Handbook of Solvents - George Wypych - ChemTech - Ventech!

1108 James L. Botsford
the test is at least an order of magnitude more sensitive to pentachlorophenol than any other
test found.
15.2.2.7 Toxicity of divalent cations
Reduction of the dye is inhibited by divalent cations in the Rhizobium system. Common
ions, calcium and magnesium, inhibit the reduction of the dye. The toxicity of the ions is
shown in Table 15.2.2.5. Mercury and cadmium, generally thought to be the most toxic
minerals were the most toxic with this assay. Calcium and magnesium are also toxic. Water
and soil samples typically contain calcium and magnesium so in order to analyze water and
soil samples for toxic organic chemicals, a method to eliminate this inhibition by metal ions
was sought.
Table 15.2.2.5. Toxicity of divalent cations
Minerals n var. m moles Minerals n var. m moles
Ba +2
6 33 0.109 Mg +2
9 20 0.404
Cd +2
12 41 0.004 Mn +2
11 35 0.045
Ca +2
11 33 0.05 Hg +2
10 23 0.0006
Co +2
11 46 0.009 Ni +2
11 11 0.452
Cu +2
13 19 0.007 Se +2
9 39 1.849
Fe +2
7 18 0.587 Zn +2
10 7 0.062
14 27 0.098
Fe +3
n = number of samples tested; var. = variation, standard deviation divided by the mean. Values reported as m moles
l -1 . All minerals except ferrous ion as chloride salts. Several were tested as both chlorides and sulfates with little
difference. When ferrous sulfate was tested, it was made up fresh each day before the assay.
EDTA is used routinely in biochemistry to chelate divalent metal ions. It was thought
this might chelate the calcium and remove it from the system. EDDA and EGTA are also
used and are thought to chelate calcium more effectively than EDTA. Neither of these
chelators affected the reaction, both were simply slightly toxic (about 3000 ppm). A series
of experiments were run and it was found that the inhibition of reduction caused by 1.4 to
1.6 μmoles of calcium was relieved by 1 μmoles EDTA. There is not a stoichiometric relationship
between EDTA and the metal ion. It is not simply chelating the metal ion.
It was found that 2.5 μmoles EDTA would eliminate the toxicity of all the ions at their
IC50, the concentration of cation that inhibited reduction of the dye 50%. Thus 2.5 μmoles
EDTA would eliminate toxicity from 5.5μmoles calcium but only 0.006μmoles mercury. It
was observed initially that the toxicity of most organic chemicals could be determined in the
presence of 2.5 μmoles EDTA. This suggested there could be two mechanisms involved in
the reduction of MTT. One is inhibited by toxic organic chemicals and the second is inhibited
by divalent cations.
15.2.2.8 Toxicity of organics in the presence of EDTA
A series of experiments were run looking at the toxicity of organic chemicals in the presence
of 0.74 μmoles calcium (25 ppm, this concentration inhibits reduction of the dye completely)
and 2.5 μmoles EDTA. Four of the 35 chemicals tested had greater toxicity with
EDTA and calcium than in the controls. Two chemicals were no longer toxic. The toxicity
of 16 of the chemicals was not affected by the calcium and EDTA. The toxicity of 13 chemicals
was decreased by at least 10% but was not eliminated by the addition of EDTA and cal-