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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
experimental period: 2009-03-25 - 2009-03-26
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
Principles of method if other than guideline:
Test was conducted according to OECD Guidelines for Testing Chemicals (1984) No. 209, method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines (OPPTS 850.6800)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test material preparation: in order to aid weighing, the test material was warmed to ca. 70 °C prior to use. Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately dispersed in ca. 250 mL of water and subjected to ultrasonification for ca. 15 minutes followed by prolonged mixing for 24 hours in order to maximise the dissolved test material concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Preparation of inoculum: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of ca. 21°C and was used on the day of collection. The pH of the sample was 7.4. The suspended solids concentration was equal to 4.2 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None.
Hardness:
Test water: ca. 140 mg/L as CaCO3
Test temperature:
21 ± 1°C
pH:
control: 7.5-7.6 (0 h), 7.9-8.0 (3 h)
test material (10-1000 mg/L): 7.6 (0 h), 7.9-8.0 (3 h)
reference material (3,5-dichlorophenol, 3.2-32 mg/L): 7.5 (0 h), 8.1-8.2 (3 h)
Dissolved oxygen:
Oxygen measurements after 3 hour contact period:
Controls: 5.7-6.2 mg O2/L (initial reading), 2.6-3.0 mg O2/L (final reading)
Test material solutions: 5.4-6.5 mg O2/L (initial reading), 2.4-3.0 mg O2/L (final reading)
Reference material solutions: 6.6-7.7 mg O2/L (initial reading), 3.7-7.0 mg O2/L (final reading)

For detailed values see below (remarks on results including tables and figures).
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/L (nominal concentrations, test material)
Details on test conditions:
Test water: laboratory tap water dechlorinated by passage through an activated carbon filter and partly softened giving water with a total hardness of ca. 140 mg/L as CaCO3.

Composition of synthetic sewage (respiratory substrate): according to OECD Guideline No. 209.

Activated sewage sludge sample: a mixed population of activated sewage sludge micro-organisms was obtained on the day of use from the aeration stage of a sewage treatment plant which treats pre-dominantly domestic sewage. The activated sewage sludge sample was maintained on continous aeration in the laboratory at a temperature of ca. 21°C. The pH of the sample was 7.4. The suspended solids concentration was equal to 4.2 g/L prior to use.

Test material preparation: in order to aid weighing, the test material was warmed to ca. 70°C prior to use. Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately dispersed in ca. 250 ml of water and subjected to ultrasonification for ca. 15 minutes followed by prolonged mixing for 24 hours in order to maximise the dissolved test material concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L.

The control group was maintained under identical conditions but not exposed to the test material.

For the purpose of the test, 3,5-dichlorophenol was used as a reference material. The reference solutions were prepared by dissolving the reference material directly in water with the aid of ultrasonification for ca. 15 minutes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
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:
Variation in respiration rates of control replicates R1 and R2 after 3 hours contact time was (+-)4%.
Results with reference substance (positive control):
Under the same conditions, the reference material (i.e. 3,5-dichlorophenol) gave a 3-Hour EC50 value of 8.7 mg/L (95% confidence limits: 7.0-11 mg/L). Further results: EC20: 2.6, EC80: 29, NOEC: 2.2 mg/L.
Reported statistics and error estimates:
Results for the reference material: confidence levels according to Litchfield and Wilcoxon (1949). The No Observed Effect Concentration (NOEC) was taken as being the EC15 value.

Validity criteria fulfilled:
yes
Remarks:
control respiration rates and reference material EC50
Conclusions:
The NOEC after 3 hours exposure was 1000 mg/L (i.e. the highest concentration tested). Under the same conditions, the reference material (i.e. 3,5-dichlorophenol) gave a 3-hour EC50 value of 8.7 mg/L.
Executive summary:

The effect of the test material on the respiration of activated sewage sludge was tested under GLP according to OECD TG 209. Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 3 hours at a temperature of ca. 21 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material (3,5-dichlorophenol).

The 3-hour EC50 value for the inhibition of the respiration rate of sewage sludge micro-organisms was >1000 mg/L. The NOEC after 3 hours exposure was 1000 mg/L. Under the same conditions, the reference material (i.e. 3,5-dichlorophenol) gave a 3-rour EC50 value of 8.7 mg/L.

Description of key information

3-hour NOEC = 1000 mg/L (activated sewage sludge, OECD TG 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The effect of the test material on the respiration of activated sewage sludge was tested under GLP according to OECD TG 209. Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 3 hours at a temperature of ca. 21 °C with the addition of a synthetic sewage as carbon source. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material (3,5-dichlorophenol).

The 3-hour EC50 value for the inhibition of the respiration rate of sewage sludge micro-organisms was >1000 mg/L. The NOEC after 3 hours exposure was 1000 mg/L. Under the same conditions, the reference material gave a 3-rour EC50 value of 8.7 mg/L, demonstrating the suitability of the sewage sludge and test system.