April 18, 1919] 



SCIENCE 



383 



2-liter glass-stoppered bottle. After the finely 

 divided undissolved oil had settled, the clear 

 liquid from the middle of the solution was 

 drawn off, and the action of this saturated 

 solution upon the recently fertilized mature 

 eggs was tested, using varying dilutions (e. g., 

 1/2, 1/4, 1/8, 1/16 saturated) and varying 

 times of exposure (from one fourtli minute to 

 an hour or more). The eggs were exposed to 

 the solutions in glass-stoppered bottles, and at 

 intervals portions were transferred by pipette 

 to dishes of normal sea-water; this water was 

 changed when the eggs had settled. The sub- 

 sequent course of cleavage and development, 

 as compared with that of untreated " control " 

 eggs, was carefully studied. 



The toxicity of " mustard " solutions pre- 

 pared in the above mannej is not constant but 

 decreases with standing, and the more rapidly 

 the higher the temperature. Solutions made 

 at room temperature (20-24°) always prove 

 strongly toxic if used immediately after prep- 

 aration; if used later the toxic action is less 

 marked, the decline of toxicity being rapid in 

 the first hour and more gradual later. This 

 decline is due to the progressive hydrolysis of 

 the " mustard," which breaks down rapidly in 

 aqueous solution, yielding HCl and residual 

 comx>ounds of low toxicity. The toxicity of a 

 " mustard " solution two days old, in which the 

 acid freed is neutralized by NaOH, is not 

 more than one fiftieth of that of the freshly 

 prepared solution, as measured by the com- 

 parative times of exposure required to pro- 

 duce a definite impairment of development or 

 a definite proportion of dead eggs in a given 

 time. The attenuation of toxicity, as thus 

 shown by the physiological action of the solu- 

 tion, exhibits a general parallelism with the 

 Tiroduction of HCl, as measured by titration 

 (with dibromocresolsulphonephthalein as in- 

 dicator). The essential toxic action is thus 

 due to the undecomposed " mustard " in the 

 solution. This conclusion was confirmed by 

 experiments in which the hydrolysis of the 

 compound was retarded by cold. The oil was 

 shaken with ice cold sea-water (below 3°), the 

 solution was filtered free from the residual 

 undissolved crystals of " mustard " (which is 



solid at this temperature), and the cold satur- 

 ated solution thus obtained was kept at 0° 

 (surrounded by ice in the refrigerator). The 

 toxic action of a portion of the solution kept 

 thus cold and brought to room temperature 

 immediately before adding the eggs was com- 

 pared with that of portions which were 

 brought to room temperature and allowed to 

 stand for varying times (e. g., 1/4 hour, 1/2 

 hour, 3 hours, 24 hours) before using. In all 

 cases solutions which were kept cold imtil just 

 before using were decidedly the most toxic, 

 15 minutes' exposure to room temi>erature re- 

 duces toxicity by about one half, and 30 min- 

 utes by two thirds or three quarters. The 

 decline in toxicity is thus at first rapid, then 

 more gradual; the same is true of the pro- 

 duction of acid as shown by titration. The 

 reaction is apparently mono-molecular. 



Our experiments favor the following con- 

 ception of the mode of action of " mustard " 

 upon the living cell. The imdecomposed 

 " mustard gas " is slightly soluble in water 

 (according to our titrations of completely 

 hydrolyzed solution to the extent of ca. .05 per 

 cent.). This dissolved "mustard" readily 

 penetrates the cell, presumably because of its 

 high lipoid-water partition-coefficient, and col- 

 lects in relatively high concentration in the 

 organic solvents of the protoplasm (cell- 

 lipoids, fats, etc.). In this situation it serves 

 as a reservoir of toxic material which con- 

 tinually enters solution in the aqueous phases 

 of the protoplasm and is continually being 

 there decomjwsed. Since by its hydrolytic 

 decomposition it yields acid, the dissolved 

 " mustard " acts destructively on the proto- 

 plasm as soon as the available buffer com- 

 pounds (which normally prevent protoplasmic 

 hyper-acidity) are exhausted. The destructive 

 action is thus due primarily to the HCl freed 

 by hydrolysis. The other decomposition-prod- 

 ucts are only slightly toxic; this we have 

 slioAvn experimentally by comparing the action 

 of partially or wholly hydrolyzed solutions of 

 the "mustard," from which the acid was re- 

 moved by neutralization with NaOH, with 

 that of the unneutralized solution. The lat- 

 ter solution is always by far the more toxic; 



