jS 593 .89 Copy 1 THE EXTRACTION AND SATURATION OF 50IL5 WITH VOLATILE ANTISEPTICS A THESIS PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF CORNELL UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY JOHANNES PETRUS DU BUISSON Reprinted from Soil Science, Vol. Ill, No. 4, April, 1917 THE EXTRACTION AND SATURATION OF SOILS WITH VOLATILE ANTISEPTICS A THESIS PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF CORNELL UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY iBxnl*'**"' BY JOHANNES PETRU5 D\J BU15SON Reprinted from Soil Science, Vol. III. No. 4. April, 1917 b THE EXTRACTION AND SATURATION OF SOILS WITH VOLATILE ANTISEI'TICS' By j. p. du buisson Introduction Thai partial sterilization cjI soils is a factor to be considered in soil fertility has l>een demonstrated beyond doulit during the last thirty years. Partial sterilization may be effected either by heating the soil or by treat- ing it with a volatile, a non-volatile, or a solid antiseptic. However, heat- ing soil and treating it with volatile antiseptics are the two methods that have especially been studied by investigators up to the present time. The effect of heat on soil was first noticed by early bacteriologists. .Since then, this phenomenon has been studied variously and extensively. Many conflicting theories have been offered as a solution for the cause of these beneficial effects. Some have attributed them to a biological, others to a chemical or inechanical, change in the soil itself, and still others to all three factors combined. The present report is limited to the study of volatile antiseptics only. The object of this investigation was to determine, if i>ossible, whether there is any essential difference in the effect of saturation as cnmi)ared with extraction of different soil types with volatile antiseptics. With this in view, the effect of both saturation and extraction, on separate saiiiples of the same soil types, was studied, in so far as these substances influ- enced plant growth, ammonification, nitrification, and the total water- soluble salts in the soils used in the experiment. A comparison of the effects of saturation and extraction was made for the purpose of testing the theory, advanced by Greig-Smith (17), that the volatile antiseptics dissolve the "agricere" that covers the surfaces of the soil particles and by so doing enables higher plants and also bacteria to obtain more nutriment. Extraction with the antiseptics .should remove the '"agricere" more completely than mere saturation, and might, there- fore, be expected to produce a condition more favorable to both higher plants and bacteria. *A thesis submitted, to the faculty of the Graduate School of Cornell tJnivcrsity, in partial fulfilment of Ihc rcquiri'mcnt* for the dcRrec of Doctor of Philosophy. Received for publication \oveml>er 27. 1916. 354 SOIL SCIENCE I. Historical 1. Increase of Productivity due to Treatment of Soil with Volatile Antiseptics The first record of an antiseptic treatment of soil seems to be that of OberHn (37). After using carbon bisulfide as an insecticide in some of his vineyards tliat were attacked by Phylloxera, he noted a marked in- crease in the growth of the vines. Girard (15) found beneficial results from this antiseptic witli sugar beet. Pagnoul (38) observed the same phenomenon with buckwheat and with mustard. His observations with mustard are corroborated by Wagner (51). Mach (32) re- ported greater yields with beets, oats, and potatoes, after the appH- cation of 200 gm. of carbon bisulfide to a square meter of soil. Wollny (53) obtained increased results in pot experiments as a result of carbon bisulfide treatment. Koch (23) found increased growth vv'ith buckwheat and mustard and beneficial results in vine- yards. In 1911 the same author (24) reported the following relative green weights for buckwheat : untreated soil, 100 ; application of 200 c.c. ether, 153; of 500 c.c, 179. Hiltner and Stormer (20) obtained increased yields for buckwheat on soil treated with carbon bisulfide. Moritz and Scherpe (35) report an increased yield with potatoes and rye with carbon bisulfide. Nobbe and Richter (36), treating soil with ether, chloroform, carbon bisulfide and benzene, report the following relative dry v/eights: 118, 114, 122, 122, respectively, against 100 for untreated. Egorov (10) noticed higher yields of oats with carbon bisulfide treatment. Darbishire and Russell (9) found an increase in the yield of buckwheat and mustard on soil treated with chloroform;, carbon bisulfide, ether and toluene. Likewise there was an increased yield of turnips on soil with formalin as an antiseptic. Russell and Hutchinson (43) obtained greater yields with wheat and rye on toluene-treated soil than on mustard soil. Sherpe (45) reports an increased yield of rye on soil treated with carbon bisul- fide. Emmerrich, Leiningen and Loew (11) noticed a beneficial effect with carbon bisulfide on cane seedlings. Stone (48) similarly obtained better growth with lettuce after treating the soil with carbon bisulfide. Gainey (14), while experimenting with toluol, carbon bisulfide and chloroform, found increased yields with oats, wheat, and buckwrheat. It is evident from these results that tlie ability of partially sterilized soils to yield larger crops is a general one. Furthermore, it holds for all the soils studied and for the various volatile antiseptics examined. Ac- cording to Russell and Hutchinson (43) this seems to be true for all plants except for those of the leguminous order. 2. The Effect of Partial Sterilization on the Ammonifying and Nitrifying Power of the Soil Here will be considered some of the investigators who noted the DU BUISSON- EFFECT OF VOLATILE A\'TLSEPT1CS 355 effect of volatile antiseptic on the nitrifying and ammonifying power of the soil. The data are very conflicting on this point. Wagner (51) found a decrease in nitrification with carbon bisulfide treatment. Coleman (8) reported an immediate inhibiting eflfect on nitrate formation on soil treated with carbon bisulfide, but after a time found an increase. Lipman (27), on the other hand, reports a beneficial effect both for ammonification and nitrification. Stomier (49) and Scherpe (45) both observed an increase in ammonification but a detrimental effect on nitri- fication. The results of tliese two investigators are corroborated by the more extensive work done in England, especially by Russell and Hutch- inson (43), who found the production of ammonia after partial steriliza- tion at first slow, then rapid, and then more or less constant. Goodey (16), Hutchinson and MacLennan (21), Buddin (5) and others report similar results. Laidlaw and Price (26) noted that in partially sterilized soil more ammonia was produced but a cessation of the nitrifying process took place. On the other hand, Chaudon de Briailles (7), Pagnoul (38), Koch (24) and Fred (13) all report that partial sterilization enhanced the process of nitrification after a considerable duration of time. Notwith- standing the fact that the reported results are somewhat conflicting, there appears to be a pronounced indication that the volatile antiseptic treatment of the soil inhibits, at least for a time after the treatment, the nitrification process. This inhibition is then followed by a marked stimu- lation. 3. The Effect of Volatile Antiseptics upon the Total Water-Soluhle Salts in the Soil The literature on this subject is very meager. Mo.st of the investi- gators have studied the availability of plant-food due to the effect of partial sterilization with heat. Koch (23) does not believe that carbon bisulfide directly liberates plant nutrient elements, but states that as car- bon hisulfiide stimulates plant growth, it is reasonable to expect that more plant-food is removed from the soil. Darbishire and Russell (9) found tliat soil treated with carbon bisulfide was able to supply the plants with 75 per cent more phosphoric acid, 40 per cent more potash and 50 per cent more nitrogen dian the untreated soil. Using plants as indicators, Russell and Hutchinson (43) showed that more nitrogen, phosphorus and potassium were remnvcd from soil treated with carbon bisulfide than from the untreated soil. The partial sterilization of the soil by heat has been more extensively studied. Frank (12), Kruger and Schneidewind (25), Lyon and Bizzell (31), Pickering (40), and Stone (48) demonstrated that soluble plant and bacterial nutrients have been increased in partially sterilized soil with such treatment. As partial sterilization with volatile antiseptics is 356 W/i. SCIENCE analogous in many respects to sterilization with heat, it would seem that more plant nutrients should become available when the soil is treated than when untreated with such volatile antiseptics. 4. Methods of Treatiiig the Soil with Volatile Antiseptics and the F.jfcct upon Biological Processes Various methods have been enij)loyed for treating the soil with vola- tile antiseptics. The general method under field conditions seems to have been the introduction of the antiseptic by means of holes bored in the soil at regular intervals. Mach (32) added 200 gm. of carbon bisul- fide per square meter. Koch (23) applied 60 c.c. of carbon bisulfide and ether respectively, to 20 kg. of soil. Hiltner and Stormer (20) applied 516 gm. of carbon bisulfide to a sc(uare meter of soil as follows: three holes were made, each 30 cm. deep, in every square meter of soil; the carbon bisulfide was poured into the holes, which were immediately filled up with soil ; the plot of soil was afterward spaded over to insure equal distribution of the antiseptic; the >^oil was not seeded nor samples taken for biological determination, as the case might be, until all odor from the antiseptic had disappeared. Koch (24) and Fred (13) used similar methods for the different antiseptics, the latter working with pot experiments. Nobbe and Richter (36) applied to the first set of experiments 62 gm. of ether to each pot of 3600 gm. of soil. To the second set having the same ciuantity of soil and known as the etlier emulsion treatment, 300 c.c. of ether and 300 c.c. of water were added. The soils were then thoroughly mi.xed vxitli the antist-ptic. put into air-ticrhl boxes with a smaJl receptacle containing ether, for a given time, and then exposed to the free atmos- phere. In a third test, hydrogen peroxide was used. A 30 per cent solution diluted with 750 c.c. of water was applied to each pot of soil as above stated. The soil was thoroughly mixed and was then considered ready for the growth of plants. The authors also report experiments with ether, carbon bisulfide, chloroform and benzene where the method of treatment was identical as under the second set described above. Darbishire and Russell (9) added 25 c.c, respectively, of carbon bisulfide, chloroform, toluene, ether and benzene to 1 kg. of soil. The pots with soil were covered and allowed to stand for a period of one week. The soil was then spread open in a thin layer and the antiseptics allowed to evaporate. The evaporation took about 3 days. Russell and Hutchinson (43) employed a similar method but used only 2 c.c. of toluene per kilogram of soil where plants were to be grown. For the determination of the ammonifying and nitrifying power of the soil, 40 gm. of toluene were added to a receptacle holding 8(X) gm. of soil. A second series was treated similarly, but the toluene allowed to evaporate at the end of 3 days, while in the first series the antiseptic remained in the soil during tlie whole period of experimentation. DU BUfSSON—EFFECT OF VOLATILE ANTISEPTICS 357 5. The Relative Effect on Crop Yields of the Different Volatile Antisep- tics in the Partial Sterilization of Soils Though several investigators have studied different antiseptics, car- bon bisulfide has been used to a greater extent than any other. Koch (23) reports that better crop yields v^-ere obtained witli carbon bisulfide as an antiseptic than with ether under similar conditions. In 1911 the same author (24) pointed out an increase of crop yield witii increase of quantities of carbon bisulfide and ether used, but not necessarily propor- tional to the amount applied. The effectiveness again was in favor of carbon bisulfide. According to the crop yields reported by Nobbe and RiclUor (36), the order of effectiveness of the antiseptics used was as follows : carbon bisulfide, benzene, chloroform, and ether. It would seem from the rela- tive weights reported for buckwheat by Darbishire and Russell (9) that carbon bisulfide is better than chloroform and the latter superior to ether as an antiseptic. For mustard tlie following order was observed by the same authors: chloroform, benzene, carbon bisulfide, and toluene, with an average relative yield of 118 against 100 for the untreated soil. From the meager literature available it is apparent that some antiseptics are more effective than others. 6. Suggested Theories in Explanation of the Effect of the Partial Sterilisation on Soil Fertility A number of views have been advanced to explain the cause of the beneficial effect that higher plants and bacterial flora derive from partial sterilization of soils by volatile antiseptics. These will be discussed in the order of their priority. Koch (23) holds that partial sterilization has a direct stimulating effect upon the higher and lower forms of plant life in the soil. He is not die only investigator to produce data substantiating this veivv. Nobbe and Richter (36), Egorov (10), Fred (13) and others are nf the same opinion. Hiltner and Stormer (20) advanced the so-called indirect selective theory of the antiseptics as related to die growdi and activity of bacterial flora. They maintain that tlie harmful organisms are suppressed, whereas the beneficial bacteria are stimulated by the changes brought about in the soil as a result of such treatment. In 1909 Russell and Hutchinson (43) announced their protozoa theory. These investigators believe that the protozoa in the soil hold in check the multiplication of tlie bacteria, especially those of the ammoni- fying type. They hold that an antiseptic destroys most of the large organisms that prey on the bacteria. The latter, although temporarily suppressed by the antiseptic, are later able to multiply unhindered and so attain numbers greatly in excess of those found in normal soils. The (iii— 27^ 358 SOIL SCIENCE greater nimiber of bacteria is assumed to cause more plant nutrients and nutrient elements to be elaborated and, consequently, larger crop yields are produced. Fred (13), Sherman (47) and others, however, have given data showing that some protozoa are not so detrimental to bacteria as Russell and Hutchinson believe. I'revious to Russell and Hutchinson's work, the existence of protozoa in toil was reported by Celli and Fiocca (6), who isolated 6 species of amoebre. Other investigators in England who have reported the presence of protozoa in the soil are Goodey (16), Martin (33), and Martin and Lewin (34). In Germany, Hiltner (19), Tsujitani (50), Emmerich, Leiningen and Loew (11 ), Killer (22) and others have noted the same phenomenon. In the United States the presence of such organisms has been observed by Gjiiney (14), Lodge and Smith (28), Rahn (41), and Sherman (46, 47). Peck (39) has observed their presence in Hawaii soil. Loew (29, 30) reported their presence in the soil of the Alps, in Japan, the Island of Borkum and in Porto Rico. Bolley (1, 2, 3) considers the parasitic fungi in the soil as the chief cause, in many cases, of poor vegetative growth. The effect of the partial sterilization is to destroy or check the parasitic fungi and, consequently, allow the plant to grow unhampered. Greig-Smitli (17) has advanced the "agricere" theory. The agricere is considered to be a waxy substance, which covers, as it were, the soil particles. When the soil is treated with antiseptics this agricere is de- stroyed and conditions are rendered more favorable for the liberation of nutrients for plants and bacteria. Greig-Smith (17) and Bottomley (4) have proved the presence of bacterio-toxins in soil. Greig-Smith foimd them to a greater degree in poor than in rich soil. He claims that the toxins check bacterial activity in the soil. Summary of Literature 1. Treatment of soil witli volatile antiseptics has a definite beneficial effect on plants subsequently grown on such soil. 2. The ultimate effect of partial sterilization is an increased produc- tion of ammonia and nitrates. 3. Heat sterilization of soil liberates plant nutrients. As the treat- ment of soil with volatile antiseptics is somewhat similar in effect to heat sterilization, a like phenomenon may probably result. 4. In general, the application of volatile antiseptics to field soil is made in holes bored for that purpose. In greenhouse and laboratory experiments the antiseptic either is allowed to volatilize after treatment or is left in the soil. 5. Some volatile antiseptics seem to be more effective sterilizing agents than others. 6. Different theories as to the beneficial effects of volatile antiseptics mav be stated as follows : DU BUISSOX—EFI-ECT Oh VOLATILE ANTISEPTICS 359 a. Koch believes that antiseptics have a direct stimulating effect on plant and bacterial life. . b. Hiltner and Stormer consider the action as a disturbance of the equilibrium of the soil flora. c. Russell and Hutchinson attribute the beneficial effects of volatile antiseptics to the suppression of the soil protozoa, which are considered to hamper ammonification. d. Bolley considers tlie checking of certain harmful parasitic fungi in many cases as the real influence of partial sterilization. e. Greig-Smidi believes that the solution of certain waxy material in the soil by volatile antiseptics afi^ords conditions for a more ready avail- ability of plant nutrients. II. Conditions and Methods of Experimental Procedure The experimental data embodied in this report were derived from two sources: greenhouse studies and laboratory investigations. Both parts of the report were carried out in the Soil Technology Department of Cornell University, beginning with the spring of 1915 and continuing through the summer of 1916. 1. Pots. Four classes of receptacles were used for die vegetative part of the experiment. ' (a) Glazed crockery pots of yi-gallon cai^acity, AY' inches in diame- ter and 6 inches deep, holding 2^ kg. of Arf soil. (b) Small ordinary unglazed flower pots of 5(X)-gm. capacity, 5 inches in diameter and 5 inches deep. These were used for the ether- treated soils described under Experiment I. (c) Unglazed clay flower pots of J^-gallon capacity, 6 inches in diameter and 6]/^ inches deep. These pots were used in Experiment XI for the growth of the wheat crop, having been previously dipped into paraffin in order to cut down evaporation and dift'usion through the sides. (d) Glazed crockery pots of 2-gallon capacity', 8 inches in diameter and 6 inches deep, each holding about 5 kg. of soil. It was noticed that the root systems which developed in the smaller pots were too crowded, and to eliminate this imfavorable condition larger pots were employed, notwithstanding the fact that greater quantities of the volatile solvents were necessarv". 2. Soils. Dunkirk clay loam and Volusia silt loam soils were used. Both are typical soils in the vicinity of Cornell ITniversity. The Dunkirk clay loam was surface soil obtained from the experimental plots of Caldwell Field. The Volusia silt loam was surface soil from the Stevens farm on Turkey Hill. 360 SOIL SCIENCE Unpublished results of bulk analysis for Dunkirk clay loam soil are as follows : FROM 9 SAMPLES OF TOMPKINS COUNTY SOIL C (organic carbon) COa KaO CaO MgO NaaO N PA 1.670 1.740 0.430 0.450 1.090 0.186 0.123 0.440 0.260 2.110 O.S30 0.690 1.280 0.082 0.126 Unpublished results of bulk analysis for Volusia silt loam soil are as follows : FROM 11 S.\MPLES OF TOMPKINS COUNTY SOIL Surface % Subsoil % C (organ COa . . . c carbon) 1.960 trace 1.630 0.270 0.240 0.850 0.169 0.153 0.650 KaO 1.970 CaO 0.240 MgO 0.250 Na«0 0.960 N 0.086 PaOo 0.127 The soil in a majority of cases was obtained in bulk. After being allowed to dry, it was reworked in order to get rid of lumps and stones, the latter being especially numerous in the Volusia silt loam. The soil was then sieved through a 2-mm. sieve, except that used in Experiment I, which was passed through a 10-mm. sieve. 3. Treatment of Soils. In order that tlie discussion of the different experiments may be easier to follow, and to avoid repetition, a description will be given of the various solvents employed witli an explanation of the different terms used. The organic solvents utilized in this experimentation were alcohol, benzene, ether, commercial gasoline, and toluene. It will be seen from the experiments which follow that the alcohol treatment was applied to both ty^pes of soil on three different occasions. Ether, gasoline, and toluene, on the other hand, were each applied twice to Dunkirk clay loam soil as a treatment and only once to Volusia silt loam. Benzene was applied only in Experiment I to Dunkirk clay loam. The treatment in general consisted in extracting and saturating each type of soil with the individual solvents. (a) Extracted soil. By extraction it is to be understood that definite quantities of the solvent were applied to definite quantities of soil. The ratio was a variable one. In case of alcohol and ether, for example, the ratio was 3 DU BUISSOK— EFFECT OF VOLATILE ANTISEPTICS 361 of solvent to 1 of the soil. With gasoline, benzene, and toluene the ratio was 4 to 1 . The previously prepared soil was placed in a receptacle and the proper amount of solvent added. The soil was thoroughly stirred three times a day as long as the extraction lasted, which varied from 1 day for alcohol, to 8 days for gasoline. At the end of tlie extraction die solvent was siphoned off. The soil thus treated was then spread out on thick paper in a well ventilated dark room until, as far as could be deter- mined by the odor, the solvent had disappeared entirely. The time varied also in this case from 3 days for alcohol to 10 days for gasoline. Moisture determinations were then made and the soil was ready for experimenta- tion. (b) Saturated soil. Each soil was saturated with the respective solvents during the same period as in the extraction above described. The soil was then spread out and the solvent allowed to evaporate. The drying continued for tlie same length of time as for the extracted soil. Moisture determinations were then made and the soil was considered ready for use. (c) Untreated soil. The soils spoken of as untreated in ihis report are the respective soils, which were worked up in the same manner as the portions taken for the two treatments just described. The soil was spread out for the same period as those described under the treatments above, and after moisture determinations were made were ready for use. 4. Moisture Control. The soil used for the vegetative work in the greenhouse was kept at 30 per cent moisture. The incubated soil was kept at 25 per cent mois- ture. Both calculations were on the moisture-free basis. The former percentage was considered to be the optimum moisture for plant growth. The 25 per cent moisture was used for all the incubated soils on which tests were run for nitrification, ammonification and total water-soluble salts. This percentage was considered the optimum moisture content for organisms. 5. Incubation. To test the ammonifying and nitrifj'ing power of the soil, incubation tests were run. These were carried out by placing 100 gm. of air-dried soil in S-ounce bottles plugged with cotton. Incubation temperature was that of the laboratory. 6. Methods Used for Determining Nitrates. Ammonia and Total Sohihle Salts. The methods used for determining nitrates and ammonia were those described in Bulletin 31 of the Bureau of Soils of the United States Department of Agriculture. 7. General Plan of Experimentation. That the data may be presented in as clear a form as possible, a gen- eral plan will be presented first and discussed in detail afterwards. The 362 SOIL SCIENCE vegetative and laboratory parts of the work were divided up into the following separate experiments : Vegetative Experiments Experiment I. Oats, spring of 1915 (followed by buckwheat of Experiment IV, summer of 1915). Experiment II. Wheat, sunimer of 1915 (followed by oats of Ex- periment V, 1915-1916). 8. Diagrauiatic Flan of Experimentation'' Experiment I Direct effect of volatile antiseptic on oats. Experiment II Direct effect of volatile iitisepttc on xvheat. Experiment III Direct effect of volatile antiseptic on oats. Experiment IV Residual effect of volatile antiseptic on buckwheat after oats (Experiment I). Experiment ]'' Residual effect of volatile antiseptic on oats after wheat (Experiment II). Experiment VI Effect of water-soluble al- coholic extract from soil treated with volatile antisep- tic as in Experiment III upon oats in water cultures. Experiment VII Direct effect of volatile antiseptic on tjlie chemical condition of the soil treated with volatile antiseptic as in Experiment II. Experiment VIII Direct effect of volatile antiseptic on the chemical condition of the soil treated with volatile antiseptic as in Experiment III. Experiment IX Residual effect of volatile antiseptic upon the chemical condition of the soil after oats (of Experiment I) and buckwheat (Experiment IV). Experiment X Residua! effect of volatile antiseptic upon the chemical condition of the soil after wheat (Experiment ID and oats (Experiment V). Experiment XI Physical condition of the soil upon certain chemical factors. Soil of Experiment III studied directly after the oats. Experiment XI I The direct effect of volatile antiseptics upon development of acid in the soil. Soil treated exactly as in Experiments I, II and III. Experiment III. Oats, spring and summer, 1916. (Soil later studied chemically. See Experiment XI.) Experiment IV. Buckwheat following oats. (See Experiment I.) Experiment V. Oats following wheat. (See Experiment II.) Experiment VI. Water culture experiments. Laboratory Investigations Experiment VII. Soil incubated for 3, 6 and 12 weeks. The soil was treated with volatile antiseptics exactly as In Experiment II. 3 It is to he noted that Experiments I, II and III are comparable, also Experiments IV and V, Experiments VII and VIII. and Experiments IX and X. Experiments VI, XI and XII are each to be considered separately. DU BUISSOK— EFFECT OF VOLATILE ANTISEPTICS 363 Experiment VIII. Soil incubated for 2, 4 and 6 months. The soil received the same treatments with volatile antiseptics as in Experi- ment III. Experiment IX. Soil incubated and studied chemically after harvest- ing the buckwheat of Experiment IV. The buckwheat followed an oats crop (Experiment I). Experiment X. Soil studied chemically directly from [X>ts after har- vesting crop of oats (Experiment V) which followed a crop of wheat (Experiment II). Experiment XL Soil studied chemically from pots and simultane- ously incubated after harvesting a crop of oats (Experiment III). Experiment XII. The study of the development of acids in soils immediately after the treatment with alcohol as an antiseptic. III. Vegetative Experiments As outlined in tlie general plan, the exp>erimental part is subdivided into different experiments which will be discussed in their numerical order. Experiment I '^ The Effect of Volatile Antiseptics Applied to the Soil upon the Following Oat Crop Both Volusia silt loam and Dunkirk clay loam soils were extracted and saturated with 90 per cent alcohol in this experiment. Furthermore, benzene, ether, toluene and gasoline, respectively, were applied to Dun- kirk clay loam alone. After the soil was treated as already described, 2 kg. of soil were weighed out in duplicate, into the J^-gallon glazed crockery pots. In case of the ether-treated soils, however, only 500 gm. of soil were used in the small flower pots. The soils stood in the pots for 8 days before seeding. On April 13, 1915, all the pots were seeded with oats, 16 seeds to a pot. When the plants were 2 inches high, all were removed except eight of uniform size. At this time, 300 gm. of clean quartz sand were spread over the surface of the soil in each pot. This was to act as a mulch and decrease the amount of water lost by evaporation. The .seeds in the treated soil in most cases germinated a day, and in the case of gasoline from 2 to 3 days, later than the seeds in the untreated soil. This, however, was not the case with the seeds in the ether-treated soil, the germination period lieing the same as that of the seeds in the un- treated soil. During the first 4 to 6 weeks of growth, the plants in all the pots with treated soil (ether being an exception again) did not show the same ainount of growth as the plants in the untreated soil. The saturated soil maintained a slight advantage over the extracted soil. From this period on, however, there was a marked increase in growth of the plants on the treated soils, as can be seen from the dry weight of the plants (Table T), 364 SOIL SCIENCE which were harvested on July 21, 1915, after the crop had passed the blooming stage and was about to ripen. Conclusions Regarding Experiment I These results indicate a decided benefit to crop growth in favor of tlie treated soils. The saturated soils responded better to the treatment than extracted soils. This is shown not only by the average data but also by every individual treatment. The 90 per cent alcoholic antiseptic seems to have affected the crop growth most beneficially, ether next, then benzene, and finally, toluene. The gasoline antiseptic had a detrimental effect, the extracted treatment even more so than the saturated treatment. T.\BLE I RESULTS OBTAINED WITH OATS GROWN' ON SOILS PREVIOUSLY TREATED WITH VOLATILE ANTISEPTICS, EXPRESSED AS AVERAGE DRY WEIGHTS (Planted April 13, 1915: harvested July 21, 1915) Soil Volatile Untreated Saturated Soil Extract ed Soil Type Antiseptic Relative Relative Relative gm. Weights gm. Weights gm. Weights VSLi Alcohol 2.50 100 2.75 110 2.50 100 DCL= Alcohol 1.70 100 .V15 185 2.90 170 DCL Ether» 1.05 100 1.65 157 1.25 119 DCL Gasoline 1.75 100 1.65 94 1.30 74 DCL Toluene 1.95 100 2.35 121 2.20 113 DCL Benzene 2.05 100 2.65 129 2.30 112 Grand Average l.SO 100 2.40 133 2.07 115 ' Volusia silt loam. 2 Dunkirk clay loam. = With ther treatment o nly 6 plants were left ir each pot to mature. All of. the antiseptics lengthened the period for germination except ether, which treatment produced no variation from the untreated soil. Experiment II The Effect of J'olatile Antiseptics Applied to the Soil upon the Following Wheat Crop This experiment was begim in August, 1915. The organic solvents used were alcohol, ether and toluene on both Dunkirk clay loam and Volusia silt loam. Two-kg. flower pots were used into which were weighed 1800 gm. of alcohol and toluene-treated soil, respectively. In the case of the ether treatments. 1400 gm. of soil were u^^ed. On September 2. 1915, these pots were seeded with Galgalos wheat, 20 seeds to a pot. The young plants were later thinned to 10 plants of uniform size. The first important consideration here was the germinating power of the seeds in these differently treated soils. Invariably, the seeds in the imtreated soil germinated 2 or 3 days sooner than on the treated soil and showed for the first 3 weeks a distinctly better growth. This was es- DU BUISSOX— EFFECT OF VOLATILE ANTISEPTICS 365 pecially noticed with the alcohol and the toluene treatments. The ether did not show this inhibiting effect. As the growing period progressed, the plants on the treated soils gradually improved, and showed a healthier color and a more vigorous growth at the end of 5 weeks. On account of a bad attack of mildew, to which wheat is especially subject when grown in the greenhouse during the summer, this crop had to be harvested on October 17, thus growing for only 6 weeks. Although these results are not so reliable and conclusive as from plants grown to maturity, nevertheless the dry weights shown in Table II show a distinct tendency in favor of the treated soil. TABLE II RESULTS OBT..\IXED WITH WIIE.^T GROWN 0\ SOILS PREVIOUSLY TREATED WITH VOLATILE ANTISEPTICS, EXPRESSED AS AVERAGE GREEN AND DRY WEIGHTS (Planted Septe 3, 1915; harvested October 17, 1915) Untreated So 1 Sat uratcd S oil Extracted Soil Weigh of Dry Weight of Dry Weight of Drv Soil Volatile Matter Matter Matter Type Antiseptic Wt. of Rela- Wt. of Rela- Wt. of Rela- Green gin. tive Green gm. tive Green gm. tive Crop Wts. Crop Wts. Crop Wts. DCL Alcohol 3.40 0.80 100 6.15 1.35 169 6.65 1.45 181 VSL Alcohol 5.70 1.20 100 7.20 1.55 127 5.75 1.35 112 DCL Ether 4.60 0.95 100 7.55 1.37 144 9.35 1.65 173 VSL Ether 5.50 1.20 100 7.30 1.32 110 8.65 1.60 133 DCL Toluene 4.60 0.95 100 5.83 1.25 131 5.35 1.20 126 VSL Toluene 5.30 1.05 100 7.45 1.40 133 7.75 1.40 133 Grand Average 4.85 1.02 100 6.91 1.37 135.? 7.25 1.44 143 Conclusions Regarding Experiment II The crop in this experiment also responded with greater yields on the treated soils than on the untreated ones. There is a general tendency in favor of the extracted treatments, aldiough there are marked excep- tions diere, also. The order of effectiveness of the antiseptics according to the results are alcohol, ether and toluene, respectively. The Dunkirk clay loam in general responded better than Volusia silt loam, except for the ether treatment, where the reverse was true. Alcohol and toluene retarded germination, while ether seemed to have no influence, either stimulating or retarding. Experiment HI The Effect of Volatile Antiseptics Applied to the Soil vpon the Folloiifing Oat Crop In tlie previous experiment die largest pots used were 3^-gallon pots. The object in Experiment III was to study the various treatments on larger quantities of soil. Four kg. of soil were used in 2-gallon pots. The Dunkirk clay loam was stock soil which had been kept for 3 years 366 SOIL SCIENCE in the store-room. The Volusia silt loam was the same as previously described. Both types of soil were extracted with 70 per cent alcohol. The gaso- line treatments on both types of soil were in the ratio of 4 parts of gasoline to 1 part of soil. Five and 10 days were allowed, respectively, for the evaporation of the alcohol and gasoline. The soils, after standing for 6 days in the pots, were seeded to oats on March 9, 1916. Twenty-five seeds were planted to a pot. but only fifteen of the resulting plants were allowed to grow. The same phenomenon was observed as in previous experiments, to wit, that the treatment had a retarding effect for the first few weeks of growth. After this period, the plants on the alcohol-treated soil began to show a more rapid growth, extending until the harvest time, June 1, 1916. The plants were in full bloom at harvesting. TABLE in RESULTS OBTAINED WITH OATS GROWN ON SOILS PREVIOUSLY TREATED WITH VOLATILE ANTISEPTICS, EXPRESSED AS AVERAGE GREEN AND DRY WEIGHTS (Planted March 9, 1916; harvested June 1 and June 30, 1916) Untreated Soil Saturated Soil Extracted Soil Weight of Dry Weight of Dry Weight of Dry Soil Type Volatile Antiseptic Matter Matter Matter Wt of Rela- Wtof Rela- Wt. of Rela- Green gni. tive Green gm. tive Green gm. tive Crop Wts. Crop Wts. Crop Wts. DCL Alcohol 91.90 16.75 100 107.45 21.20 120 133.35 26.10 155 VSL Alcohol 61.25 10.50 100 100.40 19.70 187 91.75 17.90 170 DCL Gasoline 32.50 8.95 100 35.75 8.85 98 31.00 7.05 79 VSL Gasoline 72.05 22.55 MOO 66.60 17.60 78 71.15 23.10 102 Grand Avrrage 64.45 14.69 100 77.55 16.84 120.7 81.81 18.79 126.5 m the alcohol-treated soils were harvested on June not harvested until June 30. This is one factor I the untreated soil of the Volusia silt loam. The listure. The green weights do not show this great * It should be remembered that the crops fri 1, 1916, whereas the gasoline-treated soil was that accounts for the difference in weight fro kernels were riper and thus had very little m difference. The gasoline treatment, on the other hand, did not show up die dif- ferences as early as the alcohol treatment did. After 2 months' growth, the plants on the treated soils still continued poorer than those on the untreated soil. About 10 weeks after seeding, the plants began to increase in growth on the treated soil. It may be noted that on the Dunkirk clay loam, although all the 15 plants remained alive, only 9 plants grew to full height. It seems that for some reason the gasoline treatment had a retarding effect. Conclusions Regarding Experiment III A very marked increase in plant growth in favor of the alcohol treat- ment was noted in this test. The Volusia silt loam saturated yielded DU BUISSON— EFFECT OF I'OLATILE ANTISEPTICS 367 better than the extracted treatment. Witli the Dunkirk clay loam the extracted treatment gave tlie highest yield. The gasoline treatment, as in Experiment I, shows no beneficial eft'ect. In most cases it was detri- mental. Summary of Experiments I, II and III 1. The antiseptic treatment of soil in pots has a distinctly beneficial effect on the vegetative growth of succeeding oat and wheat crops. There is a slight advantage in favor of the saturated treatments. 2. Alcohol gave better results on plant growth than ether, benzene, toluene and gasoline, respectively. Gasoline is often harmful in its effects, both on ])lant growth and on germination. Ether seems to have little or no effect in either direction. 3. A stimulating influence of volatile antiseptics on plant growth occurs for both Dunkirk clay loam and Volusia silt loam, but in different degrees. Volusia silt loam, in general, responds the better. TABLE IV RESULTS OBTAINED WITH BUCKWHEAT GROWN ON SOILS PREVIOUSLY TREATED WITH VOLATILE ANTISEPTICS AND CROPPED TO OATS, EXPRESSED AS AVERAGE GREEN AND DRY WEIGHTS (Planted August 5, 1915; harvested October 2, 1915) Untreated Soil Saturated Soil Extracted Soil Weight of Dry Weight of Dry Weight of Dry Soil Volatile Matter M atter Matter Type Antiseptic Wt of Rela- Wtof Rela- Wt of Rela- Green gm. tive Green gm. tive Green gm. tive Crop Wts. Crop Wts. Crop Wts. DCL Alcohol 34.25 7.90 100 35.75 8.10 102 32.60 7.9 100 WSL Alcohol 24.50 6.30 100 35.00 7.40 117 33.50 6.5 103 DCL Benzene 34.00 8.20 100 40.50 9.30 101 40.00 9.3 113 DCL Toluene 34,50 8.40 100 44.00 10.40 123 45.50 10.0 119 DCL Gasoline 26.00 6.45 100 24.30 6.25 97 23.25 5.3 82 Grand Average 30.65 7.45 100 35.91 8.29 108 34.97 7.8 104 'ith the alcohol treatment, there was a set ot vere obtained: green weight of crop 15.5 gm. * In this experiment on the Volusia silt loam soil fresh from the field. The following results and dry weight 3.7 gm. Experiment IV The Effect of Volatile Antiseptics upon the Second Crop (Buckivheat) Grown after the Antiseptic Treatment of the Soil — Antiseptic: Alcohol, Benzene, Ga^soline and Toluene — Crops: Oats and Buckwheat The soil from Experiment I was taken from the pots, reworked, and the oat roots, as far as possible, removed. The soil was then replaced in each corresponding pot. On August 5, 1915, all the pots, except ether treatments, which were discarded, were seeded to buckwheat, 15 seeds to 368 SOIL SCIENCE a pot. The young plants were tliinned to 10, when they were 10 inches high. Observations were made of the growth of these plants. The seeds all germinated at tlie same time and the growtli continued uniformly imtil about 5 weeks after seeding, when the plants on some of the treated soils showed a slightly better growth, as can be observed from the dry weights of Table IV. The buck-,vheat was harvested on October 2, 1915. Most of the seeds were ripe and some even ready to fall. Conclusions Regarding Experiment IV A residual eiifect of the antiseptic treatment of soil on plant growth is brought out distinctly in this experiment. It is not so marked, how- ever, as was the direct influence shown in Experiments I, II and III. The saturated treatment again averages better than the extracted. In every case, except the benzene treatment, the saturated soil gave the higher yields. The gasoline treatment, although better on the saturated soil than on the extracted, gave in both cases lower yields than the un- treated. If preference is to be given for the effectiveness in increasing crop yields, toluene seems to be slightly more effective than the other ' antiseptics. Gasoline is distinctly the least efficient. The Volusia silt loam soil responded to the treatment better than Dunkirk clay loam, as expressed in crop growth. Experiment P r/ie Effect of J^olatile Antiseptics upon the Second Crop (Oats) Grown .■Ifter the Antiseptic Treatment of the Soil — Antiseptics: Alcohol, Ether and Toluene — Crops: Wheat and Oats The soil after the harvesting of the wheat crop of Experiment II was taken out of the flower pots, reworked and transferred to >^-gallon glazed earthenware jars. Three hundred gm. of clean quartz sand were added to serve as a mulch. The moisture content was then kept constant until December 15, 1915, when the pots were seeded with oats. The oats were sterilized (52) with calcium hypochlorite powder, 10 gm. in 140 c.c. of water. Out of 20 seeds planted only 12 plants were left growing. A notable effect here was that all of the plants on treated and untreated soils made the same progress in growth. This continued to be the case for the first two months of the experiment. After this period, however, the plants in the treated soil showed a distinct improvement over the plants in the untreated soil. Hardly any difference in growth was noticed between the saturated and the extracted soils. By the middle of March, ?> months after seeding, all the plants in the treated soil began to show a very healthy dark blue color, in contrast with those on the untreated pots, which did not have this vigorous appearance; By the beginning of April there was not only a distinct difference in growth between the different treatments, but a difference was also ob- DU BUISSOX-F.FFF.CT OF VOLATILE ANTISEPTICS 369 served between the different types of soil. The Volusia silt loam invari- ably showed a better growth than the Dunkirk clay loam. This fact becomes easily apparent by a study of the results in dry weights given in Table V. In order that a fair comparison should be obtained between tlie dift'erent treatments, it was thought best to harvest the oats at this stage, although they had just begun to head. RESULTS OBT.\I.\ED WITH 0.\TS GROWN' OX SOILS PREVIOUSLY TREATED WITH VOL.VTILE ANTISEPTICS A.ND CROPPED TO WHEAT, EXPRESSED IN AVERAGE GREEN .\ND DRY WEIGHTS (Planted December 15, 1915: harvested April 17, 1916) Untreated Soil Saturated Soil Extracted Soil Weight of Dry Weight of Urv Weight jf Dry Soil Volatile \ntiseptic Matter Matter Matter Wt. of Rela- Wt. of Rela- Wt of Rela- Green gni. tive Green gm. tive Green gni. tive Crop Wts. Crop Wts. Crop Wts. DCL Alcohol 8.90 2.35 100 29.05 6.45 274 30.65 6.15 261 VSL Alcohol 35.60 6.80 100 44.90 9.15 135 40.95 8.60 126 DCL Ether 8.85 2.05 100 23.20 4.85 236 23.65 4.45 217 VSL Ether 26.50 5.-)0 100 30.10 5.70 105 27.95 5.60 104 DCL Toluene 8.95 2.15 100 20.50 4.40 204 24.45 4.90 227 VSL Toluene 23.70 5.00 100 48.65 9.50 190 39.35 7.90 158 Grand Average 18.75 3.79 100 32.73 6.67 190.6 31.16 6.26 182 Conclusions Regarding E.\periment V The residual effects from the antiseptic treatments on crop growth are brought out more markedly in this experiment than in the previous one. The saturated treatment again gave higher results than the ex- tracted, except on the Dunkirk clay loam treated with toluene. The alccv- ho! shows the highest benefit as measured in crop growtli, with toluene next and ether last. There is only one exception to this order. The rela- tive crop weights indicate a better response for the Dunkirk clay loam soil. Summary of Experiments IV and V 1. ;\ residual effect of the antiseptic treatment of soil upon the sec- ond crop is distinctly brought out in these experiments. The advantage is generally in fa\or of the saturated treatment. 2. The relative influence of the different antiseptics as measured by yields indicates that alcohol is most effective, and gasoline, the least. 3. The Volusia silt loam in general excels Dunkirk clay loam in its response to volatile antiseptics, as measured by the yield of the second crop after the antiseptic treatment. "* 370 SOIL SCIENCE Experiment VI The Hfi'ect of the Alcoholic Extract of Soils upon Oats Groivn in Water Cultures The object in this experiment was to study the effect on plant growtli of the residue of the alcohohc extract obtained in Experiment III. For this purpose the alcoholic residue obtained from recovering the alcohol extract of both Dunkirk clay loam and Volusia silt loam was evaporated to dryness on water bath. It was then taken up three consecutive times with 70 per cent alcohol, digested and filtered each time. Finally the residue was taken up with distilled water previously treated with carbon black. The water-soluble portion of this alcoholic soluble material was then determined. From the Dunkirk clay loam soil 0.5 gm. was derived, and from the Volusia silt loam soil 1.1 gm., the alcoholic-soluble residue being, respectively 2 and 3.1 gm. This water-soluble residue was used in grow- ing oats in the following way. Half of this water filtrate of both types of soil was added in the proportions of 5, 50 and 100 parts per million, re- spectively, to a full nutrient solution of the following composition : COMPOSITION OF NUTRIENTS Ca(N03)a 2. 70 gm. MgSOj 0. 60 gm. KCl 0. 75 gm. KHaPOi 1 . 50 gm. FeSO, 0.05 gm. Distilled HjO lO-OO liters Full nutrient solutions served as checks to the above. The other half of the extract was added in the same proportions as above to carbon- black-treated distilled water. Distilled water solutions served as checks in this case. Oat seedlings were grown in these solutions for one month, all of the solutions being replaced once during this period. Four plants as uniform as it was possible to obtain were grown in each 8-ounce bottle. Conclusions Regarding Experiment VI From the data it seems that a soluble substance has been removed from the soil, which in water culture, is detrimental to plant growth. Its injurious effect in all cases with one exception was brought out more in the presence of ntttrients than with distilled water alone. The toxic effect was not apparent in the soil itself as has already been shown in previous e.xperiments ; if it were, its influence would have been noticed in the com- parison of the saturated and extracted treatments of Experiments I, II and III. As this was not the case, the results seem to substantiate the work of other investigators in their conclusion that a substance, toxic when in water culture, may not be toxic in the soil itself. DU BUISSON-EFFECT OF VOLATILE ANTISEPTICS 371 H Q E < y K < r- Z-. fe a a w i;; y.oi ft e within experimental error, it seems impossible to conceive that the development of acids by the action of the antiseptic could be an im]K)rtant factor in influencing plant and bacterial action, especially in the magnitude already described in the preceding experiments. V. .Summary 1. The application of volatile antiseptics to the soils used in tliis in- vestigation gave beneficial results on tlie crops subsequently grown thereon. 38g SOIL SCIENCE 2. A beneficial, residual effect is observed for the second crop after the application of the volatile antiseptics. This, however, was in all cases less marked than with tlie first cropping. Both types of soils re- sponded to treatment, but somewhat differently. 3. The volatile antiseptics experimented with had a definite effect upon tlie ammonification and nitrification of the soil, enhancing the for- mer and inhibiting the latter. There is a tendency for the volatile anti- septics to increase the water-soluble salts of the soil. 4. The effect of the antiseptics upon the ammonifying and nitrifying processes of the soils after two crops were grown seems to disappear. 5. No marked differences were observed as to plant growth and bio- logical activity between the saturation and extraction methods of apply- ing the volatile antiseptics to the soil. 6. In these experiments the physical condition of the soil as indicated by its ammonification and nitrification does not seem to be the cause of the influences noted upon plant growth and bacterial action. 7. By tlie extraction of soil with alcohol, a substance was removed which was toxic in water cultures but not at all toxic when in the soil itself. 8. The development of acids in the soil as a result of some action or change of the alcohol was found to be too slight to account for the marked effects of volatile antiseptics upon plant growth and bacterial action. Final Conclusions The beneficial influences obtained by treating the soil with volatile antiseptics can not be ascribed to a change in physical condition, to a suppression of some toxic material, or to a development of acids from the action of the antiseptics. The method of applying the antiseptics seems to have no marked influence upon the results obtained. The closely coordinated stimulation of plant and bacterial activity due to the treatment of the soil with volatile antiseptics points strongly towards a biological interpretation, with due regard for the chemical considerations, of the effects therefrom. 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In Vrtljsrhr. Bavcr. Landw. Rat., Bd. 3. No. 3, p. 319-342. PLATE I The effect of alcohol and gasoline treatments of Dunkirk clay loam on the growth of oats at the blooming stage. (For data see Table III.) Du P.iiissnii— l-tlr.i ni \,,laiil. AiniMi.ti ^n J:=^ /rv . ■' '} ^14^ IV-;. - oil Scii-iHc \(il. Ill, No. A Du Bui-isnn— KfffCt (if \nl;itilr AntiMi.ti.-- DLL. till i Ciilc d Alcohol Saturn led 1 \lcoh>! tZxtracted Fig. 1 ^ |i(«j«iis«|| ^^wK-o"! JVwiWito- DCL. DLL. I DLL. Unii^aicd j Ether Saturated j| Ether Extracted \.il. III. Xu 4 PLATE II The effect of alcohol and ether treatments of Dunkirk clay loam soil on the growth of oats just before heading. (For data see Table V.) LIBRflRY OF CONGRESS llllllllll'illll'll lil!'ll!''ll'i illlilll" "W 002 603 498 3 •