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Ecotoxicological information

Toxicity to microorganisms

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toxicity to microorganisms
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1 Mar - 2 Mar 1988
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
other: Bewertung wassergefährdender Stoffe, III Bestimmung der akuten Bakterientoxizität, Ad-hoc-Arbeitsgruppe 1 (Obmann Dr. Niemitz), LTwS, Nr. 10, September 1979 (Assessment of hazardous substances in water, Determination of the acute toxicity in Bacteria)
GLP compliance:

Test material

Test material form:
other: liquid
Details on test material:
- Name of test material: Glycerol trioleate
- Physical state: liquid
- Analytical purity: ca. 100% ester
- Lot/batch No.: 108
- Storage condition of test material: at ambient temperature in the dark, dry conditions

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A weighed amount of test substance was dissolved in a minimum volume of ethanol absolute. A volume was brought into a test flask and after complete evaporation of the organic solvent, a solution of 1 mg/L in sterile Milli-Q water was prepared. In the same way an oversaturated test substance solution of 2 mg/L test substance was prepared. Both solutions were diluted 1.25 in sterile Milli-Q water and mixed for 10 minutes on a magnetic stirrer. Dilution series were prepared as follows: the first flask of each series contained 160 mL of test substance solution, from this task the subsequent dilution steps at a constant dilution ratio were prepared by consistently mixing 80 mL of preliminary test substance and 80 mL sterile Milli-Q water. Consequently, each flask contained 80 mL of test substance solution at the start. Each flask of the three inoculated dilution series was made up to a final volume of 100 mL by adding 5 mL of a stock solution I, 1.5 mL of a stock solution II and 10 mL of the prepared bacterial suspension from the
preliminary culture having a known adjusted extinction value. The flasks of the dilution series that were
not inoculated (series t), were made up to 100 mL by adding 5 mL of stock solution I, 5 mL of stock solution II and 10 ml of saline. Control culture flasks (series B) with 80 mL sterile Milli-Q water, 5 mL stock solution I, 5 mL stock solution II and 10 mL prepared bacterial solution were also included
- Chemical name of vehicle (organic solvent): ethanol

Test organisms

Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Source: stock cultures of Pseudomonas putida were obtained from RIVM, Bilthoven, The Netherlands
- Method of cultivation: cultures were kept on the nutrient used for stock and preliminary cultures in agar plant tubes. New stock cultures were started at intervals of 1 week. The inoculated stock cultures were incubated at 25 °C
- Preparation of inoculum for exposure: using aseptic techniques, small amounts of bacteria front a 7-day old stock culture of Pseudomonas putida were inoculated in fluid nutrient medium in Erlenmeyer flasks. The preliminary cultures were incubated at 25°C for 18±2 hours. Subsequently the extinction of the monochromatic radiation at 436 nm for a 10 mm layer of the bacterial suspension was determined by photoelectric measurement. On the basis of the values measured, the final turbidity value of the bacterial suspension was adjusted by means of sterile saline in such a way that the extinction value for a sample that has been subject to onward dilution 1 + 9 with saline corresponded with the extinction value of a Formazin standard suspension TU/F/436nm= 10. These preliminary cultures of bacterial suspensions were used for inoculation of the test flasks

Study design

Test type:
Water media type:
Total exposure duration:
18 h

Test conditions

Test temperature:
7.0 ± 1 (adjusted pH for test solutions)
Nominal and measured concentrations:
Nominal: 0.0004, 0.001, 0.002, 0.003, 0.006, 0.013, 0.025, 0.050, 0.1, 0.2, 0.4, 0.8 mg/L
Additional oversaturated solution: 1.6 mg/L
Details on test conditions:
- Test vessel: 300 mL Erlenmeyer flasks stoppered with aluminium caps
- No. of vessels per concentration (replicates): 12
- No. of vessels per control (replicates): 10

- Source/preparation of dilution water: Milli-Q water

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : extinction of the monochromatic radiation at 436 nm in a 10 mm layer

Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
18 h
Dose descriptor:
Effect conc.:
> 0.8 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: cell multiplication inhibition
18 h
Dose descriptor:
other: EC03
Effect conc.:
> 0.8 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: cell multiplication inhibition
Remarks on result:
other: Toxicity Threshold
Details on results:
No inhibitory effects on cell multiplication of Pseudomonas putida could be observed up to the highest concentration tested
Results with reference substance (positive control):
Toxicity Threshold value 14083.58 mg/L

Applicant's summary and conclusion