EXCHANGE 
 
 The Influence of Calcium, Magnesium 
 
 and Potassium Nitrates upon the 
 
 Toxicity of Certain Heavy 
 
 Metals Toward Fungus 
 
 Spores 
 
 DISSERTATION 
 
 Submitted to the Board of University Studies of the Johns 
 Hopkins University in conformity with the require- 
 ments for the degree of Doctor of Philosophy 
 
 By 
 LON A. HAWKINS 
 
 Baltimore, June, 1913 
 
 [.PHYSIOLOGICAL RESEARCHES VOL. I, NO. 2, AUGUST, 1013] 
 
The Influence of Calcium, Magnesium 
 
 and Potassium Nitrates upon the 
 
 Toxicity of Certain Heavy 
 
 Metals Toward Fungus 
 
 Spores 
 
 Submitted to the Board of University Studies of the Johns 
 Hopkins University in conformity with the require- 
 ments for the degree of Doctor of Philosophy 
 
 By 
 LON A. HAWKINS 
 
 -*-?-.- 
 
 Baltimore, June, 191. '5 
 
 [PHYSIOI.nGirAJ. RKKKARrllKS VOt. I, NO. 2, AtTl-.VST, 
 
BIO'-OGY 
 RA 
 G 
 
THE INFLUENCE OF CALCIUM, MAGNESIUM AND 
 
 POTASSIUM NITRATES UPON THE TOXICITY 
 
 OF CERTAIN HEAVY METALS TOWARD 
 
 FUNGUS SPORES. 1 
 
 LON A. HAWKINS. 
 
 ABSTRACT. 2 
 
 This study has to do with the influence of one salt in altering the toxic 
 effect of another upon fungus spores. It is shown that the toxic effect 
 of copper nitrate on the germination of Gloeosporium spores can be in- 
 hibited or modified by the addition to the medium of calcium nitrate and 
 that the molecular ratio of the quantity of the calcium salt thus required 
 to the amount of copper nitrate present increases with the concentration 
 of the latter. This effect is apparently the result of a simultaneous action 
 of the two salts upon the organism and cannot, in the cases here con- 
 sidered, be related either to formation of an undissociated double salt or 
 to depression of the ionization of the toxic salt because of the ion common 
 to the two salts. Potassium nitrate is also effective in inhibiting or 
 modifying the toxicity of copper nitrate. The influence of calcium upon 
 the toxicity of copper is of interest in connection with the problem of 
 fungicides and fungicidal action. 
 
 The toxicity of lead nitrate is similarly influenced by the presence of 
 calcium nitrate, and the molecular proportions in which these two salts 
 may be combined so as just to counteract the toxicity of the former were 
 found to be the same for the three different concentrations of lead 
 nitrate that were employed. The toxicity of this lead salt is likewise influ- 
 enced by proper concentrations of magnesium nitrate. Both calcium nitrate 
 and magnesium nitrate markedly decrease the toxicity of zinc nitrate, but 
 neither exhibits any effect on the toxicity of aluminum nitrate in the con- 
 centrations used in these experiments. 
 
 The effects produced by the various single salts upon the germinating 
 spores are of interest. Four types of response to the salt solution are 
 clearly discernible, (i) The effect of the solution may be the same as 
 that of distilled water, the spores germinating normally, with long, narrow 
 tubes. (2) A somewhat higher concentration of the toxic substance may 
 
 'Botanical contribution from the Johns Hopkins University No. 31. 
 
 2 The manuscript of this paper was received April i, 1913. This abstract was preprinted, without 
 change, from these types and was issued as Physiological Researches Preliminary Abstracts vol. i, no. 
 2, August, 1913. 
 
 57 
 
 PHYSIOLOGIC/ L RESEARCHES VOL. I, NO. 2^ SERIAL NO. 2, 
 AUGUST, 
 
 "814805 
 
58 LON A. HAWKINS 
 
 allow germination, this being abnormal, however, with the production of 
 unusual and characteristic structures. (3) Germination may be inhibited 
 for eighteen hours, the spores germinating when subsequently transferred 
 from the toxic solution to distilled water. (4) The spores may be killed 
 in eighteen hours and thus be incapable of renewed activity even when 
 transferred to water. It was usually possible to bring about these four 
 types of response by means of any one of the compounds used in this study 
 by a proper choice of concentration. An arrangement of these substances 
 in the order of their toxicity, as evidenced by the inhibition of spore 
 germination, is found to hold also for their effectiveness in bringing about 
 the other and less final changes which lead to abnormal growth. A list 
 of the substances employed, arranged in the order of their toxicity follows : 
 Cu(N0 3 ) 2 , CuS0 4 , Pb(N0 3 ) 2 , A1(N0 3 ) 3 , HNO 3 , Zn(NO 3 ) 2 , Ni(NO 3 ) 2 , 
 Mg(NO 3 ) 2 , Ca(NO 3 ) 2 , KNO 3 , sucrose. 
 
 It is shown that no one of the several hypotheses heretofore proposed to 
 account for the dynamics of the antagonistic salt action is adequate to 
 explain all the facts brought out in this study. 
 
 INTRODUCTION. 
 
 Numerous instances have been recorded of the influence of salts on the 
 toxicity exerted by various substances upon organisms. This antagonistic 
 action, as it is frequently called, of a salt upon a toxic substance, is of 
 considerable importance in influencing the behavior of organisms in a 
 given environment, or indeed in determining whether or not they may 
 exist at all in certain environments. For example, Loew 3 has shown that 
 the toxic effect on Spirogyra of a one per cent, solution of magnesium 
 nitrate is inhibited by the presence in the medium of a 0.3 per cent, solu- 
 tion of calcium nitrate, while Loeb 4 has demonstrated that the addition of 
 a small quantity of a calcium salt to a 0.625111 solution of NaCl inhibits 
 the toxic effect of the NaCl on the development of Fundulus eggs. Oster- 
 hout 5 has shown that a physiologically balanced solution, containing sodium 
 chloride, magnesium chloride, magnesium sulphate, potassium chloride and 
 calcium chloride, is necessary for the best growth of certain marine algae. 
 True and Bartlett 6 have brought out the fact that a ratio of one molecule 
 of calcium to nine molecules of magnesium inhibits the toxic effect of 
 rather high concentrations of magnesium upon roots of Canada field 
 
 3 Loew, O., Ueber die physiologischen Functionen der Calcium- und Magnesium-salze im Pflanzen- 
 organismus. Flora 75: 368-394. 1892. 
 
 4 Loeb, J., Ueber den Einflus der Werthigkeit, und moglicherweise der elektrischen Ladung von 
 lonen auf ihre antitoxische Wirkung. Archiv ges. Physiol. 88: 68-78. 1902. 
 
 5 Osterhout, W. J. V., On the importance of physiologically balanced solutions for plants. I. Marine 
 plants. Bot. Gaz. 42: 127-134. 1906. 
 
 'True, R. H., and Bartlett, H.H., Absorption and excretion of salts as influenced by concentration 
 and composition of culture solutions. U.S. Dept. Agric. Bur. Plant. Ind. Bulletin 231. 1912. 
 
TOXICITY OF HEAVY METALS 59 
 
 peas. Other cases of antagonistic salt action in combinations of salts 
 of the alkali metals or of the alkaline earths have been demonstrated, 
 and some information has been obtained regarding the influence of 
 these salts on the effect of the heavy metals, which seem to be 
 universally toxic. The latter have not received much attention, however, 
 and it seemed worth while to investigate some of these, alone and in the 
 presence of calcium and magnesium, to obtain evidence as to whether the 
 lighter metals may modify in any way the toxicity of the heavy ones. 
 The investigation described in this paper was accordingly undertaken. 
 The problem here involved will be taken up more in detail after some of 
 the literature pertinent to the subject has been considered. 
 
 One of the earlier studies of the influence of chemical compounds upon 
 the toxicity of the heavy metals was carried out by Kronig and Paul. 7 
 These authors determined the effects of various heavy metals in com- 
 bination with many salts as well as with certain acids and bases. Mainly 
 from work with Bacillus anthracis, they regard the influence of other 
 halogen compounds upon the toxicity of mercuric chloride as probably 
 due to a depression of the ionization of the latter salt. In this connection 
 they say (page no): " Die Desinfectionswirkung wasseriger Mercuri- 
 chloridlosungen werden durch Zusatz von Halogenverbindungen der Metalle 
 und von Salzaure herabgesetzt. Es ist wahrscheinlich, dass diese Vermin- 
 derung der Desinfectionskraft auf einer Riickdrangung der elektrolytischen 
 Dissociation beruht." 
 
 Clark 8 carefully studied the influence of various concentrations of 
 sodium chloride upon the toxicity of mercuric chloride as regards the 
 process of germination in various fungus spores and seeds and that of 
 growth in yeasts and bacteria. He found that the toxicity of a mercuric 
 chloride solution increased with addition of small quantities of sodium 
 chloride but decreased when high concentrations of the sodium salt 
 were used. He explained these phenomena by considering that a double 
 salt of sodium chloride and mercuric chloride was formed, such as 
 Na 2 HgCl 4 or some similar combination. He supposed that the dissociation 
 tension of this double salt was probably much higher than that of mercuric 
 chloride, a consideration which might account for the increased toxicity 
 of combinations in which small amounts of sodium chloride were employed. 
 In this connection, he suggested that the HgCl 4 ion present when such a 
 salt as Na 2 HgCl 4 dissociated at lower concentrations, might be considerably 
 more toxic than the HgCl 2 molecule to which he seems to attribute the 
 toxicity of mercuric chloride solution. 
 
 7 Kronig, B., and Paul, Th., Die chemischen Grundlagen der Lehre von der Giftwirkung und Desin- 
 fection. Zeitsch. Hygiene und Infectionskrankheiten 25: 1-112. 1897. 
 
 8 Clark, J. P., On the toxic value of mercuric chloride and its double salts. Jour. Physic. Chem. 
 5: 289-316. 1901. 
 
60 LON A. HAWKINS 
 
 In a later investigation, on the toxicity of copper in combination with 
 various chemical compounds, the same writer 9 has shown, for spore germ- 
 ination in Oedoccphalum albidum and Rhizopus nigricans that ammonium 
 nitrate, sodium sulphate, potassium sulphate, and potassium chloride all 
 markedly decreased the toxic effects of both copper chloride and copper 
 sulphate. He used relatively high concentrations, in some cases five per 
 cent., of the alkali and ammonium salts. He considers the decreased toxicity 
 just mentioned as probably due to the formation of double salts, as he 
 suggested in the case of mercuric chloride. 
 
 Le Renard 10 studied the comparative toxicities of salts of many of the 
 heavy metals upon Penicillium, the fungus being grown in various concen- 
 trations of nutrient media. He used the acetates of potassium, magnesium 
 and ammonium alone, and the acetates, formates, nitrates, phosphates, and 
 sulphates of these three metals with glucose. Also, in combination with 
 various concentrations of the salts in the nutrient medium he used several 
 concentrations of the chlorides and nitrates of zinc, nickel, cobalt and 
 copper, mercuric chloride, silver nitrate, and the sulphate and acetate of 
 copper. The presence in the nutrient medium of the lighter metals in higher 
 concentration was usually found to decrease the toxicity of the heavy 
 metals. 
 
 True and Gies 11 showed that calcium modified the toxicity of various 
 copper salts, of zinc sulphate and of mercuric chloride, in their effect 
 upon the growth of roots of Lupinus albus. In discussing their results 
 these authors say : " The stimulating action of the calcium seems to have 
 operated against the retarding action of the copper, and the result is a 
 marked diminution in the poisonous action of the copper." They thus 
 relate this influence of calcium upon copper to a mutual effect of the two 
 salts on the protoplasm. 
 
 Sziics 12 has recently shown that the toxic effect of copper sulphate on 
 the roots of Cucurbita pepo may be inhibited by aluminum chloride in 
 certain concentrations. In this case he used as index of toxicity the ability 
 of the root to react to a geotropic stimulus after it had been removed from 
 the poisonous solution. He varied the presentation time of the toxic stimu- 
 lus (solution of copper sulphate) both alone and with addition of aluminum 
 chloride, and found, for the shorter time periods, that the presence of 
 aluminum inhibited the poisonous action of copper. However, when such 
 
 9 Clark, J.F., On the toxic properties of some copper compounds with special reference to Bordeaux 
 mixture. Bot. Gaz. 33: 26-48. 1902. 
 
 10 Le Renard, Alf., Influence du milieu sur la resistance du Pdnicille crustace aux substances tox- 
 iques. Ann. Sci. Nat. Bot. IX. 16: 276-336. 1912. 
 
 11 True, Rodney, H., and Gies, W. J., On the physiological action of some of the heavy metals in 
 mixed solutions. Bull. Torr. Bot. Club 30: 390-402. 1903. 
 
 12 Sziics, Joseph, Experimented Beitra^e zu einer Theorie der antagonistischen lonenwirkungen. 
 Jahrb. wiss. Bot. 52: 85-143. 1912. 
 
TOXICITY OF HEAVY METALS 61 
 
 a combination of the two salts was allowed to act i&c longer periods, rt 
 also was toxic in many cases, and the roots lost their ability to respond 
 afterwards to the geotropic stimulus. He used concentrations of copper 
 sulphate varying from o.ooi875n to o.o75n, combined with aluminum chlor- 
 ide in concentrations varying from o.(X>5n to o.45n. The presentation time 
 of the toxic stimulus ranged from 33 minutes to 26 hours and 50 minutes^ 
 This writer also studied the effect upon Spirogyra of quinine hydrochloride, 
 methyl violet and piperidine in combination with various other substances. 
 The toxicity of quinine hydrochloride was altered by various concentrations 
 of other substances, being almost inhibited by aluminum nitrate, markedly 
 decreased by calcium nitrate, and only slightly lessened by potassium nitrate. 
 Thus, the effectiveness of these salts in reducing the toxicity of quinine 
 hydrochloride diminished with the valency of the cation. Similar results 
 were obtained with the same series of salts in combination with methyl 
 violet. The effect of combinations of various substances with piperidine 
 was, in most cases, markedly to increase its toxicity. Sziics apparently 
 considers the antagonistic action, as investigated by him, to be due to the 
 lowering of the rate of absorption of the toxic ion by the presence in the 
 solution of another ion of similar sign. He concludes, in summarizing: 
 " Die Ursache der ' antagonistischen lonenwirkungen ' liegt in alien von mir 
 untersuchten Fallen in der gegenseitigen Beeinflussung der Aufnahmege- 
 schwindigkeit zweier im gleichen Sinne geladener lonen." 
 
 From the results obtained in the investigations just considered, it is 
 apparent that the toxic effect of the heavy metals on an organism can 
 be modified, in some cases at least, by the addition of certain salts in 
 proper concentration. True and Gies, and Sziics, working with higher 
 plants, attribute this influence of one salt on the effect of another to a 
 simultaneous action of the two salts upon the organism itself, while Clark, 
 working with fungi, relates the inhibition of the toxic effect of heavy 
 metals in combination, to some modification of the salts in the solution. 
 The proportions of salts used in the investigations just mentioned were 
 widely different, and it is of course possible that the different conclusions 
 reached may have been due to this feature. Furthermore, as fungi and 
 higher plants so frequently react differently to the same stimulus, it is 
 possible that one of these two explanations might hold for one group of 
 organisms and the other for the other group. The present study, in which 
 a fungus was employed, was undertaken partly to throw light on the 
 question just suggested. 
 
 It was the purpose of this research to examine the effects of the nitrates 
 of copper, lead, zinc, nickel, and aluminum, upon the germination of 
 fungus spores, the salts of these heavy metals being used both alone and 
 in combination with the nitrates of calcium and magnesium, to see whether 
 
62 LON A. HAWKINS 
 
 the presence of the lighter metals in various concentrations might or might 
 not decrease the toxic effect of the heavy ones. It was also considered 
 worth while, when such a decrease was found to occur, to determine as 
 far as possible whether this influence might be related to a direct effect of 
 salts on each other in the solution or to some modification of the organism 
 itself. Furthermore the results obtained in these experiments should throw 
 some light on the problem of the comparative toxicity of the various sub- 
 stances here employed, when used alone, and thus upon the general physio- 
 logical problem of toxicity. 
 
 The investigation was carried out at the Laboratory of Plant Physiology 
 of the Johns Hopkins University, and the writer's sincere thanks are due 
 to Professor Burton E. Livingston for his many helpful suggestions and 
 valued assistance throughout the progress of the work. 
 
 ORGANISM. 
 
 The fungus spores used in this research were of the Gloeosporium or 
 conidial stage of Glomerella cingulata (Stonem.) S. and v. S., the fungus 
 causing the disease of the apple known as " bitter rot." Not only is the 
 fungus parasitic upon the apple, but according to Shear and Wood, 13 it 
 is also the cause of disease on other plants. On the apple fruit it produces 
 brown, sunken areas, usually nearly circular in shape, which may be covered 
 with the fruiting bodies of the fungus, the conidia being borne in acervuli. 
 In mass, the spores appear orange-colored but have a hyaline appearance 
 under the microscope. They are usually ovate or oblong in shape, the two 
 diameters being 12-16 p and 4-6 p. Shear and Wood have shown that cer- 
 tain strains of this fungus, when grown on proper artificial media, may 
 produce conidia for generation after generation, without the interpolation 
 of the ascogenous form at any time. Cultures of such a strain were secured 
 from Dr. Shear for these experiments, and conidia only were produced 
 throughout the investigation, which lasted; about eighteen months, though 
 forty or more generations must have passed. 
 
 Corn meal agar was used as a medium for the stock cultures. This 
 was prepared by adding 4 teaspoonsful of white corn meal to one litre of 
 distilled water, which was then allowed to stand at about 58 C. for one 
 hour. After filtration, 12.5 g. of agar flour was added to the infusion, 
 and the mixture was steamed for one and one-half hours. It was then 
 re-filtered and was ready to tube and sterilize. The tubes were slanted 
 and the stock cultures were inoculated in streaks. On this medium the 
 fungus produces but little mycelium, at or beneath the surface, and the 
 conidia are borne in relatively large, orange-colored masses on the surface 
 
 13 Shear, C. L., and Wood, Anna K., Studies of fungus parasites belonging to the genus Glomerella. 
 U.S. Dept. Agric. Bur. Plant Ind. Bulletin 252. 1913. 
 
TOXICITY OF HEAVY METALS 63 
 
 of the medium along the streak. The acervuli are visiWe in from two to 
 five days after inoculation. 
 
 Before the spores were used in the experiments, the stock cultures were 
 allowed to grow undisturbed from ten to fifteen days, a procedure which 
 insured a sufficient quantity of spores fro.n a single tube for the inocula- 
 tion of an entire series of experimental cultures. Preliminary tests indi- 
 cated that spores from acervuli in the same tube, but of different ages', 
 were not at all uniform in viability. Direct inoculation of the culture 
 dishes from the spore masses of the stock tubes was thus shown to be 
 unsatisfactory, since it was not only desirable that all cultures contain 
 approximately the same number of spores, but also that the percentage of 
 viability of the spores in all cultures should be as nearly alike as possible. 
 It also seemed desirable to avoid small pieces of agar and bits of mycelium 
 in the liquid cultures, for such contamination might influence the effect 
 of the salts in the solutions on the germination of the spores, as by 
 absorption or the formation of new chemical compounds. The plan was 
 therefore evolved of allowing the stock cultures to grow for from ten to 
 fifteen days, after which period the spore masses were carefully removed, 
 with a platinum needle, to a clean area on the agar surface, from which, 
 after thorough mixing in a little heap, the inoculations to the water cul- 
 tures were made. This method usually resulted in satisfactory uniformity 
 in the germination of the spores in the various controls, from which 
 fact it was concluded that all cultures thus made contained an approxi- 
 mately equal number of viable spores, and that any inhibition or modifi- 
 cation of germination must be due- to properties of the culture medium 
 rather than to differences in the spores introduced. 
 
 In comparing the effect of the various media upon the spores, the main 
 criterion was the presence or absence of germination after a period of 
 eighteen hours. In many cases, however, germination was more or less 
 modified, as in the production of swollen tubes and other abnormalities, 
 and such modifications of germinal activity needed frequently to be taken 
 into account. As has been indicated, it was seldom necessary to consider 
 the percentage of normal germination which occurred, but in many cases 
 the proportion of abnormal to normal growth was approximately 
 determined. 
 
 These Gloeosporiurh spores possess several very favorable features for 
 such an investigation as the present. They are readily wetted by water 
 and aqueous solutions and, being slightly heavier than water, they sink 
 quickly to the bottom of a hanging drop. They germinate readily in dis- 
 tilled water in from three to four hours and produce long filaments in 
 eighteen hours, a feature which is of considerable advantage here, for it 
 is quite conceivable that the influence of various chemical compounds on 
 
64 LON A. HAWKINS 
 
 each other and on the germinating spores might be considerably altered 
 if nutrient salts were present in the solution. In view of these considera- 
 tions these experiments were carried out without the use of nutrient media. 
 
 METHODS. 
 
 The salts used in these experiments were " Baker's analysed " chemicals, 
 procured in the original packages. Stock solutions of the different salts, 
 from which the experimental solutions were afterwards prepared, were 
 made up in o.2m, 14 o.5m, or molecular concentration. In preparing the 
 stock solutions the salts were weighed in glass-stoppered weighing bottles 
 directly from freshly opened packages and were dissolved in volumetric 
 flasks. These solutions were then made up to the required volume at a 
 temperature of 15 C. They were stored in Jena glass bottles which had 
 been carefully washed with saturated solution of potassium dichromate in 
 sulphuric acid, steamed for half a day, again washed with distilled water 
 and finally allowed to soak in distilled water for a month or more, to 
 remove any soluble matter which might be present. Distilled water from 
 a still with tin lined boiler and condenser was used in making the stock 
 solutions as well as in diluting them for the cultural work. Preliminary 
 tests showed that the spores germinated as well in water from this still 
 as in more nearly pure water distilled from potassium permanganate solu- 
 tion, using a hard glass flask and a block tin condenser. 
 
 The stock salt solutions were diluted to the concentrations required in 
 making up the solutions for the experiments, by pipetting out the proper 
 amount into a hard glass beaker and then adding the necessary distilled 
 water from a burette. The concentrations of these solutions were so cal- 
 culated that the culture solutions could be prepared without the measure- 
 ment of less than 0.5 cc. in any case. Thus, errors that might have arisen 
 in attempting to read hundredths of a cubic centimeter on a burette gradu- 
 ated only to tenths, were obviated. 
 
 In making up a series of cultures, the two salt solutions which were to 
 be combined were separately diluted to twice the concentration finally 
 desired, and were then placed in burettes. From these were prepared, with 
 addition of water as needed, the combinations and concentrations actually 
 used in the experiments. These mixtures, in volumes of 10 cc. or more, 
 were made in small flasks (of about 75 cc. capacity), a flask being pro- 
 vided for each of the different combinations as well as one for the control. 
 The latter solution usually contained the salt of the heavy metal alone. 
 
 From each of the flasks just mentioned a small portion of solution (about 
 
 14 The letter nt is used throughout this paper to denote molecular, a concentration of a single gram 
 molecule in a liter of solution. There seems to be considerable confusion in this important matter of 
 defining solutions, some chemists employing the letter n to denote molecular, although the latter has 
 long been used to denote chemically equivalent. 
 
TOXICITY OF HEAVY METALS 65 
 
 a cubic centimeter) was placed in a separate glass cylinder (2 cm. high 
 and 3 cm. in diameter), to which spores from a stock culture were then 
 transferred. These inoculations were made in order, beginning with the 
 weakest solution of the lighter metal. A platinum needle was used for 
 this purpose, flamed and washed to sterilize and clean it after each 
 inoculation. 
 
 In each case the tip of the needle was dipped a single time in the well 
 mixed mass of spores which had been prepared on the agar surface, as 
 already described, and the spores that adhered were washed off in the culture 
 solution. Thus, approximately the same number of spores were inoculated 
 into all of the glass dishes and the solutions were then ready for the 
 preparation of the hanging drops. 
 
 The drop cultures were made after much the same method as that 
 described by Clark. 15 Van Tieghem cells were used, small cylinders of 
 glass tubing, with ground ends, 9 mm. high and 12 mm. in diameter, which 
 were cemented to ordinary microscope slides by means of beeswax. Two 
 cells were affixed to each slide. The culture solutions in the glass dishes, 
 into which spores had already been inoculated, were thoroughly mixed 
 with a glass rod. By means of this rod, a drop of the liquid was then 
 placed upon a flamed cover glass. 
 
 A small drop of the culture solution from the corresponding flask with- 
 out spores was placed in the bottom of the Van Tieghem cell and the cover 
 bearing the drop culture was inverted over it. Duplicate drop cultures 
 were made from each concentration of solution, both cultures being placed 
 on the same slide. As has been shown by Clark, the presence at the 
 bottom of the culture cell of a small amount of the same solution as that 
 from which the hanging drop is composed prevents evaporation from the 
 drop and hence obviates marked alteration in its concentration, even if the 
 cultures remain in the thermostat for a considerable time. Without this 
 precaution the solution contained in the hanging drop is apt to become 
 markedly more concentrated during the period of an experiment, which 
 might lead to erratic results. The covers were sealed in position with 
 petrolatum. It was not found necessary to take the precaution recom- 
 mended by Clark, of first allowing the expanding air to escape through 
 a small opening in the seal, possibly because the temperature of the thermo- 
 stat here used was only a little above the temperature at which the prepara- 
 tions were made. 
 
 For ease in handling the cultures, the slides were placed in sheet metal 
 trays, which could be piled one upon another in the thermostat so as to 
 form a rack. These trays were 15 cm. wide and 20 cm. long, with vertical 
 
 15 Clark, J. P., On the toxic effect of deleterious agents on the germination and development of cer- 
 tain filamentous funtn. Bot. Gaz. 28: 289-327, 378-404. 1890. 
 
66 LON A. HAWKINS 
 
 flanges at the ends, the flanges extending upward about 1.3 cm. and down- 
 ward about 0.4 cm. They were so bent that the lower flanges of one 
 tray fitted outside the upper flanges of the next lower one, and many 
 trays could thus be arranged in a compact and rigid pile without any 
 disturbance to the slides. The bottom of each tray was perforated with 
 circular openings about 1.5 cm. in diameter and 0.5 cm. apart, to facilitate 
 circulation of air. Each tray carried fourteen slides. The slides were 
 always transferred to and from the thermostat by means of the trays, a 
 whole series of cultures being thus moved together. 
 
 The cultures were kept during germination in an electrically heated and 
 automatically regulated thermostat, in which the temperature was main- 
 tained at or near 25 C. As the temperature of the room in which the 
 thermostat was placed sometimes rose above 25, it was necessary to 
 install apparatus for absorbing heat at such times. To accomplish this, 
 several coils of thin-walled copper tubing carrying a continuous stream of 
 tap water were placed at the top of the thermostat, surrounding a small 
 motor-driven fan, the latter insuring air circulation. The air of the 
 chamber then tended to assume a temperature several degrees below that 
 of the laboratory, and the thermostat acted as though standing in a 
 cold room. 
 
 EXPERIMENTATION, 
 
 In these studies, any renewed activity in the protoplasm of the spore 
 was considered as germination. Several forms of such renewed activity 
 are exhibited by the conidia here employed. Without any alteration in 
 size or shape, a portion of the spore may become nearly or quite opaque, 
 thus appearing dark brown or black by transmitted light. A papilla may 
 be formed at any point on the surface. Such papillae may or may not 
 enlarge to form rounded bodies, and may either remain hyaline or become 
 apparently darkened. Papillae may enlarge to form irregularly shaped 
 bodies or <may extend outward as markedly thickened tubes. Lastly, the 
 growing papillae may take the form of slender tubes. The latter type of 
 germination always occurs in distilled water and the tubes attain, in a 
 period of eighteen hours, a length at least twice as great as that of the 
 spore itself. In the following treatment germination will be considered 
 " normal " wherever tubes of the last mentioned type, at least twice the 
 length of the spore, were produced in eighteen hours. 
 
 In the present section will be considered the various effects upon germ- 
 ination brought about in the presence of the following salts either alone 
 or in certain combinations : 
 
 Ca(NO 3 ) 2 Pb(NO 3 ) 2 Ni(NO,,) 2 
 
 Mg(NO 3 ) 2 Zn(NO 3 ) 2 Cu(NO 3 ) 2 
 
 KNO 3 A1(NO 3 ) 3 CuSO 4 ' 
 
TOXICITY OF HEAVY METALS 67 
 
 TOXICITY OF SALT COMBINATIONS 
 
 Calcium, Magnesium and Potassium. 
 
 It requires a relatively high concentration of calcium, magnesium or 
 potassium nitrate to inhibit the germination of these spores. With 
 Ca(NO 3 ) 2 normal germination was found in the 0.5111 solution and 
 swollen tubes were abundant in o.6m. At a concentration of o./m, how- 
 ever, none of the spores germinated in any of the four series of duplicate 
 cultures which were carried out. The concentration of this salt which 
 just prevents germination appears, therefore, to lie between o.6m and 
 o./m. Magnesium nitrate is similar to that of calcium in its effect. With 
 this salt the spores germinated normally at a concentration of 0.4111, and 
 local swellings of the spore wall and swollen tubes were produced in 0.5111 
 solution, while no germination was evident when a o.6m concentration was 
 employed. The concentration at which Mg(NO 3 ) 2 just inhibited germin- 
 ation thus seems to lie between o.5m and o.6m. In solutions of KNO 3 
 the spores germinated normally with a concentration of 0.9111 and formed 
 local swellings of the spore wall with molecular concentration. As the 
 latter was the highest concentration here used, the 
 point at which KXCX inhibits germination was not 
 determined. 
 
 The exceedingly high .concentrations of these salts, 
 which were found necessary to inhibit germination, 
 made it seem possible that they might be without toxic 
 effect upon the spores ; the inhibition of germination 
 
 which was observed in high concentrations might have 
 
 .-..-, . in? spores from i.6m 
 
 been the result of the high osmotic pressure exerted sucrose solution, x 
 
 by the medium. To obtain evidence on this point the 550. 
 germination of the spores was tested in cane sugar 
 
 solution at various concentrations. A o.2m stock solution was prepared 
 from granulated sugar which had been previously pulverized and desic- 
 cated ; the different concentrations used in the cultures were prepared from 
 this. The spores germinated normally in concentrations of cane sugar 
 up to and including i-4m, considerable germination in the form of local 
 swellings (shown in fig. i) was found in the i.6m solution, and no germina- 
 tion occurred with i.8m concentration. The concentration of cane sugar 
 which just inhibits germination lies then between i.6m and i.8m. 
 
 It will be observed that a i.6m solution of sucrose has about the same 
 effect upon the germination of these Gloeosporium spores as do o.6m, 0.5111 
 and molecular solutions of calcium, magnesium and potassium nitrates 
 
68 LON A. HAWKINS 
 
 respectively. Calculations 16 of the osmotic pressures of these four solu- 
 tions give 51.36, 29.13, 26.00 and 39.31 atmospheres, respectively, from 
 which numbers it becomes obvious that the osmotic concentration of the 
 sugar solution was much greater than that of the other three. It is there- 
 fore suggested that, unless the wall and protoplasm of the spores here 
 employed be readily permeable to cane sugar (which seems highly improb- 
 able), the inhibiting effects observed with the salt solutions are not to be 
 related primarily to their osmotic properties. 
 
 The relatively high osmotic pressure existing within the spores here 
 dealt with, as indicated by the data just given, renders it unnecessary to 
 consider osmotic pressure as a factor in bringing about the modifications 
 and inhibitions of germination which are next to be considered ; the con- 
 centrations of the various salt solutions employed were always far too 
 low to produce any removal of water from the cells. Similarly, the 
 toxicity of calcium, magnesium and potassium nitrates is so very slight 
 as not to require any consideration in connection with the combinations of 
 these salts with those of the heavy metals ; in these combinations the 
 salts of the lighter metals have not here been used in concentrations higher 
 than o.im. 
 
 The influence of the nitrates of calcium, magnesium and potassium upon 
 the toxicity of salts of the heavy metals will now receive attention. 
 
 Copper. 
 
 As was to be expected, the effect of Cu(NO 3 ) 2 upon the germination of 
 these spores was found to be widely different from that of Ca(NO 3 ) 2 . No 
 germination was ever found in concentrations of the copper salt alone 
 higher than 0.00006111, and it was only in an occasional culture that one 
 or two spores were observed producing local swellings with that concen- 
 tration. Local swellings of the spore walls and swollen tubes were fre- 
 quently found with 0.00004111 and with o.oooo26m concentration, the next 
 lower one which was here used; with 0.00002111 solution of Cu(NO 3 ) 2 
 germination was about as in distilled water. 
 
 Copper nitrate was combined with Ca(NO 3 ) 2 , at several concentrations 
 of the copper salt well above that at which the latter was non-toxic when 
 used alone. In a series of combinations of o.oooim Cu(NO 3 ) 2 with dif- 
 
 18 Morse, H. N., and Holland, W. W., The osmotic pressure of cane sugar solutions at 25. Amer. 
 Chem. Jour. 41: I-IQ. IQOQ. The osmotic pressure of molecular cane sutfar was measured directly by 
 these writers and the osmotic pressure of 1.6 m. cane sugar as given above has been calculated from their 
 tables. The result is probably low, for they found that the ratio of observed to calculated osmotic pres- 
 sure increases with the concentration. For the gas pressure formula, by which the osmotic pressure 
 of the two electrolytes is calculated, see Landolt, H., Bornstein, R., and Meyerhoffer, W., Physikalische- 
 chemische Tabellen, 3te Aufllaije. Berlin. 1005; page 24. 
 
TOXICITY OF HEAVY METALS 69 
 
 ferent concentrations of the calcium salt, ranging from 0.00003 12 5 m to 
 o.ooo625m, the spores germinated readily in those cultures where the con- 
 centration of Ca(NO 3 ) 2 was 0.0000625111 or higher. With the last named 
 concentration many local swellings were found, and also short swollen 
 tubes (see fig. 2), much as in the 0.00004111 concentration of Cu(NO 3 ) 2 
 alone. With the next lower concentration of the calcium salt, 0.00003 125m, 
 no germination occurred. 
 
 From these facts it appears that the toxicity of o.oooim Cu(NO 3 ) 2 solu- 
 tion is so reduced as to be physiologically equivalent to a 0.00004111 solution 
 of the same salt, by the addition to the former solution of five molecules 
 of Ca(NO 3 ) 2 for every eight molecules of Cu(NO 3 ) 2 present. The addi- 
 tion of the lighter metal in this proportion pro- 
 duces the same effect as though the o.oooim cop- 
 per solution had been diluted to two and a half 
 times its original volume. 
 
 Copper sulphate exhibits about the same tox- 
 icity toward these Gloeosporium spores as does 
 the nitrate. A series of combinations of this salt 
 with Ca(NO 3 ) 2 , quite similar to the series with Fig. 2. Germinating 
 Cu(NO 3 ) 2 just described, gave no germination spores from combination 
 in solutions containing 0.00003125111 concen- solution containing 
 
 trations of the calcium salt, while germina- - 000 6m Ca(NO 3 ) 2 and 
 . . ' o.oooim Cu(N0 3 ) 2 , X6 5 o. 
 
 tion occurred in some of the cultures con- 
 taining 0.0000625111 Ca(NO 3 ) 2 . In general, the effect of CuSO 4 , either 
 alone or in combination with Ca(NO 3 ) 2 , was practically the same as was 
 that of Cu(NO 3 ) 2 . This furnishes some additional evidence toward the 
 already rather firmly established conclusion, that the toxicity of copper 
 salts is due to the cations, and it also indicates the probability that con- 
 siderations bearing upon this toxicity need deal only with the cations. 
 
 A series of experiments was carried out using o.ooo/mi Cu(NO 3 ) 2 in 
 combination with concentrations of Ca(NO 3 ) 2 ranging from 0.00025111 to 
 o.O25m. The spores in these combinations germinated readily in the pres- 
 ence of Ca(NO 3 ) 2 in concentrations ranging from o.ooim to 0.025111, but 
 no germination was found in combinations with 0.0005111 or 0.00025111 of 
 the calcium salt. The form and abundance of germination with o.ooim 
 Ca(NO 3 ) 2 was here much the same as that found in 0.00004111 of the copper 
 salt alone. Here a ratio of five molecules of the calcium salt to two of 
 the copper salt, the latter occurring in the solution at a concentration of 
 0.0004111, reduces the toxicity of the Cu(NO 3 ) 2 so as to produce an effect 
 on spore germination equivalent to that exercised by a 0.00004111 solution 
 of the copper salt alone. In other words, addition to a 0.0004111 Cu(NO 3 ) 2 
 solution, of Ca(NO 8 ) a in the molecular ratio of five of the latter to two of 
 
70 LON A. HAWKINS 
 
 the copper Salt, has the same effect as diluting the Cu(NO 3 ) 2 solution to 
 ten times its original volume. 
 
 A third series of combinations of the same two salts, but with higher 
 concentrations, was carried out in a manner somewhat different from that 
 employed in the preceding series. Here the concentration of Ca(NO s ) 8 
 was the same (o.o5m) in all of the cultures, while that of the copper salt 
 varied. The concentrations of Cu(NO 3 ) 2 ranged from 0.00025111 to o.oim, 
 and the spores germinated in all the concentrations used except the highest. 
 With a concentration of o.ooSm Cu(NO 3 ) 2 germination was similar to 
 that found with 0.00004111 of the copper salt without the addition of 
 Ca(NO 3 ) 2 . From this series it appears that addition to the Cu(NO 3 ) 2 solu- 
 tion here used (o.ooSm), of about six molecules of Ca(NO 3 ) 2 for each mole- 
 cule of the copper salt present in the solution, reduces the toxicity of the 
 latter compound in the same way as though the solution had been diluted 
 to two hundred times its original volume. It appears as though the presence 
 of Ca(NO 3 ) 2 , in this molecular proportion of about 6 to i, altered the 
 relations between spores and solution so that only a two-hundredth part 
 of the copper nitrate actually present was effective to modify or retard 
 the germination processes. 
 
 A series of combinations of Cu(NO 3 ) 2 with KNO 3 was carried out, 
 in which 'a constant concentration of the potassium salt, 0.05111, was used 
 in combination with Cu(NO 3 ) 2 in concentrations ranging from 0.0002111 to 
 o.oim. The spores germinated in concentrations up to and including 0.002111 
 of the copper salt. In the last mentioned concentration, the form of 
 germination was quite similar to that found in a 0.00004111 solution of the 
 copper salt alone. Thus in a solution containing KXO 3 at a 0.05111 con- 
 centration and Cu(NO 3 ), at a 0.002111 concentration (a molecular ratio 
 of 25 of the potassium salt to i of the other), the toxicity of the copper 
 salt is decreased to a magnitude only one fiftieth as great as is that shown 
 by this concentration of the copper salt alone. 
 
 The results of the three series of combinations of copper nitrate with 
 calcium nitrate which have been described, are summarized in table I, 
 together with certain other data which require consideration. 
 
 From the data just given the question arises, whether the influence of 
 calcium nitrate in reducing the toxicity of the copper salt may be due to 
 a direct effect produced in the solution or to some change brought about 
 in the spore itself. Kronig and Paul [97], in considering an effect 
 similar to this, in which the toxicity of HgCl 2 upon Bacillus anthracis 
 was decreased by the addition of NaCl, concluded that the change thus 
 brought about is due to depression of the ionization of the salt of the 
 heavy metal. Such a supposition cannot apply in the present case, how- 
 ever; that alteration in the ionization of Cu(NO 3 ) 2 cannot be the cause 
 
TOXICITY OF HEAVY METALS 71 
 
 of the diminution of its toxicity as here observed, is clearly shown by 
 simple calculation according to the isohydric principle. The percentage of 
 dissociation of Cu(NO 3 ) 2 in the mixtures here employed should be deter- 
 mined by the concentration of the NO 3 radical. Following this generaliza- 
 tion, the data given in table I (C, a) have been obtained. Seventy-six per 
 cent, of the copper salt of the first combination is seen to be dissociated. 
 
 TABLE I 
 
 . 
 
 Series 
 I 
 
 Series 
 II 
 
 Series 
 III 
 
 A. Cu(XO 3 ) 2 present in the 
 medium, molecular. . . t 
 
 a. Common fraction 
 
 TT5 
 
 J3U<T 
 
 i<To<nr 
 
 
 b Decimal fraction 
 
 o .008 
 
 o .0004 
 
 .0001 
 
 
 B. Ca(NO 3 ) 2 required to al- 
 low same kind and 
 amount of germination 
 as occurs in 0.00004111 
 (m/2 5 ooo) Cu(X0 3 ) 2 
 
 without PafVO 1 
 
 a. Common fraction, mole- 
 cular 
 
 * 
 
 TIFffTf 
 
 T^ffff 
 
 
 b. Decimal fraction, mole- 
 cular 
 
 0.05 
 
 O .OOI 
 
 o .0001 
 
 
 
 c. Number of molecules of 
 Ca(XO 3 ) 2 for each 
 molecule of Cu(XO 3 ) 2 . 
 
 6.25 
 
 2 .50 
 
 0.63 
 
 C. Concentration of disso- 
 ciated Cu(XO 3 ) 2 pres- 
 ent in mixture, mole- 
 cular 
 
 a. According to the isohy- 
 dric principle 
 
 o .0061 
 
 o .0004 
 
 o .0001 
 
 
 b. From potentiometer 
 measurements 
 
 o .0058 
 
 o .0004 
 
 o .0001 
 
 
 
 D. Concentration of disso- 
 ciated Cu(XO 3 ) 2 in 
 physiologically equiva- 
 lent solution of this 
 salt alone, molecular . . 
 
 From conductivity meas- 
 urements (Jones' 
 tables) 
 
 o .00004 
 
 o .00004 
 
 o .00004 
 
 
 Now, if only one per cent, of the copper here actually present in the 
 mixture were ionized, the concentration of the Cu ion would correspond 
 to that in an o.ooooSm solution of Cu(NO 3 ), alone, which, as has been 
 shown, is stronger than is necessary to inhibit spore germination. It is 
 therefore apparent that the decrease in the toxicity of the copper salt shown 
 in these combinations with Ca(NO 3 ) 2 is not to be related to decreased 
 ionization of Cu(NO 3 ) 2 . 
 
 Another theory to account for a similarly decreased toxicity of HgCU 
 
72 LON A. HAWKINS 
 
 in the presence of NaCl was advanced by Clark [01], who considered that 
 a double salt was formed, such as Na 2 HgQ 4 , and supposed this to be 
 only slightly dissociated in the presence of an excess of NaCl. This writer 
 also worked with various copper salts in the presence of KNO 3 , K 2 SO 4 , 
 NH 2 SO 4 , etc., and suggested that the decreased toxicity of the copper salts, 
 which was uniformly observed in such combinations, was likewise due to 
 formation of double salts. 
 
 In view of the work of Hosford and Jones, 17 on the dissociation of 
 double salts in dilute solution, it seems very improbable that the lowering 
 of the toxicity of Cu(NO 3 ) 2 brought about by Ca(NO 3 ) 2 , as shown in 
 the present study, is due to decreased ionization of the toxic compound, 
 brought about by double salt formation. It is, however, possible to obtain 
 direct evidence on this question by comparing the concentrations of copper 
 ions in the various solutions, with and without the calcium salt. The per- 
 centage of dissociation occurring in simple solutions of Cu(NO 3 ) 2 has been 
 determined experimentally for many concentrations, and these data are 
 available in published tables. 18 From such data may be derived the con- 
 centration of copper ions present in any solution containing only Cu(NO 3 ) 2 , 
 but no published data are yet available from which might be ascertained 
 the concentrations of copper ions in the binary mixtures here dealt with. 
 
 The determinations here required of the relative concentrations of copper 
 ions in solutions of Cu(NO 3 ) 2 with and without addition of the calcium 
 salt, may be made by means of the potentiometer. 10 With this instrument 
 the solutions involved in table I were tested, and the differences in 
 electrical potential between the combination solutions of Cu(NO 3 ) 2 and 
 Ca(NO 3 ) 2 and the corresponding simple solutions of Cu(NO 3 ) 2 were 
 measured. The two solutions to be tested (for example, the simple solu- 
 tion containing o.ooSm Cu(NO 3 ) 2 and the combination solution containing 
 o.ooSm Cu(NO 3 ) 2 and o.O5m Ca(NO 3 ) 2 ) were placed, respectively, in the 
 two arms of a U-tube, separated below by a saturated solution of ammonium 
 nitrate. Into each arm of the U-tube was introduced a copper electrode 
 and these were connected with a potentiometer (Leeds and Northrup, type 
 K). The potentiometer was so calibrated that the difference in voltage 
 between the two copper solutions might be read directly on the instrument. 
 
 17 Hosford, H. H., and Jones, Harry C., Conductivity, temperature coefficients of conductivity and 
 dissociation of certain electrolytes. Amer. Chem. Jour. 46: 240-278. ign. 
 
 18 Jones, Harry C., Electrical conductivity, dissociation and temperature coefficients of conduc- 
 tivity, from zero to sixty-five degrees, of aqueous solutions of a number of salts and organic acids. 
 Carnegie Institution of Washington Publ. 170. Washington. 1012. 
 
 19 Loomis, N. E., and Acree. S. F., A study of the hydrogen electrode, of the calomel electrode, and 
 of contact potential. Amer. Chem. Jour. 46: 585-620. 191 1. 
 
 It is a pleasure to acknowledge here, that the suggestion of this method, as applicable to the prob- 
 lem in hand, is due to Professor S. F. Acree, of the Chemistry Department of the Johns Hopkins Uni- 
 versity, and that much valuable help in making the determinations was received from him and from 
 Dr. C. N. Myers. This part of the work was carried out at the chemical laboratory. 
 
TOXICITY OF HEAVY METALS 73 
 
 The copper electrodes were freshly plated and calibrated, "for subsequent 
 correction of the readings, with reference to any difference in potential 
 which might exist between them when both were placed in the simple 
 Cu(XO 3 ) 2 solution. 
 
 The difference in concentration of copper inns in the two solutions thus 
 compared was calculated from the observed difference in electrical potential 
 by the following formula : 
 
 V = 0.059 1 logio-?^ 
 *t 
 
 in which V is the observed Electromotive force, in volts ; C and Q are 
 the concentrations, respectively, of the copper ions in the two solutions, 
 and the quantity 0.0591 is a constant (Loomis and Acree [n], page 591). 
 In each of the tests with which we are at present concerned three entirely 
 separate determinations were made, for each of which the reading was 
 several times repeated, and the results were in excellent agreement. It was 
 found that the difference in electric potential between the 0.008111 solution 
 of Cu(XO :j ) 2 and the same solution containing also a 0.05111 concentration 
 of Ca(XO.j) 2 , was 0.006 volt, the simple solution having the higher 
 potential. From this observation it appears, by substitution in the formula 
 given above, that 
 
 C 
 r =1.26, 
 
 ^i 
 
 or that the concentration of copper ions in the combination solution is 
 
 ^, or 0.794, as great as that in the simple' one. Xow, from Jones' [12] 
 
 conductivity tables (page 57), it is found by interpolation that the 
 Cu(XO :! ) 2 in our simple solution (0.008111) is about 90.6 per cent, disso- 
 ciated at 25 C. It thus appears that the Cu(XO 3 ) 2 in the combination 
 solution here considered must be dissociated to an extent equal to 0.794 X 
 90.6, or 71.9 per cent. 
 
 It has been shown above (page 71) that the Ca(NO 3 ) 2 in this particular 
 combination should decrease the dissociation of the copper salt, on account 
 of the common ion, to 76 per cent. There is thus a difference of about 
 4 per cent, between the dissociation of the Cu(XO 3 ) 2 in this mixture as 
 calculated from the isohydric principle and that derived from the potenti- 
 ometer measurements. This difference may, of course, be due to the forma- 
 tion of a double salt, following the hypothesis of Clark already mentioned, 
 but it makes no difference in the present discussion whether it be 76 or 
 72 per cent, of the Cu(XO 3 ) 2 that is here dissociated; as has been stated, 
 the solution would still be toxic enough to inhibit germination in these 
 spores if only one per cent, of the total Cu(XO 3 ) 2 were dissociated. 
 
 The remaining two concentrations of Cu(XO 3 ) 2 (0.0004111 and o.oooirn, 
 
 PHYSIOLOGICAL RESEARCHES VOL. I, NO. 2, SERIAL NO. 2, 
 AUOUST, 1913. 
 
74 LON A. HAWKINS 
 
 see table I), with and without additional Ca(NO 3 ) 2 , were also subjected 
 to potentiometer determinations of the concentrations of copper ions present 
 therein. In neither case was there any difference in electrical potential 
 between the two corresponding solutions. It is therefore clear that at least 
 99 per cent, of the Cu(NO 3 ) 2 is to be considered as dissociated in these 
 solutions, whether the calcium salt be present or not (see table I, C, b). 
 
 From the foregoing considerations, it seems quite clear that the influence 
 of Ca(NO 3 ) 2 in reducing the toxic effect of Cu(NO 3 ) 2 on the germination 
 of the spores here employed is not at all to be related to any changes 
 brought about in the solution itself by the addition of the calcium salt. 
 It appears that this antitoxic or antagonistic influence must be effective 
 upon the spores themselves, so altering them that they become capable of 
 germination in solutions whose concentration of free copper ions would 
 inhibit this process were it not for the presence of the calcium salt. 
 
 Whether the copper enters the spores and exerts its toxic action directly, 
 through some alteration in the protoplasm, or whether this toxic influence 
 is exerted primarily upon the spore walls or upon the surface film of the 
 protoplasm, thus perhaps creating some disturbance in physico-chemical 
 equilibrium that may subsequently be propagated inward, is a question for 
 the answering of which no evidence is yet at hand. Nevertheless, the present 
 studies have clearly demonstrated that the presence of Cu(XO 3 ) 2 alone 
 in the medium inhibits germination if the concentration be above a very 
 low limit, produces markedly altered forms of renewed activity if the con- 
 centration is somewhat lower., and allows normal germination only when 
 the solution is exceedingly dilute. While these facts must be interpreted 
 to mean that the copper salt tends to upset the protoplasmic system in some 
 way and that it is possible for that system to be so disturbed as either to 
 inhibit germination absolutely or to allow this process to proceed in modi- 
 fied form, yet much more direct evidence of such protoplasmic disturbance 
 was frequently met with in the progress of this work. Spores in which 
 all germinational activity had been prevented by the presence of Cu(NO 3 ) 2 
 frequently possessed a characteristic, coarsely granular appearance, as 
 though a precipitate or coagulum had been formed within the protoplasm. 
 Furthermore, this same appearance was often encountered in spores which 
 exhibited modified germination under the influence of copper. From this 
 it appears that the granular appearance of the protoplasm does not neces- 
 sarily denote death, but to settle this point conclusively and to determine 
 whether the production of apparent granulation might not be a post mortem 
 effect, the following experiment was performed. 
 
 Spores which had been for eighteen hours in cultures with 0.00008111 
 solution of Cu(NO 3 ) 2 alone were employed. In this solution no germina- 
 tion has ever been found, in the more than twenty separate cultures which 
 
TOXICITY OF HEAVY METALS 75 
 
 have been observed, and the protoplasm usually appears markedly granular. 
 The spores were transferred from the poison solution to a new drop cul- 
 ture with distilled water and were then returned to the thermostat. At 
 the end of twenty-four hours, observation showed that many of the gran- 
 ular spores had germinated normally, although the granular appearance was 
 still very noticeable. Further proof that the granulated appearance with 
 which we have here to deal is not a post mortem effect of copper poisoning 
 may be derived from the fact that the germinal tubes of spores which 
 had germinated in a combination solution of o.oooim Cu(NO 3 ), and 
 o.oooo6m Ca(NO 3 ) 2 elongated considerably when the cultures were allowed 
 to remain in the thermostat for a second day, although the spores showed 
 the characteristic granulation above mentioned. 
 
 It seems probable, from these and other similar lines of evidence, that 
 the copper of the solution penetrates the spore wall and gives rise to the 
 precipitation or coagulation effect just described. 
 
 It may be noted here that the influence of calcium upon the toxicity of 
 copper is of interest in connection with the problem of fungicides and fungi- 
 cidal action. From the results obtained in the experiments described above, 
 the conclusion seems warranted that only a small portion of the soluble 
 copper in Bordeaux mixture is effective as a fungicide. Yet it is a well 
 known fact that a long list of fungous diseases, including apple bitter-rot, 
 can be controlled by the proper applications of this mixture to the host 
 plants. The use of KOH instead of Ca(OH) 2 in preparing the fungicide 
 should result in a much more toxic mixture, for, as brought out in the 
 preceding experiments, potassium is only about one fourth as effective as 
 calcium in decreasing the toxicity of copper. From the standpoint of 
 the host, however, it is quite probable that this anti-toxic effect of the 
 calcium is important in preventing " spray-injury " to the foliage and fruit, 
 while the small amount of soluble copper present, is, in most cases, suf- 
 ficient to prevent fungous growth. 
 
 Lead. 
 
 The nitrate of lead is considerably less toxic toward the Gloeosporium 
 spores here employed than is that of copper. The concentration just 
 inhibiting germination after eighteen hours was found to be about o.QO4m. 
 With a concentration of 0.002111 of the lead salt, some of the spores showed 
 slight terminal or lateral swellings. At this concentration spores were fre- 
 quently found in which one half was brown or blackened while the other 
 seemed devoid of protoplasm, appearing as if the protoplasmic contents 
 had all collected in one end and had then taken on a thicker wall. No 
 increase in size or local swelling was apparent in such cases. In a con- 
 centration of o.ooim the blackened bodies just mentioned were found, as 
 
76 LON A. HAWKINS 
 
 well as swellings, and also definite outgrowths from one or both ends of 
 the spore. The latter (shown in figure 3) are of particular interest and 
 require some attention here. They are spherical in shape, dark brown or 
 black in color and appear in all respects similar to the chlamydospores or 
 appressoria which have been frequently described as resulting under certain 
 conditions, from the germination of spores of various Gloeosporium forms. 
 Appressoria have been described by Hasselbring 20 as forming when 
 nutrient materials are absent and when germinating spores or germ tubes 
 come in contact with such hard surfaces as are furnished by the cover 
 glass in a drop culture or by the epidermis of various fruits. This writer 
 considers that they function as holdfasts and that they result from a contact 
 stimulus acting upon spores or tubes which are not well nourished. That 
 such bodies are frequently formed when germ tubes come in contact with 
 the cover glass of a hanging drop culture, has often been demonstrated 
 in the present studies. They are especially characteristic of cultures with 
 certain salt solutions, at concentrations somewhat below that at which 
 
 germination is entirely suppressed but above 
 that at which normal development occurs. Here 
 their formation does not appear to be related to 
 any contact stimulus, however. In some cases 
 
 swollen bodies are produced which have the 
 Fig. 3. Germinating form Q appressoria but which are haline 
 spores from o. 00033^1 
 
 Pb(NO ) X6so ^* e t " e usua l spores and germ-tubes of this 
 
 .fungus, thus apparently differing from the 
 
 appressoria only in not being brown or black in color. In the descriptions 
 which follow the term appressoria will be used to denote the dark colored, 
 appressorium-like bodies, and swellings of similar form but without dark 
 appearance will be termed hyaline appressoria. These terms are applied 
 here merely in a descriptive way, without intended implication that the 
 bodies thus designated may not be physiologically or otherwise different 
 from the appressoria of the mycologists. 
 
 In concentrations of Pb(NO 3 ) 2 from o.oooim to 0.00005111, most of the 
 germination observed resulted in appressoria. Some of the similar, hyaline 
 swellings also occurred in these cultures. When cultures where appressoria 
 were common were allowed to remain in the thermostat for several days 
 and were examined from time to time, these swellings continued to develop 
 successively from the same spore, until sometimes as many as four 
 appeared together at one end (see fig. 4). In the formation of chains 
 of these bodies it appeared, from the observation of different stages, that 
 a second or later swelling may be brought about either by the germination 
 of one previously formed or by enlargement of the constricted portion of 
 
 20 Hasselbring, H., The appressoria of anthracnoses. Bot. Gaz. 42: 135-142. 1906 
 
TOXICITY OF HEAVY METALS 
 
 77 
 
 the tube between one previously formed and the spore itself; in the latter 
 case possibly by new growth from the spore. In many cases a light colored 
 area quite like that considered by Hasselbring to be a pore, could be dis- 
 tinguished, and this may have been present in all cases. Spores which had 
 germinated in drop cultures by forming appressoria were transferred to 
 new cultures in distilled water and returned to the thermostat. Nearly 
 all the appressoria had germinated with normal germ tubes at the end of 
 twenty-four hours. 
 
 An occasional spore germinated normally in a concentration of 0.00005111 
 Pb(NO 3 ),, and a few normal tubes were found in the 0.00004111 concen- 
 tration, but it was only in the o.oooo2m concentration that no effect of the 
 Pb(XO 3 ) 2 upon the germination of the spores was noticeable. In the 
 latter concentration the spores germinated with long tubes, just as in simul- 
 taneous controls in distilled water. 
 
 Series including combinations of Pb- 
 (NO 3 ) 2 with Ca(NO 3 ) 2 were carried out 
 at three different concentrations of the lead 
 salt, o.oooi6m, .0.00033111, and o.ooo66m. 
 With a o.ooo66m concentration of Pb- 
 (NO 3 ) 2 no concentration of the calcium salt 
 was found which entirely suppressed the 
 formation of appressoria and in which only 
 normal germination was produced, though 
 with a concentration of 0.05111 Ca(NO 3 ) 2 
 only a few appressoria were found. This 
 combination gave practically the same re- 
 sults as were found with a 0.00004111 con- 
 centration of Pb(NO 3 ) 2 alone; i.e., some- 
 what over 50 per cent, of the germination was normal, though a considerable 
 number of swellings and appressoria were formed. In the combination of 
 the same concentration of Pb(NO 3 ) 2 (o.ooo66m) with o.oo4m of the cal- 
 cium salt the effect produced was similar to that found in cultures with 
 0.00005111 concentration of Pb(NO 3 ) 2 ; only occasional normal tubes were 
 found, these probably constituting less than I per cent, of the total germina- 
 tion. With concentrations of o.ooim, 0.002111 and 0.003111 Ca(NO 3 ) 2 , com- 
 bined with a o.ooo66m concentration of the lead salt, practically no normal 
 tubes were produced, germination resulting in appressoria and swellings. 
 These concentrations correspond to solutions of Pb(NO 3 ) 2 alone varying 
 in concentration from o.ooim to o.oooim. 
 
 In the second series of combinations of Pb(NO 3 ) 2 with Ca(NO 3 ) 2 , the 
 concentration of the first salt was o.ooo33m and the same concentrations of 
 Ca(NO 3 ) 2 were employed as in the series just described. The results in 
 
 Fig. 4. Spores after four day 
 in o.oo4m Pb(NO 3 ) 2 , X650. 
 
78 LON A. HAWKINS 
 
 these experiments were similar to those of the previous series, with some 
 differences due to the lower concentration of the lead salt. Practically no 
 appressoria were formed with the combination containing the o.o5m con- 
 centration of Ca(NO 3 ) 2 , germination being here (see fig. 5) about the 
 same as that which occurred with the o.oooo2m concentration of the lead 
 salt alone. With a combination containing o.oim Ca(NO 3 ) 2 , the same 
 effect was obtained as with 0.00004111 Pb(NO 3 ) 2 alone, and with combina- 
 tion containing o.oo2m Ca(NO 3 ) 2 about the same effect was evident as 
 with a concentration of o.oooo5m Pb(NO 3 ) 2 alone. In the combinations 
 with o.ooim and 0.0005111 Ca(NO 3 ) 2 the results were practically the same 
 as with a concentration of o.oooim Pb(NO 3 ) 2 alone; most of the germina- 
 tion here gave appressoria. 
 
 With a o.oooi6m concentration of Pb(NO 3 ) 2 in combination with the 
 same concentrations of Ca(NO 3 ) 2 as were employed in the two series 
 
 Fig. 5. Germinating spores from combination 
 solution containing 0.00033111 Pb(NO 3 ) 2 and 0.05111 
 Ca(NO 3 ) 2 X 650. 
 
 above described, the concentration of the calcium salt in which the majority 
 of the germinating spores formed normal tubes (instead of swellings and 
 appressoria), and the concentration in which only an occasional spore 
 formed a normal germ-tube, were somewhat lower than in the previous 
 series, as was to be expected. The combination containing a o.oo2m con- 
 centration of Ca(NO 3 ) 2 gave practically the same effect as did the con- 
 centration of o.oooo/mi of the lead salt alone, while that containing a o.ooim 
 concentration of Ca(NO 3 ) 2 gave results similar to those obtained with a 
 o.oooo2m concentration of the lead salt alone. Combinations containing 
 Ca(NO 8 ) 2 in concentrations of o.O333m, 0,04111 and 0.05111, respectively, pro- 
 duced the same sort of germination as was obtained with a concentration 
 of 0.00033111 Pb(NO 3 ) 2 combined with a 0.05111 concentration of the calcium 
 salt ; germination was practically all normal, only an occasional appressorium 
 or swelling being found. 
 
 From the above data the conclusion seems warranted that the same 
 molecular ratio of the calcium salt to that of lead produces practically 
 
TOXICITY OF HEAVY METALS 79 
 
 identical effects with the three different concentrations of Pb(NO 3 ) 2 here 
 used. Concentrations of o.oim, o.O2m and o.o6m Ca(NO 3 ) 2 combined, 
 respectively, with concentrations of o.oooi6m, o.ooo33m and o.ooo66m 
 Pb(NO 3 ) 2 produce practically the same effect on spore germination as does 
 the 0.00005111 concentration of the lead salt alone. It thus appears that 
 for all three very different concentrations of Pb(NO 3 ) 2 , the addition of 
 Ca(NO 3 ) 2 in the proportion of six molecules of the calcium salt to one 
 molecule of Pb(NO 3 ) 2 reduces the toxicity of the latter salt to a point 
 where it almost, but not quite, inhibits normal germination and nearly 
 restricts germinal activity to the formation of appressoria and other swell- 
 ings. To obtain this same condition of germination a simple solution of 
 Pb(NO 3 ) 2 must have a concentration of 0.00005111. 
 
 A similar series of equivalent effects upon spore germination, with the 
 three different concentrations of the lead salt, is shown by taking as the 
 index of physiological activity the prevalence of normal germ tubes in 
 the cultures, as contrasted with swellings and appressoria. This condi- 
 tion of affairs is attained in combination solutions containing, respectively, 
 o.02m Ca(NO 3 ) 2 combined with o.ooo66m Pb(NO 3 ) 2 , o.oim Ca(NO 3 ) 2 
 combined with 0.00033111 Pb(NO 3 ) 2 , and 0.005111 (Ca(NO 3 ) 2 combined 
 with o.oooi6m Pb(NO 3 ) 2 . These three combinations of the two salts all 
 produce practically the same effect on germination as does the concentration 
 of 0.00004111 Pb(NO 3 ) 2 alone. Here the addition of about 30 molecules 
 of Ca(NO 3 ) 2 to i of Pb(NO 3 ) 2 (30:1) reduces the toxicity of the lead 
 salt until it is physiologically equivalent to that of the 0.00004111 solution 
 of this salt alone. 
 
 Combination of Pb(NO 3 ) 2 with Mg(NO 3 ) 2 shows a similar influence 
 of the alkaline earth upon the toxicity of the heavy metal, though with 
 somewhat different concentrations. Equivalent physiological effects were 
 produced by o.ooo66m Pb(NO 3 ) 2 in combination with o.O2tn Mg(NO 3 ) 2 , 
 by 0.00033111 Pb(NO 3 ) 2 in combination with o.oim Mg(NO 3 ) 2 and by 
 o.oooo5m Pb(NO 3 ) 2 alone. Here the addition of about 30 molecules of 
 Mg(NO 3 ) 2 for each molecule of Pb(NO 3 ) 2 in the solution, reduced the 
 toxicity of the latter salt so that it became equivalent, in both combinations, 
 to a simple solution containing 0.00005111 Pb(NO 3 ) 2 . Under these condi- 
 tions most of the germinal activity took the form of the production of 
 appressoria and other swellings, but a small amount of normal germina- 
 tion was observed in all cases. Thus, Mg(NO 3 ) 2 appears to be only about 
 one fifth as effective in reducing the toxicity of the lead salt as is Ca(NO 3 ) 2 . 
 
 Magnesium nitrate was not employed in concentration sufficiently high 
 to inhibit all toxic effects of the lead salt. The nearest approach to normal 
 germination was obtained in a combination solution containing o.O5m 
 Mg(NO 3 )o and 0.00033111 Pb(NO 3 ) 2 , in which about half of the germina- 
 
8o 
 
 LON A. HAWKINS 
 
 tion was normal. The main points brought out by these experiments are 
 summarized in table II. 
 
 TABLE II 
 
 Type of germination 
 
 Physiologically equivalent solutions containing 
 Pb(NO 3 ) 2 alone and in combination with 
 Ca(NO 3 ) 2 or with Mg(NO 3 ) 2 , molecular 
 
 Molecular 
 ratio in 
 combination 
 Ca :Pb or 
 Mg:Pb 
 
 Simple 
 . solutions, 
 Pb(N0 3 ) 2 
 
 Combination solutions containing 
 
 Pb(NO 3 ) 2 
 
 Ca(NO 3 ) 2 
 
 Mg(N0 3 ) 2 
 
 Mainly normal. Some appres- 
 soria and other swellings . . 
 
 o .00004 
 
 o .00016 
 0.00033 
 o .00066 
 
 o .005 
 
 .01 
 .02 
 
 
 3: 
 3: 
 30: 
 
 Almost no normal germina- 
 tion, mainly appressoria 
 and other swellings 
 
 o .00005 
 
 o .00016 
 0.00033 
 0.00033 
 o .00066 
 o .00066 
 
 O .OI 
 
 o .02 
 
 o .01 
 
 .02 
 
 6: 
 6: 
 
 3: 
 6: 
 
 3: 
 
 
 o .06 
 
 
 From the results obtained with the different combinations of calcium 
 and magnesium nitrates with Pb(NO 3 ) 2 , it is evident that the decrease in 
 toxicity of Pb(NO 3 ) 2 , due to the other salt, cannot be caused by a depression 
 of the ionization of the lead salt on account of the common anion. In the 
 combination containing o.oo4m Ca(NO 3 ) 2 and o.ooo66m Pb(NO 3 ) 2 , about 
 90 per cent, of the lead salt was calculated to be in the dissociated condi- 
 tion. In the combination containing o.O2m Ca(NO 3 ) 2 or Mg(NO 3 ) 2 to- 
 gether with a o.ooo66m concentration of the lead salt, the latter should be 
 about 40 per cent, dissociated. The presence of either of these concentra- 
 tions of dissociated lead salt alone in a culture solution would either prevent 
 germination entirely or give only abnormal growth. That the decrease in 
 toxicity may have been due to the formation of a double salt remains 
 possible ; at least no direct evidence to the contrary was obtained. 
 
 With the concentrations of Pb(NO 3 ) 2 that inhibited germination no 
 granular appearance of the protoplasm, such as was found in otherwise 
 physiologically similar solutions of Cu(NO 3 ) 2 , was evident. Yet it is 
 clear that the lead salt either directly or indirectly affects the protoplasm 
 through the spore wall, as is shown by the formation of the dark bodies 
 occupying one half of the spore in many cases. This response was observed, 
 as has been mentioned, in the higher concentrations of the lead salt. A 
 general discussion of the different forms of germination will be taken up 
 below, after the effects of the combinations of calcium and magnesium 
 nitrates with the other salts have been presented. 
 
TOXICITY OF HEAVY METALS 81 
 
 Zinc. 
 
 Zinc nitrate, alone and in combination with the nitrates of calcium and 
 magnesium, was studied in much the same way as were the nitrates of 
 copper and lead. Zinc nitrate alone inhibited germination in o.25m concen- 
 tration. Many local swellings of the spore walls were found in concen- 
 trations of o.oSm and 0.04111, while local swellings and short tubes were 
 present in concentrations of 0.008111 and 0.004111. Normal germination 
 occurred with the concentration of o.oo2m. Combinations of Zn(NO 3 ) 2 
 with Ca(NO 3 ) 2 and Mg(NO 3 ) 2 were tested, using a concentration of 
 0.04111 Zn(NO 3 ) 2 . In these combinations the calcium salt was employed 
 in concentrations ranging from 0.000125111 to o.O2m. Some normal germ- 
 ination occurred with all concentrations of Ca(NO 3 ) 2 below and including 
 that of 0.0005111, but none was observed in the mixture containing a 
 o.oooi25m concentration of this salt. It thus appears that, normal germ- 
 ination of these spores may be brought about in a 0.04111 Zn(NO 3 ) 2 solu- 
 tion, by the addition of Ca(NO 3 ) 2 in the proportion of one molecule of this 
 to every 80 molecules of Zn(NO 3 ) 2 present. 
 
 The concentrations of Mg(NO 3 ) 2 which were employed in these com- 
 binations with the 0.04111 solution of Zn(NO 3 ) 2 ranged from 0.00025111 to 
 0.025111. Here it was found that no normal germination occurred until the 
 magnesium salt reached a concentration in the mixture of 0.0025111. This 
 means that to produce any normal germination in the Zn(NO 3 ), solution 
 here used, by addition thereto of Mg(NO 3 ),, it is necessary to add one 
 molecule of the latter salt for every 16 molecules of Zn(NO 3 ) 2 already 
 present. It therefore requires five times as much of the magnesium salt 
 to counteract the toxic influence of Zn(NO 3 ) 2 in the concentration here 
 used (0.04111) as is required of the calcium salt to produce the same effect. 
 
 The relatively small amounts of the calcium and magnesium salts, which 
 are required to inhibit the toxic effects of Zn(NO 3 ) 2 in 0.04111 concentra- 
 tion, preclude any possibility that the antagonistic influence of either of the 
 former salts might be related to decreased dissociation of the zinc salt, 
 brought about by their addition, so that this consideration needs no atten- 
 tion in this case. 
 
 It is interesting to recall here that the effectiveness of Mg(NO 3 ) 2 in 
 counteracting the toxicity of Pb(NO 3 ) 2 , in the two concentrations studied, 
 so as to allow some normal germination of the spores, was also found to 
 be only about one fifth as great as that of Ca(NO 3 ) 2 . The relative effec- 
 tiveness of the nitrates of calcium and magnesium thus seems to be the 
 same whether they are employed to counteract the toxicity of Pb(NO 3 ) 2 
 or that of Zn(NO 3 ) 2 . 
 
 Zinc nitrate did not seem to stimulate the spores to form appressoria, 
 though hyaline swellings were common in the more concentrated solutions. 
 
82 
 
 LON A. HAWKINS 
 
 No direct evidence was obtained as to whether or not zinc nitrate enters 
 the spores. 
 
 Aluminum. 
 
 Aluminum nitrate was used at certain concentrations, both alone and in 
 combination with magnesium and calcium nitrates. Series were carried 
 out, and several times repeated, including the combinations and concentra- 
 tions which are presented in table III. 
 
 TABLE III 
 
 Concentration of Al(NO 3 ) 3 
 molecular 
 
 Concentration of Mg(NO,) 2 
 
 or of Ca(NO 3 ) 2 , 
 
 molecular 
 
 Common 
 fraction 
 
 Decimal 
 fraction 
 
 Common 
 fraction 
 
 Decimal 
 fraction 
 
 9/2000 
 
 0.0045 
 
 I/IOO 
 
 .01 
 
 8/2OOO 
 
 o .004 
 
 2/IOO 
 
 o .02 
 
 6/2OOO 
 
 0.003 
 
 4/100 
 
 o .04 
 
 4/2000 
 
 o .002 
 
 6/100 
 
 o .06 
 
 3/2000 
 
 o .0015 
 
 7/100 
 
 o .07 
 
 2/2000 
 
 .001 
 
 8/100 
 
 0.08 
 
 1/2000 
 
 o .0005 
 
 9/100 
 
 o .09 
 
 1/4000 
 
 o .00025 
 
 95/1000 
 
 0.095 
 
 In none of the cultures with A1(NO 3 ) 3 did the addition of either 
 Mg(NO 3 ) 2 or Ca(NO 3 ) 2 bring about any alteration at all in the behavior 
 of the spores. Although many other proportions of these pairs of salts 
 might have been tried, it appears probable that, if either the calcium or 
 magnesium salt, at any concentration, exerts any influence upon the toxicity 
 of A1(NO 3 ) 3 , in any concentration, some evidence in this direction would 
 have been obtained from the experiments which were carried out. This 
 seems still more probable from the fact that different concentrations of 
 the aluminum salt alone produced markedly different effects upon the 
 activities of the spores, a matter which will now receive attention. 
 
 In the highest concentration of A1(NO 3 ) 3 used in the combinations, 
 namely 0.0045111, only a few local swellings of the spores were observed. 
 A blackening of half of the spore was frequently found here (fig. 6). No 
 normal germination occurred in this solution. In the two lowest concen- 
 trations of A1(NO 3 ) 3 (0.005111 and 0.00025111) however, much of the germ- 
 ination was normal. Aluminum nitrate appears to influence the form of 
 germination occurring in these spores, with much the same results as have 
 been described for cultures poisoned with Pb(NO 3 ) 2 . The types of renewed 
 
TOXICITY OF HEAVY METALS 83 
 
 activity which occur with A1(NO 3 ) 3 are: (i) the formation of a dark body 
 occupying about half of the spore, (2) the formation of a small local 
 swelling at one end of the spore, and (3) the development of one or more 
 appressoria, the latter sometimes having their characteristic dark color and 
 sometimes being hyaline. Unless the formation of the internal dark bodies, 
 which seem to arise directly through protoplasmic activity, may be taken as 
 evidence of a penetration of the toxic salt, no information as to whether 
 A1(NO 3 ) 3 actually enters the spores was obtained. 
 
 Nickel. 
 
 Nickel nitrate proved to be very slightly toxic to these fungus spores, its 
 effect on germination being manifest only in very high concentrations. On 
 this account no study was made of the possible influence of calcium and 
 magnesium nitrates upon this toxicity. 
 
 The toxic effects of Ni(NO 3 ) 2 
 alone may be added here. A con- 
 centration of o.5m inhibited ger- 
 mination. In a concentration of 
 0.25111, numerous local swellings of 
 the spores were observed. Both 
 local swellings and normal germina- 
 tion were present in a 0.12501 
 solution of this salt, and germina- 
 tion was perfectly normal, as in 
 the distilled water control, where 
 a concentration of o.o5m or lower was employed. Germinating spores in 
 o.25m concentration of Ni(NO 3 ) 2 frequently exhibited a granular appear- 
 ance such as has been described for spores in solutions of Cu(NO 3 ) 2 which 
 were not quite concentrated enough to inhibit all activity. 
 
 Fig. 6. Germinating spores 
 o.oo2m Al (NO 3 ) 3 , X 650. 
 
 from 
 
 TOXICITY OF SINGLE SALTS 
 
 The present section will be devoted to the relative degrees of toxicity 
 exhibited by the various substances heretofore dealt with, together with 
 certain other considerations bearing upon the problem of chemical stimu- 
 lation as evidenced in the simple solutions employed in these studies. Besides 
 the substances already mentioned, nitric acid entered into the experimen- 
 tation, and the information gained regarding the toxicity of this substance 
 will also find place here. 
 
 From what has preceded it appears possible to group the effects of the 
 different treatments into four distinct kinds of physiological response on 
 the part of the fungus spores here employed, (i) a given treatment may 
 
84 LON A. HAWKINS 
 
 be without apparent effect; under such treatments the spores germinate 
 normally within eighteen hours, just as they do in the control with dis- 
 tilled water. (2) Germinal activity may be manifest, but in other ways 
 than that which is here considered as normal. Under treatments which 
 produce this sort of response, the activities of the spores result sometimes 
 (a) in an actual decrease in the exposed surface as well as in the volume 
 of the organism, and sometimes (b) in an increase in both volume and 
 surface, the latter, however, increasing much less markedly, as related to 
 increase in volume, than is the case in normal germination. In the first 
 category (a, above) belong the phenomena involved in the production of 
 the dark bodies which have been described as forming within the spore wall 
 and occupying only a portion of its volume. These appear to have essen- 
 tially the characteristics of chlamydospores formed within the organism. 
 All other renewed activities of the spores which have been described belong 
 to the second category (b), and embrace those growth processes which 
 result in more or less restricted swellings, especially at the ends of the 
 spores, in appressoria, with either dark or hyaline wall, and in short germ 
 tubes somewhat resembling the normal tubes but of much greater diameter. 
 (3) No renewed activity may occur at all during a period of from eighteen 
 to twenty hours under the given treatment, although ability to germinate 
 if transferred from this treatment to another (as to distilled water) may 
 still be retained. Here the treatment prevents germinal activity but does 
 not destroy viability, at least within the given time limits. (4) Viability, 
 or power to germinate in water, may be destroyed within a period of from 
 eighteen to twenty-four hours; the organism is killed outright. 
 
 It has already become clear in this work that most of the substances 
 dealt with produce death within the assumed time limits, if applied at a 
 sufficiently high concentration (4). With a somewhat lower concentration 
 of the injurious material, germination is inhibited but viability is retained 
 throughout the given period (3). When the concentration is still lower 
 germinal activity becomes manifest but takes other forms than those recog- 
 nized as normal (2). Finally, when the concentration of the toxic agent 
 is still further decreased, the stimulation threshold in the present sense is 
 passed and normal germination becomes the rule (i). 
 
 Livingston 21 working with a green alga has presented a somewhat similar 
 series of responses to chemical stimuli. This writer studied the effect 
 of a large number of nitrates and sulphates on a form of Stigeoclonium, 
 adding different concentrations of the salts to a nutrient medium in which 
 the alga was grown. With a dilute nutrient solution, in which the alga 
 had a characteristic filamentous form, its response to stimulation might be 
 considered as of three types, (i) death, (2) change in phenomena of 
 
 21 Livingston, B.E., Chemical stimulation of a green alga. Bull. Torr. Bot. Club 32: 1-34. 1905. 
 
TOXICITY OF HEAVY METALS 85 
 
 growth, and (3) change in phenomena of reproduction. The response to a 
 high concentration was usually death, while addition of a toxic salt in con- 
 centration somewhat below the fatal one stimulated the production of the 
 palmella form, with spherical cells and division occurring in all directions. 
 It is interesting that, with relatively similar concentrations of the salts, 
 (that is at concentrations somewhat lower than those required to 
 inhibit germination) the spores dealt with in the present paper exhibited 
 responses similar to those which Livingston found with his alga ; the pro- 
 duction of appressoria and swellings in this fungus appears physiologically 
 similar to the production of the palmella form in Stigeoclonium. The third 
 type of response discussed by Livingston, change in phenomena of repro- 
 duction, of course finds no parallel m this investigation. 
 
 For convenience of comparison, the limits of the various responses of 
 the fungus spores here dealt with, to the different treatments employed, 
 are presented in table IV. The only nitrate occurring in the table that has 
 not already received attention is the acid. In a series of cultures with 
 HNO 3 the germination at a 0.005111 concentration was practically all normal, 
 while at o.oim and all higher concentrations, no germination took place in 
 the eighteen hour period. The acid prevented germination for the period 
 of eighteen hours, in a o.O2m concentration and killed the spores in 0.5111. 
 No concentration was found, therefore, at which the germination took the 
 form of any of the various abnormal growths found at some concentration 
 with all the other substances used, nor was there any apparent coagulation 
 of the protoplasm at concentrations below that which killed the spores. 
 From these considerations it seems, then, that the various abnormal 
 growths and effects on the protoplasm of the spore cannot be due to 
 acid present in the salt solutions, as a result of hydrolysis of the salts, 
 but must be related, either directly or indirectly, to the metals themselves. 
 
 Turning now to table IV, the different substances are there arranged in 
 the order of the concentration which just allowed normal germination in a 
 period of eighteen hours. In the first column are listed the substances 
 dealt with. The remaining columns are each double. The second column 
 presents the concentrations in which the spores are killed in eighteen hours 
 and fail to germinate later, after transfer to distilled water. Here, as in 
 many other instances, the critical concentrations must not be regarded as 
 definite in the sense of the more exact physical sciences ; the concentrations 
 employed in the experimental series were frequently rather widely separated, 
 and. were this not the case, the variability of the organism in its resistance 
 to toxic substances would render quite useless any attempt to define such 
 critical points with very great accuracy. In all such work as the present, 
 dealing with organisms, the internal conditions of the cells must be as 
 important in determining reactions as are the external ones, and we are 
 
86 
 
 LON A. HAWKINS 
 
 
 
 
 || 
 
 N CS N O 
 
 O O O <t ^ 
 
 O O O O ""> O 
 O O O O O O P< 
 OOOOOOO"'-'OO 
 
 
 
 a 8-g 
 
 s ^ s 
 
 2*j C 
 
 Q -S 
 
 OOOOOOOOOOi-i 
 
 
 
 ^i 
 
 l 
 
 Common 
 fraction 
 
 O O 
 O 
 O O O 
 - - - O O 
 
 ooo -ooooo 
 
 IT) IT) IT) Tf C< W PJ M "-I W> M 
 
 
 
 
 
 
 
 
 o 
 
 !^ P 
 
 Decimal 
 fraction 
 
 + + 
 
 vO >0 
 N M *r> 
 
 o o o "5 + 
 
 O O O O ""> M *n 
 OOOOOON<^ 
 OOOOOO | - | ' V 2 1 '>^'*' 
 
 
 
 E "2 3 
 
 
 OOOOOOOOOO" 
 
 
 o 
 
 2 
 
 normal ge 
 occun 
 18 h 
 
 Common 
 fraction 
 
 ooo 
 
 O 
 \j-> if) o O 
 
 - - -ooo 
 
 t^-r^O'OO O OO 
 rO'~OPNN"-)OQ'-'P<'- 1 '-' 
 
 
 
 
 
 
 ration 
 
 Maximum 
 
 a 
 I 
 g -5 
 
 1-2 
 3 o 
 
 vO vO 
 
 
 o o ^ 
 o o o o oo > 
 
 OOOO OM 1/ ^ V OO V O 
 
 c 
 
 
 * B S 
 
 C 3 i 
 
 25 
 
 Qi 
 
 OOOO -OOOO"- 11 - 1 
 
 
 Molecular co 
 
 
 any germinal 
 in 18 1 
 always a 
 
 Common 
 fraction 
 
 o o 
 
 
 
 
 OOOO 
 OOOO ^ 
 
 --IT5N -MTfWlOl-l'O 
 
 <3\o>-ii-' *;< oo 
 
 
 
 
 
 00 00 
 
 
 
 4 _ l 
 
 S g 
 
 
 
 
 3 -0 
 
 
 
 
 
 A J 
 
 .5 "^ 
 
 
 
 
 u * 12 
 
 
 
 
 
 > c 3 
 
 Q & 
 
 
 
 
 =i|5 
 
 
 
 
 
 t - 
 
 > o h 
 
 
 
 
 
 
 M C ^ 
 
 o S 
 
 
 
 
 
 c .2 
 
 
 
 
 
 
 
 
 
 2 
 
 Jl 
 
 
 
 
 sr-^ 
 
 2 
 
 ^\ > \ I- 1 ^^ ^s, ^X 
 
 
 
 
 
 
 
 | 
 
 
 11 
 
 + , . . . 
 
 M M fj .... 
 O O . ui f5 f> 
 
 
 f 
 
 c 
 
 
 |.S B 
 
 *-o 
 
 Q i 
 
 o o o o o o 
 
 
 Minimum 
 
 M* 
 |S 
 
 mmon 
 action 
 
 o o 
 o o .... 
 
 M - IO C r^ N 
 
 
 
 
 ci$i 
 
 
 
 
 
 
 
 
 
 Substance 
 in 
 
 medium 
 
 cf -c^c:" L.c"c :c "cr i, M 
 /.c/./.c/.Z^/.c c 
 
 O :/2 ' ' ^ ' xlj- ' v ' 
 
 3 3-Q 'f^ B rs teP < a 3 
 OOO<SSN!sSOM 
 
TOXICITY OF HEAVY METALS 87 
 
 as yet unable either to control or measure the former, excepting in a very 
 general way. 
 
 In the third column are found the concentrations at which the different 
 salts inhibit germination for at least eighteen hours. When spores from 
 these cultures were removed from the toxic solution and placed in water 
 for a day, they germinated. The fourth column gives the maximum con- 
 centration in which germination occurred. In all cases this growth was 
 abnormal, showing swellings, swollen tubes and other unusual structures, 
 such as are illustrated in figs, i to 6. In the fifth column the concentrations 
 given are the maxima in which any normal germination was observed. The 
 sixth column presents the highest concentrations in which no toxic influ- 
 ence was manifest. 
 
 From table IV it will be seen that the copper salts are by far the most 
 toxic of all the salts here studied. These two salts are very similar in 
 their effect on the germination of the spores, which is in accord with the 
 results of Clark [99], and points almost conclusively to the commonly 
 accepted idea that the toxicity of such copper salts is mainly or entirely 
 due to the copper ion. The last mentioned writer found that the concen- 
 tration of copper salts which inhibited germination were higher than those 
 producing the same effect in this investigation, a fact that may probably 
 be due to internal differences in the organisms dealt with; he found that 
 his five fungi varied markedly in their response to the same stimulus. Also, 
 Clark usually made use of nutrient media, while the present studies were 
 carried out without their employment. It is quite possible that the presence 
 of nutrient substances might have modified the effect of the poison. Dug- 
 gar 22 found the nitrate of copper considerably more toxic than the sulphate 
 and also considerably more so than it is here shown to be. In the work 
 of Stevens, 23 these two salts affected germination similarly and the concen- 
 trations required to prevent germination varied, with the different fungi 
 used, from 111/6400 to 111/200. Many other investigations of the effect of 
 copper on the germination of fungus spores have been carried out, with 
 varying results. The effect of this poison on algae has been studied by 
 Livingston [05]. Kahlenberg and True, 24 , Heald, 25 Jensen, 26 Szucs [12] 
 and others, have investigated its toxicity toward higher plants. The results 
 obtained indicate that fungus spores are considerably more resistant toward 
 the toxic effects of copper than are either the algae or higher plants. 
 
 tfar, B.M., Physiological studies with reference to the germination of certain fungus spores. 
 Bot. Gaz. 31: 38-65. 1901. 
 
 23 Stevens, F.L., The effect of aqueous solutions upon funtjus spores. Bot. Gaz. 26: 377-406. 1898. 
 
 2J Kahlenberg, Louis, and True, Rodney H., On the toxic action of dissolved salts and their elec- 
 trolytic dissociation. Bot. Gaz. aa: 81-124. 1896. 
 
 25 Heald. F.D., The toxic effect of dilute solutions of acids and salts upon plants. Bot. Gaz. 22: 
 I2S-IS3- 1896. 
 
 28 Jensen, G.H., Toxic limits and stimulation effects of some salts and poisons on wheat. Bot. Gaz. 
 43: 11-44. 1907- 
 
88 LON A. HAWKINS 
 
 In the order of their toxicity, Pb(NO 3 ) 2 follows the copper salts, though 
 not closely, with A1(XO 3 ) 3 next. The effects of the last two salts on 
 plants seems to have been studied but little. Livingston [05] employed 
 them, however, and found the same relation to hold true as is here brought 
 out. Also, Jensen [07] found that Pb(NO 3 ) 2 is somewhat less toxic toward 
 wheat seedlings than is Cu(NO 3 ) 2 . The nitrates of zinc and nickel, which 
 are next in the order of diminishing toxicity, are not exceedingly toxic 
 toward these Gloeosporium spores. It is interesting to note that Clark [99], 
 working with fungi, and Livingston [05], with an alga, found nickel salts 
 to be more strongly toxic than those of zinc, results which are opposite 
 to those obtained in this investigation. 
 
 Magnesium, calcium, and potassium nitrates and cane sugar end the list, 
 in the order named. It is noticeable that, with these last four compounds 
 at concentrations just below that required to inhibit germination, growth 
 takes the form of swollen bodies which tend to give a lower value to the 
 ratio of surface to volume of the organism than that resulting from normal 
 germination. This reaction to toxic stimuli, at concentrations just below 
 those required to inhibit growth, occurs commonly with all the salts here 
 used. That it is found with cane sugar, which is usually regarded as 
 non-toxic (see True 27 ) suggests that such reactions may be brought about 
 by high osmotic pressure as well as by chemical stimulation in the true 
 sense of this term. This suggestion is in accord with the conclusions of 
 Livingston [05], who has shown that his form of Stigeoclonium assumes 
 the palmella form in response either to a chemical stimulus or to one of 
 relatively high osmotic pressure. Whether this effect of sugar, and similar 
 effects produced by high concentrations of calcium, magnesium and 
 potassium nitrates is really due, in the present instances, to osmotic pressure 
 relations, was not determined experimentally ; the minuteness of the spores 
 must render experiments involving plasmolysis exceedingly difficult with 
 such organisms as those here employed. It is obvious, however, that, the 
 high concentrations of the calcium, magnesium and potassium salts, found 
 necessary to affect germination, must be capable of exerting an extremely 
 high osmotic pressure if the ectoplasm of the spore is not readily permeable 
 to them. 
 
 From table IV it may be seen that, with the exception of HNO 3 , the dif- 
 ferent compounds arrange themselves in the same order no matter what 
 type of reaction to the chemical stimuli may be taken as criterion. This 
 suggests that the physiological response of death, on the one hand, and 
 the various morphogenic responses considered in this paper, on the other, 
 are ultimately related to the same properties or characteristics of the various 
 
 27 True, Rodney H., The physiological effects of certain plasmolyzing agents. Bot. Gaz. 26: 407- 
 416. 1898. 
 
TOXICITY OF HEAVY METALS 89 
 
 stimulating substances which bring them about. Copper 4s the most toxic 
 metal here dealt with, whether we consider its toxicity as bringing about 
 changes that result in death or as bringing about the less final changes 
 that lead to abnormal growth. In the same way, magnesium, calcium and 
 potassium are the least toxic metals of the list, on whatever physiological 
 criterion we may base our judgment. 
 
 GENERAL DISCUSSION AND CONCLUSION. 
 
 The results of the experiments described in the foregoing pages show 
 that, in certain cases at least, the effect of a toxic salt on the germination 
 of the conidia of Glomerella cingulata may be influenced by the addition, 
 to the medium, of calcium, magnesium or potassium nitrate. That this 
 effect is not due to depression of ionization of the toxic salt has been 
 demonstrated. That it cannot be due to the formation of undissociated 
 double salts has been shown for the combination of Cu(NO 3 ) 2 with 
 Ca( NO 3 ) 2 and of Zn(NO 3 ) 2 with calcium or magnesium nitrate. The influ- 
 ence of calcium upon the toxicity of the salts of the heavy metals here 
 employed must then be related to an effect of the Ca(NO 3 ) 2 on the spore, 
 or to an effect on the contained protoplasm, as is made otherwise probable 
 from the fact that copper, lead, aluminum and nickel nitrates sensibly effect 
 the protoplasm in various ways, without producing any apparent changes 
 in the spore wall. 
 
 There are, of course, a number of logically possible explanations for the 
 antagonistic actions of salts upon the germination of fungus spores as 
 here recorded, but any such possible hypothesis must necessarily be yery 
 complex if it be able to account generally for even the small array of 
 facts so far at our disposal. 
 
 Various theories have been suggested from time to time to explain similar 
 salt antagonisms. Loeb 28 has advanced the theory of ion-proteid formation, 
 suggesting that one or both ions of a salt may combine with proteids of 
 the protoplasm to form ion-proteid compounds. This writer found, for 
 example, that a solution of NaCl is poisonous unless some calcium and 
 potassium be also present in the medium, in which case the toxicity of 
 NaCl is greatly decreased or inhibited altogether. He considers that cal- 
 cium and potassium ions may to some extent take the place of the sodium 
 ion in the ion-proteid compounds above mentioned, and suggests that the 
 organism cannot live without some of these proteid compounds containing 
 calcium and potassium as well as those containing sodium. The work of 
 Osterhout 29 on balanced solutions supports this theory of Loeb. 
 
 28 Loeb, J., On ion-proteid compounds and their role in the mechanics of life phenomena. I. The 
 poisonous character of a pure NaCl solution. Amer. Jour. Physiol. 7: 327-138. 1900. 
 
 29 Osterhout, W. J. V. [06], On the importance of physiologically balanced solutions for plants. II. 
 Fresh water and terrestrial plants. Bot. Gaz. 44: 259-272. 1907. 
 
 PHYSIOLOGICAL RESEARCHES VOL. I, NO. 2, SERIAL XO. 2 
 
 AUGUST, 191^. 
 
9O . LON A. HAWKINS 
 
 Wo. Ostwald, 30 from his studies on Gammarus, concludes that the toxicity 
 of a solution is related to the power of the protoplasm to adsorb the dis- 
 solved substance, saying " dass eine Losung um so giftiger istje starker 
 sie adsorbiert wird." Morowitz 31 somewhat similarly considers that the 
 toxic salt is adsorbed by the surface layer of the organism and diffuses 
 thence into the interior, the amount thus entering in a given time being 
 related to the rate of adsorption. He supposes that, if an otherwise indif- 
 ferent but strongly adsorbed substance be simultaneously present in the 
 medium, the adsorption of the latter decreases the power of the surface 
 layer to adsorb the toxic salt, so that a smaller amount of this enters the cell. 
 
 True and Gies [03] also relate these antagonistic effects to protoplasmic 
 properties, and seem to consider the antagonism between copper and calcium 
 to be due to an accelerating effect of calcium acting against a retarding 
 influence exerted by copper. Sziics [12], as has already been mentioned, 
 likewise supposes the influence of one salt upon the toxicity of another to 
 be due to simultaneous effects of the two salts upon the protoplasm. 
 
 Loeb 32 has rather recently advanced the theory that the influence of one 
 salt on the toxic action of another is due to changes brought about in the 
 outer colloidal membrane of the protoplasm, whereby the latter becomes 
 impermeable to the toxic salt. Osterhout, 33 working with cut disks of 
 Laminaria and his method of electrical conductivity, also comes to the con- 
 clusion that ([12], page 115)" the antagonistic action of salts is largely or 
 entirely due to the fact that they hinder or prevent one another from 
 entering the protoplasm." 
 
 It will be seen that any one of the various hypotheses just reviewed may, 
 with adequate quantitative assumptions, explain the phenomena of antagon- 
 ism as these are exhibited by any particular pair of concentrations of any 
 particular pair of salts dealt with in the present study. It seems, impos- 
 sible, however, to formulate a hypothesis, either by means of any of these 
 published suggestions or with any other logical possibilities, which may 
 simultaneously and satisfactorily account for all the cases with which we 
 have to deal. The marked differences, brought out in the present studies, 
 between the behavior of calcium when employed with copper and that of 
 the same element in combination with lead, zinc or aluminum, strongly 
 suggest the probability that valence alone does not determine the efficiency 
 of one cation in influencing the toxicity of another. It appears that other 
 properties must be taken into account if such differences are to be explained. 
 
 30 Ostwald, Wo., Ueber die Beziehungen zwischen Adsorption und Gifti^keit von Salzl<">sum?en fur 
 Susswassertiere (Gammarus). Archiv. Ges. Physiol. 120: 10-30. 1007. 
 
 31 Morowitz, Huso, Ueber Adsorption und Kolloidfalluns;. Kolloidchem. Beih. i: 301-331. IQIO. 
 
 32 Loeb, J., The mechanistic conception of life. Chicago. 1912. Page 173. 
 
 33 Osterhout, W.J.V., The permeability of protoplasm to ions and the theory of antagonism. Science 
 N.S. 35: 112-115. 1012. 
 
 .Some quantitative researches on the permeability of plant colls. Plant World 16 : T 20-144. 1013- 
 
TOXICITY OF HEAVY METALS 91 
 
 A consideration of the toxicities of the single salts,~aa brought out in 
 this investigation, also suggests that the influence of each one of these salts 
 upon the protoplasm of the spore is specific. The nitrates of lead and 
 aluminum, in concentrations somewhat below those which inhibit germina- 
 tion, frequently produce within the spore (either directly or indirectly) a 
 dark, chlamydospore-like body. The presence of copper, at a similar con- 
 centration as regards toxicity, causes a granular appearance of the proto- 
 plasm and much the same effect was observed in concentrations of nickel 
 nitrate not quite strong enough to inhibit germination. Toxic concentra- 
 tions of zinc nitrate, however, fail to produce any visible alteration in the 
 protoplasm. The effects of the different single salts upon the spores are 
 therefore not at all the same, and it seems at least reasonable to suppose 
 that the requisite antidotes may not be identical and may not be effective 
 in the same manner in all cases. A given substance at a given concentration 
 may inhibit the poisonous effects of one toxic salt and yet have no influ- 
 ence upon the toxicity of another. Thus, neither calcium nor magnesium 
 nitrate exerts any apparent influence upon the toxic effect of aluminum 
 nitrate, but both are markedly effective in counteracting the toxicity of the 
 corresponding salts of zinc and lead. 
 
 The conclusion seems unavoidable, therefore, that no simple and broadly 
 general explanation can be applied to these exceedingly various antagonistic 
 actions, and that the explanation of each particular case must involve 
 the chemical characteristics of the salts concerned and the physico-chemical 
 properties of their solutions. The exceeding complexity of the material 
 system within the cell must make possible a great variety of explanations 
 until this variety becomes further limited by increased knowledge. As has 
 been well emphasized by Osterhout [13] the future advance of our knowl- 
 edge of this intricate subject must depend solely upon further investigation 
 of a quantitative nature. 
 
 THE LABORATORY OF PLANT PHYSIOLOGY 
 OF THE JOHNS HOPKINS UNIVERSITY. 
 APRIL i, 1913. 
 
 LITERATURE CITED. 
 
 Numbers in brackets throughout the preceding pages refer to the year of publi- 
 cation and to the corresponding numbers which follow authors' names in this list. 
 
 Acree, S.F. [n]. See Loomis and Acree [n]. 
 
 Bartlett. H.H. [12]. See True and Bartlett [12]. 
 
 Clark, J.F. [99], On the toxic effect of deleterious agents on the germination and development of cer- 
 tain filamentous fungi. Bot. Gaz. 28: 280-327, 378-404. 1899. 
 
 [01], On the toxic value of mercuric chloride and its double .salts. Jour. Physic. Chem. 5: 
 
 289-316. 1901. 
 
 - [02], On the toxic properties of some copper compounds wth special reference to Bordeaux 
 mixture. Bot. Gaz. 33: 26-48. 1902. 
 
92 LON A. HAWKINS 
 
 Duggar, B.M. [bi], Physiological studies with reference to the germination of certain fungus spores. Bot. 
 Gaz. 31 : 38-65. 1901. 
 
 Gies, WJ. [03]. See True and Gies [03]. 
 
 Hasselbring, H. [06], The appressoria of anthracnoses. Bot. Gaz. 42: 135-142. 1906. 
 
 Heald, F.D. [96], The toxic effect of dilute solutions of acids and salts upon plants. Bot. Gaz 22: 
 125-153. 1896. 
 
 Holland, W.A. [09]. See Morse and Holland [09]. 
 
 Hosford, H.H., and Jones, H.C. [n], Conductivity, temperature coefficients of conductivity and dis- 
 sociation of certain electrolytes. Am. Chem. Jour. 46: 241-278. 1911. 
 
 Jensen, G.H. [07], Toxic limits and stimulation effects of some salts and poisons on wheat. Bot. Gaz. 
 43: 11-44- i97- 
 
 Jones, H.C. [n]. See Hosford and Jones [n]. 
 
 [12], Electrical conductivity, dissociation and temperature coefficients of conductivity, from 
 
 zero to sixty-five degrees, of aqueous solutions of a number of salts and organic acids. 
 Carnegie Institution of Washington Publ. 170. Washington. 1912. 
 
 Kahelenberg, L., and True, R.H. [96], On the toxic action of dissolved salts and their electrolytic dis- 
 sociation. Bot. Gaz. 22: 81-124. 1896. 
 
 Kronig, B., and Paul, Th. [97], Die chemischen Grundlagen der Lehre von der Giftwirkung und 
 Desinfection. Zeitschr. Hygiene und Infectionskrankheiten 25: 1-112. 1897. 
 
 Le Renard, A. [12], Influence du milieu sur la resistance du Penicille crustace aux substances toxiques. 
 Ann. Sci. Nat. Bot. IX. 16: 276-336. 1912. 
 
 Livingston, B.E. [05], Chemical stimulation of a green alga. Bull. Torr. Bot. Club 32: 1-34. 1905. 
 
 Loeb, J. [oo], On ion-proteid compounds and their role in the mechanics of life phenomena. I. The 
 poisonous character of a pure NaCl solution. Amer. Jour. Physiol. 7: 327-338. 1900. 
 
 [02], Ueber den Einfluss der Werthigkeit, und moglicherweise der electrischen Ladung fur 
 
 lonen auf ihre antitoxisch Wirkung. Archiv ges. Physiol. 88: 68-78. 1902. 
 
 t6], The dynamics of living matter. New York. 1906. 
 
 [12], The mechanistic conception of life. Chicago. 1912. 
 
 Loew, O. [92], Ueber die physiologischen Functionen der Calcium- und Magnesium-saize im Pflanzenorgan- 
 
 ismus. Flora 75: 368-394. 1892. 
 Loomis, N.E., and Acree, S.F. [n], A study of the hydrogen electrode, of the calomel electrode and of 
 
 contact potential. Amer. Chem. Jour. 46: 585-620. 1911. 
 
 Horowitz, H. [10], Ueber Adsorption und Kolloidfallung. Kolloidchem. Beih. i: 301-331. 1910. 
 Morse, H.N., and Holland, W.W. [09], The osmotic pressure of cane sugar solutions at 25. Amer 
 
 Chem. Jour. 41 : 1-19. 1909. 
 Osterhout, W.J.V. [06], On the importance of physiologically balanced solutions for plants. I. Marine 
 
 plants. Bot. Gaz. 42: 127-134. 1906. 
 
 [07], On tte importance of physiologically balanced solutions for plants. II. Fresh 
 
 water and terrestrial plants. Bot. Gaz. 44: 259-272. 1907. 
 
 [12], The permeability of protoplasm to ions and the theory of antagonism. Sci. N.S. 35: 
 
 112-115. 1912. 
 
 - [13]. Some quantitative researches on the permeability of plant cells. Plant World 16: 
 
 129-144. 1913- 
 Ostwald, Wo. [07], Ueber die Beziehungen zwischen Adsorption und Giftigkeit von Salzlosungen fur 
 
 Susswassertiere (Gammarus). Archiv. ges. Physiol. 120: 19-30. 1907. 
 Paul, Th. [97]. See Kronig and Paul [97]. 
 Shear, C.L., and Wood, Anna K. [13], Studies of fungus parasites belonging to the genus Glotnerella. 
 
 U.S. Dept. Agric. Bur. Plant Ind. Bulletin 252. 1913. 
 
 Stevens, F.L. [98], The effect of aqueous solutions upon fungus spores. Bot. Gaz. 26: 377-406. 1898. 
 Szucs, J. [12], Experimentelle Beitrage zu einer Theorie der antagonistischen lonenwirkungen. 
 
 Jahrb. wiss. Bot. 52: 85-143. 1912. 
 True, R.H. [96]. See Kahlenberg and True [96]. 
 
 - [98], The physiological effects of certain plasmolyzing agents. Bot. Gaz. 26: 407-416. 
 
 1898. 
 True, R.H., and Bartlett, H.H. [12], Absorption and excretion of salts as influenced by concentration 
 
 and composition of culture solutions. U.S. Dept. Agric. Bur. Plant Ind. Bulletin 231. 
 
 1912. 
 True, R.H., and Gies, W.J. [03], On the physiological action of some of the heavy metals in mixed 
 
 solutions. Bull. Torr. Bot. Club 30: 390-402.1903. 
 Wood, Anna K. [13]. See Shear and Wood [13]. 
 
VITA. 
 
 The writer was born at La Motte, Iowa, May 30, 1880. He attended 
 Morningside Academy and College, Sioux City, Iowa, and was graduated 
 with the degree of Bachelor of Science in 1906. The summer of 1904 was 
 spent in special work in Botany at the University of Chicago. From 1903 
 to 1906 he was assistant at Morningside College, Fellow in Botany at the 
 Ohio State University September 1906 to April 1907, and Scientific Assist- 
 ant, United States Department of Agriculture, April 1907 to the present (on 
 furlough October 1912 to June 1913). During the years from 1909 to 1913 
 he attended the Johns Hopkins University as a graduate student in Plant 
 Physiology, Chemistry, and Botany, and was appointed University scholar 
 in Plant Physiology for the year 1912-1913. 
 
SYRACUSE, - NY, 
 
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