II. Effect of Arsenic on the Growth of Higher Plants.

1. Toxic effect.

(a) Toxic action of arsenic compounds in water cultures in the presence of nutrients.

The poisonous action of arsenic on plants has long been recognised. [Chatin (1845)] gave accounts of tissues poisoned by strong arsenical solutions. [Nobbe, Baessler and Will (1884)] carried on water culture experiments with buckwheat, oats, maize and alder, and found that arsenic was a particularly strong poison for these plants. When small quantities of arsenious acid (As₂O₃) were added to the food solutions, growth was measurably hindered by a concentration of 1/1,000,000 As (reckoned as As). The element only appears in plants in very small quantity and can never be detected in notable quantities. The aerial organs show the effect of arsenical poisoning by intense withering, interrupted by periods of recovery, but eventually followed by death. It was also found that if plant roots were exposed to the action of arsenical solutions for a short period, say ten minutes, and then were transferred to normal food solutions, the action of the poison was delayed, but eventually hindering of growth or death occurred, according to the strength of the poison used in the first solution.

At the same time that Nobbe, Baessler and Will were establishing the great toxicity of the lower oxide of arsenic, [Knop (1884)] was carrying the matter a step further by comparing the action of arsenious and arsenic acid and their derivatives on plant growth. He established the fact that while arsenious acid is a strong poison for maize plants, arsenic acid in small quantities is not toxic to the roots and that the plants can produce flowers and fruit in its presence. Arsenic acid applied as potassium arsenate proved to be harmful to young maize seedlings if the solutions contained ·05–·1 gm. arsenic acid per litre (= 1/–2/20,000 arsenic acid). If however the plants were allowed to form 10–15 leaves in a pure food solution and then when strongly rooted were transferred to a solution of ·05 gm. arsenic acid per litre, they were found to grow strongly and develope big healthy leaves. Careful measurements indicated that the development is unchecked by the addition of the poison, though arsenic was determined in the ash of the treated plants.

Stoklasa ([1896], [1898]) tested the effect of arsenic compounds on plant growth with special attention to their comparative relation to phosphoric acid. He corroborated Knop’s statement as to the greater toxicity of arsenious acid and arsenites in comparison with arsenic acid and arsenates, stating that 1/100,000 mol. wt. arsenious acid per litre causes definite trouble in plants, while with arsenic acid 1/1000 mol. wt. per litre first shows a noticeable toxicity. Water culture experiments were made with and without phosphoric acid, in each case with and without the addition of arsenic and arsenious acid. It was found that the arsenic acid was unable to replace the phosphoric acid, the plants decaying in the flower in the absence of the latter. In the complete absence of phosphoric acid, arsenic acid causes a strong production of organic substances up to the flowering time. The following figures were obtained with maize:—

·002 gm.		As₂O₃	with P₂O₅		2·84 gm. dry wt.
·005 gm.		„	„	„	2·37	„
·01 gm.		As₂O₅	„	„	67·32	„
·40	„	„	„	„	64·13	„
·03	„	As₂O₅	without P₂O₅		39·98	„
·07	„	„	„	„	42·13	„
normal solution			„	„	12·93	„
„	„		with	„	65·84	„

Comparative experiments with the two arsenical oxides showed that varying times were required to kill different plants. Young seedlings were brought into solutions containing 1/10,000 mol. wt. arsenious acid (= ·019 gm. As₂O₃ per litre) and the plants died in a very short time.

Hordeum distichum		46 hours
Polygonum Fagopyrum		84	„
„	Persecaria	90	„

With ten times the strength of arsenic acid (1/1000 mol. wt. = ·23 gm. per litre) the plants took much longer to kill.

Hordeum distichum		24·5 days
Polygonum Fagopyrum		40	„
„	Persecaria	42	„

Various experiments have been carried on at [Rothamsted] with peas and barley. With arsenious acid on barley a depressing influence is manifest even at a concentration of 1/10,000,000, while no growth at all is possible with 1/10,000 and upwards. Apparently the toxic action on the root ceases at a higher strength than on the shoot, as with 1/1,000,000 and less the dry weight of the root remains practically constant. At this same strength the shoots look better than the controls, but this is not apparent in the dry weights (Figs. [9] and [10]). With peas the depression is again evident to 1/10,000,000, but the plants are more sensitive to the higher concentrations, as no growth can take place in the presence of 1/250,000 arsenious acid ([Fig. 11]). A striking difference is observed with arsenic acid on barley, as apparently this does not act as a toxic even with such comparatively great concentrations as 1/100,000, though possibly the shoot is slightly depressed by this strength ([Fig. 12]).

Fig. 9. Photograph showing the action of arsenious acid on barley in the presence of nutrient salts. (March 16th–May 9th, 1911.)

1.	Control.
2*.	1/50,000	arsenious	acid.
2.	1/100,000	„	„
3.	1/150,000	„	„
4.	1/200,000	„	„
5.	1/250,000	„	„
6.	1/500,000	„	„
7.	1/1,000,000	„	„
8.	1/5,000,000	„	„
9.	1/10,000,000	„	„
10.	1/25,000,000	„	„
11.	1/50,000,000	„	„

Fig. 10. Curve showing the mean value of the dry weights of ten series of barley plants grown in the presence of arsenious acid and nutrient salts. (March 16th–May 9th, 1911.)

Fig. 11. Curve showing the mean value of the dry weights of ten series of pea plants grown in the presence of arsenious acid and nutrient salts. (June 8th–July 21st, 1910.)

Fig. 12. Curve showing the mean value of the dry weights of ten series of barley plants grown in the presence of arsenic acid and nutrient salts. (Feb. 28th–April 24th, 1911.)

With sodium arsenite the dilutions were carried further, to 1/250,000,000, but this still depressed barley to some extent ([Fig. 13]). With peas the results vary somewhat in the different tests, the depression with 1/2,500,000 and less being usually slight, though occasionally it is much more strongly marked ([Fig. 14]). In a single series with sodium arsenate barley was apparently unaffected by a concentration of 1/1,000,000, but from this point down to 1/250,000,000 a constant depression showed itself, which was paralleled by a similar depression in the sodium arsenite series from 1/25,000,000 to 1/250,000,000, the curves grading downwards instead of up towards the normal. With peas sodium arsenate has little or no action, though it is just possible that the rather irregular curves indicate a very slight depression below the normal throughout.

Fig. 13. Curve showing the mean value of the dry weights of ten series of barley plants grown in the presence of sodium arsenite and nutrient salts. (Feb. 10th–April 18th, 1913.)

Fig. 14. Curve showing the mean value of the dry weights of ten series of pea plants grown in the presence of sodium arsenite and nutrient salts. (June 27th–Aug. 10th, 1911.)

(b) Toxic effect of arsenic compounds in sand cultures.

Comparatively few tests seem to have been made as to the action of arsenical solutions in sand cultures. [Stoklasa (1898)] repeated his water culture work, using sand as a medium, and found analogous results by the two methods, i.e. that arsenites are far more toxic than arsenates, and also that the degree of toxicity of a salt varies with the plant to which it is applied, as was shown by the fact that different plants lived for varying times when treated with similar strengths of solution.

(c) Toxic effect of arsenic when applied to soil cultures.

[Daubeny (1862)] watered barley plants with a solution of arsenious acid, 1 ounce in 10 gallons, five times in succession, and found that the crop arrived at maturity about a fortnight earlier than the untreated part of the crop, though the amount harvested was rather less. With turnips four waterings had no effect upon the time of maturity, but again the crop was slightly decreased. The analyses made indicated that no arsenic was taken into the tissues, but that it merely adhered to the external surfaces.

[Gorup-Besanez (1863)] mixed 30 grams arsenious acid with 30·7 litres[9] soil, growing two plants on this quantity of earth. Most of his experimental plants (Polygonum Fagopyrum, Pisum sativum, and Secale cereale) developed normally, but Panicum italicum died soon after the plants appeared above the surface, the leaves being very badly coloured. Analyses by Marsh’s test showed no trace of arsenic in 20 grams dry matter from Secale cereale, but in 148 grams Polygonum Fagopyrum the presence of arsenic was evident, though the mirror formed was weak. With such a large proportion of arsenious acid in the soil it seems hardly conceivable that the plants were not injured to some extent, and also it is probable that with more careful analyses arsenic would have been detected in those instances in which its presence was denied. Yet it must be remembered that [Davy (1859)] had treated pea plants in pots with a saturated solution of arsenious acid for a short time and had stated that the plants were uninjured. Thus both Gorup-Besanez and Davy concur in the opinion that Pisum sativum is indifferent to relatively large quantities of arsenious acid when presented in the soil, whereas the Rothamsted experiments show that in water cultures the plant is extremely sensitive even to minute traces of the substance. It is possible that the arsenic in the solution added to the soil enters into combination with other substances, forming insoluble compounds, thus being removed from the sphere of action and rendered unable to affect plant life. If this be so, the apparent immunity of certain plants to arsenious acid is explained. [F. C. Phillips (1882)], in his experiments on various flowering plants, such as geraniums, coleas and pansies, found that compounds of arsenic in the soil exercised a distinct poisoning influence, tending, when present in large amount, to check the formation of roots, so that the vitality of the plant was so far reduced as to interfere with nutrition and growth, or even to kill it outright. He also stated that traces of arsenic were found in all the plants grown upon the poisoned soil.

In this connection it is interesting to note that a certain proportion of arsenic is frequently present in the superphosphate used as manure. In view of the known toxicity of arsenical compounds to plant life the question arose as to whether superphosphate manuring would exercise a detrimental influence on account of its arsenic content. Experiments carried out by [Stoklasa (1898)], however, indicate that there is not sufficient arsenic in maximum doses of superphosphate to exercise a toxic action in the field.

(d) Physiological considerations.

The physiological action of arsenic compounds on plant life early attracted the attention of investigators. [Chatin (1845)] put forward some rather curious and unexpected considerations with regard to this action. He stated that the effect of arsenic on plant growth is determined more by the constitution and temperament of individual plants than by their age, and that apparently difference in the sex of plants is of no significance. The chief determining agent, however, is the species, and Chatin found that as a general rule Cryptogams are more sensitive than Phanerogams, and Monocotyledons than Dicotyledons, as is shown by the fact that under treatment the former perish first. Some extreme exceptions exist, though, as Mucor mucedo and Penicillium glaucum will grow on moist arsenious acid, whereas leguminous plants are killed by an arsenical solution in a few hours. Chatin held the view that elimination of the poison succeeded its absorption, and that this elimination is complete if the plant lives long enough. Here again the species exerts a great influence on the excretory functions of the plants. Lupins and Phaseolus are presumably able to eliminate in six weeks all the arsenious acid they can absorb without dying. Most Dicotyledons need 3–5 months, while Monocotyledons retain traces of poison for six months after its absorption. Lichens are said to eliminate it more slowly still. Again, woody species are longer in freeing themselves than herbaceous, and young plants carry out the elimination more easily than old plants. The excretory function is influenced by other physiological factors such as dryness and season. The toxic effects and elimination are supposed to act inversely and parallel, the absorbed arsenious acid combining with alkaline bases, making a very soluble salt which is excreted by the roots. Calcium chloride is given as the antidote to arsenious acid, all soluble acid being “neutralised” by it. This view of the elimination of arsenic apparently did not gain much support, as no further references to the matter have so far come to light. In view of the work of some modern investigators (Wilfarth, Römer and Wimmer) on the excretion of salts by plant roots, the idea may prove of fresh interest. Chatin also found that moving or still air influenced the working of the poison, indicating that the external physical conditions affect the toxic action considerably. Nearly forty years later [Nobbe, Baessler and Will] found that, if transpiration were hindered by placing plants in a dark or moist room, it was possible to keep the plants turgescent in arsenic solutions for a long time without thereby increasing the toxic effect later on. The poisonous action proceeds from the roots, of which the protoplasm is disorganised and the osmotic action hindered. Finally, in the presence of sufficient of the poison, the root dies without growth.

Stoklasa ([1896], [1898]) again found that phanerogamic plants can withstand arsenic poisoning for some time in the dark or in CO₂-free air, provided that glucose is given in the food solution. The arsenic poisoning is at its maximum during carbon assimilation by means of chlorophyll. The toxic action of arsenious and arsenic acids, especially in phanerogams, is due to injury to the chlorophyll activity. The destruction of the living molecule is far more rapid in the chlorophyll apparatus than in the protoplasm of the plant cell.

Thus it seems that the physiological cause of the toxicity of arsenic is partly a direct action on the root protoplasm, whereby its osmotic action is hindered, and partly a detrimental action upon those functions which are directly concerned with the elaboration processes of nutrition.

2. Effect of arsenic compounds on germination.

In view of the great toxicity of arsenic to plants in their various stages of development, one would naturally expect to find a similar action with regard to the germination of the seeds. [Davy (1859)] casually mentioned cases in which watering with arsenical solutions or dipping seeds in arseniated water prevented germination. [Heckel (1875)] found that arsenious acid checks germination and kills the embryo at relatively feeble doses, ·25 gm. to 90 gm. water.[10] Guthrie and Helms ([1903]–[4]–[5]) carried out a systematic series of experiments to test the effect of arsenic compounds upon different farm crops. Various amounts of arsenious acid were added to soil in pot experiments, and the seeds of the several crops were then sown. With barley, wheat and rye 0·10% arsenious acid had little or no effect on germination, while an increase in the poison exercised a retarding action. Maize could withstand 0·40% arsenious acid without retardation being perceptible. The aftergrowth with the different crops varied considerably. The wheat plants with 0·10% arsenious acid grew all right at first, but later on they developed weakly. The toxic action increased rapidly as the strength of the poison rose in the different pots. Barley proved even more sensitive than wheat, for even 0·05% arsenious acid affected the growth adversely. After a time the plants with 0·05–0·06% recovered and grew strongly, though not so well as the controls, but those with 0·10% practically died off. Rye behaved in the reverse way from wheat. The plants with 0·10% were slightly checked at first but later recovered and made growth quite equal to the check plants. Growth was stunted with 0·20% arsenious acid, and the plants were killed with 0·30%, so that rye is far less sensitive than barley. With maize the growth was slightly affected with 0·05% As₂O₃, and increasingly so with greater quantities. It was also found that the action of 0·8% As₂O₃ was strongly adverse to the germination of all plants, and that above this strength germination was altogether prevented.

The results show very clearly how impossible it is to draw any general conclusions with regard to the action of arsenic compounds on plants, as they emphasise the strong individuality of the species in their reaction.

3. Do arsenic compounds stimulate higher plants?

The question of stimulation due to arsenic does not seem to have engaged the attention of investigators to any extent. Water culture experiments at Rothamsted have so far yielded negative results, and no stimulation has yet been obtained with any plant, with the possible exception of white lupin with sodium arsenite. In a single series a stimulus was suggested, beginning to make itself felt at 1/500,000, rising to an optimum at 1/10,000,000. No stress can be laid on this result, as it is never safe to draw any certain conclusions without several repetitions of the same experiment. With arsenic acid on barley a possible stimulus is sometimes indicated to the eye, the plants being fine and of a particularly healthy dark colour, but this is not corroborated by the dry weights. Additional tests were made with peas and barley, treated with sodium arsenite and arsenate, the dilutions being carried down to 1/250,000,000, but no evidence of stimulus was obtained, so that it hardly seems possible that arsenic can act as a stimulative agent for these two plants when grown in water cultures. It had been thought that the failure to find a stimulation point hitherto might be due to the too great concentration of the toxic substance rather than to the actual inability of the poison to stimulate, but this hypothesis must now be dismissed so far as these plants are concerned.