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Title: Inorganic Plant Poisons and Stimulants
Author: Brenchley, Winifred E.
Language: English
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  CAMBRIDGE AGRICULTURAL MONOGRAPHS

  INORGANIC PLANT POISONS AND STIMULANTS



  CAMBRIDGE UNIVERSITY PRESS
  C. F. CLAY, MANAGER
  London: FETTER LANE, E.C.
  Edinburgh: 100 PRINCES STREET

  [Colophon]

  London: H. K. LEWIS, 136 GOWER STREET, W.C.
  London: WILLIAM WESLEY AND SON, 28 ESSEX STREET, STRAND
  New York: G. P. PUTNAM’S SONS
  Bombay and Calcutta: MACMILLAN AND CO., LTD.
  Toronto: J. M. DENT AND SONS, LTD.
  Tokyo: THE MARUZEN-KABUSHIKI-KAISHA

  _All rights reserved_



  INORGANIC PLANT POISONS
  AND STIMULANTS

  BY
  WINIFRED E. BRENCHLEY, D.SC., F.L.S.

  Fellow of University College, London
  (Rothamsted Experimental Station)

  Cambridge:
  at the University Press
  1914



  Cambridge:
  PRINTED BY JOHN CLAY, M.A.
  AT THE UNIVERSITY PRESS



PREFACE


During the last century great and widespread changes have been made in
agricultural practice--changes largely associated with the increase in the
use of artificial fertilisers as supplements to the bulky organic manures
which had hitherto been used. The value of certain chemical compounds
as artificial manures is fully recognised, yet many attempts are being
made to prove the value of other substances for the same purpose, with
a view to increase in efficiency and decrease in cost. The interest in
the matter is naturally great, and agriculturists, botanists and chemists
have all approached the question from their different standpoints. In the
following pages an attempt is made to correlate the work that has been
done on a few inorganic substances which gave promise of proving useful
in agricultural practice. Much of the evidence put forward by different
workers is conflicting, and it is clear that no definite conclusions can
yet be reached. Nevertheless, examination of the evidence justifies the
hope that results of practical value will yet be obtained, and it is hoped
that the analysis and coordination of the available data put forward in
this book will aid in clearing the ground for those investigators who
are following up the problem from both the academic and the practical
standpoints.

                                                          W. E. B.

    ROTHAMSTED.
    _October 1914._



CONTENTS


    CHAP.                                                       PAGE

    I. INTRODUCTION                                                1

    II. METHODS OF WORKING                                         7
      I. Discussion of Methods                                     7
        1. Water cultures                                          7
        2. Sand cultures                                           8
        3. Soil cultures in pots                                   9
        4. Field experiments                                       9
      II. Details of Methods                                      10

    III. EFFECT OF COPPER COMPOUNDS                               15
      I. Presence of Copper in Plants                             15
      II. Effect of Copper on the Growth of Higher Plants         17
        1. Toxic effect                                           17
          (a) Toxic action of copper compounds alone in water
                cultures                                          17
          (b) Masking effect caused by addition of soluble
                substances to solutions of copper salts           20
          (c) Effect of adding insoluble substances to
                solutions of copper salts                         22
          (d) Effect of copper on plant growth when present
                in soils                                          24
          (e) Mode of action of copper on plants                  25
        2. Effect of copper on germination                        27
          (a) Seeds                                               27
          (b) Spores and pollen grains                            28
        3. Does copper stimulate higher plants?                   28
        4. Action of copper on organs other than roots            30
          (a) Effect of copper sprays on leaves                   30
          (b) Effect of solutions of copper salts on leaves       32
      III. Effect of Copper on Certain of the Lower Plants        33
      Conclusion                                                  35

    IV. EFFECT OF ZINC COMPOUNDS                                  36
      I. Presence of Zinc in Plants                               36
      II. Effect of Zinc on the Growth of Higher Plants           38
        1. Toxic effect                                           38
          (a) Toxic action of zinc salts alone in water
                cultures                                          38
          (b) Effect of soluble zinc salts in the presence of
                nutrients                                         39
          (c) Effect of zinc compounds on plant growth when
                they are present in soils                         41
          (d) Mode of action of zinc on plants                    43
        2. Effect of zinc compounds on germination                43
        3. Stimulation induced by zinc compounds                  45
          (a) Stimulation in water cultures                       45
          (b) Stimulation in sand cultures                        46
          (c) Increased growth in soil                            46
        4. Direct action of zinc salts on leaves                  47
      III. Effect of Zinc on Certain of the Lower Plants          48
      Conclusion                                                  50

    V. EFFECT OF ARSENIC COMPOUNDS                                51
      I. Presence of Arsenic in Plants                            51
      II. Effect of Arsenic on the Growth of Higher Plants        52
        1. Toxic effect                                           52
          (a) Toxic action of arsenic compounds in water
                cultures in the presence of nutrients             52
          (b) Toxic effect of arsenic compounds in sand
                cultures                                          57
          (c) Toxic effect of arsenic when applied to soil
                cultures                                          57
          (d) Physiological considerations                        59
        2. Effect of arsenic compounds on germination             60
        3. Do arsenic compounds stimulate higher plants?          61
      III. Effect of Arsenic Compounds on Certain of the
             Lower Plants                                         62
        1. Algae                                                  62
        2. Fungi                                                  63
      Conclusion                                                  64

    VI. EFFECT OF BORON COMPOUNDS                                 65
      I. Presence of Boron in Plants                              65
      II. Effect of Boron on the Growth of Higher Plants          67
        1. Toxic effect                                           67
          (a) Toxic action of boron compounds in water
                cultures                                          67
          (b) Toxic action of boron compounds in sand
                cultures                                          70
          (c) Toxic action of boron compounds in soil
                experiments                                       71
        2. Effect of boron compounds on germination               72
        3. Does boron stimulate higher plants?                    73
          (a) Water cultures                                      73
          (b) Sand cultures                                       73
          (c) Soil cultures                                       74
      III. Effect of Boron Compounds on Certain of the Lower
             Plants                                               76
      Conclusion                                                  77

    VII. EFFECT OF MANGANESE COMPOUNDS                            78
      I. Presence of Manganese in Plants                          78
      II. Effect of Manganese on the Growth of Higher Plants      81
        1. Toxic effect                                           81
          (a) Toxic action of manganese compounds in the
                presence of soluble nutrients                     81
          (b) Toxic action of manganese compounds in sand
                cultures                                          82
          (c) Toxic action of manganese compounds in soil
                cultures                                          82
        2. Effect of manganese compounds on germination           84
        3. Does manganese stimulate higher plants?                84
          (a) Stimulation in water cultures                       85
          (b) Stimulation in soil cultures                        86
      III. Effect of Manganese Compounds on Certain of the
             Lower Plants                                         90
      IV. Physiological Considerations of Manganese
             Stimulation                                          90
      Conclusion                                                  92

    VIII. CONCLUSIONS                                             93

    BIBLIOGRAPHY                                                  97

    INDEX OF PLANT-NAMES                                         107

    GENERAL INDEX                                                109

    LIST OF ILLUSTRATIONS                                          x



LIST OF ILLUSTRATIONS


    FIG.                                                        PAGE

     1. Sketch illustrating water culture methods                 12

     2. Photograph. Barley grown with copper sulphate   _To face_ 20

     3. Curve. Ditto                                              21

     4. Photograph. Peas grown with copper sulphate     _To face_ 29

     5. Curve. Ditto                                              29

     6. Curve. Barley grown with zinc sulphate                    40

     7. Photograph. Peas grown with zinc sulphate       _To face_ 40

     8. Curve. Ditto                                              41

     9. Photograph. Barley grown with arsenious acid    _To face_ 54

    10. Curve. Ditto                                              55

    11. Curve. Peas grown with arsenious acid                     55

    12. Curve. Barley grown with arsenic acid                     56

    13. Curve. Barley grown with sodium arsenite                  56

    14. Curve. Peas grown with sodium arsenite                    57

    15. Curve. Barley grown with boric acid                       69

    16. Photograph. Peas grown with boric acid          _To face_ 69

    17. Curve. Barley grown with manganese sulphate               85

    18. Photograph. Ditto                               _To face_ 86

    19. Photograph. Peas grown with manganese sulphate            86



CHAPTER I

INTRODUCTION


Ever since the physiological side of botany began to emerge from
obscurity, the question of the relation between the nutrition and the
growth of the plant has occupied a foremost position. All kinds of
theories, both probable and improbable, have been held as to the way in
which plants obtain the various components of their foods. But quite early
in the history of the subject it was acknowledged that the soil was the
source of the mineral constituents of the plant food, and that the roots
were the organs by which they were received into the plant.

A new chapter in the history of science was begun when Liebig in 1840
first discussed the importance of inorganic or mineral substances in plant
nutrition. This discussion led to a vast amount of work dealing with the
problem of nutrition from many points of view, and the general result
has been the sorting out of the elements into three groups, nutritive,
indifferent, and toxic. Thus calcium, phosphorus, nitrogen and potassium
are classed as nutritive, arsenic, copper and boron as toxic, and many
others are regarded as indifferent.

Closer examination, however, shows that this division into three classes
is too rigid. Now that experiments are more refined it has become evident
that no such simple grouping is possible. It has been found that typical
nutrient salts are toxic when they are applied singly to the plant in
certain concentrations, the toxic power decreasing and the nutritive
function coming into play more fully on the addition of other nutrient
salts. For instance, Burlingham found that the typical nutrient magnesium
sulphate in concentrations above _m_/8192 (_m_ = molecular weight) is
toxic to most seedlings, the degree of toxicity varying with the type
of seedling and the conditions under which growth takes place. It will
be shown in the following pages that even such a typical poison as
boric acid may, under suitable conditions, increase plant growth just
as if it were a nutrient. A review of the whole subject leads one to
conclude that in general both favourable and unfavourable conditions of
nutrition are present side by side, and only when a balance is struck
in favour of the good conditions can satisfactory growth take place. As
indicated above, experiments have shown that the very substances that are
essential for plant food may be, in reality, poisonous in their action,
exercising a decidedly depressing or toxic influence on the plant when
they are presented singly to the roots. This toxic action of food salts
is decreased when they are mixed together, so that the addition of one
toxic food solution to another produces a mixture which is less toxic than
either of its constituents. Consequently a balanced solution can be made
in which the toxic effects of the various foods for a particular plant
are reduced to a minimum, enabling optimum growth to take place. Such
a mixture of plant foods occurs in the soil, the composition of course
varying with the soil.

While the earliest observations set forth the poisonous action of various
substances upon plants, it was not long before investigators found
that under certain conditions these very substances seemed to exert a
beneficial rather than an injurious action. The poisons were therefore
said to act as “stimulants” when they were presented to the plant in
sufficiently great dilution. This stimulation was noticed with various
plants and with several poisons, and a hypothesis was brought forward that
attempted to reconcile the new facts with the old conceptions. Any poison,
it was suggested, might act as a stimulant, if given in sufficiently small
doses. It will be seen in the following pages that this is not universally
true, such substances as copper, zinc, and arsenic failing to stimulate
certain plants even in the most minute quantities so far tested.

Of recent years investigators in animal physiology have brought into
prominence the striking effect of minute quantities of certain substances
in animal nutrition, as for example iodine in the thyroid gland (see
E. Baumann, 1895). This and other work has rendered it imperative to
re-examine the parallel problems in plant physiology.

The words “stimulant” and “stimulation” themselves need more precise
definition. As a matter of fact the “stimulation” noticed by one observer
is not necessarily held to be such by another. Stimulation may express
itself in various ways--the green weight and the general appearance of
the fresh plant may be improved, the dry weight may be increased, the
transpiration current may be hurried up, entailing increased absorption
of water and food substances by the roots, assimilation processes may be
encouraged. But these benefits are not of necessity correlated with one
another, e.g. a plant treated with a dilute solution of poison may look
much healthier and weigh far more in the green state than an untreated
plant, whereas the latter may prove the heavier in the dry state. To a
market gardener to whom size and appearance is so important, stimulation
means an improvement in his cabbages and lettuces in the green state, even
though the increased weight is chiefly due to additional water absorbed
under the encouragement of the stimulative agent, whereas to a scientific
observer, the dry weight may give a more accurate estimate of stimulation
in that it expresses more fully an increased activity in the vital
functions of the plant whereby the nutritive and assimilative processes
have gone on more rapidly, with a consequent increase in the deposition
of tissue.

While stimulation expresses itself in the ways detailed above poisoning
action also makes itself visible to the eye. Badly poisoned plants
either fail to grow at all or else make very little or weak growth. Even
when less badly affected the toxic action is well shown in some cases
by the flaccidity of the roots, and in others by the formation of a
“strangulation” near the crown of the root, which spreads to the stem,
making it into a thin thread, while the leaves usually wither and die. If
such plants as peas are able to make any shoot growth at all the roots
show signs of a desperate attempt to put forth laterals. The primary
root gets much thickened and then bursts down four sides, the tips of the
laterals all trying to force their way through in a bunch, but failing to
do so on coming in contact with the poison. Most curious malformations
of the root arise from this strong effort of the plant to fight against
adverse circumstances.

While all the inorganic substances examined in this monograph are
toxic in high concentrations, some lead to increased growth in lower
concentrations, while others apparently have no effect. In this sense all
substances could be classed as toxins, even the nutrients. Thus the old
distinction between toxin and nutrient has now lost its sharpness, but
it does not lose all its significance. The old “nutrients” had certain
definite characters in common, in that they were essential to plant
growth, the growth being in a great degree proportional to the supply,
a relatively large amount of the nutrients being not only tolerated but
necessary. The substances dealt with more particularly in this book have
none of these characters. Even those that cause increased growth do not
appear to be essential, at any rate not in the quantities that potassium,
phosphorus, nitrogen, &c., are essential, while there is no evidence that
growth is proportional to supply. The substances fall into two groups:

(1) Those that apparently become indifferent in high dilutions and never
produce any increase in plant growth.

(2) Those that cause a small, but quite distinct, increased growth when
applied in quantities sufficiently small.

The former group may be legitimately regarded as toxins; the latter
present more difficulty and even now their function is not settled. It
is not clear whether they stimulate the protoplasm or in some way hasten
the metabolic processes in the plant, whether they help the roots in
their absorbent work, or whether they are simple nutrients needed only
in infinitesimal quantities. The two groups, however, cannot be sharply
separated from one another. Indeed a substance may be put into one of
these classes on the basis of experiments made with one plant alone and
into another when a different plant is used, while it is quite conceivable
that further experiments with other plants may abolish the division
between the two groups altogether. It is even impossible to speak rigidly
of toxicity. The addition of the inorganic food salts to solutions of a
poison reduces the toxicity of the latter, so that the plant makes good
growth in the presence of far more poison than it can withstand in the
absence of the nutrients. This masking effect of the inorganic food salts
upon the toxicity of inorganic plant poisons is paralleled by a similar
action on organic toxic agents. Schreiner and Reed (1908) found that the
addition of a second solute to a solution decreases the toxicity of that
solution; further the plant itself may exercise a modifying influence upon
the toxic agent. Water culture experiments were made upon the toxicity
of certain organic compounds, with and without the addition of other
inorganic salts. Arbutin, vanillin, and cumarin were definitely toxic and
the toxicity decidedly fell off after the addition of sodium nitrate and
calcium carbonate, especially with the weaker solutions of the toxins.
Curiously enough, while weaker solutions of vanillin alone produced
stimulation, the stimulating effect of this toxic agent disappeared
entirely on the addition of the inorganic substances. The results showed
that the addition of certain inorganic salts to solutions of toxic organic
compounds was decidedly beneficial to the plant.

Another important problem has come to the front with regard to these
toxic substances--How do these substances get into the plant? Are they
all absorbed if they occur in the soil, or is there any discriminatory
power on the part of the root? In other words, do the roots perforce take
in everything that is presented to their surfaces, or have they the power
of making a selection, absorbing the useful and rejecting the useless and
harmful?

Daubeny (1833) described experiments in which various plants, as radish,
cabbage, _Vicia Faba_, hemp and barley were grown actually on sulphate
of strontium or on soils watered with nitrate of strontium. No strontium
could be detected in the ash of any of the plants save barley, and then
only the merest trace was found. Daubeny concluded that the roots _were_
able to reject strontium even when presented in the form of a solution.
“Upon the whole, then, I see nothing, so far as experiments have yet
gone, to invalidate the conclusion ... that the roots of plants do, to a
certain extent at least, possess a power of selection, and that the earthy
constituents which form the basis of their solid parts are determined as
to _quality_ by some primary law of nature, although their _amount_ may
depend upon the more or less abundant supply of the principles presented
to them from without.” Some years after, in 1862, Daubeny reverted to
the idea, stating “I should be inclined to infer that the spongioles of
the roots have residing in them some specific power of excluding those
constituents of the soil that are abnormal and, therefore, unsuitable
to the plant, but that they take up those which are normal in any
proportions in which they may chance to present themselves[1].” This,
however, was not held to apply to such corrosive substances as copper
sulphate. De Saussure had found that _Polygonum Persecaria_ took up copper
sulphate in large quantities, a circumstance which he attributed to the
poisonous and corrosive quality of this substance, owing to which the
texture of the cells became disorganised and the entrance of the solution
into the vegetable texture took place as freely, perhaps, as if the plants
had been actually severed asunder[2]. Daubeny concluded that a plant is
unable to exclude poisons of a corrosive nature, as this quality of the
substance destroys the vitality of the absorbing surface of the roots and
thus reduces it to the condition of a simple membrane which by endosmosis
absorbs whatever is presented to its external surfaces, so that whenever
abnormal substances are taken up by a living plant it is in consequence
of some interference with the vital functions of the roots caused in the
first instance by the deleterious influence of the agent employed.

    [1] This idea of a selectivity of the roots has been recently
    revived by Colin and Lavison (1910) who found that when peas
    were grown in the presence of barium, strontium or calcium
    salts no trace of barium could be found in the stem, strontium
    only occurred in small quantities, while calcium was present in
    abundance. They concluded that apparently salts of the two latter
    alkaline metals could be absorbed by the roots and transferred
    to the stem and other organs, but that this is not the case with
    salts of barium. They obtained similar results with other plants,
    beans, lentils, lupins, maize, wheat, hyacinth. Their proof is
    not rigid, and exception could be taken to it on chemical grounds.

    [2] Vide Daubeny, _Journ. Chem. Soc._ (1862), p. 210.

In spite of the enormous amount of work that has been done on this subject
of toxic action and stimulation it is yet too early to discuss the matter
in any real detail. A voluminous literature has arisen around the subject,
and in the present discussion some selection has been made with a view
to presenting ascertained facts as succinctly as possible. No attempt
has been made to notice all the papers; many have been omitted perforce;
it would have been impossible to deal with the matter within reasonable
length otherwise. A full and complete account would have demanded a
ponderous treatise. This widespread interest on the part of investigators
is fully justified, as the problems under discussion are not only of
the highest possible interest to the plant physiologist, but hold out
considerable promise for the practical agriculturist.



CHAPTER II

METHODS OF WORKING


I. DISCUSSION OF METHODS.

In the course of the scattered investigations on plant poisons and
stimulants, various experimental methods have been brought into use,
but these all fall into the two main categories of water and soil
cultures, with the exception of a few sand cultures which hold a kind of
intermediate position, combining certain characteristics of each of the
main groups.

The conditions of plant life appertaining to soil and water cultures are
totally different, so different that it is impossible to assume that a
result obtained by one of the experimental methods must of necessity hold
good in respect of the other method. A certain similarity does exist, and
where parallel investigations have been carried out this becomes evident,
but it seems to be more or less individual, the plant, the poison and the
cultural conditions each playing a part in determining the matter.


1. _Water cultures._

This method of cultivation represents the simplest type of experiment.
Its great advantage is that the investigator has absolute control over
all the experimental conditions. Nutritive salts and toxic substances
can be supplied in exact quantities and do not suffer loss or change
by interaction with other substances which are beyond control. Any
precipitates which may form in the food solution are contained within
the culture vessel and are available for use if needed. The results are
thus most useful as aids in interpreting the meaning of those from the
field experiments, the results of the one method frequently dovetailing
in with those of the other in a remarkable way. The disadvantage of the
water culture method is that it is more or less unnatural, as the roots
of the plants are grown in a medium quite unlike that which they meet in
nature, a liquid medium replacing the solid one, so that the roots have
free access to every part of the substratum without meeting any opposition
to their spread until the walls of the culture vessel are reached. The
conditions of aeration are also different, for while the plant roots meet
with gaseous air in the interstices of the soil, in water cultures they
are dependent upon the air dissolved in the solution, so that respiration
takes place under unusual conditions. It is possible that the poverty
of the air supply can be overcome by regular aeration of the solution,
resulting in decided improvement in growth, as L. M. Underwood (1913) has
shown in recent work on barley in which continued aeration was carried
out.


2. _Sand cultures._

This method has the advantage over water cultures in that the environment
of the plant roots is somewhat more natural, but on the other hand
the work is cumbersome and costly, while the conditions of nutrition,
watering, &c., are less under control than in the water cultures. Sand
cultures represent an attempt to combine the advantages of both soil and
water cultures, without their respective disadvantages. Generally speaking
perfectly clean sand is used varying in coarseness in different tests, and
this is impregnated with nutritive solutions suitable for plant growth.
The sand is practically insoluble and sets up no chemical interaction with
the nutritive compounds, while it provides a medium for the growth of the
plant roots which approximates somewhat to a natural soil. It is probable,
however, that a certain amount of adsorption or withdrawal from solution
occurs, whereby a certain proportion of the food salts are affiliated,
so to speak, to the sand particles and are so held that they are removed
from the nutritive solution in the interspaces and are not available for
plant food, the nutritive solution being thus weakened. The same remark
applies to the poisons that are added, so that the concentration of the
toxic substance used in the experiment does not necessarily indicate the
concentration in which it is presented to the plant roots. On the other
hand, undue concentration of the solution is apt to occur on account of
the excessive evaporation from the surface of the sand. The sand particles
are relatively so coarse in comparison with soil particles that the water
is held loosely and so is easily lost by evaporation, thus concentrating
the solution at the surface, a condition that does not apply in soil work.
With care this disadvantage is easily overcome as it is possible to weigh
the pots regularly and to make up the evaporation loss by the addition of
water.


3. _Soil cultures in pots._

In this case the conditions of life are still more natural, as the plant
roots find themselves in their normal medium of soil. But the investigator
has now far less control, and bacterial and other actions come into play,
while the nutrients and poisons supplied may set up interactions with
the soil which it is impossible to fathom. This method is useful in the
laboratory as it is more convenient for handling and gives more exact
quantitative results than plot experiments. Also the pots can be protected
from many of the untoward experiences that are likely to befall the crops
in the open field. The conditions are somewhat more artificial, as the
root systems are confined and the drainage is not natural, but on the
whole the results of pot experiments are very closely allied to those
obtained in the field by similar tests.


4. _Field experiments._

These make a direct appeal to the practical man, but of the scientific
methods employed the field experiments are the least under control. The
plants are grown under the most natural conditions of cultivation it
is possible to obtain, and for that reason much value has been attached
to such tests. Certainly, so far as the final practical application is
concerned, open field experiments are the only ones which give information
of the kind required. But from the scientific point of view one very
great drawback exists in the lack of control that the investigator has
over the conditions of experiment. The seeds, application of poison, &c.,
can all be regulated to a nicety, but the constitution of the soil itself
and the soil conditions of moisture, temperature and aeration introduce
factors which are highly variable. No one can have any idea of the
composition of the soil even in a single field, as it may vary, sometimes
very considerably, at every step. Further, no one knows the complicated
action that may or may not occur in the soil on the addition of extraneous
substances such as manures or poisons. Altogether, one is working quite
in the dark as to knowledge of what is going on round the plant roots. It
is impossible to attribute the results obtained to the direct action of
the poison applied. While the influence may be direct, it may also happen
that certain chemical and physical interactions of soil and poison occur,
and that the action on the plant is secondary and not primary, so that
a deleterious or beneficial result is not necessarily due to the action
of the toxic or stimulating substance directly on the plant, but it may
be an indirect effect induced possibly by an increase or decrease in the
available plant food, or to some other physiological factor. Consequently
great care is needed in interpreting the results of field experiments
without the due consideration of those obtained by other methods.


II. DETAILS OF METHODS.

Many details of the sand and soil culture methods have been published
by various investigators, e.g. Hiltner gives accounts of sand cultures,
while the various publications issued from Rothamsted deal largely with
the soil experiments. As this is the case, and as all crucial experiments
have always been and must always be done in water cultures, it is only
necessary to give here full details of these.

The great essential for success in water culture work is _strict attention
to detail_. Cleanliness of apparatus and purity of reagents are absolutely
indispensable, as the failure of a set of cultures can often be traced
to a slight irregularity in one of these two directions. Purity of
distilled water is perhaps the greatest essential of all. Plant roots are
extraordinarily sensitive to the presence of small traces of deleterious
matter in the distilled water, especially when they are grown in the
absence of food salts. Ordinary commercial distilled water is generally
useless as the steam frequently passes through tubes and chambers which
get incrusted with various impurities, metallic and otherwise, of which
slight traces get into the distilled water. Loew (1891) showed that
water which contained slight traces of copper, lead or zinc derived from
distilling apparatus exercised a toxic influence which was not evident
in glass distilled water. This poisonous effect was removed by filtering
through carbon dust or flowers of sulphur. Apparently only about the
first 25 litres of distilled water were toxic, in the later distillate
the deleterious substance was not evident.

The best water to use is that distilled in a jena glass still, the steam
being passed through a jena glass condenser. For work on a large scale,
however, it is impossible to get a sufficient supply of such water, while
the danger of breakage is very great. Experiments at Rothamsted were made
to find a metallic still that would supply pure water. While silver salts
are very injurious to plant growth it was found that water that had been
in contact with _pure_ metallic silver had no harmful action. Consequently
a still was constructed in which the cooling dome and the gutters were
made of pure silver without any alloy, so placed that the steam impinged
upon the silver dome, condensed into the silver gutter and was carried
off by a glass tube into the receptacle. Such water proved perfectly
satisfactory so long as any necessary repairs to the still were made
with pure silver, but a toxic action set in directly ordinary solder was
employed. More recently a new tinned copper still has been employed with
good results, but this is somewhat dangerous for general purposes, as in
the event of the tin wearing off in any place, copper poisoning sets in
at once. The water is always filtered through a good layer of charcoal as
a final precaution against impurity.

In the Rothamsted experiments no attempt is made to carry on the cultures
under sterile conditions. Bottles of 600 c.c. capacity are used, after
being thoroughly cleaned by prolonged boiling (about four hours) followed
by washing and rinsing. The bottles are filled with nutritive solution
and the appropriate dose of poison, carefully labelled and covered with
thick brown paper coats to exclude the light from the roots and to prevent
the growth of unicellular green algae. The corks to fit the bottles are
either used brand new or, if old, are sterilised in the autoclave to
avoid any germ contamination from previous experiments. Lack of care in
this respect leads to diseased conditions due to the growth of fungi and
harmful bacteria. Two holes are bored in each cork, one to admit air, the
other to hold the plant, and the cork is cut into two pieces through the
latter hole.

The seeds of the experimental plants are “graded,” weighed so that they
only vary within certain limits, e.g. barley may be ·05–·06 gm., peas
·3–·35 gm., buckwheat ·02–·03 gm. In this way a more uniform crop is
obtained. Great care is needed in selecting the seeds, the purest strain
possible being obtained in each case. With barley it has always proved
possible to get a pure pedigree strain, originally raised from a single
ear. In this way much of the difficulty due to the great individuality
of the plants is overcome, though that is a factor that must always be
recognised and reckoned with. The seeds are sown in damp sawdust--clean
deal sawdust, sifted and mixed up with water into a nice crumbly mass--and
as soon as they have germinated and the plantlets are big enough to handle
they are put into the culture solutions. Barley plants are inserted
in the corks with the aid of a little cotton wool (non-absorbent) to
support them, care being taken to keep the seed above the level of
the water, though it is below the cork. With peas it is impossible to
get a satisfactory crop if the seed is below the cork, as the plant is
very prone to bacterial and fungal infection in its early stages, and
damp cotyledons are fatal for this reason. Consequently the mouths of
the bottles are covered with stout cartridge paper, the pea root being
inserted through a hole in the paper, so that the root is in the liquid
while the cotyledons rest on the surface. As soon as sufficient growth
has been made the papers are replaced by corks, the remnants of the seeds
still being kept on top in the air. Other plants are treated according to
their individual needs and mode of germination (Fig. 1).

[Illustration: Fig. 1. Diagrammatic sketches showing methods of setting
up water cultures.

    ~A.~ _a._ Seedling of cereal.
         _b._ Cork bored with two holes, and cut into two pieces
                through one hole.
         _c._ Food solution.

    ~B.~ _a._ Pea seedling.
         _b._ Paper shield which supports the seedling.
         _c._ Brown paper cover over bottle of food solution.]

The constitution of the nutritive solution is important, and it is
becoming more and more evident that different plants have different optima
in this respect. For several years a solution of medium strength was used,
containing the following:

    Potassium nitrate                   1·0 gram
    Magnesium sulphate                   ·5  „
    Sodium chloride                      ·5  „
    Calcium sulphate                     ·5  „
    Potassium di-hydrogen phosphate      ·5  „
    Ferric chloride                      ·04 „
    Distilled water                      to make up 1 litre.

This is an excellent solution for barley plants, giving good and healthy
growth. While peas grew very well in it, they showed some slight signs
of over-nutrition. A weaker solution is being tested which gives very
good results. Peas grow very strongly in it and it also seems to be
sufficiently concentrated to allow barley to carry on its growth long
enough for the purposes of experiment. The solution is as follows:

    Sodium nitrate                       ·5 gram
    Potassium nitrate                    ·2  „
    Potassium di-hydrogen phosphate      ·1  „
    Calcium sulphate                     ·1  „
    Magnesium sulphate                   ·1  „
    Sodium chloride                      ·1  „
    Ferric chloride                      ·04 „
    Distilled water                      to make up 1 litre.

The latter solution was made up so that the quantity of phosphoric acid
and potash approximated more or less to the amount of those substances
found by analysis in an extract made from a good soil.

The experiments are usually carried on for periods varying from 4–10
weeks, six weeks being the average time. Careful notes are made during
growth and eventually the plants are removed from the solutions, the roots
are washed in clean water to remove adherent food salts, and then the
plants are dried and weighed either separately or in sets. In order to
reduce the error due to the individuality of the plants, five, ten or even
twenty similar sets are grown in each experimental series, the mean dry
weight being taken finally. Also the same experiment is repeated several
times before any definite conclusions are drawn.

Another method of water cultures is used by some investigators, in which
the experiments only last for a few hours or days, usually 24–48 hours.
While such experiments may not be without value for determining the
broader outlines of toxic poisoning, they fail to show the finer details.
The effect of certain strengths of poison is not always immediate. Too
great concentrations kill the plant at once, too weak solutions fail
to have any appreciable immediate action and so appear indifferent.
Between the two extremes there exists a range of concentrations of which
the effect varies with the plant’s growth. A solution may be of such a
nature and strength that at first growth is seriously checked, though
later on some recovery may be made, while it is also possible that a
concentration which is apparently indifferent at first may prove more
or less toxic or stimulant at a later date, according to circumstances.
Consequently too much stress must not be laid upon the results of the
short time experiments with regard to the ultimate effect of a poison upon
a particular plant.

       *       *       *       *       *

An examination of the various experimental methods shows that while no
one of them is ideal, yet each of them has a definite contribution to make
to the investigation of toxic and stimulant substances. Each method aids
in the elucidation of the problem from a different standpoint, and the
combination of the results obtained gives one a clearer picture of the
truth than could be obtained by one method alone. Water cultures, with
their exactitude of quantitative control lead on by way of sand cultures
to pot cultures, and these to field experiments in which the control is
largely lost, but in which the practical application is brought to the
front.



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 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.



CHAPTER IV

EFFECT OF ZINC COMPOUNDS


I. PRESENCE OF ZINC IN PLANTS.

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

    _Thlaspi alpestre_, var. _calaminaria_.

       Root       6·28% ash,  0·167% ZnO,   1·66% ZnO in ash.
       Stem      11·75%  „    0·385%  „     3·28%  „    „
       Leaves    11·45%  „    1·50%   „    13·12%  „    „
       Flowers    8·49%  „    0·275%  „     3·24%  „    „

    _Viola tricolor._

       Root       5·59% ash,  0·085% ZnO,   1·52% ZnO in ash.
       Stem      10·55%  „    0·065%  „     0·62%  „    „
       Leaves     9·42%  „    0·110%  „     1·16%  „    „
       Flowers    7·66%  „    0·075%  „     0·98%  „    „

    _Armeria vulgaris._

       Root       4·74% ash,  0·17% ZnO,    3·58% ZnO in ash.
       Stem       5·37%  „    0·02%  „      0·37%  „    „
       Leaves     9·36%  „    0·11%  „      1·17%  „    „
       Flowers    6·08%  „    0·07%  „      1·15%  „    „

    _Silene inflata._

       Root       2·71% ash,  0·02% ZnO,    0·74% ZnO in ash.
       Stem    }
       Leaves  } 11·43%  „    0·22%  „      1·92%  „    „
       Flowers }

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

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

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

    _Tussilago Farfara._

          Root          Leaf-stalk     Leaf-blade
       2·51%–3·26%     1·75%–1·63%    2·90%–2·83% ZnCO₃
     = 1·629%–2·115%   1·136%–1·058%  1·882%–1·836% ZnO.

    _Polygonum aviculare._

          Root           Stem          Leaves
       1·77%–1·93%    2·25%–2·86%   1·24%–1·49% ZnCO₃
     = 1·148%–1·252%  1·46%–1·856%   ·804%–·967% ZnO.

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

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

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


II. EFFECT OF ZINC ON THE GROWTH OF HIGHER PLANTS.


1. _Toxic effect._


(_a_) _Toxic action of zinc salts alone in water cultures._

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

    Trifolium pratense      killed in 16 days
    Spergula arvensis           „     21  „
    Hordeum vulgare             „     30  „
    Vicia sativa                „     31  „
    Polygonum Fagopyrum         „     60  „
    Beta vulgaris               „     76  „
    Onobrychis sativa           „    194  „

    [5] 44 mg. ZnSO₄.7H₂O = 10 mg. Zn = 1/22,727 ZnSO₄.7H₂O approx.

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

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


(_b_) _Effect of soluble zinc salts in the presence of nutrients._

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

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

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

Recent experiments at Rothamsted have shown that zinc sulphate is very
toxic to barley, though the plant is able to make some slight amount of
growth even in the presence of a solution of the anhydrous salt ZnSO₄
as strong as 1/5000, rapid improvement occurring as the concentration
decreases to 1/2,500,000 or less (Fig. 6). On the whole the higher
strengths of zinc sulphate are less poisonous to peas than they are
to barley. At a concentration of 1 in ¼ or 1 in ½ million in different
experiments the growth was nearly as good as with the control plants,
though it consistently lagged a little way behind until a dilution of
1/10,000,000 was reached (Figs. 7 and 8). Incidentally it is very striking
to see the desperate efforts that badly poisoned pea plants make to
reproduce themselves. Growth of the roots is nearly always checked in
advance of that of the shoots, probably on account of the contact of the
roots with the poison. In the greater strengths of such poisons as zinc
and copper sulphate root growth is checked from the outset, but usually
a very little shoot growth is made, and one frequently obtains ridiculous
little plants about an inch high bearing unhappy and diminutive flowers,
which are occasionally replaced by equally unhappy and miniature fruits.
The same thing has also been noticed when unsuccessful attempts have been
made to introduce spinach as a test plant for water cultures.

[Illustration: Fig. 6. Curve showing the mean value of the dry weights
of ten series of barley plants grown in the presence of anhydrous zinc
sulphate and nutrient salts. (March 2nd–May 8th, 1911.)]

[Illustration: Fig. 7. Photograph showing the action of anhydrous zinc
sulphate on pea plants in the presence of nutrient salts. (Sept. 30th–Dec.
20th, 1912.)

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

[Illustration: Fig. 8. Curve showing the mean values of the dry weights of
nine series of pea plants grown in the presence of anhydrous zinc sulphate
and nutrient salts. (May 18th–June 28th, 1910.)]


(_c_) _Effect of zinc compounds on plant growth when they are present in
soils._

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

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

    [6] This is equivalent to about ·1% of poison.

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

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

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

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

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


(_d_) _Mode of action of zinc on plants._

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


2. _Effect of zinc compounds on germination._

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

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

Micheels (1906) approached the matter from a totally different standpoint,
seeking to discover what influence the valency of a metal has upon the
toxicity of its salts. In each of a series of experiments 1000 c.c. of ⅝
decinormal solution of sodium chloride in pure distilled water were used,
with the addition of varying strengths of calcium sulphate. Grains of
wheat, which previously had been soaked in distilled water, were placed
in the solutions, and it was found that the stronger the calcium sulphate
solution (up to 1/64 normal--the limit of experiment), the better the
growth. The calcium sulphate was then replaced by salts of other bivalent
metals, as zinc, lead and barium, with analogous results, the quantity
necessary to obtain the maximum development varying with one and another;
with zinc, _n_/128 gave the maximum. In this case the toxic action of both
sodium chloride and zinc sulphate on germination were considerably reduced
by their mutual presence--a result which fits in perfectly with what is
known as to the masking effect of soluble substances upon toxic action.
The same fact obtains in the animal kingdom, where Loeb and others have
found that the toxicity of solutions of sodium chloride for marine animals
is reduced by the introduction of salts of the bivalent metals.


3. _Stimulation induced by zinc compounds._

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

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

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


(_a_) _Stimulation in water cultures._

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

[7] _m_ probably = gram molecular weight.

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

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

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


(_b_) _Stimulation in sand cultures._

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


(_c_) _Increased growth in soil._

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

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

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

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


4. _Direct action of zinc salts on leaves._

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

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


III. EFFECT OF ZINC ON CERTAIN OF THE LOWER PLANTS.

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

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

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

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

    (_a_) normal Raulin’s solution with zinc,
    (_b_) Raulin’s solution without zinc.

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

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

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


_Conclusion._

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

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



CHAPTER V

EFFECT OF ARSENIC COMPOUNDS


I. PRESENCE OF ARSENIC IN PLANTS.

The occurrence of arsenic as an occasional constituent of plants has
been recognised for many years. Chatin (1845) found that if a plant were
supplied with arsenical compounds at the roots arsenic was absorbed, but
that it was distributed unequally to the various tissues. The greatest
accumulation of the element was in the floral receptacle and the leaves,
while it was scarce in the fruits, seeds, stems, roots and petals. E.
Davy (1859) commented on the presence of arsenic in plants cultivated
for food. He grew peas in pots and watered them for a short time with a
saturated aqueous solution of arsenious acid, the application being then
discontinued. The plants, apparently uninjured by the treatment, flowered
and formed seeds. On analysis arsenic was readily detected in all parts
of the plant, including the seeds. Other analyses revealed the presence
of the element in cabbage plants (from pots) and turnips (from field),
both of which had been manured with superphosphate containing some amount
of arsenic. This absorption of arsenic by the roots of plants was further
established by Phillips (1882).

Various physiological workers have pointed out that this element is
frequently or usually present in animal tissues. Cerný (1901) reached
the general conclusion that minimal traces of arsenic can occur in animal
organisms, but that these play no part in the organism and indeed are not
constant in their occurrence. Bertrand (1902) established its presence in
minute quantities in the thyroid glands of the ox and pig, hair and nails
of the dog, and the feathers of the goose. Gautier and Clausmann (1904)
realised the constant presence of arsenic in human tissues and recognised
that it must inevitably be introduced into the body with the food. This
led them to estimate the arsenic present in various animal and vegetable
foods, some of their results being given in the following table.

_Arsenic per 100 parts fresh substance in µ gr._ (= thousandth part of a
milligram)[8].

    Wheat (Victoria--complete grain)  ·7
      „   (from Franche Comté)        ·85
    White bread                       ·71
    Whole green cabbage               ·2
    Outside leaves of cabbage         ·0 (absent)
    Green haricots                    ·0    „
    Turnip                            ·36
    Potatoes                         1·12

    [8] 0^{µ gr.}, 1 = 0·0001 mg.

Arsenic was also found in wine and beer and in considerable quantities
in sea water and various kinds of salt. Since it cannot be found in some
things even in the least traces, the authors conclude that it is incorrect
to say that the element is always present or that it is essential to all
living cells.

S. H. Collins (1902) found that barley is able to absorb relatively
large quantities of arsenic. The plants were grown in pots on soil which
originally contained a certain amount of the substance, and various
combinations of arsenic acid, arsenious acid and superphosphate were
added. Particulars and details are not given by the author, except
that arsenic was detected by Reinsch’s test in the grains from all the
experimental pots, and in one case (not specified) in the upper and lower
halves of the straw and in the threshed ears. The analyses of the soil at
the close of the experiments showed the presence of 7–22 parts arsenious
acid per million.

Wehmer (1911) quotes references to the occurrence of arsenic in _Vitis
vinifera_. The element was detected in the ash of the must and its
presence was attributed to treatment of the plants with arsenical
compounds. In this connection it is interesting to note the observation
of Swain and Harkins (1908), who, while acknowledging the absorption of
arsenic from the soil by many plants, yet indicate that in the case of
those plants which are exposed to smelter smoke the arsenic is deposited
on the vegetation, and is not absorbed by the latter from the soil.


II. EFFECT OF ARSENIC ON THE GROWTH OF HIGHER PLANTS.


1. _Toxic effect._


(_a_) _Toxic action of arsenic compounds in water cultures in the presence
of nutrients._

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


(_b_) _Toxic effect of arsenic compounds in sand cultures._

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


(_c_) _Toxic effect of arsenic when applied to soil cultures._

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

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

    [9] 30 grams arsenious acid to 30·7 “cubik Decimeter” soil = about
    ·1%.

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


(_d_) _Physiological considerations._

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

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

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


2. _Effect of arsenic compounds on germination._

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

    [10] In the present state of our knowledge such a concentration
    seems relatively strong!

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


3. _Do arsenic compounds stimulate higher plants?_

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


III. EFFECT OF ARSENIC COMPOUNDS ON CERTAIN OF THE LOWER PLANTS.


1. _Algae._

Loew (1883) was sceptical concerning the specific toxicity of arsenic for
plant protoplasm. He was convinced that arsenic and arsenious acid were
poisonous to algae, not because of their specific character as arsenical
compounds, but because of their acid nature, algae being peculiarly
sensitive to any acid, and he maintained that these substances were not
more poisonous than vinegar or citric acid. He placed various species
of _Spirogyra_ in solutions of ·2 gm. potassium arsenate per litre water
(1/5000), and found that the algae grew well without making any abnormal
growth in a fortnight, showing hardly one dead thread. Some of this
alga was then transferred to a 1/1000 solution of potassium arsenate.
This suited it excellently and it increased and the appearance under
the microscope was very fresh and strong, which was attributed more to
the potash than to the arsenic acid. Loew maintained that for the lower
animals and for many of the lower plants arsenic in the form of neutral
salts is not a poison. When the differentiation of the protoplasm into
certain organs reaches a specific degree in the higher plants, then the
poisonous action of the arsenic compounds comes into play.

Knop (1884) found that certain unicellular green algae grew luxuriantly
in a neutral solution supplied with potassium arsenate. Bouilhac (1894)
concerned himself chiefly with the possibility of the replacement of
phosphates by arsenates. He recognised that the influence of arsenic
is not the same on all species of plants, so he confined his attention
to certain of the algae. _Stichococcus bacillaris_ Naegeli was found
to live and reproduce itself in a mineral solution containing arsenic
acid. Even in the presence of phosphoric acid the arsenic acid favours
growth, the best dose being about 1/1000. The arsenic acid is capable of
partly replacing phosphoric acid. Other species of algae, _Protococcus
infusionum_, _Ulothrix tenerrima_, and _Phormidium Valderianum_ invaded
the original culture of Stichococcus from the atmosphere, but with no
arsenic or phosphoric acid their development was poor. The jars with
arsenic compounds were invaded by still more species which grew strongly.
Under these conditions it is evident that these algae are capable of
assimilating arsenic, and the addition of arsenic acid to a solution
free from phosphoric acid is sufficient to enable these algae to live
satisfactorily, the arsenates in this case replacing the phosphates.
Ono (1900) found that algae are favourably influenced by small doses of
poisons, the optimal quantity for algae being lower than that for fungi.
Protococcus showed a possible stimulus when grown in concentrations of
potassium arsenate varying from ·00002–·0005%. This possible stimulus is
interesting in view of the failure to observe stimulation in higher plants
by minute traces of arsenic.


2. _Fungi._

The effect of arsenic on fungi is of special interest in that it has a
direct bearing upon hygienic and commercial interests. Gosio (1892, 1897,
1901) found that certain of the fungi, _Mucor mucedo_ and _Aspergillus
glaucum_, will grow on various arsenic compounds and exercise a reducing
influence on them. These moulds attack all oxygen compounds of arsenic
including copper arsenite, and develope arsenical gases. Sulphur compounds
of arsenic are not influenced by these fungi. The same moulds would,
if cultivated in soil containing arsenic, develope hydrogen arsenide.
_Penicillium glaucum_ has such a strong and definite action on arsenic
compounds that he states that there is no doubt of the possibility of
poisoning by arsenical gas in a room hung with paper containing arsenic.
The compounds are so extraordinarily potent that if a mouse is placed
in a vessel in which the mould is strongly developed in the presence
of arsenic, it dies in a few seconds. _Penicillium brevicaule_ uses the
element in its development as a food substance. If material containing
arsenic is placed in contact with dead fungi no reaction occurs. The life
activity of the mould is evidently necessary for the reaction by which
the arsenic-containing gases are liberated. Csapodi (1894) put forward the
earlier results of Gosio and noted that the so-called arsenical fungicides
do not only fail to kill the mould fungi but actually favour their
development. This action explains why wallpaper containing arsenic is so
disadvantageous in a room. Abba (1898) severely tested Gosio’s method
of detecting arsenic by means of growths of _Penicillium brevicaule_,
whereby arsenic gases are liberated, vindicating the method completely,
and establishing the test as an exceptionally delicate one. Segale (1904)
applied the same method to the detection of the presence of arsenic in
animal tissues.

Ono (1900) grew Penicillium cultures with solutions of potassium arsenate
and found no important differences either of depression or stimulation.
Orlowski (1902–3) stated that small doses of arsenic (1/1000–1/100% Sodium
arsen--[11]) stimulate the growth of _Aspergillus niger_, larger doses up
to ⅛% retard growth, while ⅙% kills. Spores of the fungus taken from soil
containing arsenic are said to possess an immunity against arsenic, in
that they germinate in the presence of an arsenic content which rapidly
kills control fungi. This immunity is not specific for arsenic, but
extends also to other poisons. The chemical composition and water content
are not altered.

    [11] The exact compound is not specified in the abstracted paper,
    1/1000–1/100% Natr. Ars. being given.


_Conclusion._

The toxic effect of arsenic upon higher plants is much more marked
with arsenious acid and its compounds than with arsenic acid and its
derivatives. No definite evidence of stimulation has yet been obtained
with any arsenic compound, however great the dilution at which it is
applied. With certain algae a stimulus may occur, and it is possible that
arsenic acid is capable of replacing phosphoric acid to some extent under
certain conditions. With fungi the toxic effect of great concentrations
is marked with certain species, but there are others which are capable of
living happily on arsenical compounds and of liberating highly poisonous
arsenic gas.



CHAPTER VI

EFFECT OF BORON COMPOUNDS


I. PRESENCE OF BORON IN PLANTS.

The first claim to the discovery of boron in plants was put forward
in 1857 by Wittstein and Apoiger, who carried out investigations
on the Abyssinian Saoria (seeds of _Maasa_ or _Maessa picta_, N.O.
Primulaceae[12]). In the course of analyses a crystalline mass was
obtained which was found to contain chlorine, phosphoric acid, lime, and
boric acid. The discovery apparently attracted little attention and for
about another thirty years the matter was again allowed to sink into
oblivion. Then it came to the front again, and from 1888 onwards one
investigator after another demonstrated the presence of boron in various
plants.

    [12] According to Engler’s classification this plant belongs to N.O.
    _Myrsinaceae_.

In 1888 Baumert detected boron in French, German, and Spanish wines
without exception, while E. O. von Lippman (1888) demonstrated it in sugar
must and also in the leaves and root of the sugar beet. In the latter case
the reactions were so definite that the presence of more than a minimal
amount of boric acid was conjectured.

Crampton (1889) tested various fruits, but while he found boron in every
part of the watermelon, he could get no reaction with apples or with
certain samples of sugar cane. He predicted, however, that the occurrence
of boron would prove to be more general in the plant kingdom than had
previously been supposed. The next year (1890) Hotter extended the work
on fruits, testing for boron in the fruits, leaves, and twigs of certain
plants, and finding it in the apple, pear, cherry, raspberry, fig, and
others. His results indicated that fruits are relatively rich in boron.
Later on (1895) Hotter carried his experiments further, and he stated
that stone fruits are richer in boric acid than are berries and pomes.
The accumulation of boron is in the fruit itself, the other parts of the
plant containing little. The quantities of boric acid found in the ash of
the various fruits ranged from ·58% in the “Autumn Reinette” apple to ·06%
in figs. Bechi had previously (1891) detected boron in the ash of figs,
love-apple, and rubus fruits from Pitecio, but he attributed this to the
presence of boric acid or borates in the soil at the place.

Passerini (1891) found traces of boron in the stems of chickpea
plants, while in 1892 Brand determined boric acid in the ash of beer.
In consequence of this various samples of hops were ashed without the
addition of any alkali, and then the ash was distilled with sulphuric
acid and methyl alcohol. When tested all the hops showed relatively large
quantities of boric acid in comparison with beer, hence he argued that
the boric acid in beer is derived from the hops. Boron was discovered in
various parts of the hop plant--in the clusters, leaves, pedicels, and
stems.

Jay (1895) analysed many plants and plant products grown in various soils
and waters, and arrived at the conclusion that boron is of practically
universal occurrence in the plant world. Of all vegetable liquids wines
are the richest in this constituent, the amount varying from ·009 gram to
·33 gram per litre. He confirmed Hotter’s statement as to the richness of
fruits in this substance, finding from 1·50–6·40 grams in 1 kgm. of ash.
Chrysanthemums and onions, amongst other plants, are well off in this
respect, containing 2·10–4·60 grams per kgm. of ash. Jay also found that
the plants vary in their capacity for absorbing boric acid, those which do
so the least easily being Gramineae (as wheat, barley, rice), mushrooms
and watercress, the quantity in these plants never exceeding ·500 grams
per kgm. of ash.

Of all the workers upon boron, Agulhon has done the most to extend and
concentrate our knowledge of the subject. He used the most refined,
up-to-date methods for the detection and estimation of boric acid,
and so determined its presence in many plants, including angiosperms,
gymnosperms, ferns, algae, and fungi. Tobacco is so rich in boron that it
can be detected in the ash of one cigarette. Among the plants tested, the
highest percentages of boric acid were found in _Betula alba_ (1·175% of
ash) and _Laminaria saccharina_ (·682% of ash), the lowest in _Cannabis
sativa_ (·123% of ash). Generally speaking annual plants and parts of
plants seem to have the least boron in the composition of their ashes. In
one and the same plant the durable parts like bark and wood are richer
than the leaves, even in evergreen trees. He indicated that plants seem
to have a great affinity for boron, as even when plants are grown on soils
in which the boron is practically indetectable they always seem to extract
an appreciable quantity of the element.

From the foregoing results it is evident that boron is very widespread in
the vegetable kingdom, entering into the composition of many plants in all
the great classes. A general impression obtains that its distribution is
universal, and that it will ultimately prove to enter into the composition
of practically every plant, as the scope of the analyses is widened
and as methods of detection are improved. On the other hand, Agulhon is
inclined to think that boron may be a “particular element,” characteristic
of certain groups of individuals or of life under certain conditions.
The series of individuals differ among themselves as to their particular
needs of nutriment (in the widest sense) and doubtless each group has
special need of particular elements, a need that is possibly correlated
with morphological and chemical differences. It may well be that boron is
one of these elements, associated with certain vital functions in a way
as yet unexplained, though it may possibly be found to play some part in
the formation of vascular tissues, since it is most abundant in bark and
lignified parts.


II. EFFECT OF BORON ON THE GROWTH OF HIGHER PLANTS.


1. _Toxic effect._


(_a_) _Toxic action of boron compounds in water cultures._

Excessive quantities of boric acid are decidedly poisonous to plants, the
action being well marked in water cultures. Knop (1884) found that free
boric acid was poisonous in neutral food solutions when present at the
rate of ·5 gram per litre, but he was not able to detect boron in the
ash of the roots of the experimental plants. Archangeli (1885) placed
seedlings of maize, white lupins, _Vicia sativa_ and _Triticum vulgare_
in solutions of boric acid varying in concentration from 1–·05%, with
controls in spring water. In the latter case the development was normal,
with 1% boric acid the plants were killed, while it was found that the
weaker the solution (within the indicated limits) the stronger the root
and shoot growth.

Hotter (1890) stated that it was known that 1/20,000 boric acid by weight
was harmful to soy beans in nutritive solutions. He experimented with
peas and maize, placing the seedlings first in distilled water, later
in nutritive solutions. When the peas were nineteen days old they were
transferred to nutritive solutions containing 1/1000–1/100,000 boric acid
by weight per litre, and within three days the plants with 1/1000 showed
signs of injury. Two days later all the plants showed signs of poisoning
in that, even with the weakest strengths, the lower leaves were flecked
with brown, especially at the edges, while with the greater strengths
the lower leaves were dead and the flecking had extended to the upper
leaves. In eleven days from the start the plants with 1/1000 boric acid
were completely dead, while the other plants showed more or less signs of
poisoning. The dry matter and ash decreased steadily with the increase
in the boric acid, while the boric acid per 100,000 parts of dry matter
increased steadily from 8 to 557 parts. Similar experiments were carried
on with potassium borate and with borax; the results showed that, weight
for weight, borax is less toxic than potassium borate, which in turn is
less toxic than boric acid, while at a strength of 1/100,000 there is
little to choose between the three poisons. Similar results were obtained
with maize; plants treated with boric acid or potassium borate yielded
about 2300 parts boric acid in 100,000 parts dry matter. The general
conclusion arrived at by Hotter was that the effect is not so much that
of a general poisoning as of a bleaching of parts of the leaf, mere
traces of boron being harmless. The cause of injury is local inhibition
of assimilation and killing of roots in stronger concentrations. Increase
of the strength of boron raises the toxicity until 1/1000 practically
inhibits increase in dry substance. The boron was found to be fairly
evenly distributed through sound and affected organs.

Kahlenberg and True (1896) worked with seedlings of _Lupinus albus_ L.,
limiting their experiments to those of 15–24 hours in duration. Various
combinations of boron and other substances were tested. With boric acid
alone 2/25 gram molecule per litre killed the plants, with 1/25 they
were apparently just alive, while 1/100 and less had no injurious effect.
Boromannitic acid was possibly more poisonous than the boric acid, while
a combination of boric acid and cane sugar proved slightly less toxic.
The short duration of these experiments limited their scope considerably,
as with certain concentrations the toxic action would not become evident
within the prescribed limits of time.

Agulhon (1910 a) worked with sterile nutrient solutions, and found that
the higher strengths of boric acid hindered growth, 200 mg. boric acid
per litre rendering growth impossible. He supported Hotter’s idea that
the toxic action affects the roots and the formation of chlorophyll, and
he stated that the plants are less green as the dose of boron increases,
plants growing in doses of above 10 mg. per litre being yellowish. In
other experiments he found that at 100 mg. boric acid per litre life seems
impossible for the plant. The roots seem to be more adversely affected by
toxic doses than do the shoots. In control plants Agulhon determined the
stem/root ratio as 6, with a little boron as 7, while the ratio rose to
13 as the dose of the poison increased to 50–100 mg. boron per litre.

The Rothamsted experiments show that boric acid is definitely poisonous
to barley down to a strength of 1/250,000 (Fig. 15), the depressing
effect frequently being evident at much smaller concentrations, while
peas can withstand far more of the poison, the limit of toxicity being
about 1/25–1/50 thousand (Fig. 16). With the greater strengths of poison
the lower leaves of both barley and peas are badly damaged. In barley
the leaves turn yellow with big brown spots, giving the leaves a curious,
mottled appearance, while with peas the poisoning seems to begin at the
tip and edge of the leaves, spreading inwards, without, however, showing
the large spots as in barley. So far as chemical tests go at present, it
is very probable that boron is deposited in the leaves in the same way
as manganese, and that this is the cause of the degeneration. As with
manganese, the lower leaves are attacked first, and the trouble spreads
upwards, one leaf after another being involved. These observations fit
in very well with those of Hotter, and the hypothesis of direct boron
poisoning gains support from the fact that in dilutions which produce
stimulation of the shoot the leaves show hardly any sign of dying off,
even after prolonged growth in the solutions. With barley the effects of
boron can be seen in the leaves in concentrations as low as 1/2,500,000,
and it may be significant that this is the point at which the depressant
action of boric acid entirely ceases in many cases.

[Illustration: Fig. 15. Curve showing the mean value of the dry weights
of ten series of barley plants grown in the presence of boric acid and
nutrient salts. (May 1st–June 20th, 1911.)]

[Illustration: Fig. 16. Photograph showing the action of boric acid on pea
plants in the presence of nutrient salts. (Sept. 30th–Dec. 20th, 1912.)

    1.  Control.
    2.  1/5,000     boric acid.
    3.  1/10,000      „    „
    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   „    „   ]

Tests with white lupins gave no conclusive results, as for some reason
it proved very difficult to get satisfactory plants in water cultures.
When they are grown under such conditions the roots always tend to get
more or less diseased and covered with slime, probably fungal in nature.
In the presence of much boric acid the roots remain in a much healthier
condition, which suggests that the acid has in this case a strong
antiseptic action, and protects the roots. With high concentrations
the lower leaves of the plant are badly affected, just as with peas and
barley, turning brown and withering at an early date. Various experiments
have been made with yellow lupins, but these again are very difficult to
grow well in water cultures, as they are apt to drop their leaves for no
apparent reason. Generally speaking, the evidence goes to prove that boric
acid is toxic down to a concentration of about 500 parts in 25 million.
It is difficult to get a true control with which to make comparisons
as the plants without boric acid are encumbered with the slime on their
roots, which naturally interferes with normal growth, while the plants in
the presence of boric acid have the unfair advantage due to the probable
antiseptic action of the boron. The effect of the boron poisoning is again
evident in the dying off of the lower leaves, which become flaccid and
drooping and finally drop off. The lupins grown with boron are very active
in the putting forth of lateral roots, so much so that the cortex of the
roots is split along the line of emergence of the laterals, which are very
numerous and crowded.


(_b_) _Toxic action of boron compounds in sand cultures._

Agulhon (1910 a) moistened 2 kgm. pure sand with 500 c.c. nutritive
solution for each pot, and boron was added at the rate of 0, 0·1, 1, 10,
and 50 mg. boric acid per litre of nutritive solution. Twenty wheat seeds
were sown in each pot, and after twelve days the healthy plants in the
first four pots were 6–8 cm. high, but those with the maximum amount of
boron showed yellowish leaves only 3 cm. long. After three months’ growth
the plants were harvested, when those with most boron were found to have
died after making about 10 cm. growth. The toxic doses in sand proved to
be weaker than those in water cultures, probably because evaporation from
the surface of the sand caused concentration of the poisonous liquid.


(_c_) _Toxic action of boron compounds in soil experiments._

Long before any experimental work was done with boron in water cultures,
the poisonous properties of the substance were recognised with regard to
plants growing in soil. Peligot (1876) grew haricots in porous earthenware
pots, the plants being watered by rain and by solutions, each containing
about 2 grams per litre of such substances as borax, borate of potassium,
and boric acid, other pots receiving various fertilisers, as potassium
nitrate, sodium nitrate, &c. This quantity of boron completely killed
off the plants receiving it, whether it was applied as free or combined
boric acid, while the fertilised plants completed their development well.
On this account the deleterious action was attributed to the boric acid
and not to the sodium or potassium base supplied. Peligot hinted at the
improbability of a substance like boron, which is so poisonous to plants,
being really innocuous to human beings when it is used as a preservative
for foods.

Nakamura (1903) also found that borax is harmful in pot cultures if
present in large quantities, 50 mg. borax per kgm. of soil exerting a
very injurious influence, while even 10 mg. per kgm. did some damage.
Agulhon (1910 c) found that the toxic doses of boric acid in soil cultures
approached those in nutritive solutions rather than in sand cultures, a
phenomenon that he attributed to the fact that the boric acid was fixed
by the soil, probably as insoluble borate of calcium, so that the surface
concentration obtained with sand cultures was avoided. He found that the
ash of plants grown with excess of boron contained more than the normal
amount of boron, while the weight of ash per 100 dry matter was also
increased. He concluded that the plant thus suffers an over-mineralisation
and in consequence an augmentation of its hold on water, so that the fresh
weight of the plant may indicate a more favourable action of the boric
acid than does the dry weight. Other investigators (Fliche and Grandeau
1874) had found the same increase in the proportion of ash in chestnut
trees grown on too calcareous soil, so Agulhon concluded that one is
here dealing with a general reaction of plants to an excess of a useful
element.

Other experiments were carried on in the open field, maize being grown
on control plots and on plots receiving 2 gm. boron per square metre.
At first the latter plants were behind, the dose being too strong.
Eventually, however, they pulled up level and the dry weights from the two
plots proved to be nearly the same, the fresh weights being identical.
Maize is evidently far less sensitive to boron poisoning than are peas
and oats, for with these one-half the original amount of boron (= 1 gm.
per sq. metre) proved toxic.

Interesting results were obtained (Agulhon 1910 a) by repeated experiments
with the same soil containing boron. It was found that sand or soil
containing a proportion of boron which is lethal or toxic to a first
culture will allow much better growth with a second and subsequent
crops. Repeated experiments on the same soil may show the change from a
lethal dose to a toxic one, thence to an indifferent and finally to an
optimum concentration. Furthermore (Agulhon 1910 b) the very plants may
accustom themselves to greater quantities of boron, the increased power
of resistance being transmitted. He concluded from his experiments that
the progeny of the second generation of maize were able to withstand
quantities of boron that were toxic to control plants[13]. Agulhon once
again emphasised the fact that for toxic doses of boron the first symptom
is the more or less marked disappearance of chlorophyll, though the aerial
parts are not affected so soon as the roots.

    [13] “Il apparaît donc que les graines fournies par des plantes
    ayant crû en présence d’une quantité de bore élevée présentent une
    accoutumance vis-à-vis de cet élément; les plants auxquels elles
    donnent naissance semblent non seulement faire un meilleur emploi
    des petites doses de bore qui leur sont offertes, mais encore
    supportent les doses toxiques plus facilement que les plants
    témoins, issus de graines non accoutumées.”


2. _Effect of boron compounds on germination._

One of the first indications that boron compounds affect the germination
of seeds was given by Heckel (1875) who found that germination was
retarded for 1–3 days by weak solutions of borates (·25 gm. to 20 gm.
water), and was stopped altogether by stronger solutions (·60 gm. to 20
gm. water). Archangeli (1885) tested the germination of a variety of seeds
of Leguminosae, Gramineae, and of Cannabis, Iberis, Raphanus, Collinsia,
and Linum in the presence of boric acid. The seeds were placed in bowls
with solutions of ·25, ·5, and 1% boric acid at temperatures ranging from
16°–23° C. The bowls were covered with glass plates to prevent evaporation
and consequent increase of concentration, controls in spring water being
dealt with under similar conditions. 1% boric acid was found to check
germination altogether, and the weaker the concentration the less was
the process hindered. Morel soaked seeds of haricots and wheat in various
solutions of boric acid, and found that germination was generally hindered
or inhibited. The deleterious action diminishes as the strength of the
solution or the time of contact diminishes, but solutions of the same
concentration do not act equally on all seeds. Boric acid and borax proved
to be similar in their action qualitatively.

The deleterious effect of strong doses of boric acid on germination was
confirmed by Agulhon (1910 a), the higher quantities (above 10 mg. boric
acid per litre) retarding germination of wheat.


3. _Does boron stimulate higher plants?_

Of recent years a few investigators have thrown out hints as to the
stimulant action exerted by boron compounds on plants. Roxas indicated
that M/100,000 (M = molecular weight) of boric acid exercised a favourable
action on rice. Nakamura (1903) tested the point by means of pot cultures.
Peas and spinach plants were grown in soil which received 1 and 5 mg.
borax per kgm. With peas the 1 mg. exerted evident stimulant action,
as determined by the increase in height of the shoot over that of the
control, 5 mg. seeming to be slightly depressant in action. With spinach
a stimulation was observed both in weight and height with a dose of 5 mg.
borax per kgm.

                  Average weight      Average length of leaves
    5 mg. borax        10·35                    38·2
    Control             7·2                     34·0

Agulhon (1910 c and d) took the matter up still more definitely and made
many tests of various kinds, in water, sand and pot cultures.


(_a_) _Water cultures._

His water cultures were made under sterile conditions, the seeds when
possible being sterilised with corrosive sublimate, the germinating
apparatus being also sterilised. With wheat a stimulant action was
evident, maximum growth being obtained with between 2·5 and 10 mg. boric
acid per litre, though the dry weight increase did not quite keep pace
with that of the fresh weight, a fact to which previous reference has been
made. The chief improvement is in the root, the stem/root ratio falling to
5, as against 6 in the control series. Visual observation indicated that
the roots of plants receiving 5–10 mg. boric acid per litre are longer
than the others, though they are less rich in adventitious roots. The
increased dry weight due to boron may amount to as much as 30%.


(_b_) _Sand cultures._

Agulhon again observed stimulation in this case. 2 kgm. of sand were
moistened with 500 c.c. nutritive solution, varying quantities of boric
acid being added in addition. ·1 mg. boric acid per litre of N.S. (·05
mg. per pot) gave an increase of 25% fresh weight, and 7·5% dry weight.
The stimulating doses seem to be weaker than in the experiments with
liquid media, probably because the evaporation from the sand increases
the concentration of the boric acid at the surface. It was also noticed
that the increase of weight varied in experiments made at different times.
With oats the stimulating influence is greater than with wheat, showing
that some plants are more sensitive than others to the influence of boron.
With radish 1 mg. boric acid per litre exercised a stimulating effect,
the enormous average increase of 61% in fresh weight occurring with this
strength, though this only represented an average increase of 9·6% dry
weight.


(_c_) _Soil cultures._

Here again the stimulating action was evident with higher concentrations
than in sand cultures, and Agulhon obtained good results with strengths
that are toxic in sand. The evaporation from earth is not so rapid as from
sand, so that the concentration is not increased, and also some of the
boric acid is withdrawn from the solution by interaction with the soil,
so that the stimulating concentration rises in the scale.

In field experiments Agulhon found that peas were more sensitive to the
toxic action of boric acid than is maize. A strength of boric acid (= 1
gm. B per sq. metre) that poisoned peas, gave an increase of 61% fresh
weight and 39% dry weight with maize; half the strength proved to be
indifferent for peas, the improvement with maize equalling 56% increase
fresh and 50% increase dry. Curiously enough, judging by appearances in
the first experiment, an unfavourable influence was at work, though in
reality a great stimulation was being caused. Colza gave a good increase
with similar strengths, but with turnips 1 gm. B per sq. metre only
favoured the aerial parts, while ·5 gm. B per sq. metre only increased
root development. Agulhon concluded that it is as yet impossible to
determine with any precision the exact part that boron plays in the plant
economy. He suggests that boron is a “particulier” element characteristic
of a certain group of individuals or of life under particular conditions.
In his summary he argues that each series of individuals adapted to
different environments has doubtless need of particular elements, and that
perhaps chemical causes and morphological differences are very closely
connected. Boron may be of this “particulier élément” type in the higher
plants of the vegetable kingdom, and it may be useful commercially as a
manurial agent, the “catalytic manure” of Bertrand and Agulhon.

While the higher concentrations of boric acid proved definitely toxic
to both peas and barley in the Rothamsted water cultures, some evidence
of stimulation was obtained with the lower strengths. With barley the
question of stimulation is still an open one, as below the toxic limit
growth seems fairly level in most of the experimental series. The lower
limit of toxicity varies from 40–4 parts boric acid per 10,000,000
according to circumstances. Below this critical concentration the boric
acid has apparently no action, either depressant or stimulant, unless the
stimulation should prove to begin at a dilution of 1/50,000,000, but the
evidence on this point is not sufficiently well marked or consistent to be
conclusive. This failure to detect stimulation was somewhat unexpected, as
when judged by the eye the plants treated with the lower concentrations
of boric acid seemed better than the controls, and also exhibited a
particularly healthy green colouration.

Peas on the other hand are definitely stimulated with traces of boric
acid, concentrations of 1/100,000 and less causing an improvement in
growth, while under some experimental conditions even higher amounts
of boric acid were beneficial. All the stimulated plants showed the
characteristic dark green colour which seems to be associated with
the presence of minute traces of boron in the nutritive solution. An
interesting morphological feature was the strong development of small side
shoots from the base of the plants in the presence of medium amounts of
boric acid, from 1 part in 100,000 downwards. This gave rise to a certain
bushiness of growth, which was less evident as the concentration of the
stimulant decreased. The general outcome of the tests seems to be that
boric acid needs to be supplied in relatively great strength to be fatal
to pea plants, and that the toxic action gives place to a stimulative one
high up in the scale of concentration. As far as experiments have already
gone it seems as though the stimulation is not a progressive one, as the
effect of 1/100,000 boric acid is as good as that of 1/20,000,000, a flat
curve connecting the two. This, however, needs confirmation.

Yellow lupins also give some evidence of stimulation with concentrations
of about 1/50,000 boric acid, the improvement being far more strongly
marked in some sets of experiments than in others.


III. EFFECT OF BORON COMPOUNDS ON CERTAIN OF THE LOWER PLANTS.

Our knowledge of the action of boron on the lower plants is less definite
and complete than with regard to the higher plants. Morel (1892) found
that boric acid acts as a strong poison to the lower fungi and similar
organisms, their development being completely arrested by very weak
solutions of the acid. He suggested, on this account, that boric acid
might be used in the same way as copper to attack such diseases as mildew,
anthracnose, &c., which attack useful plants.

On the other hand Loew (1892) stated that such algae as _Spirogyra_
and _Vaucheria_ showed no harmful influence for many weeks when the
culture water contained as much as ·2% (= 1/500) boric acid. This may
be supplemented by a recent observation at Rothamsted, in which certain
unicellular green algae (unidentified), were found growing at the bottom
of a stoppered bottle containing a stock solution of 1/100 boric acid.

Agulhon (1910 a) dealt chiefly with yeasts and certain ferments, and found
that yeasts grown in culture solutions are not influenced favourably or
unfavourably by relatively large quantities of boric acid up to 1 gram
per litre, while all development is checked with 10 grams per litre. The
presence of boron affects the action of yeast on glucose and galactose.
Galactose alone is not attacked even after 40 days in the presence of ·66%
boric acid. When glucose is mixed with the galactose the latter is said
to be at first left untouched, but later it disappears very slowly.

Boric acid exercises an antiseptic action on lactic ferments, 5 gm.
per litre checking their action sufficiently to enable milk to remain
uncoagulated. Lactic acid is still produced even with as much boric acid
as 10 gm. per litre. The microbe is not actually killed by the boric acid,
but its development is so arrested that reproduction cannot take place.
The same phenomenon was observed with yeast. With moulds again, while no
stimulation could be obtained with small quantities of boric acid, yet
the toxic action does not begin to set in until 5 gm. boric acid per litre
are present.

Thus it appears that such lower organisms as yeast, lactic ferment and
_Aspergillus niger_ are remarkably indifferent to the action of boric
acid, as is shown by the fact that the toxic dose is remarkably high,
while stimulation effects cannot be observed even in the presence of the
smallest quantities yet tried.


_Conclusion._

Boric acid is less harmful to the growth of higher plants than are the
compounds of copper, zinc, and arsenic. Evidence exists that below a
certain limit of concentration boron exercises a favourable influence upon
plant growth, encouraging the formation of stronger roots and shoots. This
stimulation is more strongly marked with some species than with others,
peas responding more readily than barley to the action of boric acid.
Fungi are very indifferent to boron, whether it is present in large or
small quantities, and there is evidence to show that certain of the green
algae can also withstand large quantities of it.



CHAPTER VII

EFFECT OF MANGANESE COMPOUNDS


I. PRESENCE OF MANGANESE IN PLANTS

The presence of manganese as a constituent of plant tissues has been known
for many years, and in view of the close association between iron and
manganese it was natural that the early investigators should seek for the
latter element. De Saussure (1804) gives one of the earliest references to
manganese in plant ash, stating that it occurs in the seeds in less great
proportion than in the stems, and also that the leaves of trees contain
less in autumn than in spring. At first oxides of iron and manganese
were put together as “metallic oxides” and little or no attempt was made
to separate them so as to get an idea of their relative abundance. John
(1814) gives a number of rough analyses of plants and indicates the
presence of manganese in many plants, including _Solanum tuberosum_,
_Brassica oleracea viridis_ L., _Conium maculatum_, _Aesculus_ (in outer
bark), and _Arundo Sacchar_. No further references presented themselves
until 1847, as probably manganese was overlooked and always classed with
iron in any analyses made during that time. Kane (1847) found traces of
manganese in the ashes of some samples of flax, but none in others, and
examinations of the soils on which the plants were grown gave similar
results. Mayer and Brazier (1849) confirmed this result. Herapath (1849)
analysed the ashes of various culinary vegetables, finding manganese in
cauliflowers, swede turnips, beetroot, and in one variety of potato (Forty
fold).

Malaguti and Durocher (1858) tried to investigate the matter
quantitatively. The oxides of iron, manganese, and aluminium were all
classed together, and the mean percentage of the three varied from
·85%–5·06% according to the varieties of plants concerned, Cruciferae
possessing least and Leguminosae most. Different mean results with the
same plant were obtained from different soils.

Wolff (1871) made other quantitative analyses including _Trapa natans_
(·15% Mn₃O₄), _Acorus Calamus_ (1·52% Mn₃O₄), _Alnus incana_ (trace–·73%
Mn₃O₄), _Pyrus communis_ (2·15% Mn₃O₄). Many other plants were mentioned
by Wolff as containing manganese.

Campani (1876) found manganese in ash by a method in which it was detected
as phosphate of manganese, and he claimed to be the first to discover
manganese in wheat ash. Warden (1878) found traces of Mn₃O₄ in the ash of
opium from Behar.

Dunnington (1878) detected manganese in the ash of wheat, ·00144 gm. (as
Mn₃O₄?) in 300 grams of “Dark Lancaster” variety, equivalent to ·027% of
the pure ash. The ash was exhausted with nitric acid, and after separating
the iron the ammonium sulphide precipitate was found to contain manganese,
and gave by fusion with nitre and sodium phosphate a violet coloured mass.
Andreasch (1878) found slight traces of Mn₃O₄ in the flowers of _Dianthus
caryophyllus_, none occurring elsewhere, while in _Rosa remontana_ it
appeared in both leaves and flowers.

Maumené (1884) tested many food plants and concluded that some quantity
of manganese is frequently present in potato, rice, barley, carrot,
lentil, pea, beetroot, asparagus, chicory, most fruits, tea, and also in
some fodder plants, as lucerne, oats, and sainfoin. Ricciardi (1889),
Hattensaur (1891) also added to the list of plants proved to contain
manganese. Guerin (1897) studied the manganese content of woody tissues.
Sawdust was treated with distilled water containing 1% caustic potash,
expressed, and filtered after two or three days. A brown coloured liquid
was obtained, which when treated with a slight excess of hydrochloric acid
gave an abundant flocculent precipitate. This precipitate proved to be
soluble in pure water, so it was washed with slightly acidulated distilled
water, and after further purification was analysed. No trace of iron was
obtained, but about ·402% Mn was found. Guerin regarded the precipitate
as a “nucleinic” combination, which he supposed to occur generally in wood
and to contain the manganese present in the woody tissues of all plants.

Schlagdenhauffen and Reeb (1904) detected manganese in a petrol extract
of such cereals as barley, oats, and maize, and since inorganic salts
of manganese are not soluble in such liquids as ether or petrol they
concluded that the manganese must be present in the plant in organic
combination, thereby upholding Guerin’s view. Loew and Seiroku Honda
(1904) give a table of Mn₃O₄ in the ashes of certain trees. This is very
high in some cases, rising to 11·25% in the ash of beech leaves, 6·73% in
birch leaves, and 5·48% in chestnut fruits.

Gössl (1905) gives lists of the distribution of manganese in plants, both
Thallophytes and Phanerogams, indicating the presence of much or little of
the element. As a rule, he states, marsh and water plants gather up more
manganese than do land plants.

The Gymnosperms seem to be particularly rich in their manganese content.
Schröder (1878) tested for the element in firs and pines and found the
following amounts of Mn₃O₄.

    In 100 parts ash.    In 1000 parts dry matter.
       Fir    Pine             Fir     Pine
      33·18  13·46             2·76     ·77

He gave a table of detailed analyses showing the differing proportions of
manganese in the different parts of the fir.

Baker and Smith (1910) paid special attention to manganese in their
exhaustive work on the Pines of Australia. They state that “in the
anatomical investigations of the timber, bark, and leaves of the various
species, there was found to be present, in a more or less degree, a
naturally brownish-bronze coloured substance, which invariably stained
dark brown or almost black with haematoxylin.” This substance on careful
investigation proved to be a compound of manganese. The quantity present
varies with the species and also with the plant organs. The different
species of the genus Callitris show variable percentages of manganese
from a maximum of 0·230% in _C. gracilis_, to a minimum of 0·010% in _C.
robusta_. The percentage of manganese in Australian Coniferae other than
Callitris is given by the authors in the following table:

    Ash of timber of Agathis robusta                 0·145% Mn.
     „       „       Araucaria Cunninghamii          0·054%  „
     „       „       Araucaria Bidwilli              0·077%  „
     „       „       Actinostrobus pyramidalis       0·077%  „
     „       „       Podocarpus elata                0·002%  „
     „       „       Dacrydium Franklini             0·129%  „
     „       „       Athrotaxis selaginoides         0·019%  „
     „       „       Phyllocladus rhomboidalis       0·145%  „
    Air-dried black gum of Agathis robusta           0·0046% „
        „           „      Araucaria Cunninghamii    0·0038% „

Baker and Smith assume that manganese is essential to the production
of the most complete growth of Coniferae. The element is found in these
plants even when they grow on soils containing only traces of manganese
and it is suggested that possibly the excess or deficiency of manganese
in the soil helps to govern the location of certain of the Australian
Coniferae. The authors conclude that manganese may be essential to the
growth of these plants, and that its association with plant life may be
considered to date back to past geological time, as is indicated by plates
illustrating fossil woods.


II. EFFECT OF MANGANESE ON THE GROWTH OF HIGHER PLANTS.


1. _Toxic effect._


(_a_) _Toxic action of manganese compounds in the presence of soluble
nutrients._

Little work seems to have been done on the action of manganese compounds
in water cultures. Knop (1884) just indicated that manganese compounds had
no effect on maize, but gave no details. Japanese investigators touched
on the matter in the course of their extensive experiments with this
element. Asō (1902) found that the greater concentrations of manganese
sulphate exercised an injurious influence on barley. Even in solutions
with as little as ·002% manganese sulphate (= 1/50,000 MnSO₄) the roots
gradually turned brown, the lower leaves following suit. The brown colour
was concentrated at certain points of the leaves, and microscopical
examination showed that the membranes of the epidermal cells, and in some
cases the nuclei, were stained deeply brown. The greatest concentration
endured by barley without injury seemed to be about ·01 per 1000 =
1/100,000. The presence of iron in the food solutions seems to counteract
the effect of the manganese to some extent by delaying the yellowing of
the leaves. Wheat proved very similar to barley in its reactions, though
more iron is necessary to give good healthy growth. Asō states that
wheat is able to overcome the injurious action of manganese much more
readily than is barley. With peas the yellowing of the leaves was delayed,
probably on account of a sufficient supply of iron in the reserve stores
of the seeds.

Loew and Sawa (1902) found that ·25% = 1/400 MnSO₄ (anhydrous) kills pea
plants within five days and that the green colour is gradually affected
with more dilute solutions. Barley and soy beans were grown in nutritive
solutions with either iron sulphate or manganese sulphate or both (·01%
FeSO₄, ·02% MnSO₄, ·01% FeSO₄ + ·02% MnSO₄). At first the growth was
increased by the action of two salts together, but eventually the shoots
turned yellowish, and assimilation was depressed, so that decreased
nutrition led to relaxation in the speed of growth, indicating the toxic
action due to the manganese sulphate.

The Rothamsted experiments supported Asō’s work on the action of manganese
sulphate on barley, concentrations of the salt above 1/100,000 having a
retarding influence on the growth, the roots being coloured brown and
the leaves also showing discolouration. At an early stage in growth
the lower leaves of the plants receiving the most poison began to be
flecked with brown spots, which were at first attributed to an attack
of rust. Suspicion was soon aroused, however, and a closer microscopic
investigation showed that no disease was present, but that the cells
in the affected spots were dead and brown, though they retained their
shape. The dead cells at first occurred in small patches, which spread
and coalesced until ultimately the whole leaf was involved. Some of the
affected leaves were detached and fused with a mixture of sodium carbonate
and potassium nitrate. On dissolving up the resulting mass with water
a green colouration was obtained, indicating the presence of manganese
in the leaves. This shows that the manganese is taken up by the roots,
transferred to the leaves and then deposited in them, the lower leaves
being the first affected.

The presence of manganese in the nutritive solution retarded the ripening
of the grain to some extent, as when the grains from the control plants
were hard and ripe, those from plants treated with 1/10,000 MnSO₄ were
green, those with 1/100,000 were a mixture of ripe, half-ripe, and green
grains, while plants which had received 1/1,000,000 MnSO₄ possessed ripe
grains.

Peas give similar results to barley so far as the vegetative growth is
concerned, the same retardation with the higher concentrations being
observed, while the brown discoloured patches in the lower leaves are
much in evidence. All traces of manganese in the leaves disappear when the
concentration falls to 1/250,000. On the whole peas are more sensitive to
manganese poisoning than is barley, and the higher strengths of manganese
prove more deleterious to them.


(_b_) _Toxic action of manganese compounds in sand cultures._

Little work has been done on this aspect of the problem. Prince de Salm
Horstmar (1851) grew oats in sand with various combinations of nitrogenous
substances and inorganic mineral salts. He stated that until the time of
fruit formation manganese does not seem to be essential to the oat unless
iron is in excess in the substratum.


(_c_) _Toxic action of manganese compounds in soil cultures._

A large body of work has been done with manganese in soil cultures, but
the toxic effect is hardly indicated, possibly because it is less manifest
under soil conditions, possibly because the observation of the toxic
action has been almost completely overshadowed by the interest in the
stimulation observed under the same circumstances. Namba stated that ·5
gm. MnSO₄ added to 8 kgm. Japanese soil exerted a depressing influence
on the growth of various plants. The Hills Experiments (1903) indicated
some toxic effect. Various soluble and insoluble salts of manganese were
added to soil in pots at the rate of 2 cwt. per acre, wheat being sown.
On the whole the plants from untreated pots were as good as any with
manganese except those that received manganese nitrate or phosphate.
Manganese iodide distinctly retarded growth. The plants that grew did well
eventually, but development of the ear was greatly or entirely retarded.
If the seeds were soaked in the iodide, a concentration of 10% was found
to be harmful, 5% allowing normal growth. Similar experiments with barley
showed that plants treated with manganese carbonate and sulphate were both
inferior to the untreated plants; with iodide less plants were obtained
and their development was abnormal. Soaking the seeds in the iodide, even
in 10% solution, did not do damage as it did with wheat. The oxides were
apparently innocuous, but gave no increase either in corn or straw.

Kelley (1909) found that on soils in Hawaii in which excessive quantities
of manganese are present (5·61% Mn₃O₄) pineapples do not flourish,
but turn yellow and produce poor fruits, and also that if rather less
manganese is present (1·36% Mn₃O₄) the pineapples show the toxic effect
by yellowing during the winter months, but they recover completely during
the hot summer months. Kelley also observed that the deleterious effect is
hardly noticeable during the first twelve months of growth, and that after
a time a darkening occurs in the colour of the soil, which he attributes
to some change in the constitution of the manganese compounds.

Some interesting observations were made by Guthrie and Cohen (1910) on
certain Australian soils. A bowling green that was initially covered with
a healthy mat of couch grass developed a number of small patches after
about three years growth, on which the grass died off. No reason was
apparent for this phenomenon, as the cultural conditions were uniform and
to all appearances the soil over the whole area was similar in character.
Analyses of soil samples from the dead patches and from the neighbouring
healthy parts of the green showed that the chemical composition in both
cases was practically the same, except that while no manganese occurred in
the soil from the unharmed part, as much as ·254% Mn₂O₃ was found in that
from the dead patches. As no other differences were found it was argued
that the manganese, present in such large quantities, acted as a toxic
agent and killed off the grass. Other instances of manganese poisoning
in which wheat and barley were affected are quoted by these authors, the
analytical results indicating that possibly barley is able to withstand
without injury a greater quantity of manganese compounds in the soil than
is wheat.


2. _Effect of manganese compounds on germination._

Nazari (1910) rolled wheat grains in a paste of manganese dioxide,
iron sesquioxide (both with and without organic matter), and in what he
terms “artificial oxydases.” The seeds rolled in the last-named showed
the greatest energy in germination, while those with manganese gave
an appreciable acceleration. The presence of organic matter decreased
the action of manganese. The plants from the manganese seedlings gave
an increased yield in both straw and grain, while those treated with
sesquioxide of iron showed no gain over the check plants.

The Hills Experiments yielded some information as to the differing effects
of various compounds of manganese on germination. With wheat plants in
pot experiments manganese oxide (MnO₂) distinctly retarded germination
when applied at the rate of 2 cwt. per acre. With barley MnO₂, manganese
carbonate and sulphate all retarded germination, while with the iodide
50% of the seeds were entirely prevented from germinating.


3. _Does manganese stimulate higher plants?_

With manganese the evidence in favour of stimulation is more weighty than
with such poisons as copper, zinc and arsenic, and the literature on the
subject is correspondingly plentiful.


(_a_) _Stimulation in water cultures._

While Asō (1902) asserted that plants can develope normally in water
cultures in the absence of any trace of manganese, he further stated that
manganese compounds exercise both an injurious and a stimulant action on
plants. With increasing dilution of the compound the deleterious action
diminishes, while the stimulant action increases, and a dilution can be
reached in which only the favourable influence of the manganese becomes
obvious. The addition of ·002% manganese sulphate (= 1/50,000) to culture
solutions stimulated radish, barley, wheat and peas. The intensity of the
colour reaction of the oxidising enzyme of the manganese plants was found
to exceed that of the control plants, at least with regard to those leaves
on the manganese plants which had turned a yellowish colour.

Loew and Sawa (1902) obtained an initial increase of growth with
barley and soy beans in nutritive solutions + ·01% ferrous sulphate +
·02% manganese sulphate, but this initial stimulation was followed by
depression. These authors support Asō’s contention that manganese exerts
both an injurious and a stimulative action upon plants, and that the
promoting effect is still observable with manganese compounds in high
dilution, while the injurious effects disappear under this condition.

The Rothamsted experiments with barley show a decided stimulation with
1/100,000 MnSO₄ and less. Care was taken to utilise sublimed FeCl₃
to avoid error due to the introduction of manganese into the control
solution through the agency of this salt. It is interesting to notice that
concentrations that are weak enough to stimulate the vegetative growth
still show a depressing action in that they retard the ripening of the
grain, a fact which supports Loew and Sawa’s contention that manganese
exerts both a toxic and a stimulative action at one and the same time, the
balance showing itself according to the concentration (Fig. 17). In the
later experiments the plants were not allowed to form ears, but similar
results were obtained, except that when dealing with the vegetative growth
only, a definite stimulus was obtained with a higher concentration than in
those experiments in which the plants were allowed to form seed. This may
or may not be significant, as it is possible that seasonal variation and
individuality of the plants may have played some part. Barley seems to be
most extraordinarily sensitive to the action of manganese, as even 1 part
in 100,000,000 was found to exercise a beneficial action (Fig. 18). With
peas the evidence of stimulus is less well marked. No sign of stimulation
is obtained until a greater dilution is reached than is necessary with
barley. Even so the resulting curves are not sufficiently conclusive to
warrant the definite statement that manganese does act as a stimulant to
peas when present in very small quantities (Fig. 19).

[Illustration: Fig. 17. Curve showing the mean value of the dry weights
of ten series of barley plants grown in the presence of manganese sulphate
and nutrient salts. (Feb. 5th–March 29th, 1909.)]

[Illustration: Fig. 18. Photograph showing the action of manganese
sulphate on barley plants grown in the presence of nutrient salts. (Feb.
5th–March 29th, 1909.)

    1.  Control.
    2.  1/10,000      manganese sulphate.
    3.  1/100,000         „        „
    4.  1/1,000,000       „        „
    5.  1/10,000,000      „        „
    6.  1/100,000,000     „        „     ]

[Illustration: Fig. 19. Photograph showing the action of manganese
sulphate on pea plants in the presence of nutrient salts. (Oct. 2nd–Dec.
20th, 1912.)

    1.  Control.
    2.  1/5,000     manganese sulphate.
    3.  1/10,000        „         „
    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     „         „    ]


(_b_) _Stimulation in soil cultures._

Roxas carried out pot experiments with rice in soil to which was added
varying proportions of manganese sulphate, with and without the addition
of nutrient salts of ammonium, potassium, and calcium. The criterion
of stimulation was the length of the growing leaves as measured daily,
a strength of M/1000 MnSO₄ (M = molecular weight) giving a favourable
result.

In the Hills Experiments (1903) an increase of produce was obtained
with wheat by manuring with manganese phosphate, chloride, sulphate, or
oxide (MnO₂), while an increase of straw was gained with nitrate, though
this compound decreased the yield of corn. With barley no evidence of
stimulation is set forth for any compound, except that the root growth
was improved by the addition of manganese iodide, in spite of the general
unfavourable action this substance exerted upon germination and growth.

Bertrand (1905) whose work will later be considered in detail,
experimented on arable land, adding quantities of manganese sulphate (?)
equivalent to about 1·6 gm. Mn to each square metre, growing oats from
February to May. Increase of weight was found in the plants growing on
the manganese plots, the differences in favour of manganese being

    For total crops  22·5%.
     „ grain only    17·4%.
     „ straw only    26·0%.

A certain alteration in the quality of the grain was also noted from
the manganese plots, the weight per hectolitre exceeding that from the
untreated plot, the % of water and of total nitrogen being somewhat lower
than that from the untreated, while the ash and the quantity of manganese
present was the same in the grain from both plots. Bertrand suggested
that these results might indicate a new line to follow in the study of
the causes of the soil fertility.

Strampelli (1907) tested the effect of manganese dioxide, carbonate,
and sulphate, and of a manganiferous mineral from the Argentine upon
wheat, and found that while all four substances exercised a favourable
influence on the vegetation, the best result was obtained with the
sulphate. When however other manures were used in conjunction with the
manganese compounds the balance of improvement shifted. With nitrogen,
applied as nitrate of soda, manganese dioxide proved the most beneficial,
with farmyard manure the manganiferous mineral[14], and with blood the
carbonate. It was also found that a manganese compost did not increase
production when phosphatic manure was applied as basic slag.

    [14] As no analysis of the mineral is given it is obviously
    impossible to say to what constituent the increase is due in this
    case.

Feilitzen (1907) indicated that the nature of the soil plays its part in
determining whether manganese acts as a stimulant or not. His experiments
were made in the field on poor moor soil, which carried a little Sphagnum
turf and Eriophorum, and which was poor in food salts. The soil was
prepared and manured and then the plots were watered with a solution of ·1
gm. MnSO₄.4H₂O per litre at the rate of 10 kgm. sulphate per hectare, six
control plots being left untreated. Oats were sown and the soil rolled.
During growth no difference was noted between the various plots, and after
harvesting the weights of the different crops showed that the manganese
had not caused increase of crop in either grain or straw on this poor moor
soil.

The great bulk of the work on this problem has been carried out by various
Japanese investigators, whose work extends over several years. Loew and
Sawa (1902) found that small quantities of manganese sulphate in soil
cultures stimulated the growth of rice, pea, and cabbage. They suggested
that soils of great natural fertility contain manganese in an easily
absorbed condition, and that this forms one of the characteristics of such
soils.

Nagaoka (1903) dealt with plots in the rice fields which had not been
manured for the three previous years and which were then treated with
manure at the rate of 100 kgm. ammonium sulphate, 100 kgm. potassium
carbonate and 100 kgm. double superphosphate per hectare. Twelve series
were worked in triplicate and received manganese sulphate in varying
quantities, equivalent to 0–55 kgm. Mn₂O₃ per hectare, one set of three
being left untreated. The cultivation was normal and the application of
manganese was found to influence the yield of rice. 25 kgm. per hectare
gave the best result and increased the harvest of grains by one-third;
higher doses of Mn₂O₃ gave no better crop. The percentage of grain
relative to the straw was also increased. The increase in both respects
was evident all through the series from 10 to 55 kgm. Mn₂O₃ per hectare.
The conclusion was reached that the application of this salt to soils poor
in manganese would be a commercial advantage.

The next year (1904) the experiments were extended to observe the after
effects of the initial doses of manganese sulphate. The harvest of grain
was greatest in those plots that had received 30 kgm. Mn₂O₃ per hectare,
while it was approached very closely by that from the plot with 25 kgm.
Mn₂O₃, which had proved the best in the first year’s experiments. The
maximum increase of yield over the unmanured plots in the first year was
37%, while in the second year it dropped to 16·9%.

Asō (1904) also obtained an increase of one-third in produce of grain when
25 kgm. Mn₃O₄ per hectare (as manganous chloride) was applied to rice.
The development of the plants was improved and the treated plants flowered
about four days before the untreated ones.

Loew and Honda (1904) grew _Cryptomeria japonica_ in beds, treating the
soil with various manures and with iron or manganese sulphate. The latter
favoured increase in height, and within 1½ years the cubic content of the
trees had increased to double.

Fukutome (1904) grew flax in pot cultures, each pot containing 8 kgm.
soil, to which was added ·4 gm. MnCl₂.4H₂O and ·4 gm. FeSO₄.7H₂O. This
mixture had a marked effect on the growth of the flax, but the individual
salts in doses of ·4 gm. per 8 kgm. soil had but little effect.

Namba (1908) applied manganese salts to onion plants in pots with a
considerable measure of success. Pots containing 8 kgm. loamy soil were
manured and received:

    (1)  no manganese,
    (2)  ·1 gm. MnSO₄.4H₂O,
    (3)  ·2 gm. MnSO₄.4H₂O,

the manganese sulphate being applied in high dilution as top dressing. The
bulbs and leaves were considerably stimulated by small doses of manganese
sulphate, the best results being obtained from (2), which represents a
manuring of 22 kgm. MnSO₄ per hectare. An increase of the dose lessens
the beneficial effect, as the toxic action begins to come into play. The
actual figures obtained may prove of interest.

    Wt. leaves  Wt. bulbs    Total weight         Bulbs & roots
                & roots   Absolute   Relative     _____________
                                                     leaves
         gm.       gm.        gm.        gm.
    1.  29·5       8·5       38·0      100·0           ·28
    2.  38·0      22·5       60·5      159·2           ·59
    3.  35·5      16·5       51·0      134·2           ·46

Uchiyama (1907) carried on a variety of experiments with manganese
sulphate on several plants on different soils, both in the field and in
pots, and found that the compound exercised a favourable action in most
cases when applied in appropriate quantities. In summarising his results
he stated that both manganese and iron stimulate the development of
plants, different plants varying in their susceptibility to the action.
Sometimes a joint application of the two salts is the most beneficial,
sometimes an individual application is the better, in which case manganese
sulphate is generally better than ferric sulphate in its action. The
stimulating action of manganese varies greatly with the character of the
soil, and the mode of application also affects results. As a general rule
the manganese acts best when applied as a top dressing rather than when
added together with the manure. Further the stimulating action differs
greatly with the nature and reaction of the manurial mixture. Uchiyama
concludes that 20–50 kgm. per hectare of crystallised manganese sulphate
is a good general amount to apply.

Takeuchi (1909) corroborates the statements of the various writers that
plants differ in their response to the manganese manuring. Pot cultures,
in each of which 8 kgm. soil were similarly manured, received ·2 gm.
MnSO₄.4H₂O applied as a solution of 1/100 strength, the controls receiving
the same amount of water. The manganese increased the green weight of
spinach by 41%, while the dry weight of barley, peas and flax rose 5·3%,
19·4%, and 13·9% respectively above that of the untreated. The control
plants of flax were behind the manganese plants in growth and flowering,
while barley was the least stimulated of all the test-plants. Other
observations seemed to show that Leguminosae and Cruciferae are more
susceptible to manganese stimulation than are the Gramineae.


III. EFFECT OF MANGANESE COMPOUNDS ON CERTAIN OF THE LOWER PLANTS.

The information on this point is exceedingly meagre, possibly because of
the diversion of general attention to the higher plants in view of the
commercial interests involved.

Richards (1897) carried out experiments with various nutritive media
with the addition of certain metallic salts, including those of zinc,
iron, aluminium and manganese. The fungi tested were _Aspergillus niger_,
_Penicillium glaucum_ and _Botrytis cinerea_. His general conclusion was
that fungi may be stimulated, though it must not be concluded without
further investigation that all fungi react in the same degree to the same
reagent, but this conclusion is traversed by Loew and Sawa (1902). These
writers state that fungi are _not_ stimulated by manganese, and take this
as a proof that the improvement in the growth of phanerogams, induced by
manganese compounds, is not due to direct stimulation of the protoplasmic
activity, but to some other more obscure cause.


IV. PHYSIOLOGICAL CONSIDERATIONS OF MANGANESE STIMULATION.

The physiological cause of the stimulation exerted by manganese compounds
has raised much controversy. Loew and Sawa suggested that the action of
the sun’s rays upon a normal plant puts a certain check on growth, arising
out of the action of certain noxious compounds which they supposed to be
produced in the cells under the influence of light. The stimulation of the
manganese compounds may be due to a supposed increase in the oxidising
powers of the oxidising enzymes, so that destruction of the checking
compounds can be accomplished as quickly as they are formed, so enabling
growth to continue more rapidly.

Asō (1902) had previously stated that colorimetric tests for oxidising
enzymes indicate that the yellowish leaves from plants treated with
manganese compounds give reactions of higher intensity than the green
leaves from control plants, the difference between the reactions being
specially marked in barley, and less so in radish.

Bertrand has devoted much time to the consideration of this and allied
problems. In 1897 (a, b, c) he proceeded to investigate the essential
nature of manganese in the economy of the plant, his experiments showing
its constant presence in a ferment (laccase) obtained from plants. He
also extracted from lucerne a substance very poor in manganese, which
was somewhat inactive, but which regained or increased its activity on
the addition of manganese. Bertrand stated that manganese was apparently
not to be replaced by another metal, not even by iron, and that the small
quantity of it occurring was no reason for regarding it as a _secondary_
element in the composition of plants. The view was also put forward that
in the presence of certain organic substances, such as hydroquinone,
pyrogallol or similar bodies, manganese is capable of fixing free oxygen
from the air, the volume of oxygen absorbed varying according to the
compound of manganese used. Bertrand was led to conceive the oxydases
as special combinations of manganese in which the acid radicle, probably
protein in nature and variable according to the ferment considered, would
have just the necessary affinity to maintain the metal in solution, i.e.
the form the most suitable for the part it has to play. The manganese
would then be, according to his view, the true active element of oxydase,
which functions as the “activator”; the albuminous matter, on the other
hand, gives to the ferment those special characters, which show themselves
in their behaviour with regard to reagents and physical agents. From this
point of view manganese could no longer be considered as a non-essential
element, but as a substance of vital necessity to the functions of
plant-life. The name “complementary” manure was suggested for compounds of
such elements as manganese, which exert a physiological action and which
were proposed for use as manures. Later (1905) Bertrand considered that
he had still further proved the indispensable nature of manganese. The
absence or insufficiency of one essential element arrests or diminishes
growth. This applies not only to those substances which are present in the
greatest abundance, such as C, P, N, &c., but also to those elements like
manganese, boron, and iodine, which only occur in traces. These elements
are usually specialised in function, and for them the name “catalytic”
elements was suggested, in view of the work they are held to do. As late
as 1910 the rôle of manganese in the functioning of the oxidising enzymes
was again insisted on. It was concluded that manganese intervenes as a
catalytic agent in the material changes of which plants are the seat,
and that it participates in an indirect manner in the building up of the
tissues and in the production of organic matter.


_Conclusion._

Manganese exerts a toxic influence upon the higher plants, if it is
presented in high concentration, but, in the absence of great excess
of the manganese compounds, the poisoning effect is overshadowed by a
definite stimulation. As is the case with boron, manganese stimulates some
species more than others, the action on barley being more evident than
that on peas. It seems probable that manganese may prove to be an element
essential to the economy of plant life, even though the quantity usually
found in plants is very small.



CHAPTER VIII

CONCLUSIONS


In the foregoing chapters a very limited number of plant poisons have been
considered, yet there is sufficient evidence to show that even these few
differ considerably in their action upon plant-life. This action is most
variable, and it is impossible to foretell the effect of any substance
upon vegetative growth without experiments. The degree of toxicity of the
different poisons is not the same, and also one and the same poison varies
in the intensity and nature of its action on different species of plants.
While certain compounds of copper, zinc and arsenic are exceedingly
poisonous, compounds of manganese and boron are far less deleterious,
so that a plant can withstand the presence of far more of the latter
substances than of the former. Again, the tested compounds of copper, zinc
and arsenic do not seem to stimulate growth, even when they are applied
in the smallest quantities, whereas very dilute solutions of manganese
and boron compounds decidedly increase growth. But, differentiation occurs
even in this stimulative action, for while manganese is the more effective
in stimulating barley, boric acid is far more potent for peas, the shoots
being particularly improved.

A consideration of the experimental work that has been done on this
subject of poisoning and stimulation leads one to the inevitable
conclusion that it is not true to maintain the hypothesis that _all_
inorganic plant poisons act as stimulants when they are present in very
small quantities, for while some poisons do increase plant growth under
such conditions, others fail to do so in any circumstances. It is probable
that what has been found true with the few substances tested would prove
to be similarly true over a much wider range of poisons, and at any rate
the hypothesis must be dismissed in its universal application. A more
accurate statement would be that some inorganic poisons act as stimulants
when present in small amounts, the stimulating concentrations varying both
with the poisons used and the plants on which they act.

It is quite possible for a stimulation in one respect to be correlated
with a retardation in another. In the Rothamsted experiments on the action
of manganese sulphate on barley the weaker concentrations of the salt
improved the vegetative growth, as was shown by the increase in the dry
weights, but with the same strengths of the poison the ripening of the
grain was retarded, so that, while certain of the physiological functions
were expedited, others were hindered by the action of the poison.

Thus it is evident that it is exceedingly difficult sharply to
characterise either toxic or stimulant action. In neither case is the
reaction simple--many factors may come into play and many processes are
concerned, while the effect of a so-called poison may vary in respect of
each of the functions and processes concerned. If the poison is presented
in great strength the toxic action is dominant, and probably affects
many functions in the same sense, so that the action is, so to speak,
cumulative. Lower down in the scale of concentration differentiation of
action may set in, and while some processes may still be hindered, others
may be stimulated. If the two actions balance one another an apparent
indifference may be manifested, so that it seems that such strengths of
the poison have no effect on growth, either harmful or beneficial. At
still lower concentrations, with certain plants and certain poisons, the
stimulative action overpowers the toxic effect, so that in some respect
or other improvement occurs in growth.

It is quite conceivable, however, that some poisons are truly indifferent
in weak concentrations, as no stimulation makes itself evident under any
circumstances. In these cases one is inclined to suspect that the action
is somewhat more simple, in that the toxic effects gradually diminish
until no poisonous action is manifest at very weak concentrations, and as
no stimulation is present to bring the growth above the normal with these
very weak concentrations the plant is similar to those grown without any
addition of the poison.

The _modus operandi_ of these stimulative agents is not yet fully
understood. Perhaps at the present time two main theories hold the field:
(1) that they act as catalytic agents, being valueless on their own
account, but valuable in that they aid in the procuring of essential food
substances; (2) that the stimulants themselves are of integral value for
nutrition. The French school, with Bertrand at the head, hold strongly
to the catalytic theory, maintaining that manganese and boron compounds
are able to increase growth if they are present in small quantities, as
they act as “carriers” whereby the various functions of the plant are
expedited by the increased facility with which the essential nutritive
elements are supplied. The manganese in laccase, for instance, is held
to be an oxygen carrier, whereby the oxygen is first absorbed and then
released for the benefit of the plant, the manganese being regarded as
essential for the functioning of the enzyme. But, if these elements are
_essential_, this theory seems to stop short of the truth. If certain
functions are dependent for their very occurrence upon the presence
of even minute traces of any element, then surely that element is as
essentially a nutrient element, as vital to the well-being of the plant as
is such an element as carbon or nitrogen or phosphorus, even though the
latter occurs in far greater quantity. It is necessary that one should
free one’s mind from the idea that the quantity of an element present in
a plant is an index of its value to the plant. Naturally enough, in the
early days of plant physiology, the most abundant elements first engaged
the attention of investigators, and they were divided into essential
and non-essential, ten elements being classed in the former category.
More recent work is beginning to show that other elements are constantly
present in plants, but in such small quantities that the older and cruder
methods of analysis failed to reveal them, so that until lately they have
been completely overlooked in work on plant nutrition. Even yet we do
not know which of these other elements are essential and which are merely
accidental. While we do know that the ten essential elements (C, H, O, N,
S, P, K, Mg, Fe, Ca) are necessary for the well-being of all plants, it
is conceivable that these other substances which only occur in very small
quantities may be more individual in their action, and that while a trace
of a certain element may be absolutely essential to one plant, that same
element may be quite indifferent for another species. If one takes a broad
outlook, the two theories seem to be in reality only parts of one, the
“nutrition” theory carrying matters a little farther than the “catalytic”
idea, broadening its scope and extending its application.

It seems probable that all the experimental work that has been
discussed will prove to be simply preliminary to a far greater practical
application of the principle of stimulation or increased growth. While
the physiologists have been feeling their way towards the conclusions
put forth on this subject, the agriculturists have been discovering and
extending the application of artificial manures, until at the present
time such manuring is coming into its own and is receiving more of the
widespread attention that it deserves. The possibility now exists that
in some respects the two lines of work are converging and that the
more purely scientific line will have a big contribution to make to the
strictly practical line. Artificial manuring aims at improvement of the
soil and crop by the addition of food substances that are needed in a
particular soil, a result that used to be obtainable only by the use of
the bulky farmyard manure, seaweed, &c. Apart from any other aspect of
the matter the artificials, when intelligently used, are far more easy
to handle and to regulate in supply, and they yield excellent results,
especially in conjunction with a certain proportion of organic manures.
The further prospect now opened up is the possibility of utilising some
of these stimulating compounds as artificial manures. As only small
traces are beneficial, larger amounts being poisonous, it is obvious
that only small quantities would be needed, and, as the compounds are not
usually very expensive, a considerable increase of crop for a relatively
small outlay might be anticipated if no complicating factors intervened.
Very much work will be required in the field to test the value of these
substances, as their action may be influenced by the nature of the soil,
climatic conditions, general conditions of manuring, and the crops grown.
Some tests have already been made, especially in Japan, with boron and
manganese, and these indicate a promising field for investigation.

Above all, it is most important to realise that one is approaching an
entirely unexplored field, and that it is inevitable that the results of
the initial experiments will be contradictory, at least in appearance,
so that it is necessary to keep an open mind on the subject, being ready
to modify one’s ideas as circumstances require, as improved experimental
methods lead on to more accurate results.



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INDEX OF PLANT-NAMES


_The symbols after the plant-names represent the elements referred to
on the pages indicated._

    Acorus Calamus
        Mn, 79

    Actinostrobus pyramidalis
        Mn, 80

    Aesculus
        Mn, 78

    Agathis robusta
        Mn, 80

    Ageratum
        Cu, 24;
        Zn, 42

    Alder
        As, 53

    Algae
        As, 62, 64;
        B, 66, 77

    Allium
        (_see_ Onion)
        Zn, 47

    Alnus incana
        Mn, 79

    Aloe
        Cu, 26

    Amomum sp. (Paradieskörner)
        Cu, 15

    Ampelopsis
        Cu, 32;
        Zn, 47

    Anaboena
        Cu, 35

    Angiosperms
        B, 66

    Anthracnose
        B, 76

    Apple
        B, 65, 66

    Apricot
        Cu, 16

    Araucaria Bidwilli
        Mn, 80
      Cunninghamii
        Mn, 80

    Armeria sp.
        Zn, 38
      vulgaris
        Zn, 36, 37

    Arundo Sacchar
        Mn, 78

    Asparagus
        Mn, 79

    Aspergillus
        Cu, 33, 34

    Aspergillus niger (= Sterigmatocystis nigra)
        Zn, 48, 49, 50;
        As, 63;
        B, 76;
        Mn, 90

    Athrotaxis selaginoides
        Mn, 80


    Barley, 11, 13;
        Sr, 5;
        Cu, 16, 17, 20, 29;
        Zn, 37, 39, 40, 44, 46;
        As, 52, 54, 55, 57, 60, 61;
        B, 66, 69, 75;
        Mn, 79, 81, 82, 83, 84, 85, 86, 89, 90, 92, 93, 94

    Beans
        Cu, 16, 17, 26;
        Zn, 47

    Beech
        Mn, 79

    Beetroot (Beta vulgaris)
        Cu, 16, 26;
        Zn, 37, 39;
        Mn, 78, 79

    Beet, sugar
        B, 65

    Betula alba
        B, 66

    Birch
        Mn, 79

    Botrytis cinerea
        Mn, 90

    Brassica oleracea
        Mn, 78

    Buckwheat, 11;
        Cu, 16, 17, 29, 30;
        As, 53
        (_see_ Polygonum Fagopyrum)


    Cabbage
        Sr, 5;
        As, 51, 52;
        Mn, 87

    Cacao
        Cu, 16

    Callitris gracilis
        Mn, 80
      robusta
        Mn, 80

    Cannabis
        B, 72
      sativa
        B, 66

    Capsella Bursa-pastoris
        Zn, 37

    Cardamomum minus
        Cu, 15

    Carrot
        Mn, 79

    Cauliflower
        Mn, 78

    Cherry
        B, 65

    Chestnut
        Ca, 71;
        Mn, 79

    Chickpea
        Cu, 16;
        B, 66

    Chicory
        Mn, 79

    Chrysanthemum
        B, 66

    Clover
        Zn, 42, 44

    Colea
        Cu, 24;
        Zn, 42;
        As, 58

    Collinsia
        B, 72

    Coniferae
        Zn, 38

    Conium maculatum
        Mn, 78

    Colza
        B, 74

    Couch grass
        Mn, 83

    Cow pea
        Cu, 18

    Cruciferae
        Mn, 78, 89

    Cryptomeria japonica
        Mn, 88

    Curcuma longa
        Cu, 15

    Currant
        Cu, 31


    Dacrydium Franklini
        Mn, 80

    Dianthus caryophyllus
        Mn, 79


    Elodea canadensis
        Cu, 32;
        Zn, 48


    Fagopyrum esculentum
        Cu, 29

    Ferns
        B, 66

    Fig
        Zn, 42;
        B, 65, 66

    Fir
        Mn, 80

    Flax
        Mn, 78, 88, 89

    Fungi
        Cu, 33;
        Zn, 44, 50;
        As, 64;
        B, 66, 77


    Geranium
        Cu, 24;
        Zn, 42;
        As, 58

    Gramineae
        B, 72;
        Mn, 89

    Grasses
        Zn, 39, 40, 42

    Gymnosperms
        B, 66;
        Mn, 80


    Haricot
        B, 71, 72
      green
        As, 52
      white
        Zn, 37

    Hemp
        Sr, 5

    Hop
        B, 66

    Hordeum distichum
        As, 54
      vulgare
        Zn, 39, 47
        (_see_ Barley)


    Iberis
        B, 72


    Laminaria saccharina
        B, 66

    Leguminosae
        B, 72;
        Mn, 78, 89

    Lentil
        Mn, 79

    Lichen
        As, 59

    Linseed
        Cu, 16, 17

    Linum
        B, 72

    Love-apple
        B, 66

    Lucerne
        Mn, 79, 91

    Lupin
        Cu, 17, 19;
        As, 59
      white
        As, 61;
        B, 67, 70
        (_see_ Lupinus albus)
      yellow
        B, 70, 75

    Lupinus albus
        Cu, 19, 20, 22;
        Zn, 45;
        B, 68
        (_see_ White Lupin)


    Maasa picta
        B, 65

    Maize
        Cu, 16, 17, 19, 24, 26, 27;
        Zn, 37, 44;
        As, 53, 54, 60;
        B, 67, 68, 71, 72, 74;
        Mn, 79, 81

    Menyanthes trifoliata
        Cu, 35

    Mildew
        B, 76

    Molinia cærulea
        Cu, 16;
        Zn, 37

    Mould
        B, 76

    Mucor mucedo
        As, 59, 63

    Mushroom
        B, 66

    Mustard
        Cu, 17


    Nasturtium
        Cu, 17

    Nuphar lutea
        Cu, 35


    Oak
        Cu, 16;
        Zn, 42

    Oat
        Cu, 16, 17, 19;
        As, 53;
        B, 74;
        Mn, 79, 82, 86, 87

    Onion
        B, 66;
        Mn, 88

    Onobrychis sativa
        Zn, 39

    Opium
        Mn, 79

    Oscillatoria
        Cu, 35


    Panicum italicum
        Cu, 26;
        As, 58

    Pansy
        Cu, 24;
        Zn, 42;
        As, 58

    Paprika
        Cu, 16, 17

    Paradieskörner (Amomum sp.)
        Cu, 15

    Pea
        (_see_ Pisum sativum)
      sweet
        Cu, 17

    Pear
        Cu, 16;
        B, 65

    Penicillium
        Cu, 33, 34
      brevicaule
        As, 63
      cupricum
        Cu, 34
      glaucum
        Cu, 23;
        As, 59, 63;
        Mn, 90

    Phaseolus vulgaris
        Cu, 17;
        As, 59

    Phormidium Valderianum
        As, 62

    Phyllocladus rhomboidalis
        Mn, 80

    Pine
        Mn, 80

    Pineapple
        Mn, 83

    Pisum arvense
        Cu, 29
      sativum
        Cu, 17, 18, 26, 27, 29;
        Zn, 41, 47;
        As, 58
      (“Pea”), 3, 11, 13, 93;
        Cu, 17, 26, 29, 30;
        Zn, 40, 46;
        As, 51, 54, 55, 56, 58, 61;
        B, 67, 73, 74, 75, 93;
        Mn, 79, 81, 82, 85, 86, 87, 89, 92

    Plantago lanceolata
        Zn, 37

    Podocarpus elata
        Mn, 80

    Polygonum amphibium
        Cu, 35
      aviculare
        Zn, 37, 38
      Fagopyrum
        Cu, 26, 39;
        Zn, 41;
        As, 54, 58
        (_see_ Buckwheat)
      Persecaria
        Cu, 5;
        As, 54

    Poplar
        Cu, 15

    Potato
        Cu, 16, 26, 27, 30;
        As, 52;
        Mn, 78, 79

    Protococcus infusionum
        As, 62
      sp.
        As, 63

    Pyrus communis
        Mn, 79


    Radish
        Sr, 5;
        B, 74;
        Mn, 84, 90

    Raphanus
        B, 72
      sativus
        Zn, 39

    Raspberry
        As, 65

    Rice
        Zn, 47;
        B, 66, 73;
        Mn, 79, 86, 87, 88

    Rosa remontana
        Mn, 79

    Rubus
        B, 66

    Rye
        Cu, 16;
        As, 60, 61


    Sainfoin
        Mn, 79

    Secale cereale
        Cu, 26;
        Zn, 41;
        As, 58

    Silene inflata
        Zn, 36, 37

    Solanum tuberosum
        Mn, 78

    Soy beans
        Cu, 17, 19;
        B, 67;
        Mn, 81, 85

    Spinach, 41;
        B, 73;
        Mn, 89

    Spergula arvensis
        Zn, 39

    Spirogyra
        Cu, 35;
        As, 62;
        B, 76

    Stichococcus bacillaris
        As, 62

    Sterigmatocystis nigra
        Zn, 48, 49
        (_see_ Aspergillus niger)

    Sugar cane
        B, 65


    Taraxacum officinale
        Zn, 37

    Tea
        Mn, 79

    Thlaspi alpestre
        Zn, 36
      sp.
        Zn, 38

    Tobacco
        B, 66

    Trapa natans
        Mn, 79

    Trifolium pratense
        Zn, 39

    Triticum vulgare
        Cu, 17;
        B, 67
        (_see_ Wheat)

    Tropeolum Lobbianum
        Cu, 17, 18

    Turnip
        As, 51, 52;
        B, 74
      swede
        Mn, 78

    Tussilago Farfara
        Zn, 37, 38


    Ulothrix tenerrima
        As, 62

    Ustilago
        Cu, 28


    Vaucheria
        B, 76

    Vicia Faba
        Sr, 5;
        Cu, 27, 29
      sativa
        B, 67;
        Zn, 39

    Viola sp.
        Zn, 38
      tricolor
        Zn, 36

    Vine
        Cu, 31

    Vitis vinifera
        As, 52


    Watercress
        B, 66

    Water-melon
        B, 65

    Wheat
        Cu, 16, 17, 23;
        Zn, 37, 44, 46;
        As, 52, 60;
        B, 66, 70, 72, 73;
        Mn, 79, 81, 83, 84, 85, 86, 87

    Willow
        Zn, 39, 40


    Yeast
        Zn, 50;
        B, 76


    Zea japonica
        Cu, 17, 18
      Mays
        (_see_ Maize)



GENERAL INDEX


    Absorption capacity of soils for zinc, 41
      of poisons by plants, 25

    Accelerators, 45

    Action of heavy metals in mixed solutions, 20

    Adsorption, 8, 23

    Aeration in water cultures, 8

    Algae, assimilation of arsenic by, 62
      clearing ponds of, 35
      effect of arsenic on, 62
      effect of boron on, 76
      effect of copper on, 35

    Aluminium, 45, 78

    Arbutin, 4

    Arsenate, potassium, 53, 62, 63
      sodium, 55, 57, 61

    Arsenates, 53, 57

    Arsenic acid, 53, 54, 60, 61, 62, 64
      acid v. arsenious acid, 53
      acid v. phosphoric acid, 53, 62
      elimination of, 59
      gas liberated by moulds, 63
      in soil, effect of, 58
      in superphosphate, 58

    Arsenious acid, 53, 54, 57–61, 64
      immunity of plants to, 58

    Arsenite, sodium, 55, 56, 61

    Arsenites, 53, 57
      v. arsenates, 57

    Artificial oxydases, 84
      soil, 24, 46

    Assimilation, reduction in water plants, 48


    Barium, 44

    Borate, calcium, 71
      potassium, 71

    Borates, 72

    Borax, 71, 73

    Bordeaux mixture, 30
      blocking of stomata by, 33
      on assimilation, effect of, 33

    Boric acid, 1, 65–76, 93

    Boromannitic acid, 68

    Boron, antiseptic action of, 70
      colour due to, 75
      distribution in plants, 66
      poisoning, indication of, 68, 69
      rôle in plant economy, 74


    Cadmium, 31

    Calamine, 37
      plants, 38
      soils, flora of, 37

    Calcium carbonate, 4, 23, 25
      chloride, 20, 59
      sulphate, 20, 44, 45

    Carbon black, 23
      dust, 10

    Catalytic elements, 49, 91

    Chlorophyll, 44, 60

    Complementary manures, 47, 91

    Conditions of plant life, 7

    Copper, acetate, 19
      action on plant organs, 30
      bicarbonate, 26
      bromide, 19
      chloride, 19, 20
      compounds, corrosive action on plant roots, 5, 27
      distribution in tissues, 16
      mode of action on plants, 25
      nitrate, 19, 25
      oxide, 15, 25
      quantity in certain plants, 17
      salts, injection into plant tissue, 31
      sprays, effect on leaves, 30, 32
      sulphate, 5, 19, 20, 22–27, 29–35, 41

    Cumarin, 4


    Distilled water, preparation of, 10

    Double decomposition in soil, 25

    Duration of experiments, 13


    Experimental methods, comparison of, 14


    Ferric chloride, sublimed, 86
      hydrate, 23

    Fungi, effect of arsenic on, 63
      effect of boron on, 76
      effect of copper on, 33, 34
      effect of manganese on, 90
      effect of zinc on, 48


    Galactose, 76

    Glucose, 76

    Germination, effect of arsenic on, 60
      effect of boron on, 72
      effect of copper on, 27, 28
      effect of manganese on, 84
      effect of zinc on, 43
      of seeds in sawdust, 11

    Grading of seeds, 11

    Growth in copper-distilled water, 17
      of peas in water cultures, 11


    Hydrochloric acid, 22

    Hydroquinone, 91

    Hypothesis of universal stimulation, 93


    Iodine, 2, 91

    Individuality of plants, error due to, 13
      of species, 61

    Interaction between soil and poison, 9

    Iron, 31, 49
      oxide, 78
      sesquioxide, 84
      sulphate, 48, 81, 85, 88


    Laccase, 91, 95

    Lack of control over field experiments, 9

    Lead, 10, 26, 42, 44


    Magnesium carbonate, 83, 84, 87
      chloride, 20
      sulphate, 1

    Manganese as top-dressing, 89
      chloride, 86, 88
      commercial value of, 88
      cytological action of, 81, 82
      dioxide, 84, 87
      essential to Coniferae, 80
      in Australian soils, 83
      in leaves, deposition of, 82
      in organic combination, 79
      iodide, 83, 84
      manuring, after-effects of, 88
      nitrate, 83, 84
      oxide, 78, 79, 83, 84, 86, 88
      phosphate, 79, 83, 86
      retardation of ripening by, 82, 85
      sulphate, 81–89, 94

    Masking effect of inorganic food salts, 4, 20

    Mercuric chloride, 22
      oxide, 41

    Mercury, 26

    Metallic oxides, 78

    Methods;
      field experiments, 9
      sand cultures, 8
      soil cultures in pots, 9
      water cultures, 7, 11

    Mode of entry of poisons into plants, 4


    Nickel, 24, 50

    Nucleinic combination, 79

    Nutrient solutions, composition, 13


    Oligodynamic action, 28

    Over-mineralisation of plants, 71


    Phosphoric acid, 53, 54, 62, 64

    Photo-synthesis, effect of copper on, 32

    Potassium hydrate, 22

    Presence of arsenic in animals, 51
      in plants, 51
      of boron in plants, 65
      of copper in plants, 15
      of manganese in plants, 78
      of zinc in plants, 36

    Pyrogallol, 91


    Raulin’s solution, 49

    Reproduction of poisoned plants, 40


    Silver nitrate, 22

    Sodium chloride, 20, 44
      hydroxide, 22
      nitrate, 4

    Sterile cultures, 24

    Stimulation, by injection of copper solutions, 31
      by small doses of poisons, 2
      definition of, 2
      local, 47
      of Aspergillus niger, 50
      of fungi by copper, 34
      of plants by arsenic, 61
      of plants by boron, 73
      of plants by copper (negative), 28
      of plants by manganese, 84
      of plants by zinc, 45–47
      physiological considerations of manganese, 90

    Strontium sulphate, 5

    Sugar, 22, 31, 48, 49, 50, 68

    Sulphur, flowers of, 10


    Thymol, 22

    Toxic action, effect of arsenic, 52
      effect of boron, 67
      effect of copper, 17
      effect of light on, 44
      effect of manganese, 81
      effect of zinc, 38
      equivalent, 18
      limits of plant poisons, estimation of, 26

    Toxicity, of nutrient salts, 1
      of organic compounds, 4
      of poisons, cause of, 22
      of positive ions in copper compounds, 19, 22
      reduction of, 39, 44
      reduction of, by carbon black and ferric hydrate, 23
      reduction of, by insoluble substances, 22

    Toxin and nutrient, distinction between, 3

    Transmission of power of resistance, 72


    Valency, effect on toxicity, 44

    Vanillin, 4

    Variation in results on different substrata, 24


    Zinc, absorption by roots, 42
      carbonate, 38, 42, 43
      effect of, on lower plants, 48
      effect of, on plant and soil, 41
      fixation of, 49
      mode of action on plants, 43
      oxide, 37, 47
      oxide on leaves, deposition of, 47
      storage in seeds, 43
      sulphate, 38–49
      sulphide, 42


CAMBRIDGE: PRINTED BY JOHN CLAY, M.A. AT THE UNIVERSITY PRESS.



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       *       *       *       *       *


Transcriber’s Notes

Minor inconsistencies in punctuation have been standardised--in
particular, missing periods and parentheses have been supplied where
obviously required. All other original errors and inconsistencies have
been retained, except as follows:

  Page 27:  changed Just to Just’s
            (in Just’s _Bot. Jahresber._)
  Page 37:  Added missing decimal point
            (total ash, (·006% of the)
  Page 76:  changed gms. to gm.
            (in until 5 gm. boric)
  Page 97:  changed in to im
            (Vorkommen von Iod im Thierkörper.)
  Page 98:  changed and to et
            (Colin, H. et Lavison, J.)
  Page 100: changed Experiments to experiments
            (Pot experiments to determine the)
  Page 101: changed cœrulea to cærulea
            (Zusammensetzung von Molinia cærulea)
  Page 101: changed and to und
            (Hiltner, L. und Gentner, G.)
  Page 101: changed Waar. to War.
            (Nahrungsmittel Hyg. War. IX, 1–4.)
  Page 102: changed Genuss-mittel to Genusmittel
            (Nahrungs- und Genussmittel,)
  Page 102: added date to entry
            (IV, 489–92. (1901))
  Page 102: changed Rübensache to Rübenasche
            (Bestandtheile der Rübenasche. Ber.)
  Page 103: changed Magenesium-salze to Magnesiumsalze
            (Calcium- und Magnesiumsalze im)
  Page 105: changed Vorhandsein to Vorhandensein
            (das Vorhandensein von Arsen)
  Page 105: changed Pflanzen-production to Pflanzenproduction
            (der Pflanzenproduction. Casopis pro)
  Page 105: changed prumsyl to prumysl
            (Casopis pro prumysl chemicky,)
  Page 107: added entry for Cu under Fungi
            (Cu, 33;)
  Page 110: changed Photosynthesis to Photo-synthesis
            (Photo-synthesis, effect of copper on, 32)





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