Section B—Chairman's Address Biology and the Trace Elements

Transactions and Proceedings of the Royal Society of New Zealand, Volume 82, 1954-55, Page 871

Section B—Chairman's Address Biology and the Trace Elements Dr.H. O. Askew, Cawthron Institute, Nelson. During the many thousands of years that man has been an agriculturist and horticulturist he has accumulated a vast store of empirical knowledge concerning the most satisfactory methods of handling his soils and his crops and animals under the varied conditions of the countries in which he lived. Roman and Greek writers have preserved a number of these traditional methods of working. Authors in the Middle Ages and others down to our own times have continued the recording of these ways of the man on the land. For some of these methods of operation we can give a rational explanation but for others we cannot. He is a bold scientist who will glibly say that any particular method is wrong and should be changed. A very broad and complete knowledge of all the conditions under which a certain field or crop is being handled is necessary before radical changes from traditional practice are made. Pliny the Elder, in the first century of our era, said: “In every subject there are certain deep and recondite secrets, which it is left to the intelligence of each to penetrate.” The agriculturist, horticulturist and stock breeder have been trying to do this for many centuries, and latterly the scientist has entered the field. Looking at the nutrition of man and his animals we find that, the early scientific approach was on the basis of supply of energy—how many calories would the food supply—and on the proportions of the proteins and carbohydrates in the foodstuffs. But this was found to be unsatisfactory. Attention was later given to the amounts of inorganic material present and to their proportions, especially of calcium and phosphorus. Here again the theoretically satisfactory diets failed to give that good health to the recipient of the food that had been hoped for and expected. Then in 1912 the first vitamin was isolated, to be followed later by a spate of work on these minor organic constituents of plants and animals that has by no means ceased yet. In fact, we are still passing through a period in which talk of and use of vitamins is distinctly fashionable. Related, although not directly allied with the vitamins, are the hormones, but we shall not deal with them in our consideration of trace elements. Even with the addition of vitamins to the armament of the nutritionist everything was not satisfactory. A number of obscure ailments of humans and domestic animals, and also of plants, could only be classed as being due to a “physiological disturbance”. Many of these have later been shown to be due to lack or excess of inorganic elements, now classed as minor, trace or micro-nutrient elements. Just as vitamins became fashionable to discuss and to use so have these inorganic elements become. We are still in that period where it is fashionable, and sometimes even as a means of escape from any other explanation, to say that a certain ailment is due to a “trace element” deficiency.

One has only to pick up any volume of a chemical, botanical, horticultural, agricultural or biological journal nowadays to see over how great a range of inquiry concerning the life of plants or animals there is a very lively interest in those chemical elements which have come to be known as minor or trace elements, or more correctly, so far as the majority is concerned, micro-nutrients. Minor is certainly a misleading term to use because, as we shall see later on, although these elements operate in quantities small in relation to the commonly so-called major elements, such as calcium or potassium, their presence in amounts often minute is as fully necessary for a healthy life for the organism, whether an animal or plant, as those present in perhaps a million-fold greater concentration. Bertrand has said: “The body cannot be regarded as a democracy, but rather as an oligarchy in which a large amount of passive elements is ordered and governed by minute amounts of active ones.” Conversely we may also find that some of these micro-nutrients enter the organism in undesirably large amounts—although they may still only be in concentrations conveniently expressible as parts per million rather than as parts per centum. “When this occurs the animal or plant suffers more or less serious disturbances in its life processes and it becomes ill, having been forced into an abnormal manner of living. Moreover the level of intake of the micro-nutrient may be governed by lack or superabundance of a major or of another minor nutrient element. This condition may develop with or without the assistance of man himself, but the result is that the plant or animal living under those conditions is forced into departure from that normality which we call health into an abnormal or unhealthy state. Also the offending chemical element may enter the organism only at what may be perhaps called a second-degree stage. As an example we may note how a large intake of selenium by forage plants may not affect their normal development, but that that forage when eaten by a grazing animal will result in very serious disturbances to its health. Marco Polo noted such ill-effects on horses during his travels in China: it is now known that selenium was the-offender. The periodic table of the chemist with its 92 elements (we need not concern ourselves with the later additions of transuranic elements) provides a convenient means of showing the properties of those elements relative to one another. Can this arrangement based on chemical and physical properties give any indication as to the requirement of living matter for a given element'. We must, however, first satisfy ourselves whether a given element is invariably present and whether it is necessary for the growth and development of the organism, or whether it is in the organism merely because no means are available for its rigid exclusion from entry. Spectroscopic examination of plants and animals has shown that nearly every element of the periodic table has been reported as being present in some form of living material. It does not appear to be acceptable that all of these elements will be necessary for the living processes in an animal or plant, until one recollects that perhaps a thousand different enzymes may occur in animal cells and that in general an enzyme has associated with it as an activator a metallic element of our trace micro-nutrient class.

Now there are three main criteria that must be satisfied before an element can be admitted as an essential element necessary for the health of the organism. Arnon1 has pointed out that the acceptance of each element as an essential micro-nutrient has preceded knowledge of the biochemical function that it performed. For example with boron, manganese, copper and zinc, agricultural practice has preceded knowledge of the manner in which they work. Moreover, Arnon says, “The inconclusiveness of soil experiments with regard to the essentiality of micro-nutrients is in sharp contrast to the decisiveness, whenever available, of biochemical evidence on the specific function of a micro-nutrient element.” Arnon's criteria of essentiality which we shall adopt are as follows. “(a) a deficiency of it makes it impossible for the plant to complete the vegetative or reproductive stage of its life cycle, (b) such deficiency is specific to the element in question and can be prevented or corrected only by supplying this element, and (c) the element is directly involved in the nutrition of the plant quite apart from its possible effects in correcting some unfavourable micro-biological or chemical condition of the soil or the culture medium.” He goes on, “The criterion of the foremost physiological significance is the requirement of an inorganic element for the successful completion of the life cycle of a plant. This is, of course, different from merely demonstrating a favourable effect on growth.” But we are still left with another matter: if an element is shown to be essential for one plant must it also be essential for all plants? I think that we must admit the second part of this statement to be true, but in practice we may perhaps be misled owing to the varying requirements by the several species, or even varieties within a species, being so greatly different. We are in fact dealing with a question of degree rather than of absolute necessity for the element concerned. Let us consider some examples of the effects of micro-nutrient elements at a test of Arnon's criteria. We will take as our main example his work on the effect of traces of molybdenum on the tomato plant. First of all the nutrient solutions that were proposed for use were rigorously purified to ensure that only minimal amounts of any unwanted element should be added, the water was redistilled from glass so that its total metal content was less than 0001 p. p. m. The degree of purification was such that reproducible results could be obtained from minute additions of each individual metallic element under examination: for example, response of growth from zinc at a concentration of 1 part in 200 millions of water was obtained, the additional me being only 0 001 mg. per plant. Tomato seedlings grown in nutrient solutions purified in this way, and supplied with B, Mn, Zn and Cu soon developed characteristic leaf symptoms, a mottling different from any previously known symptom, followed by necrosis and incurling of the margins. In addition the blossoms fell and no fruit was set. Thus the first criterion was satisfied—the life cycle could not be completed. A supplementary solution named B7, because it contained the seven elements. Mo, V, Cr, Ni, Co, W and Ti, was added, when normal growth was obtained by separate trials the effective element was found to be molybdenum. For prevention of the symptoms only 1 part of molybdenum in 100 million parts of solution was necessary. Now, Arnon had another nutrient mixture. C13, containing a further thirteen elements. These were Al. As,

Cd, Sr, Hg, Pb, Li, Rb, Br, I, F, Se and Be. Addition of this mixture in conjunct on with the previous elements, except Mo, did not produce healthy growth; on addition of Mo all was well again. When all these were omitted and Mo was added, growth was satisfactory. Thus it was shown that the second criterion was satisfied: Mo could not be replaced by any other element tested—its function was specific. The third criterion that the effect should not be obtained by any indirect influence through the soil was tested by spraying the affected plants with a very dilute solution of molybdic acid, containing 1 p.p.m. of Mo. The symptoms disappeared and normal growth was obtained. Hence, molybdenum was required for normal development and not to overcome any abnormality of a soil. To see how the soil factor may complicate matters consider the following data on cotton, grown on the same field but in two separate experiments:2 Treatment. Yield Ib./acre. Treatment. Yield Ib./acre. (1) Featilizen NP 965 (1) Fertilizer NPK Mg 1320 (2) as (1) with K (sulphate) 1205 (2) as (1) With 5Ib MnSO4 2040 (3) as (1) With K (murnate) 1385 (3) as (1) with 10Ib CuSO4 1960 The data on the left-hand side certainly indicate that a worth while response was obtained from applications of potassium compounds. Similarly the data on the right-hand side show that the field was also deficient in available manganese and copper (although it is somewhat curious that such a small application of manganese sulphate to the soil should have had such a marked effect). In this second experiment the amounts of nitrogen and phosphate-used wire the same as in the first, but further potassium and magnesium had been added. On the face of it the soil was potassium deficient. The author-dealing with the first experiment were aware of the results of the second and therefore did not commit themselves to an unqualified potassium deficiency. The workers in the second experiment did not express an opinion on the potassium question. But obviously either experiment could be misleading. These experiments will lead us on to a consideration of the effect of one element on the absorption of another. One of the earliest tissue tests, of the type now used frequently in the diagnosis of mineral deficiencies, was Hoffer's test for potassium deficiency in maize: the test was not for potassium but for iron which he found was deposited at the nodes of the stalk when potassium supply was low. It has been found since though that even manganese may be deposited at the nodes when there is an excess of available manganese in the soil. To control the deposition of iron, applications of potassium, copper or even manganese are effective. When excessive manganese is present the uptake by the plant may be still further increased by the use of soluble fertilizer salts.3 Over a period of years a considerable amount of work has been done on the relationship between iron and manganese supply and the health of the plant. At one time it appeared that a fairly defined ratio between iron mid manganese was required.4 Excess of iron intake will produce symptoms very similar to those of a deficiency of manganese, and conversely excess manganese leads to an apparent iron deficiency. But is it not also possible that the symptoms are respectively due to toxic amounts of iron and manganese? We

shall see later that the micro-nutrient elements, especially the metals, act in the animal and plant as parts of enzyme systems. Now these systems are usually specific: that is, a given enzyme carries out only one reaction. In some cases replacement of the necessary metal by another will permit functioning of the enzyme at a reduced rate, in other cases the reaction stops when such replacement is made. So suppose that the enzyme that we are considering is activated by iron, then if an excessive supply of manganese is presented by a mass action effect some iron will be displaced from the enzyme system and manganese will take its place, with the result that the necessary chemical reaction which that enzyme performed will be reduced or stopped. The life of the organism will no longer be normal and symptoms of some kind will be manifested. As often as not the symptoms in leaves take the form of a chlorotic pattern; and most “deficiency disease” symptoms are chloroses at some stage. We have therefore produced by unbalance in the nutrient supply what is apparently a deficiency disease. Workers at Long Ashton Station in England have carried out a large amount of work with Cu, Co, Ni, Mn, Zn, Pb, Cd, etc added to nutrient solutions in toxic amounts.5 In nearly all cases the chlorotic symptoms that developed were suggestive of iron deficiency, although some elements produced symptoms characteristic to themselves. That some of these elements may occur naturally in abnormally large amount is indicated by the occurrence of striking symptoms, especially in oats, of crops grown on an Aberdeenshire soil, which, as workers at Macaulay Institute have shown, contains a large amount of available nickel.6 The identification of a disease, whether due to an excess or to a deficiency of a minor nutrient requires that the investigator should be thoroughly acquainted with the normal appearance of the animal or plant with which he is dealing, taking into account the stage of development and the effect of seasonal conditions. In short he must know what the “normal” should be. He must draw upon his experience to decide whether anything is abnormal, and then try to suggest the cause of that abnormality. If he is a chemist he analyses suitable material, perhaps of an organ of an animal or of a particular part of a plant. What he examines will be governed by his previous experience of what material proved suitable in the past. His analysis may show that a certain element is present in unusually small or large amounts, but he must be on his guard not to be misled by either condition, because a sick plant may be suffering from a deficiency of a given element and yet that element may be present in larger amount than in his comparable healthy material. Again unusual amounts of certain constituents, for example nitrates, may suggest that the cause of the trouble is lack or excess of a given metallic element. Having decided that he is dealing with a particular element he can check his conclusions by growing his plants under controlled conditions or by suitable treatment of the crop in the field. In general animals may be dealt with on similar lines. On the other hand the investigator may not be able to tie down the symptoms to any one element: he must then try the effects of several elements each alone or in combination with one or more of the others. You will see, therefore that his method of working-may be somewhat empirical. Especially with plants may the symptoms expressed be very similar as the result of under-, or over-, supply of an element. And at all times he must bear in mind criteria of essentiality similar to those proposed by Arnon, which have already been discussed.

But there is a word of warning that may perhaps be issued to us as chemists, and conversely to biologists. Pliny two thousand years ago said, “The very signs also from which we form our judgment are often very deceptive”. We have already seen that there are often similarities in the symptoms exhibited by a given plant when it is suffering from a deficiency of one or another micro-nutrient. A combination of deficiencies may even be present, but the symptoms appear to be due to absence of one element only. But I have in mind something rather different from this: it is that fungus or virus diseases, or the reaction to an insect pest, may cause a plant to exhibit symptoms that suggest the presence of a nutritional deficiency. The final result so far as the plant is concerned may even be a deficiency, or at any rate a disturbance in the action, of some chemical element, but that upset is not the primary cause of the symptoms. Therefore I would suggest that when a diseased specimen indicates a previously unknown nutritional deficiency the chemist would be well-advised to call in the assistance of his colleagues, the plant pathologist and the entomologist, before finally deciding that the cause of the unsatisfactory condition of his specimen is primarily nutritional. It has been remarked that “without enzymes there can be no life”7 and also that “There exists a common, fundamental chemical ground-plan of composition to which all animals, and very probably other living organisms also, conform”.8 And again, quoting. “Nature knows only a few fundamental principles which she cleverly adapts to different purposes and circumstances”.9 The expectation, therefore, is that we shall find the same enzyme in both plants and animals. A classification of enzymes, with their accompanying metals, has been given by Seekles, as follows (modified)10 (1)Synthests and destruction of tissue elements (mainly Poten) A. d-Pepttidases: Mn. Co. Fe, Zn, Mg. B. Argrnase — Mn. Co. N1. Ca. V. C. iso — Citric acid dehydrogenase: Mn, Mg. (2) Energy Change (oxrdation - reduction) A Haemoglobin: Fe Haemocyann: Cu Raspiratory: Enzy me Fe Cytochromes: Fe. Mn. Peroxydase: Fe. Mn. Transferen of Oxygen Catalase: Fe Oxidases: Cu B. Phosphate tiansfen: Mg, Mn. C. Enolase: Mg, Mn, Zn D. Catboxylase: Mg. Mn. Zn E. Pyruvne acid dehydhogeans: Mg. Zn F. Phosphases (vanous): Mg G. Zymase (yeast): Mn H. Carbonic acid anhydrase: Zn J. Phodphohpoid oxidation: V. K. Lipases: Ca. Mn. (3) Detixucatuib reathons A. Tyrosinase: Cu B. Polyphenoloxydases: Cu C. Diamno-oxydase: Co (4) Transference of nervous stimult A Chlinesterase: Mn, Mg, Ca, Ba.

As indicated in the above table the metals may act in two ways (1) by being bound in enzyme complexes or (2) by behaving as an activator of the enzyme, and thus indirectly exerting an influence on metabolic processes. It is interesting to note too in the above table that Cu, Fe and Mg act most frequently in the bound form in a number of enzymes. Zinc is given only in carbonic acid anhydrase in the above table, where its presence is extremely necessary, because this anhydrase governs the rate of reaction of water, carbon dioxide and carbonic acid in both plants and animals. Magnesium is required for the working of a number of enzymes, especially those concerned with the transfer of phosphate, which is so important in the respiratory cycle and other reactions. In these functions magnesium is not replaceable by any other element and is therefore an indispensable element for animals and plants. Vanadium appears only once, as an activator for oxidation of phospholipoids. However, this element is so widespread, especially in marine organisms, that it may carry on some other function in life. For example many ascidians have green pigments containing vanadium. This vanadium is not combined with a protein and is not an oxygen carrier and thus has no respiratory function. On the dry-matter basis, the vanadium content of the pigment varies from 0.04 to 0 19 per cent.11 We should also note the relatively large amounts of vanadium in many petroleums, apparently as a porphyrin complex,12 and in the ash of coal. A notable omission from the above table is molybdenum. From the very severe disturbance that occurs in plants when this element is lacking it must be very important in the normal living processes. It may play some essential part. in animal life too, judging by the recently demonstrated relationship between xanthm oxidase activity and molybdenum concentration in the liver and intestines 13. It may well be that the occurrence of xanthin calculi in sheep in tho Nelson district is due to a deficiency of molybdenum because the soils and pastures of the area in which this ailment occurs are very low in that element. It having been established that small quantities of the micro-nutrient metals are required for the working of enzymes, further interesting questions arise. Most of the quantities of micro-nutrients that have been mentioned already have sounded very minute. In what relation do they stand to the numbers of atoms available and to the number of cells among which they will be distributed. The position for a selection of cells and of elements is given in the following Table I (see also Table IV) Table I Orgarnism. No of Cells. Analytreal Basis. Atom-per Cell Milhons. Human body 1×1012 01 pp. m Fe on fresh basis 75 Fe 7 Blod (Human) 5×1012 per litre 034 0/0 Fe in Hb. 1.000 Fe Apple (100 gm. tresh weight) 5×107 10 pp. m B in D.M. 200,000 B Apple ((100 gm. tresh weight) 5×107 001 p.p.m Mo in D.M. 20 Mo water (purthed) — 0.00001 p.p.m Mo (001 gm per liter) 60,000 Mo* Per cubic centimetre.

Iron if dealt with in the first two entries. For the human body as a whole-about 75 trillions of atoms of iron are present on the average per cell, but in each red blood corpuscle more than ten times that number are present. We would expect to find a large number in the latter, but it is surprising to find so many iron atoms in the tissue cells of the body. Passing now to the element-boron, we find that in the apple fruit, where about 10 p.p.m of boron in the dry matter is necessary for healthy development, each cell contains about 200.000 millions of boron atoms—truly a remarkable number! If, from data recently obtained at the Cawthron Institute, for the molybdenum content of apple fruit, a figure of 0.01 p.p.m in the dry-matter, a level that would be associated with deficiency symptoms in vegetative material, is taken, then calculation shows that 20 millions of molybdenum atoms would be present in each cell Arnon and Wessel (Nature 1953, 172, 1039). In discussing the essentiality of V for Seenedesmus obliquus (green alga), state that the minimum requirement of Seenedesimus for Mo was about 1,200 atoms per cell, and no improvement in growth occurred with > 0·1 μ gm. Mo per litre, but growth increased with up to 100 μ gm per litre of V. To return to blood, it is reported that sheep's blood may contain 0.006 mg of molybdenum per 100 ml;14 this type of blood averages 10 × 1012 cells per litre, whence each cell is calculated to have 35,000 atoms of molybdenum per cell. This number you will note is much lower than those with which we have dealt so far. As a final example lot us consider the position of water used in nutrient experiments with micro-nutrients. Perhaps the most rigorous purification for this purpose has been earned out by workers at Long Ashton Research Station in England. Let us take one of their nutrient solutions, prepared from purified water to which 0 00001 part per million of molybdenum was added15 You will agree that this is a very minute amount to add and it is indeed a tribute to the experimental skill of those concerned that consistent results could be regularly produced with this small addition to the purified water. How many atoms of molybdenum were added per cubic centimeter? The answer is staggering—60,000 million atoms'. And one cannot grow a plant with one cubic centimetre of water. We are impressed therefore with the enormous numbers of atoms of a micro-nutrient which are supplied even when we are working at levels where symptoms of lack of an element may be only too clearly seen in the condition of the plant or animal. Why is it then that these enormous numbers of atoms are insufficient to maintain the organising in a healthy state9. One worker has suggested that the minimum amount of an element for healthy growth should be such that at least one molecule of the nutrient is provided for each cell of the organism16. The calculations, which have just been presented suggest that this statement require some modification. Perhaps the following would be more suitable the minimum amount of a micro-nutrient required for optimum healthy growth should provide one atom of the micro-nutrient element for each molecule of the enzyme for which it is an activator or is bound in a complex with the enzyme. You will note that two qualifications appear in this statement, optimum and healthy, because we must allow that according to the degree of insufficiency the enzyme will not be fully effective in its necessary duties, and thus the organism will not be in an optimum condition of health; moreover un excessive supply of the element may

lead to a disturbance in the action of the enzyme, so that again the organism will not be healthy. In order to obtain an idea of the order of magnitude of enzyme and micro-nutrient contents let us consider ascorbic acid oxidase. Assuming that it is present in apple tissue to the extent of 0 01 per cent of the dry-matter, and that it has a copper content of 0 25 per cent., then this amount of enzyme would require the presence of 0 25 p.p.m. of copper in the dry-matter of the apple. Actually 5–10 p.p.m of copper have been found. It is of course more than likely that ascorbic acid oxidase is not the only enzyme present in that tissue that will require copper; and in any event there is probably a mass action relationship between the total copper present and that existing in the enzyme. Now how are those quantities related to the individual cell2. Already it has been assumed that an apple has 5 × 107 cells whence, assuming that apple flesh has 15 per cent of dry-matter. 1 Kgm of dry-matter contains 3.3 × 109 cells. Now 0 25 mg of copper corresponds to 2 75 × 1018 atoms, whence each cell contains 8 × 108 copper atoms per cell. Working in the reverse direction, to calculate how many molecules of enzyme are present per cell, we take the molecular weight of the enzyme molecule as 150,000, then for a concentration of 0 01 per cent, in the dry-matter, there are 1.3 × 108 molecules per cell. Since each molecule of enzyme has 6 atoms of copper associated with it there are 8 × 108 atoms of copper per cell, which is the result that was obtained from the analytical data for copper (The same result must be obtained because the data used in the two calculations are not independent.) Data for the copper content of apple flesh from copper-deficient trees do not seem to be available, but on deficient trees the leaves may contain only 2 p.p m of copper in the dry-matter compared with a normal figure of at least 5 p.p.m. Even allowing that several enzymes will be in competition for the copper it appears that, unless the enzymes are present at much greater concentration than the 0.01 per cent allowed for the ascorbic acid oxidase. there should be sufficient copper present in a deficient leaf for the enzymes to be active: in deficient leaves it is probably not correct to assume however that the activity of the enzymes is reduced to zero. If this were so then the plant or other organism would collapse completely. But the relatively small range of copper content from deficiency to sufficiency demonstrates the closely defined condition of nutrition under which the enzyme is called upon to work to maintain the organism in health. All our astronomical figures for the numbers of atoms and of cells therefore reduce to a simple question of degree of supply—will it be halved (say) to produce deficiency conditions, or should it be doubled (say) to carry the organism from ill-health to good health? And with this increase in knowledge of the functions of the micro-nutrients in biological material the stage has been passed when the gibe could be made that the determination of these elements in living material was little more than a useful analytical exercise. The chemist's best representation of the relationships of the elements to one another is given by the Periodic Table. Unfortunately it is usually convenient to give this table in a planar form, which to a considerable extent obscures the gradual change in properties from the elements placed at the right-hand side to those on the left-hand side. Allowing for this, the following form of the table. Figure 1

based on that of von Antropoff, seems to be the best for the present purpose, which is to see whether a satisfactory classification, preferably in an elegant form, car be made of elements essential for the healthy development of plants and animals (Fig. I). In this diagram a full line has been drawn to include the following 26 elements, which appear to be essential to living matter: H, B, C, X, O, Na, Mg, Al, Si, P, S, Cl, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Br, Eb, Mo, and I. It is notable that most of these elements are of low atomic weight; in fact the first 14 are of atomic weight of 40 or less, but of these only B would probably be readily accepted as a micro-nutrient. Many workers in different parts of the world have shown the necessity for a supply of available boron if plants are to grow in a normal way, Schropp has given a list of 158 species for which boron has been shown to be essential17. Even for those plants for which at present boron appears to be non-essential it may well be a question of reducing the boron content of the nutrient medium to a lower value than that reached so far by any experimenter if deficiency symptoms are to be demonstrated, rather than that these plants do not require boron at all. On the otter hand animals do not seem to require boron for normal development, although it is widely distributed in animal tissues. The position of sodium as an indispensable nutrient does not appear to be firmly decided as yet. There is no question however that in many plants it acts at least as a stimulant to growth. Complete replacement of potassium by sodium is not possible, although partial replacement may lead to better growth, the beet family is outstanding in this respect. Among animals sodium has an important position because of its occurrence in relatively large amount in the blood. The concentration of sodium in body fluids varies greatly, being almost the same as in sea-water in many marine invertebrates but it is present in relatively small amounts in the fluids of earthworms aid insects. Among vertebrates the sodium content of the body fluids is

not high and does not show the same large range of values as among the invertebrates, but again the marine have more sodium than the amphibious or terrestrial organisms. Magnesium is well-known as a minor element, being present in most plant material to the extent of about ¼ to ½ per cent, of the dry-matter. Lack of sufficient supply of available magnesium leads to chlorosis and necrosis of inter-veinal areas of leaves, as in apples, hops and tobacco, or to the development of reddish colours, as in cotton and some varieties of grape. In the animal lack of magnesium leads to severe nervous symptoms. It has been suspected too that in districts where the water supply is low in magnesium, the human population is affected. These reactions are possibly due to the essential role that magnesium plays in the working of the phosphatases concerned with phosphorylation reactions of the body. Magnesium is also indispensable for the growth of moulds and yeast. Among marine animals there is the peculiar relationship that those from warm waters contain much more magnesium than those from cool waters; moreover the magnesium carbonate occurs in quantity only in those organisms where the calcium carbonate is laid down as calcite. Forms with aragonite are almost free from magnesium.18. There is a nice little physiological problem here. The next element on the list is aluminium concerning which it is difficult to form a definite opinion as to its essentiality. In some lower forms of plant life aluminium is stored in appreciable amounts. More will be said about this later. In animals aluminium is found only in small amounts. It is possible however that aluminium may be an essential element because it is the metallic constituent of succinoxidase, which is a necessary part of the oxidation systems for carbohydrate in both plants and animals. On acid soils soluble aluminium may rise to toxic levels with resultant phosphorus deficiency. Silicon is present in animals and plants in only small amounts usually, but there are some plants which build into their structures relatively large amounts of silica. For example Poa caespitosa is such a plant. In marine organisms such as radiolarians and diatoms the skeletons consist almost entirely of silica; in the siliceous sponges the spicules also consist of silica. This suggests that silica is used in the normal life processes of these organisms. Chlorine occurs in variable amounts in both animal and plant life. In the former, the body fluids of marine invertebrates have almost as much chlorine as has sea-water; marine vertebrates have only about one-third as much. Among the terrestrial forms of animal life earthworms and insects have much lower chlorine contents than the vertebrates. In plants the chlorine content of many plants is less than one per cent, of the dry-matter. It would appear that even if chlorine is not essential for the living processes of the plant yet it is very desirable to have some present even if only for regulation of the water regime. Vanadium has been discussed already but it may be added that this element is widespread in its occurrence in small quantities in both animals and plants. There is some evidence too that vanadium is important for the growth of Aspergillus niger and other fungi and for some nitrogen-fixing organisms. For higher plants its essentiality is doubtful. In plant material the average content of vanadium is about 1 part per million of the dry-matter. It has been claimed that clover, beans, beets and pine needles are relatively rich in vanadium. It is interesting to note that benefit to crops has been claimed for the vanadium in

basic slag. In the table of enzymes already presented vanadium appears as an activator for the phospolipoid-oxidizing enzyme, and as a replacement element for manganese in arginase. Chromium is widespread in soils even to the extent of being toxic to plants. It is very toxic too in the chromate form when added to nutrient solutions. In normal plant material it is found usually in amounts of less than 1 part per million. Chromium has been reported to be present in very small amounts in some marine organisms. It can substitute for aluminium in the succinic oxidase system. The next six elements, Mn, Fe, Co, Ni, Cu and Zn can be taken together. They are most important elements all of which, with the possible exception of cobalt, have been shown to be essential for healthy growth of plants; for animals only nickel may not be essential, although it is widely distributed in the animal kingdom Lack of Mn, Fe, Cu and Zn produces in plants chlorotic symptoms in leaves which are in the main characteristic for each element. All tend to be present in greater amounts in rapidly growing tissues. There are distinct differences in the quantities absorbed by plants, thus manganese and iron are present in the dry-matter at levels of 100 p.p.m. or more, copper and zinc at 10 to 50 p.p.m., and cobalt and nickel at 0.1 to 1 p.p.m. There are wide differences in uptake, especially of manganese, between different botanical families: thus grasses generally absorb much more manganese them legumes. Application of salts of these elements in aqueous solution as sprays is usually the most satisfactory way of increasing the uptake of these elements when plants suffer from a lack of them. Plant diseases due to unusually large supplies of these elements in available form are also known. Among animals these elements are very widespread in the internal organs, the liver usually being the richest in them. Thus in diseases due to deficiencies of cobalt or copper the level of these elements in the liver is probably the best indicator of the correctness of the diagnosis. There is also a tendency for manganese to concentrate in the reproductive organs. Iron has an especial importance in animals because of its presence in hemoglobin; and the respirators process; it may be stored in appreciable amounts in the spleen and liver, especially in the presence of certain diseases. Copper works in close association with iron and its presence in the foodstuff is necessary for formation of hemoglobin, although copper itself does not occur in that compound Lack of copper in farm animals may have serious consequences to them in cattle there may be unthriftiness or even sudden death and in sheep poor wool growth or paralysis. In both classes of animal low copper intake makes the animals more susceptible to the affects of any increased intake of molybdenum. Nickel is widespread in animals and is apparently accumulated to some extent by marine organisms; for example, in the Lamellibranch Cardium edule up to 23.5 p. p.m. was found in the liver. An appreciable proportion of this was apparently in combination with protein. Up to 100 p.p.m. has however been reported in marine organisms Cobalt occurs in larger amounts in marine than in domesticated animals, thus among Lamellibranchs Paulais19 found up to 1.8 p.p.m, the liver usually being the richest, while in cattle and sheep a normal liver figure would be 0 25 p.p.m. in the dry-matter. A low content of cobalt in the food of cattle and sheep, but not of horses, leads to a wasting disease known by various local names in different countries; in New Zealand Bush-Sickness is the best known local name. It has been shown recently that the function of cobalt

in the animal is to assist in the production of Vitamin-B12 by organisms in the rumen. This vitamin is necessary for the health of lower as well as of higher organisms. The last element of this group is zinc. This element is also widely distributed and is essential for healthy growth. It has already been seen that it is an essential constituent of carbonic anhydrase, the enzyme which regulates the exchange of carbon dioxide and water in animals and plants. Zinc is usually present in greater amounts in marine animals than in domesticated ones; thus in the dry-matter of oysters up to nearly 4,000 p. p.m. have been found. Bromine is readily absorbed by plants and the occurrence of this element in biological material is probably due to the absence of any selective mechanism that would exclude it. Some substitution by bromine for chlorine can occur. In cultivated plants the green parts are much richer in bromine than the roots. Usually less than 5 p. p.m. of bromine is present in plant material, but in marine organisms appreciable amounts have been found. Among mammals the thyroid and blood are somewhat richer than other organs. No functional property for bromine has been suggested as yet. Rubidium, one of the alkali metal series, is widely distributed in plants and is found in the greatest amount in young material. There is some evidence of a stimulatory effect on plant growth, especially when the supply of potassium is reduced, even though the element may not be wholly essential. An average figure for rubidium content is about 20 p.p.m on the dry basis. Some fungi apparently act as accumulators of this element, as high a figure as 2,800 p.p.m. having been reported. In general cryptogams absorb more rubidium than phanerogams. To fully discuss molybdenum would take more time than can be given to it here. Suffice it to say that molybdenum is essential for the normal growth of many plants and fungi. When the supply is sub-optimal or seriously deficient profound disturbances occur in the metabolism of the plants leading to characteristic visual symptoms and to great changes in the nitrogenous and other compounds in the plant. About 1 p.p m. of molybdenum in the dry-matter may be regarded as a normal figure for plant material. Molybdenum exerts a very marked effect on the growth of nitrogen-fixing organisms in the soil and thus on the nodulation of the roots of legumes. Legumes absorb greater -amounts of molybdenum from the soil than grasses, and thus may, on soils of high availability of molybdenum, take up such quantities of this element that, although they themselves suffer no disability as a result of that absorption, animals feeding on such material, especially in the fresh state, may be rendered seriously ill. Molybdenum and copper act in an antagonism such that administration of copper sulphate to animals suffering from excessive intake of molybdenum will restore them to health. Within the animals excess amounts of molybdenum may reduce the copper content of the liver to deficiency levels. The precise relationships between copper and molybdenum in animals remain to be worked out. Recent work in Australia shows that sulphate is important in relation to copper and molybdenum nutrition of animals. Iodine, the element of greatest atomic weight that the present classification regards as essential, is widespread in nature, being especially abundant in the sea-weeds and mid marine animals. In food plants there is usually less than 10 p.p.m. 'Of iodine in the dry-matter. The uptake of iodine by food crops is probably increased artificially through the common use of natural nitrate of soda which

contains about 0.02 per cent, of iodine; synthetic nitrate, however, is low in iodine content. There is no good evidence that iodine is essential for plant life, but the widespread occurrence of goitre in humans and animals, which is curable and controllable by administration of iodine, testifies to the necessity of this element for animal life. In Fig. 1 certain elements are enclosed by dotted lines: these are Be, F, Sc, Ti, Ga, Se, Sr, Ba, W, Ag and Au; some of these may eventually prove to be essential. Of these Ba, Se and Au will be discussed later, together with Aluminium. Of beryllium very little seems to have been reported as to its occurrence in living material. It is known, however, that beryllium will produce a form of rickets when fed to rats. The suggestion has been made that beryllium may have been included with aluminium in many analyses. Fluorine occurs in small amounts in vegetable material, but has no known function which would lead to its being considered essential; indeed some of the enzyme systems of plants are readily poisoned by small amounts of fluorine. Under some circumstances fluorine may be absorbed with formation of fluoracetate, which has been considered to be the poisonous constituent of plants causing sickness in animals in South Africa. Later work indicates that the fluoracetate is metabolized to fluoracitrate, which is the toxic agent. Among animals an excessive fluorine intake has very detrimental effects on teeth and bones. Among human brings high intake of fluorine has been considered to be a cause of mottled enamel of the teeth, while a deficiency of fluorine can be correlated with increased incidence of dental caries. It is of interest to note that tea is rich in fluorine, values as high as 355 parts per million having been reported. A single cup of tea may provide as much fluorine as the total intake per day from all other foods. Aluminium can apparently act antagonistically to fluorine and thus cause reduced absorption of the latter. Scandium and Gallium have both been claimed to be essential for the growth as Aspergillus niger. Both have been reported in very small amounts in plants, and although there is no evidence at present that they are essential for the higher plants, it has been predicted that gallium may eventually be found “to constitute the prosthetic group of some oxidative enzyme, present in low concentration, but in a number of diverse organisms.”20 Titanium occurs often in appreciable percentages in soils, but in plant material only about 2 p.p.m. is present in the dry matter; animal organs contain only minute amounts. The green parts of plants are stated to be the richest in titanium; a claim has also been made that additions of titanium to cultures of lucerne and peas increased the number of nodules and the fixation of nitrogen. Titanium has been reported to occur in all organs of the human body to the extent of up to 0.1 mg. per kg. (fresh weight?). It has been found in marine plants and animals but owing to the difficulty of cleaning these organisms the numerical values are probably doubtful. Although, as will be seen later, plants can distinguish between barium and calcium, yet they are apparently incapable of doing so between strontium and calcium. Thus in general the ratio of concentrations of the latter two elements is the same in the plant as in the soil on which the plant was growing. In Brazil nut (Bertholletia excelsa) the Ca:Sr ratio was found to be constant

Table II. Occurrence of Twenty Chemical Elements in PLant and Annual Materal. Expressed as Parts per million on the dry matter basis. B Al Ti B Cr Mn Fe Co Ni Cu Zn Ga Se Rb Sr Mo Ba Apple: leaf 30 60 150 15 30 1 Apple: fruit 20 5 0.9 5 15 0.2 10 10 0.03 3 Hop: leaf 40 200 200 14 30 0.5 Hop: cones 20 31 40 250 14 40 Tabacco: leaf lamina 20 70 17 50 0.5 88 Tamato: fruit 14 22 50 160 0.1 0.15 14 39 Grass 20 3 2 0.1 0.1 100 100 0.1 1 15 30 12 10 0.5 15 Red Clover 20 28 2 0.1 0.1 50 70 0.2 2 15 80 16 65 2 50 Lucerne 27 99 4 0.2 80 270 0.2 0.5 9 57 2 5 1 70 wheat (grain) 4 0.9 60 80 0.01 0.3 10 60 0.5 Brazil nut ash (testa) 500 800 800 400 7500 30,000 Laminaria digitata (fround) 4 0.7 1.8 < 30 410 0.22 2.1 6 71 950 0.17 18 Lycopodium labelliforme20 30 2730 3 67 37 0.2 4 13 0.03 4 4 Mcan composition of Terrestrial vegetatin20 10 20 1 1*Not included in data of reference 20. 0.2* 70 200 0.2 1 3 18* 30 30 Blood (manmalian) 0.7 0.03 0.09 0.1 2200 0.03 0.06 0.5 30 0.03 Liver (manmalian) 50 0.2 10 350 0.3 0.6 200 110 60 0.8 4 Milk (cow)† Data on liquid basis. 1 0.2 0.04 0.001 0.004 0.2 4 Pleurobrachula 100 140 140 20 1400 40 200 30 140 1100 6 Marine Animals 1 0.4

for the outer testa, inner testa and endosperm, although the amount of strontium in the ash varied respectively from 0.75 to 0.1 per cent.21 Strontium can substitute for calcium in the nitrogen-fixation processes of Azotobacter. It has been found to be present in a wide range of plant material, and also in animals. In many marine organisms the ratio Ca:Sr does not vary greatly from the value of 35 found for sea-water, so that the organisms do not show any marked discrimination between these two elements. On the other hand the skeleton of a radiolarian, Podecanelius, has been reported to consist almost wholly of strontium sulphate. Table III. Mean Composition of Terrestrial Vegetation (from Hutchinson and Wollack). Elements Parts per million (on dry basis?) Element Parts per million (on dry basis?) Silicon 1500 Aluminium 20 Magnesium 700 Boron 10 Chlorine 400 Zinc 3 Iron 200 Titanium 1 Manganese 70 Copper 1 Strontium 30 Nickel 0.2 Barium 30 Lead 0.2 Tungsten has been sought spectrographically in animal feedstuffs and animal tissues by several workers, but was not found. There is some evidence that tungsten can partially substitute for molybdenum in the nutrition of nitrogen-fixing blue-green algae. Silver has been reported to occur in the gills of mushrooms up to more than 0.05 per cent.; as much as 0.2 per cent, has been found in other fungi, while up to 50 parts per million have been found in the livers of marine Lamellibranchs. Spectroscopic examination of the wood of 26 species of trees showed the presence of silver in 17. In a series of 17 metals silver was the most active in stimulating growth of pollen tubes, but was not so active as Co, Mg, or Na in the germination of the pollen. Many of the remaining elements of the periodic outside the boundaries that we have drawn have been reported from living matter, but it is not likely that many of them are present because the organism requires them: they are there simply as impurities derived from their sources of food, in the case of animals from the plants on which they live, and in the case of plants because they have no ready means of exclusion of these elements from their nutrient supply. They will not be further considered here. In Tables II and III are given data on the normal occurrence in plants and animals of many of the elements that have been discussed, together with those for Al, Ba, and Se. Now it will be noted that the arrangement of the elements given above is simply in the sequence of the atomic weights. When the curves showing variation in atomic volumes are examined it is found that the alkali metals are at peaks and the micro-nutrient elements are in the troughs between the peaks. Other essential elements, metals and non-metals, are on the sloping portions of the curves. Again, the metallic elements have been arranged in terms of their electrode potentials, or of their atomic and ionic radii, and in terms of their electron shells. Attempts have also been made to arrange the elements according

to the magnetic properties of their atoms or ions. However, none of these arrangements is really satisfying. While the ionic radii and valencies can be very usefully employed, as was done by Goldschmidt, to explain the occurrence together of particular elements in rocks and ores they have not the same usefulness for biological purposes: the distinction lies in the fact that the micro-nutrient metals at least are probably not present as simple ions in biological material. In living matter the metals are more likely than not combined with proteins as such or with enzymes, which have also been shown to contain a protein moiety. Baudisch7 has quoted the following striking example of the effect of iron in ionic form in two combinations in bringing about chemical reactions: thus an ordinary iron ion decomposes only two molecules of hydrogen peroxide, giving water and oxygen gas, but when the same amount of iron is present in the enzyme catalase it decomposes millions of hydrogen peroxide molecules. Perhaps a too severe attitude has been taken to the usefulness of classifications of the elements based on the physical properties mentioned above. It is true that they bring together more or less successfully the biologically active elements, but they do not seem capable of offering an explanation of the reason why these elements are active. It does seem possible, however, that a consideration of the ability of an element to form inner complexes with organic compounds may lead to an acceptable explanation of the activities of the micro-nutrients and of their replacement values or antagonistic properties. One further matter is worthy of discussion. There are very definite indications that living matter may store up relatively large amounts of a certain element; those micro-nutrients which are thus accumulated in a striking manner by some plants are aluminium, barium, selenium and gold. And considering the minute concentrations of the metallic elements in sea water some remarkably large quantities are retained by many marine organisms, especially in their livers. However, we will consider more fully only the four elements mentioned above as being retained in considerable quantities by plants. A very complete survey of the occurrence of aluminium in biological material has been given by Hutchinson.22 It is shown there that particularly in the lower orders aluminium is stored by the plant; thus, in the genera Lycopodium and Symplocos more than 25 per cent, of the ash may consist of this element Culture of S. japonica in sand showed that only when the aluminium content of the culture solution was raised to 27 p.p.m was normal growth obtained. Such a high concentration of aluminium indicates that aluminium may be an essential element for this species. On the other hand so-called aluminium accumulators can grow normally without building into their tissues any unusually large amounts of aluminium, should the nutrient supply be low in this element. While barium is widely distributed in plants and animals there is at least one plant, the Brazil nut (Bertholletia excelsa), already discussed under strontium, which has the property of using comparatively large amounts of barium. Thus Webb and Fearon21 found the following percentages of barium (Ba) in the ash of the parts of the nut: woody part of testa, 3.0; inner part of testa (fibrons, next to endosperm), 8.0; endosperm, 0.03. Actually the -endosperm contained more strontium than barium. This plant is therefore

able to discriminate between calcium, strontium and barium, and it may even require barium as an essential nutrient. What seems to be a similar case of discriminatory power is provided by Foraminifera xenophyophora whose skeleton is stated to consist of barium sulphate. Comment has already been made on the undesirability of the presence of much selenium in animal fodder. On certain geological formations of the Cretaceous period in the United States, and in other countries, unusually large amounts of selenium are absorbed by native plants and sown crops. Of native plants The American vetches (Astragalus spp.) are outstanding in this respect; thus when growing on a soil containing only 1.5 p.p.m. A. pectinatus accumulated 3,890 p.p.m. of sielenium into its dry-matter.23 Species selectivity is illustrated by the following data, again for plants on a soil containing 1.5 p.p.m. of selenium: A. racemosus, 1,220 p.p.m. and A. mollissimus, 3 p.p.m. The highest figure seen for the former was 14,920 p.p.m.24 Other plants which absorb large amounts of selenium are Stanleya bipinnata. Aster multi-florus and Grindelia squarrosa. Seeing that there is a wide range in selenium content for different species of plants growing on seleniferous soil, and also that some species are not found unless the soil is seleniferous to some degree, it would seem that in these selenium-loving plants this element has a part in some vital process. Indeed selenium has been shown to be a stimulating and possibly essential element to some species of Astragalus. Uptake of selenium by grasses and many cultivated crops although relatively small may still be sufficiently great to cause illness in stock feeding upon them. Selenium in the vegetative parts of the plants is in unknown organic forms, but in seeds (wheat, etc.) the selenium is found in the protein fraction. The last to be considered here as an unusual accumulated element in plants is gold. Certain Equisetums can apparently raise the concentration of gold in their parts to very much greater levels than this element occurs in soils. Thus E. palustre returned 610 grammes of gold per ton of ash when growing on a soil containing 0.2 gm. per ton. Another accumulator was Mentha arvensis with 300 gm. per ton; but in the ash of Urtica dioica only 16–8 gm, was found. The ash of wood from Fagus silvatica, Carpinus betulus and Salix caprea contained only traces of gold. Fruits of Clematis vitalba had 600 gm. per ton, but sunflower seeds and rose fruits had none. It would appear that the absorption of gold by E. arvense is an efficient process because when grown on the soil containing 0.2 gm. of gold per ton its ash contained 576 gm., and 63 gm. per ton on a soil carrying only traces of gold.25 Now, what significance is to be attached to these unusual concentrations of certain elements in biological material? Are they due to some freak of metabolism which has arisen during their evolutionary history, or are they due to metabolic processes which arose when the nutrients available to their ancestors were in different proportions from those available at the present time ? If either of these suggestions should be true, then can it be used for checking the relationship of one genus or species to another? Consideration of these matters may perhaps begin with a related subject, namely the composition of the blood of animals. In the lower orders copper in the hemocyanins apparently takes the place of iron among the higher animals, and where hemoglobin is present in the lowly organisms it is not used in the normal respiratory process. Conversely residual amounts of copper arc present

Table IV. Mean Numbers of Various Kinds of Atoms in the Mammalian Liver Cell (from Hutchinson). Element No. of Atoms per Cell Millions. Element No. of Atoms per Cell Millions. Zinc 4200 Titanium 44 Lithium 1800 Molybdenum 43 Rubidium 680 Cobalt 14 Copper 350 Lead 10 Manganese 220 Strontium 10 Aluminium 190 Nickel 7 Tin 88 Silver 2 in mammalian blood. We also have vanadium as a blood constituent in lower animals. The question therefore arises, was there throughout the development of animal life a continuous change in the respiratory processes which involved passing from copper or vanadium as the active metal to iron, which we have at the present time, as the main respiratory metal, even though organisms still remain which use copper? Again, has a somewhat similar process occurred with cobalt? Hutchinson26 when discussing the concentrations of elements in the mammalian liver (see Table IV), says, “… in certain of the higher invertebrates and notably in the vertebrates, the cobalt content is considerably increased, as if an important new function for the latter element had been evolved in the more complex animals.” Is it possible that this is connected with the requirements of the higher, and some lower organisms, for Vitamin B12, known to be a cobalt-containing compound? Towards the end of last century and in the earlier years of the present one considerable discussion took place on the origin of the high aluminium content of certain coal ashes. One of the suggestions made was that it arose from the remains of plants which had acted during their lives as accumulators of aluminium. If this were so then possibly more aluminium was taking part in biological reactions than there is at the present time. One worker has even suggested that aluminium may be one of the elements which tends to decrease in living material with increase in morphological complexity. Others consider that the chemical composition of any species of plant or animal is a fixed specific character. It is immediately obvious that a great deal of detailed work would be required to establish either viewpoint. Hutchinson and his associates do not seem, in spite of the large amount of work that they have carried out on the Lycopodiaceae, to be prepared to say definitely whether the variation in aluminium contents of the different, species can be satisfactorily correlated with their botanical classification. They have admitted though that a good case can be made out of the distribution of the accumulators of selenium in the genus Astragalus. Hutchinson has suggested too that zinc in the Ostreidae and manganese in the sub-families of Unonidae may provide similar cases.20 This summary of some of the relations of the trace, or micro-nutrient, elements to living matter may be completed by the following quotation: “It is therefore not at all improbable that at the dilutions under consideration,” (i.e., in a quantity of the order of a few thousand molecules per cell), “a large number of substances have set limits to the structural and functional possibilities of the cell, and that physiological evolution has been determined,

albeit negatively, by a far greater variety of metals than the most enthusiastic student of the trace elements would hitherto have deemed possible.”26 References 1. Arnon, D. I., 1950. in, Trace elements in Plant Physiology, Lotsya. 3, 21 2. Willis, L. G. and Piland, J. R., 1938. J. Amer. Soc. Agron., 30, 887. 3. —— J. Amer. Soc Agron., 30. 888. 4. Shive, J. W., 1941. Plant Physiol., 16, 435. 5. Nicholas, D. J. D. and Forster, W. A., 1950 Long Ashton Ann Report, p. 96. 6. Hunter, J. G. and Vergnano, O., 1952 Ann. App. Biol., 39, 270. 7. Baudisch, O., 1943. American Scientist, 31, 211. 8. Baldwin, E., 1939, in Perspectives in Biochemistry, Camb. Univ. Press, p. 166. 9. Szent-Györgi, A., 1939. in, Perspectives in Biochemistry, Camb. Univ. Press, p. 166. 10. Seekles, L. 1950. in, Trace Elements in Plant Physiology, Lotsya, 3, 119. 11. Prosser, C. L., editor, 1950. Comparative Animal Physiology, Saunders, p. 304. 12. Skinner, D. A., 1952. Ind. Eng. Chem. 44, 1159 13. Richert, D. A. and Westerfeld, W. W., 1953. Jour. Biol. Chem, 203, 915. 14. Cunningham, I. J., 1950. in, Copper Metabolism. The Johns Hopkins Press, p. 246. 15. Agarwala, S. C. and Hewitt, E. J. (1952). Long Ashton Ann. Rpt., p. 195. 16. Nemec, B., 1950. in, Trace elements in Plant Physiology, Lotsya, 3, 39. 17. Schropp, W., 1950. Zeit, f Pfianzenernah., Dung., Bodenk, 51, 127. 18. Clarke, F. W. and Wheeler, W. C., 1922 U. S. Geol. Survey Prof. Paper. 124. p. 58. 19. Paulais, R., 1939, Recherches sur les infiniment petits chimiques minéraux chez lemollusques, Libraire le Francois. Paris, p. 123. 20. Hutchinson, G. E. and Wollack, Anne, 1943. Trans Conn. Acad. Arts and Sciences. 35, 73. 21. Webb, D. A. and Fearon, W. R., 1937. Sci. Proc. Roy. Dublin Soc., 21 (N.S.), 487. 22. Hutchinson, G. E., 1943, Quart. Rev. Biol., 18, 1, 128, 242, 331. 23. Byers, H. G. et al., 1938. U.S.D.A. Tech. Bull. No. 601, p. 16. 24. Beath, O. A., Eppson, H. F. and Gilbert, C. S., 1937. Jour. Amer. Phar. Assn., 26. 394. 25. Nemec, B., Babicka, J. and Oborsky, A. Bull. Internat. Acad Sci. Boheme: 1936 (2 papers); 1937. 26. Hutchinson, G. E., 1943, Quart. Rev. Biol., 18, 331.