DEPARTMENT OF MARINE BIOLOGY. 213 



natural sea-water, even when all other factors were kept constant, and it was 

 thought that the use of an artificial sea-water with which some preliminary 

 tests had been made would obviate this difficulty. Accordingly, a large 

 quantity of artificial sea-water was made up, following a formula based upon 

 F. W. Clarke's analysis of the sea-water at Tortugas, i. e., 100 c.c. of 0.6 m 

 NaCl+17 c.c. of 0.4 m MgCl2+3.5 c.c. of 0.4 m CaCl2+2.1 c.c. of 0.62 w KCl. 



In the report upon the experiments with this artificial sea-water last year, 

 it was stated that the behavior of the spermatozoa of Strombus in this medium 

 corresponds very closely with that in natural sea-water. This, however, is 

 not exactly the case, as was shown this year by an exhaustive series of com- 

 parative tests. It was very clear that in artificial sea-water the sperma- 

 tozoa do not become as quickly active nor do the}^ reach as great a degree of 

 activity as they do in natural sea-water. On this account it was found to be 

 impossible to use the artificial sea-water as contemplated. When a culture 

 is started under standard conditions in natural sea-water, the eupyrene 

 spermatozoa reach a maximum degree of activity within 3 to 5 minutes and at 

 the end of that time they are either largely or entirely loosened from the tufts 

 in which they He in the sperm-ducts. Eventually they all become freed and 

 swarm about freely. When a culture is started in artificial sea-water, the 

 eupyrene spermatozoa become active very much more slowly (10 to 15 minutes) 

 and only about 50 per cent become loosened from the tufts. Unless practically 

 all the spermatozoa are free and active, it is impossible to interpret accurately 

 any mass reaction on their part to a bubble of gas introduced into the culture. 

 It is interesting to note that no significant difference in the behavior of the 

 apyrene spermatozoa in natural and in artificial sea-water was observed. 



It is evident that there is something lacking in the artificial sea-water 

 which is present in natural sea-water and which causes full activation of the 

 spermatozoa. At present there is no known method whereby an artificial sea- 

 water can be made up which will resemble natural sea-water in this respect. 

 Furthermore, it has been found that, so far as the spermatozoa of Strombus are 

 concerned, the addition of a small percentage of natural sea-water to a volume 

 of the artificial solution will not rectify the latter. However, when sea-water 

 was added in increasing proportions, it was found that the behavior of the 

 spermatozoa became more and more as it is in pure natural sea-water. For 

 this reason it was suspected that the element that is so essential for the full 

 activation of the spermatozoa is not an enzyme, but rather the pecuhar chem- 

 ical balance, not fully understood, which is known to exist in natural sea-water. 



A series of experiments was consequently undertaken in order to deter- 

 mine, if possible, whether there is an actual variability in the alkalinity and 

 acidity of sea-water and, if there is, whether it corresponds to the variation 

 which had been observed in the behavior of the spermatozoa in cultures made 

 in pure sea-water under constant conditions. For this purpose samples of 

 sea-water were titrated to phenolphthalein by a special method in which the 

 procedure was always kept constant. The samples were varied as regards 

 temperature, atmospheric conditions, and locality. After titration a culture 

 was made from the sample and observations were made upon the time which it 

 took the spermatozoa to become fully active and the degree of activity reached. 

 The results of these experiments show that, under the conditions of the test, 

 sea-water does vary. It was, however, impossible to perform a sufficient 

 number of experiments to determine whether this variation is correlated with 

 changes in temperature, tide, time of day, or locality. These variations are 

 slight and it is questionable whether they could have any effect upon the 

 behavior of the spermatozoa. Such data as was obtained from the compara- 

 tive cultures would also indicate this, although hardly sufficient upon which 

 to base a definite statement. 



