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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2nd June to 24th July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Bringmann & Kuhn, ISO/TC 147/SC 5/WG 1 N 133 (March 1992)
Deviations:
yes
Remarks:
Test solutions incubated at 22 C instead of 25 C (bacterial growth too fast at 25 C). Absorption measured ay 600nm instead of 436nm.
Principles of method if other than guideline:
Cell multiplication inhibition test
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch no. and arrival date: WA 2809/0791, received at Solvay Duphar 's-Graveland on 14 August 1991 in a metal container.
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: colourless liquid
- Solubility of the test substance in water: 1000 mg/I
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Stock solution of 1250mg/l was diluted 1:1 with deionised water to avoid precipitation.
- Sampling method: 50ml was taken in duplicate for determination of the total organic carbon content (TOC).
Vehicle:
yes
Details on test solutions:
Test solutions consisted of a mixture of nutrient medium, stock solution (test substance) sterilised deionised water and inoculum of Pseudomonas putida. Solutions were prepared in the test vessels (erlenmeyers of 250 ml): 100 ml per test vessel; 5 test vessels per concentration. e-Caprolactone nominal concentrations of 0 (control), 16, 31, 63, 125, 250, 500 and 1000 mg/l were mixed by adding 10ml nutrient medium and 10 ml inoculum, to appropriate volumes of stock solution and deionised water.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: Bacteria (Pseudomonas putida ATCC 12633) used in these tests originated from a bacteria stock (freeze dried) of the Ecotoxicology Group, Solvay Duphar B.V. This culture originates from the Technical University, Delft, The Netherlands.
- Name and location of sewage treatment plant where inoculum was collected: the culture was freeze dried in ampullae at 27 February 1992 by the Microbiology Group, Solvay Duphar B.V., Weesp and is stored in a climate chamber at 4 C.
- Method of cultivation: the bacteria were resuspended in deionised water at the beginning of the experiment.
- Preparation of inoculum for exposure: inoculum was prepared under sterile conditions (biohazard cabinet). About 24 hours prior to test initiation 2ml sterile deionised water was transferred to a freeze dried ampulla with P. putida and shaken by hand to resuspend the bacteria. The bacteria suspension was transferred to a 50ml flask with 18ml preculture medium. The primary cultures were then incubated for about 16 hours at aout 22 C in a shaking incubator, at a speed of 145 rpm in the dark. Remaining preculture medium was stored at 4oC. The best growing primary cultures (assessed by turbidity) were used to prepare the seconday cultures. Primary cultures were diluted with preculture medium to obtain secondary cultures, then incubated for 7 hours as before. The secondary cultures were diluted with 'diluted' nutrient medium resulting in the inoculum with a turbidity of TE/F=50; this inoculum was used in the tests.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
16 h
Post exposure observation period:
None.
Hardness:
No information
Test temperature:
21.8- 25.5
pH:
5.7-6.9
Dissolved oxygen:
No information
Nominal and measured concentrations:
Nominal test concentrations: 0 (control), 16, 31, 63, 125, 250, 500 and 1000 mg/l
Measured only stock solutions: The stock solution of 1250 mg/L was diluted 1:1 with deionised water to avoid precipitation. A sample of 50 mL was taken for Total Organic Carbon (TOC) analysis.
The TOC measurements for the stock solution was 443.33 +/-13.5 (S.E) resulting in calucated test material concentrations of 1401 +/- 42.6 (S.E.) mg/L. Based on the variation in the measured stock concentrations all results are based on calculated concentrations.
Details on test conditions:
TEST SYSTEM
A randomised block design was used to place four test vessels (erlenmeyers of 250 ml: 100 ml per test vessel) per concentration in the shacking incubator. Tests were carried out in the dark at a shaking speed of 145rpm. Vessels were incubated for 16 hours. One test vessel per concentration was used for pH and temperature measurement at test initiation, and another test vessel was used for pH and temperature measurement at test termination.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : percentage inhibition of cell multiplication absorption at 600 nm

TEST CONCENTRATIONS
- Range finding and limit test study : In range-finding tests bacteria were exposed during 16 hours to concentrations of 0, 100 and 1000 mg/I test substance. There was no significant inhibition of cell multiplication observed at these concentrations. Based on these results, it was decided to carry out the final test as a limit test with concentrations of O and 1000 mg/I (C.SOL.51.026). Because there was a significant inhibition in this limit test a new test was carried out with two extra concentrations of 250 and 500 mg/I (C.SOL.51.026A). This test provided no NOEC, so two addditional tests were carried out with concentrations of 0, 16, 31, 63,125,250,500 and 1000 mg/I (C.SOL.51.026B and C.SOL.51.026C).
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
1 260 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
A reduction in pH was observed in all test solutions and the control from test initiation to termination. The authors discuss the possibility that this could be caued by a hydrolysis of e-Caprolactone by the bacteria, resulting in the formation of carboxylic acid.
Results with reference substance (positive control):
No positive control.
Reported statistics and error estimates:
Concentrations displaying a significant inhibition were significant at either the P<0.05 or P<0.01 level in the Williams' test one-sided.

All results were based on calculated concentrations because of the variation observed.

In the range finding test no significant inhibition of cell multiplication relevant to the control was observed. In the subsequent limit test a highly significant inhibition (24%) was observed at 1000mg/l.

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test material to Pseudomonas putida was tested in a series of studies. The results are based on calculated concentrations. Significant inhibition of cell multiplication occurred at calculated test concentrations of 63mg/l and higher. The EC50 was calculated to be 1260mg/l and the corresponding NOEC was 32mg/l.
Executive summary:

The toxicity of the test material to Pseudomonas putida was tested following the guideline ISO/TC 147/SC 5/WG 1 N 133 (March 1992). The study was run using a lower temperatiure of 20 °C instead than 25 °C, due to fast growth of the bacteria at the prescribed temperature. The study is reliable (Klimish 1) and conducted according to GLP stanbdards. The results are based on measured and calculated concentrations. Significant inhibition of cell multiplication occurred at test concentrations of 63mg/l and higher. The EC50 was calculated to be 1260mg/l and the corresponding NOEC was 32 mg/l, based on biological relevance.

Description of key information

The toxicity of the test material to Pseudomonas putida was tested in a series of studies. The results are based on calculated concentrations. Significant inhibition of cell multiplication occurred at calculated test concentrations of 63 mg/l and higher. The EC50 was calculated to be 1260 mg/l and the corresponding NOEC was 32 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
1 260 mg/L
EC10 or NOEC for microorganisms:
32 mg/L

Additional information

The toxicity of the test material to Pseudomonas putida was tested following the guideline ISO/TC 147/SC 5/WG 1 N 133 (March 1992). The study was run using a lower temperature of 20 °C instead 25 °C, due to fast growth of the bacteria at the prescribed temperature. The study is reliable (Klimisch 1) and conducted according to GLP standards. The results are based on measured and calculated concentrations. Significant inhibition of cell multiplication occurred at test concentrations of 63mg/l and higher. The EC50 was calculated to be 1260mg/l and the corresponding NOEC was 32 mg/l, based on biological relevance.