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EC number: 204-881-4
CAS number: 128-37-0
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
I = 1- (B2-Bl)/(A2-A1)
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
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.
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1-ml sample was withdrawn every 15 or 20 minutes, the OD600 was read in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.
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
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.
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
Photobacterium phosphoreum (Vibrio fischeri) is a
bacteria which is found in symbioses with marine organisms.
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