2'-methylacetoacetanilide

Toxicity to microorganisms

Administrative data

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-01-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The cell multiplication inhibition test with Pseudomonas putida was conducted according to the draft of November 1978 for the standard DIN 38412, part 8(1991), which also is largely in compliance with the standard DIN EN ISO 10712: 1996-02. Difference to the test standard: at the time of the test the use of a reference substance to control the sensitivity of the used Pseudomonas strain was not yet a standard procedure.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

2010

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:

Difference to the test standard: at the time of the test the use of a reference substance to control the sensitivity of the used Pseudomonas strain was
not yet a standard procedure.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2'-methylacetoacetanilide

EC Number:

202-267-0

EC Name:

2'-methylacetoacetanilide

Cas Number:

93-68-5

Molecular formula:

C11H13NO2

IUPAC Name:

N-(2-methylphenyl)-3-oxobutanamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Date of delivery (test substance): 03.10.1988

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data.

Sampling and analysis

Analytical monitoring:

not specified

Details on sampling:

No details available.

Test solutions

Vehicle:

not specified

Details on test solutions:

2 g / L in de-ionized water, pH adjusted from 5.4 to 7.2, the batch stirred over a period of 24 hours at room-temperature,
pH again adjusted to 7.6, and filtrated through a folded paper filter.
Analyses of the stock solution - 1105 mg dissolved organic carbon / L (DCC)
3795 mg chemical oxygen demand / L (COD)
Concentration of the stock solution - 1599 mg/L, calculated from the DCC of the solution and carbon content in the substance of 69.09%.
Used volume of the stock solution in the test vessel - 50 ml in the batch; total volume:100 ml
only one concentration was tested
Concentration of 2‘-methylacetoacetanilide in the test vessel - 800 mg/L
Control batch - mixture of dilution water, nutrient solution and inoculum without test substance
Reference substance - no data of a batch with the use of a reference substance was recorded in the laboratory manuscript
(in later tests 2,4-dichlorophenol was used in the concentrations of 30 and 90 mg/L)

Test organisms

Test organisms (species):

Pseudomonas putida

Details on inoculum:

Origin strain:
Pseudomonas putida MIGULA, DSM 50026, by Deutsche Sammlung Mikroorganismen, Mascheroder Weg ib, D-38124
Braunschweig
Pre culture and test batches:
According to DIN standard cited
State of acclimatization:
Not acclimatized to the test substance or the reference substance
Inoculum in the test vessels:
Diluted to a turbidity of 5 FAU (formazine attenuation units)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

16.5 h

Post exposure observation period:

No data available.

Test conditions

Hardness:

No data.

Test temperature:

The temperature was controlled at 21°C - 22°C according to the DIN standard method cited. In this item, there is difference to the ISO draft, in which
temperature of test batches is required to be at 25°-30°C.

pH:

7.6

Dissolved oxygen:

No data.

Salinity:

Not applicable (freshwater)

Nominal and measured concentrations:

No data.

Details on test conditions:

To keep the bacteria in suspension, the test batches were conducted in an incubation rotary shaker with a diameter of the shaking circle of 5 cm. The shaking frequency was set to about 220 rpm. The tested batches were incubated for a period of 16.5 hours. The temperature was controlled at 21C °-22°C according to the DIN standard method cited. In this item, there is difference to the ISO draft, in which temperature of test batches is required to be at 25°-30°C.
The composition of reaction medium corresponds to the DIN standard cited. The concentration of the growth substrate glucose was 2000 mg/L.
Test vessel - 300 ml Erlenmeyer flasks
Test volume - 100 ml
Sample taking - At the end of the test period (16 ± 1 hour) an aliquot of each test batch was taken and measured by a spectrophotometer
Pretreatment of the samples - Samples with high absorbance values (>0.4) were diluted, to reach a linear measure range (<0.4 abs). These values
were multiplied by the factor of dilution.
Analysis - measuring the absorbance at a wavelength of 436 nm

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Duration:

16.5 h

Dose descriptor:

EC50

Effect conc.:

> 800 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

Please refer to "Any other information on results incl. tables"

Results with reference substance (positive control):

No data.

Reported statistics and error estimates:

No data.

Any other information on results incl. tables

The results of the test, including all recorded values in the laboratory manuscript and the calculated inhibition effect were noted in a table. The range of the effect concentrations was estimated from the result.

Applicant's summary and conclusion

Validity criteria fulfilled:

no

Conclusions:

The results of testing the toxicity of 2‘-methylacetoacetanilide an the growth of bacteria in the Pseudomonas putida cell multiplication inhibition test
revealed that:
The EC10 and EC50 of 2‘-methylacetoacetanilide to Pseudomonas putida are >800 mgIL.

Executive summary:

Testing the growth inhibition of Pseudomonas putida was conducted according to the draft of November 1978 for the standard DIN 38412, part 8(1991). The test proceeded without disturbances. The turbidity of the inoculum in the blank controls increased to more than the 100-fold within the test period. The sensitivity of the used bacteria strain was not controlled with a reference substance as it was described later as a standard procedure in DIN 38412, part 8, and in the later published standard DIN EN ISC 10712: 1996-02. As no inhibition effect occurred with 2‘-methylacetoacetanilide in the highest concentration to be tested, in this test only this one concentration had been implemented. Five duplicates were performed with the blank control and two duplicates with the test substance. All available data are reported in this document. Some lacks in the laboratory protocols are mentioned in this report.