212 
slowly added, and after a few minutes the entire 
mixture is quickly filtered. The residue is then 
rinsed with an additional 100 ml. of acetone. 
The acetone extract, containing the tissue "wa- 
ter” from the digestive gland is then placed in 
the flask of a rotary evaporator, partial vacuum 
being supplied by an aspirator. As the percent- 
age of acetone is reduced by evaporation, the 
apolar fraction is thrown out of solution and 
deposited on the flask wall. When the remain- 
ing "water” has been evaporated to about 50 
cc., it is placed in a refrigerator overnight, after 
which it is refiltered and becomes what is re- 
ferred to as the crude extract. 
The routine bio-assay procedure for indicat- 
ing relative strength of extracts in the toxic 
principle consists of injecting 1 ml. of an ex- 
tract intraperitoneally into mice of similar 
weight and observing the death time to the last 
heartbeat audible with a stethoscope. The death 
time serves as a rough indication of the relative 
toxicity. 
Initial LD 50 values were obtained by con- 
ventional methods using three groups of mice 
to establish three points on a graph plotting 
percentage mortality against dose given. The 
LD 50 values were then read from the graph. 
Subsequent LD 50 approximations and those for 
the chick were determined using a minimum 
number of animals by the "up and down” 
method proposed by Dixon (1959)- Since the 
LD 1-99 range is very narrow, this latter method 
gave adequate results for the present purposes. 
EXPERIMENTAL RESULTS 
The average percentage weight of the diges- 
tive gland/ovotestis complex of A. calif ornica 
based on 15 specimens was 10.8 per cent, and 
the range was from 8.4 to 14.0 per cent. Vary- 
ing amounts of sand were found in the intes- 
tines of this species, which influenced the ac- 
curacy of the weights. The accuracy was higher 
for A. vac curia, however, since no sand was 
found in the intestines. The average percentage 
based on 5 specimens of the latter species was 
19.4 per cent, ranging from 18.0 to 20.3 per 
cent. 
The diet varied between the two collecting 
sites. At Doheney Beach the diet followed the 
predominant flora consisting of several coralline 
PACIFIC SCIENCE, Vol. XV, April 1961 
algae and Hypnea calif ornica. A considerably 
wider variety of seaweeds was noted in the 
Lunada Bay collections. The Lunada Bay sea 
hares appeared to be more toxic but this may 
have been the result of more maturity or merely 
a reflection of variation in the extraction ef- 
ficiency. No correlation is possible at the pres- 
ent state of the research. 
Aplysia vaccaria , a sea hare rather distantly 
related within the genus, possesses a similar 
toxin in its digestive gland. 
The LD 50 was determined for 23-gm. mice 
and is expressed in grams of digestive gland 
tissue. Two different batches of raw material 
were used to obtain two somewhat removed 
values. Each batch represents the material ob- 
tained from 5 to 12 sea hares, depending on the 
animals’ size. One batch collected May 4, 1959, 
had an LD 50 value of 0.65 gm. tissue for a 23- 
gm. mouse (0.028 gm/ gm body weight). An- 
other collected at the same location July 28, 
1959, had a value of 0.8 gm. per 23-gm. mouse 
(0.036 gm/ gm weight). However, the differ- 
ence in the two values may represent only dif- 
ferences in extraction efficiency rather than true 
variation in toxin concentration. 
The LD 50 for 3 -day-old baby chicks was 
found to be only slightly less than 25 per cent 
more than that for mice. 
When mice are injected intraperitoneally 
with the crude toxin of somewhat more than 
the LD 50 dosage, there is an almost instantane- 
ous hyperventilation. Ears are drooped and the 
mouse usually sticks his nose in a corner of the 
cage and salivates profusely. After a varying 
time in which hyperventilation is evident, the 
mouse starts scurrying about the cage, usually 
leaving a trail of urine. Perhaps it then returns 
to its corner or begins to demonstrate occa- 
sional muscular twitching which may turn into 
uncontrolled attempts at movement suggesting 
a convulsion. This uncontrolled movement may 
develop in waves and once begun is always 
terminal. Ataxia and inability to right itself 
usually develop before or during these uncon- 
trolled movements. The animal passes into a 
completely relaxed state. The heart continues 
for some time at a reduced pace, gradually be- 
coming weaker until it can no longer be heard 
with a stethoscope. The toxin also killed mice 
