The Organism as a Whole, from a Physicochemical Viewpoint

by Jacques Loeb

THE INFLUENCE OF ENVIRONMENT

1. The term environ­ment in rela­tion to an organism may easily assume a mystic rôle if we assume that it can modify the organisms so that they become adapted to its peculiarities. Such ideas are difficult to comprehend from a physico­chemical viewpoint, according to which environ­ment cannot affect the living organism and non-living matter in essentially different ways. Of course we know that proteins will as a rule coagulate at temperatures far below the boiling point of water and that no life is conceivable for any length of time at temperatures above 100° C., but heat coagula­tion of proteins occurs as well in the test-tube as in the living organism. If we substitute for the indefinite term environ­ment the individual physical and chemical forces which constitute environ­ment it is possible to show that the influence of each of these forces upon the organism finds its expression in simple physico­chemical laws and that there is no need to introduce any other considera­tions.

We select for our discussion first the most influential of external condi­tions, namely temperature. The reader knows that there is a lower as well as an upper temperature limit for life. Setchell has ascertained that in hot springs whose temperature is 43° C., or above, no animals or green alga are found.[247] In hot springs whose temperature is above 43° he found only the Cyanophyceæ, whose structure is more closely related to that of the bacteria than to that of the algæ, inasmuch as they have neither definitely differentiated nuclei nor chromophores. The highest temperature at which Cyanophyceæ occurred was 63° C. Not all the Cyanophyceæ were able to stand temperatures above 43° C., but only a few species. The other Cyanophyceæ were found at a temperature below 40° C., and were no more able to stand higher temperatures than the real algæ or animals. The Cyanophyceæ of the hot springs were as a rule killed by a temperature of 73°. From this we must conclude that they contain proteins whose coagula­tion temperature lies above that of animals and green plants, and may be as high as 73°. Among the fungi many forms can resist a temperature above 43° or 45°; the spores can generally stand a higher temperature than the vegetative organs. Duclaux found that certain bacilli (Tyrothrix) found in cheese are killed in one minute at a temperature of from 80° to 90°; while for the spores of the same bacillus a temperature of from 105° to 120° was required.[248]

Duclaux has called atten­tion to a fact which is of importance for the investiga­tion of the upper temperature limit for the life of organisms. According to this author it is erroneous to speak of a definite temperature as a fatal one; instead we must speak of a deadly temperature zone. This is due to the fact that the length of time which an organism is exposed to a higher temperature is of importance. Duclaux quotes as an example a series of experi­ments by Christen on the spores of soil and hay bacilli. The spores were exposed to a stream of steam and the time determined which was required at the various temperatures to kill the spores.

It took at 100°	over sixteen hours
" "  " 105–110°	two to four hours
" "  " 115°	thirty to sixty minutes
" "  " 125–130°	five minutes or more
" "  " 135°	one to five minutes
" "  " 140°	one minute

In warm-blooded animals 45° is generally considered a temperature at which death occurs in a few minutes; but a temperature of 44°, 43°, or 42° is also to be considered fatal with this difference only, that it takes a longer time to bring about death. This fact is to be considered in the treatment of fever.

It is generally held that death in these cases is due to an irreversible heat coagula­tion of proteins. According to Duclaux, it can be directly observed in micro-organisms that in the fatal temperature zone the normally homogeneous, or finely granulated, protoplasm is filled with thick, irregularly arranged bodies, and this is the optical expression of coagula­tion. The fact that the upper temperature limit differs so widely in different forms is explained by Duclaux through differences in the coagula­tion temperature of the various proteins. It is, e. g. known that the coagula­tion temperature varies with the amount of water of the colloid. According to Cramer, the mycelium of Penicillium contains 87.6 water to 12.4 dry matter, while the spores have 38.9 water and 61.1 dry substance. This may explain why the mycelium is killed at a lower temperature than the spores. According to Chevreul, with an increase in the amount of water, the coagula­tion temperature of albuminoids decreases. The reac­tion of the protoplasm influences the temperature of coagula­tion, inasmuch as it is lower when the reac­tion is acid, higher when the reac­tion is alkaline. The experi­ments of Pauli show also a marked influence of salts upon the temperature of coagula­tion of colloids.

The process of heat coagula­tion of colloids is also a func­tion of time. If the exposure to high temperature is not sufficiently long, only part of the colloid coagulates; in this case an organism may again recover.

Inside of these upper and lower temperature limits we find that life phenomena are influenced by temperature in such a way that their rate is about doubled for an increase of the temperature of 10° C., and that this temperature coefficient for 10°, Q₁₀, very often steadily diminishes from the lower to the higher temperature; so that near the lower temperature limit it becomes often considerably greater than 2 and near the higher temperature limit it becomes very often less than 2.[249] This influence of temperature is so general that we are bound to associate it with an equally general feature of life phenomena; and such a feature would be most likely the chemical reac­tions. It is known through the work of Berthelot, van’t Hoff, and Arrhenius that the temperature coefficient for the velocity of chemical reac­tions is also generally of about the same order of magnitude; namely ≧2 for a difference of 10°. In chemical reac­tions there is also a tendency for Q₁₀ to become larger for lower temperature, and coefficients of Q₁₀ about 5 or 6 have repeatedly been found for purely chemical reac­tions between 0° and 10°, e. g., for the inversion of cane sugar by the hydrogen ion. The temperature coefficient for the reac­tion velocity of ferments shows the same diminu­tion of Q₁₀ with rising temperature which is also noticed in most life phenomena. Thus Van Slyke and Cullen[250] found that the reac­tion rate of the enzyme urease “is nearly doubled by every 10° rise in temperature between 10° and 50°. Within this range the temperature coefficient is nearly constant and averages 1.91. From O° to 10° it is 2.80, from 50° to 60° it is only 1.09. The optimum is at about 55°.” The rapid fall of the temperature coefficient for enzyme action at the upper temperature limit has been ascribed by Tammann to a progressive destruc­tion of the active mass of enzyme by the higher temperature (by hydrolysis). This will, however, not account for the high value of the coefficient near the lower limit. But is it not imaginable that at low temperature an aggrega­tion of the enzyme particles exists which is also equivalent to a diminu­tion of the active mass of the enzyme and that this aggrega­tion is gradually dispersed by the rising temperature? This would account for the fact that at a temperature near 0°C life phenomena stop because the enzymes are all in a state of aggrega­tion or gela­tion; that then more and more are dissolved and the rate of chemical reac­tion increases since the mass of enzyme particles increases until all the enzyme molecules are dissolved or rendered active. Under this assump­tion three processes are superposed in the varia­tion of the value of Q₁₀ with temperature: (1) the supposed increase in the number of available ferment molecules with increasing temperature near the lower temperature limit; (2) the temperature coefficient of the reac­tion velocity which is nearly = 2 for 10°C.; (3) the diminu­tion of the number of available ferment molecules by hydrolysis or some other action of the increasing temperature. This latter is noticeable near the upper temperature limit. The reason that 1 and 3 interfere more strongly in life phenomena than in the chemical reac­tions of crystalloid substances may possibly be accounted for by the fact that the enzymes and most of the constituents of living matter are colloidal, i. e., consist of particles of a considerably greater order of magnitude than the molecules of crystalloids.[251]

We will now show the rôle of the temperature coefficient upon phenomena of development. F. R. Lillie and Knowlton[252] first determined the influence of temperature upon the development of the egg of the frog and showed that it was of the same nature as that of a chemical reac­tion. These experi­ments were repeated a year later by O. Hertwig.[253]

The time required for the eggs to reach definite stages was measured for different temperatures and it was found that the temperature coefficient Q₁₀ between 2.5° and 6° was equal to 10 or more; between 6° and 15° it was between 2.6 and 4.5; between 10° and 20° it was 2.9 to 3.3, and between 20° and 24° it was between 1.4 and 2.0. To anybody who has worked on this problem it is obvious that no exact figures can be obtained in this way, since the point when a certain stage of development is reached is not so sharply defined as to exclude a certain latitude of arbitrariness. The writer found that very exact figures can be obtained on the influence of temperature upon development of the sea-urchin egg by measuring the time from insemina­tion to the first cell division. Such experi­ments were carried out in a cold-water form Strongylo­centrotus purpuratus and a form living in warmer water, Arbacia.[254] The figures on Arbacia have been verified by different observers in different years.

TABLE X

Influence of Temperature upon the Time (in Minutes) Required From Insemination to the First Cell Division

Temperature		Arbacia				Strongylo­centrotus purpuratus
		Loeb and Wasteneys 1911		Loeb and Chamberlain 1915
°C.		Minutes		Minutes		Minutes
3						532
4						469
5						352
6						275
7		498				291
8		410		411		210
9		308		297	.5	159
10		217		208		143
11		175		175
12		147		148		131
13				129
14				116		121
15		100		100		100
16		85	.5
17		70	.5
18		68		68		187
19				65		178
20		56		56		175
21				53	.3	178
22		47		46		175
23				45	.5	Upper temperature limit
24				42
25		40		39	.5
26		33	.5
27	.5	34
30		33
31		37

These figures permitted the determina­tion of the temperature coefficients Q₁₀ with a sufficient degree of accuracy (see next table). It seemed of importance to attempt to decide what the chemical reac­tion underlying these reac­tion velocities is (if it is a chemical reac­tion). Loeb and Wasteneys[255] investigated the temperature coefficient for the rate of oxida­tions in the newly fertilized egg of Arbacia and found that the temperature coefficient Q₁₀ for that process does not vary in the same way as the temperature coefficient for cell division.

TABLE XI

Temperature Coefficients Q₁₀ for the Rate of Segmentation and Oxidations in the Eggs of Strongylocentrotus AND Arbacia

Temperature		Q10 for Rate of Segmentation in		Q10 for Rate of Oxidations in Arbacia
		Strongylocentrotus	Arbacia
°C.
3–	13	3.91		2.18
4–	14	3.88
5–	15	3.52		2.16
7–	17	3.27	7.3	2.00
8–	18		6.0
9–	19	2.04	4.7
10–	20	1.90	3.8	2.17
11–	21		3.3
12–	22	1.74	3.1
13–	23		2.8	2.45
15–	25		2.5	2.24
16–	26		2.6
17.5–	27.5		2.2	2.00
20–	30		1.7	1.96

It is obvious that the temperature coefficient of the rate of oxida­tions is remarkably constant, about 2 for 10°, for various temperatures and does not show the varia­tion from 7 or more to 2.2 for Q₁₀ for the rate of segmenta­tion.

Kanitz[256] has shown that in a graph in which the logarithms of the segmenta­tion velocities are drawn as ordinates and the temperatures as abscissæ the logarithms form two straight lines which are joined at an angle. According to the law of van’t Hoff and Arrhenius concerning the influence of temperature upon velocities of chemical reac­tions the logarithms should lie in a straight line. We are dealing therefore in these cases with two exponential curves, one representing in Arbacia the interval 7–13° and the second from 13–26°; in Strongylo­centrotus between 3–9° and 9–20°.

It was found in these experi­ments that if measurements of the Q₁₀ of later stages of development are attempted the varia­tions due to unavoidable difficulties become too great to permit an equal degree of reliability in the determina­tions.

The vast importance of this influence of temperature upon the rate of development is seen in the fact that in addi­tion to the food supply the rate of the maturing of plants and animals depends on this factor.

2. This influence of temperature upon development has been used to find the condi­tions determining fluctuating varia­tion. The reader knows that by this expression are understood the differences between individuals of a pure strain or breed. These varia­tions are not inherited, a fact contrary to the idea of Darwin, who assumed that by the selec­tion of extreme cases of fluctuating varia­tion new varieties could develop. What is the basis of this fluctuating varia­tion? The writer concluded that if fluctuating varia­tions were due to a slight varia­tion in the quantity of a specific substance—in some cases an enzyme—required for the forma­tion of a hereditary character, the temperature coefficient might be used to test the idea. We have just seen that the time required from insemina­tion until the cell division of the first egg occurs is very sharply defined for each temperature. If a large number e. g. one hundred or more eggs are under observa­tion simultaneously in a microscopic field it can be seen that they do not all segment at the same time but in succession; this is the expression of fluctuating varia­tion. Miss Chamberlain and the writer have measured the time which elapses between the moment the first egg of such a group segments and the moment the last egg begins its segmenta­tion, and found that this latitude of varia­tion is also very definite for each temperature, and that its temperature coefficient is for each interval of 10° practically identical with the temperature coefficient of the segmenta­tion for the same interval.[257] The slight devia­tions are practically all in the same sense and accounted for by a slight deficiency in the nature of the experi­ments. The two following tables give the latitude of varia­tions for different temperatures for the first segmenta­tion in Arbacia and the temperature coefficient for this latitude and the rate of segmenta­tion. These two latter coefficients are practically identical.

TABLE XII

Temperature	Latitude of Variation	Temperature	Latitude of Variation
°C.	Minutes	°C.	Minutes
19	52.5	18	12.0
10	39.5	19	12.5
11	26.0	20	19.6
12	22.5	21	18.0
13	19.2	22	17.8
14	17.5	23	18.0
15	13.0	24	18.0
		25	15.0

TABLE XIII

Temperature Interval	temperature coefficient of
	Latitude of Variation	Segmentation
°C.
19–19	4.2	4.7
10–20	3.9	3.8
11–21	3.2	3.3
12–22	2.8	3.1
13–23	2.4	2.8
14–24	2.3	2.8
15–25	2.6	2.5

If we assume that the temperature coefficient for the segmenta­tion of the egg is that of a chemical reac­tion (other than oxida­tion) underlying the process of segmenta­tion, the fluctuating varia­tion in the time of the segmenta­tions of the various eggs fertilized at the same time is due to the fact that the mass of the enzyme controlling that reac­tion varies within definite limits in different eggs. The first egg segmenting at a given temperature has the maximal, the last egg segmenting has the minimal mass of enzyme. It should be added that the time of the first segmenta­tion is determined by the cytoplasm and is not a Mendelian character, as was stated in a previous chapter.

3. The point of importance to us is that the influence of temperature upon the organism is so constant that if disturbing factors are removed it would be possible to use the time from insemina­tion to the first segmenta­tion of an egg of Arbacia as a thermometer on the basis of the table on page [295].

Facts of this character should dispose of the idea that the organism as a whole does not react with that degree of machine-like precision which we find in the realm of physics and chemistry. Such an idea could only arise from the fact that biologists have not been in the habit of looking for quantitative laws, chiefly, perhaps, because the difficulties due to disturbing secondary factors were too great. The worker in physics knows that in order to discover the laws of a phenomenon all the disturbing factors which might influence the result must first be removed. When the biologist works with an organism as a whole he is rarely able to accomplish this since the various disturbing influences, being inseparable from the life of the organism, can often not be entirely removed. In this case the biologist must look for an organism in which by chance this elimina­tion of secondary condi­tions is possible. The following example may serve as an illustra­tion of this rather important point in biological work. Although all normal human beings have about the same temperature, yet if the heart-beats of a large number of healthy human beings are measured the rate is found to vary enormously. Thus v. Körösy found among soldiers under the most favourable and most constant condi­tions of observa­tions—the soldiers were examined early in the morning before rising—varia­tions in the rate of heart-beat between 42 and 108. In view of this fact, those opposed to the idea that the organism as a whole obeys purely physico­chemical laws might find it preposterous to imagine that the rate of heart-beat could be used as a thermometer. Yet if we observe the influence of temperature on the rate of the heart-beat of a large number of embryos of the fish Fundulus, while the embryos are still in the egg, we find that at the same temperature each heart beats at the same rate, the devia­tions being only slight and such as the fluctuating varia­tions would demand.[258] This constancy is so great that the rate of heart-beat of these embryos could in fact be used as a rough thermometer. The influence of temperature upon the rate of heart-beat is completely reversible so that when we measure the rate for increasing as well as for decreasing temperatures we get approximately the same values as the following table shows.

TABLE XIV

Temperature	Time Required for Nineteen Heart-beats in the Embryo of Fundulus
°C.	Seconds
30	6.	25
25	8.	5
20	11.	5
15	19.	0
10	32.	5
15	61.	0
10	33.	5
15	18.	8
20	12.	0
25	10.	0
30	6.	0

Why does each embryo have the same rate of heart-beat at the same temperature in contradistinc­tion to the enormous variability of the same rate in man? The answer is, on account of the elimina­tion of all secondary disturbing factors. In the embryo of Fundulus the heart-beat is a func­tion almost if not exclusively of two variables, the mass of enzymes for the chemical reac­tions underlying the heart-beat and the temperature. By inheritance the mass of enzymes is approximately the same and in this way all the embryos beat at the same rate (within the limits of the fluctuating varia­tion) at the same temperature. This identity exists, however, only as long as the embryo is relatively quiet in the egg. As soon as the embryo begins to move this equality disappears since the motion influences the heart-beat and the motility of different embryos differs.

In man the number of disturbing factors is so great that no equality of the rate for the same temperature can be expected. Differences in emotions or the internal secre­tions following the emotions, differences in previous diseases and their after-effects, differences in metabolism, differences in the use of narcotics or drugs, and differences in activity are only some of the number of variables which enter.

4. As stated above the temperature influences practically all life phenomena in a similar characteristic way, e. g., the produc­tion of CO₂ in seeds[259] and the assimila­tion of CO₂ by green plants.[260] The writer would not be surprised if even the aberra­tions in the colour of butterflies under the influence of temperature turned out to be connected with the temperature coefficient. The experi­ments of Dorfmeister, Weismann, Merrifield, Standfuss, and Fischer, on seasonal dimorphism and the aberra­tion of colour in butterflies have so often been discussed in biological literature that a short reference to them will suffice. By seasonal dimorphism is meant the fact that species may appear at different seasons of the year in a somewhat different form or colour. Vanessa prorsa is the summer form, Vanessa levana the winter form of the same species. By keeping the pupæ of Vanessa prorsa several weeks at a temperature of from 0° to 1° Weismann succeeded in obtaining from the summer chrysalids specimens which resembled the winter variety, Vanessa levana.

If we wish to get a clear understanding of the causes of varia­tion in the colour and pattern of butterflies, we must direct our atten­tion to the experi­ments of Fischer, who worked with more extreme temperatures than his predecessors, and found that almost identical aberra­tions of colour could be produced by both extremely high and extremely low temperatures. This can be seen clearly from the following tabulated results of his observa­tions. At the head of each column the reader finds the temperature to which Fischer submitted the pupæ, and in the vertical column below are found the varieties that were produced. In the vertical column A are given the normal forms:

TABLE XV

0° to -20°C.	0° to +10°C.	A (Normal Forms)	+35° to +37°C.	+36° to +41°C.	+42° to +46°C.
ichnusoides  (nigrita)	polaris	urticæ	ichnusa	polaris	ichnusoides  (nigrita)
antigone  (iokaste)	fischeri	io	——	fischeri	antigone  (iokaste)
testudo	dixeyi	polychloros	erythromelas	dixeyi	testudo
hygiæa	artemis	antiopa	epione	artemis	hygiæa
elymi	wiskotti	cardui	——	wiskotti	elymi
klymene	merrifieldi	atalanta	——	merrifieldi	klymene
weismanni	porima	prorsa	——	porima	weismanni

The reader will notice that the aberra­tions produced at a very low temperature (from 0° to -20°C.) are absolutely identical with the aberra­tions produced by exposing the pupæ to extremely high temperatures (42° to 46°C.). Moreover, the aberra­tions produced by a moderately low temperature (from 0° to 10°C.) are identical with the aberra­tions produced by a moderately high temperature (36° to 41°C.).

From these observa­tions Fischer concludes that it is erroneous to speak of a specific effect of high and of low temperatures, but that there must be a common cause for the aberra­tion found at the high as well as at the low temperature limits. This cause he seems to find in the inhibiting effects of extreme temperatures upon development.

If we try to analyse such results as Fischer’s from a physico­chemical point of view, we must realize that what we call life consists of a series of chemical reac­tions, which are connected in a catenary way; inasmuch as one reac­tion or group of reac­tions (a) (e. g., hydrolyses) causes or furnishes the material for a second reac­tion or group of reac­tions (b) (e. g., oxida­tions). We know that the temperature coefficient for physio­logical processes varies slightly at various parts of the scale; as a rule it is higher near 0° and lower near 30°. But we know also that the temperature coefficients do not vary equally for the various physio­logical processes. It is, therefore, to be expected that the temperature coefficients for the group of reac­tions of the type (a) will not be identical through the whole scale with the temperature coefficients for the reac­tions of the type (b). If therefore a certain substance is formed at the normal temperature of the animal in such quantities as are needed for the catenary reac­tion (b), it is not to be expected that this same perfect balance will be maintained for extremely high or extremely low temperatures; it is more probable that one group of reac­tions will exceed the other and thus produce aberrant chemical effects, which may underlie the colour aberra­tions observed by Fischer and other experi­menters.

It is important to notice that Fischer was also able to produce aberra­tions through the applica­tion of narcotics. Wolfgang Ostwald has produced experimentally, through varia­tion of temperature, dimorphism of form in Daphnia.

5. Next or equal in importance with the temperature is the nature of the medium in which the cells are living.

It has often been pointed out that the marine animals and the cells of the body of metazoic animals are surrounded by a medium of similar constitu­tion, the sea water and the blood or lymph, both media being salt solu­tions differing in concentra­tion but containing the three salts NaCl, KCl, and CaCl₂ in about the same relative concentra­tion, namely 100 molecules NaCl : 2.2 molecules of KCl : 1.5 molecules of CaCl₂. This has suggested to some authors the poetical dream that our home was once the ocean, but we cannot test the idea since unfortunately we cannot experi­ment with the past. Plants, unicellular fresh-water algæ, and bacteria do not demand such a medium for their existence.

Herbst had shown that when sea-urchin larvæ were raised in a medium in which only one of the constituents of the sea water was lacking (not only NaCl, KCl, or CaCl₂, but also Na₂SO₄, NaHCO₃, or Na₂HPO₄), the eggs could not develop into plutei; from which he concluded that every constituent of the sea water was necessary. This would indicate a case of extreme adapta­tion to all the minutiæ of the external medium.

Experiments on a much more favourable animal for this purpose, namely, the eggs of the marine fish Fundulus, gave altogether different results. The eggs of this marine fish develop naturally in sea water but they develop just as well in fresh or in distilled water, and the young fish when they are made to hatch in distilled water will continue to live in this medium. This proves that these eggs require none of the salts of the sea water for their development. When these eggs are put immediately after fertiliza­tion into a pure solu­tion of NaCl of that concentra­tion in which this salt exists in the sea water practically all the eggs die without forming an embryo; but if a small quantity of CaCl₂ is added every egg is able to form one, and these embryos will develop into fish and the latter will hatch. This led the writer to the conclusion that these fish (and perhaps marine animals in general) need the Ca of the sea water only to counteract the injurious effects which a pure NaCl solu­tion has if it is present in too high a concentra­tion.[261] When we raise the eggs in a pure NaCl solu­tion of a concentra­tion ≦m/8 practically every egg will develop; and even in a m/4 or ³⁄₈ m many or some eggs will form embryos without adding Ca; it may be that a trace of Ca present in the membrane of the egg may suffice to counter-balance the injurious action of a weak salt solu­tion.

The concentra­tion of the NaCl in the sea water at Woods Hole (where these experi­ments were made) is about m/2, and as soon as this concentra­tion of NaCl is reached the eggs are all killed as a rule before they can form an embryo, unless a small but definite amount of Ca is added. It was found that the eggs can be raised in much higher concentra­tions of NaCl, but in that case more Ca must be added. The following table gives the minimal amount of CaCl₂ which must be added in order to allow fifty per cent. of the eggs to form embryos. (The eggs were put into the solu­tion an hour or two after fertiliza­tion.)

TABLE XVI

Concentration of NaCl		Cc. m/16 CaCl2 Required for 50 c.c. NaCl Solution
m.
3⁄	8	0.	1
4⁄	8	0.	3
5⁄	8	0.	5
6⁄	8	0.	6
7⁄	8	0.	9
8⁄	8	1.2–	1.4
9⁄	8	1.8–	2.0
10⁄	8	2.0–	2.5
11⁄	8	2.	0?
12⁄	8	3.0–	3.5
13⁄	8	6.	0

This indicates that the quantity of CaCl₂ required to counteract the injurious effects of a pure solu­tion of NaCl increases approximately in propor­tion to the square of the concentra­tion of the NaCl solu­tion.[262] The reader will notice that the eggs can survive and develop in a solu­tion of three times the concentra­tion of sea water, provided enough Ca is added.

It was found also that not only Ca but a large number of other bivalent metals were able to counteract the injurious action of an excessive NaCl solu­tion; namely Mg, Sr, Ba, Mn, Co, Zn, Pb, and Fe;[263] only Hg and Cu could not be used since they are themselves too toxic. The antagonistic efficiency of the bivalent cations other than Ca was, however, smaller than that of Ca. The following table gives the highest concentra­tion of NaCl solu­tion in which the newly fertilized eggs of Fundulus can still form an embryo.[264]

50 c.c. ¹⁰⁄₈ m NaCl+4 c.c. m/1 MgCl₂
50 c.c. ¹⁴⁄₈ m NaCl+1 c.c. m/1 CaCl₂
50 c.c. ¹¹⁄₈ m NaCl+1 c.c. m/1 SrCl₂
50 c.c. ⁷⁄₈ m NaCl+1 c.c. m/1 BaCl₂

On the other hand it was seen that in all the chlorides with a univalent cation, LiCl, KCl, RbCl, CsCl, NH₄Cl, the eggs could form embryos up to a certain concentra­tion of the salt; but that this concentra­tion could be raised by the addi­tion of Ca.

TABLE XVII

Concentrations at which the Eggs no longer Are Able to Form Embryos

In the Pure Salts			In the Same Salts with the Addition of 1 c.c. m CaCl2 to 50 c.c. Solution
LiCl	about 6/	32 m	>5/	8 m
NaCl	m/	2	>14/	8 m
KCl	>11/	16 m	>8/	8 m
	<6/	8 m
RbCl	>8/	8 m	>9/	8 m
	<7/	8 m
CsCl	>3/	8 m	>8/	8 m
	<4/	8 m

In short it turned out that the injurious action of the pure solu­tion of any chloride (or any other anion) with a univalent metal could be counteracted to a considerable extent by the addi­tion of small quantities of a salt with a bivalent metal. It was also found in the early experi­ments of the writer that the bivalent or polyvalent anions had no such antagonistic effect upon the injurious action of the salts with a univalent cation.

We therefore see that what at first sight appeared in the experi­ments of Herbst a necessity, namely, the presence of each constituent of the sea water, turns out as a special case of a more general law; the salts with univalent ions are injurious if their concentra­tion exceeds a certain limit and this injurious action is diminished by a trace of a salt with a bivalent cation.

Why was it not possible to prove this fact for the eggs of the sea urchin? Before we answer this ques­tion, we wish to enter upon the discussion of the nature of the injurious action of a pure NaCl solu­tion of a certain concentra­tion and of the annihila­tion of this action by the addi­tion of a small quantity of Ca. The writer suggested in 1905 that the injurious action of a pure NaCl solu­tion consisted in rendering the membrane of the egg permeable for NaCl, whereby the germ inside the membrane is killed; while the addi­tion of a small amount of Ca (or any other bivalent metal) prevents the diffusion of Na into the egg,[265] possibly, as T. B. Robertson[266] suggested, by forming a precipitate with some constituent of the membrane, whereby the latter becomes more impermeable. The correctness of this idea can be demonstrated in the following way. When eggs of Fundulus, which are three or four days old and contain an embryo, are put into a test-tube containing 3 m NaCl they will float on this solu­tion for about three or four hours; after that they will sink to the bottom. Before this happens the egg will shrink and when it ceases to float the embryo is usually dead. This is intelligible on the assump­tion that the NaCl solu­tion entered the egg, increased its specific gravity so that it could not float any longer and killed the embryo. When we add, however, 1 c.c. ¹⁰⁄₈ m CaCl₂ to 50 c.c. 3 m NaCl the eggs will float, the heart will continue to beat normally and the embryo will continue to develop for three days or more, because the calcium prevents the NaCl from entering into the egg.[267] For if we put a newly hatched embryo into 50 c.c. NaCl+1 c.c. ¹⁰⁄₈ m CaCl₂ it will die almost instantly; hence the membrane must have acted for three or more days as a shield which prevented the NaCl from diffusing into the egg in the presence of CaCl₂.

The same experiments cannot be demonstrated in the sea-urchin egg, first, because it can live neither in distilled water nor in very dilute nor very concentrated solu­tions; and second, because it is not separated as is the germ of the Fundulus egg from the surrounding solu­tion by a membrane which is under proper condi­tions practically impermeable for water and salts.

Nevertheless it can be shown that the results at which we arrived in our experi­ments on Fundulus are of a general applica­tion. Osterhout[268] has shown that plants which grow in the soil or in fresh water are readily killed by a pure NaCl solu­tion of a certain concentra­tion, while they can resist the same concentra­tion of NaCl if some CaCl₂ is added. Wo. Ostwald[269] has shown the same for a species of Daphnia. We, therefore, come to the conclusion that the injurious action following an altera­tion in the constitu­tion of the sea water is in some of the cases due to an increase in the permeability of the membranes of the cell, whereby substances can diffuse into the cell which when the proper balance prevails cannot diffuse. For this balance the ratio of the concentra­tion of the salts with univalent cation Na and K over those with bivalent cation Ca and Mg C_{Na+K salts}/C_{Ca+Mg salts} is of the greatest importance.

6. The importance of this quotient appears in the so-called “behaviour” of marine animals. We have mentioned the newly hatched larvæ of the barnacle in connec­tion with helio­tropism. These larvæ swim in a trough of normal sea water at the surface, being either strongly positively or negatively helio­tropic. They collect as a rule in two dense clusters, one at the window and one at the room side of the dish. If such animals are put into a solu­tion of NaCl+KCl (in the propor­tion in which these salts exist in the sea water), they will fall to the bottom unable to rise to the surface. They will, however, rise to the surface and swim energetically to or from the window if a certain quantity of any of the chlorides of a bivalent metal, Mg, Ca, or Sr, is added, but these movements will last only a few minutes when only one of these three salts is added; and then the animals will fall to the bottom again. If, however, two salts, e. g., MgCl₂ and CaCl₂, are added the animals will stay permanently at the surface and react to light as they would have done in normal sea water. These animals also can resist comparatively large changes in the concentra­tion of the sea water, and it seemed of interest to find out whether the quotient C_NaCl+KCl/C_MgCl2+CaCl2, which just allowed all the animals to swim at the surface, had a constant value. The MgCl₂+CaCl₂ solu­tion was ³⁄₈ m and contained the two metals in the propor­tion in which they exist in the sea water; namely, 11.8 molecules MgCl₂ to 1.5 molecules CaCl₂. The next table gives the result.[270] Since these experi­ments lasted a day or more each, usually two different concentra­tions of NaCl+KCl of the ratio 1 : 2 or 1 : 4 were compared in one experi­ment.

TABLE XVIII

Number of Experiment	Concentration of NaCl+KCl		C.c. 3/8 m CaCl2+MgCl2 Required		Value of CNa+K/CMg+Ca
1		m/16	0.	3	27.8
		m/8	0.4–	0.5	37.0
2		m/8	0.	5	33.3
		m/4	0.9–	1.0	35.1
3		3/16 m	0.	7	35.7
		3/8 m	1.	3	38.5
4		m/8	0.	5	36.0
		m/2	1.8–	1.9	39.2
5		m/4	0.8–	0.9	39.2
		m/2	1.6–	1.7	40.3
6		5/16 m	0.	9	46.3
		5/8 m	1.	7	49.0
7		3/16 m	0.	6	41.7
		6/8 m	2.	4	41.7

These experiments indicate that the ratio of C_Na+K/C_Ca+Mg remains very nearly constant with varying concentra­tions of C_Na+K.

In former experiments on jellyfish the writer had shown that there exists an antagonism between Mg and Ca[271], and this observa­tion was subsequently confirmed by Meltzer and Auer[272] for mammals. It was observed that in a solu­tion of NaCl+KCl+MgCl₂ the larvæ of the barnacle were also not able to remain at the surface for more than a few minutes, while an addi­tion of some CaCl₂ made them swim permanently at the surface. Various quantities of MgCl₂ were added to a mixture of m/4 or m/2 NaCl+KCl, to find out how much CaCl₂, was required to allow them to swim permanently at the surface.

TABLE XIX

	C.c. of m/16 CaCl2 Necessary to Induce the Majority of the Larvæ to Swim in
	m/2 (Na+K)		m/4 (Na+K)
50 c.c. NaCl+KCl+0.75 c.c. 3⁄8 m MgCl2			0.	2
50 c.c. NaCl+KCl+ 1.5 c.c. 3⁄8 m MgCl2	0.	4	0.	3
50 c.c. NaCl+KCl+ 2.5 c.c. 3⁄8 m MgCl2	0.	4	0.	4
50 c.c. NaCl+KCl+ 5.0 c.c. 3⁄8 m MgCl2	0.7–	0.8	0.7–	0.8
50 c.c. NaCl+KCl+10.0 c.c. 3⁄8 m MgCl2	1.	6	1.	6
50 c.c. NaCl+KCl+15.0 c.c. 3⁄8 m MgCl2	1.	8
50 c.c. NaCl+KCl+20.0 c.c. 3⁄8 m MgCl2	1.	8

In order to interpret these figures correctly we must remember that we are dealing with two different antagonisms, one between the salts with univalent and bivalent metals and the other between Mg and Ca. The former antagonism is satisfied by the addi­tion of Mg, inasmuch as enough Mg was present for this purpose in all solu­tions. What was lacking was the balance between Mg and Ca. The experi­ments in Table XIX therefore answer the ques­tion of the ratio between Mg and Ca. If we consider only the concentra­tions of Mg between 2.5 and 10.0 c.c. ³⁄₈ m MgCl₂—which are those closest to the normal concentra­tion of Mg in the sea water—we notice that C_Ca must vary in propor­tion to C_Mg. If we now combine the results of this and the previous paragraph we may express them in the form of the theory of physio­logically balanced salt solu­tions, by which we mean that in the ocean (and in the blood or lymph) the salts exist in such ratio that they mutually antagonize the injurious action which one or several of them would have if they were alone in solu­tion.[273] This law of physio­logically balanced solu­tions seems to be the general expression of the effect of changes in the constitu­tion of the salt solu­tions for marine or all aquatic organisms.

This chapter would not be complete without an intima­tion of the rôle of buffers in the sea water and the blood, by which the reac­tion of these media is prevented from changing in a way injurious to the organism. These buffers are the carbonates and phosphates. Instead of saying that the organisms are adapted to the medium, L. Henderson has pointed out the fitness of the environ­ment for the development of organisms and one of these elements of fitness are the buffers against altera­tions of the hydrogen ion concentra­tion.[274] The ratio in which the salts of the different metals exist in the sea water is another. It is obvious that the quantitative laws prevailing in the effect of environ­ment upon organisms leave no more room for the interference of a “directing force” of the vitalist than do the laws of the motion of the solar system.

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