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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4-5 June 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ISO/TC 147/SC 5/WG 1 N 133 (1992)
Version / remarks:
Microbial growth inhibition based on Bringmann & Kuehn (1977)
Deviations:
yes
Remarks:
Incubation was carried out 22°C rather than 25°C, absorption was measured at 600 nm rather than 436 nm. neither minor deviation affected the outcome of the study.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
A 50 ml of the saturated solution was taken for Total Organic Carbon analysis
Vehicle:
no
Details on test solutions:
510 mg of Capa-304 was added to 500 mL of deionised water and stirred for ten minutes with an ultra-turrax, this resulted in a turbid emulsion. The resulting turbid emulsion was filtered through a 0.45 um nylon membrane filter and produced a clear solution which was used as the test stock solution..

The test solutions consisted of a mixture of nutrient medium, test stock solution, deionised water and Pseudomonas inoculum as follows:

Control - 10 mL of nutrient medium, 80 mL of deionised water and 10 mL of inoculum
832 mg/L - 10 mL of nutrient medium, 80 mL of test stock solution and 10 mL of inoculum
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Bacteria used in the study were Pseudomonas putida, strain ATCC 12633, original source was the Technical University Delft, The Netherlands. The culture was freeze dried and stored at 4°C.

At the start of the study the bacteria were resuspended in sterilised, deionised water.

Approximately 24 hours prior to test initiation 2 mL of deionised water was transferred to a freeze dried sample of bacteria. 18 mL of preculture medium was added to the bacterial suspension and this primary preculture was incubated for approximately 16 hours at 22°C on a shaker at approximately 145 rpm. . At the end of the incubation period the absorption at 600 nm of the primary preculture was measured and was 0.8 corresponding to a turbidity of TE/F=450..

The primary preculture was diluted with preculture medium to obtain a secondary preculture with a calculated initial turbidity of TE/F=10, this was incubated for approximately 7 hours at 22°C. After the incubation period the absorption was 0.322 corresponding to a turbidity of TE/F=180. The secondary preculture was diluted with nutrient medium resulting in an inoculum with a turbidity of TE/F=50 and this was used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
16 h
Post exposure observation period:
No
Hardness:
Not applicable
Test temperature:
22 ± 1°C
pH:
Control 6.9 at test initiation, 6.5 at test end
832 mg/L - 6.9 at test initiation, 6.4 at test end
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations were 0 (control) and 1040 mg/L.

Analysis of the TOC sample provided a measured concentration of 650 mg/L. This value was multiplied by 1.6 (based on the C-content of Capa-304 of approximately 60%) resulting in a Capa-304 concentration of 1040 mg/L. The measured concentration is about 1.5 times higher than the earlier estimated solubility levle of 750 mg Capa-304/L. Based on the TOC determination the exposure level was calculated to 832 mg/L.

The results of the study were based on the calculated concentration.
Details on test conditions:
Test vessels were 250 mL Erlenmeyer flasks, filled with 100 mL of test medium, 5 replicates were used per treatment.

One vessel per treatment was used for pH and temperature measurements at test initiation. A randomised block design was used to place the four remaining test vessels in the shaking incubator. The study was carried out in the dark with a shaking speed of 145 rpm.

At the end of the 16 hour exposure period the absorption at 600 nm was measured in vessels at a pathlength of 1 cm. pH and temperature was determined in one replicate per treatment at the end of the exposure period.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
> 832 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth inhibition
Duration:
16 h
Dose descriptor:
NOEC
Effect conc.:
832 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth inhibition
Details on results:
The results of the study were based on the calculated exposure concentration. The 16 hour EC50 was observed to be >832 mg/L (based on the dissolved fraction of CAPA-304 components) and the corresponding NOEC was 832 mg/L.
Results with reference substance (positive control):
Performed annually by the laboratory using 3,5-dichlorophenol. The reported value for a test conducted in July 1992 was 21.2 mg/L which was close to the reported ring-tested value of 21.4 mg/L reported in the ISO guideline.
Reported statistics and error estimates:
The results were analysed using the Williams' test (1972) one-sided.

Absorption results

Calculated test concentration (mg/L)

Absorption at 600 nm

Mean absorption at 600 nm

Turbidity (TE/F)

Inhibition of cell multiplication (%)

0 (control)

1.017

1.063
0.932
0.959

 

0.993

 

560

 

-

832

0.873
0.993
1.006
0.885

 

0.939

 

525

 

6

 

Validity criteria fulfilled:
yes
Remarks:
The test is considered to be valid if the inoculum used in the control (TE/F=5) has multiplied by a factor of least 100 during the study
Conclusions:
The results of the study were based on the calculated exposure concentration. The 16 hour EC50 was observed to be >832 mg/L (based on the dissolved fraction of CAPA-304 components) and the corresponding NOEC was 832 mg/L.
Executive summary:

The toxicity of Capa-304 to the Pseudomonas putida was determined. The study was carried out according to the ISO/TC 147/SC 5/WG 1 N 133 (1992) guideline. Bacteria transferred from a pre-culture were exposed to Capa-304 over a test period of 16 hours. A saturated test medium was prepared by the addition of Capa-304 to water followed by 10 minutes of stirring, the resultant medium was filtered through a 0.45 µm filter and the resultant test solution was used to prepare the test medium.  Total Organic Carbon (TOC) analysis was performed on the saturated test medium batch.   Based on the TOC result the exposure concentration was calculated to be 832 mg/L. The results of the study were based on the calculated exposure concentration. Bacterial growth was determined by turbidity, measured as absorption at 600 nm with a Varian DMS 90 spectrophotometer.

 

The 16 hour EC50 of Capa-304 to Pseudomonas putida was observed to be >832 mg/L and the NOEC was 832 mg/L. 

Description of key information

The 16 hour EC50 of Capa-304 to Pseudomonas putida was observed to be >832 mg/L and the NOEC was 832 mg/L. 

Key value for chemical safety assessment

EC50 for microorganisms:
832 mg/L
EC10 or NOEC for microorganisms:
832 mg/L

Additional information

The toxicity of Capa-304 to the Pseudomonas putida was determined. The study was carried out according to the ISO/TC 147/SC 5/WG 1 N 133 (1992) guideline. Bacteria transferred from a pre-culture were exposed to Capa-304 over a test period of 16 hours. A saturated test medium was prepared by the addition of Capa-304 to water followed by 10 minutes of stirring, the resultant medium was filtered through a 0.45 µm filter and the resultant test solution was used to prepare the test medium.  Total Organic Carbon (TOC) analysis was performed on the saturated test medium batch.   Based on the TOC result the exposure concentration was calculated to be 832 mg/L. The results of the study were based on the calculated exposure concentration. Bacterial growth was determined by turbidity, measured as absorption at 600 nm with a Varian DMS 90 spectrophotometer.

 

The 16 hour EC50 of Capa-304 to Pseudomonas putida was observed to be >832 mg/L and the NOEC was 832 mg/L.