Figure 4-3. Computed activity of the cupric ion (M) versus 
total copper concentration for two Aquil recipes. 
Note: Chelated with: A) EDTA plus 10' 3 M Tris and B) 10' 5 - 3 EDTA. 
Although the forward kinetic constants of chelate formation are invariably 
very large, resulting in quasi instantaneous kinetics in simple systems, the 
situation can be very different in systems as complex as culturing media. For 
example, when copper was spiked in Aquil cultures of G. tamarensis, a 
dramatic short term toxic response was observed much above that expected for 
the calculated equilibrium activity of the cupric ion (2). This phenomenon 
which was not observed when Tris replaced EDTA as the major copper 
chelating agent, was attributed to the slow kinetics of the metal exchange 
reaction: 
Cu 2+ + CaY ^ CuY + Ca 2+ 
This appears as a reasonable explanation, since the calcium chelate is the major 
form of EDTA in Aquil and the dissociation is slow. No such phenomenon can 
occur with Tris, whose major species in culturing media are the various 
protonated forms of the ligand. This can be checked directly by monitoring the 
cupric ion activity with a mixed sulfide electrode (34, 13) in chemical systems 
similar to the culturing media. Figure 4-4 presents the results of such an 
experiment, and leaves no doubt as to the slow kinetics of copper reaction with 
46 
