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CAMBRIDGE AGRICULTURAL MONOGRAPHS

Inorganic "Plant "Poisons
and Stimulants

'By Winifred 8. 'Brencbley

CAMBRIDGE AGRICULTURAL MONOGRAPHS

INORGANIC PLANT POISONS
AND STIMULANTS

CAMBRIDGE UNIVERSITY PRESS

0. F. CLAY, MANAGER
ILontam: FETTER LANE, B.C.
100 PEINCES STREET

. H. K. LEWIS, 136 GOWER STREET, W.C.
WILLIAM WESLEY AND SON, 28 ESSEX STREET, STRAND

$rfo JPork: G. P. PUTNAM'S SONS
fcombag ant Calcutta: MACMILLAN AND CO., LTD.
Toronto: J. M. DENT AND SONS, LTD.
THE MARUZEN-KABUSHIKI-KAISHA

INORGANIC PLANT POISONS

AND STIMULANTS

WINIFRED E)V'BRENCHLEY, D.Sc., F.L.S.

Fellow of University College, London
(Rothamsted Experimental Station)

Cambridge :

at the University Press

1914

(fTamftrttrgr :

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

PRMTSD IN GhC.U »*ITA!fl

PKEFACE

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 . . -. ••*/'•.•' $'j ... 7

2. Sand cultures . . . , . . . . ...... . . 8

3. Soil cultures in pots . . i , , . . 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

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

viii Contents

CHAP. PAGE

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

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

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 cul-

tures in the presence of nutrients ... 52

(b) Toxic effect of arsenic compounds in sand cultures 57

tures 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 i, . . . 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

Contents ix

CHAP. PAGE

2. Effect of boron compounds on germination ... 72

3. Does boron stimulate higher plants? .... 73

(a) Water cultures 73

(b) Sand cultures . 73

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

tures . ... . . . . . 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 . . ... . '"' . '". .'t . .' . 93

BIBLIOGRAPHY .... . 97

INDEX OF PLANT-NAMES . , . ....... . . 107

GENERAL INDEX . 109

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 . ...t . .. . 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 . .y. . ., 86

CHAPTEB 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 tn/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

B. 1

2 Introduction

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 satis-
factory 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 con-
ceptions. 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

Introduction 3

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,

1—2

4 Introduction

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 addi-
tion 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

Introduction 5

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 themselves1." 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 asunder2. 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

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.

6 Introduction

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.

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 inter-
mediate 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 determin-
ing the matter.

1. Water cultures.

This method of cultivation represents the simplest type of experi-
ment. 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 pre-
cipitates 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

8 Methods of Working

nature, a liquid medium replacing the solid one, so that the roots have
free access to every part of the substratum without meeting any opposi-
tion 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 approxi-
mates 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.

Methods of Working 9

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 some-
what 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 applica-
tion 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, applica-
tion 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 bene-
ficial 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

10 Methods of Working

other physiological factor. Consequently great care is needed in inter-
preting 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.

Methods of Working 11

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 experi-
ments. 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. Conse-
quently 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

Methods of Working

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

--C

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

A. a. Seedling of cereal.

6. 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.

plants are treated according to their individual needs and mode of
germination (Fig. 1).

The constitution of the nutritive solution is important, and it is
becoming more and more evident that different plants have different

Methods of Working 13

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 •! „

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 sub-
stances 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

14 Methods of Working

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 contribu-
tion 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 " Paradieskb'rner1," amongst others, were tested and all yielded
copper in very small quantity. Meissner was led to conclude that
copper is widespread in the vegetable kingdom, but that it exists in
such minute traces that its determination in plants is exceedingly
difficult. In 1821 Phillips made an interesting observation as to the
effect of copper on vegetation. Some oxide of copper was accidentally
put near the roots of a young poplar, and soon after the plant began to
fail. The lower branches died off first, but the harm gradually spread
to the topmost leaves. As a proof that copper had been absorbed by
the plant the record tells that the blade of a knife with which a branch
was severed was covered with a film of copper where it had been
through the branch, and the death of the plant was attributed to the
absorbed copper.

After this preliminary breaking of the ground little more seems to
have been done for some sixty years, but from about 1880 till the
present day the association of copper with the vegetable kingdom has
been actively investigated in its many aspects. Dieulafait (1880)
showed that the quantity of copper present in the vegetation is largely
determined by the nature of the soil, which thus affects the ease with
which the element can be detected and estimated. Copper was shown
to exist in all plants which grow on soils of " primary origin " (" roches
de la formation primordiale "), the proportion being sufficient to enable

1 These are " grains of Paradise," Guinea grains, or meleguetta pepper. They are the
seeds of Amomum melegueta and A. Granum-Paradisi, N.O. Zingiberaceae.

16 Effect of Copper Compounds

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. 0. von Lippman found traces of copper in beets, beet leaves, and
beet products ; Passerini estimated as much as "082 °/0 copper in the
stem of chickpea plants, though he regarded this figure as too high;
Hattensaur determined '266 °/0 CuO in the total ash of Molinia ccerulea
(-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.

Vedrodi (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 °/0 CuO. It is assumed that copper must
play some physiological role in the plant, but no explanation of this
action is yet forthcoming. Lehmann criticised Vedrodi's figures of the
copper content of certain plant ashes, and the latter replied in a further

Effect of Copper Compounds 17

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

min. max.

200 680

190 230

10 30

10 70

40 200

150 160

110 150

60 110

70 80

70 290

60 70

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

B. 2

"Seeds"

min.

max.

Winter wheat

710

Summer wheat

190

630

Maize

Barley
Oats

80
40

120
190

Buckwheat

160

640

"Fisolen" (Beans)
Linseed

160
120

320
150

Peas

100

Soy Beans
Lupins
Mustard seed

70
80
70

100
190
130

Paprika pods

790

1350

18 Effect of Copper Compounds

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 1 J" x J") 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

Effect of Copper Compounds 19

minimum weight of salt, which, dissolved in 100 parts of water, kills
the seedling," the results were as follows :

Toxic equivalent Containing copper

Copper bromide (CuBr2) -004875 -001387

Copper chloride (CuCl2. 2 aq.) -005000 -001865

Copper sulphate (CuS04. 5 aq.) -005555 '001415

Copper acetate (Cu{C2H302}2.aq.) -005714 -001820

Copper nitrate (Cu{N03}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.

2—2

20 Effect of Copper Compounds

(6) 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).

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

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 ,,

Effect of Copper Compounds

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

gram.

Total.

Shool

^ Root

2 1-4-2

1 = 1:100,000

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 inter-
mediate 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.

22 Effect of Copper Compounds

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 physio-
logical 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 with-
stand a concentration of 1/400 copper sulphate, part of which reduction
of toxicity is attributed to the sugar.

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, hydro-
chloric 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

Effect of Copper Compounds 23

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 CuS04 . 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 pro-
portion 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

24 Effect of Copper Compounds

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 consider-
able 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 condi-
tions 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 °/0 copper
sulphate solution for f 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 con-
centrations, 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

Effect of Copper Compounds 25

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 T^th 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 treat-
ment, 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 ex-
tracting with water containing such soluble copper salts as sulphate and
nitrate, the food salts of the soil, especially those of calcium and potas-
sium, 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

26 Effect of Copper Compounds

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 307 cubic decimetres of soil, two plants separated
by a partition being grown on this quantity. The test plants were
Polygonum Fagopyrum, Pisum sativum, Secede 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 experi-
mental 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

Effect of Copper Compounds 27

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 °/0 solution of copper1, 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

1 The English translation in Just Bot . Jahresber. speaks only of a "solution of copper,"
and in no case is the specific compound mentioned.

28 Effect of Copper Compounds

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.

(6) 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, Nageli had asserted that the latter often exerted an oligo-
dynamic 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 Nageli'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.

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

3.
4.
5.

6.
7.
8.
9.
10.

Control.

1/50,000

1/100,000

1/250,000

1/500,000

1/1,000,000

1/2,500,000

1/5,000,000

1/10,000,000

1/20,000,000

copper sulphate.

Effect of Copper Compounds

if it existed. He worked with Pisum sativum, var. arvense, Pisum
arvense, Vicia Faba, var. equine Pers, and Fagopyrum esculentum
Monch, 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 °/0
(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 state-
ment that in certain soils copper sulphate acts as a stimulant when it

grn
1-4

t-2
1-0
*8
•8
•4
•2
0

Total
Shoot

Root

r. ^

-^— •—

f,'

~^^.

Uj~"t>r"i

^.*~

—

>0 10 4 2 1 -4 -2 -1 -05 0
1 = 1:1,000,000

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.)

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

30 Effect of Copper Compounds

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 2£ million (Fig. 5). In the case
of buckwheat the matter is still undecided, as in some experiments
apparent stimulation is obtained with 1 in 2 J 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.

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 stimu-
lating 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 Kriiger (1894) treated potato plants with a 2 °/0 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

Effect of Copper Compounds 31

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 accom-
modation 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 cata-
lytic 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 experi-
ments 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 Krtiger 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

32 Effect of Copper Compounds

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.

(6) 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 ^71,000,000 copper sulphate ('0000159 %)» there was a reduc-
tion 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 JVyiO,000,000 copper sulphate there was little or no reduc-
tion 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 '00001 5 °/0 copper
sulphate, but in 500 c.c. solution the shoots were killed after some
days in '0000015 °/0 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 sur-
rounding 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 con-
centrated solutions of copper sulphate applied to leaves caused scorch-
ing, 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

Effect of Copper Compounds 33

the action was probably of the nature of a stimulus to growth, and pro-
duced 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 Kriiger.

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.

34 Effect of Copper Compounds

Trabut (1895) found that on treating smutty wheat with a 2 °/0
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 9J 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 concentra-
tions is given as from '0015 °/0 — '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 °/0 and Aspergillus in
•004 °/0. 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

Effect of Copper Compounds 35

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, M&nyanthes
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.

3—2

CHAPTEE 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 °/0 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% „ 1-60 °/. „ 13-12 % „

Flowers S'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% „ M6% „ „

Flowers 7'66% „ 0-075 °/8 „ 0-98% „

Effect of Zinc Compounds 37

Armeria vulgaris.

Root 474 % ash, 0'17 % ZnO, 3'58 °/0 ZnO in ash.

Stem 5-37 °/o ,, 0'02°/0 „ 0-37°/0 „ „

Leaves 9'36°/0 „ 0-11°/0 „ 1-17 8/. „

Flowers 6'08°/0 „ 0-07% ,, 1'15°/. ,»

Silene inflata.

Root 2-71 % ash, OO2 % ZnO, 074 % ZnO in ash.

Stem j

Leaves V 11-43 °/0 „ 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°/0 °f as^1» 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 ccerulea and discovered the presence of copper, manganese,
zinc and lead, zinc oxide forming '265 % of the total ash, (006 °/0 °f the
air dried plant).

Jensch (1894) observed that the flora on calamine soils was some-
what scanty, the chief plants that came under his notice being Taraxa-
cum officinale, Gapsella Bursa-pastoris, Plantago lanceolata, Tussilago
Farfara, and Polygonum aviculare, all of which showed certain morpho-
logical 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

38 Effect of Zinc Compounds

purple red colour. The following figures are given for the quantities
of zinc carbonate (ZnCO3) in the ash of these two plants : —

Tussilago Farfara.

Boot Leaf-stalk Leaf-blade

2-51 °/0— 3-26 % 1-75 °/0— 1-63 % 2'90 %— 2-83 % ZnC03

= 1-629 70— 2-115 % 1-136 %— 1'058 % 1-882 %— 1-836 °/0 ZnO.
Polygonum aviculare.

Boot Stem Leaves

1-77 %— 1-93 •/. 2-25 °/0— 2-86 % 1-24 %— 1-49 % ZnC03

= 1-148 %— 1-252 7. 1-46 °/0— 1-856 °/0 -804 °/0— -967 70 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 con-
cerned 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).

Effect of Zinc Compounds 39

Baumann (1885) carried out further experiments and concluded that
zinc salts are far more toxic than Freytag suspected, 44 mg. zinc
sulphate per litre1 killing plants of 13 species belonging to 7 families
(Coni ferae 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 „

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.

(6) 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.

1 44 mg. ZnS04.7HsO = 10mg. Zn = 1/22, 727 ZnS04. 7H20 approx.

Effect of Zinc Compounds

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 consider-
ably, 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

gm.
!•€

100 20 10

Tohoi

Shoe*

Rool-

•OA -02

1 = 1:1,000,000

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.)

amount of growth even in the presence of a solution of the anhy-
drous 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 J or 1 in J 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

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

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

zinc sulphate.

Effect of Zinc Compounds

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 diminu-
tive flowers, which are occasionally replaced by equally unhappy and
miniature fruits. The same thing has also been noticed when un-
successful attempts have been made to introduce spinach as a test plant
for water cultures.

gm.
1-2

1-0
•8

•6
•4

Total

Root-

200 100 20 10

2 1-4

1 = 1:1,000,000

-05

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.)

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, ZnS04, HgO, were intimately mixed
with 30-7 litres ("cubik Decimeter") of soil1. 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

1 This is equivalent to about -1 °/0 of poison.

42 Effect of Zinc Compounds

soils for different poisons varies, that basic salts are absorbed, while the
acid salts may pass completely through the soil in the drainage water.

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

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

Holdefleiss (1883) stated that in spite of a soil content of 2 °/0 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 C02
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

Effect of Zinc Compounds 43

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 Bau-
mann'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

44 Effect of Zinc Compounds

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, (6) 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 con-
taining 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 hypo-
thesis 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 | 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 ^ normal — the limit of experi-
ment), 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.

Effect of Zinc Compounds 45

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 rdle 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)1, and found that while zinc sulphate alone at ra/8192 retarded
growth, yet with m/2048 ZnS04 and w/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 modifi-
cations 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.

1 m probably — gram molecular weight.

46 Effect of Zinc Compounds

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 °/0 (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 ZnS04 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).

(6) Stimulation in sand cultures.

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

Nakamura (1904) dealt with a few plants of agricultural importance,
adding '01 gram anhydrous zinc sulphate to 2300 grams air-dried soil.

Effect of Zinc Compounds 47

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 experi-
ment no increase manifested itself in the weights of the plants treated
with zinc ; with Hordeum the same quantity of zinc exercised a con-
sistently injurious action. These results with peas and barley corroborate
those obtained in the Kothamsted 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 com-
pounds 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 remark-
ably 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 de-
posited on the leaves, and Klopsch stated that the rain and dew containing

48 Effect of Zinc Compounds

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 °/e)
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 how-
ever the shoots were placed in (1) water, (2) '00008 % zinc sulphate,
(3) fresh -00008 °/0 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 °/0 zinc sulphate by
weight than in the control crops, indicating a stimulation caused by
zinc.

Effect of Zinc Compounds 49

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 Sterigmato-
cystis 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, vindi-
cating 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.

B. 4

50 Effect of Zinc Compounds

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 un-
certain 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 superphos-
phate 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. Cerny (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.

4—2

52 Effect of Arsenic Compounds

Arsenic per 100 parts fresh substance in \igr. ( = thousandth part of a milligram)1.

Wheat (Victoria — complete grain) 7

„ (from Franche Comte") -85

White bread -71

Whole green cabbage *2

Outside leaves of cabbage O (absent)

Green haricots -0 „

Turnip -36
Potatoes 1-12

Arsenic was also found in wine and beer and in considerable quanti-
ties 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 experi-
mental 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

i O'*«r', 1 =0-0001 mg.

Effect of Arsenic Compounds 53

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 (As203) 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, inter-
rupted 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 carry-
ing 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 potas-
sium arsenate proved to be harmful to young maize seedlings if the
solutions contained '05 — '1 gm. arsenic acid per litre (= I/ — 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 indi-
cated 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 phos-
phoric 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

54 Effect of Arsenic Compounds

phosphoric acid, arsenic acid causes a strong production of organic sub-
stances up to the flowering time. The following figures were obtained
with maize : —

•002 gm. As203 with P206 2-84 gm. dry wt.

•005 gm. „ „ „ 2-37

•01 gm. As206 „ „ 67-32

•40 „ „ „ „ 64-13

•03 „ As206 without P206 39-98 „

•07 „ „ „ „ 42-13

normal solution „ „ 12-93 „

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. AsjOg per litre) and the plants died in a very short time.

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

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

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

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

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

2*.

9.
10.
11.

Control.

1/50,000

1/100,000

1/150,000

1/200,000

1/250,000

1/500,000

1/1,000,000

1/5,000,000

1/10,000,000

1/25,000,000

1/50,000,000

arsenious acid.

Effect of Arsenic Compounds

With sodium arsenite the dilutions were carried further, to
1/250,000,000, but this still depressed barley to some extent (Fig. 13).

SM.

1-8
I-O
1-4.
»-2

•8
•6

•2

,'•'

'"x

»•— «

t£^~

^1 — -

JO K) 6-6 S 4 2 I -2 -1 -04 -02 O
1-1:1,000,000

Shoot

Roor

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.)

9<«.

\-\J
•8
•6
•4
•2

0
K

^ "•

Tokd
Shod-

Roc*

----.

f,'-'

—

/•-'

______ ^

^.^

~" —

—

)0 20 10 4 2 1 -4 -2 -1 O
1^1:1,000,000

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.)

With peas the results vary somewhat in the different tests, the depres-
sion 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

Effect of Arsenic Compounds

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

cm.

1-4
1-2
1-0
•8
•6
•4
•2

Tofd
Shoot

Roof

— — -

(

Xxx

„,

-'"

**«.

) 6-8 S * 2 1 -2 -1 -04 -02 0
MM.000,000

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.)

gm.
1-6

1-4
h2
1-0

•e

•6

•2

O
4

TohoJ
Shoo)

RooK

^^^

—

^^s,

^^.

~- —

///

x--

^ »• """

0 20 10 4 2 1 -4 -2 -1 -0* -Q5 -O4 O
l-|s 10,000,000

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.)

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

Effect of Arsenic Compounds

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.

(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

-*-^-

•MMBM*

^^ X

Total
Shoot

Root

•8

' f

^ ^ *

^^•V

•2
0

'^^

_.-

-*"

DO 20 10 4 2 1 -4 -2 -1 -04 -02 0
1 = 1:1000,000

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.)

which it is applied, as was shown by the fact that different plants lived
for varying times when treated with similar strengths of solution.

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.

58 Effect of Arsenic Compounds

Gorup-Besanez (1863) mixed 30 grams arsenious acid with 30'7 litres1
soil, growing two plants on this quantity of earth. Most of his experi-
mental 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 solu-
tion added to the soil enters into combination with other substances,
forming insoluble compounds, thus being removed from the sphere
of action and rendered unable to affect plant life. If this be so, the
apparent immunity of certain plants to arsenious acid is explained.
F. C. Phillips (1882), in his experiments on various flowering plants,
such as geraniums, coleas and pansies, found that compounds of arsenic
in the soil exercised a distinct poisoning influence, tending, when
present in large amount, to check the formation of roots, so that the
vitality of the plant was so far reduced as to interfere with nutrition
and growth, or even to kill it outright. He also stated that traces of
arsenic were found in all the plants grown upon the poisoned soil.

In this connection it is interesting to note that a certain proportion
of arsenic is frequently present in the superphosphate used as manure.
In view of the known toxicity of arsenical compounds to plant life the
question arose as to whether superphosphate manuring would exercise
a detrimental influence on account of its arsenic content. Experi-
ments 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.

1 30 grams arsenious acid to 30'7 "cubik Decimeter" soil = about -1%.

Effect of Arsenic Compounds 59

(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 indi-
vidual 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, Romer and Wim-
mer) 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

60 Effect of Arsenic Compounds

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 C02-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 func-
tions 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. water1. 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 experi-
ments, 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 germi-
nation, 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 con-
siderably. 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

1 In the present state of our knowledge such a concentration seems relatively
strong !

Effect of Arsenic Compounds 61

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 O'lO % 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 °/Q 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% As208 was strongly adverse to the
germination of all plants, and that above this strength germination
was altogether prevented.

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

3. Do arsenic compounds stimulate higher plants?

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

62 Effect of Arsenic Compounds

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 in-
fluence 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 Valderi-
anum 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

Effect of Arsenic Compounds 63

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 Asper-
gillus glaucum, will grow on various arsenic compounds and exercise a
reducing influence on them. These moulds attack all oxygen com-
pounds 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, de-
velope 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. Peni-
cillium 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 wall-
paper 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— l) stimulate the growth of Aspergillus

1 The exact compound is not specified in the abstracted paper, ^7 — jfo- % Natr. Ars.
being given.

64 Effect of Arsenic Compounds

niger, larger doses up to 1/8 % retard growth, while 1/6 % 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.

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 con-
centrations 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.

CHAPTEE 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. Primulaceae1).
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.

In 1888 Baumert detected boron in French, German, and Spanish
wines without exception, while E. 0. 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 king-
dom 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

1 According to Engler's classification this plant belongs to N.O. Myrtinaceae.
B. 5

66 Effect of Boron Compounds

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 210 — 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 re-
fined, 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 saliva (*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.

Effect of Boron Compounds 67

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 distri-
bution 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 Triti-
cum vulgar -e in solutions of boric acid varying in concentration from
1 — "05 %, with controls in spring water. In the latter case the develop-
ment 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

5—2

68 Effect of Boron Compounds

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
aUbus 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 concen-
trations 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 sup-
ported 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

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

Control.

1/5,000

1/10,000

1/25,000

1/50,000

1/100,000

1/250,000

1/500,000

1/1,000,000

boric acid.

Effect of Boron Compounds

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 de-
pressing 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

Root

200 100 20

I -04 -02

42 I -4 •

»« 1:1,000.000

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.)

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 de-
generation. 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

70 Effect of Boron Compounds

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.

Tests with white lupins gave no conclusive results, as for some
reason it proved very difficult to get satisfactory plants in water cul-
tures. 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 concen-
tration 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.

(6) Toxic action of boron compounds in sand cultures.

Agulhon (1910 a) moistened 2 kgm. pure sand with 500 c.c. nutri-
tive solution for each pot, and boron was added at the rate of 0, O'l,
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 cul-
tures, probably because evaporation from the surface of the sand caused
concentration of the poisonous liquid.

Effect of Boron Compounds 71

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 com-
pleted 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 indi-
cate 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

72 Effect of Boron Compounds

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 gene-
ration of maize were able to withstand quantities of boron that were
toxic to control plants1. 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.

2. Effect of boron compounds on germination.

One of the first indications that boron compounds affect the germi-
nation 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, Rapha-
nus, Collinsia, and Linum in the presence of boric acid. The seeds were
placed in bowls with solutions of '25, *5, and 1 °/0 boric acid at tempera-
tures 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 °/0 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

1 "II apparait done que les graines fournies par des plantes ayaut cru en presence
d'une quantit^ de bore eleve"e pre'sentent une accoutumance vis-a-vis de cet e'le'ment ; les
plants auxquels elles donnent naissance sembleut 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 te"moms, issus de graines non accoutume"es."

Effect of Boron Compounds 73

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 indi-
cated 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

74 Effect of Boron Compounds

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 °/0 fresh weight, and 7*5 °/0
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 °/0 dry weight.

Here again the stimulating action was evident with higher concen-
trations 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 °/0
fresh weight and 39 °/0 dry weight with maize ; half the strength
proved to be indifferent for peas, the improvement with maize equalling
56 % increase fresh and 50 °/0 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 element" type in the higher plants of the vegetable

Effect of Boron Compounds 75

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 Kothamsted 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 un-
expected, 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 con-
centrations of about 1/50,000 boric acid, the improvement being far
more strongly marked in some sets of experiments than in others.

76 Effect of Boron Compounds

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°/0 (=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 favour-
ably 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 °/0 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 unco-
agulated. 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 gms. 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.

Effect of Boron Compounds 77

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) con-
firmed 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

Effect of Manganese Compounds 79

natans ('15 % Mn3O4), Acorus Calamus (1/52 % Mn304), Alnus incana
(trace — '73 % Mn3O4), Pyrus communis (215 % Mn304). 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 pre-
cipitate was found to contain manganese, and gave by fusion with nitre
and sodium phosphate a violet coloured mass. Andreasch (1878) found
slight traces of Mn304 in the flowers of Dianthus caryophyllus, none
occurring elsewhere, while in Rosa remontana it appeared in both leaves
and flowers.

Maumene (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. Bicciardi (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 °/Q 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 Mn304 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 °/Q in chestnut fruits.

80 Effect of Manganese Compounds

Gossl (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. Schroder (1878) tested for the element in firs and pines and
found the following amounts of Mn304.

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 G. gracilis, to a minimum of
O'OIO °/Q in C. robusta. The percentage of manganese in Australian
Goniferae other than Callitris is given by the authors in the following
table :

Ash of timber of Agathis robusta 0-145 % Mn.

„ „ Araucaria Cumiinghamii 0'054 % „

„ „ Araucaria BidwiUi 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 pro-
duction 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

Effect of Manganese Compounds 81

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 man-
ganese 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. Aso (1902) found that the greater con-
centrations of manganese sulphate exercised an injurious influence on
barley. Even in solutions with as little as '002 °/Q 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 f
similar to barley in its reactions, though more iron is necessary to give •
good healthy growth. Aso 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 MnS04 (anhy-
drous) 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 % MnS04, '01 % FeS04 + "02 % MnSO4).
At first the growth was increased by the action of two salts together,
but eventually the shoots turned yellowish, and assimilation was de-
pressed, so that decreased nutrition led to relaxation in the speed of
growth, indicating the toxic action due to the manganese sulphate.

B. 6

82 Effect of Manganese Compounds

The Rothamsted experiments supported Aso'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 car-
bonate and potassium nitrate. On dissolving up the resulting mass with
water a green colouration was obtained, indicating the presence of man-
ganese 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 MnS04
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 MnS04 pos-
sessed 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 sen-
sitive to manganese poisoning than is barley, and the higher strengths
of manganese prove more deleterious to them.

(6) 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.

A large body of work has been done with manganese in soil cul-
tures, but the toxic effect is hardly indicated, possibly because it is

Effect of Manganese Compounds 83

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. MnS04 added to 8 kgm. Japanese soil exerted a
depressing influence on the growth of various plants. The Hills Ex-
periments (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 un-
treated 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 develop-
ment 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 % Mn304) pineapples do not
flourish, but turn yellow and produce poor fruits, and also that if rather
less manganese is present (1'36 % Mn304) the pineapples show the toxic
effect by yellowing during the winter months, but they recover com-
pletely 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,

6—2

84 Effect of Manganese Compounds

as much as '254 °/o 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
Mn02, manganese carbonate and sulphate all retarded germination, while
with the iodide 50 °/0 of the seeds were entirely prevented from germi-
nating.

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 Aso (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 °/Q manganese
sulphate ( = 1/50,000) to culture solutions stimulated radish, barley,

Effect of Manganese Compounds

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 4-
•02 % manganese sulphate, but this initial stimulation was followed by
depression. These authors support Aso'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.

gram

Total

Sboot.

1000 100

10 2 1 -2 -1

1 = 1:10,000,000

•01

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.)

The Rothamsted experiments with barley show a decided stimulation
with 1/100,000 MnS04 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

86 Effect of Manganese Compounds

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).

(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 MnS04 (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, ex-
perimented on arable land, adding quantities of manganese sulphate (?)
equivalent to about T6 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

>» as ^^-

& O rH «H

TH 01

- ^ O O

^ 2

S °
§§

-. o ^ '-|

PH O ^T ^T

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

Control.

1/5,000

1/10,000

1/25,000

1/50,000

1/100,000

1/250,000

1/500,000

1/1,000,000

manganese sulphate.

Effect of Manganese Compounds 87

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 mineral1,
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.

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 un-
treated. 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 man-
ganese 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.

1 As no analysis of the mineral is given it is obviously impossible to say to what con-
stituent the increase is due in this case.

88 Effect of Manganese Compounds

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 Mn203 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. Mn203 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.
Mn203 per hectare, while it was approached very closely by that from
the plot with 25 kgm. MnaOs, 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 °/Q, while in the second year
it dropped to 16'9 %.

Aso (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 l£ 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.
FeS04.7H2O. 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. MnS04.4H2O,

(3) '2 gm. MnS04 . 4H2O,

the manganese sulphate being applied in high dilution as top dressing.

Effect of Manganese Compounds 89

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 Kelative leaves

gm. gm. gm. gm.

1. 29-5 8-5 38-0 lOO'O '28

2. 38-0 22-5 60-5 159'2 '59

3. 35-5 16-5 51O 134'2 '46

Uchiyama (1907) carried on a variety of experiments with man-
ganese 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. MnS04. 4H20 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 Legumi-
nosae and Cruciferae are more susceptible to manganese stimulation
than are the Gramineae.

90 Effect of Manganese Compounds

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 Asper-
gillus 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 stimu-
lated 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 com-
pounds 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 accom-
plished as quickly as they are formed, so enabling growth to continue
more rapidly.

Aso (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

Effect of Manganese Compounds 91

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 ac-
cording 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 con-
sidered 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 insuf-
ficiency 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 man-
ganese, 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 rdle 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.

92 Effect of Manganese Compounds

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

94 Conclusions

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 charac-
terise 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 differ-
entiation 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 indif-
ferent 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 es-
sential 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

Conclusions 95

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 essen-
tially 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 over-
looked 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

96 Conclusions

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 improve-
ment 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 pro-
portion 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 con-
siderable 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 con-
ditions, 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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MAYER, J. E. and BRAZIER, J. S. Analyses of mineral constituents of the

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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. (Paradieskorner) 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

Cunningham!! 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 ; Ma
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 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

108

Index of Plant-Names

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 casrulea 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
Paradieskorner (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

Eadish Sr 5 ; B 74 ; Mn 84, 90
Kaphanus B 72

sativus Zn 39
Baspberry As 65

Kice Zn 47 ; B 66, 73 ; Mn 79, 86, 87, 88
Kosa remontana Mn 79
Kubus 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
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 zino 41

of poisons by plants 25
Accelerators 45
Action of heavy metals in mixed solutions

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

role 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

bi-carbonate 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

110

General Index

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
Photosynthesis, 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

Eaulin'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 solu-
tions 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 man-
ganese 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

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By C. E. Moss, D.Sc., F.L.S., assisted by specialists in certain genera.
Illustrated from drawings by E. W. HUNNYBUN. The work will be
completed in about ten volumes. Volume II is now ready.

The styles of binding and the prices will be as follows :

Published Price Price per volume

per volume to subscribers to

the whole work
Paper boards, with canvas back and paper label, each

volume in two parts, the first containing the

text and the second the plates ... £2 10s net £2 5s net

Quarter morocco, in two parts divided as above . £6 net £5 5s net

Paper boards, with canvas back and paper label, in

one volume, the plates mounted on guards and

bound interspersed with the text ... £3 net £2 15s net

Quarter morocco, in one volume, the plates mounted

on guards and bound interspersed with the text £6 net £5 5s net

"The appearance of Dr Moss's work has been anticipated by British botanists with
the greatest interest ; not only to them does it appeal, for its completeness and attention
to detail entitle it to rank among works of Continental importance. The Cambridge
University Press has been fortunate in securing the services of Dr Moss, than whom no
one more competent for the task could be found. By a combination as admirable as it is
rare, Dr Moss is at once an acute field botanist, a diligent investigator of herbaria, and a
student of botanical literature.... Mr Hunnybun's drawings are all made from living plants,
so that the work may be regarded as representing more fully than has been hitherto done
our knowledge of British Botany at the present day." — Journal of Botany

Genera of British Plants. Arranged according to Engler's Syllabus
Der Pflanzenfamilien (Seventh edition 1912), with the addition of the Characters of
the Genera. By H. G. CABTEB, M.B., Ch.B. Crown 8vo. 4s net.

British Rust Fungi (Uredinales). Their Biology and Classification.
By W. B. GROVE, M.A. Demy 8vo. With 290 figures. 14s net.

Types of British Vegetation. By members of the Central Committee
for the Survey and Study of British Vegetation. Edited by A. G. TANSLEY, M.A.,
F.L.S. Crown 8vo. With 36 plates and 21 figures. 6s net.

The Production and Utilisation of Pine Timber in Great

Britain. Part I. Production. By E. B. BUBDON, M.A., and A. P. LONG, B.A.

No. 1. Sample Plots of Scots Pine at Woburn. Demy 8vo. Paper covers.

Is 6d net.
No. 2. Sample Plot of Scots Pine at King's Lynn. Demy 8vo. Paper covers.

9d net.

The Journal of Agricultural Science. Edited by B. H. BIFFEN,
M.A., F.B.S., A. D. HALL, M.A., F.R.S., E. J. BDSSELL, D.Sc., and T. B. WOOD, M.A.

Vols. I, II, III, IV and V now ready. In four parts. Paper covers. 15s net per
volume. Bound in Buckram, 18s 6d net per volume.

Price to subscribers, 15s net per volume, post free, payable in advance. The price
of each part sold separately will be 5s net.

Cambridge University Press

C. F. Clay, Manager: Fetter Lane, London

5401
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