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form, and the pentavalent form is not reduced to a great extent because the alkaline reaction of the blood is less favorable to this process. This may be the reason why there is more trivalent arsenic in the sap of Valonia and less retained by the protoplasm, while the opposite is true in animals; namely, more pentavalent arsenic excreted and more trivalent retained. However, the two cases are not strictly comparable, since, in the animals investigated, the kidneys eliminated the arsenic, whereas in Valonia the arsenic diffusing through the protoplasm was held in the sap.
An interesting fact also shown by Figures 4, 5, and 6 is that the minimum amount of arsenic penetrates when the pH of the surrounding solution is near neutrality. As the reaction becomes either more acid or more alkaline, more arsenic is found inside the cell. Factors which may enter into these reactions are the greater rate of reduction of arsenic (pentavalent into trivalent) in the acid range and vice versa. Acidity favors the reduction, and alkalinity the oxidation of arsenic compounds. Differences in the solubility of the arsenic compounds in the sea water at various H ion concentrations could not be responsible for the differences in results, as no precipitate was formed in any of the solutions of the arsenical in sea water.
EFFECT OF PIIOSPHATE BUFFERS.
The effect of the presence of phosphate buffers in the surrounding solution upon the penetration of arsenic is also evident in all the curves of Figures 4, 5, and 6. Those curves, which indicate penetration of arsenic in the presence of buffer, are, generally speaking, considerably lower in the acid range than those in which no buffer was used. In the case of the sap, in all these figures, there is considerably less arsenic in the acid range when buffers are used. At neutrality the curves coincide and approach a minimum concentration.
In the case of protoplasm, the arsenic of atoxyl penetrates in decidedly larger amount in the absence of buffer than when buffer is used. With As206 there is less arsenic penetrating at pH 5 and 6 when buffer is used, and with As203 there is also a slightly less amount of arsenic penetrating in the presence of buffers.
In the case of wall, less arsenic penetrates in every case when buffer is used than when no buffer is used. This shows the great influence of the presence of phosphates upon the penetration of arsenic. Such differences were observed in a largo number of experiments. The difference in the case of trivalent arsenic is not so marked, owing probably to the small amount of arsenic penetrating from acid solutions.
Emphasis must therefore be placed on the fact that in studying the influence of the II ion concentration of the surrounding medium, 71227°—23 3
the nature of other ions present in the solution is of verj- great importance and cau by no means be neglected.
Previous observations by the writer on the penetration of arsenic into Nitella (7) from solutions of atoxyl show that in the presence of phosphate buffers, the anion (arsenic) enters more readily from alkaline solutions than from acid ones. The possibility that the ions of the buffer salts may affect the rate of penetration of arsenic was also suggested by the writer in this paper. Taking into consideration the experiments of the writer on Valonia, it now seems as if the phosphate buffers added in the case of Nitella were responsible for the lack of penetration of arsenic in the acid range. As the quantity of sap obtainable from Nitella was very small, it is possible that its arsenic content was too low to be detected by the Gutzeit method. In the alkaline range there was increasing penetration of arsenic into Nitella with increasing alkalinity; but this was probably associated with an increase in the amount of injury. Further experiments on NiteUa, involving shorter durations of time and larger amounts of sap, are necessary to settle this point.
In Valonia the amount of arsenic penetrating in the acid range was considerably less in the presence of buffer salts than when no buffer salts were used.
May not these effects of buffer salts be operative in the case of experiments reported by other writers? The results of an experiment may even be reversed by neglect of this factor, as might have happened in the case of Valonia. In the absence of control experiments to determine whether the buffer salts are responsible for any of the observed effects, conclusions as to the effect of H ion concentrations are unwarranted.
Hoagland and Davis (8), working with Nitella, used phosphate buffers to maintain a constant pH. They state that the penetration of the anion NO, into the cell sap from dilute solutions was definitely influenced by the H icn concentration, and that penetration was much more rapid from a slightly acid solution than from an alkaline one. Irwin (9) also used phosphate buffers in her experiments on the entrance and accumulation of chlorides as well as of the basic dye, brilliant cresyl blue in Nitella, and agrees with other observers that the entrance and accumulation of cations (except H) is favored by alkaline solutions, and' that of anions (except OH) by acid solutions. In this case it would also be of interest to know how shortly after the period of the experiment irreversible injury occurred. It has already been noted that in the case of Valonia this point could not be determined merely by noting the appearance of the plant immediately after experimentation; but that observations had to be made for some time after the plant had been replaced in its normal environment, and these had to he compared with observations on the viability of normal plants.
As stated before, the appearance of a cell immediately after it has been in a test solution is not a sufficient criterion for injury. In order to be sure that there is no injury, the plant must be returned to its normal environment and its length of survival compared with that of the control. It is frequently found, under these precautions, that a plant which appears in fine condition at the end of an experiment will rapidly die upon being returned to a normal environment. Certainly it can not be said of such a cell that it is not injured.
THE INFLUENCE OF NaHC03 UPON THE PENETRATION OF THE ARSENIC OF AS2Os AND Asjoj INTO LIVING CELLS.
In a previous paper (10) on the influence of bicarbonates on changes in the C03 content and alkalinity of the sap of Vaionia, the writer suggested that the permeability of the cell may have been increased by the large amount of bicarbonate in the surrounding solution.
It was shown that when cells of Vaionia were placed in a bicarbonate solution an excess of C02 accumulated in the sap. After the cells had been one hour in sea water plus bicarbonate, the pll of the aerated sap2 increased from G.8 to 8.4. This showed that, in addition to the free CO,, basic ions enter the sap. In further experiments reported in the same paper evidence was given which seemed to show that Na' and K' penetrated more rapidly from bicarbonate solutions in sea water than from similar solutions of the acetate chloride, or citrate (Table I), but this was not conclusive evidence of a special role for the bicarbonate ion as a regulator of cellular permeability to other ions.
In a later paper (11) it was shown that Na' and K' hardly penetrated at all from solutions of their hydroxides.3 Since in this experiment the solution used differed only in II' and HC03' content from those used in the bicarbonate experiments, the penetration of bases into the sap in the latter case must have been duo to one of these ions; but the relative influence of the two ions was still left unsettled.
It was therefore thought of interest, in connection with the studies on the penetration of arsenic, to find out whether previous exposure to bicarbonate solutions would affect the rate of penetration of some other substance, as, for example, arsenic, into the cell. If this were the case it would be important evidence of a special role of HCO', in regulating the permeability to other substances.
The plants were therefore placed for one hour in such a solution of NaHC()3 made up to 0.6 M in distilled water, enough of this being added to sea water to give a bicarbonate concentration of 0.0024 M. The pH of this solution was the same as that of sea water (8.6). The, cells were then transferred to solutions of arsenic in sea water (As,0, and As,05 in the same concentrations as were used in Figures 4, 5, and 6). Figure 0 shows the results after one hour in the arsenic solution. Upon analyzing the protoplasm of the cells which had been placed in a bicarbonate containing solution before treatment with the pentavalent arsenic, it was found that the amount of As was 150 per cent greater than in the cells which had not previously been exposed to the bicarbonate solution. There was also a slight increase of arsenic in the sap, amounting to about 25 per cent. In the case of trivalent arsenic, on the other hand, the amount of arsenic in the protoplasm of the cells was decreased about 25 per cent by previous treatment of the cell with bicarbonate solution, whereas the amount of arsenic in the sap was increased by about 100 per cent. Evidently, in the latter case, the permeability to arsenic diffusing into the sap was increased. It therefore appears as if the permeability of the protoplasm to both these substances was increased. Exposure of Valonia to pentavalent arsenic leads to a retention of arsenic by the protoplasm in much greater amounts, and it therefore reaches the sap at only slightly greater speed. In the case of trivalent form the rate of diffusion of arsenic into the sap is increased, even though no more arsenic is taken up by the protoplasm than when the cells have not been in bicarbonate solutions.
1 Sap in equilibrium with COrfree air.
'The slight amount of penetration ocoirrins may have been <luo to the bie:ub:>aates formc.1 in the solutions, since IhU alurayj uaiipoui un.eoo the solution; are especially protected.
Previous observations show that no injury was produced by allowing the cells to remain in the bicarbonate solutions for one hour.
The penetration of arsenic into the cell wall remains about the same in all of the experiments.
Briefly, then, these experiments show that when the plants have been placed in a solution of NaHC03 and then transferred to a solution containing arsenic, certain changes take place in the cell by which a greater amount of pentavalent arsenic is taken up by the protoplasm and a greater amount of trivalent arsenic by the sap; whereas lesser amounts of arsenic are found in the protoplasm when the trivalent form is used, and only a slightly greater amount in the sap when the pentavalent form is used.
How NaIIC03 operates in allowing more, arsenic to enter the cell can not be explained definitely. There are several possibilities. It is theoretically possible that the increase of HC0'3 or free C03 in the cell facilitates, in some unknown way, the formation of compounds of arsenic with protoplasmic constituents, thus leading to the accumulation of arsenic in the cell. It seems probable that the increase of either the bicarbonate ion or of free C02 is responsible for an increased permeability of the cell to arsenic.
1. The penetration of arsenic into the cell wall, protoplasm, and cell sap, respectively, has heen determined.
2. By far the highest concentration of arsenic was found in the protoplasm. The great amount of arsenic taken up by the protoplasm points to a combination of arsenic with protoplasm.
3. The penetration of both pentavalent arsenic (in the form of As,05 and atoxyl) and trivalent arsenic (As,03) is least when the external solution is nearly neutral. As the II ion concentration of the suspension fluid diverges from neutrality, more arsenic enters, the amount increasing as the surrounding medium becomes cither more acid or alkaline. This is true of sap and protoplasm but not of wall.
4. Trivalent arsenic does not penetrate so readily into protoplasm in the acid range as pentavalent arsenic; but it passes into the sap more readily, except in the range of neutrality.
5. The use of phosphate bufTers to regulate the pH may lead to deceptive results, and experiments in which such buffers are used must be controlled by parallel experiments without buffers.
6. The relation of the rate of penetration of arsenic acid into living and dead cells to the changes in pll of the sap has been determined.
7. Previous exposure, of Valonia for one hour in a solution of NaHCO, in sea water increases the accumulation of pentavalent arsenic in the protoplasm by 150 per cent; whereas the amount of trivalent arsenic accumulation in the protoplasm is reduced by 25 per cent. Under the same conditions the entrance of trivalent arsenic from the protoplasm into the sap is increased about 100 per cent, whereas that of pentavalent arsenic is increased only about 25 per cent.
8. It is suggested that either the bicarbonate ion or the presence of rbnormal amounts of free CO, increases the permeability of the cell to arsenic.
Adcnoivkdgmente.—The writer takes pleasure in acknowledging the courtesies afforded by the Miami Aquarium Association, where this work was done, and in expressing much gratitude to the authorities of the Carnegie Institution of Washington, D. C, who made arrangements for collecting plants, and to Dr. David Fairchild for his kind interest and encouragement.
(1) VoeRtlin, C, and Thomp?on, J. W.: Jour. Pharm. and Exp. Therap., xx, No. 2,
(2) Vocgtlin, C, and Smith, Homer W.: Jour. Pharm. and Exp. Therap., xv, Xo. 5;