Registration Dossier

Administrative data

Endpoint:
toxicity to microorganisms
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:
other: Proprietary study conducted according to guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Bringmann & Kuhn, ISO/TC 147/SC 5/WG 1 N 133 (March 1992)
Deviations:
yes
Remarks:
Test solutions incubated at 22oC instead of 25oC (bacterial growth too fast at 25oC). Absorption measured ay 600nm instead of 436nm.
Principles of method if other than guideline:
Cell multiplication inhibition test
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
e-Caprolactone, colourless liquid, batch no. WA2809/0791. Purity: >99%. Solubility in water: >1000mg/l. Stored at room temperature.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not relevant

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Stock solution of 1250mg/l was diluted 1:1 with deionised water to avoid precipitation. 50ml was taken in duplicate for determination of the total organic carbon content (TOC).

Test solutions

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

Test organisms (species):
Pseudomonas putida
Details on inoculum:
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 22oC 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Post exposure observation period:
None.

Test conditions

Hardness:
No information
Test temperature:
Measured at test initiation and test termination.

Test a - Temperature ranged between 23.8 and 24.9 (0 hours) and 21.8 to 22.1 deg C (16 hours)
Test b - Temperature ranged between 25.5 and 26.1 (0 hours) and 22.3 to 22.5 deg C (16 hours)
Test c - Temperature ranged between 24.1 and 24.4 (0 hours) and 22.6to 22.8 deg C (16 hours)
pH:
Measured at test initiation and test termination.

Test a - pH in the control ranged from 6.9 (0 hours) to 6.7 (16 hours). In the treatments pH ranged between 6.8 - 6.9 (o hours) to 6.5 to 6.2 (16 hours).
Test b - pH in the control ranged from 6.9 (0 hours) to 6.5 (16 hours). In the treatments pH ranged between 6.8 - 6.9 (o hours) to 6.5 to 5.7 (16 hours).
Test c - pH in the control ranged from 6.9 (0 hours) to 6.9 (16 hours). In the treatments pH was 6.9 (0 hours) to 6.5 to 6.3 (16 hours).
Dissolved oxygen:
No information
Salinity:
Not relevant
Nominal and measured concentrations:
Only stock solutions in tests a, b and c were measured. 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 solutions in the tests a, b and c were 480, 450 and 400 mg/L respectively resulting in calucated test material concentrations of 1517, 1422 and 1264 mg/L. Based on the variation in the measured stock concentrations all results are based on calculated concentrations.
Details on test conditions:
A randomised block design was used to place four test vessels per concentration in the shaking 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.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
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
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.

Any other information on results incl. tables

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.

Test a - 303, 605 and 1210 mg/L had 24, 36 and 48 % inhibition of cells respectively. All of the were statistically significant from the control (P<0.01, Williams test, one-sided)

Test b - 71, 143, 285, 570 and 1140 mg/L had 20, 23, 33, 40 and 55% inhibition of cells respectively. All of the were statistically significant from the control (P<0.01, Williams test, one-sided)

Test c - 16, 32, 63, 126 and 253 mg/L had 5, 9, 12, 18 and 22% inhibition of cells respctively. All apart from the 16 mg/L value of 5%

were statistically significant from the control (P<0.01, Williams test, one-sided)

The authors conclude that the first test was an outlier. The turbidity of the control increased more than 100 times and therefore the test met the validity criteria.

The Ec50 was 1260 mg/L and the NOEC was considered to be 32 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of e-Caprolactone 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 e-Caprolactone 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.