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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 May - 5 June 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in GLP compliance and in accordance with several internationally established guidelines (OECD, EEC, EPA guidelines, see below).

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
according to guideline
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
C33 H30 N4 O2 x HCl
4’-(2-Propyl-4-methyl-6-(1-methylbenzimidazolyl-2-yl)-benzimidazol-1-ylmethyl)biphenyl-2-carbonsäure Hydrochlorid
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): BIBR 277 CL
- Molecular formula (if other than submission substance): C33H30N4O2 * HCl
- Molecular weight (if other than submission substance): 514.6 + 36.5 g/mol
- Structural formula attached as image file (if other than submission substance): see Reference substance
- Physical state: solid, light brownish
- Analytical purity: 99.7 %
- Purity test date: 26 March 2007
- Lot/batch No.: 70
- Expiration date of the lot/batch: 8 March 2008
- Storage condition of test material: At room temperature, dark and dry

Sampling and analysis

Analytical monitoring:
not specified

Test solutions

Details on test solutions:
Just before the start of the test defined amounts of the test item stock solution were directly filled into each test flask. The test concentrations (10, 31, 98, 313 and 1000 mg/l) were chosen to permit the determination of the EC50. Concentrations in excess of nominal 1000 mg test item /L were not tested

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and a ratio of wet sludge to its dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.47. The activated sludge was used directly after conditioning.

Study design

Test type:
Water media type:
not specified
Limit test:
Total exposure duration:
3 d

Test conditions

Test temperature:
19.2 to 20.9°C during incubation and
19.1 to 20.8°C during oxygen measurement
from pH 5.83 to 7.22
Dissolved oxygen:
from 6.7 mg O2/L to 8.0 mg O2/L
Details on test conditions:
One test solution with a final volume of 330 ml was tested per treatment in a glass flask. 10.56 ml synthetic sewage and an adequate amount of the stock solution of the test item or an adequate volue of the stock solution of the reference item was filled up with deionised water to 198 ml before the start of the test. At the start of the test 132 ml activated sludge inoculum with a sludge concentration of 4 g/L (dry weight) was added, first to first control (C1), then in time intervals of 12 minutes (an optional interal) to the test solutions of the reference item and the test item and finally to a second control (C2).
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
3 h
Dose descriptor:
Effect conc.:
10 mg/L
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? Yes; the 3 hour EC50 of the reference item 3,5-dichlorophenol was determined to be 8.54 mg/L and thus in the range of 5 to 30 mg/L for the used activated sludge batch.

Any other information on results incl. tables

Influence of test item on oxygen consumption of activated sludge

 Flask No.     Test group     Concentration (mg/L)     Oxygen consumption (mg/L)     Inhibition (%) -    pH values     Oxygen concentration (mg O2/ L)   
 start*  end*  start*  end*
 1  Control  -  0.570  -  7.22  6.95  7.0  7.2
 10  Control  -  0.560  -  7.12  7.05  7.1  7.4
   Mean  -  0.565  -  -  -  -  -
   Deviation %  -  1.8  -  -  -  -  -
 5  Test item  10  0.560  0.9  7.04  6.93  8.0  7.2
 6  Test item  31  0.520  8.0  6.97  6.90  7.1  7.4
 7  Test item  98  0.460  18.6  6.82  6.81  6.9  6.7
 8  Test item  313  0.380  32.7  6.40  6.77  7.2  6.9
 9  Test item  1000  0.360  36.3  5.83  6.55  7.7  7.3

*start and end of 3 hour aeration

Applicant's summary and conclusion

Validity criteria fulfilled:
A activated sludge respiration inhibition of EC50 > 1000 mg/L was determined in a reliable study conducted according to an appropriate OECD test method.