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Toxicity to soil microorganisms

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Endpoint:
toxicity to soil microorganisms
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Principles of method if other than guideline:
The inhibition of cell duplication of Bacillus subtilis was measured at different doses for several compounds including BHT. The increase in the cell number per mL was measured by the increase in the optical density at 600 nm.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Details on preparation and application of test substrate:
VEHICLE:
- Chemical name of vehicle: The publications includes studies on several substances among which was BHT. The solvent used for BHT is not specified. However, the solvents used to dissolve the compounds were either ethanol (95%) (the final concen- tration was not more than 1%); dimethylsulfoxide (Sigma Chem. Co.) (the final concentration was not more than 1%); sodium hydroxide (the final concentration was not more than 9 mM); or distilled water.
Test organisms (inoculum):
other: Bacillus subtilis (60015 trpC metC)
Total exposure duration:
60 min
Test temperature:
37ºC
Details on test conditions:
TEST SYST
- Test container (type, material, size): When the bacterial culture reached an OD600 of 0.35, 1-mL samples were placed into Prewarmed small plastic tubes (automated method) or 125 mL Erlenmeyer flasks (Flask method)
- Test conditions:
In the automated method, four controls were run, two with added solvent and two with added distilled water. The tubes were located in an aluminum block and aerated at 37°C through stainless steel tubes. Sixty minutes later the OD600 of the cultures in the tubes were read automatically in a Gilford spectrometer 300N and the results fed into a computer, which calculated the inhibition index.

With the flask method a 1-mL sample was withdrawn every 15 or 20 minutes, the OD600 was read in a Gilford spectrometer 240GH and the inhibition index was determined at 60 minutes after the addition of the inhibitor.

EFFECT PARAMETERS MEASURED
Concentration in mM required for 50 % inhibition in rate of duplication (IC50).

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:
Unspecified. Different amounts of inhibitor (BHT).
Reference substance (positive control):
not specified
Duration:
60 min
Dose descriptor:
EC50
Remarks:
Based on growth inhibtion
Effect conc.:
0.1 other: mM
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth inhibition.

The inhibition of cell duplication of Bacillus subtilis was measured at different doses for several compounds including BHT. The increase in the cell number per ml was measured by the increase in the optical density at 600 nm. The cell number was determined at different times in different culture dishes (all plated at the same time) using a Cytograph. The inhibition was quantitated by an inhibition index defined as :

I = 1- (B2-Bl)/(A2-A1)

where

B is the culture value (OD600 or cell number per plate) in the presence of the inhibitor and A the culture value in the absence of the inhibitor. These culture values were determined at different times after the cells had been exposed to the inhibitor. For B. subtilis, A1 and B1 were measured immediately after addition of the compound, A2 and B2 60 minutes later.

One can characterize the potency by interpolating between the observed values and determining the concentration needed to produce a certain extent of inhibition; in this paper the concentration that produces 50% inhibition of growth was used.

Validity criteria fulfilled:
not applicable
Conclusions:
The concentration of BHT required to inhibit 50 % the growth of Bacillus subtilis was 0.1 mM equivalent to 22.04 mg/L
Executive summary:

A inhibition growth test in Bacillus subtilis was performed with BHT among other substances. The growth of the bacteria was evaluated by optical absorbance at 600 nm. Two methos were used; an automated method and a flask method. When bacterial cultures reached and OD600 of 0.35 different amounts of sample were placed in plastic tubes (automated method) or Erlen meyer flasks which already contained different amounts of BHT. In all samples the amount of solvent used to dissolve the inhibitor was the same. In the automated method, four controls were run, two with added solvent and two with added distilled water. The tubes were located in an aluminum block and aerated at 37°C through stainless steel tubes. Sixty minutes later the OD600 of the cultures in the tubes were read automatically in spectrometer and the results fed into computer which calculated the inhibition index. With the flask method a 1-ml sample was withdrawn every 15 or 20 minutes, the OD600 was read in a spectrometer and the inhibition index was determined at 60 minutes after the addition of the inhibitor. The concentration of BHT required to inhibit 50 % the growth of Bacillus subtilis was 0.1 mM, equivalent to 22.04 mg/L.

Description of key information

Toxicity of BHT to microorganisms was studied using the test organisms: P. fluorescens, Tetrahymena pyriformis and Photobacterium phosphoreum (Vibrio fischeri).

The 30min-EC50 of BHT to Photobacterium phosphoreum based on light emission is 8.98 mg/L.

In a growth inhibition test with P. fluorescens, the EC50 was greater than the highest tested concentration of 50 mg/L (Trevors et al., 1981).

A 24-h EC50 of 1.7 mg/l has been determined for the protozoa Tetrahymena pyriformis (Yoshioka et al., 1985). This is the lowest available effect value which was determined for BHT.

It can be assumed that 2,6-di-tert-butyl-p-cresol is not toxic for soil microorganism up to the limit of water solubility.This conclusion is supported by a growth inhibition test on Bacillus subtilis(soil bacteria) where the 1h-EC50 was 22.04 mg/L, well-above the water solubility of the test subtance.

Key value for chemical safety assessment

Additional information

P. fluorescens is a bacteria which may be found in soil and water

Tetrahymena pyriformis is a protozoa . Protozoa are eukariotic cells and ubiquitous in the aquatic and terrestrial environment.

Photobacterium phosphoreum (Vibrio fischeri) is a bacteria which is found in symbioses with marine organisms.