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Toxicity to microorganisms

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toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 May 1998
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
The test substance concentration is not monitored throughout the test. No analytical purity of the test substance is given.
according to guideline
other: ISO 10712 Water quality: Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test) adopted in 1996
GLP compliance:
Analytical monitoring:
Details on test solutions:
- Method: A nominal 100 g/L stock solution is prepared by adding 4.9996 g of the test substance to 50 mL of acetone. Nominal 56, 32, 18 and 10 g/L stock solutions were made by diluting the nominal 100 g/L stock solution in acetone. The test solutions were prepared by adding appropriate amount of stock solution directly into the flasks.
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (final test solution(s) including control): 0.1% v/v
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: Pseudomonas putida, strain NCIMB9494, was obtained as a freeze-dried culture from the National Collections of Industrial and Marine Bacteria Ltd, Aberdeen, UK. This was stored in a refrigerator until use.
- Method of cultivation: The freeze-dried culture was rehydrated in 10 mL of sterile nutrient broth (Oxoid Ltd). This was incubated at 25 °C for three days. A loop of the resulting suspension was streaked onto a nutrient agar (Oxoid Ltd.) slope in a universal bottle and incubated at 25 °C overnight. The original streaks were prepared on 20 Apr 1998 and re-streaked on 30 Apr 1998. These were then stored at laboratory temperature in the dark until used as the stock culture.
- Preparation of inoculum for exposure: 19 h before the start of the test, 4 mL of the growth medium concentrate were added to 46 mL of deionised water in duplicate, sterile, conical flasks. A loop of Pseudomonas putida stock culture was added to each growth medium solution and both cultures were incubated for 19 h at 25 °C in an orbital shaker at 150 rpm. After this period, replicate 1 gave the most growth and therefore was selected as the test inoculum. Absorbance in this replicate was recorded as 0.868 at 600 nm with a Uvikon 930 spectrophotometer using 4 cm quartz cells. This culture was diluted with 8% (v/v) growth medium solution, which resulted in an absorbance of 0.823. This diluted culture was used as test inoculum.
Test type:
Water media type:
Limit test:
Total exposure duration:
6 h
Test temperature:
25 ± 0.5 °C
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
- Test vessel: flask
- Fill volume: 50 mL
- No. of vessels per concentration: 2
- No. of vessels per control: 3
- No. of vessels per vehicle control: 3
- other: The test vessels were shaken at 150 rpm throughout the test period.

- Source/preparation of dilution water: deionized water
- Culture medium different from test medium: no. The growth medium concentrate were prepared by adding 26.25 g of dipotassium hydrogen phosphate, 11.25 g of potassium dihydrogen phosphate, 1.175 g of trisodium citrate dihydrate, 2.5 g of ammonium sulphate and 0.25 g of magnesium sulphate heptahydrate to 1 L of deionized water. This solution was autioclaved at 121 °C for 15 minutes and stored in a refrigerator. A glucose solution was prepared using 6.25 g of glucose dissolved in 1 L of deionized water. This solution was also autoclaved and stored in a refrigerator. Immediately prior to use equal volumes of the salt and glucose solutions were mixed in a sterile flask to form the growth medium concentrate.

EFFECT PARAMETERS MEASURED: The cell growth is recorded after 6 h test period by measuring the optical density of the contents of each flask at 600 nm using a Uvikon 930 spectrophotometer and 4 cm quartz cells. Measurements were carried out with 8% v/v growth medium solution in the reference cell.
Reference substance (positive control):
6 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
No inhibition was recorded in any of the test concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The nominal 18 mg/L solution of the reference substance gave 87% inhibition of growth indicating that the Pseudomonas putida culture was responding normally to this known toxicant.

Description of key information

EC10 (6h) > 100 mg/L (nominal) for growth inhibition of Pseudomonas putida (ISO 10712)

Key value for chemical safety assessment

Additional information

One study investigating the toxicity of Propane-1,2,3-triyl 2-ethylhexanoate (CAS No. 7360-38-5) to aquatic microorganisms is available. The GLP study was conducted under static conditions according to ISO guideline 10712 using Pseudomonas putida as test organism. Test concentrations between 10 and 100 mg/L were prepared using acetone as vehicle (0.1% v/v). No growth inhibition was observed in the control and any treatment throughout the test period of 6 h. Hence, a 6 h-EC10 is determined to be > 100 mg/L based on the nominal test concentration.