Ichthyolic acid, sodium salt

Toxicity to microorganisms

Administrative data

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

start of study: November 4th, 1993
termination of study: November 5th, 1993
duration of study 16 +- 1 h

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

Deviations:

not specified

Principles of method if other than guideline:

Determination of the inhibitory effect of the sample on Pseudomonas putida by measurement of cell growth under the influence of varying dilutions of the test sample, compared to the cell growth of a culture obtained under the same conditions, but without the test sample.
Determination of the cell concentration as optical density after a test period of 16 h ± 1 h.
The concentrations of the test sample at which cell multiplication is inhibited by 10 % and 50 % within 16 h ± 1 h. are the basis for assessment.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ichthyolic acid, sodium salt

EC Number:

215-671-7

EC Name:

Ichthyolic acid, sodium salt

Cas Number:

1340-06-3

IUPAC Name:

Identification of the UVCB substance by "Chemcial Abstracts Index Name", among others: Ichthyolic Acid, Sodium Salt. No IUPAC name known.

Test material form:

liquid

Specific details on test material used for the study:

The test substance was taken from a regular production batch of the registrant's manufacturing site (sulfonated shale oil, pale batch no. R 92/0541). The name "sulfonated shale oil, pale is a common general expression for the trademark substance ICHTHYOL PALE. A certificate of analysis is part of the study report.

Sampling and analysis

Analytical monitoring:

not specified

Test solutions

Vehicle:

no

Details on test solutions:

Test solutions consisted of inoculation medium, stock solutions and test substance solution. For preparation of the different solutions aqua dest. was used.

Test organisms

Test organisms (species):

Pseudomonas putida

Details on inoculum:

One day old stock cultures of Pseudomonas putida served as inocula for preliminary cultures. Using aseptic techniques, suspensions of bacteria in preliminary culture medium (stock solution I, III, IV; 2.5 ml stock solution I, 2.5 ml stock solution III, 5.0 ml stock solution IV; see below) were diluted to a concentration corresponding a turbidity value of TU/F = 10 (turbidity units formazin).

Stock solution I
10.0 g sodium nitrate
2.4 g dipotassium hydrogen phosphate
1.2 g potassium dihydrogen phosphate
1.0 g yeast extract
in 500 ml aqua dest.

Stock solution II
10.0 g sodium nitrate
2.4 g dipotassium hydrogen phosphate
1.2 g potassium dihydrogen phosphate
in 500 ml aqua dest.

Stock solution III
44.0 g D(+)-glucose-monohydrate
in 500 ml aqua dest.

Stock solution IV
0.01 g ferrous citrate, w(Fe) = 19%
4.0 g magnesium sulphate, heptahydrate
in 1000 ml aqua dest.

Four parallel dilution series in 250-ml Erlenmeyer flasks with sterile stoppers were prepared with a final volume of 100 ml test medium each. Three series were inoculated with bacteria. The fourth series was not inoculated and served as a blank.
The final volume consisted of 10 ml of the inoculation medium (or 10 ml of preliminary culture medium for the fourth series which was not inoculated), 10 ml of different stock solutions and 80 ml of the test substance solution. The dilution factor was 2.0. The bacterial concentration at start corresponded to a turbidity value of TU/F = 5 (TU/F: turbidity units formazin). The pH was 6.94 to 6.95.
Four control culture flasks with 80 ml sterile water, 2.5 ml of stock solution and 10 ml of preliminary culture medium (1 flask) were also incubated.
Both inoculated an non-inoculated dilution series as well as the control culture flasks were left at 20°C - 21°C in the dark for 16 +- 1 hours (shaker; 75 - 85 rpm).
After termination of the test period the extinction of the monochromatic light at 436 nm was measured in a 10-mm cell with samples from the four test substance flasks per concentration and the control flasks. Since turbidity occurred in the dilution series for chemical-physical reasons, the analogous steps of dilution of the non-inoculated series were used as photometric blank values for the turbidity of the inoculated dilution series.

Study design

Test type:

not specified

Water media type:

other: The water media type follows the provisions of the method: surface, ground, or waste water to be tested.

Limit test:

yes

Total exposure duration:

16 h

Remarks on exposure duration:

according to guideline requirements

Post exposure observation period:

according to guideline requirements

Test conditions

Hardness:

not applicable

Test temperature:

20°C - 21°C

pH:

Initial pH of all test compound solutions and the control solution: 6.94 - 6.95

Dissolved oxygen:

not applicable

Salinity:

not applicable

Conductivity:

not applicable

Nominal and measured concentrations:

Nominal concentrations apply according to study report information. Preparation of the inoculation medium is described accordingly.

Details on test conditions:

Both inoculated and non-inoculated dilution series as well as the control culture flasks were left at 20°C - 21°C in the dark for 16 +- 1 hours (shaker: 75 - 85 rpm).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

16 h

Dose descriptor:

EC10

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Effect concentrations 2

Key result

Duration:

16 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

After termination of the test period the extinction of the monochromatic light at 436 nm was measured in a 10 mm cell with samples from the four test substance flasks per concentration and the control flasks. Since turbidity occured in the diluiton series for chemical-physical reasons, the analogous steps of dilution of the non-inoculated series were used as photometric blank values for the turbidity of the inoculated dilution series.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The following values were calculated
(i) mean extinction value of the control cultures
(ii) mean extinction values of each concentration of the inoculated dilution series

The mean extinction values of each dilution step were plotted against the logarithm of the values of the concentration of the test substance. The extinction values were corrected for turbidity.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Sulfonated Shale Oil, Pale (ICHTHYOL PALE) was investigated for its ability to inhibit the cell multiplication of the bacteria species Pseudomonas putida. Cultures of the bacteria were exposed to concentrations ranging from 625 mg to 10000 mg test substance/l.
Under the present test conditions no inhibition of cell multiplication of the bacteria species Pseudomonas putida was observed. On the contrary, the sulfonated shale oil, pale (ICHTHYOL PALE) appeared to have a nutritive effect.
The highest tested concentration which is equivalent to the highest reasonable concentration (limit test) was 10000 mg test substance/l.

The following parameters were determined for Pseudomonas putida:
EC10(16 h) : > 10000 mg sulfonated shale oil, pale/l
EC50(16 h): > 10000 mg sulfonated shale oil, pale/l