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Administrative data

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1992-07-20 until 1992-07-24
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1992

Materials and methods

Test guideline
Guideline:
other: O2 consumption test (Huels method)
Principles of method if other than guideline:
The "Oxygen Consumption Test" offers the possibility of measuring chronic toxicity effects in bacteria with difficult substances e.g. those which are sparingly soluble, coloured, emulsifying or dispersive, even slightly volatile products can be tested.
Principle of the test: Glass vessels (100 ml BSB-bottles, Loh) with known volume will be completely filled with culture solution, bacterial suspension and test substance in different concentrations (emulsified if insoluble in water), bubble-free sealed with glass stoppers and incubated for 5 to 5.5 hours at 25.5 ± 1°C on a shaking machine. Test vessels without bacterial suspension and vessels without test substance serve as controls. The differences between the oxygen content in the vessels at the beginning and the end of the incubation time is a measurement for the bacterial oxygen consumption.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenol, dodecyl-, branched
EC Number:
310-154-3
EC Name:
Phenol, dodecyl-, branched
Cas Number:
121158-58-5
Molecular formula:
C18H30O
IUPAC Name:
4-(3,4,5-trimethylheptyl)phenol
Constituent 2
Reference substance name:
Dodecylphenol, branched
IUPAC Name:
Dodecylphenol, branched
Details on test material:
- Name of test material (as cited in study report): Dodecylphenol T
- Substance type: technical product
- Physical state: liquid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): not mentioned
- Composition of test material, percentage of components: not mentioned
- Purity test date: not mentioned
- Lot/batch No.: continuously production; 3633/81416
- Date of production: 1992-05-07
- Stability under test conditions: 1 year
- Storage condition of test material: under nitrogen

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The test substance was directly pipetted into the test flasks.

Test organisms

Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: yes
- Method of cultivation: not described
- Preparation of inoculum for exposure: approx. 19 h incubation of liquid culture of test organism
- Pretreatment: 200 ml stock solution I and II each were filled in an sterile 5 l-flask, containing 3520 ml deionized water and a magnetic stirrer bar, the solution were stirred for 30 min. and aerated. Thereafter 80 ml pre-culture were added and additionally 5 min stirred and areated.
- Initial biomass concentration: approx. 10 x 10E5 bacteria/ml, adjusted with sterile NaCl solution by photometrical measurement at 436 nm (extinction 0.297 - 0.302)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
5.5 h
Remarks on exposure duration:
2 experiments performed 5 and 5.5 h
Post exposure observation period:
none

Test conditions

Hardness:
not mentioned
Test temperature:
24.9 - 26.2 °C
pH:
not mentioned
Dissolved oxygen:
not mentioned
Salinity:
see "Details on test conditions"
Nominal and measured concentrations:
100, 500, 1000 and 2000 µl/l (nominal concentrations)
Details on test conditions:
INOCULUM/TEST ORGANISM
- Species/strain: Pseudomonas putida, strain Berlin
- Source: Institut für Wasser-, Boden- und Lufthygiene des BGA, Berlin, Germany
- Method of cultivation: not described
- Preparation of inoculum: approx. 19 h incubation of liquid culture of test organism
- Pretreatment: 200 ml stock solution I and II each were filled in an sterile 5 l-flask, containing 3520 ml deionized water and a magnetic stirrer bar, the solution were stirred for 30 min. and aerated. Thereafter 80 ml pre-culture were added and additionally 5 min stirred and areated.
- Cell concentration of the inoculum: approx. 10 x 10E5 bacteria/ml, adjusted with sterile NaCl solution by photometrical measurement at 436 nm (extinction 0.297 - 0.302)
TEST SYSTEM
- Number of culture flasks:
a) 4 flasks per concentration with test substance, 2 of this flasks were used for determination of autooxidation of the test substance by adding of 1 ml HgCl2 solution, final concentration in the flasks approx. 3 mg/l
b) 4 flasks without test substance but with HgCl2 addition for determination of the oxygen content at the beginning of the test
c) 5 flasks without test substance, without HgCl2 for determination of the uninfluenced bacterial oxygen consumption. 1 flask of these was used to measure the oxygen contents after 4.5 h to determine the end of incubation. This value was not used for evaluation.
- Measuring equipment: oxygen electrode
- Termination of test: 1 ml HgCl2 solution (final concentration approx. 3 mg HgCl2/l) were added to each test vessel to stop the reaction.
TEST SUBSTANCE CONCENTRATIONS: 0.5, 1.0, 1.5, 2.0 ml/l
- Preparation of test substance solutions: The test substance was directly pipetted into the test flasks.
DURATION OF THE TEST: 5.0 resp. 5.5 h
ANALYTICAL PARAMETER: O2 consumption
TEST CONDITIONS
- Composition of solutions:
a) Trace element solution (autoclaved):
0.055 g/L Al2(SO4)3 x n H2O
0.028 g/L KJ
0.028 g/L KBr
0.055 g/L TiO2
0.028 g/L SnCl2 x 2 H2O
0.028 g/L LiCl
0.389 g/L MnCl2 x 4 H2O
0.614 g/L H3BO3
0.055 g/L ZnSO4 x 7 H2O
0.055 g/L CuSO4 x 5 H2O
0.059 g/L NiSO4 x 6 H2O
0.055 g/L Co(NO3)2 x 6 H2O
b) Stock solution I (sterilized):
20 g D(+)Glucose
4.24 g Na NO3
2.40 g K2HPO4
1.20 g KH2PO4
30 ml Trace element solution (a)
1000 ml deionized water
c) Stock solution II (steril filtrated):
0.200 g FeSO3 x 7 H2O
4.00 g MgSO4 x 7 H2O, resp. 3.70 g MgSO4 x 6 H2O
made up to 1 l with deionized water
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
5 h
Dose descriptor:
EC10
Effect conc.:
> 2 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
Inhibition of the bacterial O2 consumption (mean values):
0.5 ml/l: 2.6 %
1.0 ml/l: - 0.4 %
1.5 ml/l: - 0.9 %
2.0 ml/l: 0.8 %
The EC10 could not be established, because of a missing dose-response relationship.
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
No statitistics performed

Applicant's summary and conclusion

Validity criteria fulfilled:
yes