5-methylheptan-3-one

Toxicity to other aquatic organisms

Administrative data

Endpoint:

toxicity to other aquatic vertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment. However no guideline was followed.

Data source

Reference

Reference Type:

publication

Title:

COMPARISON OF THE TOXICITY THRESHOLDS OF WATER POLLUTANTS TO BACTERIA, ALGAE, AND PROTOZOA IN THE CELL MULTIPLICATION INHIBITION TEST

Author:

Bringmann et al.

Year:

1980

Bibliographic source:

Water Research 14, pp. 231-241

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Dissolved toxic water ingredients will inhibit cell multiplication of the protozoon Entosiphon sulcatum. Thus, the count of organisms after a certain period of exposure to the toxic substance will be less than in a test culture kept under identical conditions, however, free from toxic influence. The number of protozoa was determined by a cell counter.

GLP compliance:

no

Remarks:

Study performed before the introduction of GLP

Test material

Test material information

Constituent 1

Reference substance name:

ethyl amyl keton

IUPAC Name:

ethyl amyl keton

Details on test material:

- Name of test material (as cited in study report): ethyl amyl keton
- Assigned identity of the test material: 3-Octanone (CASNr: 106-68-3)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

other: Entosiphon sulcatum

Details on test organisms:

To cultivate the protozoon Entosiphon sulcatum, it is first required to cultivate bacteria on which they are feeding: E coli. Fresh stock cultures of E.coli were thus prepared in a nutrient medium monthly. To inoculate the expected number of cultures, cell material from the stock solution of E. coli were streaked on a nutrient medium 1 in Kapsenberg culture tubes, incubated at 37°C for 48h and stored at 25°C. Preliminary cultures were prepared by inoculating different flasks containing 100 ml of nutrient solution 2 and incubated at 37°C for 24h. The cell suspensions were then centrifuged at 6000rpm. To prepare feeding for the protozoon stock or preliminary cultures, the bacterial centrifugate was resuspended with a 50% solution of stock solution 1 in sterile double distilled water to reach the initial volume and centrifuged again at 6000rpm. This step was repeated once and the turbidity of the suspension was then adjusted with a 50% solution of stock solution 1 in sterile double distilled water to reach an extinction value of the monochromatic radiation at 436nm for a 10 mm layer corresponding to the turbidity value of the Formazin standard TE/F/436nm=200.

For the feeding for the protozoon test cultures, the bacteria were prepared the same way as for the stock and preliminary cultures of protozoon except for an additional first step: the inactivation of the feeding bacteria. The feeding bacteria were inactivated by diluting the bacterial centrifugate in a 2% sulphuric acid. The suspension was stirred for 60 minutes adjusted to pH 6.9 with NaOH and centrifuged again. The remaining steps of the procedure: redilution in a 50% the solution of the stock solution 1 and bidistilled water as well as the centrigugation were repeated twice and as for the stock and preliminary cultures of protozoon the turbidity was adjusted.

The test strain of Entosiphon sulcatum were maintained by continuously inoculating the stock cultures at 72 or 96 hours intervals. Briefly, 8 ml of stock solution 1 was added to 8 ml of sterile bidistilled water in 300 ml flasks stoppered with metal caps to which 2ml of stock cultures and 2 ml of the adjusted bacterial suspensions for the protozoon stock cultures were added. The stock cultures of protozoon were stored at 25°C. Preliminary cultures of Entosiphon sulcatum were fed in the same way and were kept for 72h at 25°C before being used for the inoculation of the test culture.

Study design

Test type:

static

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

25°C

Details on test conditions:

The test strain of Entosiphon sulcatum were maintained by continuously inoculating the stock cultures at 72h or 96 hours intervals. Briefly, 8 ml of stock solution 1 was added to 8 ml of sterile bidistilled water in 300 ml flasks stoppered with metal caps to which 2ml of stock cultures and 2 ml of the adjusted bacterial suspensions for the protozoon stock cultures were added. The stock cultures of protozoon were stored at 25°C. Preliminary cultures of Entosiphon sulcatum were fed in the same way and were kept for 72h at 25°C before being used for the inoculation of the test culture.

An initial solution of the test substance (CASNr: 106-68-3) was prepared, neutralised if required. Two parallel dilutions from the initial solution of the test substance (CASNr: 106-68-3) were prepared in 300 ml flasks stoppered with metal caps. The first flask contained 16 ml of the initial solution containing the test substance (CASNr: 106-68-3). Subsequent dilutions were prepared from this first flask using a constant dilution ration of 8 ml preliminary ethyl amyl keton (CASNr: 106-68-3) dilution with 8 ml of double-distilled water. Each flask contained 8 ml of liquid and were completed with 8 ml of the stock solution 1 and inoculated with 2 ml of the preliminary protozoan cultures (approx. impulses 15000/ml) and 2 ml of the suspension of inactivated bacteria having a known extinction value. The test cultures were then stirred with a magnetic stirrer on a small rotatory shaker for 72 hours at 25°C. The test cultures were first controlled with a microscope before adding 10% of a 1.1% NaNO3 solution in bidistilled water, filtered through a membrane filter (0.2um pore size) an determine the number of protozoa by means of a cell counter.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

other: TT

Effect conc.:

256 mg/L

Nominal / measured:

nominal

Basis for effect:

other: cell number

Details on results:

Toxicity threshold (TT) obtained in growth inhibition test for Entosiphon sulcatum.

Any other information on results incl. tables

For the evalutation of the results, the mean value (A) of impulses/ml for the test cultures free from both toxic influence and stimulation of growth (with the exception of those having extinction values outside a standard deviation of < 5%) and the mean value (B) of the

impulses/ml for the test cultures having the lowest toxic pollutant concentration within the dilution series were calculated at the end of the test period.

For mathematical evaluation, the highest non-toxic poIlutant concentration (a) was plotted against (A) and the lowest toxic pollutant concentration (b) was plotted against (B) as coordinates in a semi-logarithmic coordinate system. With the assumption that a negative deviation of the mean extinction by a 5% difference against the mean impulses/ml value for all test cultures having a non toxic and non-stimulating pollutant concentraion may be used as an inicator of the beginning of inhibitory action, the pollutant concentration at which the inhibitory effect was beginning (c) was deduced from the regression line between (a; A) and (b;B) using the extinction value (A-5%) as ordinate.

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

The following results were determined for the read across ethyl amyl keton (CASNr: 106-68-3) (species: Entosiphon sulcatum): TT(72h)= 256 mg/L. The value falls in the range of the measured values for fish, aquatic invertebrates and algae. The results are not required for classification or risk, but confirm that protozoa are less sensitive than other species.

Executive summary:

Toxicity threshold of the test substance (CASNr: 106-68-3) were determined for the protozoon Entosiphon Sulcatum by a multiplication inhibition test. The toxicity threshold concentration (greater or equal 5% lower impulses/ml measured in comparison to control) can therefore be interpreted as an EC05 value which represents a worst-case for a EC10 value. The determined EC10 value for the read across substance (CASNr: 106-68-3), and by extension to the substance of interest 5 -methylheptan-3 -one, shows that protozoa are less sensitive to this substance than other species.