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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 1, 2010 to March 12, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This exception with regard to GLP is considered not to affect the purpose or integrity of the study.
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For test concentrations of 10, 32, 100, 320 and 1000 mg/L, test item was dispersed in approximately 250 mL of water and subjected to ultrasonication for approximately 15 minutes followed by prolonged mixing for 24 h in order to maximise the dissolved test item concentration. For test concentrations of 1.0 and 3.2 mg/L, test item was dissolved in acetone followed by dispersion in test medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone for test concentrations 1.0 and 3.2 mg/L (to avoid very small amounts of test item being weighed out); acetone was also added to other test concentrations vessels in order to maintain consistency.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): After 30 min. and 3 h contact time, the test concentrations of 1.0, 3.2, 10 and 32 mg/L contained a dark brown dispersion with no undissolved test item visible and the test concentrations of 100, 320 and 1000 mg/L contained a dark brown dispersion with globules of test item on the surface and dispersed throughout.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK (aeration stage)
- Laboratory culture: 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.
- Suspended solids concentration: 4.1 g/L prior to use (determined by filtering, washing and drying)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 d
Post exposure observation period:
5-10 min
Hardness:
140 mg/L as CaCO3
Test temperature:
21±1 °C
pH:
At start of exposure (0 h): 7.4-7.6
At end of exposure (3 h): 7.7-8.2
Dissolved oxygen:
1.9-8.3 mg O2/L
Salinity:
No data
Nominal and measured concentrations:
1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel details: 500 mL conical flask (containing 16 mL of synthetic sewage, 200 mL of inoculum, test item and water upto 500 mL)
- Aeration: Aeration with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 L/min.
- No. of vessels per concentration (replicates): One
- No. of vessels per control (replicates): Two
- No. of vessels per vehicle control (replicates): One

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water dechlorinated and partly softened
- Synthetic sewage composition:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCI
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in 1 L of water with the aid of ultrasonication

OTHER TEST CONDITIONS
- Light intensity: Normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The rate of respiration was measured over the linear portion of the oxygen consumption trace (where possible
between approximately 6.5 and 2.5 mg O2/L). In the case of low oxygen consumption, the rate was determined over an approximate 10 min. period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 3.2
- Range finding study: Test concentration: 10, 32, 100, 320 and 1000 mg/L;
Results used to determine the conditions for the definitive study: Significant inhibition was observed at the 10 mg/L but not at the 32 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3.2, 10 and 32 mg/L)
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
ca. 110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: -
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 140 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: -
Key result
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: -
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
ca. 110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: EC15 value was considered as NOEC
Results with reference substance (positive control):
The EC50 value for 3,5-dichlorophenol (8.4 mg/L; C.I.: 6.6-11 mg/L) lie within the acceptable range (5 to 30 mg/L) thus fulfilling the validity criteria.
Reported statistics and error estimates:
95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon.

Control results: Variation in respiration rates of two controls was ± 8% (i.e. < 15%) thus fulfilling the validity criteria.

Other: In some instances, the initial and final dissolved oxygen concentrations were below recommended values (6.5 and 2.5 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:
Under the test conditions, 3h EC50 of test material was found to be 140 mg/L (nominal) in the activated sludge respiration inhibition test. The No Observed Effect Concentration (NOEC) after 3 h exposure was 110 mg/L (nominal).
Executive summary:

A study was conducted to assess the effect of the test substance on the respiration of activated sewage sludge according to OECD Guideline 209, Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Guideline OPPTS 850.6800. Activated sewage sludge was exposed to the test substance at concentrations of 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L for a period of 3 h at a temperature of 21±1 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 h contact time and compared to data for the control and a reference substance, 3,5-dichlorophenol. Under the test conditions, 3h EC50 of test substance was found to be 140 mg/L (nominal) in the activated sludge respiration inhibition test. The NOEC after 3 h exposure was 110 mg/L (nominal) (Clarke, 2010).

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
140 mg/L
EC10 or NOEC for microorganisms:
110 mg/L

Additional information

A study was conducted to assess the effect of the test substance on the respiration of activated sewage sludge according to OECD Guideline 209, Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Guideline OPPTS 850.6800. Activated sewage sludge was exposed to the test substance at concentrations of 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L for a period of 3 h at a temperature of 21±1 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 h contact time and compared to data for the control and a reference substance, 3,5-dichlorophenol. Under the test conditions, 3h EC50 of test substance was found to be 140 mg/L (nominal) in the activated sludge respiration inhibition test. The NOEC after 3 h exposure was 110 mg/L (nominal) (Clarke, 2010).