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THE OSMOTIC PRESSURE OF SEA WATER AND
OF THE BLOOD OF MARINE ANIMALS.
INCLUDiNG SOME OBSERVATIONS ON THE PERMEABILITY OF ANIMAL MEMBRANES.'
WALTER E. GARREY.
Experimental work on the relation of salts and other substances in solution to the life processes of marine animals requires an accurate knowledge of the osmotic pressure of both the sea water and the body fluids of the animals. This knowledge should be supplemented by definite information relative to the permeability of the membranes of the animals under investigation. Thus far this field has been neglected by American investigators. It has been assumed that local conditions are similar to those existing along the European sea board. Owing to the character of the work in our Marine Laboratories it seemed advisable to make some observations which would place our knowledge of local conditions on a firmer basis.
METHOD. Until recently our knowledge of sea water and animal fluids has been based solely on quantitative chemical analyses. F. Bottazzi' at Naples in 1897 calculated the osmotic pressure from the depression of the freezing point. This method has since been used,by several investigators working in the same field (Rodier, Quinton, Frédéricq)and was adopted as most con venient for our purpose. Employing the Beckmann apparatus the freezing point is determined. For aqueous solutions a freez ing point below that of distilled water signifies the presence of some substance in solution which is exerting an osmotic pres sure. This pressure calculated for 00 C. is equal to about twelve
‘¿ Theseinvestigations were made at the Marine Biological Laboratory, Woods Holl. Mass. •¿ Theywere reported to the Biological Seminar, August 12, 1904. Investigations conducted on the Pacific Coast will be reported later as they are still in progress. 257
258 WALTER E. GARREY.
atmospheres for a depression of one degree centigrade (@ @0C.). The depression of the freezing point is designated 4. Owing to the super-cooling which takes place in solutions as strong as those with which we are dealing, inconstant results are obtained unless freezing is induced by innoculation with a tiny crystal of ice as soon as supercooling of about three tenths of a degree has taken place. A few earlier experiments in which this technique was neglected have not been recorded here. Invertebrate blood clots slowly and the first clot is easily broken up so that the freezing point of fluid as a whole may be determined. Teleost blood was whipped before freezing but no attempt was made to remove the corpuscles in as much as it has been found that they exert no appreciable effect upon the freezing point (Hamburger “¿ ,Hedin 12).
OSMOTIC PRESSURE OF SEA WATER. The animals worked with at Woods Hole were obtained from so many different localities that it seemed advisable to determine the freezing point of the water from the same sources. The re sults of these determinations are given in Table I.
TABLE I.
@ Buzzard's Bay. Basin the U. Laboratory Tap. “¿ EelPond.―
Maximum @X —¿ 1.835° —¿ 1.84° —¿ 1.84° —¿ 1.82° Minimum @. —¿ i.8i —¿ i.8o5 —¿ i.8z —¿ 1. Average .@. —¿ i.8x8 —¿ 1.82 —¿ 1.82 —¿ 1. Eleven samples. Twenty-three Forty samples. Eight samples. sampies. _______ The slight variations noted in the concentration of the different samples of sea water are not due to errors in observation for each sample of sea water was tested repeatedly and the results were checked by the use of three thermometers. The variations may be explained by the more or less land-locked condition of the bodies of water, the concentration being continually, though slightly, altered by the tides and by the continued advent of fresh water. After one extremely heavy rain the water of the labora tory tap showed considerable dilution 4 being —¿1.78°. “¿ Eel Pond― water also was diluted till the freezing point was only —¿ 1.700.
26o WALTER E. GARREY.
The osmotic pressure of the blood and body fluids of inverte brates is due exclusively to the salts which are in solution, the proteid molecules being so large that they exert no appreciable osmotic pressure: Analyses made by L. Frédéricq7show that the salts in the blood of a large number of invertebrates are pres ent in the same concentration as in the sea water. Although the blood of selac/zians has a freezing point approximately the same as that of the sea water, the salts are present in much smaller amount, i.6 per cent. to 2.3 per cent. according to different analyses. The high osmotic pressure is maintained by the presence of a large and variable amount of urea (2—3per cent., V. Schroeder, Quinton,'° Rodier,2' Frédéricq9). Teleosts. —¿ The blood of all teleosts examined showed a low osmotic pressure which, in round numbers, approximated one half that of the sea water.* In Table III. are given the extreme variations in the freezing point for individuals of each species.
TABLE III. Source of Blood. Maximum .@. Minimum @. i. Sword fish Heart after death. —¿o.q6° —¿0.90° -2. Tautog. (Thutciça onzi!is) Branchial artery. —¿ o.86 -— o.
- Squeteague (Ci'noscion reçzlis) “¿ “¿ —¿ 0.864 —¿ o.
- Conger eel “¿ “¿ —¿ 0.82 —¿o.8o
- (.Tominon eel (Anç-nil/a chiysi'pa)@ “¿ “¿ —¿ 0.90 —¿ 0.
The results of all these investigations on marine animals agree with those of F. Bottazzi' and of Frédéricq9at Naples. These investigators found that invertebrate blood froze at —¿2.03° as did also that of selachians, while teleost blood showed I = —¿ 1.04°. The slightly greater depression of the freezing point found by these authors is to be accounted for by a greater con centration of the sea water at Naples than at Woods Hole.
VARIATIONS IN OSMOTIC PRESSURE OF THE BLOOD DUE TO CHANGES IN THE CONCENTRATION ON THE EXTERNAL MEDIUM. The analyses of L. Frédéricq7showed that the concentration of the salts in the blood of invertebrates varied with the concentration *** Incidentally it was observed that the red corpuscles of teleosts were crenated by** ‘¿ eawater.
OSMOTIC PRESSURE OF SEA \VATER. 26!
of the salt water from which the animals were taken. This was most strikingly shown by the blood of Can-inns meenus taken from brackish water and from sea water. In the course of our investigations it was found that differences in the freezing point of the blood of invertebrates accompanied differences in the freez ing point of the sea water from which they were taken; thus the blood of lobsters taken from the “¿ basin―traps showed 4 = —¿ 1.820, but when taken from the “¿ eel pond― 4 = —¿ 1.77°. A decrease in the concentration of the water from the laboratory tap, due to severe rains, caused exactly the same changes in the freezing point of lobster's blood, I became —¿1.78°. An increase ill the osmotic pressure of the blood of Lizizulus was induced by a two days' exposure to the drying influence of the atmosphere. The freezing point went down to —¿1.90°. The blood of a Limulus kept alive for two weeks in a damp cellar froze at —¿ 2.030, the normal I = —¿ 1.82°. Both diurnal and seasonal changes occur in the concentration of the water of San Francisco Bay, Cal. (taken near the Golden Gate), and the perivisceral fluid of starfish shows exactly the same changes; thus on March 17, I904, I = —¿ 1.470 at high tide but at low tide only —¿ 1.3850. On September 23, 4 = —¿1.800. With these facts as a starting point it was decided to test the freezing point of the blood when the animal was subjected to a large decrease or increase in the osmotic pressure of the external medium. Dilution of the sea water was first tried and after a longer or a shorter immersion the animal was removed and the freezing point of the blood was determined. The changes which are thus induced are given in Table IV. In nearly every experiment the animals were kept in the dilute medium until collapse set in, but in a majority of cases they were able to revive when replaced in normal sea water. Limulus and Sycotypus are particularly hardy and it is noted that the freezing point of their blood changes very quickly until in some cases it is approximately equal to that of the external medium. When the external medium is very dilute death may occur before this equalization takes place; this was particularly true in the case of Homarus, w-hich is very susceptible to a change in the con- /
OSMOTIC PRESSURE OF SEA WATER. 263
in the osmotic pressure, viz., the interchange of water, and of salts. The entrance of water is proven by the enormous increase in weight, and the swollen appearance of those animals which have been placed in diluted sea water. This swelling has already been referred to (Table IV.) as noticeable in Asterias and the two worms Nereis and Clia/topterus. In one experiment in which a Limulus was kept for sixteen hours in fresh water the animal became so swollen that the gills burst and the water of the aquarium be came blue from the hi@emocyanin of the exuded blood. The blood of the animals subjected to diluted media became notice ably less viscous and owing to its increased volume and the high internal pressure, was easily obtained from the animal. When subjected to concentrated sea water it was often difficult to secure sufficient blood from the lobster to make the desired determi nations. That an exchange of salts also takes place, although far less rapidly than the exchange of water, is shown by the fact that when the animals are placed in distilled water, chlorides are eliminated and a copious precipitate is obtained upon the addi tion of silver nitrate. No quantitative chemical analyses of the aquarium water were made but in one such case an increase in. the osmotic pressure was indicated by the change in the freezing point; Limulus was the animal experimented with and after twelve hours' immersion the freezing point of the aquarium water had been lowered from —¿0.02° to —¿0.23°. Q uinton,'8' 19 experimenting with Aplysia, has also found an in crease in weight when the animal is subjected to dilute sea water and a loss in weight in concentrated solutions, and he has further, by chemical analyses, found loss and gains in the amount of salts in the blood of this animal just equalling the respective gain or loss from the aquarium water. Frédéricq3,7,8, made similar analyses. There are many other proofs of the permeability of the inverte brate membranes to various salts. Loeb's ‘¿ @experiments on the rhythmic contractions of medus2e (Gonionernus) indicade the per meability to NaC1, CaC1,, and KC1. The death of invertebrates is easily induced, by acids and the salts of the heavy metals. Frédéricq has placed potassium ferrocyanide and nitrates in the
2')4 \VALTER E. GARREY.
aquarium water and later obtained positive tests for these sub stances in the blood of Carcinus rna'nus. We may conclude then that the membranes of marine invertebrates as a class are per meable both to water and to salts and act exactly “¿ likea dialyzer membrane.― The path taken by the exchanged material is not so certainly known. Frédéricq6 assumes that the branchial membranes of Carcinus iius'nus are the permeable structures but publishes no evidence supporting the view. Quinton 19 takes it for granted that it is the external wall which is permeable, and this seems to be true for Aplysia, the form with which he worked, as has been shown by Ph. Bottazzi and P. Enriquez.' That the outer wall is the permeable structure of worms may be shown by a very simple experiment performed by the author on Nereis and Clia/topterus. Ligatures were passed about the animals close to either end and drawn tight thus completely closing the alimentary canal; care was taken to avoid abrasion of the integument. When placed either in fresh water or dilute sea water swelling and increase in weight was obtained. Limulus is an animal on which the permeability of the gills may be easily demonstrated. These structures are borne on the ab dominal segment which may be bent ventrally to an angle of about 90°. When placed in this position and so propped up in the aquarium that only the abdomen is under the surface of the water, the mouth parts may be as much as fifteen centimeters above the surface. No water can enter the alimentary canal, nevertheless in equal parts of fresh water and sea water (I = 1.03°), six hours sufficed to render the integument and gills tense and swollen. A freezing-point determination showed that I of blood had changed from —¿1.82° to —¿1.32°. In another experiment with Lizizulus the animal was placed astride a narrow dish of fresh water and so supported that only the gills dipped beneath the surface with each rhythmic oscillation. After eight hours enough water had been absorbed to bring 4 down to —¿1.4 I°,the freezing point of the water had also changed from —¿ 0.02°to —¿0.20°, and a copious precipitate of silver chloride was obtained. The gills of Limulus are permeable both to water and to salts. Metals in proteid combination, e. g., copper of hzemo
266 WALTER E. GARREY.
different tests made with animals taken from the same medium. In all these experiments it was necessary to sacrifice several ani mals to get even a minimum amount of blood for making determi nations, so that the results were on the whole unsatisfactory; still, they suffice for the conclusion that only a slight, if any, change is induced by a change in the osmotic pressure of the external medium. The animals are “¿ homoiosmotic.― Fundulus Jieteroclitus is a hardy little fish \velI suited to this form of experimentation, although the quantity of the blood is too small to admit of making cryoscopic determinations. It was found that if care was taken to select individuals which were not injured in catching, about eighty per cent. lived in fresh water for six weeks when the experiment was discontinued. This is as high a percentage as can be kept alive in the sea water aquaria of the laboratory. When placed in external media of concentra tions varying from fresh water to sea water concentrated to twice its normal strength they showed the same hardihood. It is reasonably certain that the osmotic pressure of the blood does not change to any marked degree for an examination of the blood corpuscles did not show either laking in the dilute media or crenation in the media of higher concentration. The integu ment and gills are therefore impermeable. Loeb 14,16has found that Fundulus embryos will live in distilled water and in sea water to which 5 per cent. NaCl has been added. The view that the membranes of these fish are completely per @ meable (Brown) is not tenable, at least concerning adult Fun dulus, as is shown by the following series of experiments which were repeated often enough to assure the verity of the results. A large number of healthy specimens were selected and about one half the body surface denuded of scales by gentle scraping with the edge of a scalpel, or the skin was removed over an area of one square centimeter on each side; then they were divided into three lots and placed respectively into fresh water, sea water diluted with an equal volume of distilled water, and normal sea water. Of those kept in fresh water in every experiment from eighty to ninety per cent. died within twenty-four hours while all died in less than thirty-six hours. In normal sea water the fish suffered a similar fate although death did not intervene so soon.
OSMOTIC PRESSURE OF SEA WATER. 267
But of those kept in sea water of one half its normal concentra tion only three per cent. were dead at a time when all those in the other two media had died, and seventy per cent. were kept alive for four weeks, when the wounds were all healed and the experiments discontinued. In these experiments therefore, no deleterious effects obtain when the internal and external media are approximately isotonic—in spite of the injuries and free inter change between blood and aquarium water. In the hypotonic and hypertonic solutions, however, distinct changes resulting in death, take place. In the strong solutions (normal sea water) microscopic examination showed that the blood corpuscles were crenated. In fresh water the fish became greatly swollen indi cating the absorption of watel-. Whether laking or swelling of the corpuscles takes place was not determined in this series of experiments. * From these experiments we may conclude that in all proba bility the blood of Fzindu/zis does not suffer much if any change in concentration when the fish is transferred from salt water into fresh water or vice versa, provided the membranes are uninjured. If these experiments admit of general application to migratory toleosts they would indicate that these animals also are in some way protected from changes in the osmotic pressure of the blood and tissues and that the principal protective factor probably lies in a lack of permeability of their membranes. We may further conclude that in case of serious abrasion to the integument the membranes become permeable and a change of osmotic pressure of the blood results, a change which may induce the death of the animal. The great mortality of the salmon after spawning in the head waters of California's streams, is a well-known fact (Rut terz@). Whether the generally battered condition of these fish at the spawning season bears any relation to changes in the osmotic pressure of the blood has not been investigated. It is not impossi ble that the actual cause of death lies in a decrease in the osmotic pressure of the blood and that the injuries are responsible for death only in so far as they permit the entrance of water and de *The ha@matocrit would doubtless prove a valuable aid in making experiments of this sort when it is impossible to obtain sufficient blood for freezing point letermina. tions.
OSMOTIC PRESSURE OF SEA WATER. 269
the sea water, the deficit of salts in the blood of this latter group is compensated by the osmotic pressure of the urea in the blood.
- The osmotic pressure of teleost blood is about half that of the sea water (4 = o.8° to —¿0.96° C.).
- A dilution or concentration of the aquarium water always causes an equivalent change itt the blood of invertebrates, and osmotic equilibrium between “¿ internaland external media― is established. Their membranes are completely permeable. This permeability is proven for the integument of worms and the gills of Limulus.
- Dilution or concentration of the aquarium water causes a change in the same sense in the blood of selachians, but death ensues before osmotic equilibrium is established. The mem branes of selachians are semi-permeable.
- Only slight if any change takes place when teleosts (Anguilla) are transferred from salt to fresh water and vice versa. Normal Funditlus Iteteroclitus will live in water varying in osmotic pres sure from that of the tap to sea water concentrated to double its strength. The membranes of teleosts are impermeable, or the fish possess some regulative mechanism which keeps the osmotic pressure of the blood nearly constant. Extensive abrasion of the skin of Fundulus results in death in aquarium water of less
or greater osmotic pressure than that of their blood, for example
they die in fresh water and in normal sea water but not in sea water diluted with an equal volume of distilled water.
PHYsIoLoGIcAL LABORATORY, COOPER MEDICAL COLLEGE, SAN FRANCISCO.
LITERATURE. i. Bottazzi, F. ‘¿ g7 Archives ital. de biologic, 1897, XXVIII., p. 61.
2. Ph. Bottazzi and Enriquez. **‘¿ oi Archiv. f. Anat. u. Physiol., 1901, p. 109.
- Frédericq, L. ‘¿ 82 Bull. de I' Acad. roy. de Belgique, 1882.** @. Fredericq, L. **‘¿ g8 Bull. de l'Acad. roy. de Belgique, 1898, p. 831.
- Frédéricq,L. Bull. de l'Acad. roy. de llelgique, 3 sér,Tom. IV.**
@
r
270 WALTER E. GARREY.
6. Frédéricq,L. ‘¿ 01 Bull. de 1'Acad. roy. de Belgique, 1901, p. 68. 7. Fredericq, L. ‘¿ 85 Archiv. de Zool., 1885.
- Frédéricq, L. ‘¿ 84,‘¿ giArchly. Zool. Exp., 1884 and 1891, p. 117. g. Frédéricq,L. **‘¿ 04 Archly. de Biologic., 1904, XX., p. 709.
- Garrey. ‘¿ 04 Seminar Reports, Marine Biol. Lab. Woods Hole, 1904. II. Hamburger. ‘¿ 97 Zentral f. Physiologic, 1897, Vol. ii.**
- Hedin. - ‘¿ g5 Skand. Arch. f. Physiol., 1895, p. 328, p. 377. ‘¿ 3.Höber. **‘¿ 02 Physikal. Chemie der Zelle u. der Gewebe, 1902, p. 25.
- Loeb, J. ‘¿ oo Amer. Jour. Physiol., 1900, III., p. 327.**
- Loeb, J. - **‘¿ 00 Amer. Jour. Physiol., 1900, III., p. 385. i6. Loeb, J. ‘¿ 94 Archiv. fur d. ges. Physiol., 1894, p. 530
- Mosso. ‘¿ 90 Biol. Centralbl., 1890, Vol. 10, p. 570.** i8. Quinton, R. ‘¿ 00 Compt. rend. de l'Acad. des sciences, 1900, Vol. 131, p. 905. ig. Quinton, R. ‘¿ 00 Compt. rend. de l'Acad. des sciences, 1900, Vol. 131, p. 952.
- Quinton, R. **‘¿ gg Compt. rend. de la Soc. de biol., 1899, p. 197.
- Rodier. ‘¿ 99 Traveaux des Lab. d'Arcachon, 1899, cit. Hôber, p. 25.
- Rodier. ‘¿ 01 Compt. rend. de l'Acad. des sciences, 1901, Dec. 10.
- Rutter. @o2 Reports of U. S. Fish. Com., and Popular Sc. Monthly, July, 1902.
- V. Schroeder. ‘¿ 90 Zeitschrift Physiol. Chemie, 1890, XXIV., p. 576.**
- Brown, 0. H. ‘¿ 03 Amer. Jour. Physiol., 1903, IX., p. III.
