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Ecotoxicological information

Toxicity to microorganisms

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Administrative data

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
publication
Title:
Application of Bacterial Growth Kinetics to in Vitro Toxicity Assessment of Substituted Phenols and Anilines.
Author:
M. NENDZA AND J. K. SEYDEL
Year:
1990
Bibliographic source:
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 19, Pg. no. 228-24 1, 1990.

Materials and methods

Principles of method if other than guideline:
Toxicity to micro-organisms study was conducted on E. coli ATCC 11775 and Mycobacterium smegmatis M169.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): 4-methoxyaniline
- Molecular formula (if other than submission substance): C7H9NO
- Molecular weight (if other than submission substance): 123.1541 g/mol
- Smiles notation (if other than submission substance): COc1ccc(N)cc1
- InChl (if other than submission substance): 1S/C7H9NO/c1-9-7-4-2-6(
8)3-5-7/h2-5H,8H2,1H3
- Structural formula attached as image file (if other than submission substance): No data available
- Substance type: Organic
- Physical state: Solid
- Analytical purity: No data available
- Impurities (identity and concentrations): No data available
- Composition of test material, percentage of components: No data available
- Isomers composition: No data available
- Purity test date: No data available
- Lot/batch No.: No data available
- Expiration date of the lot/batch: No data available
- Radiochemical purity (if radiolabelling): No data available
- Specific activity (if radiolabelling): No data available
- Locations of the label (if radiolabelling): No data available
- Expiration date of radiochemical substance (if radiolabelling): No data available
- Stability under test conditions: No data available
- Storage condition of test material: No data available
- Other: The test chemical of highest grade of purity was used for the study.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data available

Sampling and analysis

Analytical monitoring:
not specified
Details on sampling:
No data available

Test solutions

Vehicle:
not specified
Details on test solutions:
No data available

Test organisms

Test organisms (species):
other: E. coli ATCC 11775 and Mycobacterium smegmatis M169
Details on inoculum:
- Laboratory culture: The test bacteria E. coli ATCC 11775 was obtained from the American Type Culture Collection.
Mycobacterium smegmatis M169 was also used for the study.

- Method of cultivation: Portions of 150 ml of culture from diluted broth were transfered to 1 liter Erlenmeyer flasks. 30 minutes later when the bacteria were in the logarithmic growth phase the chemical was added. Samples of the incubated cultures were taken every 45 min. Particle-free saline (0.85%) and formaldehyde (2%) solution were added to stop multiplication as well as to dilute the solution to about 1,000- 10,000 organisms per count. Counting was achieved with a Coulter counter Model ZB equipped with a 0.030-mm orifice; counts per 0.050 ml were obtained. The instrument settings were: l/aperture current, 1; l/amplification, 4; matching switch, 40 K; gain, 10; lower threshold, 7; upper threshold, maximum.

The MIC was determined by the standard procedures.

Along with the Coulter counter experiments samples were drawn from the cultures under toxicant treatment. Culture suspension (0.5 ml) was diluted with saline. The solution, now containing 10 - 100 organisms/ml, was pipetted onto each of five petridishes. After mixing the saline suspension with 5 ml of melted agar, the bacteria were allowed to multiply for 48 hr at 37°C. The resulting colonies were counted.

- Preparation of inoculum for exposure: The stock cultures were maintained on agar slants at room temperature.

The culture broth used was dextrose-salts-casamino acids (vitamin-free), pH 6.9. This broth culture was inoculated from an agar culture and the bacteria allowed to grow for 12 to 16 hr at 37°C. From this broth, cultures were prepared by dilution with medium to obtain 104 cells/ml.

- Pretreatment: No data available

- Initial biomass concentration: 104 cells/ml

Study design

Test type:
not specified
Water media type:
not specified
Total exposure duration:
48 h
Post exposure observation period:
Every 45 min samples were taken and the number of organisms was counted.

Test conditions

Hardness:
No data available
Test temperature:
37°C
pH:
6.9
Dissolved oxygen:
No data available
Salinity:
No data available
Nominal and measured concentrations:
No data available
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask (1 l)
- Type (delete if not applicable): No data available
- Material, size, headspace, fill volume: 1 liter
- Aeration: No data available
- Type of flow-through (e.g. peristaltic or proportional diluter): No data available
- Renewal rate of test solution (frequency/flow rate): No data available
- No. of organisms per vessel: No data available
- No. of vessels per concentration (replicates): No data available
- No. of vessels per control (replicates): No data available
- No. of vessels per vehicle control (replicates): No data available
- Biomass loading rate: No data available

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: No data available
- Total organic carbon: No data available
- Particulate matter: No data available
- Metals: No data available
- Pesticides: No data available
- Chlorine: No data available
- Alkalinity: No data available
- Ca/mg ratio: No data available
- Conductivity: No data available
- Culture medium different from test medium: No data available
- Intervals of water quality measurement: No data available

OTHER TEST CONDITIONS
- Adjustment of pH: No data available
- Photoperiod: No data available
- Light intensity: No data available

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : No data available

TEST CONCENTRATIONS
- Spacing factor for test concentrations: No data available
- Justification for using less concentrations than requested by guideline: No data available
- Range finding study: No data available
- Test concentrations: No data available
- Results used to determine the conditions for the definitive study: No data available
Reference substance (positive control):
not specified

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
other: MIC
Effect conc.:
357.1 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
other: Decrease in colony count
Remarks on result:
other: The MIC value is for E. coli ATCC 11775
Duration:
48 h
Dose descriptor:
other: MIC
Effect conc.:
62.8 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
other: Decrease in colony count
Remarks on result:
other: The MIC value is for Mycobacterium smegmatis M169
Duration:
48 h
Dose descriptor:
other: I50
Effect conc.:
1 231.5 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
other: Reduction in generation rate of bacteria to 50%.
Remarks on result:
other: The I50 value is for E. coli ATCC 11775
Details on results:
No data available
Results with reference substance (positive control):
No data available
Reported statistics and error estimates:
No data available

Any other information on results incl. tables

Table:Bacteriotoxic activity of aniline derivatives (MM/l).

 

Test chemical

MIC (E.coli)

MIC (M. M169)

I50(E.coli)

4-methoxyaniline

2.9

0.51

10

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
The MIC and I50 value for E.coli was found to be 357.1 mg/l and 1231.5 mg/l, respectively.

The MIC value for Mycobacterium smegmatis M169 was found to be 62.8 mg/l.
Executive summary:

Toxicity to micro-organisms study was conducted on E. coli ATCC 11775 and Mycobacterium smegmatis M169.

The test bacteria E. coli ATCC 11775 was obtained from the American Type Culture Collection.

 

The stock cultureswere maintained on agar slants at room temperature.

 

Portions of 150 ml of culture from diluted broth were transfered to 1 liter Erlenmeyer flasks. 30 minutes later when the bacteria were in the logarithmic growth phase the chemical was added. Samples of the incubated cultures were taken every 45 min. Particle-free saline (0.85%) and formaldehyde (2%) solution were added to stop multiplication as well as to dilute the solution to about 1,000- 10,000 organisms per count. Counting was achieved with a Coulter counter Model ZB equipped with a 0.030-mm orifice; counts per 0.050 ml were obtained. The MIC was determined by the standard procedures.

 

Along with the Coulter counter experiments samples were drawn from the cultures under toxicant treatment. Culture suspension (0.5 ml) was diluted with saline. The solution, now containing 10 - 100 organisms/ml, was pipetted onto each of five petridishes. After mixing the saline suspension with 5 ml of melted agar, the bacteria were allowed to multiply for 48 hr at 37°C. The resulting colonies were counted.

 

Every 45 min samples were taken and the number of organisms was counted.

 

Based on decrease in colony counts,the MIC forE.coliandMycobacterium smegmatisM169 was found to be 357.1 mg/l and 62.8 mg/l, respectively.

 

Based on reduction in generation rate of bacteria to 50%, the I50value forE.coliwas found to be 1231.5 mg/l.