34 U. S. BUREAU OF FISHERIES. 
manipulations required for this experiment, consumed much time. 
Consequently the end of the breeding season for oysters came before 
there was opportunity to work out the relations of all these factors 
in an entirely quantitative manner. 
Results may be summarized as follows: 
With regard to the condition of the eggs, it was observed that 
embryos from eggs of which 50 per cent or more developed quickly 
to a free swimming stage withstood deprivation of oxygen better 
than those from eggs of which only a small proportion developed. 
With respect to age, it appeared that younger larve endured com- 
parative oxygen deprivation slightly longer than older ones, but the 
experiments were not conclusive. 
Tt was found that embryos show some effect of oxygen lack in 
water containing 25 per cent of saturation of oxygen at 24° C.and a 
markedly increased mortality in 10 per cent of saturation. Killing 
time varied from 12 to 30 hours according to extent of oxygen de- 
pletion. At 28 to 30° C. embryos die much more rapidly from oxygen 
starvation than at 22 to 24° C. . 
Carbon dioxide seems to have a toxic effect other than that due to 
hydrogen ion concentration. Embryos died earlier in water con- 
taining sufficient oxygen but excessive CO, than in water of the same 
oxygen content and with the same Py obtained by adding lactic acid. 
Investigation of the effect of pollution on satisfactory development 
of eggs in gonads was also conducted. Results showed that from 
oysters, kept 7 to 10 days in water polluted so as to markedly lower 
its oxygen content, only an abnormally small proportion of eggs 
could be made to develop after artificial fertilization, conducted ac- 
cording to standardized technique, and that the embryos so obtained 
did not develop as far as controls. 
An incidental observation proved of considerable interest. Oysters 
which had been in cold storage 24 days for the purpose of studying 
greening were found to have very full gonads. Eggs fertilized with 
sperm from some of the same lot of oysters developed satisfactorily, 
and many of the embryos reached the primitive shell stage. Some 
of them, indeed, were used in oxygen-requirement tests. 
Observations on the cause of copper greening confirmed previous 
conclusions by showing that not only the amount of copper obtain- 
able by the oysters, but also conditions of habitat determine whether 
or not visible green spots occur. Marked appearance of greenness 
after one or more weeks in cold storage was observed. It has been 
twice noted that with gonads filled a smaller proportion of oysters 
become green during storage than in other conditions. ‘Two-year- 
old spent oysters, set and reared in Wareham River, did not become 
green during four weeks in cold storage. This is in contrast to 
oysters which at some time during their previous history have been 
in waters known to produce greenness (near Bridgeport and New 
Haven). 
Experiments both in the laboratory and in the field indicated a 
positive influence of diminished oxygen supply in causing the ap- 
pearance of green spots. Repeated observations of marked greening 
of oysters in polluted tanks of sea water were also made, thus con- 
firming the similar results obtained during the previous summer in 
the Seekonk River. 
