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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 1992 to 11 December 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study generally followed guideline, and met validity criteria. However, due to the insolubility of the test substance in the test medium, a constant factor between the test concentrations could not be achieved. It was not possible to determine an absolute EC50 given that no inhibition was observed over the concentrations tested.
Qualifier:
according to guideline
Guideline:
other: 87/302/EEC page 118-122 Activated sludge respiration inhibition test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
No samples were taken for analysis of the test substance concentration.
Vehicle:
no
Details on test solutions:
TEST CONCENTRATIONS
21.0, 9.1, 3.3, 1.4, 0.8 and 0.3 mg of the test substance were weighed and added to the test medium. The volume was adjusted to 200 ml with water and aerated for 3 hours. This produced test solutions of nominal concentrations of 105.0, 45.5, 16.5, 7.0, 4.0 and 1.5 mg/l.

The sludge concentration in the test bottles was 1.60 g/l (dry weight) .

A reference compound (3,5-dichlorophenol) was also tested at 3 concentrations (32.0, 10.0, and 3.2 mg/L).
Test organisms (species):
activated sludge
Details on inoculum:
Activated sludge collected from the sewage treatment plant of CH-4153 Reinach on 28/09/92. The pH after collection was 7.0. The preparation was carried out according to the method described in the guideline. The sludge was separated from the aqueous layer by settling instead of centrifugation. The pH of the sludge before use was 7.8
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
none
Hardness:
Not stated
Test temperature:
Temperature was maintained at 20 ± 2 ºC.
pH:
pH was measured in blank (2 samples), reference standard (three samples) and test concentrations (six samples). The pH in blank number 1 was measured as 7.8 and in all other sameples was measured as 8.3.
Dissolved oxygen:
Oxygen consumption per hour in mg/liter was measured with an ORION - Electrode Type 97-08, ORION Microprocessor Ionalizer 901 and plotted on a recorder.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations of test subsatnce were 105.0, 45.5, 16.5, 7.0, 4.0 and 1.5 mg/l of the test substance. Actual concentrations were not measured.
Details on test conditions:
DESIGN AND PROCEDURE
Vessels: 250 ml BOD flasks with gas inlet
Water: Dechlorinated drinking water
Temperature: 20 ± 2°C
Duration: 3 hours
Nutrient solution: 16g Peptone, 11g Meat extract, 3.0g Urea, 0.7g NaCl, 0.4g CaCl2 • 2H2O, 0.2g MgSO4 • 7 H2O, 2.8g K2HPO4 are dissolved and made up to one liter with dechlorinated drinking water.

Deviations from guideline: Instead of a centrifuged sludge a settled sludge was used. Due to the poor solubility of the test substance at test concentrations, no stock solution was prepared: The test substance was given directly into the medium. Therefore, a constant factor between the test concentrations could not been achieved.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
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:
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:
other: EC80
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
The calculated EC50 (3 h) for the reference substance, based on nominal concentrations, was 12.6 mg/l.
Reported statistics and error estimates:
The inhibitions were calculated on the basis of the measured time depend oxygen consumption of a Blank and Test solution. The Results ( ECxx Values ) were calculated with linear regression.

Table 5. Rates of inhibition (%) for control and test solutions.

Sample Conc (mg/l) Consumption rate (mg/l/h) Inhibition (%)
Blank 1 0.0 52.8 3
Blank 2 0.0 56.3 -3
Reference 1 32.0 11.0 80
Reference 2 10.0 31.8 42
Reference 3 3.2 50.4 8
Test conc. 1 105.0 50.8 7
Test conc. 2 45.5 53.9 1
Test conc. 3 16.5 55.7 -2
Test conc. 4 7.0 54.3 0
Test conc. 5 4.0 55.5 -2
Test conc. 6 1.5 61.1 -12
Validity criteria fulfilled:
yes
Remarks:
Study met the "Quality Criteria" given in the guideline.
Conclusions:
Study met the validity (quality) criteria of the guideline, though it deviated from the method in some minor ways (used settling instead of centrifugation for sludge, and test concentrations were not spaced by an equal factor). Because no significant inhibition of oxygen consumption was found over the test concentrations compared to the controls, resulting EC50 was >= 100 mg/L (the highest test concentration). Study provides no direct discussion of whether solubility prevented testing higher concentrations, though this is likely given that elsewhere in the report the poor solubility of the test compound is noted.
Executive summary:

Study: Determination of the inhibitory concentration of a chemical substance on the respiration of aerobic waste water bacteria

Test system: Activated sludge from a sewage treatment plant.

Duration:3 hours

Guideline: 87/302/EEC page 118-122 Activated sludge respiration inhibition test

Deviations: Instead of a centrifuged sludge a settled sludge was used. Due to the poor solubility of the test substance at test concentrations, no stock solution was prepared. The test substance was given directly into the medium. Therefore, a constant factor between the test concentrations could not been achieved.

Results: Calculated based on nominal concentrations:

EC50 (3h) = > 100 mg/l

EC20 (3h) = > 100 mg/l

EC80 (3h) = > 100 mg/l

Description of key information

No significant inhibition of oxygen consumption was found over the test concentrations compared to the controls, resulting in an EC50 of >= 100 mg/L (the highest test concentration).

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

MOTE inhibitory potential toward the respiration of aerobic waste water bacteria has been investigated by Baumann (1992). Activated sludge from a sewage treatment plant were exposed for 3 hours following the procedure described in guideline: 87/302/EEC page 118-122 Activated sludge respiration inhibition test.

Study generally followed guideline, and met validity criteria. However, due to the insolubility of the test substance in the test medium, a constant factor between the test concentrations could not be achieved. It was not possible to determine an absolute EC50 (result is >= 100 mg/L) given that no inhibition was observed over the concentrations tested

The results were calculated based on nominal concentrations:

EC50(3h) = > 100 mg/l

EC20(3h) = > 100 mg/l

EC80(3h) = > 100 mg/l