Registration Dossier

Administrative data

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4th - 26th June 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ISO/TC 147/SC 5/WG 1 N 133 (based on Bringmann & Kuhn)
Deviations:
yes
Remarks:
The test solutions were incubated at 22°C instead of at 25°C (as stated in the ISO guideline), because at 25°C the bacteria grew too fast and were no longer in the exponential growth phase after 16 hours incubation. The absorption was measured at 600 nm i
Principles of method if other than guideline:
ISO document: ISO/TC 147/SC 5/WG 1 N 133 (1992), Water -quality - determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Capa 305 is a colourless liquid. Batch no. 79H. Stored at room temperature. Solubility ~ 400 mg/l. Purity: mixture of polymers.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The saturated stock solution of Capa-305 was diluted 1:1 with deionised water to avoid precipitation. A sample of 50 ml was taken for determination of the total organic carbon content (TOC). The TOC was determined by Centrilab, Soest, The Netherlands. The sample was taken in duplicate.

Test solutions

Vehicle:
no
Details on test solutions:
A saturated stock solution of Capa-305 in deionised water was prepared. Deionised water was sterilised at 121°C for 10 minutes. Solutions were: 500mg of Capa-305 added to 500ml deionised water, and 750 g of Capa-305 added to 750 ml deionised water. Solutions were stirred in an ultra-turrax for 10 minutes resulting in a turbid emulsion. A clear and sterile solution was obtained by filtration of portions of the turbid emulsion through 0.45 µm filters under suction and combining the filtrated solutions to one saturated stock solution.

Test organisms

Test organisms (species):
Pseudomonas putida
Details on inoculum:
The bacteria in this study (P. putida ATCC 12633), originated from a bacteria stock (freeze dried) at the testing laboratory. The culture originated from the Technical University, The Netherlands. Freeze dried cultures were stored in a climate chamber at 4°C. The bacteria were resuspended in deionised water at the beginning of the experiment.
Preparation of the inoculum was carried out under sterile conditions. About 24 hours before test initiation 2 ml sterile deionised water (water for injection, Lansberg) was transferred to a freeze dry ampulla with P. putida using a hypodermic syringe. The ampulla was shaken by hand to resuspend the bacteria. The suspension was transferred to a sterile erlenmeyer of 50 ml with 18 ml preculture medium using a hypodermic syringe. This was done in duplicate. The two resulting primary precultures were incubated for about 16 hours at about 22°C in a shaking incubator, at a shaking speed of 145 rpm in the dark. After the incubation period the absorptions of the primary cultures were measured at 600 nm. The best growing primary cultures with absorptions of 0.800 and 0.815 in the successive tests were used to prepare the secondary precultures. The primary precultures were diluted with preculture medium to obtain the secondary precultures with a calculated initial turbidity of TE/F=10. The secondary precultures were incubated for about 7 hours under the same circumstances as described for the primary cultures.
The secondary precultures were diluted with 'diluted' nutrient medium resulting in a inoculum with a turbidity of TE/F=50, this was used in the tests.

Study design

Test type:
static
Limit test:
no
Total exposure duration:
16 h
Post exposure observation period:
Not examined

Test conditions

Hardness:
Not determined
Test temperature:
22±1°C
pH:
The pH in the control decreased from 6.9 at test initiation to 6.6 and 6.4 after 16 hours in the two tests. The pH in the highest concentration decreased from 6.9 at test initiation to 6.5 after 16 hours.
Dissolved oxygen:
Not determined
Salinity:
Not determined
Nominal and measured concentrations:
Nominal concentrations of 0, 20, 40 and 80% of the saturated stock solution were used. Based on the results of the TOC-determinations the following exposure concentrations were calculated: 0 and 538 mg/l in the limit test and 0, 167, 333 and 666 mg/l in the definitive test.
Details on test conditions:
A range finding test was conducted in which bacteria were exposed over 16 hours to concentrations of 0, 8 and 80% of the saturated stock solution of Capa-3050. There was no significant inhibition of cell multiplication observed at these concentrations. A limit test was carried out with concentrations of 0 and 80% of the saturated solution. This test provided no NOEC so an additional test was carried out with concentrations of 0, 20, 40 and 80%.
Deionised water was sterilised at 121°C for 10 minutes. Glass material was dry sterilised at 170°C for 1 hour. The tests were carried out in a Biohazard cabinet.
Test solutions consisted of a mixture of nutrient medium, saturated stock solution, deionised water and inoculum. Test solutions were prepared in 250ml erlenmeyers: 100 ml per test vessel, 5 test vessels per concentration. Test vessels contained 10 ml nutrient medium, 10 ml TE/F=50 inoculum, and 80 ml of the relevant test solution (made up with saturated Capa-305 solution and deionised water).
One test vessel per concentration was used for pH and temperatire measurement. A randomised block design was used to place 4 test vessels per concentration in the shaking incubator. The temperature of the shaking incubator was maintained at 22±1°C. Tests were carried out in the dark, at a shaking speed of 145 rpm. The absorption at 600 nm was measured after 16 hours incubation.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
Duration:
16 h
Dose descriptor:
NOEC
Effect conc.:
670 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: TOC of saturated solution
Basis for effect:
other: cell multiplication inhibition
Details on results:
The TOC of the saturated stock solution was 260 mg/l, resulting in the Capa-305 concentrations in the saturated stock solution of 672 and 832 mg/l.In the limit test, the 80% test solution (calculated concentration 538 mg/l) caused a significant inhibition of cell multiplication (14%) compared to the control. In the main test Capa-305 did not show any cell multiplication inhibition even at higher concentations.
Results with reference substance (positive control):
Yearly toxicity tests with 3,5-dichlorophenol were carried out at the testing laboratory. The most recent test conducted in July 1992 resulted in a 16h EC50 of 21.2 mg/l in good agreement with an international round robin test involving 21 laboratories.
Reported statistics and error estimates:
In the limit test, the 80% test solution (calculated concentration 538 mg/l) caused a significant increase of 14% inhibition of cell multiplication compared to the control, p<0.01, Williams test one-sided.

Any other information on results incl. tables

In the limit test there was a small significant inhibition of cell multiplication.There was no inhibition of cell muliplication at test concentrations of 20, 40 and 80% Capa-305.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The turbidity of the control increased by more than 100 times
Conclusions:
No significant inhibition of cell multiplication occurred at the highest test concentration during the 16h incubation period. The NOEC was 670 mg/l.
Executive summary:

The toxicity of Capa-305 to the bacteria P. putida was tested in the cell multiplication inhibition test, at nominal concentrations of 0, 20, 40 and 80% of the saturated stock solution. No significant inhibition occurred at the highest test concentration during the 16 hour incubation period. The NOEC was 670 mg/l.