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Some effects of sulphur dioxide on coniferous trees Hughes, Norah Louise 1934

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U . B . C . LIBRARY! •I ICC". 1 ^ . - J J L ^ ^ b ^ E ^ ^ . j SOME EFFECTS OF SULPHUR DIOXIDE ON CONIFEROUS TREES No rah. Louise Hughes, £. A . A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ARTS IN THE DEPARTMENT of BOTANY THE UNIVERSITY OF BRITISH COLUMBIA MAY, 1934 gable of Contents. Page Acknowle dgement s. Statement of the Problem and Scope of the present study 1 Review of the Literature — • — — 2 Material and Methods i n Experimental work • 6 Experimental work — - — — — • — — 7 Fumigations v v t P ~ — — — — - — — ~ ™ 7 Material and Methods •*•»•»•—-—--— — — - ~ — • — - 7 Method of analysis o f gas samples — 7 MeMiod of Fumigation — — • — • — — — — — 11 General r e s u l t s — - — • — — — — 11 S u p e r f i c i a l symptoms of injury — — — — • — — 11 Specific rate of in j u r y — — — 12 Factors involved i n determining the rate of injury — — — — 13 Histo l o g i c a l studies — 19 Material and methods — - — • — • — — 20 Study of leaf sections — - — — • 21 Normal specimens - — — — — — - — — 21 Fumigated specimens — • — 23 Physiological studies — • — • — — • 31 Food storage • — • 31 Material and methods •—• —* 31 gable of (W . m ^ . g . Acknowledgemsnts. Page Re s u l t s — — 3 3 D i s c u s s i o n o f r e s u l t s — 44 Dessication Studies — — — — 53 M a t e r i a l and mathods — • 53 Results — — . — ~ — ~ 54 Summary. — — — — — - — - 56 Some suggested problems f o r further study a r i s i n g out of the above experiments — — — • — 57 Explanation of plates Plates (1) to (XT) Bibliography. AOZN'OWLEDGEMEHTS The writer wishes to express her deep indebtedness to Dr. A. H. Hutchinson, Head of the Departnsnt o f Botany at the University of B r i t i s h Columbia, f o r h i s u n f a i l i n g i n t e r -est i n the problem, and f o r his many h e l p f u l and encouraging suggestions. Thanks are also due to Dr. Hutchinson and to Dr. F. Dickson, Associate Professor a t the U n i v e r s i t y of B r i t i s h Columbia f o r the loan of books and p e r i o d i c a l l i t e r a t u r e . Gratitude i s also expressed for the kindness of Mr. Garnish at the University Greenhouse .who cared for the specimens used i n the experiments. SOME STUDIES IK IHE EFFECTS OF SULPHUR DIOXIDE • U P O E CONIFEROUS TREES  Statement of the Problem and, the Sco-pe of  the present Study. Much attention has been drawn to the problem of the injurious effects of smelter fumes containing sulphur dioxide upon the vegetation of areas surrounding metallurgical workss, and there has been a great deal of d i s -cussion of t h i s subject. The problem i s a very wide one. i t i s necessary to show, f i r s t that any appreciable amount of sulphur dioxide i s given out by the smelter i n question, secondly that the vegetation i s actually injured by t h i s sulphur dioxide* t h i r d l y the nature of the injury, and l a s t l y how i t i s brought about. The f i r s t part of the problem involves sampling and analyzing fumes given o f f by the smote staclc. In order to demonstrate that the vegetation i s actually Injured by the sulphur dioxide, two lines of investigation might be followed. By a thorough study of the d i s t r i c t affected i t might be possible to eliminate a l l other sources of injury, or on the other hand, by subjecting plants to toiown quantities of sulphur dioxide under controlled conditions injury may be obtained. This injury should be similar to that shown in the area surrounding the smelt-er. The t h i r d and fourth l i n e s of investigation could be c a r r i e d on i n the f i e l d or i n connection with the controlled fumigation experiments and would include h i s t o l o g i c a l and physiological research. In the present study no p a r t i c u l a r smelter or area was being i n -vestigated so that the f i r s t l i n e of investigation was not involved and no f i e l d researches were ca r r i e d on. The l i n e followed was that of controlled fumigations with sulphur dioxide* Stress was l a i d on the factors which affected the degree and rate of injury of a l l the species and on the varying rate and degree of injury shown by the f i v e species under observation. In an attempt to determine the actual form of the injury, h i s t o l o g i c a l and physiological investigations were carried out. The study was l i m i t e d to f i v e species of coniferous trees. Review of the Literature There has been a great deal of work done on t h i s problem p a r t i c u l -a r l y i n the United States of America and in Germany. Most of the l i t e r -ature deals with the investigation of conditions around metallurgical works and i n i n d u s t r i a l areas and the results of controlled fumigation experiaents. In the United States o f America one of the foremost workers i s J . K. 1 Haywood. In his paper "Injury to Vegetation by Smelter Fumes. Haywood gives the results of a series of controlled fumigation experiments. Work-ing with Pines he found that a f t e r th£ee, one hour periods in an atmos-phere containing one part i n one hundred of sulphur dioxide' the leaves become greyish and began to droop, while a f t e r s i x hours most of the leaves had f a l l e n and those remaining were yellowish brown. Again, Pines sub-jected to three one hour fumigation periods i n an atmosphere containing one part of sulphur dioxide In one thousand had some leaves drooping and yellowish, while a f t e r nine hours a l l the leaves appeared yellowing and drooping. In a concentration of one part of sulphur dioxide i n ten 1. Haywood J . K., Injury to Vegetation by Smelter Fumes, Dept. of Agric-ulture, Bureau of Chemistry, B u l l e t i n 89. Government P r i n t i n g Office, Washington, U. S. A. 1905. thousand the leaves dropped and became greyish i n twenty f i v e hours and i n f i f t y hours many leaves became brownish and a l l looked unhealthy. Great injury resulted from eighteen hours of fumigation with one part of sulphur dioxide i n t h i r t y thousand. With cedar two hours i n an atmos-phere containing one part of sulphur dioxide i n one thousand resulted 1 i n leaves becoming brown and drooped.. In another a r t i c l e Haywood states that Scrub Pine showed d i s t i n c t injury a f t e r t h i r t y four hours of fumig-ation with one part of sulphur dioxide i n f i f t y thousand, a l l the leaves becoming brown and dry. Ninety-six one hour fumigations also affected a l l the leaves so that the young leaves died and the mature ones were 2 dead at the t i p s . Haywood obtained similar r e s u l t s with Cowpeas , and with 3 Black oak, White oak and Chestnut. Ebough working with A l f a l f a found that w i l t i n g resulted from two fumigations with one part of sulphur diox-ide i n ten thousand, w i l t i n g and bleaching from three fumigations of that strength and complete bleaching from seven fumigations. Four periods i n ftn atmosphere of one part of sulphur dioxide i n twenty thousand caused w i l t i n g but not bleaching while a l i t t l e bleaching was noticed after eight 4 fumigations. Holmes, Franklin and Gould i n the report of the Selby Smelter 1. Haywood, J . K«, Injury to Vegetation and Animal l i f e by Smelter Wastes; U. S. Dept. of Agriculture, Bureau of Chemistry, B u l l e t i n No. 113 (revised). Gov. P r i n g i n % Office, Washington, U. S. A. 1910. 2. Haywood, J . K. Injury to Vegetation by Smelter Fumes; U. S. Dept. of Agriculture, Bureau of Chemistry, B u l l e t i n No. 89. Government P r i n t -ing Office, Washington, U. S. A. 1905. 3. Ebough, E. W. Gases vs. Solids: An investigation of the injurious i n -gredients of smelter smoke. Journal of the American Chemical Society v 29. Pages 951 - 970. The Chemical Publishing Co. Easton, Pa. 1907. 4. Holmes, J. A., Franklin, E. C. and Gould, A., Report of the Selby Smelter Commission, U. S. Department of the Interion, Bureau of Mines, B u l l e t i n 98., Gov. P r i n t i n g Office, Washington. U. S. A. 1915. - 4 -Commission state that they found one part i n 190009OCO- of sulphur dioxide was only s l i g h t l y injurious to barley a f t e r two seven hour fumig-ations and also that s i x seven hour, or ninety 10 minute fumigations gave 1 l i t t l e effect. Zimmerman and Crocker performed a series of experiments with garden plants and weeds. They abtained the following r e s u l t s . One to four hours i n an atmosphere containing three or four parts in a mil l i c z i of sulphur dioxide resulted i n injury to such plants as tomato, coleus, p o t e n t i l i a , nasturtium, chickweed and s a l v i a . Four to eight parts i n © m i l l i o n f o r three to f i v e hours injured castor beans, Sorbus Aucupasia, snap dragon,, peach. I l e x apaca, white carnations* gardenias and Bhodaden~ drons were injured by four hours i n eight to ten parts per m i l l i o n of 2 sulphur dioxide. Sbaugh demonstrated that sulphur dioxide i s injurious to vegetation but does not think that quantities s u f f i c i e n t l y large to be harmful would be found near a smelter. He emphasizes the importance of f l u dust and suggests that this i s the main factor i n causing injury. While not denying the importance of the sulphur dioxide content smelter 3 fumes A. L. Bakke stresses the effect of the tarr y deposit l e f t upon the leaves by smoke and the consequent plugging of the stomata. Holmes, 4 Franklin and Gould i n th e i r report of the Selby Smelter Commission state that they do not believe the concentration of sulphur dioxide i n the a i r around a smelter to be harmful i f the out put i s less than f o r t y tons 1. Zimmerman, P. W . and Crocker, W . , Sulphiu* Dioxide injury to plants. Proceedings of the Amsrican Society for H o r t i c u l t u r a l Science. Vo&. 27 pages 51 to 52. Published by the Society, Geneva, New York, U.S.A 1931. 2. ©p. C i t . 3. BakkB, A. S., The effect of Smoke and Gases on Vegetation, Contrib-ution of the Botanical Departments Iowa State College, Ames., No. 54. 4. ©p. C i t . of sulphur a day. J . V/. Blenkinship who assisted i n the work of the Selby 1 Smelter Commission states i n the report, "In a l l my t r i p s through the sm-oke zone I have only once been able to distinguish the odor of sulphur dioxide, and then only f o r a few minutes and very f a i n t l y , at a point two and a half miles northeast of the smelter. Sulphur dioxide to do material damage i n dry weather to vegetation i n h a l f an hour must be so strong as to be almost unbearable, or i f diluted must be very frequently repeated or almost constant over the area injured i n order to a f f e c t 2 vegetation at a l l . " Haywood on the other hand, in replying to Ebaugh's a r t i c l e states"...the w r i t e r wishes to explain that he i s firmly of the opinion that the s o l i d emanations which arise from a smelter (Including perhaps, soluble copper, arsenic and lead compounds) are injurious to vegetation i n so f a r as they reach i t , but that such emanations reach as f a r as sulphur dioxide or have so injurious an action appears to be de-cidedly doubtful and certainly has not been proven by the paper published 3 by Professor Chough." P. Prazer investigated the causes of injury to vegetation i n an urban v i l l a near a large i n d u s t r i a l establishment. He found that cloths saturated with a l k a l i n e carbonates absorbed from the atmosphere a large percentage of their weights, varying from 6.06 to 10.85 percent of sulphur t r i o x i d e . He also showed that there was an increased 1. Holmes, J . A., Franklin, E. C , and Gould, A., Report of the Selby Smelter Comnission, Dept. of the Inter i o r , Bureau of Mines, Government P r i n t i n g Office, Washington U. S. A. 1315, Page 397. 2. Haywood, J . K. Smelter Smoke. Science Vol. 26., McMillan Co., New York, 1907, page 476. 3. Frazer, P. Search for causes of Injury to Vegetation i n an Urban V i l l a near a large Industrial Establishment. Transactions of the American Institute of Mining Engineers. Volumes 38. Published by the Institute New York 1907, pages 498 to 555. sulphur t r i o x i d e content i n the leaves of trees growing near the i n -i. d u s t r i a l plant. F. W. Oliver found that the sulphur dioxide content of c i t y fog was s u f f i c i e n t to be injurious to greenhouse plants. A great deal has been written on some phases of injury due to sulphur dioxide but t h i s l i t e r a t u r e w i l l (be dealt with as each part of the experimental work i s presented. Material and Methods i n Experimental work. The experimental method followed was that of controlled fumig-ations with known proportions of sulphur dioside. The following f i v e species were used i n the present study. Pseudatsuga t a x i f o l i a . (Lambefct), Br i t t o n , (Douglas F i r ) . Pioea sitohensis. Oarr. (Sitka Spruce), Tsugfe hetorauhylla, Sarg., (Western Hemlock), Pinus monticola'i Dougl., (Western White Pine) and Thuja t i l i o a t a . Donn., (Western Red Oedar) The trees were four or f i v e years old. Specimens for immediate use were planted i n ten or twelve inch pots and placed under cover i n an open shed, while the remaining trees v/e re planted d i r e c t l y i n the s o i l in the same shed. As trees were need-ed for fumigation purposes they were placed in the greenhouse for some time i n order to become acclimatized to greenhouse conditions before being subjected to treatment. Fumigations were made p e r i o d i c a l l y and material was collected for h i s t o l o g i c a l and physiological studies. 1. F. W. Oliver. Urban Fogs. Journal of the Royal H o r t i c u l t u r a l Society, Volume (XVI). Spottiswold and Co., London, 1893. EXPERIMENTAL WORK 1. Fumigations a. Material and Methods s (The fumigations were carried out in a glass sided cabinet having a capacity of approximately 800 l i t r e s . A l l the j o i n t s between the glass and the wooden frame were c a r e f u l l y sealed with putty. The door occupied the whole of one side of the chamber and could be raised and lowered at w i l l when putting the plants into the chamber and removing them. Sulphur dioxide was introduced by burning Carbon bisulphide i n alcohol. Carbon bisulphide s u f f i c i e n t to give the necessary concentration of sulphur dioxide was added to a few cubic centimetres of ninety-five per cent me tho 1 alcohol and the mixture ignited in the chamber. A small e l e c t r i c fan was used to c i r c u l a t e the a i r and make sura that the sulphur dioxide was evenly d i s t r i b u t e d throughout the chamber. In order to f i n d out i f the desired concentration vras being abtained, samples of a i r from the cabinet were analyzed. Method of Analysis of Gas Samples Apparatus • Two 8 l i t r e aspirator bottles were used for taking samples of the gas mixture. Each was closed at the top with a rubber stopper f i t t e d with a s a a l l piece of glass tubing to which a piece of rubber tubing was attached. A pinchoock made i t possible to close the tube. The lower aperture was f i t t e d with a one-holed rubber stopper into which a small piece of glass rod was inserted. Three wide-mouthed glass j a r s , a l l of clear white glass and with a capacity of about 550 cubic centimeters^ Shsss fcstti&K)were f i t t e d with rubber stoppers containing three holes. Two of the j a r s ware f i t t e d up as testing jars i n the following manner. A piece of bent tubing having one arm about two and a h a l f inches long and the other about f i v e inches Icaig was inserted with the short end i n the stopper i n such a way that the end of the tubing was just l e v e l with the inside of the cork. Two straight pieces of tubing were inserted i n the other two holes. One piece about three inches long was put i n so that the end was l e v e l with the inner edge of the stopper, the other piece reached to within a quarter of an inch of the bottom of the b o t t l e . Both the straight tubes were closed with rubber tubing and pinchcocks. Around the lower part of each bottle was pasted a piece of white paper leaving a s t r i p about three quarters of an inch wide through which the colour of the solution in- the j a r could be seen. One hundred and f i f t y cubic cejil&ffleters of water was c a r e f u l l y measured into each b o t t l e and the l e v e l marked upon the white paper. The t h i r d j a r was f i t t e d up as a mixing b o t t l e . The f i t t i n g of this b o t t l e was the same as for the testing bottles except that a small piece of rubber tubing was attached to the bent glass tubing and that marks for one hun-dred and f i f t y and three hundred cubic centimeters were made on the white paper. In order to be able to discharge the l i q u i d from a burette well inside the testing bottles the burette was f i t t e d with a fine drawn out point which would pass through the short straight piece of glass tubing inserted i n the rubber stopper. Solutions needed: An 1J/500 Iodine Solution ,1% Starch Solution. As i t was needed about 100 ccs. of 1% starch solution was made up. This was diluted to form .1% starch. Soluble starch was used and a drop of toluene used as a preservative. Procedure: Before actual sampling of the gas took place the aspirators were well washed, f i r s t with water and then with starch-iodine solution. About 300 ccs. of .1% starch were added to mixing bottle and iodine solution added from the burette u n t i l a l i g h t blue t i n t was abtained. The mixture was then divided between the mixing bo t t l e s i n such a way that the solution was not i n contact with the a i r . In order to accomplish t h i s , the bent tube of the test bottle was inserted into the rubber tubing at the end of the bent tube of the mixing bottle and the l a t t e r inverted. By releasing the plnchcock on the long tube of the mixing b o t t l e and the short tube of the test bottle the mixture could be made to flow from the mixing bottle to the testing bottle u n t i l i t reached the mark in d i c a t i n g 150 ccs. These bottles were shaken thoroughly. Th© solution from each te s t i n g bottle was introduced into one of the aspirators also in such a manner as to prevent contact with the a i r . The glass rod was removed from the lower rubber stopper of the aspirator and the bent tube of one of the a«-pir&toss inserted. The testing bottle was then inverted and the pinchcock of the long glass tube and that of the tube i n the upper stopper of the aspirator released so that the starch-iodine solution flowed into the aspirator. The test-bottle was then removed, the stopper replaced and the upper pinchcook closed. B o t h aspirators were thoroughly shaken and the solution returned to the test bottles i n a manner s i m i l a r to that des-cribed f o r passing the solut i o n from the mixing bottle to the tes t - b o t t l e . If the color of the solution i n the two bottles was the same the aspirators were judged ready for use. I f the colour wa? not the same the solutions were mixed and the procedure repeated u n t i l the same colour was abtained for both. When t h i s condition had been reached the solutions could be poured out of the aspirators and sampling could be begun. Both stoppers were removed from the aspirators. One was placed i n the cabinet i n which fumigation was to be carried on and the other was exposed to the normal gas mixture of the a i r . Care was taken i n both cases to obtain free c i r c u l a t i o n of the a i r . While the aspirators were being exposed, fresh starch iodine solutions were prepared and poured into the testing bottles as previously described. When the sulphur dioxide had been well mixed with the a i r i n the chamber both aspirators were corked and the one removed from the fumigation cabinet. The starch-iodine solutions were introduced into the aspirators and these were shaken thoroughly. The solutions were then poured back into the testing bottles and compared. It was found that the solution from the aspirator exposed to sulphur dioxide was much l i g h t e r i n colour than that from th© other. This colour difference was due to the acti o n of the sulphur dioxide i n the gas of the cabinet. Iodine was then t i t r a t e d into the li g h t e r solution very c a r e f u l l y u n t i l t h i s solution was brought up to the same coluur as that from the aspirator exposed only to the a i r . Since l c . c of fl/500 Iodine solution is equal to .0224 c.cs. of S0j> at 0°G and 760 mm. pressure i t was possible to calculate the proportion of SOg i n the 8 l i t r e sample under e x i s t i n g conditions. By this means i t w as determined that with the apparatus being used the actual con-centration of sulphur dioxide being abtained was approximately 98% of that expected. This method i s adapted from that used by the Selby Smelter 1 Commission. Method of Fumigation: The potted trees ware placed i n the cabinet. A small casserole containing the mixture of alcohol and carbon bisulphide was placed on a shelf i n the cabinet and the door nearly closed leaving only enough room to insert a lighted match to ignite the alcohol. The cabinet door was shut t i g h t l y and the e l e c t r i c fan set i n motion. The fan was allowed to run f o r about ten minutes and then stopped. The trees were allowed to remain i n the chamber for a further t h i r t y - f i v e minutes. If several fumigations were to be carried out i n quids: succession at least f i f t e e n minutes of thorough a i r i n g was allowed between fumigations and the trees were removed from the chamber a f t e r the f i n a l fumigation f o r the day was completed. If only one fumigation was being performed the trees were re-moved after f o r t y - f i v e minutes fumigations b. general results S u p e r f i c i a l symptoms of injury. In a l l cases the f i r s t sign of injury i s a general greying of the fo l i a g e . The next stage i s a general browning of the leaves* usually 1. Holmes, J . A., Franklin, E. C, and Gould, A. Be port of the Selby SmeIter Commission, Dept. of the Interiors, Bureau of Mines. Gover-nment Printing Office, Washington, U. S. A., 1915. pages,*-s t a r t i n g from the t i p of the needle hut somstimss i n i r r e g u l a r patches over the surface. Young leaves wither at the t i p s and droop very quickly. In some trees such as the Douglas F i r the needles f a l l very readily even a f t e r a l i t t l e exposure to sulphur dioxide funas. The brovaiing i s f o l l -owed by a bleaching action which causes the needles to become a light tan shade. Yihen t h i s stage i s reached there i s usually no recovery of the tree even i f i t i s removed from the fumigation chamber. Buds seem to be very resistant,even s l i g h t l y open buds of the Douglas F i r and Pine showed growth a f t e r the plant had been exposed to fourteen doses of sulphur dioxide i n a concentration of 1 part of SOg i n 10,000, and had appeared completely dead. It was noticeable i n the case of the Douglas F i r that a few of the young leaves were p a r t i a l l y exposed and of these the t i p s were withered while the base continued normal development about four weeks a f t e r the apparantly dead plant had been removed from the cabinet. Specific rate of in j u r y . ' One of the f i r s t things noticed about the f i v e species used was that sulphur dioxide fumes did not effect a l l of them at the same rate. (Tables 1 to 5 ) . In each fumigation series the f i r s t species to show marked effect was the Douglas F i r which showed very d i s t i n c t graying of the leaves a f t e r only one or two periods of exposure to sulphur dioxide. (Tables 2 and 3). In t h i s species also leaves were lost or f e l l at a touch a f t e r two fumigations i n Series 2 (Table 2)and Series 3 (Table 3) and a f t e r three periods i n Series 4 (Table 4). The l a s t to show injury was the cedar which exhibits only greying when the Douglas F i r shows loss of leaves. (Tables 1 and 3). A f t e r injurious effects commence to become evident there i s a very rapid browning and withering of the leaves i n the Cedar (Table 4). The Pine shows browning at the t i p s of the leaves e a r l i e r than does the cedar, but the progress of t h i s condition i s very much slower i n the Pine than i n the cedar (Tables 3 and 4). The pine needles while brown at the t i p s and showing greying of the rest of the needles remains i n t h i s condition for some time before t o t a l browning occurs. Hemlock and spruce are intermediate between the Douglas F i r and the Pine and Sedar. Hemlock shows injury a l i t t l e sooner than does the Sprvce (Tables 2, 5 and 4). The order i n which injury i s evinced i s as follows, Douglas f i r , hemlock, spruce, pine and cedar, while the cedar usually shows t o t a l browning sooner than does the pine. (c) Factors involved i n determining the rate of injury. It w i l l be noticed from the accompanying tables that not only do the species vary i n t h e i r rate of injury by sulphur dioxide, but that the time of the f i r s t clear indication of injury to any species varies i n different fumigation series. In Series 1, injury i s evident a f t e r four periods while i n Series 3 one fumigation produces the same r e s u l t . The factors which are involved in t h i s d i f f e r i n g rate of injury w i l l be considered i n the next few paragraphs. The f i r s t factor to be discussed w i l l be the time factor. It w i l l be noticed that i n those cases which do not show noticeable injury u n t i l a f t er the fourth fumigation the fumigation periods were close together, in fact were given i n groups of several i n one day. Table 1 shows a condition where four fumigations were given i n one day and Tables 3 and 4 where only one fumigation was given i n one day. In each case obser-vations were taken on the morning following the fumigation* Examination - 14 -of the tables shows that the degree of injury i s very similar i n both cases. Injury while not evident at once develops quite markedly within twenty four hours and i t s degree i s not apparantly increased i n propor-tion by the addition of three or four fumigation periods during the day. Temperature and humidity have been found to have a very decided effect upon the rate of injury i n plants exposed to sulphur dioxide fumes. 1 Holmes, Franklin and Gould found that sulphur dioxide fumes were less 2 injurious i n a condition of low humidity and Zimmerman and Crocker found plants more resistant i n dry a i r . Under the conditions of the experiment the temperature and humidity varied considerably from day to day but similar conditions obtained i n each fumigation series. The humidity ranged from 50% to 75%, averaging about 60%. In a l l but the l a s t two series (Series 4 and 5) the temperature range was from 48°0 to o o 80 C with an average temperature of 60 C. Series 4 and 5 were run during a period of exceptionally f i n e spring weather so that the temperature o o range i s somewhat higher i n t h i s case being from 52 C to 32 C and averaging o 65 0. The humidity, however, remained p r a c t i c a l l y the same as f o r the previous experiments* With this wide range of humidity i t was impossible to draw any conclusions as to the effect of t h i s fact or upon the rate or degree of injury r e s u l t i n g from fumigation. Temperature appears to have a very great effect upon the rate of injury. One experiment (unrecorded because incomplete) was commenced i n August 1933 and i t was found that a f t e r one exposure to one part of sulphur dioxide i n ten thousand the Douglas f i r lost nearly a l l i t s leaves while hemlock, spruce and pine showed almost complete browning and a considerable portion of the cedar had turned brown. The temperature 1. Qp. c i t . 2. Qp. c i t . Date no. a ( J2 Cone. Time Notes. 20.4.33 i 1:10,000 11.15am tt 2 •i 12.30am I I 3 tt 1.40am »i 4 H 2.45am D.F.all leaves grey to brown dropping 'H and C greying and brocn P&S s l i g h t " g r e y i n g . 21.4.33 0 K 9.45am 6 It 1.45am 1 : tt 2.50am x/.jr.cteciaea Drowning M. browning.P&S &6 browning at t i p s increased S unre-gular brovmine and bleach i r e . 23.4.33 8 It 9»15am 9 I I 10.20am 10 tt 11.20ara D.P.appears dead. H. many leaves en-t i r e l y brown, and S.browning and mott-l&ng.C&P distince browning. 26.10.33 11 tt 10.00am L2 tt 11.00am r. nearly dead S. nearly dead. G. h a l f dead. 28.10.33 13 tt 10.00am 14 tt : lM i 11.00am A l l trees appear dead'. Abbreviations used throughout tables H'.. - Hemlock S - Spruce DP - Douglas F i r C - Cedar P - Pine Table 2. Record o f Fumigation Series No. P. Date Ho. SO OoSc. Time Notes 30.10,33 1 :•' 1 20,000 2.00pm 30.10.33 2 •t 3.00pm D.F. distinct greying leaves f a l l at touch. 31.10.33 3 tt 2.30pm D.F. leaves browning f a l l at slightest jarring. S greying ends of branches drooped. 31,10.33 4 tt 3.30pm 1.11.33 5 ii 9.45am' G. H & P some yellowing 1.11.33 6 it 10.45am 6.11.33 7 it 1.15pm D. F. many needles f a l l e n H. yellowing. C.P.and S. some yellowing and browning. 7.11.33 8 it 9.25am H - nearly a l l leaves brown c - browning. DP- most leaves fallen P&S browning. gable 3 Record of Fumigation Series Fo 3. Date Cone. Time •.. Notes. 22.1.34 1 10,000 5pm D.F.distinct greying. P.S.H. Some greying. 3. s l i g h t greying. 23.1.34 2 ti 4.30pn D.F.very grey, leaves drop readily, H. greying d i s t i n c t P & S greying more evident 0 greying D.F. most leaves f a l l e n . 27.1.34 •. 3 ;• it 5.15pm H & S very brown and grey C & P browning at ends of leaves C more than P. gable 4. Record of Fumigation Series No. 4. Date No. Cone. Time Notes 28.4.34 : I 1 10,000 4.30pm D.F.greying noticeably Others s l i g h t l y greying C - l i t t l e greying 29.4.34 2 1 10,000 5.30pm D.Fvery grey H greying P &S some greying C l i t t l e greying 2.4.34 3 1 »o,ooo 5 pm DF leaves f a l l readily H&S bromiing S P & C browning. 3.4.34 4 5.15pm D F leaves f a l l i n g H & S brown. C very brown P nearly a l l brown. Table 5 Record of Fumigation Series No 5„ Bate No. Gone. Time Notes 12.4.34 . , 1 10,000 4.55pm t i p s of leaves greying 15.4.34 2 « 4.00pm Tips of leaves browning 14.4.34 : 3 i t 5.30pm Leaves brown except at base 15.4.34 4 i t 4.20pm Most leaves brown some green at base. 0 at t h i s time ranged as high as 96 0 i n the shade and t h i s together with the humid atmosphere of the greenhouse produced a condition i n ©Men very rapid injury was obtained. Another important factor i n determining the rate of injury i s the co d i t i o n of the plant at the time of fumigation. In those cases where the trees used had been i n the greenhouse some time and were i n the rapidly growing condition injury was rapid (Tables 2, 3 and 4). Much less rapid effects resulted when trees were allowed to remain i n the green-house only long enough to become used to the change i n atmospheric condition (Table 5). Series 4 and 5 are p a r t i c u l a r l y interesting since the temperature and humidity conditions were almost the same for both. In series 4. however9 the trees used had been i n the greenhouse for some time and were i n the rapidly growing condition while i n Series 5 the Douglas F i r used had been i n the greenhouse f o r only a few days. In series 4 the Douglas f i r showed loss of leaves a f t e r the t h i r d fumig-ation while i n Series 5 the leaves did not f a l l even a f t e r the fourth 1 period. If as F. W. Oliver suggests, injury occurs through the en-trance of sulphur dioxide into the leaf by the stomata, the growing condition of the plant may affect the rate at which i t may be injured through the physiological condition of the stomata. On the other hand, 2 Haselhoff and Lindau as quoted by Frazer state that the gas i s absorbed by the whole leaf surface. In t h i s case the condition of the stomata would have l i t t l e effect upon the rate of injury. It i s possible that when the trees are i n a rapidly growing' condition, the chemical oontents of the leaves may be such that the sulphur dioxide on entering the leaves may most readily reach with and destroy some essential constituent of the c e l l . Whatever the cause, i t seems clear that trees are more e a s i l y i n -jured i n the rapidly growing condition than they are when p a r t i a l l y or wholly dormant. 2. H i s t o l o g i c a l studies. Very l i t t l e l i t e r a t u r e on t h i s phase of the subject was available. In most investigations analysis rather than histology has been staessed i n the study of t h i s problem and of those accounts which do deal with 3 the histology only one or two were obtainable. Frazer quotes i n tran-s l a t i o n from Hoselhoff and Lindon's," In the i n t e r i o r of the c e l l plas-molysis i s induced; the grains of chlorophyll are destroyed, and f i n a l l y form with the plasma and other materials a brown amorphous mass. At the 1. ikp. c i t 2. Op. c i t . 3. Frazer, P. Search for causes of Injury to Vegetation i n an Urban V i l l a near a large Industrial Establishment. Transactions of the American Institute of Mining Engineers. Vol. 38. Published by the I n s t i t u t e , New York 1907, page 500. S&.ES time i n most cases, especially i f injury nas been gradual, tannin separates out as can be demonstrated by brown or black nodules i n the I c e l l s . " I n "Urban Fogs" Oliver gives two main stages i n the effect of the gas on leaves, f i r s t the destruction o f chlorophyll and second the formation of a d e f i n i t e layer of separation I n the p e t i o l e . Either of these effects may occur separately or they may occur in the same leaf . Other writers mention plasraolysis as c h a r a c t e r i s t i c of c e l l s injured by sulphur dioxide. In the present study p a r t i c u l a r attention was paid to the starch grains and to various points which arose in connect ion with the general disorganization of the c e l l s , a. Material and Methods. A preliminary experiment was run i n which samples were taken a f t e r the f i f t h , ninth, eleventh and thirteenth fumigations. In sectioning these i t was found that even as soon as the f i f t h fumigations especially i n Douglas f i r , spruce and hemlocks, there were such great changes i n the c e l l contents that l i t t l e structure could be made out s a t i s f a c t o r i l y * I t was then considered necessary to take samples a f t e r each fumigation and t h i s was done. Samples were taken i n each case on the morning f o l l -owing the fumigation. The k i l l i n g and f i x i n g solution used was B. C. F r i a t i v e having the following formula. Water ( d i s t i l l e d ) , 100c.cs. Methol alcohol 95% 500 c c s . Formalin 4-0% 25 c c s . G l a c i a l acetic a c i d 25 c c s . 1. Op. Oit. The specimens were dehydrated i n metho1 alcohol and f i n a l l y embedded from S y l o l . Sections v/e re cut on a rotary microtome. The material was found to be somewhat b r i t t l e and e a s i l y torn so that most of the sec-tions had to be cut at between 8 and 15 u\ Gum arable and potassium dichroroate were used as f i x a t i v e s . Two types of staining of the sections were used. F i r s t safranin i n 50% methol alcohol with l i g h t green i n clove o i l as a counterstain and then aqueous safranin with alcoholic gention v i o l e t (50% methol alcohol was used) as a counter stain. The l a t t e r method was found to be the more effe c t i v e . The sections were mounted in Canada balsam. In fumigation Series 5, fresh material was taken for free-hand sec-tions and these were stained d i r e c t l y with Iodine. Study of the leaf sections: Normal Specimens. In order to describe the e f f e c t s produced by Sulphur diosd^O i t w i l l be necessary f i r s t to out l i n e b r i e f l y the normal condition of the leave s o In the normal Douglas F i r leaf the palisade c e l l s are tight l y packed and the spongy mesophyll c e l l s are rather loosely packed leaving large sub-stomatal c a v i t i e s . The starch grains are numerous and large and are placed peripherally around a large central vacuole. (Plates (I) and 31a) The epidermis i s one layer of c e l l s t h i c k with a heavy smooth c u t i c l e . The stomata are i n shallow, p i t s . In the hemlock the palisade layer i s s l i g h t l y less compact than i n the Douglas f i r . The spongy mesophyll c e l l s are large and loosely packed leaving big and rather i l l - d e f i n e d sub-stomatal c a v i t i e s . The starch - 2 2 -p l a s t i d s are large and are so closely packed i n a peripheral layer round the central vacuole that they appear hexagonal i n outline. (Plates (11) and(Xll)a.) ?he surface of the heavy c u t i c l e i s f i n e l y serrated. The epidermis i s one c e l l i n thickness except at the leaf margins where a second layer of schlerenchyma c e l l s may be present. The stomata are i n shallow pibs. The palisade layer i n the pine i s very compact while the mesaphyll c e l l s are somewhat loosely packed. The sub-stomatal c a v i t i e s are small and quite d e f i n i t e l y outlined by large spongy mesaphyll c e l l s . The starch grains are found i n a compact peripheral layer giving a hexagonal appear-ance to the i n d i v i d u a l grains. Two layers of c e l l s are present i n the epidermis. The outer layer consists of small c e l l s with very heavy walls, and very s n a i l lumen. In the inner layer the c e l l s are large and do nfct possess a very t h i c k w a l l . At the angles of the leaves there i s occasion-a l l y a t h i r d layer of c e l l s s i m i l a r to those i n the second layer. The c u t i c l e i s t h i c k and smooth. The deep stomata1 p i t s are protected by the heavy overhanging c u t i c l e . (Plates 1? and XI7a.) No very d e f i n i t e palisade layer i s discernable i n the spruce the en-t i r e mesaphyll region being f i l l e d w ith a f a i r l y compact mass of rather i r r e g u l a r c e l l s . The starch grains are massed i r r e g u l a r l y i n the c e l l s (Plates 111 and X l l l a . ) i n strong contrast with the d e f i n i t e p e r i p h i r a l arrangement found i n the Douglas f i r , hemlock and pine. A large number of r e s i n c e l l s are present and show dark brown when stained. The upper epid-ermis i s d e f i n i t e l y two layered. The outer c e l l s are large and t h i c k walled and the inner c e l l s are sclerenchyma with heavy walls and small lumen. D i r e c t l y below the vascular bundle the lower epidermis has two - 2 3 -layers of c e l l s s i m i l a r to those i n the upper epidermis. Between this region and the margin of the leaf the lower epidermis i s only one layer of c e l l s thiclc. The c u t i c l e i s heavy and smooth. Stomata are situated i n rather deep p i t s but have not the overhanging protective c u t i c l e found i n the pine. The palisade c e l l s i n the cedar are shorter and wider than those i n the other species studied. They form a compact layer. The spongy mes-aphyll c e l l s are rather loosely paclsBd but the sub-stomatal cavities are small. The starch grains are arranged peripherally. A large number of heavily staining r e s i n c e l l s are present. The upper epidirmis consists of two layers of heavy walled c e l l s , the inner of which are d e f i n i t e l y schlerenchyma c e l l s with very heavy walls and small lumen. The lower epidirmal c e l l s vary i n size and are nonvex on the outer surface giving, i n section, a scalloped appearance to the lower edge of the l e a f . The c u t i c l e i s very thiclc on both upper and lower epidermis. The scalloped appearance of the lower epidermis i s accentuated by the fact that the c u t i c l e i n t h i s region i s frequently thickened at the convex surface of the c e l l so that a protruberance of the c u t i c l e results (Plates Y and XVa). Stomata are situated only on the lower surface which i s closely applied to the stem and they are found i n deep p i t s . I t i s evident i n a l l these conifers that the starch grains are con-tained within the chloraplasts, the l a t t e r showing as a l i g h t e r unstained ring around the former. Fumigated Specimens: In examining sections of leaves from trees which have been subjected to fumigations there are two main features which are very evident. The - 24 » f i r s t of these features i s the decreased size of the starch grains. Table 6 shows the results of a series of measurements made from material which had been k i l l e d and f i x e d i n B. G. f i x a t i v e embedded and stained with safranin and gentian v i o l e t . Measurements i n Table 7 are taken from Douglas f i r material which was sectioned by hand i n the fresh condition and stained with iodine. A l l measuremants were taken with the a i d of a camera lu c i d a . In each case the figures refer to diameter i n microns. The f i v e species w i l l be discussed individualy. In Douglas f i r most of the starch grains are large but there i s a great deal of v a r i a t i o n i n size, the smallest being 2.5 H i n diameter and the largest 11.0 U,. Although only subjected to two fumigations the rapid decrease i n the size of the starch grains i s very noticeable being a re-duction from 6.21 U to 2.04 U.. In t h i s species the amount of the de-crease i s almost the same during the f i r s t and the second periods. The size of the smallest grains appears to remain stationary during the two fumigations but t h i s i s probably only apparent. The average size of tho smallest grains i n the normal leaf i s 2.5 Hand the average of the small-est grains a f t e r the f i r s t fumigation i s 1.3 U, a decrease of 1.2 U, i n diameter. If a corresponding decrease i n size occured between the f i r s t and second fumigation tho se starch grains which a f t e r the f i r s t fumigati oi measured 1.3 IX, i n diameter would be reduced to about. 1 IX. or destroyed altogether. In Table 7 the reduotion i n the size of the starch grains i s less marked than i n Table 6. In t h i s case the reduction i s from 4.91 U. to 3 .o i u;. Table 6. Diameters of starch grains of Douglas F i r , Spruce, Pine, Hemlock and Cedar from needles of trees exposed to SO . Material k i l l e d and f i x e d i n B. C. f i x a t i v e . Tree Fumig at ion Ho. Average diameter of Starch grains Range i n diameter of §tarGli'-graiu.s.»co Douglas F i r . : 0 6.21 IX 2.5 XI - l i n 1 4.01 U 1.3 a - 9.6 IT • • 2 ' 2.04 JU 1.3 IX - 4.5 n 3 Spruce . 0 5.03 U, 2.6 IX - 9.6 II • • • • 1 ': 3.29 T\ 1 . 3 1 - " . 5.8 II 2 2.34 1.3 IX - 3.2 IX 3 3.02 R 1.3 7X - 6.5 H Hemlock 0 6.50 tt. 3.9 n - io .3 a ' 1 -:- 4.01 Uv 2.6 II - 6.5 TI 2 3.98 a. 1.9 jDL - 6.5 U. 3 3.67 D< 1.3 U. - 5.8 TX Pine 0 4.13 Ii, 1.9 H - 7.8 U, 1 2.71 DL 1.3 JX - 4.5 IX a 2.29 H 1.3 B, - 3.9 0. . s 2.21 II 1.3 II - 3.9 U Oedar 0 ! 4.80 a 2.6 H - 13.6 IT . 1 i 3.00 DL 1.9 D, - 4.5 II 2 i 2.79 JX 1.9 II - 5.8 11 • -S t 2.05 II .65U - 3.2 II Table 7. Diameter of stardh grains of Douglas F i r from needles of trees exposed to SO. Material sectioned fresh and stained with Iodine. Tree Fumigation Ho. Average diameter of Starch Grains Range i n diameter of Starch grains. Douglas F i r 0 4.91 & 3.2 xx - 7.0 11 1 4.21 II 2.6 Ik - 5.8 Ik 2 3.34 IX 1.9 Xk - 4.5 IX 3 3.02 U, 1.9 IX, - 5.2 II 4 , 3.01 IX 1.3 - 5.2 AX - 26 -The amount of the change which takes place at each fumigation i s the same for the firstIftvo fumigations, The f i r s t period caused a reduction of .7 u and the second of ,87 TJ. This result compairs with that shown i n Table 6 except that i n the l a t t e r case the degree of reduction i s greater. As has been previously stated that the Douglas f i r used in fumigation Series 5 was not injured nearly as speedily as were those used the previous experiments. As this i s the case, reduction in the size of the starch grains would not be expected t o be as rapid as It was In the previous cases. The hemlock shows reduction from 6.5 U to 3,67 II and reduction i n range from 3.9 H- 10.3 11 to 1.5 1^  - 5.8 U. The average reduction i s about one ha l f the normal sise and the reduction i n the smallest grains i s two thirds of the normal while the largest are reduced by four f i f t h s . In t h i s case the amount of reduction is not nearly equal for each fumig-ation as i t was i n the Douglas f i r . The f i r s t period resulted in the reduction of 2.94 i n the diameter of the grains* the second caused only .03 I\ reduction, an.amount which i s p r a c t i c a l l y n e g l i g i b l e , while the t h i r d caused a loss of .31 II diameter. Reduction from 5.03 IJ^to 2.34 U i s found i n the spruce. Here as i n hemlock the greatest decrease occurs i n the f i r s t fumigation where reduction i s 1.74 Q- the smallest grains are reduced to one ha l f and the largest grains to one t h i r d of t h e i r normal s i z e . There i s an apparent increase i n the size of the starch grains i n the l a s t fumig-ation. 2his appearance may be due to the toltal destruction of many of the smaller starch grains. The pine starch grains show decrease i n size from 4.13 L\_to 2.33 II, - 27 -and a reduction i n range from 1.9 IX.- 7.8 U, to 1.3 3.9U. This species resembles the hemlock and the spruce i n that the greatest re-duction i n diameter of the grains takes place i n the f i r s t fumigation. The loss i n t h i s case i s 1.41 D\ i n the f i r s t psriod and .42 IX i n the second while i n the t h i r d i t i s .08 U. It i s noticeable that the smal-lest starch grains i n the pine are considerably smaller than those found i n the other species. A f t e r the second fumigation no s i g n i f i c a n t re-duction appears to take place. The cedar shows the greatest v a r i a t i o n i n the size of the p l a s t i d s . The range i n t h i s species i s from 2.6 II to 13.6 II and the average diameter i s 4.811. The small size of the average grains as compared with the max-imum size shows that the large grains must be small i n numbers. As i n a l l the species, but the Douglas f i r , the greatest reduction i n size i s i n the f i r s t period where the decrease i s 1.8 U. The following period shows a reduction of only .11 and the t h i r d of .64 U. The range of var-i a t i o n i s reduced from 2.6 l{ - 13.6 1^  to .65 L\to 3.2 U, the reduction of the larger grains i s p a r t i c u l a r l y noticeable. Unlike the pine and hemlock, the cedar shows de f i n i t e decrease i n the size of the grains i n the t h i r d fumigation. In comparing the results obtained, as sho\wi i n Table 6, It w i l l be seen that the degree and rate of the decrease in the size of the starch grains i n each species corresponds almost exactly with the order i n which the species show injury. The greatest t e t a l reduction i s shown i n the Douglas f i r where the grains are reduced by about two thirds of t h e i r normal size a f t e r two fumigations, i f the other species are also com-pared a f t e r the second fumigation i t i s seen that Spruce, hemlock and pine are reduced by approximately one h a l f of t h e i r normal size while cedar i s reduced by three f i f t h s . These results suggest that the cedar should show response to fumigations before the hemlocks spruce and pine, but these species seem to have reached almost the l i m i t of reduction at the second period while the cedar shows definite continued decrease in the t h i r d period. The second noticeable change in the c e l l s a f t e r exposure to sulphur dioxide i s the general disorganisation of the c e l l contents, and in some cases of the c u t i c l e . In Douglas f i r fthere i s a noticeable collapse of the spongy mesa phy11 and pain sade c e l l s a f t e r one or two fumigations (plates (VI) and [XI). The c e l l walls become irregular and i n some cases collapsed. The reduc-t i o n i n the size of the starch grains has already been, mentioned (Table 6 ) . Decrease i n size i s accompanied by change i n the shape of the grains which become more l e n t i c u l a r i n shape or even i r r e g u l a r i n outl i n e . The hexagonal shape due to the compact arrangement of the p l a s t i d s , as men-tioned i n the discussion of the normal histology of the le a f , i s lost and the plastids become widely separated in the c e l l s . In many c e l l s the nucleus i s the only d e f i n i t e structure v i s i b l e a f t e r two fumigations. The c e l l contents have become changed to a mass of f i n e l y granular mat-e r i a l which stains r e a d i l y with alcoholic gentian v i o l e t (Plate (XI). Ho effect i s v i s i l l e i n the epidermis and the c u t i c l e . Plate ( 11) shows the general collapse of the mesaphyll c e l l s in the hemlock leaf a f t e r one fumigation. The effect of the close packing of the starch p l a s t i d s as seen i n the hesagonal shape produced i s not ent-i r e l y lost although the spacing of the grains i s d i s t i n c t l y wider than i n the normal condition. (Plates (11) 711 and XL1). The reduction i n the p l a s t i d s i s not acconrpanied by such narked change in shape as i s evinced by the Douglas f i r , and only a few of the grains show irregular outlines, i n the hemlock a very d e f i n i t e effect i s produced upon the c u t i c l e which becomes loosened from the uppsr epidermis p a r t i c u l a r l y , and i s e a s i l y torn away i n sectioning (Plate 711). The pine shows rapid disorganisation of the leaf t i s sue. Plasmolysis i s very evident especially i n sorts of the c e l l s , and ths c e l l walls are frequently collapsed. The hexagonal appearance of the starch grains i s e n t i r e l y lost a f t e r one fumigation. The grains become very much smaller d i s t i n c t l y l e n t i c u l a r and show a dark staining central portion surrounded by a comparatively wide l i g h t e r area. The light area i s very d e f i n i t e l y defined i n some cases and i n others i t i s d i f f i c u l t to determine i t s l i m i t s . The nucleus does not show any effect but the remaining c e l l con-tents form an amorphous moss in contrast with the granular contents of the c e l l s of the Douglas f i r . Further fumigations only increase the e f f e c t s already described. There seems to be a continuous breakdown of the c e l l walls u n t i l no very d e f i n i t e walls can be distinguished a f t e r three fum-igations (Plate 217.) The epidermis and c u t i c l e show no effect. Disorganisation of the leaf tissue i s less evident i n the spruce than i n the preceding species. The p l a s t i d s are reduced i n size and appear scattered throughout the c e l l rather than massed together as in the n o r -mal specimens, or i n some cases they appear pushed to the periphera where they form an irregular layer (Plate XL1I). A few c e l l s show the contents as a granular mass s i m i l a r to that found i n the Douglas f i r . A few very i n d i s t i n c t p l a s t i d s may he found i n these granular c e l l s . In s t i l l other cases the starch grains appear to have p a r t i a l l y disintegrated in such a way as to form threads or a network of p l a s t i d material which stains red with safranin (Plate X I I I ) . After two fumigations there i s evidence of break down of the c e l l walls resembling the condition found i n Douglas f i r and pine. The cedar does not show much effect a f t e r the f i r s t fumigation. After three psriods, however, there is a very definite break down of the leaf tissue. The c e l l walls are collapsed. The p l a s t i d s while being reduced i n size show no p e c u l i a r i t y of shape. Many of the c e l l s show d i f i n i t e plasmalysis but no injury i s shown by the epidermis and c u t i c l e . The stomata i n almost a l l cases were found closed in the fumigated specimens suggesting that exposure to sulphur dioxide causes these c e l l s to collapse. This condition might be expected i n view of the general disorganisation of the other chloraphast containing tissue of the l e a f . Lack of material made i t impossible to f o l l o w t h i s line of investigation to a satisfactory conclusion so that i t is possible only to suggest this as a possible r e s u l t of fumigation. The general collapse of the t i s sue suggests that dessication i s one of the effects produced and this i s substantiated &y the dry weight det-erminations made and which w i l l be recorded l a t e r . The disorganisation of the tissue also suggests that some chemical reaction has taken place so that the no una 1 contents of the c e l l s are either altered or p a r t i a l l y des-troyed. In some species, p a r t i c u l a r l y i n the spruce and cedar, there are a number of c e l l s f i l l e d with a dark amorphous mass which i s probably a mucilaginous degeneration product. - 31 -3. Physiological s t a d i a s The l i n e of study persued i n the section of the work was suggested by the h i s t o l o g i c a l studies already carried out. It has already been shown that there i s a great reduction i n the size of the starch grains a f t e r exposure to sulphure dioxide and this discovery suggested an i n -vestigation of the effect of the gas upon the food storage of the le a f . P a r t i c u l a r stress was l a i d upon the sugar and starch relations in this regard. No l i t e r a t u r e wa-& found upon t h i s subject so that a l i n e of attack was worked out based upon the s o l u b i l i t y of available sugar in the leaf and the i n s o l u b i l i t y of the starch, (a) Pood Storage Material and Methods. The material used for fumigation was taken on the morning a f t e r the fumigation had been made. The fresh leaves were used and exactly .5 grams taken. The leaves were cut up f i n e l y and ground i n a mortar with quartz sand. To this material was added 25 c c s . of d i s t i l l e d water and the whole allowed to stand f o r an hour with occasional shaking. The l i q u i d was then f i l t e r e d o f f and tested f o r sugar. A ten cubic centimeter sample of the f i l t e r e d liquidwas boiled with 50 c c s . of a solution of copper sulphate i n potassium carbonate and pot-assium bicarbonate f o r ten minutes and codled thoroughly. The precip-i t a t e of cuprous oxide was f i l t e r e d out by suction through an asbestos mat i n a gooch crucible and washed v/ith d i s t i l l e d water. 30 c c s . of hot d i s -t i l l e d water were poured upon the asbestos mat containing the precipitate and the whole thoroughly heated. 10 c c s . of hot f e r r i c sulfate solution i n sulphuric a c i d was added to the mixture to disolve the pr e c i p i t a t e . The solution was then t i t r a t e d with a solution of potassium permanganate i n which 1 c c . of the permanganate solution was equivalent to 11.7 mg. of copper. The method described i s a modification of the Wood-©st copper 1 carbonate method as described by Cole . The solutions used are as follows Copper carbonate solution potassium carbonate 250 gms. potassium bicarbonate 100 gms. Dissolve in 600 c.cs. of warm d i s t i l l e d water. C r y s t a l l i n e cdpper sulphate (pure) 23.5 gms. Dissolve i n 200 c.cs. of d i s t i l l e d water. Add the copper sulphate solution to the potassium carbonate and bicarbonate solution slowly mixing the two thoroughly. Make the volume up to 1 l i t r e and f i l t e r . F e r r i c Sulphate (acid) solution. Add slowly 250 c.cs. of pure concentrated sulphuric acid to 750 c.cs. of d i s t i l l e d water. Add f e r r i c sulphate to the solution and heat. Repeat t h i s u n t i l the presence of a small amount of precip-i t a t e shows that the solution i s saturated. Potassium permanganate solution. Dissolve 6 gms. of potassium permanganate in 1100 c.cs. of cold d i s t i l l e d water and standardize the solution with ammonium oralate. Cole, S. V/. P r a c t i c a l Physiological Chemistry. Seventh Edition, W. Heffer and Sons, Ltd., Cambridge, England. 1926, pages 176 to 179. - 33 -. In determining the starch content the method used was modified from that given i n the United States Department of Agriculture, Bureau of Ch-1 emistry B u l l e t i n 107 . Ihe material from which the solution to he t e s t -ed for sugar was f i l t e r e d was used f o r the starch determination. This residue was placed i n an Eslenmeyer f l a s k with 100 c.cs. of 10^ HCL s o l -u t ion and heated on a water both f o r three hours. The l i q u i d remaining' was measured and 10 c.cs. taken for analysis. In t h i s sample the acid was almost neutralized with sodium hydroxide and the sugar content determined as described i n the previous paragraph. Results i In a l l the r e s u l t s tabulated below the figures used refer to milligrams of copper reduced. Leaves from fumigation Series 3, 4, and 5 were taken for analysis. Material was taken on the morning after fumigation i n each case. The r e s u l t s abtained from series 3 are shown in Tables 8 and S and graphically i n Figures 1 to 5. Table 8. Sugar content of .5 gms. of leaves taken from specimens used i n fumigation i n each case. Species Fumigation numbers 0 1 2 3 Pine 24.86 43.87 14.62 21.94 Hemlock 23.4 45.34 17.55 Spruce 23.4 43.87 24.86 14.62 Oedar 29.25 48.26 29.25 Douejlas F i r 34.85 76.05 4.38 leaves f a l l e n Association of o f f i c i a l A g r i c u l t u r a l Chemists, O f f i c i a l and Provisional Methods of Analysis, U. S. Dept. of Agriculture. Bureau of Chemistry B u l l e t i n 107 (revised). Government Pr i n t i n g Office, washington,U.S.5. 1912, page 53, 8 (a) - 34 -Table 9. Starch content from the same material as used, in table 8< Species Fumigation number 0 1 2 3 ...Pine. •> ; 122.85 99.91 145.08 150.67 Hemlock 90.09 147.42 187.20 153.97 Spruce 131.62 51.48 87.75 159.47 Cedar 109.69 131.62 — 148.47 Douglas Fir Xe3X ©62 131.62 119.06 leaves f a l l e n The f i r s t st±i«king thing about these results is the fact that each species shows a very marked increase in the sugar content after the f i r s t fumigation (Figure 5) and that the decrease i s a linos t as sufi&en after the second period as was the rise after the f i r s t . Another interesting feature Is the apparent corelation between the rate of injury and the increase of the sugar content. In the Douglas f i r which shows the most rapid injury two and a half times the normal sugar was found after one fumigation while in the cedar only one and a half times the normal was found in the same period. By examining the rise in the sugar content as shown in Table 8 the following order of in-crease w i l l be shown. Douglas f i r shows the greatest followed by hem-lock, spruce, pine and cedar in descending order. This l i s t w i l l be found to be the same as that given on pagelS where the trees are listed in the order in which they show injury. FIGURE U Showing the relation of sugar and starch in Pine leaves during fumigation with 1 part of sulphur dioxide i n 10,000, .5 gm. of leaves from specimen used i n fumigation Series 3 were analysed in each oase. - 36 -FIGURE 2 . Showing the relation of sugar and starch in Hemlock leaves during fumigation with 1 part of sulphur dioxide in 10,000. .5 gm. of leaves from specimen used in fum-igation Series 3 were analysed in each case. - 56 -FIGURE 3. Showing the r e l a t i o n of sugar and starch i n Spruce leaves during fumigation with 1 part of sulphur dioxide i n 10,000. .5 gni. of leaves from the specimen used i n fumigation Series 3 were analysed i n each case. - 37 -FIGURE 4. Showing the relation of sugar and starch in Douglas F i r leaves during fumigation with 1 part of sulphur dioxide i n 10»000. .5 gm. of leaves from the specimen used in fumigation Series 3 were analysed in each case. '- 38 -FIGURE 5, Showing the v a r i a t i o n i n the sugar content of leaves of Douglas F i r , Hemlock, Spruce and Pine, during fumigation with 1 part of s u l -phur dioxide i n 10,000. .5 gm. of leaves from specimens used i n fumigation series 3 were analysed i n each case. In comparing the effect of fumigation on the starch and sugar content of the leaves i t w i l l he well to discuss each species separately. The sugar and starch r e l a t i o n s h i p i n the pine i s shown graphically i n Figure 1 and tabulated i n Sables 8 and 9. The f i r s t fumigation caused a d i s t i n c t r i s e in the su§ar content from 24.86 mg. to 45.87 mg. and a corresponding f a l l i n the starch content from 122.85 mg. to 99.91 mg. At the second fumigation the result i s reversed and an increase i n the starch from 99.91 mg. to 145.08 mg. i s accompanied by a decrease i n the sugar from 45.87 mg« to 21.94 mg. while fete starch remains almost the same during the t h i r d period the sugar increases to 21.94 mg. The source of th i s additional sugar i s not certain but the apparent d i s -appearances of much of the c e l l w a l l s as diown i n plate (IX) suggests the p o s s i b i l i t y that some of the cellulose may be hydrolyzed to form sugars of some sort and thus serve as the source of t h i s increase i n sugar. Another possible source of the sugar i s the f a t s stored i n the c e l l s which may be broken down into sugars. In this series the sugar and starch reactions of the p:me are the inverse of each other. During the f i r s t period the starch and sugar both increased i n the hemlock (Figure 2, Tables 8 and 9). During the second period however a condition s i m i l a r to that i n the Pine i s found, that i s an increase i n the starch from 147.42 mg. to 187.2 mg. i s accompanied by a corres-ponding decrease i n the 45.34 mg. to 26.32 mg. During the last period both sugar and starch are shown to decrease although the rate of de-crease i s lessened f o r the sugar i n the t h i r d period i n comparison with the second. The behaviour of the sugar i n the hemlock i n the last period suggests a condition s i m i l a r to that found i n the pine but very much less - 40 marked and with p o s s i b i l i t i e s of s i m i l a r sources of the additional sugar. In the Spruce (Figure 3 and Tables 8 and 9) the sugar and starch are very c l e a r l y the inverse of each other. As the sugar increases from 23.4 mg. to 45.34 mg. i n the f i r s t period the starch decreases from 131.52 mg. to 51.48 mg. In the second period the reverse occurs and the starch increases to 87.75 mg* while the sugar decreases to 24.86 mg. During the t h i r d pariod the starch shows a very marked increase while the sugar although showing an actual decrease shows a sli g h t r e l a t i v e increase. In Douglas f i r the most marked r i s e and f a l l i n the sugar content takes place (Figure 4). During the rapid increase of the sugar from 34.85 mg. to 76.05 mg, shown i n tha f i r s t period the starch content re-mains p r a c t i c a l l y the saae. The rapid f a l l i n the sugar content during the second fumigation i s accompanied by a reduction of the starch from 131.62 mg. to 119.06 mg. which i s not at a 11 i n proportion with the sugar reduction. The results of analysis from fumigation Series 4 are tabulated i n Tables 10 and 11 and shown graphically i n figures 6 to 11. Table 10. Species Fumigation number 0 1 2 3 4 Pine 18.95 45.27 49.7 32.17 48.2 Hemlock 13.10 20.47 24.8 59.73 27.2 Sprues 14.62 17.55 26.32 36.5 26.32 Oedar 16.02 23.4 67.27 33.57 26.32 Douglas F i r 26.32 48.2 33.58 40.95 32.17 Numbers r e f e r to mg. of copper reduced. Table 11 —*- -*o " O O U Xil X « Die XUa Fumigation number Species 0 1 2 3 4 Pine 167 171.05 258.45 145.1 182.28 Hemlock .137.59 226.39 232.48 200 196.56 Spruce 193.8 231.46 205.9 142.9 107.54 Cedar 159.12 164.97 146.01 157.95 181.2 Douglas F i r 136.89 ; 117.93 150.9 164.7 163.8 As lias already been shown i n the discussion of Table 4 the specimen s i n fumigation Series 4 did not show such rapid injury as, did those i n Series 3. I t i s interesting to notice that the period of maximum sugar content i s also delayed in a l l except the Douglas f i r . (Figure I I ) . Cedar and pine show maximum sugar content at the second period and hem-lock and spruce at the t h i r d . The Douglas f i r , although i t does not show the greatest sugar increase i n this series i s the f i r s t to reach i t s maximum. In the pine the sugar shows an increase from 18.95 mg, to 45.27 ng. i n the f i r s t period while the starch shows a r e l a t i v e decrease although a s l i g h t actual increase was noticed. Daring the second period the rate of sugar increase i s very much less than i n the f i r s t while the starch shows a marked increase from 111.05 mg. to 258.45 ag. Both the sugar and starch decrease i n the thi r d period and increase i n the fourth, but the r e s u l t s of these periods may not be of much value, especially those from the fourth period since the trees were almost dead by t h i s time. - 42 -In the cedar, the analysis shows the expected results a f t e r the f i r s t period. During the second p e r i o d the sugar increases rapidly from 23.4 mg. to 67.27 mg. while the starch decreases from 164.97 mg. to 145. 01 mg. She. sugar shoWB a decrease from 67.27 mg. to 33.57 mg. i n the t h i r d period and a further decrease to 26.32 mg. i n the fourth. The starch on the other hand shows a s l i g h t increase i n the fourth p e r i o d from 157.95 mg. to 191.2 mg. (Figure 7). In the Hemloclc (Figure 8 and Tables 10 and 11) the sugar and starch content i s increased during the f i r s t two periods although the starch shows a greater inorease during the f i r s t period than does the sugar and a smaller increase than the sugar i n the second period. Daring the t h i r d and fourth periods the usual results a r e abtained. The increased sugar content from 24.8 mg. to 59.73 mg. i n the t h i r d period i s accompan-ied by a decrease i n the starch content from 232.48 mg. to 200 mg. while i n the fourt h period the opposite occurs when the starch shows a re l a t i v e increase while the sugar shows a decrease to 27.75 mg. (Figure 8). The sugar and starch both show an increase in the f i r s t period in the spruce. During the sedond and t h i r d periods the sugar shows actual increase from 17.55 mg. to 25.32 mg. and in the t h i r d period to 36.5. The starch shows a corresponding decrease from 231.46 mg. to 205.9 mg. i n the second fumigation and from 205.9 mg. to 142.9 mg. i n the t h i r d . During the fourth period the sugar shows a decrease from 36.5 mg. to 26,32 mg. while the rate of starch decrease i s s l i g h t l y l e s s . These re-sults suggest the inverse relations between starch and sugar found i n the other species. During the f i r s t period the starch and sugar both show an increase i n the Douglas f i r . (Figure 6 and Tables 10 and 11). During the second period decrease i n sugar content from 48.2 mg. to 38.58 mg. i s accom-panied by increased starch from 117.93 mg. to 150.9 mg. In the t h i r d fumigation the starch shows a r e l a t i v e decrease while the sugar increases to 40.95 mg. I t i s doubtful i f the r e s u l t s of the fourth period are valuable as stated i n the case of the pine sine© by this time many of the leaves had f a l l e n or were i n a condition to f a l l at the slightest touch or j a r r i n g . The results show the inverse r e l a t i o n between the starch and sugar to soma extent but i t i s not as d e f i n i t e i n the Douglas f i r as i n some of the other species. Results of analysis of specimens from fumigation Series 5. The only species used in this series was the Douglas f i r . The tree was i n the greenhouse long enough to become acclimatised but was not i n a r a p i d l y growing condition as were those i n Series 3. Injury effects i n t h i s case were much slower in showing themselves since the leaves were not lost even a f t e r four fumigations whereas i n Series 3 and 4 the Douglas f i r showed loss-fOf' leaves i n the second and t h i r d periods respectively. The r e s u l t s are shorn i n Table 12 and Figure 12. Table 12. Sugar and starch content of .5 gms. of leaves taken from Douglas f i r used i n fumigation Series 5. Material taken on the morning a f t e r the fumigation i n each case. Furoiga tion number ; o 1 • 2 3 4 Stegar 35.1 43.87 49.72 35.1 Starch 204.75 245.7 215.38 246.6 285.01 - 44 -numbers ref e r to mgs. of copper reduced. The climax i n sugar increase i s reached at the t h i r d fumigation i n this case which shews delayed effects since i n Series 5 and 4 Douglas f i r showed the climax at the f i r s t period of fumigation. In the second period the sugar increases from 35.1 mg. to 43.8 mg. and the starch de-creases from 245.7 mg. to 215.38 mg. while during the t h i r d period both increase. During the l a s t fumigation the r e s u l t s are the opposite of those found in the second, the sugar decreases from 43.72 mg. to 35.1 mg. and the starch r i s e s from 246.6 mg. to 285.01 mg. The usual inversed effect of the starch and sugar are observable i n t h i s series as i n those previously described although there i s a s l i g h t i r r e g u l a r i t y introduced i n the t h i r d period where both starch and sugar show increase. Discussion of Results. It i s notticeable i n each case recorded that there i s a very rapid and marked increase i n the sugar content of the leaves a f t e r one or two fumigations with one part of sulphur dioxide i n ten thousand. This con-d i t i o n suggests that one of the primary effects of the sulphur dioxide gas i s the a c t i v a t i o n of the enzyme re sponsible f o r the production of sugar. The period over which the activation i s effective varies with the condition under which fumigation i s carried out. The re s u l t s of fumig-ation Series 3 (Figure 5) show that the peak of a c t i v a t i on is reached a f t e r one fumigation i n a l l species. In Series 4 (Figure 11) the maximum was reached at the f i r s t fumigation i n the case of Douglas f i r , the second i n the case of cedar and pine and the t h i r d i n the case of Spruce and hemlock. I t i s c l e a r that in this series some factor causes a delay i n - 45 ~ FIGURE 6* Showing the r e l a t i o n o f sugar and starch i n Pine leaves during fumigation with 1 part Sulphur dioxide i n 10,000. .5 gm. of leaves from specimen used i n fumigation Series 4 were analysed i n each case. - 46 ~ FIGURE 7. Showing the r e l a t i o n of sugar and starch i n Cedar leaves during fumigation with 1 part of Sulphur dioxide i n 10„000. .5 gm. of leaves from specimen used in fumigation Series 4 were analysed i n each ca.se o FIGURE 8. Showing the r e l a t i o n of sugar and starch i n Hemlock leaves during fumigation with 1 part of sulphur dioxide i n 10,000. .5 gm. of leaves from specimen used i n fumigation Series 4 were analysed i n each case. FIGURE 9. Showing the r e l a t i o n of sugar and starch i n Spruce leaves during fumigation with 1 part of sulphur dioxide in 1^,000. .5 gm. of leaves from specimen used i n fumigation Series 4 were analysed i n each case. » 49 -FIGURE 10. Showing the r e l a t i o n of sugar and starch i n Douglas F i r leaves during fumigation with 1 part of sulphur dioxide in 10,000. x5 gm. of leaves" from specimen used i n fumigation Series 4 were analysed i n each case. FIGURE 11. Slowing the v a r i a t i o n i n the sugar content of leaves of Douglas F i r , Hemlock, Spruce, Pine, and Cedar during fumigation with 1 part of sulphur dioxide i n 10,000. z5 gm. of leaves from specimsns used in fumigation Series 4 were analysed i n each case. FIGURE 12. Showing the r e l a t i o n of sugar and starch i n Douglas F i r leaves during fumigation with 1 part of sulphur dioxide in 10,003. x5 gm. of leaves from specimens used i n fumigation series 5 were analysed i n each case. - 52 -the usual action. Once the maximum is reached there i s a sharp cirop i n the sugar content which suggests the p a r t i a l or complete destruction of the enzyra©. It has "been suggested previously that the delayed action of the sulphur dioxide was due to the physiological condition of the •fersae-at-• at the time of fumigation. In Series 3 the trees were i n a rapidly grow-ing condition owing to having been i n the greenhouse for a considerable time before being subjected to treatment, while in Series 4 they had been i n the greenhouse only a short time. Tables 9, 10, 11 and 12, show that i n f vunigation Series 4 the starch content of the leaves was n o t i c -eably greater than in those from Series 3 i n a l l cases except the Douglas 1 f i r . According to Haas and H a l l this condition i s correlated with the amount amount of diastase in the leaves for in their book on the chemis-t r y of plant products they make the following statement regarding diastase? nThe amount present i n any p a r t i c u l a r organ varies with the conditions obtaining; thus when the temperature and other factors are most favorable for growth and for the germination of starchy seeds, diastase i s much more abundant than when growth and germination are sluggish. Also, the amount of diastase i s always greater in starch leaves than i n sugar leaves, and the same holds for isolated leaves containing much starch, as compared with shaded leaves containing l i t t l e or no starch." If this correlation between starch content and diastase content holds good i t would suggest an explan-ation of the delayed injury observed i n fumigation series 4. These leaves showed a higher starch content and should, therefore, also have a higher diastase content than those of Series 3. It seems feasible that the 1. Haas, P. and H»11,T.G., An Introduction to the Chemistry of Plant Prod-ucts, Longmans, Green & Co., London 1913,pages 358 and 359. destruction of the smaller amount of diastase in the leaves of the trees used i n Series 3 would be more rapid than the destruction of the larger amount i n the leaves of the trees used i n Series 4. This theory seems to be born out by the action of Douglas f i r . The starch Gontent of t h i s species i n both series 3 and 4 i s almost the same, which suggests equal diastase content and should be accompanied by similar reaetions to s u l -phur dioxide. Examination of Figures 4 and 10 reveal that while the other species show delayed reaction i n Series 4, the Douglas f i r attains maximum sugar a f t e r the f i r s t fumigation i n both Series 3 and 4. In Series 5 however, Douglas f i r shows a much larger proportion of starch to sugar than i n the previous cases and the maximum sugar content i c not reached u n t i l the t h i r d fumigation period. Another point of inter-, est i s shown by the results obtained with spruce, hemlock,cedar and pine. The la s t two species named show a smaller starch increase than do the f i r s t two. Figure 11 shows that in the pine and cedar the maximum sugar is only delayed u n t i l the second fumigation while in the Spruce and hem-lock which show the greatest starch increase the maximum sugar i s de-layed u n t i l the t h i r d period. (b) Dessication Studies. Material and Methods. Material for these studies consisted of leaves taken from a Douglas f i r on the morning after fumigation in each case. Two experiments were carried out. In the f i r s t experiment approximately .5 gms. of leaves were f i n e l y cut up and accurately weighed in a orucible. On the second experiment branches which had shown the same rate of growth the -previous - 54 -fear were selected and one hundred loaves frora each taken f o r weighing. In each case was placed i n an oven which was kept at 100°C f o r four days and was then reweighed i n order to determine the amount of water lost from the leaves during drying. Results. The data obtained from these two experiments upon the dessication effects of sulphur dioxide are given tolow in Tables 15 and 14. Table 15. Results of experiment to investigate the dessicatlon effects of S,0g. Experiment 1. Fumigation Ho. V/t. of green l v s . Loss of wt. on drying $ loss i n wt. 0 •3550 gm. .2144 gm. 58.74$ 1 .5779 gm. .3347 gm. 57.39$ 2 .4754 gm. .2265 gm. 47.51$ 3' .4486 gm. .1886 gm. 42.04$ 4 .3600 gm. .1213 gm. 33.07$ Table 14. Results of experiment to investigate the dessication effects of S, 0^  Experiment 2. Fumigation Ho. Wt. of green l v s . Loss of Wt. on drying % loss i n V/t. © .6212 gm. .3790 gm. 61.01$ •1 .5959 gm. .3246 gm. 54.47$ 2 .6061 gm. .2790 gm. 46.03$ 3 .4716 gm. .1917 gm. 40.64$ - 55 -The re s u l t s for the two experiments, as given above, agrees i n showing a steady decrease i n the water content of the leaves as fumigation proceeds. According to the "mass law" l o s s of water i n the leaves should r e -s u l t i n reduction of sugar and increase i n starch but th i s does not actually occur u n t i l a f t e r the sugar has reached i t s maximum increase. These r e s u l t s suggest that there i s some f a c t o r involved which i n t e r -feres w i t h the usual working o f the "mass law". In the discussion of the results obtained from work on sugar and starch analysis i t i s suggested that the i n t e r f e r i n g f a c t o r i s the a c t i v a t i o n of an enzyme or enzymes. When t h i s a c t i v a t i o n ceases due to the destruction of the en-zyme or of the activator the "mass law" asserts i t s e l f and there i s an immediate increase i n the starch with corresponding decrease i n tha sugar i n the leaves (Figures 1 - 4 and 6 ~ 10'). S U M M A R Y 1. The species used f o r these experiments show d e f i n i t e injury when exposed to an atmosphere containing sulphur dioxide gas. 2. The order i n which the species show injury is as follows. Pseudotsuga t a x i f o l i a (Lambert) B r i t t o n . (Douglas F i r $ . Tsuga heterophylla, Sarg., (Western Hemlock) Picea sitchensis, Carr., (Setka Spruce ) Pinus monticola, Dougl., (Western Y/hite Pine) Thuja p l i c a t a , Donn., (Western Had Oedar) 3. Injury shows externally by greying of the foliage followed by bleaching and browning and i n sons cases by loss of leaves. 4. Environmental factors such as temperature affect the r a t e of injury. 5. I f one fumigation i s given d a i l y the resultant injury appears the same as i f several fumigations a r e given i n one day. 6. Injury shows i n t e r n a l l y by the reduction i n the s i z e of the starch grains followed by the gene r a l degeneration of the c e l l con-tents and the collapse of the c e l l w alls. 7. Physiological injury i s shown by the great increase i n the sugar content of the leaves followed by a very sudden decrease i n sugar a f t e r the maximum has been reached. 8. The rate of injury is related to the physiological condition of the trees, trees with a high sugar content i n comparison with the starch show more r a p i d injury than those i n which the reverse i s the case. 9. There i s a steady decrease i n the water content of the leaves dtTring fumigation. - 57 -SOME SUGGESTED PROBLEMS FOR  FURTHER STUDY ARI3IKG OUT OF THE ABOVE EXPERIMENTS. 1. While the starch grains continue to decrease i s size (Tables 6 and 7) the starch test shows an actual increase i n the starch content of the leaves (Tables 9 and 11). Again the sudden rise i n sugar content i s out of proportion to the decrease i n the size of the starch g r a i n s . The source of this increase i n starch and sugar has not been investigated and might prove a valuable l i n e of research. 2. Another interesting problem arises out of the correlation of the physiological condition of the tree and the rate at which i t shows injury. Involved i n t h i s question i s the suggested connection between the pro-portion of starch i n the leaves, and consequently of the amount of diast-ase present, with the rate of iasjury. 3. The method by which the sulphur dioxide enters the leaf i s another l i n e of investigation suggested. 4. The investigation of the a c t i v a t i o n and destruction of the enzymes i n the leaf would be another interesting study. 5. The general loss of colour in the leaves suggests that one of the f i r s t r e s u l t s of fumigation i s the p a r t i a l or complete destruction of the chlorophyll. Spectroscopical analysis of chlorophyll from fumigated leaves would be a f r u i t f u l subject for research. EXPLANATION OP PLATES PLATE 2. PLATE I I . PLATE I I I . PLATE 17. PLATE 7. PLATE 71. PLATE 71II. PLATE 71II. PLATE IX. PLATE A . PLATE S I . Cross section of a normal Douglas F i r l e a f . Cross section of a normal Hemlock le a f . Cross section of a normal Spruce lea f . (This material was very b r i t t l e and easily torn. The section shows the general arrange-ment of the t i s s u e and the massed condition of the chloroplasts.) Cross section of a normal Pine leaf. Cross section of a normal Cedar lea f . Cross section of a Douglas F i r leaf a f t e r two fumigations with one part of sulphur dioxide i n 10,000. Cross section of a leaf of Hemlock a f t e r two fumigations with 1 part of sulphur dioxide i n 10,000. Cross section of a leaf of Spruce after three fumigations with 1 part of sulphur dioxide in 10,000. Cross s e c t i o n of a leaf of Pine a f t e r two fumigations with 1 part of sulphur dioxide i n 10,000. Cross section of a leaf of Cedar a f t e r two fumigations w i t h 1 part of sulphur dioxide i n 10,000. Douglas F i r . (a) Part of a section of a normal leaf showing the arrangement of the starch grains and the nature of the upper epidermis. (b-c) Parts of a section of a leaf a f t e r two fumig-ations with 1 part of sulphur dioxide i n 10,000. (bJ Palisade c e l l s in which the contents have degenerated to form a granular mass, (c) Palisade and spongy mesophyll c e l l s showing the general d i s o r g a n i z a t i o n of the tissue.=The starch grains are re-duced i n s i z e and i n numbers. EXPLANATION OF PLATES (d) Section of a normal sterna. PLATE XII. Hemlock. (a-b) Parts of a section of a normal leaf. (a) Palisade and spongy mesophyll c e l l s showing the arrangements of the starch grains. The upper epi.demis is alsa shown. (b) Section of a normal stoma. (c) Part of a section of a leaf a f t e r two fumigations with 1 part of sulphur dioxide i n 10,000. Palisade c e l l s and spongy mesophyll c e l l s showing the reduc-t i o n i n the size and number of the starch grains. PLATE X I I I . Spruce. (d-g) Parts of a section of a leaf a f t e r two fumigations with 1 part of sulphur dioxide i n 10,000. (d) A mesophyll c e l l i n which the starch grains have p a r t l y disintegrated and have formed a network of material. (e) A mesophyll c e l l i n which the contents have degenerated into a granular mass. (f) A mesophyll c e l l i n which the starch grains are reduced in number and are scattered throughout the c e l l . PLATE XIV. Pine (a-b) Parts of a section of a normal leaf. (a) Part of a section showing the nature of the upper epidermis and the arrangement of the starch grains in the palisade and sp&ngy mesophyll c e l l s . (b) Section of a normal stoma. (o-d) Parts of a section of a leaf a f t e r two fumigations with 1 part of sulphur d i o x i d e i n 10,000. (c) Spongy mesophyll c e l l in which the contents have degenerated into a granular mass (d) Palisade and spongy mesophyll c e l l s showing the reduction and nature of the starch grains a f t e r fumigation. ESPLAIATOT OF PLATES PLATE XT". Cedar. {a - b) Parts of a section of a normal leaf. (a) Part of a section showing the arrangement of the s t a r c h g r a i n s i n the p a l i s a d e and spongy mesophyll c e l l s . The tws layered epidermis i s a l s o shown. (cj Part of a section of a l e a f a f t e r two fumigations w i t h 1 part of sulphur dioxide i n 10,000. Some o f the c e l l s show the reduction i n the number and size of the starch grains while i n others the contents have degener-ated into a granular mass. Magnification of Plates I to X - X 210 M a g n i f i c a t i o n of PlatSs XI to XV - X 190. PLATE II PLATE I ? PLATE VI PLATE 711I PLATE X. PLATE X I I PLATE XV Association of O f f i c i a l A g r i c u l t u r a l Chemists Bakke, A. L. BIBLIOGRAPHY O f f i c i a l and Provisional Methods of Analysis. U. S. Dept. of Agriculture Bureau of Chemistry, B u l l . No. 107 (revised!. Government P r i n t i n g Office s Washington, U. S. A. 1912. - The Effect of City smoke on Vegetation. A g r i c u l t u r a l Experiment Station, Iowa State College of Agriculture and the Mechanic Arts. Ames, Iowa. 1913 - The Effect of Smoke and Gases on Vegetation, Contribution of the Botanical Department, Iowa State College, Ames, Iowa. - P r a c t i c a l Physiological Chemistry, Seventh Edition, W. Heffer and Sons, Ltd., Cambridge, England. 1926. Crowther, C and RustonA.- The Nature, Di s t r i b u t i o n and Effects upon Vegetation of Atmospheric impurities in and near an i n d u s t r i a l Town. Journal of A g r i c u l t u r a l Science Vol. 4, University Press, Cambridge, England, 1911. Cole, S. W. Ebaugh, E. W. Frazer, P. Gases Vs. Solids * An investigation of the injurious ingredients of smelter smoke, Journal of the American Chemical Society, Vol. 29. The Chemical Publishing Co. Easton, Pa. 1907. Search f o r causes of Injury to Vegetation i n an Urban V i l l a near a large Industrial Establish-ment . Transactions of the American Institute of Mining Engineers, Vol. 38. Published by the Ins t i t u t e , New York, 1907. Haas,P. and Hill,T.G. Hamberiandt, G., Haywood, J . K., An Introduction to plant Chemistry. Green and Go. London, 1913. Longmans Physiological Plant Anatomy, McMillan and Co. Ltd., London, 1914. Injury to Vegetation by Smelter Fumes, Dept. of Agriculture, Bureau of Chemistry, B u l l 89. Government Printing Office, Washington, U. S. A. 1905. Injury to Vegetation and Animal l i f e by smelter wastes, U. S. Dept. of Agriculture, Bureau of Chemistry, B u l l . No. 113 (revised) Government Prin t i n g Office, Washington, U. S . A. Haywood, J . K.s Heald, P. G., Holmes J.,Franklin E.s and Gould, A. McLell'iuid. E. H. d u l l e r s M. 0. et a l , Oliver, F. W. Sorauer, P., Zimmerman, P. W., and Crocker, W. Smelter Smoke, Science Vol. 26. M c M i l l a n Co., New York, 1907 Manual of Plant Diseases, McGraw - H i l l Book Co. Inc., Hew York, 1926. Report of the Selby Smelter Commission U. S. Dept. of the Interior, Bureau of Mines, B u l l . 98., Gov. Pr i n t i n g Office Washington, U. S. A. 1915. Bibliography of Smoke and Smoke preventions University of Pittsburgh, Pittsburgh, Pa., Injury through Smoke and Flu Dust, U. S. Dept. of Agriculture Experiment Station Record, Vol. 25. Government P r i n t i n g Office, Washington, 1912. Urban Fogs. Journal of the Royal Hortic-u l t u r a l Society, Vol. 16. Spottiswold and Co., London. 1893. Manual of Plant Diseases, (Third Edition), Vol. 1, B e r l i n , Translated by F. Dor ranee, 1922. Sulphur Dioxide Injury to Plants. Procod-ings of the American Society for Hort-i c u l t u r a l Science. Published by the Society, Geneva, New York, 1931. 

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