Seay Sg sete Seietyieesie? : i : 19 tS heiny intent sabe yaaa Taipan PTs abet) hase ise i i y TF eae ay Basa a e ane f ; at Sop De mea ne me palit men 4 aS Dil earner ee ae ee © pnteyeecsere: cis or ie ap re or el ange “= se ee sea beey a sete nari bt a it ues in eis Ace aie - ALBEAT & Re whaghoass Petey shueaspepaceanel Fa ipincoaunece Seat ee pens if ih Bae is at ote Pinuaer bere ee hess, sian ein ii or ( atiotes et Cece iteses ar Se AT tine min bane rere art : oy aes Pig petnpcne ra ILE DALE fee eT H Hy et c Peter ttgn yeas etre tities a eee) rks aptus panaoi ensiee easmrai oe pir ose ae oe Oe Te sR fe? fe oteha; ei 5 Ba pepe - ae apne meinem P soos sis) iv pats chai ina si singyaN Wise cael ay aie teem nee “ Seaenth “ ay ; ea, eae ne pe ide idea erated . : on ; ie AA BAR iljtiestitjshs op rte sorcerer Pieler glares : et icliarece t yes Wie thatosce ai eammsiea st Sel lel sea peat ep trmeter fot = se E re pate teenies on < Hive af je itiistijananinest tinh Fo niche Jr wees Holeeenh Oe ay Ceginetg aay ¥ fait bance ee pide he ed nf pie eer ieee lec eames reer =o es . see ef Wetlhcesosinisaers cenit ene eaeegqenne ee Sete fs ise iy ester cote peares I - ‘ Seer re Af te 2 roth alee Serrated pers ee arirerac cla iy Lede lh Sede Slotted Ge Cirhie SME PoP OS nf ie aa a i Peet ie po eS oa poppe a tc pli mine ALBERT R. MANN LIBRARY NEw YorkK STATE COLLEGES OF AGRICULTURE AND HoME ECONOMICS AT CoRNELL UNIVERSITY CORNELL UNIVERSITY LIBRARY Cornell University The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. htto://www.archive.org/details/cu31924081073300 CAMBRIDGE AGRICULTURAL MONOGRAPHS INORGANIC PLANT POISONS AND STIMULANTS CAMBRIDGE UNIVERSITY PRESS C. F. CLAY, Manager London: FETTER LANE, E.C. dinburg): 100 PRINCES STREET London: H, K. LEWIS, 186 GOWER STREET, W.C. Donvon: WILLIAM WESLEY AND SON, 28 ESSEX STREET, STRAND few Work: G. P. PUTNAM’S SONS Bombay ant Calcutta: MACMILLAN AND CO., Lrp. Toronto: J. M. DENT AND SONS, Lrp. Tokyo: THE MARUZEN-KABUSHIKI-KAISHA All rights reserved INORGANIC PLANT POISONS AND STIMULANTS BY WINIFRED E. BRENCHLEY, D.Sc., F.L.S. Fellow of University College, London (Rothamsted Experimental Station) Cambridge : at the University Press IgI4 Cambridge: PRINTED BY JOHN CLAY, M.A. AT THE UNIVERSITY PRESS PREFACE AD eee! 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. I. INTRODUCTION II. Mergsops oF WorkKInG I. Discussion of Methods 1. Water cultures. 7 2. Sand cultures . F ‘ 8. Soil cultures in pots 4, Field experiments II. Details of Methods . III. Erect or Coprer Compounps I. Presence of Copper in Plants II. Effect of Copper on the Growth of Higher 3 Plants 1. Toxic effect (a) Toxic action of copper compounds alone in water cultures (b) Masking effect awtiwed by ndlaition of soluble substances to solutions of copper salts ‘ (c) Effect of adding insoluble substances to solutions of copper salts (d) Effect of copper on es growth wien pieaand in soils . ‘ . F - i (e) Mode of action of nase on latiby 2. Effect of copper on ee (a) Seeds (b) Spores and halle erated: 3. Does copper stimulate higher plants ? 4, Action of copper on organs other than roots (a) Effect of copper sprays on leaves . ‘ (b) Effect of solutions of copper salts on leaves. III, Effect of a on Certain of the Lower Plants Conclusion : : : ; , Viil Contents CHAP. IV. Errect or Zinc Compounps I. Presence of Zine in Plants II. Effect of Zinc on the Growth of Higher Plants . 1. Toxic effect (a) Toxic action of zinc gilts alone in onal ciate (b) Effect of soluble zinc salts in the presence of nutrients (c) Effect of zinc oompenils on “plant growsh sdien ; they are present in soils ‘ . . (d) Mode of action of zinc on plants . 2. Effect of zinc compounds on germination 3. Stimulation induced by zinc compounds (a) Stimulation in water cultures (b) Stimulation in sand cultures . (c) Increased growth in soil 4. Direct action of zinc salts on leaves III. Effect of Zinc on Certain of the Lower Plants Conclusion . VY. Errect or Arsenic CompounDs I. Presence of Arsenic in Plants II. Effect of Arsenic on the Growth of Higher Plants 1. Toxic effect (a) Toxic action of arsenic oman poatils in water dal. tures in the presence of nutrients (b) Toxic effect of arsenic compounds in sand edlpiiese (c) Toxic effect of arsenic when applied to soil cul- tures (d) Physiological conibidendttanis 2. Effect of arsenic compounds on germination . 3. Do arsenic compounds stimulate higher plants? III. Effect of Arsenic Compounds on Certain of the Lower Plants 1. Algae 2. Fungi Conclusion VI. Ervrect or Boron Comprounps . I. Presence of Boron in Plants . II. Effect of Boron on the Growth of Higher Plants 1. Toxic effect (a) Toxic action of poner don patindat in water culaires, (b) Toxic action of boron compounds in sand cultures PAGE 36 36 38 38 38 39 41 43 43 45 45 46 46 47 48 50 51 51 52 52 52 57 57 59 60 61 62 62 63 64 65 65 67 67 67 70 Contents OHAP. (ec) Toxie action of boron compounds in soil experi- ments 2. Effect of boron compounds on germination 3. Does boron stimulate higher pene (a) Water cultures 3 (b) Sand cultures . (c) Soil cultures III. Effect of Boron Compounds on Certain of the Lower Plants Conclusion ae Ip. i ‘ VII. Errect or Manganese Compounns . I. Presence of Manganese in Plants . Il. Effect of Manganese on. the Growth of Higher Plants 1. Toxic effect : (a) Toxic action of cravieaneee Giepouuis in the presence of soluble nutrients ‘ (b) Toxic action of manganese eas aaa in saad cultures : (c) Toxic action of samt dea gameeanian in il a tures 2. Effect of manganese compounds on germination 3. Does manganese stimulate higher plants ? (a) Stimulation in water cultures (b) Stimulation in soil cultures III. Effect of Manganese Compounds on Certain of the Lower Plants ‘ IV. Physiological Considerations of Manganese Stimulation Conclusion VIII. Concriustons BrsLioGRAPHY . InpEx oF PLANT-NAMES GENERAL INDEX List oF ILLUSTRATIONS . pera gerpwwed EL cd nc Pan rarre WOES LIST OF ILLUSTRATIONS Sketch illustrating water culture methods Photograph. Barley grown with copper sulphate Curve. Ditto . . Photograph. Peas grown with copper gaits Curve. Ditto . Curve. Barley grown with zinc liste Photograph. Peas grown with zine sulphate Curve. Ditto . a ‘ 5 5 Photograph. Barley grown with arsenious acid . Curve. Ditto . . : ; Curve. Peas grown with arsenious acid : é Curve. Barley grown with arsenic acid Curve. Barley grown with sodium arsenite . Curve. Peas grown with sodium arsenite Curve. Barley grown with boric acid . Photograph. Peas grown with boric acid Curve. Barley grown with manganese sulphate . Photograph. Ditto. Photograph. Peas grown with. manganese saiphats . To face 29 - To face 40 . To face 54 - To face 69 . To face 86 PAGE 12 . To face 20 21 29 40 41 55 55 56 56 57 69° 85 » 86 CHAPTER I INTRODUCTION EVER since the physiological side of botany began to emerge from obscurity, the question of the relation between the nutrition and the growth of the plant has occupied a foremost position. All kinds of theories, both probable and improbable, have been held as to the way in which plants obtain the various components of their foods. But quite early in the history of the subject it was acknowledged that the soil was the source of the mineral constituents of the plant food, and that the roots were the organs by which they were received into the plant. A new chapter in the: history of science was begun when Liebig in 1840 first discussed the importance of inorganic or mineral substances in plant nutrition. This discussion led to a vast amount of work dealing with the problem of nutrition from many points. of view, and the general result has been the sorting out of the elements into three groups, nutritive, indifferent, and toxic. Thus calcium, phosphorus, nitrogen and potassium are classed as nutritive, arsenic, copper and boron as toxic, and many others are regarded as indifferent. Closer examination, however, shows that this division into three classes is too rigid, Now that experiments are more refined it has become evident that no such simple grouping is possible. It has been found that typical nutrient salts are toxic when they are applied singly to the plant in certain concentrations, the toxic power decreasing and the nutritive function coming into play more fully on. the addition of other nutrient salts. For instance, Burlingham found that. the typical nutrient magnesium sulphate in concentrations above m/8192 (m= molecular weight) is toxic to most seedlings, the degree of toxicity varying with the type of seedling and the conditions under which growth takes place. It will be shown in the following pages that even such a typical poison as boric acid may, under suitable conditions, increase plant growth just as if it were a nutrient. A review of the 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, sapere 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 (1838) described experiments in which various plants, as radish, cabbage, Vicia Faba, hemp and barley were grown actually on sulphate of strontium or on soils watered with nitrate of strontium. No strontium could be detected in the ash of any of the plants save barley, and then only the merest trace was found. Daubeny concluded that the roots were able to reject strontium even when presented in the form of a solution. “Upon the whole, then, I see nothing, so far as experiments have yet gone, to invalidate the conclusion...that the roots of plants do, to a certain extent at least, possess a power of selection, and that the earthy constituents which form the basis of their solid parts are determined as to quality by some primary law of nature, although their amount may depend upon the more or less abundant supply of the principles presented to them from without.” Some years after, in 1862, Daubeny reverted to the idea, stating “I should be inclined to infer that the spongioles of the roots have residing in them some specific power of excluding those constituents of the soil that are abnormal and, therefore, unsuitable to the plant, but that they take up those which are normal in any proportions in which they may chance to present themselves'.” This, however, was not held to apply to such corrosive substances as copper sulphate. De Saussure had found that Polygonum Persecaria took up copper sulphate in large quantities, a circumstance which he attributed to the poisonous and corrosive quality of this substance, owing to which the texture of the cells became disorganised and the entrance of the solution into the vegetable texture took place as freely, perhaps, as if the plants had been actually severed asunder*. 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 or MeETHops. 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. Deraits or MetrnHops. 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 cc. 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 ‘83—'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 12 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 Fig. 1. Diagrammatic sketches showing methods of setting up water cultures, A. a. Seedling of cereal. Cork bored with two holes, and cut into two pieces through one hole. ¢. Food solution. B. «u. Pea seedling. 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 ig becoming more and more evident that different plants have different Methods. of Working 138 optima in this respect. For several years a solution of medium strength was used, containing the following: Potassium nitrate wowed gram Magnesium sulphate ie, 38 5, Sodium chloride ... age we CB 5 Calcium sulphate ... Bis ‘By, Potassium di-hydrogen phosphate 3 yy Ferric chloride... . 04 ,, Distilled water... aes ss 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... me ae 5 gram Potassium nitrate 2 Potassium di-hydrogen phosphate. L. 5 Calcium sulphate .. : ty Magnesium wullphate ts dts lg Sodium chloride rn sis A 55 Ferric chloride... awa one 04 ,, Distilled water... ae eg 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. CoprER 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 Curcwma longa, and of “ Paradieskérner’,” 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. Q. von Lippman found traces of copper in beets, beet leaves, and beet products; Passerini estimated as much as ‘082°/, copper in the stem of chickpea plants, though he regarded this figure as too high ; Hattensaur determined ‘266 °/, CuO in the total ash of Molinia cerulea (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, jinseed, salads, apricots and pears; cocoa and chocolate. He found that only in those plants which are grown on soil rich in copper does thé 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. Vedrédi (1893) tackled the problem at about the same time as Lehmann but from a rather different standpoint. He ratifies the statement as to the absorption of copper by plants, and going still further he states that in some cases the percentage of copper found in the seed may be four times as great as that occurring in the soil on which the plants.grow, quoting one instance in which the soil contained 051 °/, CuO and the seed -26°/, CuO. It is assumed that copper must play some physiological réle in the plant, but no explanation of this action is yet forthcoming. Lehmann criticised Vedrédi's figures of the copper content of certain plant ashes, and the latter replied in a further Effect of Copper Compounds Ty paper (1896) in which he brings most Interesting facts to light. The quantity of copper in any species of plant varies with the individuals of that species, even when grown on the same soil, in the same year, and under similar conditions. The copper content of certain plants is put forward as a table, the years 1894 and 1895 being compared, and enormous differences are to be noticed in some cases. A quotation of the table will illustrate this more clearly than any amount of explanation. Milligrams of copper in 1 kilog. dry matter. 1894 1895 =—_— — “Seeds” min. max, min. max. ‘Winter wheat 80 710 200 680 Summer wheat 190 630 190 230 Maize 60 90 10 30 Barley 80 120 10 70 Oats 40 190 40 200 Buckwheat 160 640 150 160 “Fisolen” (Beans) 160 320 110 150 Linseed 120 150 110 150 Peas 60 100 60 110 Soy Beans 70 100 70 80 Lupins — 80 190 70 290 Mustard seed 70 1380 60 70 Paprika pods 790 1350 230 400 II Errecr oF CopPpER ON THE GROWTH OF HIGHER PLANTS. 1. Tome effect. (a) Toaic 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 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 muesdasie was sun owing to the lack of nutriment. In every 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 aula 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 i 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 eed “Other experiments ‘showed that barley roots are peculiarly sensitive j 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 14” x }”) for an hour, and even such water exercised ‘a very considerable retarding influence upon the root-growth, checking it entirely in in some instances. L Some years before True and Gies published their r results, Coupin (1898) had grown wheat seedlings i 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 (CuBr,) 004875 001387 Copper chloride (CuCl, . 2aq.) 005000 001865 opper sulphate (CuSO,.5aq.) “005555 001415 Copper acetate (Cu{C,H3Og}e. aq.) 005714 001820 Copper nitrate (Cu{NOs}.. 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, zine 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 (b) Masking effect caused by addition of soluble substances to solutions of copper salts. Experiments were carried on with barley, in which the plants were grown in the various grades of distilled water indicated above, both with and without the addition of nutrient salts. It was found that the presence of the nutrients exercises a very definite masking effect upon the action of the poisonous substance, so that the deleterious properties of the toxic substance are materially reduced. Later work, in which known quantities of such toxic salts as copper sulphate were added to pure distilled water showed that in the presence of nutrient salts a plant is able to withstand the action of a much greater concentration of poison. For instance, a concentration of 1 : 1,000,000 copper sulphate alone stops all growth in barley, but, if nutrient salts are present, a strength of 1 : 250,000 (at least four times as great) does not prevent growth, though the retarding action is very considerable (Figs. 2 and 8). 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. 4 3 ig, Photograph showing the action of copper sulphate on barley in the presence of nutrient salts. (March 5th—April 19th, 1907.) 4 5 6 Glass distilled water. Copper distilled water. 1/12,500 1/25,000 1/50,000 1/100,000 1/250,000 1/500,000 1/1.000,000 ” a ” a” ” copper sulphate. ” 7 8 | Effect of Copper Compounds 21 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 (Le. below toxic concentration) the neutralising action of the added salts was markedly less, and the growth rate never exceeded that of the gram. Total. 225 Ws | Shooi a I] i. PAL = Af Zi 8 4 2 1 4 2 41 @o 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.) Nore. 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 01 =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, eg. 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. (c) Effect of adding insoluble substances to solutions of copper salts. Other investigators have shown that the presence of insoluble substances has a similar effect in reducing toxicity to an even greater degree. True and Oglevee (1904, 1905) again used Lupinus albus as a test plant in the presence of solutions of various poisons in pure distilled water, copper sulphate, silver nitrate, mercuric chloride, 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 Liffect 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 CuSO,.5H,O 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. (a) 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, &., 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 °/, copper sulphate solution for ? 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: j,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, zine and mercury were intimately mixed with soil, 30 grams of the poison being added to 30-7 cubic decimetres of soil, two plants separated by a partition being grown on this quantity. The test plants were Polygonum Fagopyrum, Pisum sativum, Secale cereale and Panicum italicum, and all the plants developed strongly and normally except the last named. The Panicum developed very badly coloured leaves in an arsenic-containing soil, and the plants were killed soon after they started in soils containing copper. After harvesting, the crops were analysed and no trace of copper was found in any one of the 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°/, solution of copper’, 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. speake 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. (b) Spores and pollen grains. Miani (1901) brought fresh ideas to bear upon the problem of the action of copper on living plant cells, in that he sought to attribute the toxic or stimulant effects to an oligodynamic action, ie. 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. = or eB Us PD 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 (Oct. B3rd—Dee. 20th, 1912.) copper sulphate, . ” .) ” ” Effect of Copper Compounds 29 if it existed. He worked with Pisum sativum, var. arvense, Pisum arvense, Vicia Faba, var. equine Pers, and Fagopyrum esculentum Ménch, which were grown in glass distilled water, without any food salts, so that the plants were forced to live on the reserves in the seeds, which were carefully graded to ensure uniformity of size. It was found that in water cultures copper sulphate solutions down to 00000249 °/, (about 1 in 40,160,000) are harmful to peas, and still further down to ‘0000000249 °/, (about 1 in 4,016,000,000) the copper salts act as a poison rather than as a stimulant. Against this, however, is the state- ment that in certain soils copper sulphate acts as a stimulant when it gm 14 Total 12 b 10 PA 4choot yo qu /-- a ed Cn *8 4 ‘ /, i a +4 AL4 Cc 2 yr" a Root ~_—_— =i” ens ae for 90 10 4 2 1 4 2 -1 05 O 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 24 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} 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 °/, 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 Kriiger 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. (b) Effect of solutions of copper salts on leaves. Treboux (1908) 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 V/1,000,000 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 4/10,000,000 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 10cm. long in 100 cc. solution the plants were only slightly affected by -000015°/, copper sulphate, but in 500 cc. solution the shoots were killed after some days in 0000015 °/, copper sulphate, a concentration only: one-tenth as great. While it is evident that copper sprays have a definite action upon green leaves, whether favourable or unfavourable, the question arises as to the means whereby the copper obtains access. to the plant in order ° to take effect. Dandeno found that solutions of copper sulphate were absorbed by the leaves of Ampelopsis, forming a brown ring. Generally speaking inorganic salts in solution are absorbed through both surfaces of the leaves, whether the leaves are detached or not, provided the 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. Errect 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, B. 3 34 Effect of Copper Compounds Trabut (1895) found that on treating smutty wheat with a 2°/, solution of copper sulphate he obtained a mass of flocculent white mycelium, whose surface was soon covered with aerial branches bearing pale rose-coloured spores, and he gave the provisional name of Penicillium cupricwum 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 94 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. glaucwm 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 °/,—012°/,, the biggest crop being obtained. with both moulds in the strongest of these solutions. Hattori gives the optimum as being considerably lower for the two fungi mentioned, Penicillium being at its best in a solution of ‘008 °/, and Aspergillus in 004 °/,. A. Richter (1901) opposes this absolutely so far as Aspergillus niger is concerned. In his experiments copper appears invariably as a depressant, all concentrations from 1/150 to 1/150,000,000 giving growth below the normal, no stimulative action ever being observed. Zinc however proved to be a definite stimulant and in a mixture of copper and zinc salts in appropriate concentrations the toxic effect of the copper was completely paralysed by the stimulating action of the zine, 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, Menyanthes trifoliata, and Polygonum amphibium grew in the water unharmed by the addition of the poisonous substance. For some unexplained reason it seems that “the concentration of copper sulphate necessary to kill off the algae in the laboratory is five to twenty times as great as that needed to destroy the same species in its natural habitat.” Conclusion. Altogether, after looking at the question from many points of view, one is forced to the conclusion that under most typical circumstances copper compounds act as poisons to the higher plants, and that it is only under particular and peculiar conditions and in very great dilutions that any stimulative action on their part can be clearly demonstrated. CHAPTER IV EFFECT OF ZINC COMPOUNDS J. Presence or 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 “zine 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 zine, which rises as high as 20°/, in places. The flora of the soil is very diversified and zinc has been determined qualitatively in most and quantitatively in some of the plants. Viola tricolor and Thlaspi alpestre are most characteristic under such circumstances, both showing such constant habit changes that they resemble new species, while other plants such as Armeria vulgaris and Silene inflata are peculiarly luxuriant. Risse’s figures of the zinc content of these four plants may prove of interest. The figures are based on the dry weights, air dried. Thiaspi alpestre, var. calaminaria. Root 6:28 °/, ash, 0°167 °/, ZnO, 1°66 °/, ZnO in ash. Stem 11°75°/, 0'385°/, 45 3°28°/, 5 ” Leaves 11'45°/, ,, 150°/, 5 13°12°/, ,, ” Flowers 8°49°/, ,, 0°275°/, 5 324°), ” Viola tricolor. Root 5°59 °/, ash, 0:085 °/, ZnO, 1:52°/, ZnO in ash. Stem 10°55 °/, 5, 0:065°/, 5 0°62°/, 5 ” Leaves 9°42°/. ,, 0110°/, 5, 116°}, 3) 5 Flowers 7°66 "le ” 0-075 “lL. ”» 0°98 “ihe ” Effect of Zinc Compounds 37 Armeria vulgaris. Root 4°74°/, ash, 0°17 °/, Zn0, 3°58 °/, ZnO in ash. Stem 537°], 0°02°/, 0°37°/, ” Leaves 9°36°/, ,, 011°/, 5, 117°/, 455 5 Flowers 6°08°/, ,, 0:07 °/, 5 115°/, 55 a Silene inflata. Root 2°71 °/, ash, 0-02 °/, ZnO, 0°74°/, ZnO in ash. Stem Leaves 11:43°/, ,, 0°22°/, 4 192°/, 3s 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—10°/, of ash, except in the abnormal case of plants growing on calamine. Lechartier and Bellamy (1877) demonstrated the presence of zinc in such food substances as wheat, American maize, barley and white haricots, but they failed to find it in maize stems and beetroot, so they cautiously concluded that if it does occur in the latter cases it must be far less in quantity than in the former. Hattensaur (1891) analysed the ash of Molinia cerulea and discovered the presence of copper, manganese, zinc and lead, zinc oxide forming ‘265 °/, of the total ash, (006 °/, of the air dried plant). Jensch (1894) observed that the flora on calamine soils was some- what scanty, the chief plants that came under his notice being Tarawxa- cum officinale, Capsella 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 Zine Compounds purple red colour. The following figures are given for the quantities of zine carbonate (ZnCO,) in the ash of these two plants :— Tussilago Farfara. Root Leaf-stalk Leaf-blade 2°51 °/,—3:26 °/, 1°75 °/,—1°63 °/, 2°90 °/,—2°83°/, ZnCO3 =1°629°/—9-115°/, 1186 °/,—1°058°/, «1882 °/, 1836 °/, ZnO. Polygonum aviculare. Root Stem Leaves 1°77 °/,—1:93 °/, 2°25 °/,—2°86 °/, 1:24 °/,—1°49 °/, ZnCO; = 1°148 °/,—1°252 °/, 1:46 °/,—1°856 °/, "804 °/ — 967 °/, ZnO. Other analyses of plants from zinc soils as against controls from normal soils indicated the high water and high ash content of the zinc plants, though the dry matter was low, and it is suggested that the increase of the ash may be connected with a stimulation caused by the zinc salts, unless it is due to phosphoric-acid hunger, since the calamine soils 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 zine occurs in some quantity. Javillier, however, is of opinion that zine 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 zine works, and showed that the plants evidently absorb smal] quantities of zinc from their surroundings. He also regarded zinc as a normal constituent of certain plants. I. Errecr or Zinc on THE GRowrH or HIGHER PLANTS. 1. Tose effect. (a) Tosxic action of zine 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 Zine Compounds 39 Baumann (1885) carried out further experiments and concluded that zine salts are far more toxic than Freytag suspected, 44 mg. zinc sulphate per litre? killing plants of 13 species belonging to 7 families (Coniferae excepted). The various plants withstand the action of the zinc salts in different degrees, ‘the same concentration killing off the species in different times. With the 44 mg. zinc sulphate the following results were obtained :— Trifolium pratense killed in 16 days Spergula arvensis 3 21, Hordeum vulgare ss 30 ,, Vicia sativa 5 31 ,, Polygonum Fagopyrum __s,, 60 ,, Beta vulgaris s 76 4, 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. (b) Effect of soluble zine 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 zine remaining quite healthy. Certain grasses took longer to kill than parley, thosé with 4 gm. zinc sulphate per litre dying in about seven weeks, while 18 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 44mg, Zn80,.7H,O=10 mg. Zn=1/22,727 Zn8O,.7H,0 approx. 40 Effect of Zine 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 h the plant is able to make some slight gm. 6 Total Ce e. , eee ene _a|—-—j]~-—| Root ° 100 20 10 4 2 1 a 2 a 704 =-02 o 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 ZnSO, 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. Ata concentration of 1 in} or lin} 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 zine sulphate on pea plants in the presence of nutrient salts. Control. 1/5,000 1/10,000 1/50,000 1/100,000 1/250,000 1/500,000 1/1,000,000 1/2,500,000 (Sept. 80th—Dec. 20th, 1912.) zine sulphate. Liffect of Zinc Compounds 41 in advance of that of the shoots, probably on account of the contact of the roots with the poison. In the greater stretigths of such poisons as zine and copper sulphate root growth i 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. ga. 2 1-0 aan Total 8 - 7, 2 a >= ===> T= | shoot <6 3 // 4 Li EG C4 Ms wo (oon = SSS Le ee Root “— i oft 200 = 100 20 10 4 2 ( 4 2 1 05 0 1=1:1,000,000 Fig. 8. Curve showing the mean values of the dry weights of nine series of pea plants grown in the presence of anhydrous zinc sulphate and nutrient salts. (May 18th— June 28th, 1910.) (c) Effect of zine compounds on plant growth when they are present in soils. As soon as the presence of zinc in members of the vegetable kingdom was established the question arose as to its effect upon both the plant and the soil. Gorup-Besanez (1863) grew plants in soil with which 30 grams of metallic poisons such as CuSO,, ZnSO,, HgO, were intimately mixed with 30°7 litres (“cubik Decimeter”) of soil, 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°/, 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 zine compounds as they filter through the soil, by decomposition by the salts of the soil. For field earth the limit of absorption of zinc oxide from zinc sulphate is between ‘21 °/,—'24°/, of the earth. F. C. Phillips (1882) corroborated Freytag’s statement as to the absorption of small quantities of zinc by the roots of plants, but he states as a fact that both lead and zinc may enter plant tissues without causing any disturbance in the growth, nutrition or functions of the plants, a conclusion that is obviously incorrect or at least incomplete in view of later work on the subject. His choice of plants was certainly unusual, including geraniums, coleas, ageratums and pansies, the poison used being zinc carbonate. Holdefleiss (1883) stated that in spite of a soil content of 2°/, zinc the vegetation was not in any way harmed, clover fields and meadow lands on zinc soil presenting a normal appearance. This observation was quite inconclusive, as the author proceeds to say that of the plants that were able to absorb zine 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 CO, 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 zine 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 Zine 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 (1888) noticed that the direct poisonous action of zine 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. (ad) Mode of action of zine 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, ie. 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 zine. 2. Effect of zinc compounds on germination. In the course of his investigations on the influence of zine 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 Zine 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, (b) water with ‘9 gm. ZnO per litre, which was frequently changed. These seeds were then placed in the dark on filter papers soaked respectively with water and with the solution 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 zine 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 cc. 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 Zine Compounds 45 3. Stimulation induced by zine 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 réle 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)}, and found that while zinc sulphate alone at m/8192 retarded growth, yet with m/2048 ZnSO, and m/256 calcium sulphate growth was more than twice as rapid as in controls grown in water, indicating a marked stimulation. The presence of the calcium exercised a definite ameliorating influence, reducing the toxicity of zinc to one-sixteenth at most. The hypothesis put forward is that interior physiological 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—ie. 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 Zine Compounds Kanda (1904) found that peas were stimulated in dilute solutions of zine sulphate in the absence of nutrients, the optimum concentration being between ‘00000287 °/, and ‘000001435 °/, (about 1 in 34,840,000 and 1 in 69,700,000), higher concentrations being poisonous when the solutions were changed every four days. Jensen (1907) stated that he obtained no stimulation at all with water cultures, even in a solution as dilute as n/100,000 (about 1 in 1,289,000), 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 zine 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 ZnSO, approximates closely to that of the controls. Beyond this the growth varies in different experiments, In some cases lower concentrations from 1/5,000,000 to 1/50,000,000 seem to cause some slight improvement in comparison with the normal, indicating a possible stimulus, but this improvement is not at all well marked. In other cases these great dilutions are apparently indifferent, neither a poisonous nor a stimulative action being exerted on the growth of the plant (Fig. 6). With peas some increase has been obtained with 1/20,000,000, and although the rise is only slight, yet it is possible that it may indicate the setting in of a stimulus which would make itself more strongly felt with still weaker concentrations (Fig. 7). (b) Stimulation in sand cultures. While Jensen denied stimulation in wheat grown in water cultures even when the solutions were as dilute as n/100,000 zinc sulphate, yet he found increase of growth with the same plant in artificial soil (quartz flour) to which much stronger solutions of zinc sulphate, from 5n/10,000—n/10,000, had been added. (c) Increased growth in soil. Nakamura (1904) dealt with a few plants of agricultural importance, adding ‘01 gram anhydrous zinc sulphate to 2300 grams air-dried soil. 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 Rothamsted experiments with water cultures in that zinc sulphate proved to be less toxic to peas than to barley. Kanda found that both peas and beans when grown in soil as pot cultures were improved by larger quantities of zinc sulphate than when they were treated as water cultures—a result in full accordance with current knowledge. Wheat is evidently peculiarly sensitive to the effects of zinc 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 zine fumes. Zinc oxide from the fumes is de- posited on the leaves, and Klopsch stated that the rain and dew containing 48 Effect of Zine 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 °/,) 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°/, zine sulphate, (8) fresh ‘00008 °/, zinc sulphate, (4) water again, it was found that while the first solution of zinc sulphate had apparently no effect on assimilation, yet during the second immersion a gradual reduction in assimilation set in, which reduction was continued after the return to pure water, so that the toxic action of the zinc sulphate upon the shoots was clearly demonstrated. III. Errect 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, (8) 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 zine cannot replace iron, as both are essential. Ono (1900) determined the relation between the weight of the mould crop in grams and the quantity of sugar used up in the presence of varying amounts of zinc sulphate. The amount of sugar used was always greater in the crops with -0037—0297°/, zinc sulphate by weight than in the control crops, indicating a stimulation caused by zine. 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 zine 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 zine 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 zine 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, (6) Raulin’s solution without zinc. The ratio of crops a/b varied from 2:°3—8"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 zine present in dilute solutions being taken up, only part being utilised in stronger solutions, The value of accordance between the quantity of zine fixed and the quantity supplied decreases rapidly with increase of concentration. Sterigmatocystis is able to fix without harm a quantity of zine 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 Lffect of Zine 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 O—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. Ifa 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 p gr. (= thousandth part of a milligram)". Wheat (Victoria—complete grain) “7 » (from Franche Comté) "85 White bread “71 Whole green cabbage 2 Outside leaves of cabbage ‘0 (absent) Green haricots 0 »y Turnip “36 Potatoes 112 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, Errect oF ARSENIC ON THE GrowTH oF HIGHER PLANTS. 1. Tomic 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 1 oM8T:, 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 (As,O;) 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 (= 1/—2/20,000 arsenic acid). If however the plants were allowed to form 10—15 leaves in a pure food solution and then when strongly rooted were transferred to a solution of ‘05 gm. arsenic acid per litre, they were found to grow strongly and develope big healthy leaves. Careful measurements 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. As,0, with P20, 2°84 gm. dry wt. 005 gm. ,, » 9 2°37 ” ‘01 gm. As,05 ” ” 67°32 ” 40 » 1» » oO” 64:13 ” 03 «=, As,O, without P.O; 39°98 es 07 yy oy ” ” 42:13 ” normal solution ,, 5 12°93 = “5 ‘5 with ,, 65°84 9 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. As,O, 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 __,, 5 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).