+ 

 O 



o 



Figure 4-3. Computed activity of the cupric ion (M) versus 

 total copper concentration for two Aquil recipes. 



Note: Chelated with: A) IQ-^-^M EDTA plus IQ-^M Tris and B) lO'^-^ 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 equihbrium 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-"^ + 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 resuhs of such an 

 experiment, and leaves no doubt as to the slow kinetics of copper reaction with 



46 



