404 
PACIFIC SCIENCE, Vol. XIV, October I960 
During this period the environmental tempera- 
ture was 25-30° C The tolerance of toad per- 
itoneal cavity to distension by the volume of 
injected homogenate was estimated at about 
8 ml. of homogenate for 100 g. of toad. This 
set the upper limit of the doses at 8 /d. of sea 
anemone per gram of toad. 
Survival time was measured as the elapsed 
time, in hours, from the moment of injection 
until death. In recording this, actual survival 
time was rounded out from the half-hour mark 
to the nearest full hour. Observations were not 
extended beyond 48 hours after injection. The 
lowest dose which caused death in a high pro- 
portion of toads within this period of time was 
found to be 1 gl/g. 
EXPERIMENTS AND RESULTS 
1. Basic experiments showed the following 
results : ( 1 ) Toads of different weights, ranging 
from 30 to 100 g., were equally susceptible to 
the poison. (2) Various amounts of diluent 
added to the homogenate did not affect the dose 
effect curve. (3) Toads did not die after in- 
jection of any of the following: (a) a clear 
fluid which spurts from the sea anemone upon 
touch and slight pressure; (b) a jelly-like sub- 
stance which the sea anemone secretes upon 
exposure to air; (c) sea water. (4) Homoge- 
nates which were dialyzed with running chlo- 
rinated rain water of pH 6 for 12 hours at 
22° C. yielded the same dose effect curves as 
the fresh material of the batches from which 
they had been taken. 3 It was noticed that the 
pungent smell of the original material disap- 
peared during the dialysis. ( 5 ) Homogenates 
which had been heated in a boiling water bath 
for 15 minutes caused no mortality among in- 
jected toads. 
2. For evaluation of "fresh” homogenates, 
twelve batches of R. howesii, harvested on nine 
irregularly spaced days, were assayed. Figure 1 
summarizes the dose effect curves of these 
assays. 
A total of 225 toads was injected with doses 
of 2, 4, and 8 /xl/g. Eight of them, with the 
three doses distributed at random, survived for 
3 pH was estimated with nitrazine paper. 
more than 48 hours. A total of 75 toads was 
injected with 1 gl/g, and 20 of these survived 
for more than 48 hours. Only the data from 
toads which died within less than 48 hours 
were used for Figure 1. 
The data of Figure 1 are derived from dose 
effect curves from batches which were harvested 
during all seasons of the year over a period of 
14 months. Included were batches from a yellow- 
brown and a dark blue variety of R. howesii, 
both of which occur on the reefs of American 
Samoa; from each of two different colonies of 
sea anemones which grew a mile apart from 
each other; and from a colony which had been 
transplanted from its normal habitat on the 
reef to the shore line of the lagoon and kept 
there for 6 months. The narrow range of var- 
iability of the dose effect curves indicates that 
the poison content of R. howesii is not subject 
to seasonal variations and that it is the same in 
varieties of two different colors, in colonies of 
different locations of the area, and in colonies 
transplanted under the experimental conditions. 
3. Evaluation of effects of preserving proce- 
dures: Two portions of a given batch of sea 
anemone were assayed to establish dose effect 
curves of the fresh material (controls). Two 
other portions of the same batch were preserved 
under a given condition and assayed after 8-14 
days of preservation ( experimental series ) . Dose 
effect curves of the experimental series were 
compared with those of the controls. The sig- 
nificance of differences of mean survival time 
at any one dose (Burn et al . , 1950) was esti- 
mated by the t test. The 2 per cent level of P 
was taken as the limit of significance. 
4. Experiments with preserved homogenates: 
( 1 ) Sea anemones were mixed with their weight 
of sodium chloride. A 5 per cent sodium car- 
bonate solution was added until the mixture 
showed a pH of about 8. The mixture was kept 
at 25-30° C. for 8 days, after which it was 
dialyzed and assayed. It was found that the 
survival times, as compared with the controls, 
were significantly prolonged at doses of 2-8 
fxl/g and that there was no mortality at the 
dose of 1 /xl/g. (2) The same experiment was 
done with a 2 per cent HC1 solution added un- 
til the mixture showed a pH of about 5. It gave 
the same results as the experiment at alkaline 
