1918 * Shelf ord; on Acidity Affecting Fishes 99 



2. Materials and methods 



The material used in the experiments was collected by seining in 

 the vicinity of the Biological Station. Four species of fish, the surf smelt 

 (Hypomesus pretiosus [Gir.]), herring {Clupea pallasi C. & V., vivi- 

 parous perch {Cymatogaster aggregatus Gib.) and flat fish (Psettichthys 

 melanosticius Gir.) were the principal ones used. After seining, these 

 were kept in a float with a screen bottom where an abundance of plankton 

 organisms supplied them with food. In addition to the work by the 

 writer, Miss Hall (1918) undertook the study of the effects of acids on 

 the eggs of the large red sea-urchins, while Dr. Powers undertook a 

 survey of the hydrogen ion concentration of the water in the vicinity 

 of the Biological Station, and also a careful study of the differences in 

 hydrogen ion concentrations the fishes could recognize, when produced 

 by the addition of carbon dioxide. Work was done with both sulphuric 

 and sulphurous acids. 



Experiments were performed to determine the resistance and reactions 

 of the fishes to these acids. They were performd in two ways: (1) In 

 standing water, to which the acids were added and which was kept cool 

 by running water surrounding the containers. (b) In running water. 

 Most of the standing water experiments, of which there were a large 

 number, were performed in section dishes with a capacity of about 3 

 liters, and a few experiments in bottles holding from ll^ to 3 liters. 

 These experiments were finally used, however, only as a general guide 

 and the more accurate work was done with running water. In the running 

 water experiments a flow of sea-water was introduced into a bottle through 

 a 3-hole rubber stopper. A flow of acid was introduced into the same 

 bottle through another tube. The general arrangement was as shown in 

 Figure 1. Here a 1 cc. pipette projected into the large chamber of a 

 50 cc. pipette. The 1 cc. pipette was connected with the siphon in the 

 acid bottle and by means of a pinch-cock the flow was adjusted to any 

 desired number of drops per minute. Thus the acid and water were 

 introduced into the bottom of a 200 cc. mixing bottle and left at the top 

 through a tube in the third hole of the stopper. With the water com- 

 paratively free from floating particles, the flow of water and of acid 

 could be maintained for 24 hours or more with little or no adjustment. 

 The tests of fish resistance were made in a large bottle (Fig. 1, D) 

 connected with the mixing bottle and at a temperature only 1° C. or less 

 above that of sea-water. 



The reaction experiments were performed with water to which the 

 acids were added by the same method. The gradient tank used is shown 

 in Figure 2. Water of two kinds were introduced, one at one end and 

 one at the other, and flowed out at the middle so that a gradient in 



