312 L. H. HYMAN. 



at the same hydrogen ion concentrations. As these conditions 

 did not obtain in my experiments, I considered it unlikely that 

 the findings of Clowes applied to them. It seemed necessary to 

 me, however, that the matter should be tested by experiment. 

 A new series of experiments was therefore begun in the fall of 

 1924 using carbonate-free water. The results obtained con- 

 vinced me that the depressing action of acidified water is largely 

 due to the carbon dioxide liberated in it. 



In connection with these experiments a large number of trials 

 in ordinary carbonate-containing water were carried out as 

 controls. These constituted repetitions of the work of the 

 previous year. It was very puzzling to me to find that all acids 

 were less effective than had been the case in the preceding year. 

 This was particularly noticeable at the lower concentrations, 

 pH 7.5 to 7.0. The mineral acids produced about the same 

 depressing effect as previously at the higher concentrations, 6.5 

 to 5.0. The organic acids (acetic and butyric were the only 

 ones tried) were also markedly less effective than had previously 

 been the case, at all concentrations tried. I am unable to 

 explain this state of affairs except on the assumption that the 

 carbonate content of the water had decreased in the meantime. 

 The fact that in many cases the controls gave a smaller percentage 

 of depression than previously made it difficult to arrive at 

 conclusive results. 



i. Preparation of Carbonate-free Water. Carbonate-free water 

 was prepared according to the method of Smith and Clowes 

 ('24&). Two cc. of concentrated hydrochloric acid were added 

 to eight liters of the well water in a large bottle. Air from the 

 compressed air system was passed through the water in the 

 bottle for 24 hours or more. This treatment was found to remove 

 the carbonate from the water completely. Several times liter 

 samples of this water were evaporated to dryness and the residue 

 tested for carbonate with entirely negative results. In some 

 cases borax was added to the carbonate-free water to serve as 

 buffer but as the results were not affected by this procedure, it 

 was generally omitted. The oxygen consumption of the worms 

 in this carbonate-free water, with or without borax, was re- 

 peatedly compared with that in the normal unaltered well water. 

 No difference was found. 



