Handbook of Solvents - George Wypych - ChemTech - Ventech!

1098 James L. Botsford
Figure 15.2.2.1. Cyanazine is in units of μl. Cyanazine is a herbicide. The toxicity of cyanazine is: IC50 = [Y/2
-B]/m; Y = 0.35, 0.37 = 0.36, 0.36/2 = 0.18; B = 0.3487 m = 3.5058x10 -3 ; IC50 = 48.1; 48.1μl = ([48.1 x 10 -6 l]/test
tube) x([3300 mg]/l) x [1 test tube]/3.3 ml)= 48.1 μg/ml = 48.1 mg/l = 48.1 ppm.
Since alternative tests don’t involve killing test animals, results are reported as IC50
values (inhibitory concentration, 50%), the concentration that results in death of 50% of the
cells, in 50% inhibition of oxygen consumption, 50% inhibition of reduction of the dye in
the Rhizobium assay.
Cells are grown overnight in a simple defined medium with 0.1% casamino acids.
Cells are collected by centrifugation in a refrigerated preparative centrifuge. Cells are
washed once with 0.01 M (pH 7.5) potassium phosphate buffer and are then diluted in this
same buffer. Volumes of the toxin (usually 5 to 1000 μl) are put into test tubes. The volume
of the toxin is made up to 2.1 ml with water. 0.1 ml of 0.1 M Tris or Bicene buffer, pH 7.5, is
added. The absorbancy at 550 nm is determined. 0.1 ml MTT (the tetrazolium dye) is added
(3.2 mM). The tubes are incubated 20 minutes at 30 o C. The absorbance is read a second
time. The difference in the absorbance at time = 0 and at time = 20 min is determined. The
results are plotted, the difference in absorbance of each tube with a different volume of the
toxic chemical on the X axis versus the absorbance on the Y axis (Figure 15.2.2.1). A regression
line is fit to the plot and if the regression coefficient is less than 0.8, the experiment
is rejected. From the average value of the controls (tubes with no toxin), the Y intercept
forms the regression equation and the slope, the volume of toxin inhibits reduction of the
dye 50% can be readily calculated. For some toxins, better results are obtained if the log of