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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: ISO/TC 147/SC 5/WG 1 N 133
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The saturated stock solution of Capa-203 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, one was analysed and the other was a spare sample.
Vehicle:
no
Details on test solutions:
In order to simulate an accidental spill a saturated solution of Capa-203 was made by adding 750 mg Capa-203 to 750 ml of deionised water and stirring by an ultra-turrax for 10 minutes at level 1.5. This resulted in a turbid emulsion.

A clear and sterile solution was obtained by filtration of portions of the turbid emulsion through four 0.45 p. m filters (Nalgene filter units) under vacuum and combining the four solutions to one saturated stock solution.

The test solutions consisted of a mixture of nutrient medium, saturated stock solution, deionised water and inoculum of Pseudomonas putida.

The test solutions were prepared in the test vessels (erlenmeyers of 250 ml): 100 ml per test vessel; 5 test vessels per concentration.

One test vessel per concentration was used for pH and temperature measurement at test initiation. A randomised block design was used to place four test vessels per concentration in the shaking incubator. In this incubator the temperature was intentionally maintained at 22 ± 1°C. The test was carried out in the dark.

The shaking speed in the incubator was about 145 rotations per minute.

After 16 hours of incubation the absorption at 600 nm was measured in all test vessels using a spectrophotometer (Varian DMS 90) and a cuvette with a pathlength of 1 cm.

The pH and temperature at test termination were measured in one test vessel per concentration.

The saturated stock solution of Capa-203 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, one was analysed and the other was a spare sample.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Bacteria (Pseudomonas putida ATCC 12633) used in this test 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. 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.

The bacteria were resuspended in deionised water at the beginning of the experiment.

The next procedures were carried out under sterile conditions (Biohazard cabinet). About 24 hours before test initiation 2 ml sterile deionised water (water for injection, Lansberg) was transferred to a freeze dry ampulla with Pseudomonas putida by means of a hypodermic syringe. The ampulla was shaken by hand so that the bacteria were resuspended. The bacteria suspension was transferred to a sterile erlenmeyer of 50 ml with 18 ml preculture medium (Table 1) by means of the 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. The remainder of the preculture medium was stored at 4°C.

After the incubation period the absorptions at 600 nm (Varian DMS 90 spectrophotometer; cuvette with pathlength of 1 cm) of the primary precultures were measured. The best growing primary preculture with an absorption of 0.715 was used to prepare the secondary preculture.

The turbidity (in TE/F units) of the primary preculture was 400. This was determined from a formazin calibration curve (Figure 1) in which the absorption at 600 nm (y-axis) is plotted against the turbidity in TE/F (x-axis).

The primary preculture was diluted with preculture medium to obtain the secondary preculture with a calculated initial turbidity of TE/F=10.
The secondary preculture was incubated for about 7 hours under the same circumstances as described for the primary preculture.

After the incubation period the absorption at 600 nm of the secondary preculture was 0.310 corresponding with a turbidity of TE/F=175.
The secondary preculture was diluted with 'diluted' nutrient medium, resulting in the inoculum with a turbidity of TE/F=50.
This inoculum was used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
21 ± 1˚C
pH:
pH 6.5 to 7.0
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0, 20,40 and 80% of an initial saturated stock solution (with TOC determined concentration of 576 mg/L)
Calculated concentrations: 0, 115, 230 and 461 mg/L
Details on test conditions:
In order to simulate an accidental spill a saturated solution of Capa-203 was made by adding 750 mg Capa-203 to 750 ml of deionised water and stirring by an ultra-turrax for 10 minutes at level 1.5. This resulted in a turbid emulsion.

A clear and sterile solution was obtained by filtration of portions of the turbid emulsion through four 0.45 p. m filters (Nalgene filter units) under vacuum and combining the four solutions to one saturated stock solution.

The test solutions consisted of a mixture of nutrient medium, saturated stock solution, deionised water and inoculum of Pseudomonas putida.

The test solutions were prepared in the test vessels (erlenmeyers of 250 ml): 100 ml per test vessel; 5 test vessels per concentration.

The initial concentration of the inoculum in the test solutions was TE/F=5.

One test vessel per concentration was used for pH and temperature measurement at test initiation. A randomised block design was used to place four test vessels per concentration in the shaking incubator in darkness. In this incubator the temperature was intentionally maintained at 22 ± 1°C to exponential growth was maintained.

The test was carried out in the dark. The shaking speed in the incubator was about 145 rotations per minute.

After 16 hours of incubation the absorption at 600 nm was measured in all test vessels using a spectrophotometer (Varian DMS 90) and a cuvette with a path length of 1 cm.

The pH and temperature at test termination were measured in one test vessel per concentration.
Reference substance (positive control):
yes
Remarks:
Once per annum test conducted with 3,5-dichlorophenol
Key result
Duration:
16 h
Dose descriptor:
NOEC
Effect conc.:
461 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Remarks:
Total organic carbon content of saturated stock
Basis for effect:
growth inhibition
Details on results:
The TOC of the 1:1 dilution was 180 mg/L. This value was multiplied by 2 (dilution factor) and by 1.6 (according to the sponsor the C-content of Capa-203 is 60%) (stated as 61% in sister zebrafish study), resulting in the Capa-203 concentration in the saturated stock solution of 576 mg/L.

The measured concentration is well in agreement with the estimated solubility level of 600 mg Capa-203A

Based on the results of the TOC-determinations the following exposure levels were calculated : 0, 115, 230 and 461 mg/L.

At calculated concentrations of 0 (control), 115, 230 and 461 mg/L mean absorbance at 600 nm was 1.067, 0.947, 1.047 and 1.012 respectively.
This indicated that Capa-203 did not inhibit cell multiplication at the highest calculated concentration of 461 mg/L, this was considered to represent the no observed effect concentration.

(note: conclusion section of report states NOEC=460 mg/L, however table suggest highest calculated concentration was actually 461 mg/L).
Results with reference substance (positive control):
Once a year a toxicity test with Pseudomonas putida and the reference substance 3,5-dichlorophenol was conducted. The most recent test was conducted in July 1992. The 16h-EC50 found in this reference test was 21.2 mg/L (study number C.REF.51.005).

An international round-robin test with participation of 21 laboratories revealed a mean EC50 for 3,5-dichlorophenol of 21.4 mg/L (ISO-document) which shows a good agreement between the results of our laboratory and the results of the round-robin test.
Reported statistics and error estimates:
The EC50, the concentration at which the percentage cell multiplication is 50%, is determined from the figure which expresses the percentage of cell multiplication against the logarithm of the test substance concentration.

The results were analysed by Williams' test (Williams, 1972), one-sided. For the experiments with only one concentration next to the control, this is equivalent to the t-test.

The NOEC was determined as the highest concentration that does not show a statistically significant response in comparison to the control.

Results of toxicity test with Capa-203.

Calculated test concentration (mg/L)

Absorption at 600 nm

Mean Absorption at 600 nm

Turbidity (TE/F)

Inhibition of cell multiplication (%)

0 (Control)

1.063

1.067

595

0

0.997

1.058

1.147

115

0.967

0.947

530

11

0.91

0.999

0.91

230

1.09

1.047

585

2

1.035

1.011

1.05

461

1.009

1.012

565

5

0.98

0.939

1.119

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of Capa-203 to the bacteria Pseudomonas putida was tested according to ISO-document ISO/TC 147/SC 5/WG 1 N 133 (1992) at concentrations of 0%, 20%, 40% and 80% of the saturated stock solution. A sample of the saturated stock solution was taken at test initiation and the total organic carbon content was determined. The calculated concentration was 576 mg/L. No significant inhibition of cell multiplication occurred at the highest test concentration during the 16 h incubation period. The NOEC was 460 mg/L.
Executive summary:

The toxicity of Capa-203 to the bacteria Pseudomonas putida was tested at calculated concentrations of 0, 115, 230 and 461 mg/L.

The absorption (spectrophotometer Varian DMS 90, cuvette with pathlength of 1 cm) results indicated that at calculated concentrations of 0 (control), 115, 230 and 461 mg/L mean absorbance at 600 nm was 1.067, 0.947, 1.047 and 1.012 respectively. This indicated that Capa-203 did not inhibit cell multiplication at the highest calculated concentration of 461 mg/L, this was considered to represent the no observed effect concentration.

Description of key information

As there was no inhibition found at the highest concentration, the NOEC is considered to be 461 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
461 mg/L

Additional information

The toxicity of Capa-203 to the bacteria Pseudomonas putida was tested at calculated concentrations of 0, 115, 230 and 461 mg/L.

The absorption (spectrophotometer Varian DMS 90, cuvette with path length of 1 cm) results indicated that at calculated concentrations of 0 (control), 115, 230 and 461 mg/L mean absorbance at 600 nm was 1.067, 0.947, 1.047 and 1.012, respectively. This indicated that Capa-203 did not inhibit cell multiplication at the highest calculated concentration of 461 mg/L, this was considered to represent the no observed effect concentration.