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Toxicity to microorganisms

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toxicity to microorganisms
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not conducted to GLP or standard guideline. Methodology was sound and data can be considered reliable with restrictions.

Data source

Reference Type:
Report Date:

Materials and methods

Test guideline
other: Comparison of protzoan toxicity tests
Principles of method if other than guideline:
This paper compares 2 protazoa toxicity test methods using multiple xenobiotic test chemicals. A flask method and a microplate technique using the protazoan species Tetrahymena Pyriformis. Monochloro acetic acid was one of the chemicals chosen for testing. The methodology of the microplate method is summarised here.
GLP compliance:

Test material

Details on test material:
Specific information on test chemical purity was not provided. However all organic xenobiotics used for testing were obtained from Fluka chemicals.

Sampling and analysis

Analytical monitoring:

Test organisms

Test organisms (species):
other: Tetrahymena Pyriformis (TP)

Study design

Test type:
Water media type:

Test conditions

Details on test conditions:
Type: aquatic

Results and discussion

Effect concentrationsopen allclose all
9 h
Dose descriptor:
Effect conc.:
83 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: Flask Method
36 h
Effect conc.:
16 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: Microplate Method

Any other information on results incl. tables

Experimental Procedures.

Microplate Technique

15 -hr culture of TP in exponential growth phase was first prepared in normal PPYS. For experiments, TP were collected by centrifugation for 5 min at 800 rpm. The PPYS sumatant was discharged and the TP pellet was carefully suspended in some fresh PPYS m to a cell concentration of 50000 cells/ml. After a 1 hr acclimitization of TP in this 2 ml aliquots of TP cultures were treated with tested substances in sterile tubes. Then; the treated samples were mixed thoroughly and dispensed (200µl/well) into a 96 well microplate (Clusters Costar 3599). One control culture and five concentrations of test substance were distributed in the eight wells of one column of the microplate, leaving an empty column between each of the columns filled with TP-treated cultures; distribution in this manner provided some.reserves of air for TP cultures. The absorbance of the wells were then measured against the blanks with a microplate reader. The system was linked to a computer that calculated the optical densities and measured the absorbance periodically up to 36 hours. The doubling time was calculated and the concentration level causing 50% increase in the doubling time (DT) (reproduction inhibition) was calculated as the endpoint. (IC 50).

Flask Technique

Test cultures were prepaired by innoculating TP from a stock culture into 100ml of PPYS into 500ml fernbach flasks. 15 hours later the cells were treated with the test substances 1 ml samples were removed immediately and then every hour for 9 hours. The samples were fixed with formaldehyde in isotonic buffer and the cellular density was determined using a coulter counter. The doubling time was calculated as graphically by the plotting of the results and then determining the concentration that increases DT by 50%.

Applicant's summary and conclusion

This study provides useful data as to the sensitivity of protozoans to monochloroacetic acid. and can be considered reliable with restrictions.
Executive summary:

Sufficient methodological explination was given and practical aspect of this research can be considered reliable. However this test was not conducted to GLP or to a standard guideline and was lacking the detail of a substance specific study. Therefore can be considered reliable with restrictions.