygen:
- Combined limit/range-finding test: 6.9-8.7 mg O2/L
- Final test: the oxygen concentration at the start of measurement was above 5 mg O2/L in the test bottles, corresponding to more than 60% saturation (6.4-9.1 mg O2/L), except in 2 test bottles at 560 mg/L (3.7 and 4.7 mg O2/L) and in 1 bottle at 1000 mg/L (4.7 mg O2/L). As sufficient information was obtained to calculate the respiration rates, the study integrity was not adversely affected by these deviations.
Details on test conditions:
TEST CONCENTRATIONS
- Combined limit/range-finding test: 20, 200 and 2000 mg/l expressed as solid content
- Final test: 100, 180, 320, 560 and 1000 mg/l expressed as solid content

Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol at 5.0, 12 and 30 mg/l (1 replicate) in the combined limit/range-finding test as well as in the final test
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
560 mg/L
Nominal / measured:
nominal
Conc. based on:
other: expressed as solid content
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:
other: expressed as solid content
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Combined limit/range-finding test: No inhibition of the respiration rate was observed at 20 mg/l and 200 mg/l expressed as solid content. At 2000 mg/l expressed as solid content the average inhibition of the respiration was 70%. There was no significant oxygen uptake from abiotic processes and the results at 2000 mg/l expressed as solid content with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. Therefore, N-allylthiourea was not used in the final test.
- Final test: No statistically and biologically significant inhibition of the respiration rate of the sludge was recorded at 560 mg/L expressed as solid content (Bonferroni t-Test: α=0.05 Toxstat). At 1000 mg/l expressed as solid content 34% inhibition (mean value) of the respiration rate was observed.
Results with reference substance (positive control):
EC50 of 3,5-dichlorophenol:
- Combined limit/range-finding test: 5.6 mg/L
- Final test: 6.8 mg/L
Validity criteria fulfilled:
yes
Conclusions:
In accordance with OECD 209 (2010) and according to GLP principles the influence of the substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The substance was not toxic to waste water (activated sludge) bacteria at a concentration of 560 mg/L (NOEC, based on solid content).
Executive summary:

In accordance with OECD 209 the influence of the substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours. In a combined limit/range-finding test concentrations of 20, 200 and 2000 mg/l based on solid content were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at the concentrations 20 and 200 mg/L. At 2000 mg/l, the average inhibition of the respiration was 70%. Therefore, a final test was performed at 100, 180, 320, 560 and 1000 mg/l based on solid content. No statistically and biologically significant inhibition of the respiration rate of the sludge was recorded in this test at 560 mg/L (NOEC, based on solid content). At 1000 mg/L, the average inhibition of the respiration was 34%. The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The validity criteria were all fulfilled.

Description of key information

In accordance with OECD 209 (2010) and according to GLP principles the influence of the substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The substance was not toxic to waste water (activated sludge) bacteria at a concentration of 560 mg/L (NOEC, based on solid content).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
560 mg/L

Additional information

In accordance with OECD 209 the influence of the substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours. In a combined limit/range-finding test concentrations of 20, 200 and 2000 mg/l based on solid content were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at the concentrations 20 and 200 mg/L. At 2000 mg/l, the average inhibition of the respiration was 70%. Therefore, a final test was performed at 100, 180, 320, 560 and 1000 mg/l based on solid content.No statistically and biologically significant inhibition of the respiration rate of the sludge was recorded in this test at 560 mg/L (NOEC, based on solid content). At 1000 mg/L, the average inhibition of the respiration was 34%. The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The validity criteria were all fulfilled.