Inorganic Plant Poisons and Stimulants – Effect of Copper Compounds

CHAPTER III
EFFECT OF COPPER COMPOUNDS

I. Presence of Copper in Plants.

Copper has been recognised as a normal constituent of certain plants for at least a century, so much so that in 1816Meissner brought out a paper dealing solely with the copper content of various plant ashes. The ash of Cardamomum minus, of the root of Curcuma longa, and of “Paradieskörner,” amongst others, were tested and all yielded copper in very small quantity. Meissner was led to conclude that copper is widespread in the vegetable kingdom, but that it exists in such minute traces that its determination in plants is exceedingly difficult. In 1821 Phillips made an interesting observation as to the effect of copper on vegetation. Some oxide of copper was accidentally put near the roots of a young poplar, and soon after the plant began to fail. The lower branches died off first, but the harm gradually spread to the topmost leaves. As a proof that copper had been absorbed by the plant the record tells that the blade of a knife with which a branch was severed was covered with a film of copper where it had been through the branch, and the death of the plant was attributed to the absorbed copper.

After this preliminary breaking of the ground little more seems to have been done for some sixty years, but from about 1880 till the present day the association of copper with the vegetable kingdom has been actively investigated in its many aspects. Dieulafait (1880) showed that the quantity of copper present in the vegetation is largely determined by the nature of the soil, which thus affects the ease with which the element can be detected and estimated. Copper was shown to exist in all plants which grow on soils of “primary origin” (“roches de la formation primordiale”), the proportion being sufficient to enable it to be recognised with certainty in one gram of ash, even by means of the ammonia reaction. Samples of white oak from the clay soils, and plants from the dolomitic horizons also gave evidence of copper in one gram of ash, though less was present than in the first case considered, but with plants grown on relatively pure chalk 100 grams of ash had to be examined before copper could be recognised with certainty.

E. O. von Lippman found traces of copper in beets, beet leaves, and beet products; Passerini estimated as much as ·082% copper in the stem of chickpea plants, though he regarded this figure as too high; Hattensaur determined ·266% CuO in the total ash of Molinia cærulea (·006% of total plant, air-dried).

After this Lehmann (1895, 1896) carried out more exhaustive studies on the subject of detecting and estimating the copper in various articles of food: wheat, rye, barley, oats, maize, buckwheat, and also in various makes of bread; potatoes, beans, linseed, salads, apricots and pears; cocoa and chocolate. He found that only in those plants which are grown on soil rich in copper does the copper reach any considerable value, a value which lies far above the quantity present in an ordinary soil. Plants from the former soils contained as much as 83–560 mg. Cu in 1 kilog. dry substance, whereas ordinarily the plants only contained from a trace to 20 mg. Apparently the species of the plants concerned seems to be of less importance for their copper content than is the copper content of the soil. The deposition of copper (in wheat, buckwheat and paprika) is chiefly in the stems and leaves, little being conveyed to the fruits and seeds, so that a high content of copper in the soil does not necessarily imply the presence of much copper in the grain and seed. The metal is variously distributed among the tissues, the bark of the wood being the richest of the aerial parts in that substance. The form in which the copper exists in the plant is uncertain and it is suggested that an albuminous copper compound possibly exists.

Vedrödi (1893) tackled the problem at about the same time as Lehmann but from a rather different standpoint. He ratifies the statement as to the absorption of copper by plants, and going still further he states that in some cases the percentage of copper found in the seed may be four times as great as that occurring in the soil on which the plants grow, quoting one instance in which the soil contained ·051% CuO and the seed ·26% CuO. It is assumed that copper must play some physiological rôle in the plant, but no explanation of this action is yet forthcoming. Lehmann criticised Vedrödi’s figures of the copper content of certain plant ashes, and the latter replied in a further paper (1896) in which he brings most interesting facts to light. The quantity of copper in any species of plant varies with the individuals of that species, even when grown on the same soil, in the same year, and under similar conditions. The copper content of certain plants is put forward as a table, the years 1894 and 1895 being compared, and enormous differences are to be noticed in some cases. A quotation of the table will illustrate this more clearly than any amount of explanation.

Milligrams of copper in 1 kilog. dry matter.

  1894   1895
/ \ / \
    “Seeds”     min.     max.     min.     max.
Winter wheat 80 710 200 680
Summer wheat 190 630 190 230
Maize 60 90 10 30
Barley 80 120 10 70
Oats 40 190 40 200
Buckwheat 160 640 150 160
“Fisolen” (Beans) 160 320 110 150
Linseed 120 150 110 150
Peas 60 100 60 110
Soy Beans 70 100 70 80
Lupins 80 190 70 290
Mustard seed 70 130 60 70
Paprika pods 790 1350 230 400

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

1. Toxic effect.

(a) Toxic action of copper compounds alone in water cultures.

The method of water cultures has been largely applied to determine the relation of copper compounds to plants. Twenty years ago (1893) Otto discovered the extreme sensitiveness of plants to this poison when grown under such conditions, as he found that growth was very soon checked in ordinary distilled water which on analysis proved to contain minute traces of copper. Controls grown in tap water gave far better plants, but this superiority was attributed partly to the minute traces of mineral salts in the tap water, and not only to the absence of the copper which occurred in the distilled water.

Tests made at Rothamsted have carried this point still further. Pisum sativum, Phaseolus vulgaris, Triticum vulgare,Zea japonica, Tropeolum Lobbianum, sweet pea (American Queen), nasturtium, and cow pea—the first three of these being the species used by Otto—were grown in (1) ordinary distilled water, which was found to contain traces of copper, (2) glass distilled water, for about a month, till no more growth was possible owing to the lack of nutriment. In every single case the root growth was checked in some degree in the ordinary distilled water, the roots seeming to the eye to be less healthy and less well developed. In Pisum, Tropeolum and Zea, the shoot growth of the coppered plants appeared stronger than that of the controls, and this was borne out when the dry weights of the plants were obtained. In every other case the coppered plants were inferior, root and shoot, to those grown in the pure water. With the first three plants it appears that while the toxic water has a bad effect on the roots, yet the growth of the shoots is increased. The idea suggests itself that this apparent stimulation is in reality the result of a desperate struggle against adverse circumstances. The roots are the first to respond to the action of the poison, as they are in actual contact; their growth is checked, and hence the water absorption is decreased. No food is available in the water supply from the roots, so the plant is entirely dependent on the stores laid up in the seed and on the carbon it can derive from the air by photo-synthesis carried on by the green leaves. The result of the root checking in these particular cases seems to be so to stimulate the shoots by some physiological action or other, that this process of photo-synthesis is hastened, more carbon being converted into carbo-hydrates, so that the shoot development is increased, yielding a greater weight of dry matter. In each of the other cases observed the shoot was obviously not stimulated to increased energy by the poison, and so the whole plant fell below the normal.

Other experiments showed that barley roots are peculiarly sensitive to the presence of minute traces of copper, as very little root growth took place in the copper distilled water, and root growth was also entirely checked by the presence of one part per million copper sulphate in the pure glass distilled water. Yet again, one litre of pure distilled water was allowed to stand on a small piece of pure metallic copper foil (about 11/2? × 1/2?) for an hour, and even such water exercised a very considerable retarding influence upon the root-growth, checking it entirely in some instances.

Some years before True and Gies published their results, Coupin (1898) had grown wheat seedlings in culture solutions with the addition of copper salts for several days in order to find the fatal concentrations of the different compounds. Taking toxic equivalent as meaning “the minimum weight of salt, which, dissolved in 100 parts of water, kills the seedling,” the results were as follows:

Toxic equivalent   Containing copper
Copper bromide (CuBr2) ·004875 ·001387
Copper chloride (CuCl2?.?2aq.) ·005000 ·001865
Copper sulphate (CuSO4?.?5aq.) ·005555 ·001415
Copper acetate (Cu{C2H3O2}2?.?aq.) ·005714 ·001820
Copper nitrate (Cu{NO3}2?.?6aq.) ·006102 ·001312

These numbers appear to be very close, so Coupin considered that it might be permissible to regard the differences as due to the impurities in the salts, and to the water of crystallisation which may falsify the weights, so that under these conditions one may believe that all these salts have the same toxicity. This is considerable, and is evidently due to the copper ion, the electro-negative ion not intervening with such a feeble dose. A recalculation of these toxic equivalents to determine the actual amount of copper present in each, gives results that are fairly approximate, but it is difficult to accept this hypothesis in view of other work in which different salts of the same poison are proved to differ greatly in their action on plant growth.

Kahlenberg and True (1896), working with Lupinus albus, found that the various copper salts, as sulphate, chloride and acetate, were similar in their action upon the roots. Plants placed in solutions of these salts of varying strengths for 15–24 hours showed that in each case 1/25,600 gram molecule killed the root, while with 1/51,200 gram molecule the root was just alive. These workers discuss their results from the standpoint of electrolytic dissociation, and concur in the opinion that the positive ions of the toxic salt are exceedingly poisonous.

The toxicity of the positive ion was again set forth by Copeland and Kahlenberg (1900). Their water culture experiments were carried on in glass vessels coated internally with paraffin to avoid solution of glass, and in tests with seedlings of maize, lupins, oats and soy beans it was found that such metals as copper, iron, zinc and arsenic were almost always fatal to the growth of plants. As a general rule those metals whose salts are toxic, themselves poison plants when they are present in water. The assumption made was that the injury to plants when cultivated in the presence of pure metals depends on the tendency of the metal to go into solution as a component of chemical compounds and on the specific toxicity of the metallic ion when in solution.

(b) Masking effect caused by addition of soluble substances to solutions of copper salts.

Experiments were carried on with barley, in which the plants were grown in the various grades of distilled water indicated above, both with and without the addition of nutrient salts. It was found that the presence of the nutrients exercises a very definite masking effect upon the action of the poisonous substance, so that the deleterious properties of the toxic substance are materially reduced. Later work, in which known quantities of such toxic salts as copper sulphate were added to pure distilled water showed that in the presence of nutrient salts a plant is able to withstand the action of a much greater concentration of poison. For instance, a concentration of 1:1,000,000 copper sulphate alone stops all growth in barley, but, if nutrient salts are present, a strength of 1:250,000 (at least four times as great) does not prevent growth, though the retarding action is very considerable (Figs. 2 and 3).

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Fig. 2. Photograph showing the action of copper sulphate on barley in the presence of nutrient salts. (March 5th–April 19th, 1907.)

1. Glass distilled water.
2. Copper distilled water.
3. 1/12,500 copper sulphate.
4. 1/25,000
5. 1/50,000
6. 1/100,000
7. 1/250,000
8. 1/500,000
9. 1/1,000,000

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Fig. 3. Curve showing the dry weights of a series of barley plants grown in the presence of copper sulphate and nutrient salts. (March 13th–May 3rd, 1907.)

Note. In each scale of concentrations represented in the curves a convenient intermediate strength is selected as a unit, and all other concentrations in the series are expressed in terms of that unit. Thus, with 1/1,000,000 as the unit a scale of concentrations might run thus:

10 1/100,000
  4 1/250,000
  2 1/500,000
  1 1/1,000,000
  0·5 1/2,000,000
  0·1 1/10,000,000
  0·05 1/20,000,000
  0· Control.

These later Rothamsted results fit in very well with those obtained ten years ago (1903) by True and Gies in their experiments on the physiological action of some of the heavy metals in mixed solutions. Plants of Lupinus albus were tested for 24–48 hours with different solutions in which the roots were immersed. Given the same strength of the same poison, the addition of different salts yielded varying results. For instance, with copper chloride as the toxic agent, the addition of magnesium chloride did not affect the toxicity, calcium chloride decreased it, while sodium chloride slightly increased the poisonous action. Calcium sulphate with copper sulphate enabled a plant to withstand four times as much copper as when the latter was used in pure solution. Calcium salts in conjunction with those of copper proved generally to accelerate but not to increase growth, but with silver salts they did not cause any improvement. Perhaps this amelioration is in inverse proportion to the activity of the heavy metals. With a complex mixture consisting of five salts—copper sulphate and salts of sodium, magnesium, calcium and potassium, all except calcium being present in concentrations strong enough to interfere with growth if used alone—it was shown that “as a result of their presence together, not only is there no addition of poisonous effects, but a neutralisation of toxicity to such degree as to permit in the mixed solutions a growth-rate equal to or greater than that seen in the check culture.” If the concentration of the copper salts was increased, the other salts remaining the same, the poisonous activity of the copper became greater than could be neutralised by the other salts. If the copper remained the same and the other salts were diminished by half (i.e. below toxic concentration) the neutralising action of the added salts was markedly less, and the growth rate never exceeded that of the control. This was apparently due to the action of the unneutralised copper. The indications are that the conspicuously effective part of the molecule is the cation or metal, and that the anion plays little or no part in causing the toxicity; in such great dilutions the metals act as free ions. The hypothesis is put forward 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; in other words, the growth rate represents the physiological sum of oppositely acting stimuli or of antagonistic protoplasmic changes where mixtures of salts occur. This is really an extension of Heald’s idea that the toxic effect of a poison is due partly to changes in the turgescence of the cell, a sudden decrease causing retardation or inhibition of growth, and partly to a direct action on the protoplasm, which differs in different plants with the same salt. Heald (1896) went so far as to suggest that the poisonous action is a mere matter of adaptation and adjustment, since toxic substances are not usually present in soil, but this assertion is too sweeping to be accepted in its entirety, although it probably holds good to a certain extent with some species of plants.

Kahlenberg and True (1896) found that the addition of an organic substance produced the same effect as the addition of some nutrient salt, in that it reduced the toxicity of the copper salt, e.g. in the presence of sugar and potassium hydrate the lupins were able to withstand a concentration of 1/400 copper sulphate, part of which reduction of toxicity is attributed to the sugar.

(c) Effect of adding insoluble substances to solutions of copper salts.

Other investigators have shown that the presence of insoluble substances has a similar effect in reducing toxicity to an even greater degree. True and Oglevee (1904, 1905) again used Lupinus albus as a test plant in the presence of solutions of various poisons in pure distilled water, copper sulphate, silver nitrate, mercuric chloride, hydrochloric acid, sodium hydroxide, thymol and resorcinol all coming under consideration. Clean sea sand, powdered Bohemian glass, shredded filter paper, finely divided paraffin wax and pure unruptured starch grains were respectively added to the solutions, and seedlings were suspended over glass rods so that their roots were in the solutions for 24–48 hours. The solids varied in their action on the different poisons; while the toxic influence of mercuric chloride was reduced by sand and crushed glass, the action of silver nitrate was modified by nearly all the solids. Lupin roots proved unable to withstand an exposure of 24 hours to a concentration of copper sulphate of 1 molecular weight in 60,000 litres of water (i.e. about 1 part by weight CuSO4?.?5H2O in 240·4 parts water), but the addition of solids caused a great decrease in toxicity. When the amount of copper was diminished an advantage was regularly obtained in favour of the cultures containing the solid bodies. On the whole the ameliorating action of solids is more clearly marked with dilute solutions of strong poisons than with relatively concentrated solutions of weaker poisons. As a general rule, filter paper and potato starch grains exert a more marked modifying action than the denser bodies, such as sand, glass or paraffin.

Breazeale (1906) tested the same point with extracts of certain soils which proved toxic to wheat seedlings grown in them as water cultures. The toxicity was wholly or partly removed by the addition of such substances as carbon black, calcium carbonate or ferric hydrate. Other experiments showed that the toxic substances of ordinary distilled water are removed by ferric hydrate and carbon black, and further that the latter substance will take out copper from copper solutions, rendering them far less poisonous.

Further corroboration of True and Oglevee’s work was obtained by Fitch (1906) who worked in a similar way with fungi, arriving at the general conclusion that insoluble substances in a solution act as agents of dilution or absorption whereby poisonous ions or molecules are in some way removed. He found that n/256 of copper sulphate in beet concoction exercised a stimulating effect on Penicillium glaucum, but the addition of fine glass to the solution increased the stimulation, while large or medium sized pieces did not have the same effect.

This action of solid bodies in reducing the deleterious effects of poisonous solutions is attributed to the process of “adsorption” whereby a layer of greater molecular density is formed on the surfaces of solids immersed in solutions. The solids presumably withdraw a certain proportion of poisonous ions or molecules from the body of the solution (retaining them in a molecularly denser layer over their own surfaces), so that the toxic properties of the solution are reduced owing to the withdrawal of part of the poison from the field of action. In some cases this reduction may be so great as to relieve the solution of its toxic properties, or even to cause an abnormal acceleration to replace a marked retardation. Also, if the solution is of such a dilution as to cause acceleration of growth in plants, the addition of insoluble substances may increase this acceleration. The progressive addition of quantities of solids causes progressive dilution of the toxic medium, the underlying cause of these results being the gradual removal of molecules or ions from the solutions by the insoluble body present.

Fitch’s results are also in accordance with the well-known fact that the physical condition and properties of the added solid play a considerable part in determining its efficacy as an adsorbing agent.

(d) Effect of copper on plant growth when present in soils.

As has already been shown the toxic property of copper with regard to plants was recognised almost as soon as that element was found to occur in the vegetable kingdom, but little notice was taken of the discovery for many years. In 1882 F. C. Phillips asserted, as the result of experiments with various cultivated flowering plants, including geraniums, coleas, ageratum, pansies, &c., that under favourable conditions plants will absorb small quantities of copper by their roots, and that such compounds exercise a distinctly retarding influence even if in very small amount, while if large quantities are present they tend to check root formation, either killing the plants outright or so far reducing their vitality as seriously to interfere with nutrition and growth. Two years later Knop confirmed both the absorption and the toxicity of copper by his experiments on maize.

Jensen (1907) worked with “artificial” soils, under sterile conditions, using finely ground quartz flour for his medium and wheat for a test plant, parallel experiments being carried on with solutions. Every precaution was taken to ensure sterility—the corks were boiled first in water and then in paraffin, the seeds were sterilised in 2% copper sulphate solution for 3/4 hour, washed in sterilised water, planted in sterilised sphagnum, the transplanting being done in a sterile chamber into sterilised solutions. The criteria used to determine the toxic and stimulation effects were the total transpiration, average length of sprout, the green weight and dry weight of plants. The results obtained with the different substrata showed that it does not follow that a salt highly toxic in solution is equally so in soil, or that one which holds a relatively high toxic position in soil should occupy the same relative position in solution cultures. For instance, while in soil cultures nickel compounds were the most toxic of all the substances tried, in solution cultures silver compounds were more poisonous than nickel. The range of concentrations, both fatal and accelerating, was found to be much greater in solution than in soil cultures.

In the sand cultures the toxicity of the copper sulphate was found to decrease as the ratio of the quartz sand to the poisonous solution increased, provided that a water content suitable for growth was present. Jensen states that the fatal concentration of copper sulphate in solution cultures is approximately 1/10th that of the fatal concentration in his artificial soil.

When copper salts are added to soil a complication at once sets in due to the double decomposition which is always likely to occur when any soluble salt is added to soil. The reaction may be graphically expressed as follows, in a much simplified form—

AB + CD = AC + BD.

Haselhoff (1892) extracted several lots of 25 kgm. soil, each with 25 litres of water in which quantities of mixed copper salts varying from 0–200 mg. had been dissolved, the mixture consisting of three parts copper sulphate and one part copper nitrate. This operation was repeated 15 times, the soils being allowed to drain thoroughly after each treatment, so that altogether each 25 kgm. soil was extracted with 375 litres water. The drainage waters were analysed, so that the amount of copper absorbed by the soils could be estimated. It was found that by extracting with water containing such soluble copper salts as sulphate and nitrate, the food salts of the soil, especially those of calcium and potassium, were dissolved and washed out, copper oxide being retained by the soil. In this way a double action was manifest, whereby the fertility of the soil was reduced by the loss of plant food, while its toxicity was increased by the accumulation of copper oxide. So long as the soil contained a good supply of undissolved calcium carbonate the harmful action of the copper-containing water was diminished, but as soon as the store was exhausted by solution and leaching, the toxic influence became far more evident.

(e) Mode of action of copper on plants.

Quite early in the investigations on the effect of copper on plants the question arose as to its mode of activity—whether the toxicity was merely due to some mechanical action on the root from outside, whereby the absorptive power of the root was impaired, or whether the poisonous substance was absorbed into the plant, so acting directly on the internal tissues.Gorup-Besanez made definite experiments towards ascertaining the truth of these theories as far back as 1863, endeavouring first of all to see whether the plants take up any appreciable quantity of poisons which exist in the soil as mixtures or combinations and which are capable of solution by the cell-sap. Salts of arsenic, copper, lead, zinc and mercury were intimately mixed with soil, 30 grams of the poison being added to 30·7 cubic decimetres of soil, two plants separated by a partition being grown on this quantity. The test plants were Polygonum Fagopyrum, Pisum sativum, Secale cereale andPanicum italicum, and all the plants developed strongly and normally except the last named. The Panicum developed very badly coloured leaves in an arsenic-containing soil, and the plants were killed soon after they started in soils containing copper. After harvesting, the crops were analysed and no trace of copper was found in any one of the experimental plants by the methods adopted. Also the absorption capacity of different soils for different poisons was shown to vary, for basic salts are absorbed, while acids may pass completely through the soil into the drainage water.

These results obtained by Gorup-Besanez are possibly not altogether above criticism, for later workers showed that copper was absorbed to some extent by plants grown in water cultures, and if that is so it seems unlikely that no absorption should take place from soil. Nevertheless, the absorption is very slight, for apparently living protoplasm is very resistant to copper osmotically. Otto showed that beans, maize and peas can have their roots for a long time in a relatively concentrated solution of copper sulphate, and yet take up very little copper indeed, but analyses do reveal slight traces after a sufficient interval of time of contact has elapsed. Berlese and Sostegni indicate that the roots of plants grown in water culture in the presence of bicarbonate of copper showed traces of copper.

Verschaffelt (1905) devised an ingenious method of estimating the toxic limits of plant poisons, though it is rather difficult to see how the method can be put to practical use with water culture and soil experiments. Living tissues increase in weight when put into water on account of the absorption of water. Dead tissues do not, as they have lost their semi-permeable characteristics, so a decrease in weight takes place owing to part of the water passing out. This principle is applied by Verschaffelt to determine the “mortal limit” of external agents in their action on plant tissues. Root of beetroot, potato tuber, aloe leaves, and parts of other plants rich in sugar all came under review. The parts were cut into small pieces weighing about 3–5 grams, dried with filter paper, weighed, and plunged into solutions of copper sulphate of varying strengths from ·001–·004 gm. mol. per litre, and left for 24 hours. After drying and again weighing all were heavier owing to the absorption of water. The pieces were then immersed in pure water for another period of 24 hours, when after drying and weighing, those from the weaker strengths of copper sulphate (·001–·002) had absorbed yet more water, while those from higher concentrations (·003–·004) had lost weight. So the author assumes that for such pieces of potato the limit of toxicity lies between ·002 and ·003 gm. mol. copper sulphate per litre.

These experiments may possibly give some indication as to the action of copper salts on plant roots. So long as the solution of copper salt is dilute enough, the absorption layer of the root, acting as a semi-permeable membrane and upheld by the resistant protoplasm, is able to keep the copper out of the plant and to check its toxicity. As soon as a certain limit is reached the copper exercises a corrosive influence upon the outer layer of the root whereby its functions are impaired, so that it is no longer able efficiently to resist the entry of the poison. As the concentration increases it is easy to conceive that the harmful action should extend to the protoplasm itself, so that the vital activities of the plants are seriously interfered with and growth is entirely or partially checked, death ensuing in the presence of sufficiently high concentrations.

2. Effect of copper on germination.

The action of copper on the germination of seeds, spores and pollen grains has attracted a certain amount of attention, and although the results are apparently contradictory this is probably due to the different plant organs with which the observers have worked.

(a) Seeds.

Miyajima (1897) showed that the germinating power of such seeds as Vicia Faba, Pisum sativum, and Zea Mays was partly destroyed by a 1% solution of copper[4], Zea Mays being the most resistant and Vicia Faba the least resistant of the three. Micheels (1904–5) stated that water distilled in a tinned copper vessel was more favourable for germination than water from a non-tinned vessel. He suggests that this is due to copper being present in the water in a colloidal form in which the particles are exceedingly small and maintain themselves in the liquid by reason of a uniform disengagement of energy in all directions, to which energy the influence on germinating seeds must be attributed, the nature of the suspended substance determining whether the influence be favourable or not. It is questionable, however, whether Micheels was really dealing with a true colloidal solution of copper or with a dilute solution of some copper salt produced by oxidation of the copper vessel from which his distilled water was obtained.

(b) Spores and pollen grains.

Miani (1901) brought fresh ideas to bear upon the problem of the action of copper on living plant cells, in that he sought to attribute the toxic or stimulant effects to an oligodynamic action, i.e. spores and pollen grains were grown in hanging drop cultures in pure glass distilled water with the addition of certain salts or traces of certain metals. While the salts are known to be often disadvantageous to germination, Nägeli had asserted that the latter often exerted an oligodynamic action. In some cases pure copper was placed for varying times in the water from which the hanging drop cultures were eventually made, or tiny bits of copper were placed in the drop itself. Various kinds of pollen grains were tested, and as a rule, pollen was only taken from one anther in each experiment, though occasionally it was from several anthers of the same flower. It was generally found that the germination of pollen grains or Ustilago spores was not hindered by the use of coppered water or by the presence of small bits of copper in the culture solution. The only cases in which some spores or pollen grains were more or less harmed were those in which the water had stood over copper for more than two weeks, and even so the deleterious effect was chiefly noticeable when the pollen itself was old or derived from flowers in which the anther formation was nearly at an end. As a rule germination was better in the presence of copper, whether in pure water or food solution, the stimulus being indicated both by the greater number of germinated grains and by the regular and rapid growth of the pollen tubes. Miani attributes this favourable action to the mere presence of the copper, corroborating Nägeli’s idea of an oligodynamic action.

3. Does copper stimulate higher plants?

From the foregoing review it is evident that it is the toxic action of copper that is most to the front, so far as the higher plants are concerned, and that little or no evidence of its stimulative action in great dilution has so far been discussed. Kanda dealt with this question, with the deliberate intention of obtaining such evidence, if it existed. He worked with Pisum sativum, var. arvense, Pisum arvense, Vicia Faba, var. equine Pers, and Fagopyrum esculentum Mönch, which were grown in glass distilled water, without any food salts, so that the plants were forced to live on the reserves in the seeds, which were carefully graded to ensure uniformity of size. It was found that in water cultures copper sulphate solutions down to ·00000249% (about 1 in 40,160,000) are harmful to peas, and still further down to ·0000000249% (about 1 in 4,016,000,000) the copper salts act as a poison rather than as a stimulant. Against this, however, is the statement that in certain soils copper sulphate acts as a stimulant when it is added in solution. Jensen again could obtain no stimulation with copper sulphate.

The Rothamsted experiments go to uphold Kanda’s statements as to the failure of copper sulphate to stimulate plants grown in water cultures. Peas are perhaps slightly more resistant to the greater strengths of copper sulphate than are barley and buckwheat, for while 1/100,000 proves mortal to the latter, peas will struggle on and fruit in 1/50,000, though this strength is very near the limit beyond which no growth can occur (Fig. 4). As a general rule, with barley the depression caused by the poison is still evident with 1/5,000,000 and 1/10,000,000, though occasionally these doses act as indifferent doses, no sign of stimulation appearing in any single instance. With peas again, even 1/20,000,000 copper sulphate is poisonous, although to the eye there is little to choose between the control plants and those receiving poison up to a concentration of one part in 21/2 million (Fig. 5). In the case of buckwheat the matter is still undecided, as in some experiments apparent stimulation is obtained with 1 in 21/2 or 1 in 5 million copper sulphate, while in others a consistent depression is evident, even when the dilution is carried considerably below this limit. The reason for the variation with this particular plant is so far unexplained.

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Fig. 4. Photograph showing the action of copper sulphate on pea plants in the presence of nutrient salts. (Oct. 3rd–Dec. 20th, 1912.)

  1. Control.
  2. 1/50,000 copper sulphate.
  3. 1/100,000
  4. 1/250,000
  5. 1/500,000
  6. 1/1,000,000
  7. 1/2,500,000
  8. 1/5,000,000
  9. 1/10,000,000
10. 1/20,000,000

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Fig. 5. Curve showing the mean values of the dry weights of four series of pea plants grown in the presence of copper sulphate and nutrient salts. (Oct. 3rd–Dec. 20th, 1912.)

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. glaucumwhich 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 forP. 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.