T H E T O X I C ACTION O F ACIDS A N D SALTS ON SEEDLINGS1
BY F. K. CAMERON AND J. F. BREAZEALE
Activity in the investigation of the action of solutions of various electrolytes on living organisms has been pronounced within the past few years, stimulated largely by the fact that the hypothesis of electrolytic dissociation has seemed to offer a guiding principle in this work. T h e interest of the chemist in this line of investigation has been especially augmented by the well-known work of Kahlenberg' and his associates, from which it has been concluded that while some of the observed phenomena support, nevertheless the weight of the evidence is against the validity of the hypothesis of electrolytic dissociation, at least in the form which it is now generally understood. Perhaps the most important work in this line of investigation, from the point of view of dissociation studies, has been that involving the action of solutions of acids, the action of tlie hydrogen ion being relatively so large in comparison with other ions that it has been supposed the effect of the latter might safely be disregarded as of vanishing importance when the former was present. Similar assumptions have sometimes been made regarding the action of the hydroxyl ion as compared with the other ions. But it has been repeatedly demonstrated that when comparing the action of other dissociation products of electrolytes the effect of both cathion, anion and the undissociated electrolyte must be considered. In the course of an investigation being carried on in this Published by permission of the Secretary of Agriculture. Kahlenberg and True. Bot. Gaz. 22, 81 (1896) ; Heald. Ibid. 22, 125 (1896) ; True. Am. Jour. Sci. (4) 9, 183 (1900); Kahlenberg and Austin. Jour. Phys. Chem. 4, 553 (1900); Kahlenberg and Mehl. Ibid. 5, 113 (1901); Kahlenberg. Ibid. 5, 339 (1901). For a more extended bibliography, see Report 71, U. S.Department of Agriculture, 1902,pp. 56-60.
2
F. K.Cameron andJ R Breazeale
laboratory, the death or toxic " limit of concentration in aqueous solution of a number of acids and the corresponding" potassium and calcium salts, as well as mixtures, were carefully determined for certain plant seedlings. It is believed that this work has sufficient interest as a contribution to the study of the dissociation hypothesis to justify publication from that point of view, although the work was undertaken with quite a different purpose. I n work of this character great difficulty has been encountered in deciding what criteria should be taken as determining the death of the plant. T h e concentration required to kill a seedling or disorganize an entire radicle varies widely from that required to completely hinder any growth or elongation, or that which will permit some elongation but will kill the root tip and of course ultimately produce death. All these criteria have been used by various investigators, so that a coinparison of their results is often exceedingly difficult if not,inipossible.l To illustrate this poiiit the following table is presented, showing a comparison of the relative concentrations required to kill the entire wheat seedling and to just permit life without injuring the tip of the radicle. TABLEI. -Solute
Whole seedling
Tip of radical
Sulphuric acid N/ I 5000 N / I 8000 Hydrochloric acid Nitric acid N / I5000 Acetic acid N/20000 Oxalic acid N / I 5000 Succinic acid N/20000 Calcium chloride N/4 Calcium nitrate N/5 T h e criterion which apparently has found the most favor For instance, the figures given in this' paper for wheat are in no way comparable to those which have already been obtained by Coupin, since the manipulation and criteria used were so widely different.
Toxic Action o f Acids a d Sadis
012
Seedzings
3
and which has been used in former studies in this laboratory,' was the obvious death and flaccid condition of the tip of the radicle for the first 15 millimeters from the apex. But in some of the experiments with solutions of potassium salts, the apices of the radicles instead of becoming flaccid when the toxic limit was reached, became excessively hard, brittle and transparent.' T h e determination of the toxic limit was much more difficult in these cases, requiring nice judgment and much experience. Bot while the results obtained with potash salts may possibly lack the accuracy of those obtained with acidsor lime salts, owing to the necessary use of criteria which are far from being sharp and decisive, yet they are believed to be sufficiently near the truth to justify the use of them in the argument of this paper. Generally, before the death of the extreme 15 millimeters the radicle had somewhat elongated from plumule to the apex. These death criteria usually became apparent within 24 hours and definite results were practically always obtainable after this interval of time. Frequently, however, the experiments were prolonged for a week or more. In these latter cases there was often a development of new roots. But whenever these new roots penetrated the solution the death phenomena were observed in their tips, so that it seems perfectly safe to say that the concentration of solution producing death of the tip of the radicle would ultimately prove prohibitory to the growth of the plant, and, therefore, that safe and reliable criteria were used. Solutions insufficient in concentration to prohibit growth might however seriously retard it. I t was generally observed that the radicles immersed in solutions of a concentration approaching the death limit tended to curl u p in an apparent effort to leave the solution, seeking a more congenial environment. This curving of the radicles away from the injurious solution was noticed in the former studies3 in this laboratory upon the white lupine and alfalfa. It is also noteworthy that Report 7 1 , U. S.Department of Agriculture, 1902. Similar observations were made by Heald (loc. cit.) when working with salts of nickel, cobalt, etc. Report 71, U. S. Department of Agriculture, 1902.
4
fi. K. Cameron and]. F: Breazeale
the stimulating effects of concentrations much below the toxic limits frequently observed and discussed by other investigators was especially marked in solutions of acids. It was further observed that the absolute length of the radicle from plumule to apex, if the seedlings did not vary too much in age, appeared to have no effect upon the result, death occurring in equal strengths of solution within 2 4 hours, no matter what length of radicle was immersed in the solution when first introduced or at the end of 2 4 hours. I t has been found in all investigations of this character that the amount of sunlight accessible to the seedling during the experiments has a marked effect upon the strength of the solution which they can withstand. T h e experiments here recorded were conducted in a room to which there was no access of direct sunlight, and in which the illumination was quite feeble, though sufficient to enable one to conduct this work readily. T h e experiments were all made in the late winter so that the illumination probably exerted a very feeble effect, though the conditions were not strictly comparable with those obtaining in a dark closet. Check experiments carried on in the dark showed that in some instances the seedlings would stand slightly higher concentration than the results herein given. But the differences were small and always insufficient to justify abandoning the ad. vantage of watching the develepment of the plants from time to time, which was secured by working in a feebly lighted room, These differences were only of a noticeable character with wheat, barely observable with clover, and practically did not exist with corn. In almost every experiment made there were found to be individual idiosyncrasies of the seedlings, some being very much less affected than others and surviving in concentration sufficient to kill the majority. T h e figures given here represent the concentrations just permitting growth to continue in upwards of 60 percent of the seedlings in from 3 to 8 duplicate experiments, each experiment involving 4 seedlings of corn, 6 to 8 of wheat, or 8 to IO of clover. I n narrowing down the limits
Toxic Action o f Acids and Salts on Seedlings
5
between which these duplicates were made, from 2 0 to 30 experiments were required. A practically constant tetnperature of about 2 5 O C was maintained in the room where the experiments were conducted. T h e seedlings were sprouted in a specially prepared box between new blotting papers, moistened with distilled water. They were kept at a temperature of about 25' until the primary radicle had reached the desired length and then removed from the germinating box and transferred as quickly as possible to the vessels containing the solution. These vessels were cylindrical vials about 9 centimeters high and 4 centimeters in diameter. They were repeatedly washed before being used, and it seems improbable that the solubility of the glass could have affected the results. Attempts to prevent any such solution by coating the interior of the vial with paraffine, etc., mere found unsatisfactory and, as tested by a few check experiments, probably unnecessary. T h e seedlings were picked u p as tenderly as possible, the radicle inserted through a loop in a platinnm wire and the plants let down into the solution until the greater part of the radicle was immersed. T h e platinum wire passed through a tight-fitting cork with a small slit in it to allow ventilation. This furnished a support for the plants and allowed freedom of movement up or down, so that a desired immersion of the radicle could be readily obtained. T h e vials under observation were placed under glass bell-jars in which there was a small open vessel containing distilled water. A separate vial in which the radicles were immersed in distilled water was carried along with each set as a check. T h e plants used in this investigation were wheat (Triticum vulgaris), Indian corn (Zea mais), clover ( Tr$%Ziumjraetensea), cowpeas (Vzgna catjang * *) , cotton (Gossyjium herbacium), and white lupine (Lujinus albus). With the cowpeas and cotton it was found impossible to obtain satisfactory results under the conditions described, no matter what concentrations were used, and they will not, consequently, be given here. T h e work
6
F. K. Camevon a n d J I;: Bveazeale
with the white lupine was only preliminary ih character and was done for the purpose of acquiring experience in manipulation, and to test whether the procedure and criteria used would furnish results in satisfactory agreement with those formerly obtained by Kahlenberg and True, and in the previous work of this laboratory. With wheat, 594 experiments were made, about 2600 seedlings being used. T h e small size of the seedlings of this plant made the work quite difficult. T h e radicles moreover are normally, nearly transparent or translucent, so that the effect of the solution was often uncertain. With clover, 688 experiments were made with about 5000 seedlings. These were even more difficult to handle than the wheat, not only on account of their size, but also because of their extreme sensitiveness to the action of the dissolved electrolytes. With corn, 550 experiments were made, using about 2500 seedlings. This proved to be a very satisfactory plant for this kind of work, the only difficulty encountered being the tendency for a mold to appear quickly and accumulate in the vials in which the seedlings of this plant were being tested. T h e large number of experiments cited were occasioned by the fact that we have attempted to work within narrower limits than has hitherto been attempted. I n the case of corn in solutions of acids the results given are within O.OOOOI N of the strength of solutions on either side of that determined as representing the toxic limit. With wheat and clover the absolute value of the probable experimental error is even smaller, though considered on a percentage basis this might not be so. T h e solutes used were carefully examined to secure purity, in many cases the substances being prepared by ourselves. Great care was exercised in making up the various concentrations and duplicate preparations were made, to guard against errors as far as possible, since no analytical methods of sufficient delicacy were available to check this work. T h e results obtained in solutions containing a single solute are given in Table 11. They are stated in terms of the dilution,
I
Toxic Aciioi2 o f Acids and Salis on Seedlings
7
that is, the number of liters containing one grain equivalent of the solute. TABLE11. Toxic Limits for Seedlings in Solutions of Single Solutes -~ I
Name of solute
Hydrogen sulphate " potassium sulphate " chloride " nitrate " acetate " oxalate ' I succinate " malate sulphate chloride nitrate acetate oxalate succinate malate carbonate Calcium " "
chloride nitrate acetate
1
Toxic limits for
Corn
I
3000 3000 3000 2250
850
'750
1
Wheat
-
1
Clover
-
I 8000 15000 20000 I 5000
20000 20000
20000 20000
600 I2jO 22.5
I5
12.5 12.5
12.5
'5
25
I5
25
55 1 2 .j
3;
* *
9:
IO
85
95
140
4 5
5 5
20
4 1 4
9 *
*
*
The results obtained with corn are generally in very good accord with those published by Heald' as the result of his work with this plant. But Heald worked between linfits of concentration far wider than we employed, and in consequence we have felt justified in stating our results in detail. Heald found that LOC.cit. I n his tabulation of results, Heald gives the toxic limit for acetic acid as N/4oo, but from the description of his experiments it would appear that it should be N'800. Loew (Science, 18, 304 (1903)) gives the toxic limit for sulphuric and hydrochloric acids as N,51z, although no reason is apparent for this wide discrepancy from the results of Heald and those presented here. See also Science, 18,4 1 1 (1903).
s
I? K.
Cameron a n d J E;: Breazede
corn seedlings would just survive in the same relative strength of nitric acid as of sulphuric or hydrochloric acid, natnely, N/3200, while we found by repeated experiments that the seedlings would survive in much stronger solutions of nitric than of the two other acids. In fact an N/3000 solution, which in the case of sulphuric or hydrochloric acid would barely permit growth, was in the case of nitric acid by no means sufficient to seriously retard growth.' I n the case of clover the uniformity of the figures for the limits in acid solutions is very striking. I t might be supposed that this uniformity was only apparent owing to the difficiilties inherent to work with such a sensitive organism and with such highly dilute solutions. But great care was exercised in determining these limits, and it was conclusively shown that an N/175oo solution of any of these acids would kill or absolutely inhibit the growth of upwards of 60 percent of clover seedlings. T h e figures obtained for wheat were determined within as narrow limits as those for clover. T h e spaces (*) in the table under the columns for wheat and clover are occasioned by the fact that it was impossible to prepare the solutes with a purity sufficient to obtain satisfactory results. A very slight excess of base or acid would totally invalidate,the results owing to the extreme sensitiveness of wheat or clover to these solutes. I t was found to be practically impossible to prepare solutions of the salts indicated which would neither be alkaline nor contain free acid sufficient to make an N/2o,ooo solution with respect to it. In saturated solutions of the slightly soluble sulphate, oxalate, succinate, malate or carbonate of calcium, seedlings of corn, wheat or clover all thrive vigorously. I n distilled water nearly saturated with respect to carbon dioxide, it was found that a large majority of corn seedlings sur1 Kahlenberg and Mehl, loc. cit., working with young bass, found decided differences in the toxic effect of equivalent solutions of these acids.
\
Toxic Action of Acids ana? Salts on Seedlings
9
vived, but that seedlings of wheat' or clover were invariably and quickly killed. In the work formerly done in this laboratory it was found that dissolved carbon dioxide had no effect upon seedlingsof white lupine other than a slightly stimulating one. Whateve1 value these results niay have for other studies, they can not be considered to have any for the mooted question as to the actual existence of carbonic acid and its dissociation in aqueous solution. T h e results obtained with clover seedlings in acids would indicate that the toxic action was due to the hydrogen ion alone; for it must be assumed that with a dilution N/20,000practically complete dissociation existed for each and every one of the acids. But the results obtained with the wheat seedlings ' are not in harmony with this view, since the dilutions here were also such as to insure practically complete dissociation, although quite different concentration of the several acids gave the toxic limit. Curiously enough, from this point of view, acetic, succinic, and malic acids were found to be more toxic than sulphuric, hydrochloric or nitric acids, although the former are weaker acids than the latter. With the corn seedlings in acid solution the results are quite as difficult to interpret. They cannot be regarded as due to the effect of the hydrogen ion alone, as the concentration with respect to this substance in the solutions representing the toxic limits varies considerably. T h e approximate amount in grams of hydrogen as ion in some of these solutions are as follows : This is in apparent opposition to the observation of Jentys, Bull. Internat. Acad. Sci. Cracovie, 1892, 306 (1893),that wheat in a pot was practically unharmed by the passage of carbon dioxide through the soil. But it should be borne in mind that seedlings or plants which have received even serious injuries may make a fair growth when the cause of the injury is removed, as by the subsequent aeration of the pot through which the carbon dioxide had been passed. In illustration of this survival of injuries it might be cited that seedlings of wheat, the roots of which had been allowed to stand 24 hours in a Niro solution of sulphuric or nitric acid, and thus very badly damaged, when transferred to distilled water, a nutrient solution, or soil, frequently throve, sending out new roots in place of those which had been destroyed. Computed from conductivity measurements, Physikalisch-Chemische Tabellen, Landolt and Bornstein, z Aufl., pp. 496-501 (1894).
ro
R K. Cameron and]. F. Breazeab
TABLE111. Corresporiding Concentrations with Respect to Acids and Ionized Hydrogen -
I solute
Sulphuric acid Hydrochloric acid Nitric acid Acetic acid Succinic acid Malic acid
~
Dilution
Grams H-ion per IOO,OOO liters solution
N/3000 N/3000
33 33
N/2250
N/850 N/6oo N/1250
44
I3
26
40
Assuming, as has generally been done in previous work of this kind, that the action of the suiphuric and hydrochloric acid solutions were entirely due to the hydrogen ions, it would be necessary to assume that the acetic and succinic acids, the anions or the undissociated solute or both had also a similar toxic effect which was not of a negligible magnitude ; but that with nitric and malic acids, the anions and undissociated solutes had a retarding or ameliorating effect. T h e necessity of such assumption would seem to absolutely invalidate the use of such organisms and criteria as we have been considering for the testing of the dissociation hypothesis in any quantitative way. I n considering the figures in Table I1 obtained for the potassium salts, difficulties are encountered quite as serious. If the toxic effects are to be ascribed mainly or alone to the ions present, it is not easy to see why clover seedlings should be able to withstand a twelfth-normal solution of potassium chloride, while a thirtieth-normal potassium nitrate or a fiftieth-normal potassium sulphate solution marks the limit. And in the case of the wheat seedlings it is no more explicable why they should be able to stand a more concentrated solution of the sulphate and chloride than of the nitrate or oxalate, which latter, if anything, are the less dissociated. Without citing further details, a casual inspection of the figures obtained in the corn seedlings would show them to be quite as confusing. It does not seem possible to consider these results as pro-
Toxic Action of Acids and Salts on Seedlings
I1
duced mainly by the cathions and modified bj7 the anions, in view of the results obtained with the wheat seedlings, where, for instance, nitric acid was found to be less toxic than hydrochloric or sulphuric acid, but potassium nitrate to be more toxic than the corresponding potassium salts of the other acids just tnentioned. Moreover, with the clover seedlings all the acids possessed the same relative toxicity, while the potassium salts varied very widely. T h e results obtained with lime salts, while interesting as compared with those obtained with the corresponding potassium and hydrogen compounds, do not seem to throw any additional light upon the value of this method for studying dissociation phenomena and need no further discussion here. I n Tables IV and V are given results obtained in solutions of acids when in addition to the free acid the indicated amounts of a corresponding potassium or calcium salt, respectively, were also present. TABLEIV. Death Lrrnits for Seedlings in Solutions of Acids Containing Various Amounts of the Corresponding Potassium Salts ~
_
_
Toxic limits with respect to free acid in solutions containing Name of plant
Corn
Name of solute
Sulphuric acid Hydrochloric acid I1 Nitric I1 Acetic Oxalic Succinic I < Malic
3000 3000
Sulphuric Hydrochloric Nitric
( 1
I
