Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 2007
Reliability:
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, see below).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
480-420-2
EC Name:
-
Cas Number:
144702-27-2
Molecular formula:
C33 H29 N5
IUPAC Name:
4'-{[4-methyl-6-(1-methyl-1H-1,3-benzodiazol-2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl}-[1,1'-biphenyl]-2-carbonitrile
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): BIBR 277 Nitril
- Physical state: Solid
- Analytical purity: 99.7%
- Purity test date: 20 March 2007
- Lot/batch No.: 80A
- Expiration date of the lot/batch: 08 March 2008
- Stability under test conditions: yes
- Storage condition of test material: At room temperature, dark and dry

Sampling and analysis

Analytical monitoring:
not specified

Test solutions

Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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.

- Controls: Two controls (deionised water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.

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:
static
Water media type:
not specified
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Details on test conditions:
Preparation of Test Flasks:
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 volume 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 interval) to the test solutions of the reference item and the test item and finally to a second control (C2).

Measurement of Respiration Rate:
For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after exactly 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with a stirring O2 electrode and was recorded for about ten minutes. The oxygen consumption (in mg O2 L-1 minute1) was determined from the most linear part of the respiration curve.
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorphenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
3 h
Dose descriptor:
EC100
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Results with reference substance (positive control):
3-hour EC50 of Reference Item: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 8.54 mg/L with 95 % confidence limits of 6.53 to 11.17 mg/L.

Any other information on results incl. tables

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited, but slightly stimulated at the nominal concentrations of 31, 313 and 1000 mg/L (the respiration rates were: -2.7, -4.4 and -0.9, respectively). As well at the concentrations of 10 and 98 mg/L no significant inhibition was observed, 2.7 % being based on biological variation. At the nominal concentration of 1000 mg/L, the respiration rate was not inhibited, but slightly stimulated (-0.9 %).

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 values were higher than 1000 mg/L.

Based on measured inhibition rates the 3-hour EC20, EC50 and EC80 and their 95 %-confidence limits were not calculated statistically because of the lack of sufficient effects.

Based on the results of the performed experiment the NOEC was determined to be 1000 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, the following parameters for BIBR 277 Nitril with regard to Activated Sludge Respiration Inhibition were determined:
3h EC20 > 1000mg/L; 3h EC50 > 1000mg/L; 3h EC100 > 1000mg/L; 3h NOEC = 1000mg/L.