330 HELEN DEAN KING 
conditions to which the eggs were subjected during the early 
stages of their development. 
The results obtained in this experiment might seem to indicate 
that increasing the amount of water in the egg at the time of 
fertilization has no influence whatever on the process of sex- 
determination; but there is another possible interpretation of 
them which seems worth considering. The ripe eggs of the toad 
are surrounded by two membranes and embedded in a thick, 
jelly like substance. It is therefore possible that when eggs are 
fertilized in distilled water the osmotic pressure on them is, for 
some little time, practically the same as that to which eggs are 
subjected when they are fertilized under natural conditions. If 
this be so, the results of this experiment give no evidence what- 
ever regarding the effects on the sex ratio of increasing the water 
content of the eggs during the period of fertilization. 
Although the sex-determining mechanism was not affected by 
the distilled water, some change was produced in the eggs which 
had a decided influence on their later development. The tad- 
poles belonging to this lot were very small, and their development, 
although apparently normal, was so retarded that they were the 
last of all of the individuals in the various series of experiments 
to undergo metamorphosis. 
None of the experiments in which an attempt was made to in- 
crease the water content of the zygote have given results that 
could be considered as conclusive. It is suggestive, perhaps, that 
in every instance a relatively low percentage of females has been 
obtained; but other methods of experimentation will have to be 
employed before it will be possible to determine whether increas- 
ing the amount of water in the eggs at the time of fertilization 
really leads to an alteration of the sex ratio. 
With loss of water 
It would seem to be an easy matter to reduce the water content 
of the zygote by fertilizing the eggs in a hypertonic solution and 
allowing them to remain in the solution for a considerable length 
of time. Unfortunately the spermatozoa of Bufo are very easily 
injured, and even 1 per cent solutions of salt or of sugar render 
the great majority of them incapable of fertilizing the eggs. In 
