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 1816 Meissner 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[3],” 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.
[3] These are “grains of Paradise,” Guinea grains, or meleguetta
pepper. They are the seeds of _Amomum melegueta_ and _A.
Granum-Paradisi_, N.O. Zingiberaceae.
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 1½″ × ½″)
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 Containing
equivalent 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).
[Illustration: 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 „ „ ]
[Illustration: 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 ¾ 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_ and _Panicum 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.
[4] The English translation in Just’s _Bot. Jahresber._ speaks only
of a “solution of copper,” and in no case is the specific compound
mentioned.
(_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 2½ million (Fig. 5). In the case of buckwheat
the matter is still undecided, as in some experiments apparent stimulation
is obtained with 1 in 2½ 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.
[Illustration: 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 „ „ ]
[Illustration: 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 9½ 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.