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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:
other: Guideline-conform GLP study without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: A mixed population of activated sewage sludge micro-organisms was obtained on 30 November 201 1 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Laboratory culture: no
- Method of cultivation: not applicable
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection. The pH of the sample was 7.3.
- Pretreatment: no
- Initial biomass concentration: 2.7 g/L prior to use
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C.
pH:
initial: 7.3 - 7-4
after 3 hours: 7.6 - 8.0
Nominal and measured concentrations:
Nominal: 1.0, 10, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: volumetric flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: not reported
- Aeration: aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute
- Renewal rate of test solution (frequency/flow rate): no renewal
- No. of vessels per concentration (replicates):
1 mg/L: 1
10 mg/L: 1
100 mg/L: 3
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the test was deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: normal laboratory lighting
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : O2 consumption rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: In the range finding study, a NOEC of 100 mg/L was observed. It was therefore not considered necessary to perform the main test and the range finding study is reported as the main experiment.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 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.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid?: yes
- Relevant effect levels: EC50=8.1 mg/L

Validity criterion

- The coefficient of variation of oxygen uptake in the control vessels was 3.7% and the specific respiration rate of the controls was 22.08 mg oxygen per gram dry weight of sludge per hour. The validation criteria has therefore been satisfied.

- The validation criteria for the reference item EC50 values were also satisfied.

- In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (7.0 mg O2/L and 2.0 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.
Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD TG 209.

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 1.0, 10 and 100 mg/L (3 replicates) for a period of 3 hours at a temperature of 20 ± 2 °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 item, 3,5-dichlorophenol.

The reference item gave a 3-Hour EC50 value of 8.1 mg/L, 95% confidence limits 6.6 - 10 mg/L, and confirmed the suitability of the inoculum.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD TG 209.

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 1.0, 10 and 100 mg/L (3 replicates) for a period of 3 hours at a temperature of 20 ± 2 °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 item, 3,5-dichlorophenol.

The reference item gave a 3-Hour EC50 value of 8.1 mg/L, 95% confidence limits 6.6 - 10 mg/L, and confirmed the suitability of the inoculum.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

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