Yet, in spite of all the accumulated evidence as to the consistent toxicity of copper salts in great dilution, the possibility still remains that the limit of toxicity has not yet been reached, and that a stimulating concentration does exist, so that it is still uncertain whether beyond the limits of toxicity copper salts act as indifferent or stimulative agents.

4. Action of copper on organs other than roots.

The bulk of the work on the relations of copper with the life-processes of plants has dealt with those cases in which the metal has been supplied to the roots in some form or other, and many of the results may be said to apply more strictly to the theoretical, or rather to the purely scientific aspects of the matter, than to the practical everyday life of the community. This statement is hardly correct, in that the two lines of work are so inextricably interwoven that the one could not be satisfactorily followed up without a parallel march of progress along the other. In practice, copper has proved remarkably efficient as a fungicide when applied as sprays in the form of Bordeaux mixture to infested plants and trees. Observations on the action of the fungicide have shown that the physiological processes of the treated plants are also affected to some degree, and a number of interesting theories and results have been put forward.

(a) Effect of copper sprays on leaves.

Frank and Krüger (1894) treated potato plants with a 2% Bordeaux mixture, and obtained a definite improvement in growth, which they attributed to the direct action of the Bordeaux mixture upon the activities of the plant. The effect of the copper was most marked in the leaves, and was chiefly indicated by increase in physiological activity rather than by morphological changes. The structure of the sprayed leaves was not fundamentally changed but they were thicker and stronger in some degree, while their life was lengthened. Apparently, treatment increased the chlorophyll content, and, correlated with this, was a rise in the assimilatory capacity, more starch being produced. Rise in transpiration was also observed. While the leaves were the organs most affected, a subsidiary stimulation occurred in the tubers, since the greater quantity of starch produced required more accommodation for its storage. In different varieties the ratio of tuber formation on treated and untreated plants was 19:17 and 17:16. In discussing the meaning of this stimulation these writers, following the custom then in vogue, were inclined to hold that it was due to a catalytic rather than to a purely chemical action, an idea similar to one which later on came much into prominence in connection with the work of Bertrand’s school on manganese, boron and other substances.

The imputed increase in photo-synthesis seems to have met with approval and acceptance, but nevertheless it did not pass unchallenged. Ewert (1905) brought forward a detailed discussion and criticism of the assumption that green plants when treated with Bordeaux mixture attain a higher assimilation activity than untreated plants. His experiments were made to test the effects of differing conditions of life on plants treated in various ways, and his conclusions lead him to assert that “instead of the organic life of the plant being stimulated by treatment with Bordeaux mixture it is rather hindered.”

While Frank and Krüger indicated a rise in transpiration when copper compounds were applied to the leaves as sprays, Hattori (1901) attributed part of the toxic effect of copper salts, when applied to the roots, to a weakening action on the transpiration stream, and he maintained that the toxic effect of the copper salts is therefore connected with the humidity of the air. No further confirmation or refutation of this statement has so far come to light.

In certain plants the application of cupric solutions as sprays causes a slight increase in the quantity of sugar present in the matured fruits. Chuard and Porchet (1902, 1903) consider that such a modification in the ripe fruit during the process of maturation occurs in all plants which ripen their fruits before leaf-fall begins. Injection of solutions of copper salts into the tissues of such plants as the vine causes more vigorous growth, more intense colour and greater persistence of the leaves; in other words the copper acts as a stimulant to all the cells of the organism. A similar effect is produced by other metals such as iron or cadmium. By injecting small quantities of cupric salts into the branches of currants an acceleration of the maturation of the fruits was caused, identical with that obtained by the application of Bordeaux mixture to the leaves. If the quantity of copper introduced into the vegetable organism was augmented, the toxic action of the metal began to come into play. These investigators attributed the stimulus, as shown by the earlier maturation of the fruits, to a greater activity of all the cells of the organism and not to an excitation exercised only on the chlorophyll functions.

(b) Effect of solutions of copper salts on leaves.

Treboux (1903) demonstrated the harmful action of solutions of copper salts on leaves by means of experiments on shoots of Elodea canadensis. The activity of photo-synthesis was measured by the rate of emission of bubbles of oxygen. On placing the shoots first in water, then in N/1,000,000 copper sulphate (·0000159%), there was a reduction from 20 to 15 or 16 bubbles in 5 minutes. On replacing in water there was an increase to 18, but not to 20, indicating a permanent injury. With N/10,000,000 copper sulphate there was little or no reduction in the number of bubbles. This experiment had an interesting side issue in that it was noticed that not only the concentration, but also the quantity of fluid was concerned in the toxic action, indicating that both the proportion and the actual amount of poison available play their part. For instance, with a shoot 10 cm. long in 100 c.c. solution the plants were only slightly affected by ·000015% copper sulphate, but in 500 c.c. solution the shoots were killed after some days in ·0000015% copper sulphate, a concentration only one-tenth as great.

While it is evident that copper sprays have a definite action upon green leaves, whether favourable or unfavourable, the question arises as to the means whereby the copper obtains access to the plant in order to take effect. Dandeno found that solutions of copper sulphate were absorbed by the leaves of Ampelopsis, forming a brown ring. Generally speaking inorganic salts in solution are absorbed through both surfaces of the leaves, whether the leaves are detached or not, provided the surrounding atmospheric conditions are favourable, the absorption being usually more ready through the lower surface. Dilute solutions applied in drops stimulate the leaf tissue in a ring, whereas if the solutions are concentrated the entire area covered by the drop is affected. Too concentrated solutions of copper sulphate applied to leaves caused scorching, but if this was avoided while the solution was still strong enough to cause a darkening of green colour after a time, Dandeno considered that the action was probably of the nature of a stimulus to growth, and produced a better development of chlorophyll and protoplasm in the region where the tissues appeared dark to the naked eye, a conclusion which tallies very closely with that of Frank and Krüger.

Amos (1907–8) experimented to see whether the application of Bordeaux mixture affected the assimilation of carbon dioxide by the leaves of plants, and whether any stimulation was produced. Brown and Escombe’s methods and apparatus were used and the summarised results indicate that the application of Bordeaux mixture to the leaves of plants diminishes the assimilation of carbon dioxide by those leaves for a time. The effect gradually passes off, whatever the age of the leaves may be. The suggestion is made that the stomata are blocked by the Bordeaux mixture, so that less air diffuses into the intercellular spaces and less carbon dioxide comes into contact with the absorptive surfaces. If this hypothesis is correct, the physiological slackening of assimilation is not due to the toxic action of the copper in the Bordeaux mixture, but to a mechanical hindrance due to blocking of the stomata.

III. Effect of Copper on Certain of the Lower Plants.

On turning to the lower plants, especially to some species of fungi, one notices a striking contrast in their behaviour to that of the higher plants. Some species of fungi have the power of living and flourishing in the presence of relatively large quantities of copper compounds, or even of copper or bronze in the solid state. Dubois (1890) found that concentrated solutions of copper sulphate, neutralised by ammonia, which were used for the immersion of gelatine plates used in photography, showed white flocculent masses resembling the mycelium of Penicillium and Aspergillus, which grew rapidly and fructified in Raulin’s solution, but which remained as mycelium in cupric solutions. The mould proved capable of transforming copper sulphate into malachite in the presence of a piece of bronze, but it was found that the presence of the latter was not essential for the conversion into basic carbonate. The same result was obtained if the culture liquid was put in contact with a body which prevented it from becoming acid, fragments of marble acting in this way. Copper sulphate solution in the presence of the mould produced a green deposit on the marble, while without the fungus the solution simply evaporated leaving a blue stain of copper sulphate.

Trabut (1895) found that on treating smutty wheat with a 2% solution of copper sulphate he obtained a mass of flocculent white mycelium, whose surface was soon covered with aerial branches bearing pale rose-coloured spores, and he gave the provisional name of Penicillium cupricum to the species. On preparing nutritive solutions by steeping a handful of wheat in water for 24 hours, and then adding various amounts of copper sulphate to them, Penicillium was found to vegetate quite well until the amount of copper sulphate reached 9¹⁄₂ grams in 100 c.c., after which the seedings with spores did not develope at all. De Seynes tested this Penicillium more exhaustively with different culture media under various conditions and decided that Trabut was right in only assigning the name P. cupricum provisionally, as the mould reverts to the form P. glaucum when seeded in a natural medium, indicating that P. cupricum has not an autonomous existence, but is P. glaucum which modifies the colour of its conidia under the influence of copper sulphate, in the same way that it often modifies them in other media. It is noticeable that the mycelium arising from the germination of conidia of P. cupricum in a normal medium has a very poor capacity for producing reproductive organs, but this diminished activity is attributed not to a special deleterious action of the copper sulphate but to the impulse given to the vegetative functions, at the expense of the reproductive, when the spores are seeded in a richer medium than the solutions of copper sulphate which serve as the soil for P. cupricum.

Ono found that Aspergillus and Penicillium are retarded in growth in the higher concentrations of copper sulphate, but that they are stimulated by weaker strengths. The range of stimulating concentrations is given as from ·0015%–·012%, the biggest crop being obtained with both moulds in the strongest of these solutions. Hattori gives the optimum as being considerably lower for the two fungi mentioned, Penicillium being at its best in a solution of ·008% and Aspergillus in ·004%. A. Richter (1901) opposes this absolutely so far as Aspergillus niger is concerned. In his experiments copper appears invariably as a depressant, all concentrations from 1/150 to 1/150,000,000 giving growth below the normal, no stimulative action ever being observed. Zinc however proved to be a definite stimulant and in a mixture of copper and zinc salts in appropriate concentrations the toxic effect of the copper was completely paralysed by the stimulating action of the zinc, 1/200,000 zinc salt paralysing or overcoming the copper salt at 1/1125.

Ono states that the optimal quantity of such poisons as copper salts is lower for algae than for fungi, copper failing to stimulate algae at dilutions which were the most favourable to the growth of fungi. Bokorny indicates that silver and copper salts work harm in unusually dilute solutions.

Attempts have been made to utilise the poisonous action of copper on algae in clearing ponds of those plants. Lindsay (1913) describes experiments carried on in a reservoir infested with Spirogyra. A quantity of copper sulphate sufficient to make a solution of 1/50,000,000 was found necessary to kill off the Spirogyra, but it is suggested that the solution was probably weaker before it reached the algae, owing to the currents of fresh water. Anaboena needed 1/10,000,000 before it was killed off, while Oscillatoria is less sensitive still, 1/5,000,000 usually representing the mortal dose, though 1/4,000,000 was necessary in some instances. Algae seem to be peculiarly sensitive to the copper sulphate, far more so than the higher plants, as Nuphar lutea, Menyanthes trifoliata, and Polygonum amphibium grew in the water unharmed by the addition of the poisonous substance. For some unexplained reason it seems that “the concentration of copper sulphate necessary to kill off the algae in the laboratory is five to twenty times as great as that needed to destroy the same species in its natural habitat.”

Conclusion.

Altogether, after looking at the question from many points of view, one is forced to the conclusion that under most typical circumstances copper compounds act as poisons to the higher plants, and that it is only under particular and peculiar conditions and in very great dilutions that any stimulative action on their part can be clearly demonstrated.

CHAPTER IV
EFFECT OF ZINC COMPOUNDS

I. Presence of Zinc in Plants.

The presence of zinc in the ash of certain plants has been recognised for many years, especially in so far as the vegetation of soils containing much zinc is concerned. Risse, before 1865, stated that most plants when grown on such soils prove to contain greater or less quantities of zinc oxide. He states that the soil at Altenberg, near Aachen, is very rich in zinc, which rises as high as 20% in places. The flora of the soil is very diversified and zinc has been determined qualitatively in most and quantitatively in some of the plants. Viola tricolor and Thlaspi alpestre are most characteristic under such circumstances, both showing such constant habit changes that they resemble new species, while other plants such as Armeria vulgaris and Silene inflata are peculiarly luxuriant. Risse’s figures of the zinc content of these four plants may prove of interest. The figures are based on the dry weights, air dried.

Freytag (1868) carried out various experiments on the influence of zinc oxide and its compounds on vegetation, and found that all plants are capable of absorbing zinc oxide by their roots when grown on soils containing such oxide. Generally speaking the zinc is deposited chiefly in the leaves and stems, very little being found in the seeds, such minute traces occurring that he stated that the seeds must be harmless for men and animals. The general content of ZnO in plants is given as ·5–1·0% of ash, except in the abnormal case of plants growing on calamine.

Lechartier and Bellamy (1877) demonstrated the presence of zinc in such food substances as wheat, American maize, barley and white haricots, but they failed to find it in maize stems and beetroot, so they cautiously concluded that if it does occur in the latter cases it must be far less in quantity than in the former. Hattensaur (1891) analysed the ash of Molinia cærulea and discovered the presence of copper, manganese, zinc and lead, zinc oxide forming ·265% of the total ash, (·006% of the air dried plant).

Jensch (1894) observed that the flora on calamine soils was somewhat scanty, the chief plants that came under his notice being Taraxacum officinale, Capsella Bursa-pastoris, Plantago lanceolata, Tussilago Farfara, and Polygonum aviculare, all of which showed certain morphological peculiarities. Generally speaking the growth of these plants on the calamine soils was weak and poor, the stems and leaves being very brittle. Jensch found that the roots were deformed and showed a tendency towards a plate-like superficial spread of root. The leaves of Tussilago were uneven in shape and lacked the white hairs on the under side, the flower stalks were twisted, while the flowers themselves were a deep saturated yellow colour. The stems of Polygonum aviculare were much thickened at the nodes, the leaves weak and rolled in character, while the flowers were long-stalked, the calyces being usually of a purple red colour. The following figures are given for the quantities of zinc carbonate (ZnCO₃) in the ash of these two plants:—

Other analyses of plants from zinc soils as against controls from normal soils indicated the high water and high ash content of the zinc plants, though the dry matter was low, and it is suggested that the increase of the ash may be connected with a stimulation caused by the zinc salts, unless it is due to phosphoric-acid hunger, since the calamine soils concerned are very deficient in phosphorus.

Javillier (1908 c) corroborated the early statements of Risse as to the presence of considerable quantities of zinc in certain species of Viola, Thlaspi and Armeria, and also he cited a list of other plants in which zinc occurs in some quantity. Javillier, however, is of opinion that zinc oxide, like the oxides of iron and manganese, is very common in plant ash, being present in all plant organs. Zinc is specially abundant in Coniferae, where it is probably characteristic, as is the presence of manganese in the ash and manno-cellulose in the wood. The so-called “calamine” plants show great powers of accommodation to large amounts of zinc.

Klopsch (1908) analysed 17 species of plants grown on soil in the vicinity of zinc works, and showed that the plants evidently absorb small quantities of zinc from their surroundings. He also regarded zinc as a normal constituent of certain plants.

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

1. Toxic effect.

(a) Toxic action of zinc salts alone in water cultures.

In comparison with copper little work has been done with regard to the action of soluble zinc salts alone on higher plants when grown in water cultures. Freytag (1868) stated that zinc salts must be very dilute if the plants are not to be harmed, and that for zinc sulphate the concentrations must not be more than 200 mg. per litre (= 1/5000). Baumann (1885) carried out further experiments and concluded that zinc salts are far more toxic than Freytag suspected, 44 mg. zinc sulphate per litre[5] killing plants of 13 species belonging to 7 families (Coniferae excepted). The various plants withstand the action of the zinc salts in different degrees, the same concentration killing off the species in different times. With the 44 mg. zinc sulphate the following results were obtained:—

With still less poison, 22 mg. zinc sulphate per litre, all the species mentioned were eventually killed with the exception of Onobrychis sativa, while 4·4 mg. zinc sulphate seemed to be harmless for all the plants tested except Raphanus sativus, which is evidently exceptionally sensitive to this toxic substance.

Jensen (1907) again indicated the poisonous action of zinc salts and also found that a relatively small reduction of toxicity was obtained by the addition of finely divided quartz to the solutions.

(b) Effect of soluble zinc salts in the presence of nutrients.

Krauch (1882) grew various plants in the presence of nutrient solutions and quantities of zinc sulphate varying from ·1 to ·8 gm. per litre (= 1/10,000 to 8/10,000). Barley proved to be very sensitive, even to the weakest strength of the poison, as the plants soon showed reddish flecks, while all were dead within six weeks, the control plants without zinc remaining quite healthy. Certain grasses took longer to kill than barley, those with ·4 gm. zinc sulphate per litre dying in about seven weeks, while 13 weeks elapsed before the others were killed. Even after this length of time the plants with ·1 gm. zinc sulphate per litre still survived, although in a very sickly condition. With willow, again, even ·1 gm. zinc sulphate per litre made the plants very sickly after four weeks, growth being weak, the leaves yellow, and the roots brownish. In this case the solutions were renewed, but the plants treated with zinc compounds were dead within eight weeks from the start, the controls being very healthy.

The next year (1883) Storp repeated these experiments made by Krauch and corroborated his results fully. Barley and grasses (timothy and others) grown in solutions of zinc sulphate, both with and without nutrients, soon lost their green colour and became covered with rusty brown flecks, the barley dying within 14 days, and the grasses soon after. With willow, too, the toxic action was again manifested.

True and Gies (1903) showed that the addition of calcium salts in appropriate concentrations reduced the toxicity of zinc salts considerably, a result similar to that which they obtained for copper.

Recent experiments at Rothamsted have shown that zinc sulphate is very toxic to barley, though the plant is able to make some slight amount of growth even in the presence of a solution of the anhydrous salt ZnSO₄ as strong as 1/5000, rapid improvement occurring as the concentration decreases to 1/2,500,000 or less (Fig. 6). On the whole the higher strengths of zinc sulphate are less poisonous to peas than they are to barley. At a concentration of 1 in ¹⁄₄ or 1 in ¹⁄₂ million in different experiments the growth was nearly as good as with the control plants, though it consistently lagged a little way behind until a dilution of 1/10,000,000 was reached (Figs. 7 and 8). Incidentally it is very striking to see the desperate efforts that badly poisoned pea plants make to reproduce themselves. Growth of the roots is nearly always checked in advance of that of the shoots, probably on account of the contact of the roots with the poison. In the greater strengths of such poisons as zinc and copper sulphate root growth is checked from the outset, but usually a very little shoot growth is made, and one frequently obtains ridiculous little plants about an inch high bearing unhappy and diminutive flowers, which are occasionally replaced by equally unhappy and miniature fruits. The same thing has also been noticed when unsuccessful attempts have been made to introduce spinach as a test plant for water cultures.

(c) Effect of zinc compounds on plant growth when they are present in soils.

As soon as the presence of zinc in members of the vegetable kingdom was established the question arose as to its effect upon both the plant and the soil.

Gorup-Besanez (1863) grew plants in soil with which 30 grams of metallic poisons such as CuSO₄, ZnSO₄, HgO, were intimately mixed with 30·7 litres (“cubik Decimeter”) of soil[6]. On analysing the ash of Secale cereale, Polygonum Fagopyrum, and Pisum sativum after six months growth he failed to detect the presence of zinc in any one of the three. As the results varied with different poisons on different plants he concluded that the absorption capacity of the various kinds of soils for different poisons varies, that basic salts are absorbed, while the acid salts may pass completely through the soil in the drainage water.

Freytag (1868) stated that zinc is retained by the soil in the form of oxide, which is derived from dilute zinc compounds as they filter through the soil, by decomposition by the salts of the soil. For field earth the limit of absorption of zinc oxide from zinc sulphate is between ·21%–·24% of the earth.

F. C. Phillips (1882) corroborated Freytag’s statement as to the absorption of small quantities of zinc by the roots of plants, but he states as a fact that both lead and zinc may enter plant tissues without causing any disturbance in the growth, nutrition or functions of the plants, a conclusion that is obviously incorrect or at least incomplete in view of later work on the subject. His choice of plants was certainly unusual, including geraniums, coleas, ageratums and pansies, the poison used being zinc carbonate.

Holdefleiss (1883) stated that in spite of a soil content of 2% zinc the vegetation was not in any way harmed, clover fields and meadow lands on zinc soil presenting a normal appearance. This observation was quite inconclusive, as the author proceeds to say that of the plants that were able to absorb zinc salts without disadvantage the most luxuriant were the so-called zinc plants—the exceptions that prove the rule. Two years later Baumann showed that such insoluble zinc salts as the carbonate and sulphide in the soil cannot hurt plants. These salts are certainly dissolved to some extent by water containing CO₂ but solution is hindered by the constitution of the soil. He also found that the various kinds of soil act differently upon zinc solutions, the absorptive power of pure humus soils (“reinem Humusboden”) for zinc solutions being the strongest. Clay and chalk soils also decompose such solutions energetically, while poor sandy soils have only a weak power of absorption. This selectivity of absorption may account for the difference in the toxicity of zinc salts to plants in the various soils.

Storp (1883) experimented to determine the changes in the various characters of the soil by the action of zinc salts on it, and he makes the remarkable statement that in some soils the presence of zinc generates free sulphuric acid, which is particularly injurious to plant life. Grasses, young oaks and figs showed a decrease in dry weight, nitrogen and fat, as the quantity of zinc compounds increased in the water added to the soil. Both the quality and the quantity of the crop were adversely affected. This decrease in the dry weight due to the presence of zinc was confirmed by Jensch later on, and also by Nobbe, Baessler and Will (1884), who state that both lead and zinc compounds work disadvantageously to vegetation even when they are present in such small quantities that the plants are outwardly sound, the harmful action appearing in the decrease of dry weight. Contrary to Baumann’s opinion, zinc carbonate is said to be one of the salts that exercises this insidious poisonous action. Storp (1883) noticed that the direct poisonous action of zinc compounds is largely destroyed by their admixture with soil, but he suggests that a secondary cause of harm is introduced by the accumulation of insoluble zinc salts, so that the fertility of the soil is impaired to the detriment of the vegetation.

Ehrenberg (1908) throws out a suggestion that zinc is specially harmful to plant life when it occurs in conjunction with ammonia, but no further evidence has come to light.

(d) Mode of action of zinc on plants.

The reason for the toxicity of zinc salts when present in soil forced itself upon the attention of some of the early investigators in this field. Freytag (1868) put forward the hypothesis that the zinc oxide is partly or exclusively absorbed by the roots on account of the cell walls of the root being corroded by the very thin layer of zinc salts lying in contact with it—the same theory as has been held with regard to copper. He stated also that the quantity of zinc oxide taken up by the plant through its roots is strictly limited, not being proportional to the quantity occurring in the soil, but varying between narrow limits. Krauch (1882) found himself unable to accept another hypothesis which at one time found favour, i.e. that the zinc salts kill the plants by coagulating the protoplasm. If this were so, he argued, no plants at all could grow upon soils containing zinc, and he was content to leave the cause as one yet to be explained. Even at the present time, thirty years after, we know very little more about the physiological cause of the toxicity of zinc.

2. Effect of zinc compounds on germination.

In the course of his investigations on the influence of zinc on vegetation Freytag just touched upon the question of seed germination. According to his statement the presence of zinc oxide in the soil does not exercise much influence upon germination and the growth processes of plants. Little zinc is stored up in seeds and on this account seeds originating from plants containing zinc germinate quite normally and do not seem to be affected by the peculiar nutritive conditions of the parent plants.

In certain cases light seems to have something to do with the harm zinc compounds work on plants. Storp found that when clover seeds were germinated in the dark on filter paper moistened with water containing ·025 gm. ZnO per litre (added in the form of zinc sulphate) no deleterious action was observed. Barley seeds were soaked for four days in (a) distilled water, (b) water with ·9 gm. ZnO per litre, which was frequently changed. These seeds were then placed in the dark on filter papers soaked respectively with water and with the solution containing ZnO. So long as no light was admitted, for a period of eleven days, germination was uniform in both sets, but directly the covers were removed the growth of the seeds with zinc ceased almost entirely, and they did not assume the green colour taken on by the unpoisoned seedlings. With maize the germination was retarded by zinc even in the dark, but the harmful action of light on the plants with zinc was again established. These results seem to indicate that the formation and activity of chlorophyll is impaired by the toxic agent, and this hypothesis is borne out by the fact that in many fungi and non-assimilating higher plants the toxic action of zinc is not evident.

Micheels (1906) approached the matter from a totally different standpoint, seeking to discover what influence the valency of a metal has upon the toxicity of its salts. In each of a series of experiments 1000 c.c. of ⁵⁄₈ decinormal solution of sodium chloride in pure distilled water were used, with the addition of varying strengths of calcium sulphate. Grains of wheat, which previously had been soaked in distilled water, were placed in the solutions, and it was found that the stronger the calcium sulphate solution (up to ¹⁄₆₄ normal—the limit of experiment), the better the growth. The calcium sulphate was then replaced by salts of other bivalent metals, as zinc, lead and barium, with analogous results, the quantity necessary to obtain the maximum development varying with one and another; with zinc, n/128 gave the maximum. In this case the toxic action of both sodium chloride and zinc sulphate on germination were considerably reduced by their mutual presence—a result which fits in perfectly with what is known as to the masking effect of soluble substances upon toxic action. The same fact obtains in the animal kingdom, where Loeb and others have found that the toxicity of solutions of sodium chloride for marine animals is reduced by the introduction of salts of the bivalent metals.

3. Stimulation induced by zinc compounds.

While the toxic action of zinc on the higher plants is so obvious that it forced itself upon the attention of investigators at an early date, the question of possible stimulus is so much more subtle that it has only come into prominence during the last twelve years, during which time an extraordinary amount of experimental work has been done with regard to it. One investigator, Gustavson, was somewhat in advance of his time, for as long ago as 1881 he hinted at the possibility that zinc, aluminium and other substances might act as stimulants or rather as accelerators. He indicated that the rôle of certain mineral salts in the plant economy is to enter into combination with the existing organic compounds, the resulting product of the reaction aiding in the formation of yet other purely organic compounds which ordinarily require for their formation either a very high temperature or a long time—in other words, such a mineral salt acts as a kind of accelerator.

This work was apparently not followed up immediately, but it evidently contains the germ of the “catalytic” hypothesis of which so much has been made during recent years.

The work dealing with zinc as a stimulant to plant growth has yielded such various and apparently contradictory results that the question cannot yet be regarded as settled—it is even still more or less uncertain whether zinc compounds act as stimulants, or whether they are merely indifferent at concentrations below the toxic doses.

(a) Stimulation in water cultures.

True and Gies (1903) suspended seedlings of Lupinus albus for 24–48 hours with their roots in solutions of zinc sulphate and calcium sulphate (m/256)[7], and found that while zinc sulphate alone at m/8192 retarded growth, yet with m/2048 ZnSO₄ and m/256 calcium sulphate growth was more than twice as rapid as in controls grown in water, indicating a marked stimulation. The presence of the calcium exercised a definite ameliorating influence, reducing the toxicity of zinc to one-sixteenth at most. The hypothesis put forward is that interior physiological modifications are responsible for the observed differences in growth rate, the cell processes being so affected as to bring about different results on cellular growth—i.e. that where mixtures of salts are concerned growth rate represents the physiological sum of oppositely acting stimuli or of antagonistic protoplasmic changes.

Kanda (1904) found that peas were stimulated in dilute solutions of zinc sulphate in the absence of nutrients, the optimum concentration being between ·00000287% and ·000001435% (about 1 in 34,840,000 and 1 in 69,700,000), higher concentrations being poisonous when the solutions were changed every four days. Jensen (1907) stated that he obtained no stimulation at all with water cultures, even in a solution as dilute as n/100,000 (about 1 in 1,239,000), but he suggested that it was quite possible that in proper concentration the zinc sulphate might prove to be a stimulant.

Javillier (1910) grew wheat in nutritive solutions with quantities of zinc salts containing from 1/5,000,000–1/250,000 zinc, and found that the dry weight of the plant was increased in so far as the stems and leaves were concerned, though it remained uncertain whether a similar increase occurred in the grain.

A consideration of the Rothamsted experiments shows that up to the present time there is no conclusive evidence that zinc sulphate acts as a stimulant to barley grown in water cultures. As a general rule the growth of those plants with 1/5,000,000 ZnSO₄ approximates closely to that of the controls. Beyond this the growth varies in different experiments. In some cases lower concentrations from 1/5,000,000 to 1/50,000,000 seem to cause some slight improvement in comparison with the normal, indicating a possible stimulus, but this improvement is not at all well marked. In other cases these great dilutions are apparently indifferent, neither a poisonous nor a stimulative action being exerted on the growth of the plant (Fig. 6). With peas some increase has been obtained with 1/20,000,000, and although the rise is only slight, yet it is possible that it may indicate the setting in of a stimulus which would make itself more strongly felt with still weaker concentrations (Fig. 7).

(b) Stimulation in sand cultures.

While Jensen denied stimulation in wheat grown in water cultures even when the solutions were as dilute as n/100,000 zinc sulphate, yet he found increase of growth with the same plant in artificial soil (quartz flour) to which much stronger solutions of zinc sulphate, from 5n/10,000–n/10,000, had been added.

(c) Increased growth in soil.

Nakamura (1904) dealt with a few plants of agricultural importance, adding ·01 gram anhydrous zinc sulphate to 2300 grams air-dried soil. The marked individuality in the response of the various plants to the poison is very striking. Allium showed signs of increased growth throughout; Pisum was apparently improved in the early stages of growth, but when the dry weights were taken at the end of the experiment no increase manifested itself in the weights of the plants treated with zinc; with Hordeum the same quantity of zinc exercised a consistently injurious action. These results with peas and barley corroborate those obtained in the Rothamsted experiments with water cultures in that zinc sulphate proved to be less toxic to peas than to barley.

Kanda found that both peas and beans when grown in soil as pot cultures were improved by larger quantities of zinc sulphate than when they were treated as water cultures—a result in full accordance with current knowledge.

Wheat is evidently peculiarly sensitive to the effects of zinc compounds under differing conditions. Javillier (1908 c) pointed out that while wheat is very susceptible to the toxic action of zinc, yet it can benefit by the presence of sufficiently small quantities of the compounds of the metal. Rice is another cereal that is said to respond to the action of zinc sulphate, as Roxas, working in pot cultures with soil both with and without the addition of nutritive salts, obtained an acceleration of growth on the addition of m/1000 zinc sulphate, a quantity so remarkably great that it might be expected to act as a toxic rather than as a stimulant.

With phanerogams the zinc question is not only concerned with the effect of the metal upon germination, but also with its effect upon the later growth of the green plants, and on the physiological functions involving the construction of substances at the expense of mineral elements and the carbon dioxide of the air. Javillier holds that the indications are that zinc would prove to be profitable if applied to crops as a “complementary” manure.

4. Direct action of zinc salts on leaves.

Dandeno (1900) applied zinc sulphate in drops to the leaves of Ampelopsis, and found that the solution was not all absorbed by the leaf, but that a slight dark ring of a yellow colour was produced, and he was induced to think that some local stimulation was produced if the salt was presented in sufficient dilution.

Klopsch (1908) discussed the effect on plant growth of zinc derived from industries producing zinc fumes. Zinc oxide from the fumes is deposited on the leaves, and Klopsch stated that the rain and dew containing dissolved zinc compounds find entrance to the tissues by way of the stomates and work injury to the plants. Against this, however, it must be remembered that these same fumes also contain other substances which are admittedly harmful to plant life, and so the deleterious effect may be partly or even chiefly due to these substances rather than to the zinc. Yet it is probable that at least some of the depreciation is due to the zinc. Treboux (1903) tested the effect of zinc sulphate on shoots of Elodea canadensis. If the shoots were placed in n/100,000 (= ·000016%) zinc sulphate no reduction of assimilation (as observed by counting the number of oxygen bubbles emitted per minute) took place, and replacement in water apparently had no effect either way. When however the shoots were placed in (1) water, (2) ·00008% zinc sulphate, (3) fresh ·00008% zinc sulphate, (4) water again, it was found that while the first solution of zinc sulphate had apparently no effect on assimilation, yet during the second immersion a gradual reduction in assimilation set in, which reduction was continued after the return to pure water, so that the toxic action of the zinc sulphate upon the shoots was clearly demonstrated.

III. Effect of Zinc on Certain of the Lower Plants.

Among the fungi, one species stands out in special prominence on account of the great amount of work that has been done on it with regard to its reactions to zinc salts. Aspergillus niger = Sterigmatocystis nigra van Tgh was used as a test plant by Raulin (1869), who evidently considered that zinc was an essential primary constituent of the food solutions of the fungi, ·07 parts zinc sulphate being added to each 1500 parts of water. In his experiments he tested (1) ordinary nutritive solution, (2) nutritive solution with various salts added, as zinc sulphate, (3) nutritive solution and salts (as 2) and also powdered porcelain. (2) gave a crop of Aspergillus about 3·1–3·5 times better than (1), while (3) was even better still. Sulphate of iron also proved stimulating in its action, but Raulin stated that zinc cannot replace iron, as both are essential.

Ono (1900) determined the relation between the weight of the mould crop in grams and the quantity of sugar used up in the presence of varying amounts of zinc sulphate. The amount of sugar used was always greater in the crops with ·0037–·0297% zinc sulphate by weight than in the control crops, indicating a stimulation caused by zinc.

Richter (1901) carried out rather similar experiments. When grown in solutions without and with 1/700,000 gram molecule zinc sulphate the dry weights of the mould were practically the same for the first two days, then the dry weight of the zinc crop shot ahead for a day or two, a depression setting in on the fifth day. Without zinc a less increase took place, and a similar drop was noticeable about the sixth day. The conclusion drawn is that the stimulation due to the zinc occurs chiefly in the first few days and also that the rise in the sugar consumed is more rapid at first with the moulds treated with zinc. Concentrations above 1/600 are harmful, but in weaker solutions zinc is a definite stimulant.

Coupin (1903) re-investigated some of Raulin’s work under more antiseptic conditions in order to see what substances were really needed by the mould and whether certain elements declared essential were really so. He concluded that iron and zinc are of no use in the nutrition of Sterigmatocystis nigra, but that the zinc retards the development of mycelium when food is abundant, killing it if it is badly nourished. This denial of stimulation was controverted by Javillier (1907) who re-tested Raulin’s solution with extreme care, growing Sterigmatocystis in

• (a) normal Raulin’s solution with zinc,
• (b) Raulin’s solution without zinc.

The ratio of crops a/b varied from 2·3–3·1 in four experiments, vindicating the favourable action of zinc. With regard to the optimum value for zinc the mould seemed to be perfectly indifferent to the presence of medium quantities but very sensitive to extremes, the maximum weights being reached in dilutions between 1/10,000,000 and 1/250,000, while quantities above 1/25,000 were toxic in their action. At a dilution of 1/50,000,000 stimulation was still evident, though in a less degree than with the optimal concentrations.

Javillier maintains that zinc is fixed by the fungus, the whole of the zinc present in dilute solutions being taken up, only part being utilised in stronger solutions. The value of accordance between the quantity of zinc fixed and the quantity supplied decreases rapidly with increase of concentration. Sterigmatocystis is able to fix without harm a quantity of zinc equal to more than 1/1100 of its weight. Zinc is regarded as a catalytic element, as essential to the well-being of the plant as are the more obvious nutrients, carbon, sulphur, phosphorus, &c., in spite of the minute traces in which it occurs.

A few tests on yeasts made by Javillier showed that with vegetative yeasts zinc has a specific action, a consistent increase occurring in the amount of yeast formed and in the amount of sugar consumed as the quantity of zinc increased from 0–1/10,000,000–1/10,000. With ferment yeast, however, zinc exerted no appreciable action. These results lend force to the conclusion of Richards (1897) who carried out experiments on fungi with various nutritive media with the addition of certain salts of zinc, nickel, manganese, iron, &c. He considered that his general results showed that the fact of a chemical stimulation of certain metallic salts upon the growth of fungi is established, although it must not be considered without further investigations that all fungi react in the same degree to the same reagent.

Conclusion.

As matters stand at the present day, it appears that it is still uncertain whether higher plants grown in water cultures are susceptible to stimulation by zinc salts. If a stimulus does exist, it must be at exceedingly great dilutions, but further evidence is needed. In soil cultures, however, the fact of increased growth seems to be more firmly established, certain species responding to zinc salts when used as manure, though no increase has been obtained with other species. It must always be remembered that the action may be an indirect one. The soil is very complex in its constitution, and it is impossible to determine the exact action of the added poison upon it, so that a stimulating effect need not necessarily be due to a direct action of a substance upon the plant, but it may be the result of more favourable conditions for life induced by the action of the substance upon the soil.

Among the fungi the stimulation of Aspergillus niger by minute traces of zinc compounds seems to be well proved, though again it does not necessarily follow that all fungi will react in the same way to zinc.