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A study of organo-mineral complexes in some gleysolic soils : their isolation and the mineralization… Hinds, Aston Alexander 1974

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A STUDY OF ORGANO-MINERAL COMPLEXES IN SOME GLEYSOLIC SOILS: THEIR ISOLATION AND THE MINERALIZATION OF NITROGEN, SULPHUR AND PHOSPHORUS by ASTON ALEXANDER HINDS B.Sc, University of the West Indies (St. Augustine), 1967 M.Sc, University of the West Indies (St. Augustine), 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of SOIL SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1974 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that publication, in part or in whole, or the copying of this thesis for financial gain shall not be allowed without my written permission. ASTON ALEXANDER HINDS Department of Soil Science The University of British Columbia Vancouver V6T1W5, Canada i i ABSTRACT A detailed study of the factors affecting the efficiency of ultrasonic dispersion of five Gleysolic soils by the Biosonik BP-III probe type ultrasonic vibrator was conducted. Various soil/water ratios, lengths of vibration time and intensity settings were investi-gated and i t was found that a soil suspension (1:10 or 1:5 soil/water ratio) not exceeding a total volume of 125 ml could be effectively dispersed by vibration for 20 minutes at a probe setting of 80. The efficiency of soil dispersion was reduced by 30-40 percent unless there was a "cooling off" period for the probe of 25-30 minutes between consecutive dispersions. The condition of the probe tip exerted a major influence on the effectiveness of dispersion and severe pitting which developed after about 70 hours of use, reduced the output of energy from the probe by nearly 70 percent. The heating effects of sonication was found to be a useful index of the energy output of the probe, and a technique for comparing the energy delivered to a suspension, which was equally effective and simpler than radiation pressure measurements, is described. A major finding which emerged from this study was that relatively large amounts of Fe, A l , S i , and C were solubilized during the vibration of soils in water, and that this effect could be suppressed by dispersing soils in a dilute electrolyte solution, notably 0.01 M CaCl 9. i i i A fractionation scheme for the isolation in bulk of organo-mineral complexes was developed based on ultrasonic dispersion Qf the soil and a system of continuous flow centrifugation. The method reduces considerably the time involved in obtaining various particle-size fractions, and gives quantitative separation of the size groups, (an average of 98 percent of the soil material was recovered). The amounts of C, N, S, and P in the soils and their particle-size fractions (2-50um, l-2um, 0.2-lum, and <0.2ym) were determined. There was an increase in the contents of C, N, S, and P and a narrow-ing of the C/N, C/S, and C/P(organic) ratios with decreasing particle size. An aerobic incubation technique was developed, based on a Factorial Experiment designed to determine the effects of moisture, sand, inoculum, and nutrients plus their interactions, on the mineraliz-ation of N, S, and P from organo-mineral complexes. The percent of organic C, N, and S in the particle-size separates of the five soils were found to be positively and significantly correlated with ammonium-N, nitrate-N, total-N, and sulphate mineralized during eight weeks of incubation, but not with phosphate. Total-P and organic-P were also positively correlated with N and S mineralized, but negatively correl-ated with phosphate. The percentage of total N, S, and P mineralized after six weeks of incubation ranged between 3.6-18.6 percent for N; 8.3-21 percent for S and 0.2-60.8 percent for P. The rate of mineralization as well as the percent of the total N, S, and P mineralized was generally highest in the <0.2um fractions. iv On the basis of the rates and amounts of N, S, and P mineraliz-ed from the particle-size fractions, (especially the <0.2um fraction) i t was concluded that the bonding of mineral and organic colloids in these soils was unlikely to be directly responsible for the observed biological stability of the organic moiety, however, the aggregation of such complexes could occlude relatively large amounts of organic matter thus rendering i t inaccessible to soil microbes. A colorimetric method for the determination of total and ammonium-N in soils and soil extracts was developed during this study, The method is based on the reaction of ammonia with phenol and hypochlorite in alkaline solution to form a blue complex with an absorbance maximum at 625 nm. The proposed method has the advantage over other standard methods of being quite simple yet precise and better suited to routine laboratory analysis. The distribution of exchangeable and "organically" bound Mn, Cu, and Zn, in the various soil size fractions was determined and their possible role in organo-mineral complexes discussed. V TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE 5 1. ISOLATION AND CHARACTERIZATION OF ORGANO-MINERAL COMPLEXES 5 1.1 Introduction 5 1.2 Properties of Clays and Organic Matter Which Influence the Formation of Complexes . . 6 1.2.1 Clay Minerals 6 1.2.2 Soil Organic Matter 8 1.3 Bonding Mechanisms • 10 1.3.1 Ionic Interactions • . 10 1.3.2 Hydrogen Bonding . . . . . 12 1.3.3 Ion-Dipole Interactions . 13 1.3.4 Van der Waals Forces 14 1.3.5 IT - Bonding 15 1.3.6 Covalent Bonding 15 2. STUDIES OF NATURALLY OCCURRING ORGANO-MINERAL COMPLEXES 17 2.1 Isolation of Complexes 18 2.1.1 Introduction 18 2.1.2 Methods of Soil Dispersion . . . . . . 20 2.1.2.1 Pre-Treatments and Dispers-ing Agents 20 2.1.2.2 Methods of Shaking and Mixing 20 vi Page 2.1.2.3 High Speed Stirrers . . . . . . 21 2.1.2.4 Air Jetting 22 2.1.2.5 Cation Exchange Resin Methods 23 2.1.3 Ultrasonic Dispersion Methods 24 2.1.3.1 Extraction of Organic Matter 24 2.1.3.2 Dispersion of Soil Suspensions and Minerals 26 2.1.3.3 Factors Affecting Dispersion. . 28 2.1.3.4 Effects of Ultrasonic Vibra-tion on Soil Components . . . . 31 2.3 Conclusion 37 THE RELATIONSHIP BETWEEN CARBON, NITROGEN, PHOSPHORUS, AND SULPHUR IN SOILS 39 3.1 Introduction 39 3.2 Incubation Methods in the Study of Complexes 42 3.3 Incubation Methods for Measuring Nitrogen Mineralization 43 3.3.1 Soil Nitrogen 44 3.3.2 Soil Particle-Size and Nitrogen Mineralization 49 3.3.3 Biological Stability of Organic Nitrogen 51 3.4 Organic Phosphorus in Soil and its Mineralization 55 3.4.1 The Nature of Organic Phosphorus and its Determination 55 3.4.2 Mineralization of Organic Phosphorus . . 58 vii Page 3.5 Organic Sulphur in Soil and its Mineraliz-ation 62 3.5.1 Organic Sulphur 62 3.5.2 Sulphur Mineralization 66 3.6 Conclusion 71 EXPERIMENTAL STUDIES 72 4. ULTRASONIC DISPERSION TECHNIQUES AND THEIR EFFECTS ON SOIL CONSTITUENTS 72 4.1 Objectives 72 4.2 Materials and Methods 72 4.3 Results and Discussion 78 4.4 Conclusion 115 5. INCUBATION STUDIES I . . . 117 5.1 Objectives 117 5.2 Materials and Methods 118 5.2.1 Selection of Factors and Factor Levels . 118 5.2.2 Incubation Method 122 5.3 Results and Discussion . . . . . . . 125 5.4 Conclusion 134 6. INCUBATION STUDIES II 135 6.1 Objectives 135 6.2 Materials and Methods 135 6.2.1 Selection of Extractant 136 6.2.2 Analytical Procedures 138 vi i i Page 6.3 Results and Discussion . . . . . . . . . . . . 139 6.4 Conclusion . . . . . 166 7. GENERAL SUMMARY AND CONCLUSIONS 168 REFERENCES 172 APPENDIX I - The Determination of Total and Ammonium Nitrogen in Soils by the Phenol-Hypochlorite Method 195 Introduction 195 Materials and Methods 199 Results and Discussion 202 APPENDIX II - Extractable Manganese, Copper, and Zinc in Soil Particle-Size Separates 205 Introduction 205 Materials and Methods 207 Results and Discussion . . . . 207 APPENDIX III - A Comparison of the Dissolution Effects of Shaking Soils in Water and Ultrasonic Dispersion (of Soils) in Different Solutions 211 APPENDIX IV - Ammonium-N, Nitrate-N, Sulphate and Phosphate Extracted (At Bi-Weekly Inter-vals) From Soils, During Eight Weeks of Incubation 217 APPENDICES REFERENCES 222 ix LIST OF TABLES Table Page 1. Characteristics of Soils 79 2. Effect of Ultrasonic Vibration on Soil pH 85 3. Effect of Time and S/W Ratio on Dispersion of Hazelwood Soil 87 4. Percent Clay Obtained by Dispersion of Soil in Different Solutions . . . . . 89 5. Correlation Coefficients (r) for Relationships Between the Peroxide-Calgon and Ultrasonic Methods of Soil Dispersion . . . . . 92 6. A Comparison of Different Dispersion Methods for the Particle - Size Analysis of Soils 94 7. Dissolution Effects of Ultrasonic Vibration of Soils in Water . 96 8. Dissolution of Iron by Dispersing Soils in Different Solutions 99 9. Dissolution of Aluminum by Dispersing Soils in Different Solutions 102 10. Dissolution of Silicon by Dispersing Soils in Different Solutions 103 11. Dissolution of Carbon by Dispersing Soils in Different Solutions 105 12. Recovery of Sand, S i l t and Clay by the Bulk Fractionation Scheme 114 13. Experimental Design 119 14. Treatment Designations 123 15. Some Characteristics of the Langley 126 16. Nitrogen, Phosphorus and Sulphur Extracted with 0.1 N CaCl0 128 X Table P a 9 e 17. Significant Treatment Effects and Interactions 130 18. Nitrogen, Phosphorus and Sulphur Extracted with 0.5 N NaHC03 pH 8.5 132 19. Significant Treatment Effects and Inter-actions 133 20. Comparison of 0.1 N CaClp, 0.5 N NaHCO- and 1 N KC1 as Extractants for mineralized N, P, and S . 137 21. Carbon, Nitrogen, Sulphur and Phosphorus in Soils and Particle-Size Separates 140 22. Ratios of C, N, P, and S in Soils and Particle-Size Separates 141 23. Nitrogen, Phosphorus and Sulphur Mineralized after 6 Weeks Incubation as a Percent of Total N, P and S in the Fraction 151 24. Nitrogen: Sulphur Ratio of Soils and Particle-Size Separates Compared to Ratio of N:S Mineralized at Six Weeks 158 25. Correlation Coefficients for Relationships Between C, N, P, and S 1 6 3 26. Correlation Coefficients for Relationships Between C, N, P, and S, and NH--N, N0 -^N, Total-N, Sulphate and Phosphate . . 7 1 6 4 xi LIST OF FIGURES Figure Page 1. Biosonic BP-III Ultrasonic Vibrator and Acrylic Plastic Reservoir for cooling samples during vibration 74 2. Heating effects of ultrasound treatment . . . . . . 80 3. Heating effects of ultrasound treatment . . . . . . 82 4. Comparison of percentage clay values obtained for ten horizon samples dispersed by ultrasonic (water) and chemical methods . 93 5. Comparison of percentage clay values obtained for ten horizon samples dispersed by ultrasonic (CaC^) and chemical methods 95 6. Fractionation scheme for bulk separation of organo-clay complexes . . . . 107 7. Sorvall RC2-B supercentrifuge with continuous flow apparatus 109 , 8. Cross-sectional view of the KSB system showing the flow of suspension through the system . . . . . I l l 9. Sharpies laboratory model supercentrifuge 112 10-12. Ammonium-N mineralized from particle-size separates during eight weeks incubation . . . . . . 144 13-15. Nitrate-N mineralized from particle-size separates during eight weeks incubation . . . . . . 148 16-17. Sulphur mineralized from particle-size separates during eight weeks of incubation . . . . . 156 18-19. Phosphorus mineralized from particle-size separates during eight weeks of incubation 160 xii ACKNOWLEDGEMENTS The author wishes to thank Dr. L.E. Lowe for his supervision throughout this study and Dr. L.M. Lavkulich for his helpful advice. Suggestions by Dr. Mary Barnes, Dr. T.H. Blackburn and Dr. C.A. Rowles are also gratefully acknowledged. Thanks are also due to Mr. B. von Spindler for the drafting of the figures and to Mrs Maryse El l is for typing the manuscript. This study was financed in part, by a grant from the National Research Council of Canada. 1 INTRODUCTION The importance of organo-mineral complexes in soils has long been recognized and voluminous literature has accumulated over the past thirty years on the nature of art i f ic ia l ly prepared organo-clays. The characteristics of naturally occurring organo-mineral complexes, however, are s t i l l imperfectly understood and there have been comparatively few studies on such materials. Jacks (1963) stated that, "the union of mineral and organic matter to form the organo-mineral complex is a synthesis as vital to the continuance of l i fe as, and less understood than, photosynthesis." This may well be so because, organo-mineral complexes play a very important role in the genesis of soils, from which most of man's food and fibre ultimately derive. Perhaps the major obstacle in the study of naturally occurring complexes is the difficulty involved in their isolation from the soil in an unaltered state. Recently, however, the application of ultra-sonic vibration techniques have contributed greatly to their isolation and has paved the way for a virtual "renaissance" in studies dealing with the isolation and characterization of organo-mineral complexes in soils. Despite the current widespread use of ultrasonics for soil dispersion, very rarely have different workers used the same set of operating conditions, thereby making direct comparison of results extremely diff icult . Also, there have been no difinitive studies of the effects of ultrasonic vibration on soil constituents. 2 It was therefore an objective of this investigation to determine the optimum conditions for the dispersion of a group of soils by a probe type ultrasonic vibrator; further, to develop a fractionation scheme for isolating various particle-size (P-S) fractions from such dispersed soils, which would effectively decrease the large number of hours required for P-S fractionation by methods currently in vogue. Because of the dearth of information concerning the dissolu-tion of soil constituents during dispersion by ultrasonics, a quantita-tive study of the solubilization of both mineral and organic matter was undertaken to determine the extent of such solubilization and to develop procedures for circumventing this problem where i t exists. It has been demonstrated that much of the organic matter in soils forms a part of the organo-mineral complex. However, this organic matter contains most of the soi l 's reservoir of nitrogen and sulphur and in many soils much of the phosphorus as well; hence i t must be assumed that most of nitrogen, sulphur and perhaps phosphorus, exist as part of the organo-mineral complex. In order to become available for plant nutrition organic-N, -S, and -P (in organo-mineral complexes) must be mineralized, and the development of an incubation technique for the study of the mineraliz-ation of N, P, and S from complexes was an important aspect of this work. It should be pointed out that there has been only one previous study of the mineralization of N from soil particle-size separates, (utilizing an anaerobic incubation technique) and none concerning S, or P mineralization. It was therefore necessary to develop an 3 aerobic incubation method capable of determining not only the release of NH -^N, but NO3-N, sulphate and phosphate as well. Such a method was devised, based on the results of a factorial experiment. Subsequently, a study of the extent to which organic -N, -S, and -P, in organo-mineral complexes (particle-size separates) were mineralized under optimal conditions during eight weeks of incubation was undertaken. A comparison was also made of the rates of mineraliz-ation of N, S, and P in the various size fractions and the soil as a whole. During this investigation a colorimetric method for the determination of ammonia in solution, which had not previously been used in soil studies, was discovered. The method was adapted to the determination of total and ammonium -N is soils and soil extracts. It was found to be highly sensitive and precise. It was also more rapid than the standard micro or semi-micro Kjeldahl procedures, and thus better suited to routine analysis. Certain micronutrient elements (viz. Cu, Zn, Mn) are believed to occur in organo-mineral complexes either as bridging cations or as organo-metallic complexes. There is , however, very l i t t l e information concerning the distribution of these elements in soil particle-size separates, therefore, a study of the distribution of these elements in soil size-fractions was conducted; the results appear in an appendix. The five soils used throughout this investigation belonged to the Gleysolic Order (Canadian System of Soil Classification), and included a representative of each of the great-groups. These soils were selected because, apart from their agricultural importance, 4 they are of world-wide distribution; also there is significantly less information available about these soils, compared to other soil orders. 5 R E V I E W OF L I T E R A T U R E 1. ISOLATION AND CHARACTERIZATION OF ORGANO-MINERAL COMPLEXES 1.1 Introduction It has been recognized for many years that the organic and inorganic colloids in soils interact with each other, and that a "clay-organic complex" is an important constituent of most soils, [Greenland, 1971]. Just how important such complexes are in soils was emphasized in a review by Greenland [1965a] that indicated the large proportions of the total organic matter in numerous types of soils existing as part of the clay-organic complex. The importance of clay-organic complexes was further highlighted by McLaren and Peterson [1965] who stated that: "Complexes between clays and organic materials are of importance from many points of view, . . . and much of the mystery that is soil remains the mystery of the true nature of these complexes." The experimental approaches that have so far been used in the study of complexes are: 1. Studies of the reactions of organic compounds of known structures with pure clay minerals (for historical review see Grim, 1968; also Mortland, 1970). 6 2. Investigations of reactions between organic matter extracted from soils and purified clay mineral. 3. Attempts to isolate and characterize naturally occurring complexes from soils by physico-chemical means. Of the three approaches mentioned the f i r s t two have received by far the most attention, especially the f i r s t , and much of our understanding of the reaction mechanisms between mineral and organic substances derives from these studies. 1.2 Properties of Clays and Organic Matter which Influence the  Formation of Complexes Current understanding of the modes of bonding between clays and organic matter in soils i s based largely upon intensive investi-gations within the past few decades into the structural and surface chemistry of clay minerals, and parallel studies in the chemistry of humus substances. 1.2.1 Clay Minerals In the last forty years X-ray diffraction and electron micro-scopy studies of clays have been greatly developed, and with the advent of differential thermal analysis (DTA) and infra-red spectro-scopy (IR), a powerful set of tools were placed at the disposal of researchers. This has led to major advances in the understanding of clay minerals and associated amorphous materials. Nevertheless, there is s t i l l considerable controversy over the classification, 7 nomenclature, and indeed, certain structures of clay minerals -- a situation in part due to the diverse fields of those engaged in their study. Three properties of clay minerals assume special importance in the adsorption of organic compounds onto their surface. First, exchangeable metal cations on a clay surface are hydrated to a degree determined by the size of the cation and its charge. Successive hydration shells of water molecules are oriented with the negative end of their dipoles towards the cation to give a hydrogen-bonded network extending out from the silicate surface [Anderson, Brown and Buol, 1967]. This must be disrupted before polar organic molecules can compete with water and co-ordinate to the cation. Second, smectites are able to expand in a direction perpen-dicular to the sil icate sheets allowing molecules to adsorb in the inter-layer spaces. Such changes in basal spacing can be determined from X-ray diffraction data. Finally, sil icate surfaces are not f lat , as often depicted, but consist of octahedral "holes" in the tetrahedral silicate sheets. Thus, the possibility arises of adsorbed molecules "keying" into the pyramidal surface like eggs in an egg-box. The clay fraction of a soil is normally defined as the mineral material <2um in effective spherical diameter. Commonly i t contains not only clay minerals, but also some primary minerals and amorphous oxides or hydroxides, particularly of Fe and Al , which may be relatively discreet or present in combination with the clay minerals. Further, although one clay mineral species may predominate, soils usually contain several others and all can vary in structure from true 8 c r y s t a l l i n e forms t o h i g h l y amorphous m i x t u r e s . A l s o , e l e c t r o n m i c r o -scopy e l e c t r o n m i c r o - p r o b e a n a l y s e s have shown t h a t v o i d s u r f a c e s may have v e r y d i f f e r e n t p r o p e r t i e s from t h e s o i l c l a y m a t r i x . Thus, extreme c a r e must be e x e r c i s e d when r e s u l t s o b t a i n e d w i t h pure c l a y m i n e r a l s ; a r e e x t r a p o l a t e d t o s o i l s y s t e m s . 1.2.2 S o i l O r g a n i c M a t t e r The o r g a n i c m a t t e r o f s o i l s c o n s i s t s o f a m i x t u r e o f p l a n t and animal p r o d u c t s i n v a r i o u s s t a g e s o f d e c o m p o s i t i o n ; o f s u b s t a n c e s s y n t h e s i z e d b i o l o g i c a l l y and/or c h e m i c a l l y from t h e breakdown p r o d u c t s , and o f m i c r o o r g a n i s m s and s m a l l a n i m a l s and t h e i r decomposing r e m a i n s . To s i m p l i f y t h i s v e r y complex system, o r g a n i c m a t t e r i s u s u a l l y d i v i d e d i n t o two g r o u p s : (a) non-humic s u b s t a n c e s and (b) humic s u b s t a n c e s , [ S c h n i t z e r and Khan, 1972]. Non-humic s u b s t a n c e s i n c l u d e compounds t h a t e x h i b i t s t i l l r e c o g n i z a b l e c h e m i c a l c h a r a c t e r i s t i c s . I n c l u d e d i n t h i s c l a s s o f compounds a r e c a r b o h y d r a t e s , p r o t e i n s , p e p t i d e s , amino a c i d s , f a t s , waxes, r e s i n s , pigments and o t h e r l o w - m o l e c u l a r w e i g h t o r g a n i c sub-s t a n c e s . These compounds a r e g e n e r a l l y v e r y s u s c e p t i b l e t o m i c r o b i a l d e g r a d a t i o n and have a r e l a t i v e l y s h o r t s u r v i v a l t i m e . The b u l k o f the o r g a n i c m a t t e r i n most s o i l s c o n s i s t s o f humic s u b s t a n c e s . These a r e amorphous brown t o b l a c k , h y d r o p h i l i c , a c i d i c , p o l y d i s p e r s e s u b s t a n c e s o f m o l e c u l a r w e i g h t s r a n g i n g from s e v e r a l hundreds t o t e n s o f t h o u s a n d s , [ F e l b e c k , 1965]. Based on d i f f e r e n t i a l s o l u b i l i t y i n a l k a l i and a c i d , humic s u b s t a n c e s a r e 9 u s u a l l y d i v i d e d i n t o t h r e e main f r a c t i o n s : (a) humic a c i d (HA), (b) f u l v i c a c i d ( F A ) , and ( c ) humin. T h i s l a t t e r f r a c t i o n i s n e i t h e r s o l u b l e i n a l k a l i nor a c i d , however, t h e r e i s i n c r e a s i n g e v i d e n c e t h a t i t s c h e m i c a l s t r u c t u r e and p r o p e r t i e s a r e s i m i l a r t o t h o s e o f HA, and t h a t i t s i n s o l u b i l i t y a r i s e s from the f i r m n e s s w i t h which i t combines w i t h i n o r g a n i c s o i l c o n s t i t u e n t s . Data a v a i l a b l e , s u g g e s t t h a t s t r u c t u r a l l y t h e t h r e e humic f r a c t i o n s a r e s i m i l a r t o one a n o t h e r , but t h a t they d i f f e r i n m o l e c u l a r w e i g h t , u l t i m a t e a n a l y s i s and f u n c t i o n a l group c o n t e n t . The FA f r a c t i o n has a lower m o l e c u l a r w e i g h t but h i g h e r c o n t e n t o f o x y g e n - c o n t a i n i n g f u n c t i o n a l groups per u n i t w e i g h t than HA and the humin f r a c t i o n . The i n f o r m a t i o n c u r r e n t l y a v a i l a b l e on the s t r u c t u r a l and ch e m i c a l c h a r a c t e r i s t i c s o f HA have been c o l l a t e d by F e l b e c k [1971] and Haworth [1971]. F e l b e c k [1971] c o n c l u d e d t h a t a p p r o x i m a t e l y 50 t o 55 p e r c e n t o f the t o t a l humic m o l e c u l e has been i d e n t i f i e d as b e i n g made up o f the f o l l o w i n g f r a g m e n t s : amino a c i d s .(10%), hexosamines ( 2 . 5 % ) , p o l y c y c l i c a r o m a t i c s ( 1 0 % ) , and oxygen c o n t a i n i n g f u n c t i o n a l groups ( 2 6 % ) . The r e m a i n i n g unknown f r a c t i o n appears t o c o n s i s t o f e a s i l y o x i d i z e d h e t e r o c y c l i c compounds. A breakdown o f the oxygen-c o n t a i n i n g f u n c t i o n a l groups i n d i c a t e d t h a t t h e c a r b o x y l a t e , c a r b o n y l , and a l c o h o l i c groups (both p h e n o l i c and a l c o h o l i c ) a c c o u n t f o r n i n e t y -f o u r p e r c e n t o f the t o t a l / , t h e s e groups a r e a l l i n v o l v e d i n the v a r i o u s b o n d i ng mechanisms which have been d e m o n s t r a t e d . Based on e l e c t r o n s p i n r e s o n a n c e (ESR) d a t a , Haworth [1971] s t a t e d t h a t HA c o n t a i n s o r r e a d i l y g i v e s r i s e t o a complex a r o m a t i c c o r e , t o which a r e a t t a c h e d c h e m i c a l l y o r p h y s i c a l l y (a) p o l y s a c c h a r i d e s , (b) p r o t e i n s , ( c ) s i m p l e p h e n o l s , and (d) m e t a l s . 10 It is clear from the foregoing that with only 50 percent of the HA fraction characterized (and this is s t i l l open to question) much remains to be uncovered about such substances before a clear understand-ing of the nature of organo-mineral complexes can be achieved. 1.3 Bonding Mechanisms When an organic molecule is adsorbed onto a clay surface any changes in strength of its individual bonds result in changes in the frequency at which infra-red radiation is absorbed. Thus, using IR spectroscopy in conjunction with X-ray and chemical studies, mechanisms ranging from strong covalent bonds to weak van der Waals attraction have been observed. Mechanisms governing reactions between clays and organic compounds have recently been comprehensively reviewed by Mortland [1970] and Greenland [1971], however, because of the relevance of the modes of bonding between mineral and organic matter, to the present discussion, they will be described herein. 1.3.1 Ionic Interactions Greenland [1971] emphasized the roles of iron and aluminum at the clay surfaces which readily form polyhydroxy complexes, with which organic substances associate. Since positive sites normally exist on aluminum and iron hydroxides, at least below pH8, organic anions can be associated with these charges by Coulombic attraction. The adsorption of the organic anion is readily reversed by exchange with chloride or nitrate. The organic anion can also be displaced by 11 raising the pH to 8 or 9 when the positive charge of the hydroxide is neutralized. This type of interaction has been termed, anion exchange. Another type of ionic interaction has been described as a "ligand exchange" reaction by Hingston ejt a]_. [1967, 1968]; Greenland [1971]. The anion involved penetrates the co-ordination shell of an iron or aluminum atom in the surface of the hydroxide and becomes incorporated into the surface hydroxyl layer. The anion cannot be displaced by leaching with chloride, and is sensitive to pH but not to electrolyte concentration. An adsorption.maximum or an inflection in the adsorption-pH curve occurs at or near the pH corresponding to the pk value of the acid species of the anion. Cation exchange reactions at clay surfaces involving organic cations have also been widely investigated [Mortland, 1970]. For example, at low pH, amines are N-protonated and may be adsorbed by cation exchange [Theng, Greenland and Quirk, 1967]: CLAY - M+ + RN+H3 • CLAY - H+H^R + M+ Protonation of already adsorbed species at the clay surface is also possible, hydrogen being derived from several possible sources; viz. exchangeable H+ ions, a proton transferred from another cation already adsorbed on the clay, or from partially dissociated water in associ-ation with a highly electronegative cation. Where there is excess base two or more molecules may compete on an equal basis for the same proton. If two molecules are involved a "hemisalt1 is formed: 12 [ BASE H BASE ] + 1.3.2 Hydrogen Bonding Several workers have observed that the increased basal spacing of montmorillonite is slightly less than the calculated minimum thickness of a monolayer of adsorbed organic molecules. This has been attributed to [C-H • • • • O-Si] hydrogen bonds between methylene protons of the organic molecule and oxygens of the sil icate surface [Bradley, 1945; MacEwan, 1948; and Talibudeen, 1955]. However, Greenland [1965a] and Theng et al_. [1967] point out that uncertainties in van der Waals radii of the atoms involved and keying into the sil icate surface [Glaeser, 1951; Greenland et al_., 1962] may account for the phenomenon and IR spectroscopy failed to give unequivocal evidence of direct hydrogen bonding. On the other hand, hydrogen bonding of organic molecules to other organic molecules already adsorbed on the clay surface, for example: + / N R 1 R 2 CLAY - N - H • • • • 0 = C has been observed for a number of systems using IR spectroscopy. Mortland 1968b; Doner and Portland, 1969]. Lailach and Brindley [1969] have suggested co-adsorption of purines and pyrimidines onto montmorillonite as a hydrogen bonded complex on the basis of X-ray diffraction data. For example, thymine was only adsorbed in the presence of adenine, hypoxanthine or 2, 6-diaminopurine. 13 A c c o r d i n g t o M o r t l a n d [1970] hydrogen bonding i s an e x t r e m e l y i m p o r t a n t bonding p r o c e s s p a r t i c u l a r l y i n l a r g e m o l e c u l e s o r polymers where a d d i t i v e bonds o f t h i s t y p e combined w i t h h i g h m o l e c u l a r w e i g h t s may produce r e l a t i v e l y s t a b l e complexes. Of p a r t i c u l a r i m p o r t a n c e a r e the f o r m a t i o n o f "water b r i d g e s " l i n k i n g p o l a r o r g a n i c m o l e c u l e s t o e x c h a n g e a b l e metal c a t i o n s t h r o u g h a water m o l e c u l e i n the p r i m a r y h y d r a t i o n s h e l l i n the f o l l o w i n g manner: OH n+ 1 CLAY - M n • • • 0 - H 0 = C i R where M n + i s the metal c a t i o n and RC00H the o r g a n i c m o l e c u l e . T h i s t y p e o f bond i s most l i k e l y where t h e c a t i o n has a h i g h s o l v a t i o n energy and so r e t a i n s i t s p r i m a r y h y d r a t i o n s h e l l . I t i s a l s o l i k e l y t h a t f o r r e l a t i v e l y s i m p l e s o i l o r g a n i c compounds such as f r e e amino a c i d s , amino sug a r s , mono- and d i - s a c c h a r i d e s and a l i p h a t i c a c i d s con-t a i n i n g d i s s o c i a t e d -C00H, -OH, C = 0 and -NH2 g r o u p s , hydrogen bonding c o u l d a l s o be o f i m p o r t a n c e i n both o r g a n i c - i n o r g a n i c as w e l l as o r g a n i c - o r g a n i c i n t e r a c t i o n s . 1.3.3 I o n - D i p o l e I n t e r a c t i o n s When the s o l v a t i o n energy o f t h e e x c h a n g e a b l e c a t i o n i s low, i n s t e a d o f hydrogen bonding v i a a water b r i d g e , d i r e c t c o - o r d i n a t i o n o f a p o l a r m o l e c u l e may o c c u r [ P a r f i t t and M o r t l a n d , 1968]: CLAY - Na 0 = C \ 14 P e s t i c i d e . h y d r o l y s i s on c l a y s u r f a c e s i s c a t a l y z e d by C u ( I I ) a f t e r c o - o r d i n a t i o n through N and S groups [ M o r t l a n d and Raman, 1967] and i t has been p o i n t e d o u t [ Y a r i v je_t a j _ . , 1966] t h a t the c u p r i c i o n has a tendency t o form s t r o n g c o - o r d i n a t e bonds w i t h N. M o r t l a n d [1970] s u g g e s t e d t h a t i o n - d i p o l e a t t r a c t i o n s assume g r e a t e r i m p o r t a n c e i n anhydrous c o n d i t i o n s when t h e r e i s l e s s c o m p e t i t i o n from water m o l e c u l e s . For example, M o r t l a n d and M e g g i t t [1966] found t h a t EPTC ( e t h y l N, N - d i - n - p r o p y l t h i o l c a r b a m a t e ) a d s o r b e d on montmor-i l l o n i t e was u n a f f e c t e d by a t m o s p h e r i c h u m i d i t y but q u a n t i t a t i v e l y r e l e a s e d when immersed i n water. T h i s work i s i m p o r t a n t because i t shows t h a t the water c o n t e n t o f a c l a y - o r g a n i c system can be a l i m i t i n g f a c t o r i n complex f o r m a t i o n . 1.3.4 van der Waals F o r c e s A l t h o u g h van d e r Waals f o r c e s a r e v e r y weak, t h e y become i m p o r t a n t when l a r g e m o l e c u l e s a r e a d s o r b e d onto a c l a y s u r f a c e w i t h many p o i n t s o f c o n t a c t . Such a d s o r p t i o n i s accompanied by a l a r g e e n t r o p y i n c r e a s e due t o d e s o r p t i o n o f a l a r g e number o f s m a l l s o l v e n t m o l e c u l e s . Some workers have s u g g e s t e d t h a t i f m o l e c u l e s w i t h l o n g a l k y ! c h a i n s a r e p r e s e n t i n h i g h c o n c e n t r a t i o n they a r e s t a c k e d a t an a n g l e t o the c l a y s u r f a c e and a t t r a c t i o n s between the c h a i n s assume more importance than t h o s e w i t h the c l a y s u r f a c e [ J o r d a n , Hook and F i n l a y s o n , 1950; G r e e n l a n d and Q u i r k , 1962; W e i s s , 1963]. A l t h o u g h s o i l humic compounds a r e p r o b a b l y t o o l a r g e t o be a d s o r b e d i n t e r n a l l y [ G r e e n l a n d , 1965b], a d s o r p t i o n by van d e r Waals f o r c e s c o u l d o p e r a t e 15 on the o u t e r s u r f a c e s o f c l a y s . N o n - p o l a r o r g a n i c compounds on the o t h e r hand, might be a b l e t o p e n e t r a t e t h e i n t e r l a m e l l a r spaces o f expanding m i n e r a l s and be a d s o r b e d . 1.3.5 TT - Bonding R e c e n t l y , d o n a t i o n o f TT e l e c t r o n s from o r g a n i c compounds to u n f i l l e d o r b i t a l s o f t r a n s i t i o n m e t a l s has been o b s e r v e d [Solomon, 1968]. Doner and M o r t l a n d [1969b] have o b s e r v e d v e r y s p e c i f i c n-bonding between benzene, x y l e n e , t o l u e n e and c h o l o r o b e n z e n e and C u ( I I ) montmor-i l l o n i t e f o l l o w i n g d e h y d r a t i o n o f m o n t m o r i l l o n i t e by e v a c u a t i o n o v e r ^2^^- ^hey  a ^ s o showed t h a t bonding o n l y o c c u r r e d i f t h e s u r f a c e n e g a t i v e c h a r g e on t h e c l a y was due t o isomorphous s u b s t i t u t i o n i n the o c t a h e d r a l l a y e r , hence, benzene and i t s d e r i v a t i v e s o n l y gave complexes w i t h m o n t m o r i l l o n i t e o r h e c t o r i t e , and n o t w i t h v e r m i c u l i t e . T h i s mechanism c o u l d c o n t r i b u t e t o o r g a n o - m i n e r a l complexes i n s o i l s o f u n u s u a l l y h i g h t r a n s i t i o n metal c o n t e n t , o r where h i g h a c i d i t y c a uses r e l e a s e o f Al from C l a y l a t t i c e s . 1.3.6 C o v a l e n t Bonding A c e t y l and phenyl d e r i v a t i v e s o f m o n t m o r i l l o n i t e have been made by Slabaugh [1952] and Spencer and G i e s e k i n g [1952] whereas r e c e n t l y F r i p i a t and M e n d e l o v i c i [1968] have p r e p a r e d methyl d e r i v a t i v e s o f c h r y s o t i l e . A l t h o u g h f a i r l y d r a s t i c c o n d i t i o n s a r e n e c e s s a r y , as M o r t l a n d [1970] p o i n t s o u t , t h i s i s an a c c e p t a b l e approach because i n g e o l o g i c a l t i m e s s i l t and s h a l e d e p o s i t s have been s u b j e c t e d t o h i g h 16 pressures and temperatures. Covalently bound organic compounds would thus be present in soils within particles derived from sedimentary rocks by weathering. In summary it can be concluded that the dominant factors determining the nature of clay-organic interactions are: (a) the properties of the organic compound (b) the water content of the system (c) the nature of the exchangeable cation on the clay surface (d) the properties of the particular clay mineral. Greenland [1971] feels that although hydrous oxides of Fe and Al are the most important materials involved in interactions between clays and organic compounds in soils, s i l ica and even quartz are not necessarily inert in this respect. It appears that the main stumbling block to progress in understanding interactions between clays and humic substances lies in our inadequate knowledge of the chemical structure of humic materials, which makes i t diff icult to interpret experimental data with confidence. 17 2. STUDIES:OF NATURALLY OCCURRING ORGANO-MINERAL COMPLEXES . There have been r e l a t i v e l y few s t u d i e s o f n a t u r a l l y o c c u r r i n g o r g a n o - m i n e r a l complexes d e s p i t e t h e f a c t t h a t some o f the p i o n e e r i n g work i n t h i s f i e l d d a t e s back t o the m i d - t h i r t i e s . W i t h i n r e c e n t t i m e s , however, more a t t e n t i o n has been f o c u s e d on t h i s most i m p o r t a n t component o f the s o i l system. B a r b i e r [1935] s t a t e d t h a t t h e a c t u a l c o l l o i d s i n the s o i l a r e not a mixture! o f o r g a n i c and m i n e r a l c o l l o i d s but the p r o d u c t o f d e e p - s e a t e d r e a c t i o n s between them; t h e f i n a l r e s u l t o f t h i s i n t e r a c t i o n b e i n g , the f o r m a t i o n o f s p e c i f i c o r g a n o - m i n e r a l g e l s , found o n l y i n s o i l . In 1937, T y u l i n a f f i r m e d t h a t t h e key t o t h e u n d e r s t a n d i n g o f many o f the p r o p e r t i e s o f s o i l c o l l o i d s was t o be sought i n the s t r u c t u r e o f o r g a n o - m i n e r a l g e l s and, p a r t i c u l a r l y , i n t h e l o c a l i z a t i o n o f o r g a n i c m a t t e r i n the e n t i r e mass o f g e l s . T y u l i n o b s e r v e d t h a t o r g a n i c m a t t e r e x t r a c t e d from g e l s was r i c h i n n i t r o g e n and phosphorus and p r o f f e r e d an e x p l a n a t i o n on the b a s i s o f f e r t i l i t y e x p e r i m e n t s t h a t b o t h t h e M and P were p r o b a b l y i m p o r t a n t from t h e s t a n d p o i n t o f s o i l f e r t i l i t y . U n f o r t u n a t e l y , however, w i t h the s o l e e x c e p t i o n o f C h i c h e s t e r ' s [1969] s t u d y o f t h e m i n e r a l i z a t i o n o f n i t r o g e n from s o i l o r g a n o - m i n e r a l s e d i m e n t a t i o n f r a c t i o n s , t h e r e has been no o t h e r i n v e s t i g a t i o n a l o n g t h e s e l i n e s . Developments i n the s t u d y o f o r g a n o - m i n e r a l complexes were p a r t i c u l a r l y slow p r i o r t o t h e a d v e n t o f u l t r a s o n i c d i s p e r s i o n methods, l a r g e l y due t o the d i f f i c u l t i e s o f d i s p e r s i n g s o i l i n t o p r i m a r y 18 components w i t h o u t o x i d a t i o n o f o r g a n i c m a t t e r and the a p p l i c a t i o n o f d i s p e r s i n g a g e n t s . A l s o , i t must be remembered t h a t the c h e m i s t r y o f i n d i v i d u a l s o i l components i s n o t f u l l y u n d e r s t o o d , and t h a t , i n any s t u d y o f n a t u r a l complexes, o n l y a h e t e r o g e n o u s , i l l - c h a r a c t e r i z e d s t a r t i n g m a t e r i a l i s a v a i l a b l e , making r e s u l t s much h a r d e r to i n t e r p r e t , and problems i n c h e m i c a l a n a l y s i s more p r o b a b l e . 2.1 I s o l a t i o n o f Complexes 2.1.1 I n t r o d u c t i o n The i d e a l method o f f r a c t i o n a t i n g s o i l m i n e r a l s i s one which c o m p l e t e l y s e p a r a t e s d i s c r e t e p a r t i c l e s w i t h o u t c a u s i n g e i t h e r a d e c r e a s e o r i n c r e a s e i n p a r t i c l e - s i z e . I t seems i m p r o b a b l e t h a t such a method w i l l e v e r be d e v i s e d . [Edwards and Bremner, 1967] In o r d e r t o i s o l a t e the c l a y o r any s i z e f r a c t i o n o f a s o i l i t i s ' f i r s t n e c e s s a r y to d i s r u p t f o r c e s which h o l d the i n d i v i d u a l p a r t i c l e s t o g e t h e r i n t h e form o f " a g g r e g a t e s . " T h i s may i n v o l v e the b reakage o f c h e m i c a l bonds, d i s r u p t i o n o f e l e c t r i c a l f o r c e s o f a t t r a c t i o n o p e r a t i n g between a d j a c e n t p a r t i c l e s and groups o f p a r t i c l e s , o r p h y s i c a l d i s e n t a n g l e m e n t o f p l a n t m a t e r i a l . E l e c t r i c a l f o r c e s a r i s e p r i m a r i l y because c l a y m i n e r a l s c a r r y e x c e s s n e g a t i v e c h a r g e s and e x c h a n g e a b l e c a t i o n s which a r e d i s t r i b u t e d o v e r t h e i r s u r f a c e s . P o l y v a l e n t c a t i o n s may a t t r a c t c l a y p a r t i c l e s t o form m i c r o a g g r e g a t e s , o r c l a y p a r t i c l e s may a l i g n t h e m s e l v e s i n d e p e n d e n t l y by v i r t u e o f i o n i c i n t e r a c t i o n s . F u r t h e r , s o i l water may c o n t a i n d i s s o l v e d s a l t s such t h a t i t behaves as a weak but complex e l e c t r o l y t e s o l u t i o n . 19 However, most aggregates in soils are bound together by organic or inorganic "cementing agents," principally soil organic matter, in various stages of humification; amorphous or semi-crystalline hydroxides of Fe, Si , A l , Mn and T i ; and alkaline earth carbonates and sulphates, usually Ca and Mg. Also, there is con-siderable evidence that soil micro-organisms directly bind soil particles together, and that roots or other plant debris entangle soil materials into larger aggregates or crumbs. After partial destruction of the cementing agents by chemical pretreatments, soil particles must be dispersed and maintained in a dispersed state until fractionation is complete. This is usually effected by shaking or stirring an aqueous suspension in the presence of a chemical dispersing agent. Normally soil dispersion is carried out as a prelude to determination of the mineral particle-size d istr i -bution. Similarly, when a soil fraction is separated for further mineralogical analysis a "clean" sample devoid of organic or amorphous materials is usually required. However, when i t is desired to isolate fractions with the associated organic matter intact, pre-treatments and dispersing agents must be avoided i f possible, and purely physical methods of dispersion employed. Within recent years the application of ultrasonic vibration methods for soil dispersion, [Fanning, 1965; Edwards and Bremner, 1967; Saly, 1967; Watson, 1970; Parasher and Lowe, 1971] appears to be an efficient well-suited method. 20 2-1.2 Methods o f S o i l D i s p e r s i o n 2.1.2.1 P r e - t r e a t m e n t s and D i s p e r s i n g Agents O r g a n i c m a t t e r can be removed by r e a g e n t s such as [ R o b i n s o n , 1922a], NaOBr [ T r o e l 1 , 1931], KMnO^ o r p e r s u l p h a t e s . As noted by Bauer [ 1 9 3 0 ] , o x i d a t i o n i s o f t e n by no means complete as 11-44 p e r c e n t o r g a n i c m a t t e r remained u n o x i d i z e d i n h i s samples. D i l u t e m i n e r a l o r o r g a n i c a c i d s a r e u s u a l l y employed t o d i s s o l v e c a r b o n a t e s and s o l u b l e s a l t s , whereas f r e e i r o n o x i d e s a r e removed by r e d u c t i o n w i t h sodium d i t h i o n i t e . The degree o f p r e -t r e a t m e n t n e c e s s a r y depends on the c o m p o s i t i o n o f the p a r t i c u l a r s o i l under s t u d y . For example, i t i s u n n e c e s s a r y t o p e r o x i d i z e a sample w i t h an e x t r e m e l y low o r g a n i c c o n t e n t o r t o l e a c h an a c i d i c s o i l w i t h d i l u t e a c i d . However, i n c o r p o r a t i o n o f a d i s p e r s i n g agent as an a i d t o m e c h a n i c a l d i s p e r s i o n i s e s s e n t i a l u n l e s s the c l a y c o n t e n t i s m i n i m a l . G e n e r a l l y d i l u t e s o l u t i o n s o f s a l t s o f monovalent c a t i o n s + + + a r e used e.g. Na , L i , NH^ , which exchange w i t h o t h e r c a t i o n s on the exchange complex and r e s u l t i n a s t r o n g r e p u l s i o n between i n d i v i d u a l p a r t i c l e s . The most w i d e l y used d i s p e r s a n t s are p r o b a b l y NaOH, N a 2 C 0 3 and c a l g o n ( N a P 0 3 ) 6 . 2.1.2.2 Methods o f Shaking and M i x i n g The e a r l i e s t methods o f s o i l d i s p e r s i o n i n v o l v e d s i m p l e s t i r r i n g o f t h e sample w i t h w a t e r , b o i l i n g , r u b b i n g w i t h a s t i f f b rush o r r u b b e r p e s t l e and s h a k i n g , H a l l [ 1 9 0 4 ] . These methods, 21 however, with the exception of the latter have long been superseded by more efficient chemical and/or physical methods. Shaking methods are s t i l l widely used, though rarely under the same operating conditions, which makes comparison of results, diff icult . Efficiency is largely determined by the type of shaker, usually rotary or reciprocating, speed of operation, soil/water ratio, type and concen-tration of dispersing agent and shaking time. Further attempts at improving the efficiency of shakers have included the addition of rubber balls or sand grains to the soil suspension [Puri, Dyal and Rai, 1944]. More recently Bourget and Rousseau [1967] reported that almost the same results were obtained using four types of shaker, each at three different dispersion times. They concluded that two hours was sufficient for each of the shakers provided dispersion was carried out in calgon solution after ful l a pretreatment of the soil samples with an oxidizing agent and dilute acid. 2.1.2.3 High Speed Stirrers Although Puri and Keen [1925] were the f i rst to use an egg-whisk for soil dispersion, high speed stirrers were developed mainly by Bouyoucos [1927]. He used a drink mixer at 9,000-14,000 rpm, and found that with baffles on the sides of the container, dispersion time could be reduced from three hours to nine minutes. He also observed, however, that there was a rapid abrasion of coarser particles and suggested that sandy soils be mixed for a shorter time than clay soils. 22 While such r a p i d s o i l d i s p e r s i o n i s c o n v e n i e n t , the t e c h n i q u e has s e r i o u s d i s a d v a n t a g e s when u t i l i z e d f o r more a c c u r a t e m e c h a n i c a l a n a l y s i s o f s o i l s where the a b r a s i o n o f p a r t i c l e s i s an i m p o r t a n t c o n s i d e r a t i o n . Lemieux [1964] compared the B o u y o u c o u s d r i n k m i x e r and a r e c i p r o c a t i n g s h a k e r u s i n g a hard g r a n i t e sand and a s l a t e sandy loam. C a r e f u l m e c h a n i c a l a n a l y s i s and p h o t o m i c r o g r a p h s c o n f i r m e d the a b r a s i v e a c t i o n o f t h e d r i n k m i x e r , p a r t i c u l a r l y on t h e s o f t e r sandy loam. Lemieux c o n c l u d e d t h a t f o r a c c u r a t e m e c h a n i c a l a n a l y s i s , s h a k e r s s h o u l d be used and t h a t t h e Bouyoncos m i x e r i s o n l y u s e f u l f o r r o u t i n e a n a l y s i s t o d e t e r m i n e broad t e x t u r a l c l a s s e s . I n t e r e s t i n g l y enough, Lag [1953] d e m o n s t r a t e d t h a t a b r a s i o n may a l s o be e n c o u n t e r e d w i t h s i m p l e s h a k i n g methods where s o i l s a r e d e r i v e d from s o f t r o c k . 2.1.2.4 A i r J e t t i n g The e a r l y a t t e m p t s a t u s i n g a i r j e t t i n g ( c o l d b o i l i n g ) as a means o f d i s p e r s i o n were u n s u c c e s s f u l , [ P u r i e_t a l _ . , 1944; P u r i , 1949]. Wintermeyer [1948] i n t r o d u c e d compressed a i r (2 l b s / i n ) i n the base o f a Column h o l d i n g a s o i l / w a t e r s u s p e n s i o n and a l l o w e d a i r b u b b l e s t o r u s h up t h r o u g h the s u s p e n s i o n and e s c a p e p a s t b a f f l e s t h r o u g h a v e n t a t the t o p . He c l a i m e d t h a t 20 m i n u t e s was s u f f i c i e n t f o r good d i s p e r s i o n o f c l a y and t h a t the a b r a s i v e a c t i o n on sand was l e s s than t h a t produced i n o n l y one minute by a h i g h speed s t i r r e r . Chu and D a v i d s o n [1953] used a s i m p l e r a p p a r a t u s and a l s o found t h a t d i s p e r s i o n was r e l a t i v e l y e f f i c i e n t w i t h o u t c a u s i n g s i g n i f i c a n t d e g r a d a t i o n o f c o a r s e r p a r t i c l e s . More r e c e n t l y T h e i s e n , Evans and Harward [1968] r e p o r t e d t h a t a f i v e minute a i r j e t d i s p e r s i o n o f Oregon s o i l s was j u s t as e f f e c t i v e as s i x hours on t h e r e c i p r o c a t i n g s h a k e r . 23 2.1.2.5 Cation Exchange Resin Methods Edwards and Bremner [1965] proposed the use of monovalent cation exchange resins to displace polyvalent cations on the soil exchange sites and thereby disperse organo-mineral colloids. In practice the method consists of shaking soil with small mesh size resin beads in the Na+, NH^+ or K+ form and after exchange is complete, the resin is removed by sieving and flotation in water. For resins in the Na+ form i t was found that an iminodiacetic acid resin required only two hours shaking, a carboxylic acid resin ten hours and a sulphonic acid resin twenty hours. A wide range of surface and sub-soils were found to give clay percentages comparable with the NaOBr method of Troell [1931], after weight corrections for associated organic matter. Parasher and Lowe [1970] compared the effectiveness of a chelating resin, a simple dispersion technique [Arshad and Lowe, 1966] and an ultrasonic dispersion method (tank type unit with a power output of 80 watts) for the isolation of organo-clay complexes on 26 horizon samples. They found that the resin method gave consistently higher yields of complexes than the other two methods; the percent of total soil carbon recovered in the complexes was also higher with resin dispersion. Unfortunately replacement of polyvalent by monovalent cations not only gave a high degree of aggregate dispersion but also solubilized much organic material. Thus the method, although an improvement upon those requiring drastic peroxide treatments-, has serious limitations for any study of organo-mineral complexes. 24 2.1.3 Ultrasonic Dispersion Methods Ultrasonic vibrations are sound waves with a frequency higher than about 16 KHz. Ultrasonics has found wide application in Industry and Medicine^particularly for cleaning surfaces, sterilization of liquids and detection of flaws in metals or plastics. Edwards and Bremner [1964, 1967] demonstrated that a wide range of soils could be dispersed by ultrasonic vibration of soil-water suspensions, and this method of soil dispersion has been used extensively during recent years for research in which i t is essential to disperse soils without the use of chemical reagents. The literature on ultrasonic vibration as a method of soil dispersion has been comprehensively reviewed by Watson [1971] and the reader is referred to that review for such aspects of ultrasonic dispersion methods not discussed herein. 2.1.3.1 Extraction of Organic Matter Felbeck [1959] was the f i rst to.extract humic acid from soils by ultrasonic (40 KHz) dispersion in 0.5 M sodium hydroxide and 0.1 M sodium pyrophosphate solutions. He found that these reagents extracted 20-48 percent more organic matter in three hours with ultra-sonic vibration, and that in the case of sodium pyrophosphate more organic matter was removed than by extraction with the reagent for eight days. '; These results were confirmed by Edwards and Bremner [1967a] who, however, found that the organic matter extracted was not s ignif i-25 cantly increased i f suspensions were vibrated for one hour prior to shaking for 24 hours, nor i f the soils were suspended in water and vibrated for one hour before treatment with alkali or pyrophosphate. Greenland and Ford [1963] studied the use of ultrasonic dispersion (in bromoform/petroleum spirit mixture) as a means of disentangling non-humified organic materials within surface soils. After standing, the suspensions were centrifuged at 2000 xg for thirty minutes to obtain "light" and "heavy" organic fractions. It was claimed that organic material not combined with the clay fraction was separated quantitatively from the remainder of the soi l . More recently Ford, Greenland and Oades [1969] have recommended addition of a surfactant to the mixture or dispersion in nemagon (1, 2-dibromo, 3-chloropropane; S.G. 2.06) for bulk separations. Ultrasonic dispersion in aqueous acetyl acetone was found by Halstead, Anderson and Scott [1966] to increase the efficiency of this reagent for extracting organic-P and -S compounds from soils. More recently Sowden [1970] and Halstead and Anderson [1970] have used the same extraction method in studies of soil organic nitrogen and organic phosphorus, respectively. As a result of the latter work i t was suggested that the determination of soil organic-P by the method of Mehta, Legg, Goring and Black [1954] gives an underestimation of total organic-P. Ultrasonic vibration has also been used to extract organic matter from crushed sedimentary rocks [Mclver, 1962, Han and Calvin, 1969]. Mclver [1962] reported that ten minutes of ultrasonic vibration replaced six hours of conventional treatment with the hot solvent (70 percent benzene and 15 percent each of acetone and methanol). 26 In addition, analyses employing ultrasonics were characterized by greater precision and higher yields of soluble organic matter. Recently attempts have been made to extract DDT and similar persistent insecticide residues from soils by ultrasonic vibration. Johnsen and Storr [1967, 1970] reported that the insecticides can be extracted far more readily from a recently treated soil than from an aged sample, since they are held by soil particles and soon become incorporated into soil flora and fauna. 2.1.3.2 Dispersion of Soil Suspensions and Minerals The effects of sound waves on the dispersion of soil particles was f i rst investigated over f i fty years ago, however, serious research into the use of ultrasonic vibration as a method for soil dispersion started in the late nineteen f i f t ies . Vasil'eva [1958] claimed that a greater yield of colloidal particles was obtained from clay soils by ultrasonic dispersion than by the standard physico-chemical methods. This was confirmed by Barkoff [1960] who found 20-28 percent more clay-size material in a clay soil when i t was subjected to ultrasonic vibration in a peptising solution of calgon and ^CO^, than when simply shaken for five hours in water. Undoubtedly the most important works are those of Edwards and Bremner [1964, 1967] and Saly [1967] who have studied in some detail the optimum conditions for the instruments they have used, and in addition have investigated the effect of ultrasonic waves on various minerals in an attempt to assess the possible effects on soil particles. Edwards and Bremner found that ultrasonic vibration of a wide-range 27 of soils and several minerals (including calcite, dolomite, biotite, microcline, quartz, kaolinite, i l l i t e and bentonite) had no significant effect on pH and conductivity of the suspensions and dissolved only trace amounts of organic and inorganic materials, except in the case of biotite. Saly [1967] also investigated possible structural deterioration of clay minerals by ultrasonification and concluded on the basis of X-ray and DTA data that the method was superior to others commonly used for clay separation. The advantages of ultrasonic dispersion of soils over other physical and physico-chemical methods has resulted in widespread adoption of the technique for particle-size separation of soils, and studies of soil organo-mineral complexes [Kaila, 1967; Juo and El l is 1968; Kaila and Ryti, 1968; Chichester, 1969, 1970; Kyuma, Hussain, Kawaguchi, 1969; Leenheer and Moe, 1969; Parasher and Lowe, 1970; Lowe and Parasher, 1971; Rostad and St. Arnaud, 1970; McKeague, 1971; Staoh and Yamane, 1972a,b]. There have been reports, however, of failure to disperse soils effectively or to obtain stable soil suspensions by this technique without the use of a dispersing reagent [Olmstead, 1931; Saly, 1967; Bourget, 1968; Emerson, 1971]. This has created some residual doubt about the applicability of the technique to a wide range of soils. Gehrich and Bremner [1972a] re-evaluated the ultrasonic dispersion method by applying the technique to a group of soils which embraced nine of the ten orders of the U.S.D.A.. 7 t h approximation and had a diversity of carbon and mineral content. They concluded from their study that the technique was applicable for the dispersion of all 28 their soils and that the suspension of dispersed soil was stable for at least four days except in the case of a high carbonate so i l , which was stable for about two days. A further study, Genrich and Bremner [1974] confirmed the usefulness of ultrasonification for the isolation of particle-size fractions. 2.1.3.3 Factors Affecting Dispersion Vibration Frequency In an investigation of the factors affecting dispersion by vibration, Edwards and Bremner [1967a] suggested that since the two instruments they used, operating at different frequencies (9 KHz and 18-20 KHz) gave similar results, vibration frequency had l i t t l e effect on the efficiency of soil dispersion. However, their sonic oscillator was of the sample cup type whereas ultrasonic vibrations were transmitted by a titanium probe. Thus, a comparison is not strictly justied and as these authors themselves point out a similar result using one instrument at different frequencies would have to be obtained before the effect of frequency could be confirmed. Indeed, since Mathieu-Sicaud and Levavasseur [1949] found that the optimum dispersion fre-quency for clay minerals varied, one might expect a similar effect for soils, although in the heterogenous soil system the effect might be less marked. Sound Intensity It has been demonstrated that dispersion efficiency increases with the intensity of ultrasonic waves, Saly [1967]. Edwards and 29 Bremner [1967] studied the effect indirectly by varying the amount of soil dispersed in a fixed volume of water and found that a decrease in the amount of soil gave an increased clay percentage by virtue of the increased sound intensity. It might be added that Weissler [1953] has observed that for some sonochemical reactions there is an optimum intensity above which a decrease in yield may occur. Time of Vibration An increase in the time of vibration generally leads to an increase in clay recovered and a corresponding decrease in sand and s i l t material. Edwards and Bremner [1967a] showed that the higher clay values do not necessarily reflect breakdown of primary particles. On the other hand Saly [1967] suggests that a mere 4-5 minutes exposure to ultrasonic waves is sufficient for effective dispersion and that a longer duration of about 15 minutes may lead to partial flocculation. This apparent disparity in observations can perhaps be attributed to differences in the frequencies and power outputs of the instruments used by those workers. Temperature Effects The effect of cavitational heating during ultrasonic vibration of soil suspensions has not been extensively studied. Watson [1971] obtained a slight increase in clay recovery as temperature was increased from 10° to 60°C and suggested that the increase was probably due to more frequent and violent collisions between aggregates, as the viscosity of the suspending medium decreased. In contrast to this 30 view, Weissler [1953] suggested that the collapse of cavitation bubbles may be less violent when the bulk liquid is hot, because the vapour pressure of water is greater; consequently decreased yields might be expected at higher temperatures. Probe Condition Perhaps the most significant factor affecting ultrasonic dispersion of soils by probe-type instruments is the condition of the probe or more specifically the probe tip, during dispersion. Although Genrich and Bremner [1972b] were the f i rst to report that pitting of the probe tip occurred after approximately 10 hours of operation i t must be assumed that this phenomenon is widely observed. Genrich and Bremner also reported that pitting decreased the output of ultra-sonic energy from the probe and increased the vibration time needed for dispersion of soils. They have suggested that pitting can be eliminated by gentle polishing of the probe tip for 5-10 seconds with fine (400-grit) emery paper after every 30 minutes of probe operation. The author was unable to confirm this observation, but noted that polishing of the probe tip did retard the onset of pitting. The length of the probe has also been cited as a critical factor in the design of probe-type ultrasonic vibrators. Genrich and Bremner [1972b] found that a probe after 10 hours of operation "was noticeably shorter" than a similar probe operated for 10 hours but polished after every 30 minutes. Unfortunately i t was not stated whether or not this change in probe length significantly affected the afficiency of ultrasonic dispersion. 31 2.1.3.4 Effects of Ultrasonic Vibration on Soil Components ' Abrasion of Mineral Particles The abrasive effect of sonic dispersion on soil minerals was investigated by Edwards and Bremner [1967a] who found that fragile minerals such as biotite were more easily degraded by insonation than by shaking in water. For most other minerals, however, sonic vibration for up to one hour was less abrasive than 10 hours shaking in water. Insonation was also significantly less damaging to fine particles than treatment with peroxide followed by shaking in calgon. It would have been interesting to see whether similar results would have been obtained by Edwards and Bremner had they investigated the abrasive effects of ultrasonic vibration on minerals, considering that the frequency of operation is double that of the sonic instrument used and the energy output significantly higher. Adams and Stewart [1969] estimated abrasion of sand grains in soils derived from sedimentary rocks by comparing the effects of ultrasonic vibration on fine sand isolated from the parent material and fine-sand-sized soil particles. They concluded that sand grains in soils derived from sedimentary rocks were susceptible to ultra-sonic disintegration. The approach used by Adams and Stewart could however, lead to an overestimation of abrasion because the presence of smaller particles and especially organic matter in soils would perhaps reduce the effects on sand grains. Other studies dealing with the effects of sonic and ultra-sonic waves on clay minerals have recognized three stages of alteration. 32 1. Simple removal of contaminants from clay surfaces as observed for kaolinite by Koms'ka, Del in, Fes'ko [1966]. 2. Splitting of the original clay lattice which is accom-panied by an increase in cation exchange capacity and variations in X-ray diffraction patterns [Gata, 1964]. 3. Complete fragmentation of the existing structures and a regrouping of the freshly formed surfaces into new mineral aggregates, [Kruglitskii, Ovcharenko, Simirov, Nichiporenko and Barshchevskaya, 1966]. Effects on Soil Organic Matter Ultrasonic waves may promote several types of chemical reactions viz. oxidation, reduction, hydrolysis, free radical polymerization, depolymerization, molecular rearrangements and photochemical reactions, [El'Piner, 1964]. It is not surprising therefore that concern has been expressed by some workers on the possible effects of ultrasonics on soil organic matter. Srivastava and Berkowitz [1960] reported that humic acids are stable in sonic and ultrasonic fields even after three hours, whereas linear polyelectrolytes such as krilium, and bovine albumin plasma, a tightly coiled protein, are readily degraded. Also Yankovskii [1968] found that apart from differences in the 1710-1930 cm ^ region of the infra-red spectrum, lignin extracted from pinewood with the aid of ultrasonic vibration, had the same properties as that extracted.without. 33 These results suggest that well humified organic materials are relatively resistant to ultrasound. However, long peripheral side chains could well be susceptible to degradation. It is also possible that low molecular weight soil organic compounds are vapour-ized in cavitation bubbles and altered chemically, and that weak hydrogen bonds might be cleaved as already demonstrated for proteins. At the present state of knowledge, i t would be diff icult to draw any precise conclusions about possible effects of ultrasonic vibration on soil organic matter because as yet, there are large gaps in our understanding of its constituents; also, the study of organo-mineral complexes, a product of its interaction with soil minerals is s t i l l in its infancy. 2.2 Characterization of Organo-Mineral Complexes The data available from studies of naturally occurring complexes seem to indicate that the mineralogy of the soil is probably of the greatest importance for complex formation. Arshad and Lowe [1966] found that more organic carbon was associated with the coarse clay fraction (2-0.2 urn) consisting mainly of kaolinite than with finer clay where montmorillonite was dominant. Similarly, Dudas and Pawluk [1969] reported that the organic carbon content was highest in the coarse clay fraction and decreased as clay size decreased. After comparing peroxidized and non-peroxidized material by CEC measure-ments and physical methods, they (Dudas and Pawluk) concluded that there was more intimate clay-organic complexing in the fine, highly 34 montmorillonitic clay (0.2 - 0.08 urn). They also inferred that com-plexed organic matter occupied edges and surfaces of clay minerals, and did not observe any interlamellar adsorption of organic matter. Kyuma et al_., [1969] also collected organo-mineral size-fractions (coarse sand, fine sand, s i l t and clay) from four Japanese soils after ultrasonic dispersion. The size fractions from peroxidiz-ed samples were also collected for comparison. Although the C/N ratio of organo-mineral complexes was found to decrease with particle-size, they claimed that the organic material associated with the clay was less humified than that in the sand fractions. This could be explained i f sand grains were coated with well-humified organo-clay skins, but the authors themselves point out that "the coarse sand-complex is not a complex in a true sense, but is a mixture of coarse sand grains, and a small amount of coarse organic debris with some stable aggregates of finer-size fractions." Also, the amount of non-humified organic material in the clay fraction was negligible when determined by Greenland and Ford's [1964] heavy liquid separation. Nevertheless, the results obtained did demonstrate that total C and especially total N were relatively concentrated in the clay complex of three out of four soils. Chichester [1969] also isolated a series of particle-size separates (2 sand, 2 s i l t , and 4 clay fractions) by ultrasonic dis-persion of the soil and centrifugal and gravity separation in water. He found that total C, and total N of the size fractions increased and C/N ratio decreased with decreasing particle-size: Chichester also determined extractable N forms and related these to the mineraliz-35 a b l e N i n the v a r i o u s s i z e f r a c t i o n s . ( F o r f u l l d i s c u s s i o n see S e c t i o n 3.3.2). McKeague [1971] i n v e s t i g a t e d t he o r g a n i c m a t t e r i n p a r t i c l e -s i z e and s p e c i f i c g r a v i t y f r a c t i o n s o f the Ah h o r i z o n s o f a number o f Canadian s o i l s . C o n s i s t e n t w i t h C h i c h e s t e r ' s [1969] r e s u l t s , he found t h a t f o r most s o i l s t h e C/N r a t i o d e c r e a s e d w i t h a d e c r e a s e i n p a r t i c l e - s i z e but i n c o n t r a s t t o many p r e v i o u s r e p o r t s , . h e noted t h a t much o f the ca r b o n i n many Ah h o r i z o n s was a s s o c i a t e d w i t h t he f i n e s i l t and even the medium s i l t f r a c t i o n , and not t h e c l a y f r a c t i o n . Hence, he proposed t h a t s t u d i e s o f n a t u r a l l y o c c u r r i n g c o m b i n a t i o n s o f . o r g a n i c and i n o r g a n i c s o i l c o n s t i t u e n t s s h o u l d i n c l u d e the c o a r s e r f r a c t i o n s as w e l l as the c l a y . The r e l a t i v e l y l a r g e amounts o f o r g a n i c m a t t e r found i n the f i n e and medium s i l t f r a c t i o n s by McKeague may i n p a r t be due t o i n c o m p l e t e s e p a r a t i o n o f c l a y from the s i l t f r a c t i o n s . As McKeague h i m s e l f p o i n t e d o u t the f i n e s i l t and c o a r s e s i l t f r a c t i o n s c o n t a i n e d 33 p e r c e n t and 70 p e r c e n t c l a y r e s p e c t i v e l y . P a r a s h e r and Lowe [1970] and Lowe and P a r a s h e r [1971] i s o l a t e d c l a y - s i z e complexes by s o n i c d i s p e r s i o n from some B r i t i s h Columbia s o i l s . They o b s e r v e d t h a t complexes from t he Bf and Bhf h o r i z o n s o f p o d z o l i c s o i l s c o n t a i n e d up t o 46 p e r c e n t o f amorphous i n o r g a n i c m a t e r i a l e x t r a c t a b l e by c i t r a t e - d i t h i o n i t e s o l u t i o n . F u r t h e r , 52-68 p e r c e n t o f the a s s o c i a t e d o r g a n i c m a t t e r was e x t r a c t a b l e by s u c c e s s i v e t r e a t m e n t s w i t h 0.1 M p y r o p h o s p h a t e and NaOH s o l u t i o n s , and 30-100 p e r c e n t o f t h e e x t r a c t e d o r g a n i c m a t t e r b e l o n g e d t o t h e f u l v i c f r a c t i o n . Lowe and P a r a s h e r [1971] a l s o i n f e r r e d from X-ray d a t a 36 t h a t t h e r e were i n t e r ! a m e l l a r complexes p r e s e n t , a s s o c i a t e d w i t h m o n t m o r i l l o n i t e and v e r m i c u l i t e . I n f r a - r e d d a t a a l s o s u g g e s t e d t h a t o r g a n i c m a t t e r was bonded t o s i l i c o n atoms o f t h e c l a y m i n e r a l s v i a oxygen l i n k a g e s . Satoh and Yamane [1972a] i s o l a t e d 4 sand, 4 s i l t and 5 c l a y f r a c t i o n s from a v o l c a n i c ash s o i l , f o l l o w i n g u l t r a s o n i c d i s p e r s i o n . They found t h a t o v e r 56 p e r c e n t o f the o r g a n i c m a t t e r was c o n t a i n e d i n t he <2ym f r a c t i o n s , and t h a t C/N r a t i o and A l o g K v a l u e s ( f o r the e x t r a c t e d o r g a n i c m a t t e r ) d e c r e a s e d w i t h a d e c r e a s e i n p a r t i c l e s i z e . In an e f f o r t t o f u r t h e r c h a r a c t e r i z e t h e complexes Satoh and Yamane [1972b] i s o l a t e d e i g h t d i f f e r e n t d e n s i t y f r a c t i o n s i n a Bromoform-e t h a n o l system r a n g i n g between <1.2 t o >2.4 g/ml. These workers r e p o r t e d t h a t i n t h e c a s e o f t h e s i l t and c o a r s e c l a y f r a c t i o n s , o r g a n i c m a t t e r and c l a y m i n e r a l c o n t e n t i n c r e a s e d as p a r t i c l e d e n s i t y d e c r e a s e d . However, i t was a l s o p o i n t e d o u t t h a t complete s e p a r a t i o n o f the com-p l e x e s c o u l d not be o b t a i n e d because o f f l o c c u l a t i o n o f the f i n e r . p a r t i c l e s i n t h e heavy l i q u i d s used. T h e r e s t i l l seems to be a d i v e r g e n c e o f views among r e s e a r c h e r s as t o whether s o i l o r g a n i c compounds can e n t e r the i n t e r l a m e l l a r spaces o f expanding c l a y m i n e r a l s . T h i s i s p r o b a b l y i n p a r t due t o c o n f u s i o n between humic a c i d s and the more g e n e r a l term " s o i l o r g a n i c m a t t e r . " As i n t i m a t e d by G r e e n l a n d [1965b] the a v a i l a b l e e v i d e n c e s u g g e s t s t h a t humic a c i d s c o n s i s t o f a p p r o x i m a t e l y s p h e r i c a l r i g i d p a r t i c l e s w i t h m o l e c u l a r w e i g h t s i n t h e range o f 20,000 - 50,000. Such m a t e r i a l can h a r d l y be e x p e c t e d t o a d s o r b on i n t e r l a m e l l a r s u r f a c e s and i t i s not s u r p r i s i n g t h a t X-ray d i f f r a c t i o n has f a i l e d t o r e v e a l such a phenomenon [Evans and R u s s e l l , 1959]. 37 On the o t h e r hand, i t s h o u l d be p o s s i b l e f o r s i m p l e r s o i l o r g a n i c compounds such as f r e e amino a c i d s , s ugar and r e l a t i v e l y s m a l l p r o t e i n and p o l y s a c c h a r i d e m o l e c u l e s t o be a d s o r b e d i n i n t e r -l a y e r s p a c e s . However, whereas some o f the e a r l i e r i n v e s t i g a t i o n s f a i l e d t o demonstrate t h i s [ A r s h a d and Lowe, 1966; Kyuma. e t a l _ . , 1969; Dudas and Pawluk, 1969] t h e r e i s mounting e v i d e n c e t h a t i n t e r l a m e l l a r a d s o r p t i o n , o c c u r s i n n a t u r a l l y o c c u r r i n g complexes, [Lowe and P a r a s h e r , 1971; Satoh and Yamane, 1971, 1972b]. R e c e n t l y , Kodama and S c h n i t z e r [1971] p r e s e n t e d DTA. d a t a which s t r o n g l y s u g g e s t s t h a t t h e r e was i n t e r l a m e l l a r a d s o r p t i o n o f f u l v i c a c i d m a t e r i a l i n the <0.2um f r a c t i o n i s o l a t e d from a podzol Ae h o r i z o n , and S c h n i t z e r and Khan [1972] have p r e s e n t e d q u i t e c o n c l u s i v e e v i d e n c e t h a t f u l v i c a c i d i s o l a t e d from a s o i l w i l l a d s o r b on t o the i n t e r n a l s u r f a c e s o f m o n t m o r i l l o n i t e . H a r t e r and S t o t z k y [1973] o b s e r v e d t h a t p r o t e i n s i n t e r c a l a t e d s m e c t i t e s when the w e i g h t r a t i o o f p r o t e i n t o c l a y exceed 1:5, however, they a l s o i n d i c a t e d t h a t t h e ex c h a n g e a b l e c a t i o n on the c l a y might i n f l u e n c e a d s o r p t i o n . 2.3 C o n e ! u s i o n I t i s c l e a r from t he f o r e g o i n g r e v i e w o f t h e l i t e r a t u r e t h a t , w h i l e f a r from b e i n g c o m p l e t e , a. tremendous body o f knowledge now e x i s t s , about c l a y s , s e s q u i o x i d e s , and o r g a n i c m a t t e r i n s o i l s , and t h e i r i n t e r a c t i o n s . I t i s a l s o a p p a r e n t t h a t the s t u d y o f organo-m i n e r a l complexes o c c u r r i n g s o i l s i s s t i l l i n i t s i n f a n c y , and o f the 38 many studies reported in the literature, rarely has the same set of dispersion and fractionation procedures been used by different workers. The development of ultrasonic vibrators applicable for soil dispersion has provided a tremendous impetus to the study of organo-clays because vibration obviates the need for oxidizing agents and chemical reagents to effect complete dispersion of soils. Unfortunately, however, no general consensus has yet been reached as to the relative importance of several factors pertaining to ultrasonic dispersion of soils. Perhaps, more importantly, very l i t t l e quantitative data is available in the literature regarding the dissolution effects of ultrasonic vibration on both the organic and inorganic constituents of soils. This is paradoxical considering that the primary objection to the conventional methods of soil dispersion (e.g. peroxide-calgon method) was that they solubilized and degraded soil constituents. Some progress has been made in the characterization of organo-mineral complexes. It appears that complexing occurs largely in the sub-clay-size fractions although by no means exclusively so. The approaches used have been largely chemical (e.g. extraction of organic matter by NaOH or physical (viz. X-ray, DTA, IR and electron microscopy) and there is a need for further studies along these lines. A "microbiological approach" to the characterization of organo-mineral complexes was used by Chichester [1969], which indicated the extent and the rate at which organic N compounds could be mineralized from organo-mineral complexes. Mineralizable N was also correlated with various extractable N forms. It appears that this approach offers the greatest possibilities for the study of not only nitrogen, but sulphur and phosphorus as well. 39 3. THE RELATIONSHIP BETWEEN CARBON, NITROGEN, PHOSPHORUS AND SULPHUR IN SOILS 3.1 Introduction Nitrogen and phosphorus are closely related in many compounds important in metabolism and reproduction. The relationship is so widespread that in living tissue there is found a consistent ratio between the contents of the two elements, which persists into the well decomposed organic fractions in soil [Thompson and Black, 1950; Thompson et al_., 1954; Walker and Adams, 1958]. These two elements along with carbon and sulphur are apparently stabilized together in the soi l . Soil organic matter contains most of the soi l 's reservoir of nitrogen and sulphur and in many soils is also an important source of phosphorus. In most soils, humic substances constitute the major component of soil organic matter as a whole, and they, in turn, are important contributors to the pool of soil nitrogen, sulphur and phosphorus. However, these organically bound nutrient elements must be mineralized before they can be utilized for plant growth. Although the mechanism of this mineralization process is not fully understood, i t is reasonable to suppose that the humification process (i .e. the transformation of plant materials and the breakdown of the transformed products) is involved in the release of nutrients. If this were not so then humus formation and its subsequent transformation would result in ever increasing amounts of organically combined and consequently unavailable nutrients. 40 An i n c r e a s e o r d e c r e a s e i n one o f t h e s e elements has o f t e n been found t o be accompanied by a p a r a l l e l i n c r e a s e o r d e c r e a s e i n the o t h e r elements [Walker and Adams, 1958]. Such a r e l a t i o n s h i p s u g g e s t s t h a t t h e measure o f b i o l o g i c a l s t a b i l i t y e x h i b i t e d by s o i l o r g a n i c m a t t e r must be a c h i e v e d t h r o u g h r e a c t i o n s o f l a r g e o r g a n i c a g g r e g a t e s o r m o l e c u l e s c o n t a i n i n g C, N, P and S i n a p p r o x i m a t e l y t h e same p r o p o r t i o n as t h e s e elements a r e found i n m i c r o b i a l t i s s u e s . The e x t r a c t i o n o f o r g a n i c m a t t e r from s o i l s by a l k a l i o r m i n e r a l a c i d produces changes i n the c h a r a c t e r o f the o r g a n i c f r a c t i o n , and o r g a n i c p r e p a r a t i o n s so d e r i v e d may not t r u l y r e p r e s e n t o r g a n i c m a t t e r . as i t e x i s t s i n the s o i l . The h y d r o l y s i s o f ph o s p h a t e , s u l p h a t e and c e r t a i n N c o n t a i n i n g g r o u p s , t h e r e b y c o n v e r t i n g them t o the i n o r g a n i c form, i s a d i s t i n c t p o s s i b i l i t y . One approach t o the problem o f d e f i n i n g t he r o l e o f o r g a n i c m a t t e r i n s o i l s has been t o s t u d y o r g a n o - m i n e r a l complexes, o r p a r t i c l e -s i z e s e p a r a t e s , i s o l a t e d w i t h o u t t he a i d o f c h e m i c a l r e a g e n t s . I t s h o u l d be p o i n t e d o u t t h a t much o f the o r g a n i c c a r b o n and n i t r o g e n i n s o i l s o c c u r i n the form o f o r g a n o - m i n e r a l complexes [ G r e e n l a n d , 1965b] and hence such complexes might be a u s e f u l s t a r t i n g p o i n t i n the s t u d y o f t h e r e l a t i o n s h i p s between c a r b o n , n i t r o g e n , phosphorus and s u l p h u r . Walker and Adams [1958} found t h a t t he ave r a g e r a t i o o f C:N:S: o r g a n i c P i n twenty s o i l s measured t o a de p t h o f 20 i n was 120:10:1.3:2.7. O t h e r r e p o r t s from w i d e l y s e p a r a t e d p a r t s o f t h e w o r l d [Barrow, 1961a,b; B l a c k and G o r i n g , 1953] have i n d i c a t e d r e l a t i o n s h i p s between t h e s e f o u r element's, but whereas i n most c a s e s t he mean p r o p o r t i o n s o f C, N and S found a r e s i m i l a r (C:N:S: c a . 140:10:1.3) t h o s e o f o r g a n i c phosphorus 41 are more variable. The mean ratio of organic-P relative to 10 parts of N has been found to range from 0.33 - 3.0 [Williams, 1966], ( i .e. N/P(organic) Ratio of 3-30:1). In general the C:N ratios in the vast majority of agricultural soils fal l within the range 10-20:1. Even less variation is evident for the N:S ratios, either between soil groups or as a result of cultural treatments, and most soils have N:S ratios in the range 6.6-10:1. It would thus appear that the association between N and S in the soil organic matter is closer than their association with carbon. Although many investigators have reported positive correlations between organic-P and other components of soil organic matter [Barrow, 1961a; Black and Goring, 1953], the mean values found for C:P, N:P, or S:P have varied quite widely and according to the results of [Walker and Adams, 1958; Williams and Steinbergs, 1958; Williams et a]_., 1960] the correlations of organic-P with C, N and S have generally been poorer than those between C, N and S. The reasons for the higher variability in the amounts of P in the soil organic matter are not yet understood but contributing factors could be; either the unreliability of present methods for measuring organic-P or the presence of inositol phosphates. These compounds contain no N or S and have a narrow C:P ratio. Variations in the amounts present could therefore influence the phosphorus content of the total organic matter. The limited quantitative data available,however, [Williams, 1966] indicate that the inositol phosphates generally account for less than 50 percent of the total organic P and so would be insufficient to account for all of the variability known to occur. 42 It is clear from the foregoing discussion that all four elements C, N, S, and P are intimately associated in the organic fraction and that changes in the amount of organic matter, whether by accumulation or by mineralization, will involve concurrent changes in the contents of these elements. 3.2 Incubation Methods in the Study of Complexes From the discussion dealing with the characterization of organo-mineral complexes (Section 2.2) i t is clear that chemical and physical techniques have been used almost exclusively in attempts to characterize these substances. Chichester [1969] was the f i rst to use an incubation technique to determine the susceptibility to mineral-ization of N in organo-mineral sedimentation fractions. Further, by comparing the results obtained, with data on the forms of N extractable before and after mineralization, he obtained some indications as to the relative stability ( i .e. to microbial degradation) of the various N fractions. Chichester's anaerobic incubation technique precluded the measurement of nitrate formation or of measuring the susceptibility to mineralization of S and P in the various fractions. The author is unaware of any studies reported in the l itera-ture, on the susceptibility to mineralization of sulphur and phosphorus, in organo-mineral complexes or of the mineralization of N in organo-mineral complexes under aerobic conditions. 43 3.3 Incubation Methods for Measuring N Mineralization The amount of N mineralized from a wide range of soils under optimum conditions of moisture/aeration, pH and temperature has been found to be well correlated with N uptake by plants in numerous studies, [Harmsen and Van Schreven, 1955; Gasser, 1961]. N mineralization therefore is accepted as a valid means of assessing the ability of a soil to supply N to crops. Greenland [1965] presented data from several sources to show that as much as 55-98 percent of the total soil C may be present as a "clay-organic" complex, and i t is therefore reasonable to assume, that some of the C as well as some of the associated N, P and S in the organic matter will be mineralized with time from such complexes and contribute to the nutrition of soil organisms. This hypothesis has not hitherto been specifically tested, although Chichester [1969] reported that N was mineralized from organo-mineral sedimentation fractions when they were anaerobically incubated. No data are available at present on the relative amounts of phosphorus and sulphur present in organo-mineral complexes and the extent to which these nutrients are mineralized and thus added to the pool of available nutrients in the soi l . It seems quite clear therefore that such information is urgently needed i f we are to fully understand the transformation of organic matter in the soil and its role, both in soil genesis and soil fer t i l i ty . The literature dealing with the mineralization of N, P and S in turn will now be reviewed at some length. 44 3.3.1 Soil Nitrogen Numerous factors affect the mineralization of N, P and S and techniques for their determination vary widely. Fitts et aj_. [1955] found that nitrate production took place over a wide range of moisture contents, 25-35 percent, depending on the texture of the soi l . They concluded, however, that 100 cm tension was optimal for NOg production. In a subsequent study Stanford and Hanway [1955] used a similar two week incubation period to measure NOg""production in soils and also added vermiculite to the soil to facil itate moisture control and enhance aeration. Munson and Stanford [1955] concluded from their experiments that NO^ -N released during a two week period of incubation was highly correlated with N uptake by plants grown on the soi l . Waring and Bremner [1964a] cited some of the problems associated in the NOg production methods and argued that the total mineral N produced during incubation should be determined. They proposed a method involving the estimation of the NH^ -N produced by incubation of soil under water-logged conditions, and found a close relationship between the amount of NH^ -N produced (by their method) and the amount of (NH4 + UQ^ + N0g)-N produced by incubation under aerobic conditions (r = 0.96). Waring and Bremner [1964b] also high-lighted the importance of soil mesh size as a factor affecting the amount of N mineralized during incubation. They recorded a 25-124 percent increase in the amount of N mineralized in 52 soils by decreasing mesh size (through grinding) from <10 to <80 mesh. The increase was ascribed to the fact that some of the organic matter 45 in soil aggregates was not susceptible to microbial decomposition until the aggregates were disrupted by grinding or other processes that rendered this organic matter physically accessible to micro-organisms. Many chemical methods of obtaining an index of the avail-ability of soil N to plants have been proposed but they are empirical and appear to have limited value [Bremner, 1965]. Further evidence to support this assertion was provided by Keeney and Bremner [1966], who attempted to characterize the mineralizable N in soils. They studied the changes in total N, fixed NH^-N and hydrolysable and non-hydro-lysable-N, that occurred when soils were incubated under favourable conditions. They found that prolonged incubation led to a marked decrease in all forms of N determined, except fixed NH -^N, and stated that fixed NH^+ is practically unavailable to soil micro-organisms. They also found that there were marked differences among soils with regard to the nature of the N that is most readily mineralized. Consequently, they concluded that any chemical method of obtaining an index of soil N availability, based solely on the determination of a hydrolysable or non-hydrolysable form of soil N, will likely prove unsatisfactory. Keeney and Bremner [1966b] intimated that both aerobic and anaerabic incubation methods, over a 14 day period provided a good index of the availability of N to rye-grass in Iowa soils. They also affirmed that the results obtained with these soils by a chemical procedure involving the extraction of N by boiling water and ^SO^ solution, were closely related to N uptake by rye-grass. They further suggested that this procedure deserved consideration as a routine 46 method o f o b t a i n i n g an in d e x o f s o i l N a v a i l a b i l i t y . A i r d r y i n g and a i r s t o r a g e o f s o i l samples t h e y o b s e r v e d , had marked e f f e c t s on the r e s u l t s o b t a i n e d by the i n c u b a t i o n methods s t u d i e d b ut had l i t t l e e f f e c t on the r e s u l t s o b t a i n e d by the c h e m i c a l method. An i n c u b a t i o n method o f e s t i m a t i n g m i n e r a l i z a b l e s o i l N which does not r e q u i r e p r i o r a n a l y s e s t o d e t e r m i n e t he amount o f r^O r e q u i r e d f o r i n c u b a t i o n was d e s c r i b e d by Keeney and Bremner [1 9 6 7 ] . They found t h a t t h e method was a p p l i c a b l e w i t h o u t m o d i f i c a t i o n , t o s o i l s h a v i n g w i d e l y d i f f e r e n t t e x t u r e s and i s based on t h e f i n d i n g t h a t the amount o f water r e q u i r e d f o r maximal a e r o b i c m i n e r a l i z a t i o n o f N d u r i n g i n c u b a t i o n was p r a c t i c a l l y t h e same f o r d i f f e r e n t s o i l s ( c a . 0.6 ml/g o f s o i l ) , i f the s o i l was mixed w i t h t h r e e t i m e s i t s w e i g h t o f 30-60 mesh q u a r t z sand, b e f o r e i n c u b a t i o n . The method i n v o l v e s t h e d e t e r m i n a t i o n o f t h e (NH^ + N0^ + NOgJ-N pr o d u c e d , when 10 gm s o i l was mixed w i t h 30 gm sand then t r e a t e d w i t h 6 ml 1^0 and i n c u b a t e d o f 30°C f o r 14 days under a e r o b i c c o n d i t i o n s . Many workers have e x p e r i e n c e d d i f f i c u l t y i n a d h e r i n g t o the l a t t e r s t i p u l a t i o n because some b a l a n c e must be m a i n t a i n e d between l o s s o f m o i s t u r e and "adequate" a e r a t i o n . O n l y r e c e n t l y , by the use o f t h i n p o l y e t h y l e n e f i l m s has i t been p o s s i b l e t o a t t a i n a s e e m i n g l y s a t i s f a c t o r y compromise [Bremner, 1972]. An i m p o r t a n t a s p e c t o f n i t r o g e n m i n e r a l i z a t i o n s t u d i e s ( i n f r e q u e n t l y r e p o r t e d ) i s t h e method o f s t o r a g e o r t r e a t m e n t o f sample p r i o r t o i n c u b a t i o n . Robinson [1967] c o n d u c t e d e x p e r i m e n t s w i t h t r o p i c a l s o i l s , t o d e t e r m i n e t he e f f e c t s o f d i f f e r e n t methods o f s t o r i n g s o i l s and s o i l e x t r a c t s so as t o p r e s e r v e t h e m i n e r a l N 47 status. He observed that, neither the addition of chemicals, (toluene, chloroform, amyl alcohol) at the rates commonly used, nor storage at sub-zero temperatures were able to preserve the mineral N status of the soils, completely unchanged, over short periods of time. Further-more, evidence was found which indicated that storage at sub-zero temperatures can affect subsequent mineralization of N during incubation. Robinson did not speculate on possible causes for this, however, he noted that changes occurring in the mineral-N content of both soils and soil extracts during storage were not consistent, and in soils, appeared to be related to biological activity, the concentration of mineral N present, and possibly to other factors as well. The characterization of organic N in soils has been based largely on the extraction of various nitrogenous compounds with different reagents. Recently attempts have been made to correlate the N mineral-ized during incubation with one or more of these N forms (viz. extractable hydrolysable, or non-hydrolysable-N). Stanford [1968] conducted studies on two soils to determine the relationship between the amounts of N extracted by Na-pyrophosphate solutions and the reduction in the capacities of the soils to mineralize N under aerobic conditions. By varying the concentration of pyrophosphate, pH, number of extractions, temperature and duration of extraction, a relationship was established between extractable-N and the N-mineralizing capacity of both soils; despite the fact that the actual amounts of N differed appreciably. Acid hydrolysis of one soil with 6N H^SO^ for 12-16 hours reduced mineralization capacity to zero. Stanford concluded that these findings may have important implications in defining criteria for selecting the 48 c o n c e n t r a t i o n o f an e x t r a c t a n t t o be used i n a s s e s s i n g t h e N s t a t u s o f s o i l s . S t a n f o r d [1968b] a l s o found a c l o s e r e l a t i o n s h i p between t h e d i s t i l l a b l e N e x t r a c t e d from s o i l s ( e i t h e r by Na-pyrophosphate o r M/100 C a C l 2 ) and m i n e r a l i z a b l e N. Freney and Simpson [1969] s t u d i e d t h e m i n e r a l i z a t i o n o f l a b e l l e d and i n d i g e n o u s - N i n s o i l s o f d i f f e r e n t t o t a l N c o n t e n t s f o l l o w -i n g l o n g p e r i o d s o f e q u i l i b r a t i o n w i t h added l a b e l l e d NH^-N and no a d d i t i o n a l c a r b o h y d r a t e s o u r c e . They found a s u b s t a n t i a l m i n e r a l i z -a t i o n o f l a b e l l e d - N i n a l l s o i l s , b e i n g g r e a t e s t i n the n o n - d i s t i l l a b l e a c i d - s o l u b l e f r a c t i o n d u r i n g the f i r s t s t a g e s o f i n c u b a t i o n . In the i n d i g e n o u s N, the a c i d - i n s o l u b l e f r a c t i o n a p p e a r e d t o be the most a c t i v e as d e t e r m i n e d by i t s p e r c e n t a g e change, but i t c o u l d not be i d e n t i f i e d as the main c o n t r i b u t o r t o m i n e r a l N. In one s o i l t h e e f f e c t s o f l e a c h i n g , d r y i n g and h e a t i n g t r e a t m e n t s b e f o r e i n c u b a t i o n , were a l s o d e t e r m i n e d on each f r a c t i o n . The removal o f m i n e r a l N by l e a c h i n g , and d r y i n g t h e s o i l a t 50°C o r 100°C produced some s m a l l e f f e c t s on t h e r a t e o f m i n e r a l i z a t i o n o f both l a b e l l e d and i n d i g e n o u s N i n subsequent i n c u b a t i o n . The p r o p o r t i o n o f the l a b e l l e d N which was m i n e r a l i z e d was g r e a t e r than t h a t o f the i n d i g e n o u s N o v e r t h e same p e r i o d and th u s t h e r e appeared t o be an a c t i v e f r a c t i o n w i t h i n t h e s o i l N. In a d d i t i o n , i n a l l t h e o r g a n i c f r a c t i o n s t h e l a b e l l e d N underwent a g r e a t e r p e r c e n t a g e change than the i n d i g e n o u s N, and thus i t a p p e a r s t h a t t h e r e was an a c t i v e phase w i t h i n each o r g a n i c N f r a c t i o n . A s t u d y o f the m i n e r a l i z a t i o n o f N from s e l e c t e d H i s t o s o l s from W i s c o n s i n , [ I s i r i m a h and Keeney, 1973], r e v e a l s t h a t much o f 49 the m i n e r a l i z a b l e N came from the a c i d s o l u b l e p o r t i o n , m a i n l y hexo-samine-N, amino-N and an u n i d e n t i f i e d N f r a c t i o n . They no t e d a con-s i d e r a b l e m i c r o b i a l t u r n o v e r o f h y d r o l y s a b l e -N t o r e f r a c t o r y -N. T h i s l a t t e r o b s e r v a t i o n i s an i n t e r e s t i n g a l t e r n a t i v e h y p o t h e s i s t o the proposed c l a y m i n e r a l p r o t e c t i o n t h e o r y o f n o n - h y d r o l y s a b l e -N. 3.3.2 S o i l P a r t i c l e - S i z e and N i t r o g e n M i n e r a l i z a t i o n I t has a l r e a d y been mentioned t h a t the f r a c t i o n a t i o n o f s o i l samples i n t o s i z e groups l e s s than 2 mm i n c r e a s e d m i n e r a l i z a b l e N v a l u e s . Robinson [1967] undertook an i n v e s t i g a t i o n t o d e t e r m i n e whether the s t a n d a r d a i r d r y s i z e f r a c t i o n (<2 mm), p r e v i o u s l y used i n i n c u b a t i o n work to p r o v i d e a p r a c t i c a l i n d e x ,of p o t e n t i a l l y a v a i l a b l e N, c o u l d be improved upon, by c o n s i d e r i n g o n l y a l i m i t e d s i z e range o f s o i l p a r t i c l e s . He s t u d i e d f r a c t i o n s r a n g i n g i n s i z e from 2 mm t o <0.089 mm by g r i n d i n g samples t o pass the p r e s c r i b e d mesh s i z e s . He f o u n d no e v i d e n c e t o s u g g e s t t h a t r e f i n e d s o i l f r a c t i o n s o f <2 mm would be an advantage f o r the d e t e r m i n a t i o n (by i n c u b a t i o n methods), o f m i n e r a l i z a b l e N. D i f f e r e n c e s were more n e a r l y r e l a t e d t o s i m p l e p h y s i c a l f r a c t i o n a t i o n o f t h e s o i l . O r g a n i c -C, t o t a l N and s i l t c o n t e n t s o f the v a r i o u s f r a c t i o n s were p o s i t i v e l y and s i g n i f i c a n t l y c o r r e l a t e d w i t h m i n e r a l i z a b l e N; c o a r s e p l u s f i n e sand c o n t e n t o f the d i f f e r e n t f r a c t i o n s was n e g a t i v e l y c o r r e l a t e d w i t h m i n e r a l i z a b l e N. S i l t but not c l a y ( w i t h the NaOH method o f d i s p e r s i o n ) was shown t o be a s s o c i a t e d w i t h t h e a c t i v e o r g a n i c m a t t e r i n terms o f m i n e r a l i z -a b l e N v a l u e s i n the s o i l s s t u d i e d . 50 The approach used by Robinson [1967] was taken a few steps further by Chichester [1969] who investigated N mineralization in sand, s i l t (coarse, medium and fine), and clay (2-lum, 1-0.5um, 0.5-0.25pm and <0.25um) fractions. He reported that the total C and total N of the soil size fractions increased and C/N ratios decreased with decreas-ing particle-size. The percent N mineralized was greater for the finer size fractions, both in surface and subsoil samples. Differences in N mineralized for the various fractions was attributed mainly to the presence of different portions of readily versus diff icult ly extractable chemical forms of N. The amount of N mineralized was correlated highly with the water extractable portion of the total N, (r = 0.978 and 0.962 for surface and subsoil respectively). Chichester [1970] further 15 supported his earlier findings by measuring the mineralization of N incorporated into the various size fractions. He observed that although tagging was highest in the >53um fraction, total N and the amount of N mineralized after 2 weeks of water-logged incubation was greatest in the finer fractions. Craswell and Waring [1972a,b] also investigated the effects of grinding on the mineralization of N from a range of soil types and found an increase in N mineralized when 2 mm soil was ground to <0.18 mm and <0.05 mm mesh sizes. The increased mineralization was highest in soils containing 2:1 clay. They attributed their results to an increase in the supply of available organic matter to soil microbes, an effect similar to that obtained when an art i f ic ia l substrate is added to the soi l . Swift and Posner [1972] investigated the distr i-bution of soil organic-N as a function of particle-size and found that 51 organic matter intimately associated with clay minerals had a higher N content than uncomplexed organic matter. They also found that most of the soil N was extracted by pyrophosphate and alkali . 3.3.3 Biological Stability of Organic Nitrogen Bremner [1967] has reviewed the literature on nitrogenous compounds in the soil and the reader is referred to that article for a more exhaustive discussion. However, some of his observations on their biological stability are pertinent to the discussion and will be mentioned here. The biological stability of organic nitrogen in soils is indicated by the fact that only 1-3 percent of this N is mineralized by soil organisms during the growing season. It is diff icult to account for this observation because the organic N compounds added to soils as plant and animal residues are not particularly resis-tant to decomposition and nitrogenous complexes similar to these in plant and microbial products (viz. chitin, proteins, nucleic acids, etc.) are degraded fairly rapidly in soils. It should not be over-looked that the apparently high resistance of nitrogenous complexes to microbial decomposition has considerable practical significance as i t affects the amount of organic N made available for plant growth during the growing season. Two theories, which attempt to explain the stability of organic N in the soil have received wide attention. The f irst argues that organic-N compounds in soils are stabilized by reacting with other organic constituents while the second states that such compounds are stabilized through adsorption by clay minerals. 52 Waksman and Iyer [1932] postulated that 1ignin-protein com-plexes were formed in soils by the reaction of the carbonyl groups in lignin with the amino groups of proteins. They showed that protein in complexes obtained by acidification of alkaline solutions of lignin and protein, was highly resistant to mineralization. This finding has been substantiated by several workers, however, no lignin-protein complex has to date been isolated from the so i l , which brings their existence into question. The possibility that inorganic forms of N in soils may be converted to relatively stable organic forms through reaction with soil organic constituents is of interest because i t has been shown that both NH^  and NC^  can react with soil organic matter under field conditions to form compounds which are highly resistant to mineralization, Bremner and Fuhr [1966], Broadbent and Burge [i960]. Vail is and Jones [1973] found that the mineralization of leaf and leaf l i t ter of two legumes mixed with soil was related to the polyphenol content of the materials, and not to their lignin content. Materials containing the higher polyphenol content had the lower net mineralization. This seems to support the hypothesis of Davies [1971] who suggested that the formation of polyphenol-protein complexes in senescing leaves was responsible for the resistance to microbial decomposition of l i t ter from certain shrubs and trees. It has also been shown that tannins can protect protein against decomposition by both soil and rumen organisms [Basaraba and Starkey, 1966]. The second theory orginated with the work of Ensminger and Gieseking [1942] who showed that enzymatic hydrolysis of proteins was 53 markedly reduced by the presence of clay minerals. This finding has since been substantiated by numerous workers and clays have been shown to have a protective effect on plant residues and retard enzymatic hydrolysis or microbial decomposition of organic phosphorus compounds including nucleic acids. It is noteworthy that in all investigations conducted thus far, montmorillonitic clays have been reported to have a greater protective effect than other clays, while kaolinite exhibits l i t t l e or no protective effect. Indeed, Estermann et al_. [1959a,b] have shown that kaolinite can actually increase the rate of bacterial decomposition of protein, presumably by acting as a concentrating surface for adsorbed substrate and exoenzymes. The two theories discussed have gained considerable acceptance, but other explanations of the biological stability of the N compounds in organic matter have emerged from tracer studies of the decomposition of plant materials in soils. Broadbent and Norman [1947] and Broad-bent [1948] found that by adding an energy rich material to the so i l , the rate of decomposition of soil organic matter and the rate of mineralization of soil N increased. They concluded that the resistance of organic N complexes to microbial degradation was "more apparent than real" and results largely from the absence of enough energy material to support a vigorous microbial population. Subsequent work by Jenkinson [1966] found the much-discussed "priming action" can be positive or negative and has l i t t l e practical significance. Jenkinson [1965a,b] studied the decomposition of labelled rye-grass and found that the amount of C lost by the decomposition of this material in one year.was not greatly influenced by soil pH, or texture or by the 54 organic matter already present in the soi l . This led Jenkinson to the conclusion that, when plant residue are added to soil " it is primarily the chemistry (and biochemistry) of the plant residue that determines its stability, not the nature of the soil in which it is decomposing," [Bremner, 1967]. This latter statement is not altogether acceptable as soil conditions can markedly influence microbial popu-lations which in turn must affect the decomposition processes. It has been proposed that clay minerals may be partly respon-sible for the non-hydrolysable N fraction in soils. Freney and Miller [1970] investigated the stability of certain clay minerals and synthetic clay mineral-nitrogen complexes in boiling 6N HC1, to determine whether clay minerals in soil could protect N compounds or ions from solution. They concluded from their study that a clay mineral protection theory for non-hydrolysable N was tenable. It seems clear from the preceding discussion that the question of the biological stability of soil organic -N is s t i l l unresolved. The 1ignin-protein complex theory of Waksman and Iyer [1932] has lost ground within recent times, while the recently proposed polyphenol-protein complex theory [Vailis and Jones, 1973] is s t i l l to be put to the test. The long-standing clay mineral protection theory has been bolstered by much substantive evidence, however, unequivocal proof is s t i l l lack-ing. Perhaps the solution to the problem is to be found in a study of the organo-mineral complex occurring in the so i l . By observing the rate of mineralization of N from such substances, some light might be shed on this perplexing question. 3.4 Organic Phosphorus in Soil and Its Mineralization 55 3.4.1 The Nature of Organic Phosphorus and Its Determination In most mineral soils one-half to two-thirds of the total phosphorus is organic; but proportions varying from 4 percent to 90 percent have been reported [Williams and Steinbergs, 1958]. The major groups of organic phosphorus compounds that have been identified in soil are, phospholipids, nucleic acids, necleotides and the inositol phosphates. There is evidence that both phosphoproteins and a sugar phosphate (glucose-l-phosphate) may also exist [Anderson, 1967], The relative content of the latter is however considered very low. Phospholipids account for about one percent of the total organic phosphorus [Hance and Anderson, 1963] and nucleic acid-phosphorus not more than 5-10 percent. The inositol phosphates may account for as much as 60 percent of the total organic phosphorus but a wide range of values have been reported [Anderson, 1967]. This therefore means that a significant and in many instances,.major protion of the total organic phosphorus is s t i l l unidentified. Present indications are that i t is likely to be of microbial origin [Cosgrove, 1967], Early work dealing with the nature of organic P showed that the amount present in a soil tended to be related to soil N and organic C. Black and Goring [1953] suggested that organic matter in mineral soils contained C, N and P in the approximate ratio 110:9:1; the ratio was found to be wider in organic soils. Recent studies suggest, how-ever, that there is much variability in the amount of organic P in the soil and i t appears that a better correlation exists between the 56 amounts of organic S and organic N, which has been found to be quite constant for a wide range of soils. This observation is also supported by the reports of Barrow [1961] and Williams [1966], that although C, N and S ratios usually fall within broad limits the organic P ratio with respect to any one or all of these elements has a much greater variability. Further evidence from Canadian soils [McKercher, 1966] indicated that organic P is not closely related to either N or C and suggests that organic P accumulation may be largely independent of soil organic matter build up. There are no direct methods at the present time for the determination of organic P, and McKercher [1968] cites this as one of the major difficulties in its study. In support of McKercher's observation, the author is of the opinion that variability in the organic P content of soil is strongly influenced by the imprecision of the methods used for its determination. There are two basic methods of organic-P determination in use currently; the f i rst is based on the measurement of P in an extract such as the method of [Mehta et al_., 1954]. In the second method, organic P is measured by difference between inorganic P extracted from ignited and unignited soil samples as described by Saunders and Williams [1955]. Both these methods are subject to error. The most probable source of error in the extraction methods is thought to be the partial mineralization of organic P during extraction, thus giving low values; whereas, in the ignition methods, the ignition is believed to increase the solubility of inorganic P containing constituents, giving high values. 57 The factors affecting the P content of organic matter have been reviewed by Barrow [1961]. He listed the following as being of importance: (a) method of analysis, (b) method of calculating P content of organic matter, (c) the effect of sampling depth, (d) the effect of alkaline soil conditions, (e) the effects of climate and of period of soil formation, (f) drainage conditions, (g) cultivation, (h) parent material, and (i) clay content. The possible role of clays and in particular the role*of organo-clays in stabilizing organic P, has received very l i t t l e attention. Hussain and Kyuma [1969] studied the charge characteristics and phosphate fixation capacity of soil organo-mineral complexes and found, not surprisingly, that organic matter suppressed the P (inorganic) fixing capacity of clays; unfortunately, however, they did not attempt to measure how much organic P was retained by the organic matter complexed by the clays. Greaves and Wilson [1969] found that nucleic acids were strongly adsorbed by montmorillonite, (ca. 16 mg NA/mg monmorillonite from a concentrated solution) and that lattice expansion increased linearly with increased o o adsorption of nucleic acid up to 18-19 A for DNA, and 27 A for RNA. One mechanism of bonding proposed was the bridging of clay via polyvalent ions to the negatively charged phosphate group of the nucleic acids. 58 It has not been determined how much and to what extent nucleic acids adsorbed by clays are susceptible to mineralization, and whether or not a clay-mineral protection theory, akin to that proposed for organic N (non-hydrolysable) is tenable. 3.4.2 Mineralization of Organic Phosphorus It is generally believed that organic P is of l i t t l e or no direct value to plants and must be mineralized to become available, despite the fact that Estermann and McLaren [1961] showed that lecithin and phytin were able to supply barley with P under sterile conditions. The mineralization of phosphorus as a means of supplying P to plants has been a subject of much research. More recently investi-gations into the mineralization of organic P have taken one of two general approaches. The f i rst seeks to measure the phosphatase activity of organisms isolated from so i l , while the second measures the phosphatase activity of the soil as a whole, which is composite of the activities of soil microflora and any free enzymes present, [Cosgrove, 1967]. Greaves, Anderson and Webley [1963] estimated that as much as 50 percent of the microorganisms present in soil and in plant roots possess an enzyme system capable of hydrolyzing sodium phytate. When used as the sole source of P under sterile conditions, phytin was found to be more effective than lecithin; the activities of root phosphatases mediating the hydrolysis. Some workers have found derivatives of phytin (viz. inositol phosphates) quite resistant to hydrolysis and in view of the conflicting evidence so far available 59 i t can only be concluded that there exists in soil a wide range of microorganisms capable of dephosphorylating all known organo-phosphates of plant origin. Precise investigations of the processes involved in the mineralization of soil organic P have been hampered by a lack of knowledge of its chemical nature. It has been found that an increase in inorganic P in soils following incubation, agreed closely with a decrease in organic P, [Thompson, Black and Zoellner, 1954; Van Diest and Black, 1959]. The work of Thompson and Black [1949] and Thompson et al_. [1959] indicated that the rate of organic P mineralization was positively correlated with the rates of N, and C mineralizations. Further, the amounts of mineralized organic P, N, and C corresponded roughly to the proportions in which these constituents occurred in soil organic matter. The behaviour of organic P, however, is not always analogous to that of N and C and for 50 unlimed soils, Thompson et al_. [1954] found that the mineralization of organic P increased markedly with pH but that of organic C and N did not. The release of P from organic matter as a result of liming has received some attention. Hinds [1970] found that liming increased the availability of P in some acid tropical soils largely through the mineralization of organic matter. Another factor which influences mineralization is temperature, and Van Diest and Black [1959] noted that mineralization rate increased markedly above 30°C. 60 Workers in the Soviet Union have attempted to exploit the microbial release of P to increase crop yields by inoculating seed with Bacillus megatherium var. Phosphaticum (phosphobacterin), [Cooper, 1959]. Oats, wheat, millet, corn and soyabeans, i t is claimed, have benefitted from inoculation. In extensive field trials in different climatic regions of the United States, however, phosphobaterin failed to have any effect on wheat yields; although a 1.5 percent increase in tomato yields due to inoculation was recorded by Smith et_ a]_. [1959]. In the init ial stages of decomposition most plant remains seem to contain sufficient inorganic P to support the metabolic and synthetic processes of the soi l 's microbial population. It thus appears that l i t t l e mineralization of plant organic P takes place during the f i rst three months of decomposition [Cosgrave, 1967]. Investigations to determine the crit ical level or balance point between immobilization and mineralization of P in natural carbonaceous materials indicate that 0.2 percent P is the approximate crit ical level, Fuller et_ a]_. [1956]. If the system contains less than 0.2 percent P, net immobilization takes place, and both plant and native soil inorganic P are utilized by the microorganisms. There is evidence that a large portion of the organic P of animal manure is resistant to mineralization, although in some instances a readily mineralized fraction has been observed. Eventually, most i f not a l l , the P is mineralized, but the nature of the more resistant fraction is unknown Korableva [1951]. A more complete understanding of the factors affecting P mineralization is perhaps contingent upon further characterization of P in organo-mineral complexes, and more importantly, the role of clays and sesquioxides in complexing organic P. 61 Williams and Saunders [1956] studied the distribution of phosphorus in particle-size fractions of some Scottish soils and found that the clay and s i l t fraction contained over 80 percent of the total P. The content of organic P was highest in the clay fraction which suggests possible interaction between clay and P-containing organic compounds. It is also probable that during the "humification" process, mineralized phosphate becomes sorbed by organic colloids, thereby rendering P unavailable. This P-containing organic colloid could then become adsorbed or bonded to a mineral species to form an organo-mineral complex. The complexation reaction could possibly occlude the adsorbed phosphate, thus giving rise to the "difficultly mineralized," uncharac-terized portion of the organic Pl fraction. This hypothesis appears tenable in the light of the results of Mayer and Thomas [1970], who fractionated organic matter extracted from soil into three molecular weight ranges by Sephadex gel f i l trat ion. The f i rst fraction which had an approximate lower molecular weight limit of 50,000 contained 36 percent of the total organic phosphorus in the extract. The second fraction with intermediate molecular weight in the range 1,000 - 50,000 contained 31 percent of the P; whereas the third fraction, with less than 1,000 molecular weight range, contained only 7.8 percent of the organic P in the extract. Inositol phosphates were detected in the second and third fractions but not in the f i rst . The authors concluded that the inositol phosphates in the third fraction were in the free state while those in the second fraction were either in a polymer form or bound to other organic compounds. Stability studies on similarly fractionated organic matter by Veinot and Thomas [1972] 62 indicated that changes in molecular weight distribution could occur even with mild extraction techniques and that inositol phosphates may be present in the high molecular weight fraction even though i t could not be detected. 3.5 Organic Sulphur in Soil and Its Mineralization 3.5.1 Organic Sulphur The increasing number of publications on soil sulphur and its transformations within the soil environment attests to the increas-ing awareness of, and the rising concern about sulphur deficiency in agricultural crops. Of the major nutrient elements required by plants, sulphur has perhaps received the least attention and many aspects o f its chemistry and metabolism in the soil are yet to be elucidated. Comparatively l i t t l e work has been done on the organic sulphur content of soils, however, the results of many workers indicate that most of the sulphur, in the surface horizons of soil under pasture in humid and semiarid regions is in the organic form, [Walker and Adams, 1958; Williams and Steinbergs, 1958; Lowe, 1969]. This was deduced from the close relationships found between total S, and total C and between total S and total N. In many of the soils studied by Williams [1962], these elements were in the approximate ratio of 140:10:1.3 for C, N and S respectively. This conclusion is further supported by the small quantity of inorganic S found in the soils, [Freney, 1961; and Williams and Steinbergs, 1962]. Tabatabai and Bremner [1972a,b] in their study of the distribution of total and available sulphur of Iowa soils reported 63 that 95-98 percent of the total S in those soils was organic. Data from soils representative of the major soil series in Iowa showed their C:N, N:S, and C:N:S ratios averaged 10.9:1, 6.5:1, and 109:10:1.5 respectively. Similar results were obtained by Bettany, Stewart and Hal stead [1973] for a large group of Saskatchewan soils. They found that C:N:S ratios ranged from 58:6.4:1 in the arid chernozemic brown soils to 129:10.6:1 in the leached grey wooded soils. Tabatabai and Bremner [1972a] observed that inorganic sulphur represented only a minor fraction (an average of 3 percent) of total S and occurred entirely as sulphate. Analysis of profile samples revealed that the N:S ratio tended to decrease with depth and was less than four in several subsoils. In drier areas gypsum (CaSO^  • 2H20) and epsomite (MgSO^  • 7H20) can accumulate in the soil profile and minor amounts of insoluble minerals such as sphalerite (ZnS) and chalcopyrite (CaFeS2) may occur. Pyrite and Marcasite (FeS2) occur frequently in shales and other sedimentary rocks including limestone. Inorganic sulphate may also occur as a co-crystallized impurity in CaCO^  of calcareous soils and as adsorbed sulphate in many acid subsoils and some surface soils, [Williams et al_., I960]. The factors affecting the adsorption of sulphate by soil have been summarized by Harward and Reisenauer [1966] and the mechanisms by Beaton [1968]. It has been found that clay mineral particularly the kaolinite types, and the hydrous oxides of iron and aluminum have a marked tendency to retain sulphate. The equilibrium pH of the soil is also a factor, retention of sulphate increasing as pH decreases. The concentration of sulphate and the 64 pre s e n c e o f o t h e r a n i o n s a l s o a f f e c t a d s o r p t i o n . S u l p h a t e i s - g e n e r a l l y c o n s i d e r e d t o be weakly h e l d , w i t h t h e s t r e n g t h o f r e t e n t i o n i n the o r d e r ; phosphate > s u l p h a t e > n i t r a t e = c h l o r i d e . The a s s o c i a t e d o r exc h a n g e a b l e c a t i o n a l s o p l a y s a r o l e which f o l l o w s t h e l y o t r o p i c + 2+ 2+ 2+ 2+ + + + + + series; H > Sr > Ba > Ca* > Mg*. > Rb > K • > NH 4 > Na > L i S o i l o r g a n i c s u l p h u r c o n s i s t s o f two f r a c t i o n s : (a) carbon-bonded s u l p h u r ( C - S ) , and (b) non-carbon-bonded s u l p h u r . The l a t t e r group i s co m p r i s e d o f the e s t e r s u l p h a t e s , where S i s sep-a r a t e d from C by a n o t h e r atom, u s u a l l y oxygen, (C-O-S) and i n some i n s t a n c e s n i t r o g e n (C-N-S). The carbon bonded f r a c t i o n (as d e t e r m i n e d by r e d u c t i o n w i t h Raney n i c k e l , Lowe and DeLong, 1963] i n c l u d e s a l l forms o f o r g a n i c S o t h e r than c o v a l e n t s u l p h a t e s , and most a l k y ! s u l p h o n e s . The S c o n t a i n i n g amino a c i d s and p r o t e i n s c o n s t i t u t e a p a r t o f the C-bonded f r a c t i o n , and the t y p e s o f o r g a n i c S l i n k a g e s i n c l u d e d i n t h i s group c o n s i s t s . o f the f o l l o w i n g : ( i ) d i s u l p h i d e (R-S-S-R), ( i i ) s u l p b y d r y l (R-S-H), ? ( i i i ) s u l p h o x i d e (R-S-R) o ( i v ) s u l p h i n i c a c i d (R-S-OH) o (v) s u l p h o n e (R-S-R) 4- . 0 0 i ( v i ) s u l p h o n i c a c i d (R-S-OH) o 65 R e s u l t s o b t a i n e d from r e d u c t i o n w i t h h y d r i o d i c a c i d , and h y d r o l y s i s w i t h a c i d and a l k a l i [ F r e n e y , 1961, 1965; Lowe and DeLong, 1961; Lowe, 1960] s u g g e s t t h a t an average 51 p e r c e n t o f the t o t a l S i s p r e s e n t as o r g a n i c s u l p h a t e s w h i l e i n o r g a n i c s u l p h a t e a c c o u n t s f o r 7 p e r c e n t . A p p r o x i m a t e l y 41 p e r c e n t appears t o be p r e s e n t i n the C- bonded form. Some o f the o r g a n i c s u l p h a t e s a r e a p p a r e n t l y q u i t e l a b i l e , b e i n g e a s i l y c o n v e r t e d t o i n o r g a n i c s u l p h a t e , by h y d r o l y s i s [ S p e n c e r and F r e n e y , 1960; W i l l i a m s and S t e i n b e r g s , 1959], h e a t i n g [ B a r r o n , 1961] and by g r i n d i n g [ F r e n e y , 1961]. Few o r g a n i c S compounds i n the f r e e s t a t e have been i s o l a t e d from s o i l . Shorey [1913] e x t r a c t e d t r i t h i o b e n z a l d e h y d e from s o i l and p o s t u l a t e d t h a t i t was formed by the r e a c t i o n o f h^S from b a c t e r i a w i t h b e n z a l d e h y d e from decomposed l i g n i n . Putnam and Schmidt [1959] found t r a c e s o f f r e e c y s t i n e i n e t h a n o l e x t r a c t s o f a n o n - r h i z o s p h e r e s o i l , w h i l e Paul and Schmidt [1961] found m e t h i o n i n e s u l p h o x i d e , c y s t i n e , and m e t h i o n i n e , i n s o i l s i n c u b a t e d w i t h g l u c o s e and KNO^. S e v e r a l workers have shown t h a t c y s t i n e and m e t h i o n i n e do o c c u r i n s o i l s i n combined form and a r e r e l e a s e d upon h y d r o l y s i s [Sowden, 1955-58, S t e v e n s o n , 1956]. The s u l p h a t e e s t e r s , which can be reduced t o H^S by h y d r i o d i c a c i d o r r e a d i l y h y d r o l y z e d t o i n o r g a n i c S, may e x i s t as s u l p h a t e d p o l y s a c c h a r i d e s , p h e n o l i c s u l p h a t e s , c h o l i n e s u l p h a t e s o r s u l p h a t e d l i p i d s , [ F r e n e y , 1967]. I t was f u r t h e r n o t e d by Freney t h a t the p e r c e n t a g e o f t o t a l S which o c c u r s as e s t e r s u l p h a t e i n c r e a s e d , and t h a t as carbon-bonded S d e c r e a s e d w i t h i n c r e a s i n g sample d e p t h ; and t h a t e s t e r s u l p h a t e may a c c o u n t f o r more than 70 p e r c e n t o f the t o t a l S i n s u b s o i l s . 66 3.5.2 Sulphur Mineralization Contributions to the sulphur nutrition of plants may be derived directly from sulphur dioxide (SG^ ) uptake from the atmosphere, from rain-water which has absorbed SG^  from the atmosphere, or sulphate applied directly to the soils as a fert i l izer. Apart from these sources plants are dependent upon the conversion of organic S in soil organic matter and plant and animal residues, to inorganic sulphate, to meet their requirements. It is generally accepted that plants take up S in the form of sulphate, although they can assimilate i t in the form of amino acids Bardsley [I960]. Parker [1927] suggested that the conversion of organic S to sulphate was initiated by enzymes excreted by plant roots, but i t is now widely believed that this transformation is mainly accomplished by soil microbes. This means therefore, that part of the S released from the organic matter is used by the micro-organisms for cell synthesis during proliferation, while other portions are oxidized to sulphate and is released for plant growth. Evidently mineralization and immobilization are concurrent processes, the net result being determined by numerous factors, principally the composition of the substrate. Studies of the decomposition of pure organic substances in sand inoculated with a soil extract showed that mineralization of S was mainly related to the concentration of S in the mixture, Barrow [I960]. Similar relationships appear to hold when plant material is incubated with soi l . Barrow [1960, 1961] found that no S was mineral-67 i z e d from p l a n t m a t e r i a l i f i t c o n t a i n e d l e s s than 0.13 p e r c e n t S; above t h i s l i m i t i n g v a l u e t h e r e was a p o s i t i v e c o r r e l a t i o n ( r = 0.821, p < 0.001) between p e r c e n t S o f the m a t e r i a l i n c u b a t e d and the m i n e r a l S r e l e a s e d . Barrow [1961] a l s o noted a broad r e l a t i o n s h i p between S m i n e r a l i z e d from o r g a n i c m a t e r i a l s w i t h i n 12 weeks and t h e C:S r a t i o R a t i o o f C t o S < = 200 200 - 400 > = 400 Only M i n e r a l i z a t i o n E i t h e r M i n e r a l i z a t i o n Only I m m o b i l i z a t i o n o f o f S Found o r I m m o b i l i z a t i o n o f o f S Found S o f t h e s e m a t e r i a l s , (see t a b l e a b o v e ) . N e l s o n [1964] found a s i g n i f i -c a n t r e l a t i o n s h i p between S m i n e r a l i z e d from n a t i v e s o i l o r g a n i c m a t t e r a f t e r s i x months and the o r g a n i c S c o n t e n t o f a s o i l , however, o t h e r workers have f a i l e d to d e monstrate such a r e l a t i o n s h i p . Indeed, W i l l i a m s [1967] found t h a t f o r a number o f E a s t e r n A u s t r a l i a n s o i l s t he amount o f S m i n e r a l i z e d by i n c u b a t i o n a t 30°C c o u l d not be r e l a t e d t o any s i n g l e s o i l p r o p e r t y . S i m i l a r l y , Haque and Walmsley [1972] showed t h a t the amount o f S m i n e r a l i z e d i n a group o f West I n d i a n s o i l s was u n r e l a t e d t o the t o t a l amounts o f C, N o r S: w h i l e T a b a t a b a i and Bremner [1972b] found t h a t f o r Iowa s o i l s , m i n e r a l i z a b l e S was not s i g n i f i c a n t l y c o r r e l a t e d w i t h t o t a l S , s u l p h a t e -S, o r g a n i c c a r b o n , t o t a l N, m i n e r a l i z a b l e N and a r y l s u l p h a t a s e a c t i v i t y . Thus, i t appears t h a t f o r S m i n e r a l i z a t i o n f a c t o r s o t h e r than the above may be more i m p o r t a n t , p o s s i b l y the c o m p o s i t i o n o f the s o i l m i c r o b i a l p o p u l a t i o n . I t has been s u g g e s t e d t h a t i t i s the S c o n t e n t o f the r e c e n t l y added o r g a n i c m a t t e r which d e t e r m i n e s the amount o f S m i n e r a l i z -68 ed rather than the S content of the total soil organic matter [Barrow, 1961]. However, there has been l i t t le supporting evidence for this theory among recent publications. Foremost among the factors affecting sulphur mineralization in soils are, temperature, moisture content, pH, and plant growth. Williams [1967] examined the effects of drying, soil temperature, soil moisture and the addition of antiseptics and lime (calcium carbonate) on the mineralization of S, and compared i t with the effects on the mineralization of nitrogen. He found that decreasing soil temperature from 35°C resulted in decreased mineraliz-ation of S until at 10°C no sulphate was released from the soil organic matter. At low moisture levels (<15 percent) mineralization of.sulphur (N also) was considerably retarded. Formation of sulphate was also reduced at high moisture levels (>40 percent), approaching saturation. At these moisture levels nitrification was retarded and mineral N accumulated in the NH -^form. Calcium carbonate had a positive effect on S mineralization whereas formaldehyde and toluene suppressed mineralization. It was concluded from the study that the effects of temperature, soil moisture, toluene, formaldehyde and the addition of calcium carbonate to soils, on the mineralization of S were similar to their effects on the mineralization of N, [Williams, 1967]. Freney and Spencer [1960] showed that in the presence of plants a soil released more inorganic sulphate than when left fallow. Two possible explanations were proffered to account for the increased mineralization of S. Firstly, increased microbial activity due to an increase in the number of microorganisms associated with the rhizosphere of the plants and secondly that enzymes excreted by the 69 plant roots catalyzed the decomposition of the soil organic matter. The measurement of the mineralization of sulphur from soil organic matter in incubation studies may be obscured by at least two distinct mechanisms: 1. Failure to extract the released sulphate, due to adsorption -2 -2 of SO^  on soil colloids or the precipitation of SO^  by 2+ 2+ large amounts of Ca or Ba present. 2. The inclusion of fine roots and other organic residues of low sulphur content with the so i l , resulting, in the immobiliz-ation of any sulphate released from native soil organic matter. When sulphate is extracted from soi l , the extract, (depending upon the extractant) almost invariably includes, soluble sulphates plus varying proportions of adsorbed and organic-bound sulphates. This has led to the overestimation of available sulphur from soils Barrow [1961]. Kowalenko [1973] found that of four widely used extractants of sulphate-S from soils, [viz. 0.15 percent CaC^, Williams and Steinbergs, 1959; MaOAc pH 4.8, Jordan and Bardsley, 1958; 0.5 M NaHC03 pH 8.5 Kilmer and Nearpass, 1960; and 0.5 M Na-Phosphate buffered at pH 7.0, Bart, 1969], the calcium chloride extractable sulphate was the most closely related to microbial activity for the group of soils studied. Within this same group of soils i t was also shown that an increase in the solution to soil ratio, resulted in the extraction of significantly larger amounts of sulphate from two of the soils. Mineral sulphur in the form of hydrogen sulphide may be formed in soil under reducing conditions due to the decomposition of proteins 70 or the reduction of sulphate, Vamos [1964]. The accumulation of sul-phide in Japanese paddy soils has been noted, [Takijima, 1962], but the evolution of free hydrogen sulphide from these soils was slight. The presence of free hydrogen sulphide was found to be dependent upon the soil pH and the iron and manganese content. Swaby and Fedel [1973] examined a large number of Australian soils for their ability to produce sulphate from sulphur, and sulphide from added sulphur, sulphate and cystine. They found that almost one half of the soils oxidized sulphur very slowly or not at a l l , due to the absence of Thiobacilli (esp. Th. thiooxidans). Fifty to seventy percent of the soils were able to reduce sulphur and cystine to sulphide, however, the reduction of sulphate was very rare. They did not speculate on the possible cause of this, but the level of NO^ -N might have been a factor. No significant correlations were found between soil properties, init ial microbial counts, classes of microorganisms detected and metabolic activities; although, certain trends were noted. The conclusion by Swaby and Fedel was that any of the soil properties measured could affect the occurrence or activities of the organisms capable of producing sulphate or sulphide. Of the numerous studies on sulphur and its transformations in the soil no attention has yet been given to possible role of organo-mineral complexing in the sulphur metabolism of soils. No data are currently available on the distribution of S in particle size separates or of the percent S in naturally occurring organo-mineral complexes. In view of the fact that as much as ninety to ninety five percent of total S in soils is in the organic form, this would seem to be a fertile area for research. 71 3.6 Conclusion From the foregoing discussion i t can readily be concluded that there is currently a large body of information concerning the nature of the N, P and S in soils and their relationship in soil organic matter. However, i t is equally clear that our knowledge is far from complete, and that there are many fractions of the soil organic N, P and S which are yet to be characterized. This is particularly true of the organic matter closely associated with the mineral portion of the soi l , giving rise to soil organo-mineral complexes. It seems reasonable to suggest that the diff icult ly extractable portions of organic N, P and S may in large measure be due to the existence of organo-mineral complexes, and much useful information could be gained by a more extensive study of these substances. Also, the rate and extent of mineralization of organic compounds containing these elements may be directly or indirectly influenced by the nature of the bonding between mineral and organic matter. Recently the development of ultrasonic dispersion methods has provided a means whereby organo-mineral complexes can be isolated from soils with a minimum of alteration. Consequently a study of the distribution of N, P and S in organo-mineral complexes and the extent to which they can be mineralized appears to be warranted. 72 E X P E R I M E N T A L S T U D I E S 4. ULTRASONIC DISPERSION TECHNIQUES AND THEIR EFFECTS ON SOIL CONSTITUENTS 4.1 Objectives The objectives of this investigation were as follows: 1. To determine the optimum conditions for the ultrasonic dispersion of soils and to study the dissolution effects of ultrasonic vibration on soil constituents. 2. To determine the effect of probe pitting on the output of ultrasonic energy and to investigate the effect of prior use on the dispersion efficiency of the probe. 3. To develop a rapid and effective procedure for the bulk isolation of organo-mineral complexes by the use of ultrasonic dispersion and a system of continuous flow centrifugation, and to characterize the complexes as far as possible by chemical and physical methods. 4.2 Materials and Methods 4.2.1 Soils Five soils belonging to the Gleysolic order, from the Lower Fraser Valley region of British Columbia were selected for the study. 73 Each horizon A through C was sampled for analysis, however, the data presented are mainly for the A and B horizons. The samples were air dried and ground to pass a 2 mm sieve prior to analysis. Chemical characteristics of the soils are presented in Table 1. 4.2.2 Dispersion The source of ultrasonic radiation used in the present study was a Biosonik BP-III ultrasonic vibrator manufactured by the Bronwill Scientific Company, (Figure 1). Vibrations from this instrument are transmitted to the sample by a titanium probe having a tip diameter of 19 mm. It operates at a frequency of 20 Kc and has a maximum power output of 300 watts. A survey of some of the factors affecting the efficiency of ultrasonic dispersion of soil was conducted in order to develop a standard method for soil dispersion. Various soil/water ratios, lengths of vibration time and intensity settings were investi-gated. The procedures and results are discussed in a following section. Unless otherwise stated the probe was operated at a setting of 80 on the intensity dial; the range being from zero to 100. 4.2.3 Effects of Probe Pitting and Prior use on Dispersion  Efficiency The dispersion efficiency of two identical titanium probe tips, one new and the other badly pitted after approximately 20 hours use were compared. The heating effects and dosimetric measurements for both probe tips were recorded. 74 F i g u r e 1 B i o s o n i c BP - 111 U l t r a s o n i c V i b r a t o r and A c r y l i c R e s e r v o i r f o r c o o l i n g samples d u r i n g v i b r a t i o n P l a s t i c 75 Preliminary investigation of the characteristics of the probe indicated that the dispersion efficiency or energy output was reduced when consecutive dispersions were done without a rest period between each dispersion. Data are presented to demonstrate this effect. 4.2.4 Particle Size Analysis The particle-size distribution of the soils were determined by peroxide-calgon dispersion and hydrometer analysis as described by Day [1965]. 4.2.5 Effect of Electrolyte Solutions on the Dissolution of Soil  Constituents, and the Dispersion Efficiency of Ultrasonic  Vibration Lowe [unpublished data, 1971] found that the release of mineral elements (particularly Fe, Al and Si) upon ultrasonic vibration of soils could be suppressed by dispersing soils in 0.01 M calcium chloride, instead of water. This effect was further investigated by comparing the effects of 0.01 M CaCl 2, 0.01 M BaCl 2, 0.1 M NaCl and 0.03 M NaCl on the dissolution of both organic and inorganic soil constituents and in addition the effects on particle-size distribution. Procedure Quadruplicate 10 g samples of soil were weighed out in 250 ml beakers; 100 ml of water or electrolyte solution added and the suspen-sion thoroughly mixed and then allowed to stand for one hour. Each 76 sample was insonated for 20 minutes at the predetermined setting with temperature of the suspension controlled by a water bath constructed of acrylic plastic fitted with inlet and outlet tubes, (Figure 1). The dispersed soil was washed quantitatively into 250 ml polypropylene centrifuge bottles and centrifuged at 20,000 g for 25 minutes in a Sorvall RC2-B supercentrifuge to remove all particles from suspension. The clear supernatant solution was poured into 200 ml volumetric flasks, made up to mark and analyzed for Na, K, Ca, Mg, Mn, Fe, Al , Si and organic carbon, where present in measurable amounts. The soil remaining in the centrifuge bottles were resuspended in water and duplicates quantitatively transferred to one l i tre cylin-ders and made up to mark. Where the soil was vibrated in an electro-lyte solution, i t was f i rst washed free of any excess, by repeated re-suspension and centrifugation before being transferred to the l i t re cylinder. The cylinders were equilibrated in a constant temperature room and the particle-size distribution determined by the hydrometer method, Day [1965]. 4.2.6 Bulk Isolation of Organo-Clay Complexes Based on the results of Sections 3.1.2 and 3.1.3 the conditions for the operation of the probe were standardized. Further tests were conducted to determine the maximum amount of soil that could be dis-persed at one time without impairing dispersion efficiency. The fractionation of the dispersed soil by sieving and continuous flow centrifugation was investigated to develop a more rapid 77 method for achieving the isolation of particle-size separates. Sand (>53um) was first removed by wet-sieving through a 270 mesh screen and the s i l t (2-53um) and coarse clay (l-2um) separated by're-cycling the suspension through the Sharpies Supercentrifuge, Jackson [1956], The medium clay fraction (0.2-lum) was separated from the suspension by re-cycling through the Sorvall KSB (Szent-Gyorgyi and Blum) tube type continuous flow system and the fine clay fraction (<0.2um) flocculated with calcium chloride and collected by centrifugation. A flow sheet for the sequence of operations plus the percent recovery of the various fractions are presented. The methods of characterization of the particle-size separates are described in Sections 5.2 and 6.2. 4.2.7 Analytical Methods Total carbon was determined by dry combustion in the Leco carbon analyzer while total N was measured by a semi-micro Kjeldahl method. Soil pH was measured in a suspension (1:2.5 soil/water ratio) by the Sargent-Welch model NX pH meter. The following elements; Na, K, Ca, Mg, Mn, Fe, Al and Si were all measured in solution by atomic absorption spectroscopy using the Perkin-Elmer model 306 instrument. Vibrated solutions containing dissolved organic carbon were evaporated to a small volume in the Blichl Model-R Rotovapor at 80°C and carbon determined by the titration method of Walkley and Black 78 [1934]. Carbon in the vibrated NaCl solutions was determined by the Beckman 915 total-organic carbon analyzer. 4.3 Results and Discussion Table 1 shows some chemical characteristics of the soils used in the present study. The soils represent a wide range in total carbon, total N and percent clay. The pH of the samples varied from neutral to moderately acid, with values for the B-horizon being gener-ally higher than for the A. Edwards and Bremner [1967a,b] pointed out that upon ultra-sonic vibration of a soil suspension there was a considerable heat build-up unless some device was used for cooling the suspension. It appeared that the heating effects of ultrasound treatment, i f repro-ducible, could be used as a quick method of estimating the output of ultrasonic energy from the probe, thereby, ensuring the maintenance of the optimum operating conditions. Figure 2 shows the temperature rise curves for the vibration of 100 g water and a soil suspension containing 10 g soil plus 100 g water, using a probe tip which had logged about 10 hours of use, with no obvious pits on the surface. The change in temperature between zero and ten minutes was quite rapid and thereafter the percentage increase in temperature with time decreased sharply, especially after 20 minutes. The curves were found to be reproducible only i f there was an adequate rest period of approximately 25-30 minutes between runs. 79 TABLE 1 Characteristics of Soils Soil pH % Clay % C %N C/N Ratio Classification* Langley Ah •5.5 28 8.0 0.52 15.4 H.E.G. Bt 6.3 43 0.65 0.09 7.2 Hazelwood Ah 5.6 40.1 5.3 0.34 15.6 O.H.G. Bg 6.0 45.8 2.34 0.15 15.6 Hatzic Ap 5.3 51.2 3.8 0.25 15.2 O.G Bg 5.8 49.8 1.32 0.13 10.2 Cloverdale Ap 5.9 18 5.5 0.44 12.5 H.E.G. Btg 7.0 32 0.24 0.02 12.0 Delta Ap 6.1 19.9 3.4 0.21 16.2 S.O.H.G. Bg 6.5 14.9 0.32 0.04 8 Canadian System of Soil Classification. H.E.G. = Humic Eluviated Gleysol. O.H.G. = Orthic Humic Gleysol. O.G. = Orthic Gleysol. S.O.H.G. = Saline Orthic Humic Gleysol. 81 After the probe tip used in preparing the curves of Figure 2 had logged over 70 hours of use and had become badly pitted, its "dispersing efficiency" was compared to that of a new probe tip, by plotting the temperature increase of soil suspensions (10 g soil plus 100 g water) for both tips. Temperature measurements were done on triplicate samples at five minute intervals from zero up to 30 minutes. Following this the old tip was refurbished on a lathe and a similar curve prepared. The results shown in Figure 3 demonstrates quite clearly the effect of probe pitting on the thermal effects of ultrasonic vibration. Assuming that the temperature increase is directly related to the out-put of energy from the probe during the early phase of heating, after five minutes the reduction in energy output due to pitting was approximately 67 percent and after 10 minutes about 30 percent. The curves levelled off after about 15 minutes of vibration, and the difference between the curves thereafter remained constant. It is also apparent that the energy output of the refurbished probe tip was less than that of the new tip, and this was further substantiated by dosimetric measurements, although the difference was small. For the f i rst ten minutes of vibration the difference in energy output between the refurbished and the new tip averaged 13.4 percent. This difference is probably due to the difference in the length of the two tips, which was was approximately 3 mm less for the refurbished tip. Interestingly enough this reduction in the length of the probe tip is slightly in excess of the "maximum allowable tip erosion" stated by the manufacturers, which is 3/32 in. or 2.38 mm. 83 Genrich and Bremner [1972b] adopted a simplified version of a technique described by Wells et al_. [1963] for assessing the amount of ultrasonic energy delivered by a probe. The technique is based on the balance measurement of the radiation pressure (dosimetry) that sound waves exert when they are travelling through a liquid. In this method a 400 ml beaker containing 200 ml of water is placed on the pan of a balance and the ultrasonic vibrator positioned so that the tip of its probe is 2 cm below the surface of the water in the beaker. The weight on the balance pan is then determined with the vibrator turned on and turned off and the output of ultrasonic energy from the probe is assessed from the difference between the two values obtained. Genrich and Bremner [1972b] using this technique obtained values of 15.0 - 15.4 g for new and polished tips and only 3.2 - 7.8 g for pitted ones. The radiation pressure exerted by the Biosonik BP-III vibrator in 100 g of water contained in a 250 ml beaker with the probe tip inserted to a depth of 1.5 cm, was measured for both the new and refurbished tips. The values obtained, were 5-6 g and 3-4 g respec-tively, at the intensity setting previously stated. It was also noted during the tests on the energy output of the probe that following each 20-30 minutes of operation i t was necessary to rest the instrument for about half-an-hour to obtain consistent results. When consecutive runs were conducted without the "cooling off period" there was a reduction in the energy out-put during the second run of approximately 30-40 percent. A similar observation has been made by Watson [1970] for a Radyne auto-tuned 84 ultrasonic vibrator, operating at a frequency of 20 KHz with 600 watts of power. Failure to observe the "cooling off period" between dis-persions could result in erratic results and might partially explain the failure of some workers, quoted in the literature review, to achieve satisfactory soil dispersion by this technique. 4.3.1 Ultrasonic Vibration and Soil Suspension pH The efficiency of the waterbath for cooling the vibrated suspensions and the changes in pH accompanying ultrasonic dispersion were measured on duplicates of ten horizon samples, Table 2. Ten g samples of soil in 100 ml of water were vibrated for 20 minutes, and temperature and pH recorded before and after vibration. The average temperature change was less than one degree centigrade with a range of (-1.8-+3.9°C). The variation in the temperature of the vibrated suspensions perhaps reflect changes in water pressure which determined the rate of flow of water through the cooling reservoir. pH changes were very small but positive. The average was 0.28 pH unit and the range 0.1 - 0.5 pH unit. 4.3.2 Effect of Vibration Time and Soil/Water Ratio on Dispersion Quadruplicate 10 g samples of the Hazelwood Ah., were insonated for 20 minutes in 100 ml of water in a 250 ml beaker, which was cooled in a water bath during the dispersion. Pairs of dispersed suspensions were then combined in one l i tre cylinders diluted to mark and the percent clay determined by hydrometer analysis. The results are TABLE 2 85 Effect of Ultrasonic Vibration on Soil pH Soil Before After °C pH °C pH AT°C A pH LA Ah a 20.9 5.50 20.8 5.85 -0.1 0.35 b 21.0 5.50 20.0 5.90 -1.0 0.40 Bt a 20.8 6.30 20.5 6.61 -0.3 0.31 b 21.0 6.32 21.0 6.70 0.0 0.38 HAZ Ah. a 20.8 5.15 21.8 5.42 1.0 0.27 i b 20.8 5.10 19.5 5.35 -1.3 0.25 Bg a 20.8 6.15 19.0 6.44 -1.8 0.29 b 20.8 6.20 20.8 6.50 0.0 0.30 HAT Ap a 20.0 5.30 23.0 5.80 3.0 0.50 b 20.1 5.25 20.8 5.65 0.7 0.40 Bg a 20.0 5.80 23.0 6.13 3.0 0.33 b 20.0 5.80 22.5 6.10 2.5 0.30 CLOV Ap a 20.0 5.90 19.5 6.00 -0.5 0.10 b 20.0 5.90 21.0 6.15 1.0 0.25 Btg a 20.0 7.01 19.5 7.20 0.5 0.19 b 20.1 7.20 24.0 7.30 3.9 0.10 DELTA Ap a 20.5 5.80 20.5 5.91 0.0 0.11 b 20.5 5.82 21.5 5.93 1.0 0.11 Bg a 20.5 6.50 24.0 6.80 3.5 0.30 b 20.5 6.53 18.0 6.81 2.5 0.28 AVG - - - 0.88 0.28 86 p r e s e n t e d i n T a b l e 3. An a n a l y s i s o f v a r i a n c e o f the r e s u l t s r e v e a l e d t h a t the t r e a t m e n t e f f e c t was v e r y h i g h l y s i g n i f i c a n t (p < 0.005) and the l e a s t s i g n i f i c a n t d i f f e r e n c e was 4.14 p e r c e n t (p < 0.01). I t s h o u l d be n oted t h a t the p r e s e n c e o f o r g a n i c m a t t e r a s s o c i -a t e d w i t h the v a r i o u s s i z e f r a c t i o n s and e s p e c i a l l y the <2ym f r a c t i o n p r o b a b l y i n f l u e n c e d the p a r t i c l e - s i z e d i s t r i b u t i o n by a f f e c t i n g p a r t i c l e d e n s i t y . Baver [1956] s t a t e d t h a t : I t i s q u e s t i o n a b l e whether the a c t u a l d e n s i t y o f a p a r t i c l e as i t f a l l s i n water can be a c c u r a t e l y d e t e r m i n e d . In the l i g h t o f the e f f e c t s o f p a r t i c l e - s i z e , shape, and d e n s i t y upon the a p p l i c a b i l i t y o f S t o k e s ' law t o mechan-i c a l a n a l y s i s , i t s h o u l d be remembered t h a t any p a r t i c l e which i s s e p a r a t e d upon the b a s i s o f i t s s e t t l i n g v e l o c i t y does not n e c e s s a r i l y have the e x a c t s i z e c a l c u l a t e d . I t s e f f e c t i v e o r e q u i v a l e n t r a d i u s c o r r e s p o n d s t o a g i v e n s i z e g r o u p i n g , i n t h a t , a l l p a r t i c l e s w i t h i n such a group have the same v e l o c i t i e s o f f a l l . W hile i t i s p o s s i b l e t o make c o r r e c t i o n s f o r the e f f e c t o f o r g a n i c m a t t e r ( c o n t a i n e d i n the sample) on the p a r t i c l e - s i z e d i s t r i b u -t i o n o b s e r v e d , such an e x e r c i s e appears to be somewhat dubious f o r low o r g a n i c m a t t e r s o i l s , i n the l i g h t o f B a ver's pronouncement. A l s o , i t seems u n l i k e l y t h a t the p e r c e n t o r g a n i c m a t t e r p r e s e n t i n most m i n e r a l s o i l s w i l l s i g n i f i c a n t l y a f f e c t the r e s u l t s o f p a r t i c l e - s i z e d i s t r i b u t i o n a n a l y s i s . From the data p r e s e n t e d i n T a b l e 3 i t i s e v i d e n t t h a t a s o i l / w ater r a t i o o f 1:20 was i n a d e q u a t e f o r complete d i s p e r s i o n o f the s o i l due t o the l a r g e volume o f the s u s p e n s i o n which r e d u c e d t h e i n t e n s i t y o f u l t r a s o n i c r a d i a t i o n . There was, however, no s i g n i f i c a n t d i f f e r e n c e between the 1:5 and 1:10 s o i l / w a t e r r a t i o s f o r the f i r s t t e n m inutes 87 TABLE 3 Effect of Time and S/W Ratio on Dispersion of the Hazel wood Soil S/W Ratio 10 g soil Time (min) 10 - % Clay -15 20 1:5 1:10 1:20 19.1 20 10 22.5 24.2 13.2 25 30 21 33 38 25 38.2 40.4 30 LSD> 0 1 = 4.14 88 of vibration. The difference became significant after 15 minutes, but after 20 minutes of vibration the results were similar for both treatments. Prolonged vibration beyond 20 minutes did not significantly improve dispersion. It thus appears that a vibration time of 20 minutes is adequate for soil dispersion with Biosonik probe, using a soil/water ratio of 1:5 or 1:10, providing the total volume of the suspension is not much in excess of 100 ml. This hypothesis was tested by dispersing 25 g samples of the Hazelwood Ah1 in 100 ml, 125 ml, 150 ml, and 200 ml of water. The percent clay obtained by hydrometer analysis was 43 percent, 41 percent, 38 percent and 33 percent, respectively. 4.3.3 Effect of Electrolyte Solutions on the Dissolution of Soil  Constituents and Dispersion Efficiency of Ultrasonic  Vibration 4.3.3.1 Dispersion Efficiency The degree of effectiveness of soil dispersion by ultrasonic vibration of soils in different electrolyte solutions was assessed by measuring the percent clay in the suspension following vibration, and comparing this amount with the percent clay obtained by the peroxide-calgon method. The data are presented in Table 4, and the statistical analysis in Table 5. It can be seen that the vibration method of dispersion regard-less of the solution, gave clay values which were similar to those obtained by the conventional method; whereas, shaking the soil in water did not. The means for the various electrolyte solutions suggest that overall, calcium chloride dispersion was closest to the peroxide-89 TABLE 4 % Clay Obtained by Dispersion of Soil in Different Solutions Soil * P.C. Shaking in Water Ultrasonic Vibration Water 0.01 CaCl2 0.01 BaCl2 0.1M NaCl 0.03M NaCl LANGLEY Ah 30.0 6.7 28 31.3 33.3 33.4 34.1 Bt 47.1 32.3 42 48.0 50.8 50.4 53.1 HAZELWOOD Ah] 40.1 6.3 39.8 44.0 43.6 45.5 -Bg 45.8 25.2 44 49.6 49.5 51.4 -HATZIC Ap 51.2 13.1 40.2 43.8 54.5 53.9 57.5 Bg 49.8 29.8 53.2 50.2 57.8 54.8 52.2 CLOVERDALE Ap 26.0 4.8 22.2 26.0 30.4 26.8 -Btg 38.5 17.1 40.0 37.5 40.8 41.0 -DELTA Ap 19.9 9.5 17.5 19.0 19.9 20.2 -Bg 14.9 11.0 12 16.1 17.6 16.5 -MEAN 36.3 15.6 33.9 36". 6 39.8 39.4 -* P.C. = Peroxide-Calgon Method. 90 c a l g o n method, however, the d i f f e r e n c e s among the e l e c t r o l y t e s o l u t i o n s t h e m s e l v e s , were not s i g n i f i c a n t . M/100 c a l c i u m c h l o r i d e has the added advantage o v e r o t h e r s o l u t i o n s o f b e i n g w i d e l y used i n s o i l a n a l y s e s , e s p e c i a l l y f o r pH d e t e r m i n a t i o n s and i t s i o n i c s t r e n g t h i s p u r p o r t e d l y i s o t o n i c w i t h t h a t o f the d i s p l a c e d s o i l s o l u t i o n , S c h o f i e l d [1949]. C a l c i u m c h l o r i d e d i s p e r s i o n , however, does s u f f e r from one d i s a d v a n t a g e , compared t o d i s p e r s i o n i n w a t e r , i n t h a t the amount o f f i n e c l a y (<0.2.um) r e c o v e r e d f o l l o w i n g v i b r a t i o n , was s i g n i f i c a n t l y l e s s . • That t h i s s h o u l d be s o , may not be c o n s i d e r e d s u r p r i s i n g i f i t 2+ i s argued t h a t Ca b e i n g d i v a l e n t causes f l o c c u l a t i o n o f the f i n e r c l a y p a r t i c l e s . The q u e s t i o n then a r i s e s whether M/100 c a l c i u m c h l o r i d e i s s u f f i c i e n t l y c o n c e n t r a t e d t o cause f l o c c u l a t i o n s i n c e i t s i o n i c s t r e n g t h i s a p p r o x i m a t e l y t h a t o f the s o i l s o l u t i o n ; a l s o , l e s s t o t a l c l a y would be e x p e c t e d i n C a C ^ than i n water i f t h i s were t r u e . On the o t h e r hand i t i s p o s s i b l e t h a t some degree o f f l o c c u -l a t i o n o f the f i n e c l a y p a r t i c l e s might o c c u r . These p a r t i c l e s a r e c h e m i c a l l y more r e a c t i v e (due t o l a r g e r s u r f a c e a r e a and a h i g h e r charge to mass r a t i o ) than c o a r s e r p a r t i c l e s and w i l l have a h i g h e r m o b i l i t y (Brownian movement) as w e l l . The p o s s i b i l i t y f o r c o l l i s i o n s w i t h 2+ Ca i o n s i n s o l u t i o n a r e t h e r e f o r e g r e a t e r , and c o u l d l e a d t o t h e f o r m a t i o n o f v e r y t i n y f l o c c u l e s , which would reduce t h e i r m o d i l i t i e s and p o s s i b l y s t a b i l i z e t he "new p a r t i c l e s " i n the s u s p e n s i o n . T a b l e 5 shows the c o r r e l a t i o n c o e f f i c i e n t s f o r t h e p e r o x i d e -c a l g o n (P-C) and o t h e r d i s p e r s i o n methods. There i s a h i g h degree o f c o r r e l a t i o n between the P-C method and the v i b r a t i o n methods o f d i s p e r -s i o n , the r - v a l u e s r a n g i n g between (0.95-0.99). The r e g r e s s i o n l i n e s 91 for the percent clay obtained by vibration in water and calcium chloride versus the peroxide-calgon method are shown in Figures 4 and 5. The complete particle-size analysis data are presented in Table 6. The effect of ionic strength of the solution on the dispersion of soils by ultrasonic vibration was investigated by dispersing four samples in 0 . 0 3 M NaCl and 0.1M NaCl. The percent clay recovered averaged 49.2 percent and 48.1 percent for the respective solutions. 4.3.3.2 Dissolution Effects It has been stated previously that there is very l i t t le data currently available on the dissolution of soil constituents during the dispersion of soils by ultrasonic vibration. Table 7 shows the differences in the amounts of Na, K, Ca, Mg, Fe, A l , Si and C in solution, between shaking a soil suspension for 20 minutes at 120 strokes per minute (reciprocating shaker) and vibrating the suspension for 20 minutes with an ultrasonic probe. The actual values obtained are presented in Appendices III A and III B. The averages for the aforementioned elements for the A and B horizons are also presented in Table 7. It is evident that there was more dissolution of Fe, Al , Si and C than for the alkali and alkaline earth metals. There was also more Fe, Al and Si dissolved from the B horizons than from the A. The reverse was true for carbon. The amounts of Fe and Al in solution following vibration were markedly similar, averaging 49.6 and 48 ppm for the A horizon and 286 and 275 ppm for the B, respectively. In contrast much larger 92 TABLE 5 Correlation Coefficients (r*) for Relationships Between the Peroxide-Calgon and Ultrasonic Methods of Soil Dispersion Variable P-C Shaking in Water U/S Water U/S CaCl2 U/S BaCl2 U/S . NaCl 1 U/S „ NaCl 1 1 P-C 1. 0000 Shaking/Water 0. 6454 1.0000 U/S Water 0. 9556 0.6848 1.0000 U/S CaCl2 0. 9698 0.6772 0.9720 1.0000 U/S BaCl2 0. 9930 0.6576 0.9636 0.9709 1.0000 U/S NaCl1 0. 9957 0.6441 0.9665 0.9845 0.9920 1.0000 U/S NaCl 1 1 0. 9851 0.5680 0.7187 0.8561 0.9332 0.9641 1.0000 r Q 1 = 0.765 NaCl1 = 0.1M NaCl 1 1 = 0.03M U/S = ultrasonic vibration F I G U R E 4. 50 K < 5 | 40 </> a in o z o io 30 <. fx >-< o 55 20--COMPARISON OF PERCENTAGE - CLAY VALUES OBTAINED FOR TEN HORIZON SAMPLES DISPERSED BY ULTRASONIC (WATER) AND CHEMICAL METHODS OO —r— 20 30 I 40 % CLAY - PEROXIDE-CALGON DISPERSION TABLE 6 A Comparison of Different Dispersion Methods for the Particle-Size Analysis of Soils Ultrasonic Vibration Soil P.C. Water1 Water .OlM CaCl2 .OlM BaCl2 0.1M NaCl .03M NaCl SA. Si . C* SA. Si . C. SA. Si . C. SA. Si . C. SA. Si. C. SA. Si . C. SA. Si . C. LA. Ah Bt 5.5 3.1 65.5 49.7 30 47.1 50.1 2.0 43.2 65.7 6.7 32.3 4.5 1 67.5 57 28 42 3 1 65.7 51 31.3 48 2.4 0.5 63.3 48.4 33.3 50.8 3.2 1 63.4 48.6 33.4 50.4 3.6 1 62.3 46 34. 53 1 HAZ. Ah, Bg1 3.0 2.2 56.9 52 40.1 45.8 33 14 60.7 60.8 6.3 25.2 6 10 54.2 46 39.8 44 3 8 53 42.4 44 49.6 6 0.5 50.4 50 43.6 49.5 7.5 1 47 47.6 45.5 51.4 --HATZItyAp 1.3 0.4 47.5 49.8 51.2 49.8 44.1 10.9 42.8 59.3 13.1 29.8 3.5 2 56.3 44.8 40.2 53.2 10 1 46.2 48.8 43.8 50.2 5.5 0.2 40 42 54.5 57.8 4.5 0.2 41.6 44.9 53.9 54.8 1.4 1 41.1 46.9 57. 52. 5 1 CLOV. Ap Btg 6.4 4.9 67.6 56.6 26 38.5 24.1 5.4 71.1 77.5 4.8 17.1 5 2 72.8 58 22.2 40 4 4 70 58.5 26 37.5 3.3 0.6 66.2 58.6 30.4 40.8 3.5 0.8 67.7 58.2 26.8 41 - -DELTA. Ap 1 Bg 5.6 11.9 73.1 73.2 21.3 14.9 14.4 8.3 76.1 80.7 9.5 11.0 10 4 72.5 84 17.5 12 4 5 77 78.9 19 16.1 7.7 6.5 72.4 75.9 19.9 17.6 6 4 73.8 79.5 20.2 16.5 - -1 = Shaking 10 g soil in 100 ml H90 for 20 minutes. P.C. = Peroxide-Calgon. SA = Sand % { Si = Si l t C = Clay F I G U R E 5. 50-4 frt^!tIi ,8Sffai?i^B5 ,?B-S , T^O B ~ G L A Y V A L U E S OBTAINED FOR TEN HORI20N SAMPLES DISPERSED BY ULTRASONIC (CoCI 2) A N D CHEMICAL METHODS o m U oo 4<H cc UJ u z 8 30-< cc (--J 3 I > < - I o as 20 H l o ~ To" % CLAY - PEROXIDE - CALGON DISPERSION I SO 96 TABLE 7 Dissolution Effects of Ultrasonic Vibration of Soils in Water* Soil Na K Ca Mg Fe Al Si C yyui - - -LANGLEY Ah 3 -1.5 -2.4 0.6 19 21 390 420 Bt 10 16.8 0 13.3 430 365 2300 90 HAZELWOOD Ah1 6.2 -1.1 -12.8 7.2 19 15 100 250 Bg 6.4 3.5 -5.2 2.7 86 93 470 200 HATZIC Ap -2.3 -10.2 -22.6 -6.2 28 28 170 710 Bg 4.7 4.7 4 5.3 172 170 760 150 CLOVERDALE Ap 19.9 -5.9 -38.2 4.9 16 22 60 250 Btg 5.7 73.1 0 13.6 533 549 2600 120 DELTA Ap 67.2 17.5 -12.4 -109.4 166 156 810 590 Bg 24.4 17.5 -1.6 5.0 208 198 1330 120 AVERAGE A l 18.8 -0.24 -17.7 -20.5 49.6 48.4 612 672 B(g) 10.2 23.1 -0.56 8.0 285.8 275 1492 136 * Corrected for amounts released by shaking soils in water j ( p p m a i > dried soi l) . 97 quantities of Si were released especially from the B horizon samples, which were lower in organic carbon. It thus appears that ultrasonic vibration of soil suspensions can lead to considerable dissolution of some mineral elements, notably Fe, Al and Si. It is equally apparent that vibration also leads to the dissolution of organic carbon, especially from the A horizon samples, however, the presence of organic matter seems to reduce the dissolution of mineral elements either by reducing attrition of mineral particles or by absorbing some of the shock from the ultrasonic waves, thereby preventing the rupture of minerals. It has been found that ultrasonic vibration of clays can lead to structural rearrangements, or degradation of the minerals, and in a subsequent section these possibilities are investigated. Such phenomena i t should be pointed out have rarely been observed with soils, but i t is obvious that the Si in solution must be derived from a Si-containing mineral, perhaps quartz. 4.3.3.3 Relationship Between Fe and Organic-C in Solution The supernatant solutions from the vibrated suspensions in water were all highly colored and the absorbance spectra revealed a small peak at 500 nm. The color of the solutions were attributed mainly to the presence of dissolved iron and organic matter and from the data in Appendix IIIB the correlation coefficient (r) was calculated for Fe and organic-C in solution. It was found that there was a highly significant inverse relationship between the two (r = -0.7, P < 0.01) 98 which was s u r p r i s i n g because i t was o r i g i n a l l y assumed t h a t a d i r e c t r e l a t i o n s h i p e x i s t e d and t h a t Fe and o r g a n i c - C were s o l u b i l i z e d t o g e t h e r f o r m i n g an o r g a n o m e t a l 1 i c complex i n s o l u t i o n . 4.3.4 D i s s o l u t i o n E f f e c t s o f D i s p e r s i n g S o i l s i n D i f f e r e n t S o l u t i o n s 4.3.4.1 Ir o n T a b l e 8 shows the amount o f Fe found i n s o l u t i o n a f t e r s h a k i n g the s o i l s in water, and following ultrasonic treatment in water and e l e c t r o l y t e s o l u t i o n s . The d a t a i n d i c a t e t h a t t h e r e was a marked d i s s o l u t i o n o f Fe upon u l t r a s o n i c v i b r a t i o n o f s o i l s u s p e n s i o n s i n w a t e r , compared to the e l e c t r o l y t e s o l u t i o n s o r s h a k i n g i n water. T h i s o b s e r v a t i o n was p a r t i c u l a r l y t r u e f o r the B . h o r i z o n samples. The d i f f e r e n c e i n the d i s s o l u t i o n o f Fe between A and B ho r i z o n s can e a s i l y be e x p l a i n e d i f we assume t h a t Fe i n the A h o r i z o n i s i n the o x i d i z e d s t a t e whereas i n the g l e y e d B h o r i z o n the Fe i s i n the r e d u c e d s t a t e . It s h o u l d be remembered, however, t h a t a l l the samples were a i r - d r i e d p r i o r t o a n a l y s i s thus most o f the Fe i n the B h o r i z o n s h o u l d a l s o be i n t h e o x i d i z e d s t a t e . An a l t e r n a t i v e e x p l a n a t i o n i s t h a t Fe i n the A h o r i z o n o c c u r s as r e l a t i v e l y i n s o l u b l e i r o n humates w h i l e Fe i n the B h o r i z o n o c c u r s m a i n l y as h y d r a t e d i r o n o x i d e s , which a r e more s u s c e p t a b l e t o d i s s o l u t i o n , o r s o l u b l e complexes w i t h f u l v i c a c i d . A p p r o x i m a t e l y the same amount o f Fe i s r e l e a s e d from the A ho r i z o n samples f o r each s o l u t i o n and t h i s s u g g e s t s t h a t t h e r e might be c e r t a i n w a ter s o l u b l e i r o n compounds p o s s i b l y o r g a n o - i r o n compounds, 99 TABLE 8 Dissolution of Iron by Dispersing Soils in Different Solutions Ultrasonic Vibration Soil Shaking in Water Water 0.01M CaCl2 0.01M BaCl2 0.1M NaCl 0.03M NaCl LANGLEY Ah 9 28 0 0 0 0 Bt 36 466 0 0 0 0 HAZELWOOD Ah1 9 28 27 46 6 -Bg 12 98 0 6 0 -HATZIC Ap 10 38 0 26 29 3 Bg 10 182 0 0 0 0 CLOVERDALE Ap 8 24 16 56 0 -Btg 29 562 0 0 69.6 -DELTA Ap 8 174 0 0 53.8 -- • Bg 14 218 0 25 69.6 -AVERAGE A l 8.8 58.4 8.6 25.6 17.8 1.5 B(g) 20.2 305.2 0 6.2 27.8 0 100 which are a common constituent of the samples. In the case of the B horizon samples, quantities of dissolved Fe are much larger and variability much greater between samples. The amount of Fe in solution appears to be influenced by the accumulation of clay-size material in the horizon, as evidenced by the Langley Bt and Cloverdale Btg horizons. It seems, however, that the dispersing medium exerts the strongest effect on the dissolution of Fe. Again, differences in the dissolution of Fe from A horizon samples following vibration in water and electrolyte solutions are not as marked as those of the B, possibly due to the presence of small amounts of soluble organo-iron compounds in the A horizon samples. The suppression of the dissolution of Fe in the electrolyte solutions is perhaps due to a suppression of the ionization of water by the "comparatively" high concentration of other ions present. El'Piner [1964] noted that ultrasonic waves promoted a large number of chemical reactions and i t is believed that water in an ultrasonic f ield can become highly dissociated. This means that soil particles suspended in water could be subject to the effect of high concentrations of Hg0 + and OH" ions which, depending on the init ial pH and exchange properties of the so i l , could lead to considerable localized dissolution of some constituents. In an electrolyte solution, however, the effects of hydroxyl and hydronium ions would tend to be countered by ions of the opposite species present in solution. 101 4.3.4.2 Aluminum The pattern of dissolution of Al (Table 9) resembles that of Fe. Maximum dissolution of Al occurred when the soils were vibrated in water whereas very l i t t le Al was found in the vibrated electrolyte solutions. Indeed, as observed for Fe, more Al is dissolved simply by shaking the soils in water than by ultrasonic vibration in a salt solution, for the same time period. Also as observed for Fe, more Al was released from the Bt(g) horizons where there is an accumulation of clay and possibly sesquioxides. 4.3.4.3 Silicon The dissolution of Si from soils vibrated in water, as was the case for Fe and Al , was significantly greater than that following vibration of soils in electrolyte solutions or shaking in water, (Table 10). There was relatively l i t t l e difference in the amounts of Si dissolved from the A-j horizons by vibration in water or the electrolyte solutions except for the Delta Ap (lowest in organic matter); however, these quantities were significantly greater than those obtained by simply shaking soils in water. Extremely large amounts of Si were dissolved from the B horizon samples vibrated in water compared to the other treatments. Again, the dissolution was most marked in the Bt(g) samples, enriched with clay and possibly amorphous material. While i t must be concluded that the dissolution of Si derives from crystalline or non-crystalline minerals present in the soils i t should be pointed out that vibration 102 TABLE 9 Dissolution of Aluminum by Dispersing Soils in Different Solutions Shaking in Water Ultrasonic Vibration Soil Water 0.01M CaCl2 0.01M BaCl2 0.1M NaCl 0.03M NaCl rr 1" LANGLEY Ah 11 32 2 11 6 0 Bt 39 404 0 0 0 0 HAZELW00D 9 - 24 6 20 11 -Bg 17 110 12 25 12 -HATZIC Ap 10 38 4 13 13 3 Bg 14 184 0 0 0 0 CLOVERDALE Ap 4 26 0 0 25 -Btg 33 582 0 0 0 -DELTA Ap 8 164 4 0 25 -Bg 16 214 0 0 0 -AVERAGE A l 8.4 56.8 3.2 8.8 16 1.5 B(g) 23.8 298.8 2.4 5.0 2.4 0 103 TABLE 10 Dissolution of Silicon by Dispersing Soils in Different Solutions Ultrasonic Vibration Soil Shaking in Water Water 0.01M CaCl2 0.01M BaCl2 0.1M NaCl 0.03M NaCl LANGLEY Ah 30 120 60 100 150 30 Bt 140 2440 170 200 200 80 HAZELWOOD Ah1 40 140 80 120 130 -Bg 60 530 100 120 130 -HATZIC Ap 30 200 50 100 150 50 Bg 60 820 140 120 170 70 CLOVERDALE Ap 10 70 70 70 70 -Btg 80 2680 100 140 n o -DELTA Ap 20 830 120 70 60 -Bg 60 1390 100 130 110 -AVERAGE A l 26 272 76 92 112 40 B(g) 80 1572 122 142 144 75 104 o f the s o i l s o c c u r r e d i n 250 ml g l a s s beakers and the p o s s i b l e s o l u b i l -i z a t i o n o f some S i from t he g l a s s due t o e r o s i o n cannot be i g n o r e d d e s p i t e t he f a c t t h a t the b l a n k s (100 ml H^O v i b r a t e d f o r 20 m i n u t e s ) c o n t a i n e d no S i . 4.3.4.4 Carbon . T a b l e 11 shows the d a t a f o r the d i s s o l u t i o n o f carbon by the v a r i o u s t r e a t m e n t s . The p a t t e r n o f maximum d i s s o l u t i o n by v i b r a t i o n o f s o i l s i n water and s u p p r e s s i o n o f d i s s o l u t i o n i n the e l e c t r o l y t e s o l u t i o n s i s a g a i n r e p e a t e d , e x c e p t t h a t t he t r e n d n o t e d f o r the A and B h o r i z o n s i s r e v e r s e d , more C b e i n g r e l e a s e d from the A-| h o r i z o n s . In sharp c o n t r a s t t o the water and NaCl e x t r a c t s the s u p e r -n a t a n t CaCl2 and B a C ^ s o l u t i o n s were v i r t u a l l y c o l o r l e s s and no C was d e t e c t e d by the W a l k l e y and B l a c k method. Because o f p o s s i b l e c h l o r i d e i n t e r f e r e n c e and the low l e v e l o f C i n s o l u t i o n , ( v e r y p a l e c o l o u r ) the NaCl e x t r a c t s were a n a l y z e d f o r o r g a n i c c a r b o n u s i n g the Beckman 915 T o t a l - O r g a n i c c a r b o n a n a l y z e r . F a i r l y l a r g e amounts o f o r g a n i c - C were found i n some o f the s o l u t i o n s , p o s s i b l y due t o the d i s p e r s i n g a c t i o n o f N a + i o n s ; however, t h e s e were s i g n i f i c a n t l y l e s s than the amounts i n the v i b r a t e d - w a t e r e x t r a c t s . Thus i t i s a p p a r e n t t h a t the d i l u t e e l e c t r o l y t e s o l u t i o n s i g n i f i c a n t l y r e d uced the d i s s o l u t i o n o f C d u r i n g u l t r a s o n i c v i b r a t i o n o f a s o i l s u s p e n s i o n . 4.3.5 Bulk I s o l a t i o n o f Or g a n o - M i n e r a l Complexes M o d i f i c a t i o n s o f the "beaker" o r d e c a n t a t i o n method have g e n e r a l l y been used i n the p a s t f o r b u l k s e p a r a t i o n o f s o i l p a r t i c l e -105 TABLE 11 Dissolution of Carbon by Dispersing Soils in Different Solutions Ultrasonic Vibration Soil Shaking in Water Water 0.01M CaCl2 0.01M BaCl2 0.1M NaCl 0.03M NaCl LANGLEY Ah 60 420 N.D.* N.D. 250 210 Bt 15 90 II n 9 6 HAZELW00D Ah] 43 250 I I II 80 -Bg 18 200 I I H 43 -HATZIC Ap 20 170 II I I 58 50 Bg 7 150 n n 30 36 CLOVERDALE Ap 38 250 I I II 165 -Btg 2 120 I I n 36 -DELTA Ap 30 590 H H 201 -Bg 18 120 n M 77 -AVERAGE A l 38.2 336 - - 150.8 130 B(g) 12 136 - - 39 21 N.D. = Not detected by Walkley-Black Method - Extracts were virtually colorless. 106 s i z e s e p a r a t e s . Coarse sand i s removed f i r s t by s i e v i n g , then c l a y s e p a r a t e d by r e p e a t e d d e c a n t a t i o n and f i n a l l y , s i l t d e c a n t e d from f i n e sand. Yamada, Sakuma and Aoki [1966] have r e c e n t l y d e s c r i b e d a p p a r a t u s i n which s t i r r i n g , s e d i m e n t a t i o n and d e c a n t a t i o n are a u t o m a t i c a l l y o p e r a t e d . More r e c e n t l y G e n r i c h and Bremner [1974] d e s c r i b e d a method f o r the i s o l a t i o n o f s o i l p a r t i c l e - s i z e f r a c t i o n s based upon u l t r a s o n i c d i s p e r s i o n o f the s o i l and s i e v i n g , c e n t r i f u g a t i o n and f i l t r a t i o n p r o c e d u r e s . A l l t h e s e methods, however, s u f f e r from the d i s a d v a n t a g e o f b e i n g e x c e e d i n g l y time consuming, thus any t e c h n i q u e which c o u l d s i g n i f i c a n t l y reduce the time i n v o l v e d i n the f r a c t i o n -a t i o n o f p a r t i c l e - s i z e s e p a r a t e s would a i d m a t e r i a l l y i n t h e i r s t u d y . With t h i s i n mind a f r a c t i o n a t i o n scheme based on u l t r a s o n i c d i s p e r s i o n o f s o i l samples and a system o f c o n t i n u o u s f l o w c e n t r i f u g a -t i o n f o r the i s o l a t i o n o f the s e p a r a t e s was d e v e l o p e d . I t was found t h a t c o n t i n u o u s f l o w c e n t r i f u g a t i o n r e duced a l m o s t by o n e - h a l f , the time i n v o l v e d i n o b t a i n i n g s e p a r a t e s . For the f i v e s o i l s s t u d i e d t h e t o t a l r e c o v e r y o f p a r t i c l e s a v e r a g e d 98 p e r c e n t . The c h e m i c a l and p h y s i c a l c h a r a c t e r i s t i c s o f s e v e r a l b a t c h e s o f s e p a r a t e s were compared and the r e s u l t s d e monstrated t h a t the method gave h i g h l y r e p r o d u c i b l e s e p a r a t i o n s . 4.3.5.1 Pr o c e d u r e The f r a c t i o n a t i o n scheme d e v e l o p e d f o r the i s o l a t i o n o f s o i l p a r t i c l e - s i z e s e p a r a t e s i s o u t l i n e d i n F i g u r e 6. R e p l i c a t e 25 g samples o f s o i l were soaked i n 125 ml water f o r a t l e a s t an hour, then d i s p e r s e d u l t r a s o n i c a l l y . The v i b r a t e d s u s p e n s i o n s were f i l t e r e d 107 1. 25 g s o i l soaked i n 125 ml d i s t i l l e d water i n 250 ml g l a s s beaker f o r a t l e a s t 1 hour. 2. V.ibrate s u s p e n s i o n f o r 20 m i n u t e s a t s e t t i n g o f 80. 3. Pour s u s p e n s i o n onto 270 mesh s i e v e (53ym) -- Wash t h o r o u g h l y . S i e v e D r i e d i n Oven. Sand Weighed and S t o r e d S i l t R e t a i n e d (2-53um) Resuspended i n H^O; R e c e n t r i f u g e d to Remove C l a y . (- 3 C y c l e s ) S i l t C o l l e c t e d i n 250 ml P o l y p r o p y l ene B o t t l e s , Freeze-D r i e d , S t o r e d S u s p e n s i o n s from R e p l i c a t e s Combined S h a r p i e s S / C e n t r i f u g e (R) Flow Rate = 1500 ml/min (S) Speed =. 4600 rpm C l a y (<2um)Suspension S h a r p i e s S / C e n t r i f u g e R - 1500'ml/min S = 9500 rpm 1-2'urn R e - c y c l e d F r e e z e - D r i e d and S t o r e d <1.0ym S u s p e n s i o n S o r v a l l RC2-B S / C e n t r i f u g e , R = 120 ml/min S = 15,000 rpm 0.2-1 ym R e - c y c l e d F r e e z e - D r i e d , and S t o r e d <0.2 ym S u s p e n s i o n F l o c c u l a t e d w i t h S a t u r a t e d CaCl Washed, Freeze-D r i e d , and S t o r e d 2' F i g u r e 6 F r a c t i o n a t i o n Scheme f o r Bulk S e p a r a t i o n o f Organo-Clay  Complexes 108 through a 270 mesh sieve and the sieve carefully washed until all traces of finer particles removed. The sieve was then dried in an oven at 105°C and the sand collected, weighed, and stored in glass containers. The remaining suspensions were combined in 5 l i tre polypropylene beakers, diluted to about four and one half l itres and recycled through the Sharpies supercentrifuge to separate the s i l t (2-53um) and coarse clay (l-2ym) and coarse clay (l-2um) fractions. The bowl of this centrifuge has a total volume of 285 ml and a diameter of 4.4 cm. A complete description of the principle of operation for the Sharpies supercentrifuge and nomographs for the centrifugation of a range of particle-sizes are given by Jackson [1956]. With this instrument (Sharpies) the soil suspension instead of being placed in tubes, is fed into a hollow cylinder called a bowl, which is rotated at high speed. Depending on the speed of rotation of the bowl and the flow rate of the suspension into the bowl, particles of a certain limiting diameter is collected on the wall of the cylinder and the supernatant liquid is displaced out the top of the cylinder by incoming suspension. The flow rate is calculated on the basis of the time required for the sedimentation of particles of a certain limiting diameter and time (T) is in turn calculated from the following equation based on Stoke's law, [Jackson, 1956, p. 127]: (63.0 x 108)n(log10R/S) N2D2 (S p - S 1) where T is time for sedimentation in minutes, n is the viscosity of water in poises, R is the radius of rotation of the top of the 109 no sediment (cm), S i s the r a d i u s o f r o t a t i o n o f the s u r f a c e o f the s u s p e n s i o n (cm), N i s the speed o f the c e n t r i f u g e i n rpm, D i s the p a r t i c l e d i a m e t e r i n m i c r o n s , Sp i s the s p e c i f i c g r a v i t y o f the s o i l p a r t i c l e s and S'-j i s the s p e c i f i c g r a v i t y o f water. J a c k s o n [1956] p o i n t e d o u t t h a t the d e n s i t y o f h i g h l y h y d r a t e d c l a y p a r t i c l e s i s f a r l e s s than the r e s p e c t i v e m i n e r a l s p e c i f i c g r a v i t y i n the anhydrous s t a t e . F o r example J a c k s o n [1956] c a l c u l a t e d t h a t the s p e c i f i c g r a v i t y o f the <0.2um f r a c t i o n i n the h y d r a t e d s t a t e was 1.65, and not 2.5 as was f o r m e r l y assumed. A s i m p l e r e l a t i o n s h i p between f l o w r a t e through t he bowl and. the time o f s e d i m e n t a t i o n o f a p a r t i c l e i s g i v e n by the f o l l o w i n g e q u a t i o n . V ^ml/min ~ ~^  min where F i s the f l o w r a t e i n m i l l i l i t r e s per min u t e , V i s the volume o f th e bowl and T i s the time i n minutes r e q u i r e d f o r s e d i m e n t a t i o n o f the p a r t i c l e . T h i s e q u a t i o n , however, does not r e c o g n i z e d e n s i t y g r a d i e n t c o n t r o l o p e r a t i n g i n the bowl. The b u l k s u s p e n s i o n c o n t a i n i n g p a r t i c l e s w i t h an e f f e c t i v e s p h e r i c a l d i a m e t e r l e s s than one m i c r o n was t r a n s f e r r e d t o a tank a t t a c h e d t o the i n l e t tube o f the S o r v a l l RC2-B ( r e f r i g e r a t e d ) con-t i n u o u s f l o w system. T h i s system c o n s i s t s o f an a n g l e head c e n t r i -f uge c o n t a i n i n g e i g h t s t a i n l e s s s t e e l c e n t r i f u g e tubes o f 50 ml c a p a c i t y each. The maximum c e n t r i f u g a l f o r c e a t t a i n a b l e by the F i g u r e 8 C r o s s - S e c t i o n a l View o f the KSB System Showing the Flow o f the S u s p e n s i o n Through the System 112 Figure 9 Sharpies Laboratory Model Supercentrifuge 113 c e n t r i f u g e i s 48,200 x g. T h i s system has an advantage o v e r t h e S h a r p i e s system i n t h a t the temperature o f the s u s p e n s i o n f l o w i n g t h r o u g h the tube can be a c c u r a t e l y m a i n t a i n e d t h r o u g h o u t the o p e r a t i o n , a l s o , t h i s system i s f r e e from l e a k s and thus l e s s m a t e r i a l i s " l o s t " d u r i n g o p e r a t i o n . F o l l o w i n g the s e p a r a t i o n o f the p a r t i c l e s (0.2 - lym) from the s u s p e n s i o n , t h e s u p e r n a t a n t l i q u i d was t r a n s f e r r e d t o 6 - l i t r e s e d i m e n t a t i o n g l a s s c y l i n d e r s and the <0.2um p a r t i c l e s f l o c c u l a t e d w i t h s a t u r a t e d C a C ^ . The f l o c c u l a t e d p a r t i c l e s were then washed f r e e o f e x c e s s CaClg and c o l l e c t e d i n c e n t r i f u g e t u b e s . When a l l t h e f r a c t i o n s had been c o l l e c t e d they were a l l f r e e z e - d r i e d , weighed and s t o r e d i n g l a s s j a r s p r i o r t o a n a l y s i s . T a b l e 12 shows the r e c o v e r y o f the v a r i o u s p a r t i c l e - s i z e f r a c t i o n s based on the d i s p e r s i o n o f 500 g samples o f each o f the f i v e g l e y s o l i c s o i l s . The p a r t i c l e - s i z e a n a l y s i s d a t a o b t a i n e d by the p e r o x i d e - c a l g o n method (hydrometer a n a l y s i s ) i s a l s o g i v e n f o r comp a r i s o n . I t i s a p p a r e n t t h a t the proposed b u l k f r a c t i o n a t i o n scheme g i v e s e f f e c t i v e and q u a n t i t a t i v e s e p a r a t i o n o f the v a r i o u s s o i l f r a c t i o n s . The two p e r c e n t o f s o i l m a t e r i a l not r e c o v e r e d can be a c c o u n t e d f o r p a r t i a l l y on the b a s i s o f d i s s o l u t i o n o f some m a t e r i a l s , w h i l e s p i l l a g e o f some o f the s u s p e n s i o n which i n v a r i a b l y o c c u r s when d e a l i n g w i t h such l a r g e q u a n t i t i e s o f l i q u i d ; p l u s l e a k a g e o f s u s p e n s i o n from the S h a r p i e s s u p e r c e n t r i f u g e ^ a l s o c o n t r i b u t e d to t h e l o s s . TABLE 12 Recovery o f Sand ., S i l t and C l a y by t h e Bulk F r a c t i o n a t i o n Scheme (%) S o i l Sand S i l t C l a y * % A B A B A B R e c o v e r y ( l - 2 u ) ( 0 . 2 - l u ) (<0.2u) B LANGLEY Ah 5.5 4.7 65.5 63 1 30 12.6 13.8 2.8 97 HAZELWOOD Ah 1 3.0 2.8 56.9 58 40. 1 18.1 15.5 3.1 97.5. HATZIC Ap 1.3 2.7 47.5 51 5 51. 2 22 17.7 4.6 98.5 CLOVERDALE Ap 6.4 6.6 67.6 64 4 26 12.5 11.5 2.0 97 DELTA Ap 5.6 7.7 73.1 69 8 2.1. 3 8.0 10.0 2.5 98 A = P e r o x i d e - C a l g o n method. B = B u l k f r a c t i o n a t i o n scheme ( F i g u r e 6 ) . * = Based on the d i s p e r s i o n o f 500 g s o i l . 115 4.4 Conclusion A detailed study of the characteristics of the Biosonik BP-III ultrasonic vibrator with regards to the dispersion of soils and its effects on soil constituents was undertaken. On the basis of the results obtained i t was concluded that a soil suspension (1:10 or 1:5 soil/water ratio) not exceeding a total volume of 125 ml could be effectively dispersed by vibration for 20 minutes at a probe setting of 80. It was found that the efficiency of soil dispersion was reduced by about 30-40 percent unless there was a rest period of approximately 25-30 minutes between consecutive dispersions. The condition of the probe tip was found to exert a major influence on the efficiency of dispersion, and the polishing of the probe tip with fine (400-grit) emery paper after each 20 minutes operation retarded the onset of pitting. After about every 30 hours of operation i t is recommended that the probe tip be machined on a lathe to restore its surface to a smooth condition. The heating effects of ultrasound treatment was found to be a good index of the output of ultrasonic energy although soil suspensions should be cooled during dispersion to eliminate possible thermochemical reactions. Mechanical analysis of vibrated soil suspensions, confirmed the findings of many other workers that ultrasonic vibration, without the aid of chemical reagents could effectively disperse soils. A major observation from this study was that large amounts of Fe, A l , Si and C were solubilized during the vibration of soils in water. This effect was, however, suppressed when dispersion occurred in a dilute electrolyte solution, 0.01 M CaC^ being very effective. A fractionation- procedure for the isolation of soil particle-size fractions was developed based on ultrasonic dispersion of the soil and a system of continuous flow centrifugation. It was found that continuous flow centrifugation reduced almost by one-half the time involved in obtaining separates. The technique gave effective and quantitative separation of the various soil size fractions, with an average of 98 percent of the soil material recovered. 117 5. INCUBATION STUDIES I This investigation was undertaken because, as stated pre-viously, there are no reports in the literature on the susceptibility to mineralization (or lack thereof) of the sulphur and phosphorus bound in organo-mineral complexes. Nor have any reported attempts been made to measure the possible release of nitrate, sulphate and phosphate from such substances during incubation. As a f i rst step towards obtaining such information i t was necessary to study the factors which might affect the mineralization of N, P and S, in order to determine the optimum conditions for carrying out the study. Thus, a factorial experiment was designed to carefully examine the effects of moisture, sand, inoculum and nutrients plus their interactions, and select the optimum combination of these factors for subsequent experiments. 5.1 Objectives 1 . To determine the relative importance of the factors which might affect the mineralization of nitrogen, phosphorus and sulphur in soil particle-size separates. 2. To develop an effective incubation technique which could be used for further studies of the mineraliz-ation of N, P and S in organo-mineral complexes. 118 3. To obtain some indication of the mineralization potential of the soil particle-size separate under different conditions. 5.2 Materials and Methods The 2-50um fraction of the Langley Ah was selected for the study because i t had properties intermediate between the <2mm soil and the clay fraction; also, because i t was the single most abundant fraction of the particle-size (P-S) separates, and could be easily isolated from the soi l . 4 A 2 factorial experiment was designed to observe the effects of moisture, sand, inoculum and nutrients, as well as their interaction on the mineralization of N, P , and S. The actual design is shown in Table 13. There were a total of 16 treatments, each replicated four times, one pair of which was extracted with NaHCO^  and the other with CaCl 2. 5.2.1 Selection of Factors and Factor Levels 5.2.1.1 Moisture Fitts, Bartholomew and Heidel [1955] and more recently Stanford and Epstein [1974] showed that moisture was one of the critical factors affecting the mineralization of organic nitrogen. The same apparently holds true for sulphur [Williams, 1967]. In the case of phosphorus, however, a higher level of moisture appears to 119 TABLE 13 Experimental Design 4 2 Factorial Factors Levels I II Moisture (M) 20% 40% Sand (S) - + Inoculum (I) - + Nutrients (N) - + Extractants 0.5 N NaHC03 pH 8.5 0.1 N CaCl2 Factor absent + Factor present 120 favour the accumulation of phosphate [Williams and Saunders, 1956]. Nitrate and sulphate formation are apparently aerobic processes and have been shown to occur over a wide range of moisture contents. Fitts et al_., [1955] found that 100 cm tension provided the optimum moisture for the production of NO^ , and this resulted in a 25-30 percent moisture content depending on the texture of the samples. Keeney and Bremner [1967] reported, however, that the amount of water required for maximal aerobic mineralization of nitrogen during incubation of soil was practically the same for different soils (ca. 0.6 ml/g soil) regardless of texture, i f the soil was mixed with three times its weight of 30-60 mesh quartz sand before incubation. Craswell and Waring [1972a] reported that aerobic N mineralization occurred at moisture contents between 6 and 26 percent with the maximum occurring near the higher end of the range. Their soils were mixed in a ratio of 1:3 with quartz sand. On the basis of this evidence two moisture levels were selected, 20 percent and 40 percent, (weight basis) the former being nearly optimum (100 cm tension) and the latter, at the high end of the moisture scale. One reason for selecting the higher level of moisture was to observe its effect on P mineralization. 5.2.1.2 Inoculum It was considered necessary to add an inoculum because of the ultrasonic, fractionation and freeze-drying procedures used in the isolation of the particle-size separates. The inoculum was 121 o b t a i n e d from the P r e s t s o i l , a Humic G l e y s o l , which had d e m o n s t r a t e d i n l a b o r a t o r y e x p e r i m e n t s a l a r g e c a p a c i t y t o m i n e r a l i z e both o r g a n i c N and S and a l s o some c a p a c i t y t o m i n e r a l i z e P [Kowalenko, 1973]. 5.2.1.3 P r e p a r a t i o n o f Inoculum F o r t y gm o f P r e s t s o i l was m o i s t e n e d and m a i n t a i n e d a t c a . 100 cm s o i l w a t er t e n s i o n f o r s e v e r a l days i n an i n c u b a t o r a t 35°C. Two hundred ml water was s u b s e q u e n t l y added t o the s o i l , the s u s p e n s i o n s t i r r e d and f i l t e r e d . Sub-samples o f the s u p e r n a t a n t s o l u t i o n were used f o r the i n o c u l a t i o n s . 5.2.1.4 N u t r i e n t S o l u t i o n A s t o c k m i c r o n u t r i e n t s o l u t i o n was p r e p a r e d c o n t a i n i n g the f o l l o w i n g : 500 ppm B, 500 ppm Mn, 50 ppm Zn, 20 ppm Cu, 10 ppm Mo, and 20 ppm Co. F i v e ml o f t h i s s t o c k was added t o a s o l u t i o n con-t a i n i n g , 200 ppm each o f K and Mg and 100 ppm Ca i n 1 l i t r e o f s o l u t i o n , and a d j u s t e d t o pH 7.0. Samples o f t h i s s o l u t i o n were employed i n the "N +" t r e a t m e n t s . 5.2.1.5 Sand Sand has found wide a p p l i c a t i o n i n i n c u b a t i o n e x p e r i m e n t s because i t improves s o i l a e r a t i o n and has the e f f e c t o f c a u s i n g near maximum a e r o b i c m i n e r a l i z a t i o n o f N o v e r a wide range o f m o i s t u r e c o n t e n t s . C o n s e q u e n t l y i t was i n c l u d e d as a f a c t o r i n the e x p e r i m e n t . 122 5.2.1.6 Extractant 0.1 N CaCl2 and 0.5 N NaHCOg (pH 8.5) were selected as ex-tractants because of the demonstrated ability of the former to extract mineral N and S and the later to extract available P, [Jackson, 1958]. 5.2.2 Incubation Method One gm of Langley s i l t (2-50ym) was added to each of 64, four ounce glass jars and 3 gm of acid-washed Ottawa sand (0.25-1 mm) added to the appropriate treatments. The sand and s i l t samples were thoroughly mixed. The treatments were brought to the correct moisture contents by adding varying amounts of inoculum, nutrient solution and water, according to the treatment designation, as shown in Table 14. Immediately following this, the jars were covered with a piece of 1.5 mil (1 mil = 10 in) polyethylene film and secured around the edge of the jar with a rubber band, all the jars were weighed prior to being placed in an Isotemp incubator set at 37.5°C. At the end of one week the jars were reweighed and the moisture lost, restored. At the end of the second week, extraction of mineral N, P and S was conducted with the appropriate extractant. Twenty ml of extractant was added to each jar; the jar stopped with a close fitting polypropylene cap (snap on) and placed on a mechanical reciprocal shaker at -120 strokes per minute for 30 minutes. The contents of the jars were transferred to 50 ml centrifuge tubes and the suspension centrifuged at 12,000 xg for 20 minutes. The supernatant solution was poured into 50 ml 123 TABLE 14 Treatment D e s i g n a t i o n s M o i s t u r e A d d i t i o n s (ml) Treatment Composi t i o n Inoculum N u t r i e n t s Water 0.4 0.4 -M 1 I + N " 0.4 - 0.4 M-,rN+ - 0.4 0.4 - - 0.8 M 2 I + N + 0.4 0.4 0.8' M 2 I + N " 0.4 - 1.2 |M 2I"N + - 0.4 1.2 M 2I"N~ - • - 1.6 124. v o l u m e t r i c f l a s k s and made up t o volume. F i n a l l y , t h i s s o l u t i o n was f i l t e r e d Whatman No. 30 f i l t e r p a p e r ) i n t o 50 ml p o l y p r o p y l e n e b o t t l e s , 2-3 drops o f t o l u e n e added and s t o r e d a t 4°C p r i o r t o a n a l y s i s . 5.2.2.1 A n a l y t i c a l Methods  Ammoniurn-N NH^-N i n the e x t r a c t s was d e t e r m i n e d by the p h e n o l - h y p o c h l o r i t e method d e s c r i b e d i n d e t a i l i n Appendix I. N i t r a t e - N NO^-N was d e t e r m i n e d by the c h r o m o t r o p i c a c i d method d e v e l o p e d by West and Ramachandran [1966] and m o d i f i e d by Kowalenko [ 1 9 7 3 ] ; h e r e a f t e r r e f e r r e d t o as the CTA method. S u l p h a t e - S SO^-S was d e t e r m i n e d by HI r e d u c t i o n u s i n g t h e Bismuth s u l p h i d e c o l o r i m e t r i c f i n i s h as d e s c r i b e d by Kowalenko and Lowe [1973). Phosphorus M i n e r a l phosphate i n the e x t r a c t s was d e t e r m i n e d by the molybdenum b l u e c o l o r i m e t r i c p r o c e d u r e i n HC1 system as d e s c r i b e d by [ J a c k s o n , 1958, Method I I ) . 125 Total carbon and sulphur were determined by dry combustion in the Leco Analyzer while total N was determined by a modified micro-kjeldahl digestion procedure and phenol-hypochlorite colorimetric method. Total phosphorus and organic phosphorus (P(Q)) w e r e determined by the ignition method of Saunders and Williams [1955]. 5.3 Results and Discussion Some chemical characteristics of the Langley 2-50um fraction are shown in Table 15. It should be noted that this fraction was quite high in organic carbon and nitrogen, and had C/N, N/S and N/P^ ratios falling within the limits generally reported for mineral soils. The accumulation of mineral N, P and S after two weeks incubation is shown in Tables 16 and 18. NaHCOg generally extracted more NH -^N, NO -^N, phosphate and especially sulphate, than did calcium chloride. The differences being very highly significant (P < .005) in the case of sulphate. This is presumably due to the extraction of variable proportions of adsorbed and particularly organic sulphate, in addition to mineral sulphate released during the incubation. These extracts were highly colored compared to the CaCl2 extracts. Surprisingly, the difference between phosphate extracted by the two reagents was quite small, as was the total amount of phosphate released during the two week period of the experiment. The difference in mineral N extracted by the two reagents was significant only for some treatments, NaHCOg extracting significantly more NHA-N on the M-, treatments, (Nos. 1-7). 126 TABLE 15 Some C h a r a c t e r i s t i c s o f the L a n g l e y Ah 2-50 urn F r a c t i o n pH ( 1 : 25 H 2 0 ) 5 .66 % C % N 7 .30 0 . 50 % S 0 .05 % P ( T o t a l ) 0 .095 % P ( 0 ) 0 .073 C/N R a t i o 14 . 2 N/S 10 . 3 N / p ( o ) 7 . 0 C:N:S:P ( 0 ) •• 1 4 2 : 1 0 : 1 : 1 . 4 127 5.3.1 Mineralized N, P, and 5 Extracted by CaClp Moderate amounts of NH -^N, NG^ -N and sulphate, were released over the two week period, whereas only very small quantities of P were detected, (Table 16) NH4~N accumulation was generally larger for the M2 treatments, being largest where neither sand, inoculum nor nutrients were added to the fraction. This suggested that at 40 percent moisture, aeration within the incubation jar was inadequate. This conclusion is further supported by the fact that N03-N mineralization was generally greater for the M-j treatments, especially those treatments in which sand was included. The approximate total N (NH4-N + N0.J-N) mineralized for the experiment showed an interesting departure from what would normally be expected, in that the total N for the sum of the M2 treatments exceeded that of the M-j treatments. The explanation for this anomaly is perhaps two-fold. Firstly, under the M2 regimen, the rate of N mineralization was quite likely more rapid, as Waring and Bremner [1964a] reported that under waterlogged conditions N mineralization was more rapid than under aerobic conditions. Secondly, the relatively high temperature of the incubation may have also favoured NH^ -N production, and indeed, there was more NH^ -N in the M-j treatments than NO -^N. Against this should be weighed the fact that the population of nitrifying organisms may not have reached its maximum level of development and perhaps with a prolonged incubation, all or most of the NH^ -N would be converted to NO-j-N. Sulphate mineralization followed a similar pattern to nitrate mineralization, being greater for the M-| treatments, with the 128 TABLE 16 N, P and S E x t r a c t e d w i t h 0 .1 N C a C l 9 (ppm) T r e a t m e n t N H 4 - N N 0 3 - N T o t a l N 2 -S 0 4 PO4" 1 M l S + l V 24 1 5 . 5 3 9 . 5 2 3 . 0 2 . 5 2 S + l V 23 1 3 . 0 36 1 8 . 7 -3 S + I ~ N + 30 1 2 . 5 4 2 . 5 1 1 . 5 -4 S + I " N " 27 8 . 5 3 5 . 5 9 .1 2 . 5 5 s-iV 26 5 . 5 3 1 . 5 4 . 3 1.5 6 S " I + N " 21 6 . 0 27 3 . 0 1 .5 7 S ~ I ~ N + 21 4 . 0 25 2 .1 1 .5 8 S " I ¥ 20 3 . 5 2 3 . 5 0 . 2 -9 S + I + N + 27 1 1 . 0 38 1 9 . 6 1.3 10 S + l V 28 4 . 5 3 2 . 5 19 .1 1 .2 11 S + I - N + 37 4 . 0 41 8 . 9 1.3 12 S + I " N " 25 7 . 0 32 7 . 2 1.3 13 s-iV 26 4 . 5 3 0 . 5 3 . 0 1.8 14 S ~ I + N ~ 24 5 . 5 2 9 . 5 1.7 -15 S " I " N + 3 0 4 . 0 34 - 0 . 6 1.3 16 S"I"N" 38 5 . 0 43 - 1 . 5 1 .0 L.S.D. .01 8 . 3 4 . 2 4 . 6 1.8 129 largest increase occurring where near "optimum" levels of all factors were present (i .e. M-j S +I +N +). It should be noted that there was no significant difference between the M-j and treatments when sand and inoculum were added to both. This raises the question of whether increased moisture or a higher temperature, per se, was responsible for what appeared to be an inhibition of the conversion of NH^ -N to NO^ -N or whether these factors merely provided favourable conditions for denitrification. While i t is not possible to answer this question directly from the available data, the fact that there were no significant differences between the total N for treatments one (M-jS+I+N+), nine (M,,S+I+N+) and sixteen (M^S'TiN"") suggests that dentrification was perhaps minimal. In the case of P mineralization, there were no significant differences for treatments where measurable amounts of P were extant. Table 17 shows the treatment effects and interactions with respect to the mineralization of ammonium, nitrate, sulphate and phosphate. For NH^ -N the higher moisture level appears to be the most important factor for accumulation whereas, inoculum had the opposite effect. The inoculum presumably provided a source of nitrifying organisms. For the mineralization of nitrate the roles of M2 and I are reversed, both being highly significant. The use of sand in the incubation medium had a positive and highly significant effect on nitrate production. There were also other interactions of less significance. Sulphur mineralization appears to be strongly influenced by sand (improved aeration) and inoculum (increased micro-bial activity) as well as the addition of nutrients (N). This latter 130 TABLE 17 S i g n i f i c a n t T r e a t m e n t E f f e c t s and I n t e r a c t i o n s E f f e c t Means (0.1N C a C l 2 ) F a c t o r s NH 4-N N0 3-N 2-SO4 PO4" M 2 ** 5.38 ** -2.9 S ** 4.75 ** 12.44 M2S ** -2.9 I ** -3.63 ** 2.13 ** 6.3 M2I * -2.63 S I ** 3.3 M2SI ** 3.38 N ** 2.56 M2N S N * 1.5 M2SN I N M2IN S I N ** -3.63 * 1.13 M2SIN * S i g n i f i c a n t (P < 0.05). * H i g h l y S i g n i f i c a n t (P < 0.01). 131 effect was somewhat surprising, as it is oftentimes assumed that ade-quate amounts of micro-nutrients are present in soils to meet at least microbial, i f not plant requirements. 5.3.2 Mineralized N, P, and S Extracted by NaHCOg Table 18 shows the results of the bicarbonate extraction of mineral N, P, and S. The extracts were highly coloured, indicating the presence of soluble organic matter, and were treated with phosphate free activated carbon prior to the determination of phosphorus. There were fewer significant differences between the various treatments for NH^ N extracted (except for the control, treatment 8). Also, the difference between the M-j and M2 treatments for NH^ -N was not significant. NOg-N released, showed a pattern similar to that of the CaCl2 extracts; more NO^ -N being present where sand and inoculum were added. The amounts of sulphate (HI reducible) found in the extracts were not considered indicative of sulphate mineralized, as apparently labile organic sulphur compounds were extracted. The overall increase in extractable sulphate does, however, suggest that there were transformations within the organic-S fractions rendering some consti-tuents more soluble. Phosphorus present in these extracts showed a small increase over the CaCl2 extracts, however, there were very few significant differences among treatments; the most noteworthy being an increase in extractable P for the M2 treatments. Table 19 shows the effects and interactions of the factors on NHA-N, NCu-N, sulphate and phosphate 132 TABLE 18 N, P and S E x t r a c t e d With 0.5 N NaHCO. pH 8.5 (ppm) T r e a t m e n t NH 4-N N0 3-N T o t a l N so 4-s P O 4 - P 1 M 1 S + I + N + 46 27 73 225.2 2.0 2 S + l V 50 27.5 77.5 202.2 3.3 3 S V N + 47 26 73 228.5 3.8 4 S V N " 50 17 67 227.5 4.8 5 S - I V 55 16.3 71.3 166.3 3.8 6 S " I + N " 43 15.8 58.8 163.5 4.1 7 S " I " N + 41 15.5 56.5 159.5 3.6 8 S " I " N " 17 12.7 29.7 139.8 3.3 9 M 2 S + l V 43 25 68 200 4.8 10 S + l V 51 21.5 72.5 191 4.4 11 S + I " N + 46 21.0 67 185 4.1 12 S V N " 41 10 51 173.5 4.6 13 s - i V 37 10.4 47.4 130.5 4.1 14 s"iV 36 10.4 46.4 137.5 3.7 15 S " I " N + 37 11.2 48.2 132.5 5.0 16 S ~ I " N " 48 10.0 58 131 5.7 L .S ' .D . .01 7.1 10.0 39.1 2.5 133 TABLE 19 S i g n i f i c a n t T r e a t m e n t E f f e c t s and I n t e r a c t i o n s E f f e c t Means (0.5N NaHCO 3 pH 8.5) F a c t o r s NH 4-N N0 3-N SO 2" PO4" M 2 ** 7.5 ** -4.8 ** -29 * 2.7 S ** 9.1 ** 59.1 M2S I ** 3.78 M2I SI ** -2.75 * 3.0 M2SI ** 7.5 ** 10.9 N - * 2.0 * 3.4 M2N SN ** -4.5 M2SN ** 6.25 IN M2IN SIN M2SIN * S i g n i f i c a n t (P < 0.05). * H i g h l y S i g n i f i c a n t (P < 0.01). 134 mineralization, and their levels of significance. The major effects and low order interactions are similar to those for the CaC^ extracts. 5 .4 Conclusion Readily measurable amounts of mineral N, P, and S were detected following the aerobic incubation of a soil particle-size separate. Aerobic conditions seemed to prevail throughout the experiment at the lower moisture level, and was enhanced by mixing the soil fraction with three times its weight of quartz sand. The addition of an inoculum appeared to be of crit ical importance to the production of nitrate and sulphate, while the inclusion of supplementary nutrients also proved to be efficacious for nitrate and sulphate production. The accumulation of mineral phosphorus was favoured by the higher moisture level. On the basis of these results the most favourable combination of factors were selected for further investigation of the mineraliz-ation of N, P, and S from organo-mineral complexes and soil particle-size separates. 135 6. INCUBATION STUDIES II 6.1 Objectives This investigation was undertaken to determine the distribution and amounts of carbon, nitrogen, phosphorus and sulphur in the particle-size separates of five Gleysolic soils. Also, to measure the biologic-al stability of the organic component in the separates and to ascertain the extent to which organic N, S, and P could be converted to inorganic forms, assimilable by plants. A further objective of this experiment was to establish whether the breakdown of organic matter associated with the sub-clay-size fractions occurred at a faster or slower rate than organic matter in the soil as a whole. 6.2 Materials and Methods The soils and particle-size separates used in this study were the five gleysolic soils described previously and the four separates isolated from each, by the procedures outlined in detail elsewhere in this treatise. The incubation method was the same as described for treatment (M^S+I+N+) in the preceding section. Each sample was mixed with three times its weight of quartz sand, and inoculum, nutrients and water added to elevate the moisture content to 20 percent by weight. Each sample was prepared in duplicate and incubated at 37.5°C for eight weeks. Corrections for loss of moisture was done at weekly intervals while subsampling for analysis was done fortnightly. 136 F i v e gm samples o f the <2mm a i r d ry s o i l , 2~50um, l-2ym and 0.2-1 urn f r a c t i o n s and 1 gm o f the <0.2ym f r a c t i o n were used i n the i n c u b a t i o n . A t the time o f a n a l y s e s the e q u i v a l e n t o f 1 gm ( f o r t h e f r a c t i o n s >0.2ym) and 0.2 g f o r <0.2ym f r a c t i o n s was weighed o u t , and e x t r a c t e d (1:25, s o i l : s o l u t i o n r a t i o ) , b y I N KC1 pH 2.0. 6.2.1 S e l e c t i o n o f E x t r a c t a n t Because o f the d u p l i c a t i o n o f e f f o r t , and amounts o f m a t e r i a l s + - -2 r e q u i r e d when two s e p a r a t e e x t r a c t i o n s (one f o r NH 4, NO^ and S0^ , and -3 • a n o t h e r f o r P0^ ) a r e c o n d u c t e d , t he l i t e r a t u r e was s u r v e y e d and com-p a r i s o n s made i n the l a b o r a t o r y t o f i n d a s i n g l e r e a g e n t which would q u a n t i t a t i v e l y e x t r a c t a l l f o u r i o n s . P o t a s s i u m c h l o r i d e , one o r two Normal and a t d i f f e r e n t pH v a l u e s , i s w i d e l y used f o r the e x t r a c t i o n o f a l l forms o f m i n e r a l n i t r o g e n [Bremner, 1965], and was thus s e l e c t e d f o r com p a r i s o n w i t h t h e o t h e r two e x t r a c t a n t s p r e v i o u s l y used. One Normal KC1 was p r e f e r r e d t o 2 Normal because i t would r e q u i r e l e s s s i l v e r s u l p h a t e t o remove c h l o r i d e i n t e r f e r e n c e i n the c o l o r i m a t r i c d e t e r m i n -a t i o n o f n i t r a t e by CTA. The e f f i c i e n c y o f e x t r a c t i o n o f IN KC1 a t t h r e e pH v a l u e s (pH 1.0, pH 2.0 and pH 4.8) was d e t e r m i n e d and IN KC1 pH 2.0 found t o y i e l d the b e s t r e s u l t s o v e r a l l . A t pH 1.0, some e x t r a c t s were h i g h l y c o l o u r e d , more so th a n a t pH 2.0, w h i l e , phosphate i n t h e pH 2.0 e x t r a c t s was s i g n i f i c a n t l y g r e a t e r , and more c l o s e l y r e l a t e d t o P e x t r a c t e d by both 0.5 N NaHC0 3 pH 8.5 and 0.002 N H 2 S 0 4 pH 3.0, th a n 1 N CK1 a t pH 4.8. IN KCT ph 2.0 was t h e r e f o r e s e l e c t e d f o r comparison w i t h C a C l 0 and NaHC0 o. T a b l e 20 shows the amounts o f m i n e r a l N, P, and S 137 TABLE 20 Comparison of 0.1N CaCl 2, 0.5N NaHCOg and IN KC1 pH 2.0 As Extractants for Mineralized N, P and S 0.1N 0.5N IN CaCl0 NaHC03 KC1 r C pH 8.5 pH 2.0 NH4-N 24.0 ± 1.2 46.0 ± 3.6 45.6 ± 3.1 N03-N 15.5 ± 1.71 27.0 ± 4.24 22.9 ± 1.98 Total-N 39.5 73 68.5 so4 23.0 ± 1.8 225.2 ± 25.2 20.1 ± 1.2 P O 4 2.5 ± 2.2 2.0 ± 0.28 6.0 ± 1.3 138 e x t r a c t e d by the t h r e e r e a g e n t s from t h e L a n g l e y Ah 2-50um f r a c t i o n , f o l l o w i n g two weeks i n c u b a t i o n . Based on t h e s e d a t a , KC1 was used i n s u b s e q u e n t e x p e r i m e n t a l work as the e x t r a c t i n g s o l u t i o n . 6.2.2 A n a l y t i c a l P r o c e d u r e s T o t a l c a r b o n and s u l p h u r were d e t e r m i n e d by combustion methods w i t h t h e Leco i n d u c t i o n f u r n a c e . T o t a l N by the m i c r o k j e l d a h l (A.O.A.C. methods o f a n a l y s i s ) and phenol h y p o c h l o r i t e methods, and t o t a l phosphorus by t he i g n i t i o n method o f Saunders and W i l l i a m s [1955]. Ammonium-N was d e t e r m i n e d by the p h e n o l - h y p o c h l o r i t e method (no d i g e s t i o n was n e c e s s a r y ) and NOg-N by the c h r o n o t r o p i c a c i d method [Kowalenko, 1973] f o l l o w i n g p r e c i p i t a t i o n o f c h l o r i d e by s i l v e r s u l p h a t e . NOg-N was a l s o d e t e r m i n e d ( f o r purposes o f comparison) by a u t o - a n a l y z e r . In t h i s p r o c e d u r e NOg-N i s r e d u c e d t o n i t r i t e by a copper-cadmium r'eductor column and the n i t r i t e r e a c t s under a c i d c o n d i t i o n s w i t h s u l p h a n -i l a m i d e t o form a diazo-compound. T h i s compound then c o u p l e s w i t h N - l - N a p t h y l e t h y l e n e - d i a m i n e d i h y d r o c h l o r i d e t o form a r e d d i s h - p u r p l e azo dye which i s q u a n t i t a t i v e l y measured by c o l o r i m e t r i c a n a l y s i s . S u l p h a t e i n the e x t r a c t s was d e t e r m i n e d by HI r e d u c t i o n and s u l p h i d e measured c o l o r i m e t r i c a l l y as Bismuth s u l p h i d e . S u l p h a t e was a l s o d e t e r m i n e d c o l o r i m e t r i c a l l y by A u t o - A n a l y z e r based on the method o f L a z a r u s , H i l l and Lodge [ 1 9 6 6 ] . In t h i s method, the e x t r a c t i s f i r s t p a s sed t h r o u g h a c a t i o n exchange column t o remove i n t e r f e r i n g 2+ 3+ 3+ i o n s ( e . g . Ca , Fe , Al ). The e x t r a c t i s next mixed w i t h B a C l ^ 2+ a t a pH o f 2.5-3.0 t o form BaSO^. Excess Ba i o n s i n the m i x t u r e then r e a c t s w i t h methylthymol b l u e t o form a b l u e c h e l a t e , t he a b s o r b -139 ance o f which i s measured a t 460 nm. Phosphate i n the e x t r a c t s was d e t e r m i n e d by the molybdenum-bl u e c o l o r i m e t r i c method i n HC1-system as d e s c r i b e d by J a c k s o n [ 1 9 5 8 ] , method I I . 6.3 R e s u l t s and D i s c u s s i o n 6.3.1 Carbon, N i t r o g e n , Phosphorus and S u l p h u r i n S o i l P a r t i c l e - S i z e F r a c t i o n s T a b l e 21 shows t h e d i s t r i b u t i o n o f c a r b o n , n i t r o g e n , phosphorus and s u l p h u r i n the p a r t i c l e - s i z e c l a s s e s o f f i v e g l e y s o l i c s o i l s . The C:N, N:S,-N:P^QJ, C:S, S : P ^ , and C : N : S : P ^ r a t i o s a r e p r e s e n t e d i n T a b l e 22. A l l f i v e s o i l s a r e c h a r a c t e r i z e d by a marked i n c r e a s e i n the c o n t e n t s o f C, N, S, and P as p a r t i c l e - s i z e d e c r e a s e s . S i m i l a r l y , t h e r e a r e some o b s e r v a b l e t r e n d s i n the r a t i o s o f t h e s e elements w i t h r e s p e c t t o p a r t i c l e - s i z e . I t i s noteworthy t h a t the o r g a n i c phosphorus c o n t e n t o f t h e v a r i o u s f r a c t i o n s w h i l e g e n e r a l l y f o l l o w i n g the e s t a b -l i s h e d p a t t e r n f o r C, N, and S, i s q u i t e v a r i a b l e when e x p r e s s e d as a pe r c e n t a g e o f the t o t a l P. T h i s v a r i a t i o n ranges from as l i t t l e as 18 p e r c e n t i n t h e D e l t a Ap l-2ym f r a c t i o n (17 p e r c e n t f o r t h e whole s o i l ) , to 95 p e r c e n t i n the <0.2um f r a c t i o n o f the L a n g l e y Ah. The C/N r a t i o shows a p r e d i c t a b l e d e c r e a s e as p a r t i c l e -s i z e d e c r e a s e s and the p a t t e r n i s r e p e a t e d f o r the C/S and C / P ^ r a t i o s . I n d i c a t i o n s a r e t h a t the N/S r a t i o tends to d e c r e a s e w i t h p a r t i c l e - s i z e , however, t h e r e a r e many d i s c r e p a n c i e s , p a r t i c u l a r l y i n t he <0.2ym f r a c t i o n s where t h e r e i s a l a r g e i n c r e a s e i n n i t r o g e n 140 TABLE 21 Carbon, Nitrogen, Sulphur and Phosphorus in Soils and Soil Particle-Size Separates Sample %C %S % p ( o ) P(0) a s % Total P LANGLEY Ah <2 mm 8.0 0.52 0.088 0.099 94 2-50 um 7.3 0.51 .050 .073 77 1-2 8.0 0.78 .083 .140 85 0.2-1 9.6 0.73 .154 .146 93 <0.2 11.3 1.48 .200 .241 95 HAZELW00D Ah <2 mm 5.3 0.34 0.046 0.074 62 2-50 um 3.8 0.21 .040 .052 57 1-2 5.7 0.42 .056 .080 67 0.2-1 5.7 0.68 .100 .164 94 <0.2 11.8 1.58 .188 .207 77 HATZIC Ap <2 mm 3.8 0.25 0.040 0.075 60 2-50 um 2.5 0.22 .043 .034 38 1-2 2.8 0.29 .053 .061 58 0.2-1 4.4 0.32 .083 .108 65 <0.2 7.5 0.69 .137 .156 70 CLOVERDALE Ap <2 mm 5.5 0.44 0.078 0.058 87 2-50 um 3.8 0.29 .040 .035 54 1-2 6.6 0.71 .084 .107 70 0.2-1 7.0 0.82 .092 .133 67 <0.2 10.4 1.13 .182 .155 54 DELTA Ap <2 mm 3.4 0.21 0.054 0.022 17 2-50 ym 0.9 0.07 .011 .021 21 1-2 5.9 0.46 .077 .030 18 0.2-1 6.5 0.58 .097 .125 48 <0.2 9.4 0.96 .183 .103 25 141 TABLE 22 Ratios of C, N, P and S In Soils and Particle-Size Separates Sample (urn) C/N N/S N / P ( 0 ) C/S c / p ( o ) C:N:S:P ( ( ) ) LANGLEY Ah < 2 mm 15.3 5.9 5.3 90.6 80.5 153:10:1.7:1.9 2-50 14.2 10.3 7.0 145.2 99.5 142:10:1:1.4 1-2 10.3 9.4 5.6 96.6 57.3 103:10:1.1:1.8 .2-1 13.1 4.7 5.0 62.1 65.5 131:10:2.1:2.0 < 0.2 7.6 7.4 6.1 56.4 46.8 76:10:1.4:1.6 HAZELW00D Ah < 2 mm 15.6 7.4 4.6 115.2 71.6 156:10:1.4:2.2 2-50 17.8 5.3 4.1 95 73.1 178:10:1.9:2.4 1-2 13.7 7.4 5.2 101.8 71.3 137:10:1.4:1.9 .2-1 8.4 6.8 4.2 57 34.8 84:10:1.5:2.4 < 0.2 7.5 8.4 7.6 62.8 57 75:10:1.2:1.3 HATZIC Ap < 2 mm 15.2 6.3 3.3 95 50.7 152:10:1.6:3.0 2-50 11.4 5.1 6.5 58 73.5 114:10:2.0:1.6 1-2 9.8 5.4 4.7 61 45.9 98:10:1.9:2.1 .2-1 13.8 3.9 3.0 53 40.7 138:10:2.6:3.3 < 0.2 10.9 5.1 4.4 54.7 48.1 109:10:2.0:2.3 CLOVERDALE Ap < 2 mm 12.5 5.6 7.6 70.5 94.8 125:10:1.8:1.3 2-50 13.3 7.1 8.1 95 108.6 133:10:1.4:1.2 1-2 9.3 8.5 6.6 78.6 61.7 93:10:1.2:1.5 .2-1 8.5 8.9 6.2 76.1 52.6 85:10:1.1:1.6 < 0.2 9.2 6.2 7.3 57 67.1 92:10:1.6:1.4 DELTA Ap < 2 mm 16.2 3.9 9.6 63 154.6 162:10:2.6:1.1 2-50 12.9 6.4 3.3 81.8 42.9 129:10:1.6:3.0 1-2 12.8 6.0 15.3 76.6 196.7 128:10:1.7:0.65 2-1 11.3 6.0 4.6 67 52 113:10:1.7:2.2 < 0.2 9.8 5.3 9.3 51.4 91.3 98:10:1.9:1.1 142 relative to carbon. There are no discernable trends in the N/P^ ratios with respect to particle-size, possibly due to the variability in organic-P content. The relative enrichment of N, P, and S in the finer fractions raised the question of what mechanisms or agencies are responsible for this enrichment. There seems to be two possible explanations. Firstly, i t is probable that N, P, and S containing functional groups are directly involved in the bonding of organic matter to mineral particles. Thus, these elements would be more closely held to clay surfaces and perhaps less susceptible to mineralization. It should be noted, however, that these would generally be of secondary importance to the comparatively large amounts of carboxyl and hydroxyl groups present in organic matter. Secondly, the enrichment may occur subsequent to the reaction of organic matter with clay, due to the loss of carbon by biological oxidation. The f i rst hypothesis is particularly attractive because there is much evidence (for reviews see Mortland, 1970; Greenland,1965,a,b, 1971] to indicate that clays will bond to N containing functional groups of large molecules. Unfortunately, very l i t t l e work has been done on the bonding between clays and hydrous oxides of iron and aluminum and organic sulphur and phosphorus compounds. However, Somasundaram and Fuerstenau [1966] have shown that sodium dodecyl sulphonate is adsorbed by alumina, while Flegmann [1963] has shown that i t is similarly bonded at the edges of Kaolinite particles. Since positive sites normally exist at the edges of clays and on aluminum and iron hydroxides below pH 8.0, organic compounds containing negatively charged phosphate 143 groups (due t o h y d r o l y s i s , e.g. o f [R-0-P]-0-R bonds) can be a s s o c i a t e d by c o u l o m b i c a t t r a c t i o n , o r a d s o r b e d by the c l a y s u r f a c e v i a a c a t i o n b r i d g e . T h i s i s a l i k e l y mode o f bonding f o r the su g a r phosphates arid p o s s i b l y some n u c l e i c , a c i d d e r i v a t i v e s , a l t h o u g h i n the l a t t e r c a s e t h e r e a r e o t h e r p o s s i b i l i t i e s . T here i s a l s o s u p p o r t i n g e v i d e n c e f o r t h e second h y p o t h e s i s , m a i n l y from t h e R u s s i a n workers [Kononova, 1966], who have r e p o r t e d t h a t t h e h u m i f i c a t i o n p r o c e s s l e a d s t o a n a r r o w i n g o f t h e C/N r a t i o o f s o i l o r g a n i c m a t t e r . W h i l e i t c a n n o t be assumed, a p r i o r i , t h a t the t r a n s f o r m a t i o n o f o r g a n i c m a t t e r o c c u r s a f t e r bonding t o m i n e r a l s u r f a c e s , t h e h i g h a f f i n i t y o f c l a y s f o r p r o t e i n a c e o u s m a t t e r [ H a r t e r and S t o t s k y , 1971] and amino a c i d m e t a b o l i t e s [ S o r e n s e n , 1972] s u g g e s t s t h a t t h e m e t a b o l i s m o f c a r b o n r e l a t i v e t o n i t r o g e n might o c c u r s u b s e -quent t o a s s o c i a t i o n w i t h c l a y s . 6.3.2 Ammoniurn-N The amounts o f ammonium-N, n i t r a t e - N , s u l p h a t e and phosphate m i n e r a l i z e d d u r i n g e i g h t weeks i n c u b a t i o n were measured i n f i v e s o i l s and t h e i r p a r t i c l e - s i z e f r a c t i o n s . The d a t a f o r NH^-N a r e p r e s e n t e d i n F i g u r e s 10-12 (se e a l s o Appendix I V ) . I t s h o u l d be n o t e d t h a t t h e da t a f o r the <0.2ym f r a c t i o n o f the Hazelwood and C l o v e r d a l e s o i l s were not i n c l u d e d because the measurements were not d u p l i c a t e d , due t o i n s u f f i c i e n t m a t e r i a l , and hence c o u l d n o t be i n c l u d e d i n t h e s t a t i s t i c a l a n a l y s i s o f the e x p e r i m e n t a l d a t a . The t r e n d s were n e v e r -t h e l e s s the same as f o r the o t h e r <0.2um f r a c t i o n s . 144 L A N G L E Y A h D E L T A A p < 2000 u. 2-50 1-2 0.2-1 < o.2 g 800-j T I M E (weeks) Figure 1 0 -NH^ -N Mineralized from Particle-Size Separates During Eight Weeks "of Incubation 145 HAZELWOOD Ah, CLOVER DALE Ap < 20CC u 2-50 1-2 0.2- I 8 0 0 i TIME (weeks) F i g u r e 11 NH^-N M i n e r a l i z e d from P a r t i c l e - S i z e S e p a r a t e s D u r i n g E i g h t Weeks o f I n c u b a t i o n 146 HATZIC Ap _ 800-1 I TIME (weeks) F i g u r e 12 NH^-N M i n e r a l i z e d from P a r t i c l e - S i z e S e p a r a t e s D u r i n g E i g h t Weeks o f I n c u b a t i o n 147 The amounts o f NH^-N m i n e r a l i z e d i n c r e a s e d as p a r t i c l e - s i z e d e c r e a s e d f o r each o f the s o i l s s t u d i e d . The b e h a v i o u r o f the s i l t f r a c t i o n , however, d i d not v a r y much from the u n f r a c t i o n a t e d s o i l , r e l e a s i n g somewhat l e s s NH^-N i n the L a n g l e y and D e l t a s o i l s , and s i g n i f i c a n t l y l e s s i n the H a t z i c , Hazelwood and C l o v e r d a l e s o i l s . The r a t e o f r e l e a s e o f NH^-N was a l s o h i g h e s t i n the-two f i n e r f r a c t i o n s s u g g e s t i n g e i t h e r a g r e a t e r a c c e s s i b i l i t y o f the o r g a n i c - N o r the p r e s e n c e o f a more l a b i l e form o f o r g a n i c - N . The l a t t e r p r o p o s a l i s p r o b a b l y the c a s e as Kyuma, H u s s a i n , and Kawaguchi [1969] found s o i l s c o n t a i n e d more h y d r o l y s a b l e N7 S i m i l a r l y , C h i c h e s t e r [1969] found t h a t the f i n e r f r a c t i o n s o f the C h e s t e r s i l t loam s u r f a c e s o i l , ( v i z . <0.25ym, 0.5-0.25um, 1.0-0.5um, and 2.0-1.0um) showed a s e v e r a l f o l d i n c r e a s e i n the m i n e r a l i z a t i o n o f NH^-N o v e r the c o a r s e r f r a c t i o n s which was a t t r i b u t e d m a i n l y t o the p r e s e n c e o f d i f f e r e n t p r o p o r t i o n s o f r e a d i l y v e r s u s d i f f i c u l t y e x t r a c t a b l e c h e m i c a l forms o f n i t r o g e n . The maximum m i n e r a l i z a t i o n o f N t o the ammonium form was r e a c h e d a f t e r 4-6 weeks w i t h o n l y a s m a l l p e r c e n t a g e change t h e r e a f t e r , e x c e p t f o r t h e <0.2ym f r a c t i o n s o f the D e l t a and H a t z i c s o i l s . For t h e s e two f r a c t i o n s t h e r e was a marked d e c l i n e i n NH^-N a f t e r 4 weeks, which was p o s s i b l y due t o the o x i d a t i o n o f NH^-N t o NO^-N f o l l o w i n g a c e s s a t i o n o f NH^-N p r o d u c t i o n . -The r e l e a s e o f NH^-N seems to v a r y d i r e c t l y w i t h the p e r c e n t N i n t h e f r a c t i o n s and i n v e r s e l y w i t h the C/N r a t i o . The amount o f NH^-N m i n e r a l i z e d d u r i n g the e x p e r i m e n t e x p r e s s e d as a p e r c e n t o f the t o t a l N i n each f r a c t i o n i s shown i n T a b l e 23. I t can be seen t h a t o n l y a s m a l l p e r c e n t a g e o f the t o t a l N i s . r e l e a s e d as NH.-N f o r the 148 L A N G L E Y Ah D E L T A A p 8001 <2000 u 2-50 1-2 0.2- I <0.2 8 0 4 TIME (weeks) 8 F i g u r e 13 NO^-N m i n e r a l i z e d f r o m P a r t i c l e - S i z e S e p a r a t e s D u r i n g E i g h t Weeks o f I n c u b a t i o n HAZELWOOD Ah, CLOVERDALE Ap F i g u r e 14 NO^-N M i n e r a l i z e d f r o m P a r t i c l e - S i z e S e p a r a t e s D u r i n g E i g h t Weeks o f I n c u b a t i o n 150 HATZIC Ap 8001 E o. Q. O L U N -J < L U 600 *= 400H I ro o 200 ,<* <0.2 \i •••• 0.2 - I p < 2000 M TIME (weeks) F i g u r e 15 M0 3-N M i n e r a l i z e d f r o m - P a r t i c l e - S i z e S e p a r a t e s D u r i n g E i g h t Weeks o f I n c u b a t i o n TABLE 23 N, P and S M i n e r a l i z e d A f t e r 6 Weeks I n c u b a t i o n a s a P e r c e n t o f T o t a l N, P & S i n t h e F r a c t i o n NH 4 -N NH 4-N + NH 4 + F r a c t i o n NH 4-N as % N0 3 -N N0 3-N % N0 3-N N0 3 -N % so4 ppm T o t a l N ppm T o t a l N ppm T o t a l N ppm LA<2,000 101.2 1.9 145.2 2.8 246.4 4.7 79.1 2-50 58.7 1.1 128.1 2.5 186.8 3.6 63.1 1-2 97.5 1.2 195 2.5 292.5 3.7 143.5 0. 2-1 301.3 4.1 360 4.9 661.3 9.0 182 <0'.2 656 4.4 696.9 4.7 1353 9.1 217 HAZ<2,000 194.3 5.7 241 7.1 435.3 12.8 122.1 2-50 69.3 3.3 154.9 7.4 224.2 10.7 58 1-2 63.8 1.5 152 3.6 215.8 5.1 67 0. 2-1 446.1 6.5 480.1 7.0 926.2 13.5 191 HAT<2,000 250.6 10.0 269 10.7 519.6 20.7 66 2-50 80.7 3.6 101.6 4.6 182.3 8.2 72 1-2 103.2 3.5 197.4 6.8 300.6 10.3 80 0. 2-1 243.2 7.6 353 11.0 596.2 18.6 205 <0.2 525 7.6 632 9.1 1157 16.7 197.2 CL0V<2,000 235.7 5.3 261 5.9 496.7 11.2 134.8 2-50 100 3.4 139.8 4.8 239.8 8.2 89.6 1-2 285.6 4.0 365 5.1 650.6 9.1 194 0.2-1 603.8 7.3 735 8.9 1339 16.2 251 DEL<2,000 46.3 2.2 92.2 4.4 138.5 6.6 -0.3 2-50 23.6 3.3 76 10.8 99.6 14.1 -6.1 1-2 137.5 3.0 194 4.2 331.5 7.2 56.1 0.2-1 252.5 4.3 322 5.5 574.5 9.8 147 <0.2 556 5.8 705.1 7.3 1261 13.1 240 S 0 4 as % T o t a l S P 0 4 ppm P 0 4 a % T o t a l P (0) 8.0 8.6 10.2 12.4 9 16.5 11.1 8.3 11.6 8.8 21.0 13.1 19.0 12.6 23.2 25.6 18.1 18.8 18.7 11.7 23.3 2.0 2.0 4.7 2.7 -12.2 -6.2 -24.5 29 18.4 23.5 10.8 74.8 3.1 -15.5 6.3 -1.6 -3.3 15.5 3.1 47 182.3 348.7 -41.5 0.2 0.3 0.3 0.2 3.6 1.1 3. 3. 12. 0. 1.1 1.2 1.4 22.4 60.8 152 various fractions studied, with the exception of the Hatzic (unfraction-ated) soi l . There is also, a noticeable increase in the N mineralized percentage as particle-size decreases. 6.3.3 Nitrate-N The mineralization of nitrogen to the nitrate over the same period, is shown in Figures 13-15. Not surprisingly, there is a similarity in the rate and amounts of NOg-N produced with respect to particle-size. One exception being the Hazelwood so i l , where the s i l t (2-50um) and coarse clay (l-2um) fractions had a similar rate of NOg-production, with slightly less nitrate being produced in the coarse clay. Once again the unfractionated soil showed a pattern of mineraliz-ation intermediate between those of the various fractions, indicating a greater contribution from the finer fractions in some of the soils. Unlike NH^ -N production, which was favoured by the high in-cubation temperature (37.5°C) NOg-N production did not reach a maximum at six weeks and showed only a slight tendency to level off even after eight weeks of incubation. This was to be expected, however, considering the large amounts of NH^ -N present. Nitrate released was directly related to total N in the fraction and inversely with C/N ratio. For the purpose of comparison the nitrate released after six weeks of incubation is expressed as a percent of total N in the fraction, (Table 23) and found to be higher than the corresponding values for NH^ -N released. Also, percentages increased as particle-size decreased. 153 6.3.4 Total-N The sum of the ammonium and nitrate-N produced was taken as the total-N mineralized because very l i t t le nitrite has been found to exist in soils, and under aerobic conditions i t is usually rapidly converted to nitrate. The total mineral N produced during incubation is of importance because i t is often used as an index of a soi l 's capacity to supply nitrogen to a crop, and more recently has been correlated with some of the extractable portions of soil organic-N, [Freney and Simpson, 1969; Chichester, 1969; Keeney and Bremner, 1966b]. The total mineral-N produced for each fraction during six weeks of incubation, and this amount, expressed as a percentage of the total N for each fraction is shown in Table 23. Relatively large amounts of mineral-N was produced by both unfractionated soils and particle-size separates. The <0.2um fraction mineralized an average of 1257 ppm or 13 percent (Average for three soils) of the total N in that fraction. This is somewhat less than the 1907 ppm or 32.3 percent M mineralized, reported by Chichester [1969] for the <0.25um fraction of the Chester surface soi l . There is , however, an important difference between the latter's study and the present investigation. The difference is that Chichester used a waterlogged incubation system similar to that proposed by Waring and Bremner [1964a,b] while the present study utilized an aerobic system. This might partially explain the observed difference, because Bremner and his co-workers have shown that mineralization of N is more rapid under anaerobic conditions. 154 A l s o worthy o f note i n the p r e s e n t i n v e s t i g a t i o n i s the r e l a t i v e l y l a r g e p e r c e n t a g e o f the t o t a l s o i l N m i n e r a l i z e d by the Hazelwood and e s p e c i a l l y the H a t z i c u n f r a c t i o n a t e d s o i l s . In a p a r a l l e l s t u d y the Hazelwood, H a t z i c and C l o v e r d a l e s o i l s were i n c u b a t e d under i d e n t i c a l c o n d i t i o n s e x c e p t t h a t no i n o c u l u m was added. , The v a l u e s f o r t o t a l N m i n e r a l i z e d a f t e r s i x weeks i n t h e s e s o i l s were 5.3 p e r c e n t , 6.1 p e r c e n t and 8.0 p e r c e n t , r e s p e c t i v e l y , compared t o 12.8 p e r c e n t , 20.7 p e r c e n t and 11.2 p e r c e n t f o r t h e same s o i l s w i t h i n o c u l u m . The d i f f e r e n c e s were s i g n i f i c a n t , P < 0.05 f o r C l o v e r d a l e . These f i n d i n g s once a g a i n b r i n g i n t o q u e s t i o n t h e w i d e l y h e l d view t h a t the i m p o r t a n t m i c r o o r g a n i s m s c a p a b l e o f m i n e r a l i z i n g N, P,-and S a r e u b i q u i t o u s i n s o i l s . The h i g h l y s i g n i f i c a n t e f f e c t o f the i n o c u l u m on the "whole" s o i l s s u g g e s t s not j u s t the a d d i t i o n o f more " a i t i m o n i f i e r s " o r " n i t r i f i e r s " per s e , but r a t h e r , the i n t r o d u c t i o n o f s p e c i e s which a r e more e f f i c i e n t f o r t h e p u r p o s e , under the c o n d i t i o n s o f t h e i n c u b a t i o n . The l a c k o f u b i q u i t y i n s o i l s o f c e r t a i n i m p o r t a n t s p e c i e s o f m i c r o o r g a n i s m s i s f u r t h e r s u p p o r t e d by Swaby and F e d e l [1973] who found t h a t about o n e - h a l f o f the f i f t y -s i x A u s t r a l i a n s o i l s t h ey examined o x i d i z e d added s u l p h u r v e r y s l o w l y o r not a t a l l . T h i s b e h a v i o u r was a s c r i b e d t o , "the absence o f T h i o b a c c i 1 j , p a r t i c u l a r l y Th. t h i o o x i d a n s . " F u r t h e r t o t h i s Cosgrove [1967] r e p o r t e d t h a t workers i n the S o v i e t Union have e x p l o i t e d the m i n e r a l i z a t i o n o f o r g a n i c - P by B a c i l l u s megatherium ( p h o s p h o b a c t e r i n ) t o enhance c r o p growth on s o i l s where the o r g a n i s m c o u l d not be i s o l a t e d , by i n o c u l a t i n g seed w i t h the b a c t e r i u m . 155 6.3.5 Sulphur Figures 16 and 17 show the mineralization of sulphur from the soils and particle-size separates. The pattern of mineralization with respect to particle-size is repeated. The shapes of the curves are somewhat similar to those of nitrate except that by the eighth week the curves have levelled off and in some instances there is a negative rate of increase. There is a net immobilization of sulphate in the Delta unfractionated soil and its 2-50um fraction. This was a saline so i l , in i t ia l ly high in soluble salts including sulphate which probably explains the net immobilization of sulphate. In addition the total S is only 0.01 percent in the 2-50um fraction. The sulphate produced after six weeks of incubation expressed as a percentage of the total S in all the fractions is shown in Table 23. There is no clear trend with respect to particle-size for sulphur mineralization and this suggests that either there is a relatively uniform distribution of the various organic-S compounds throughout the various fractions or the labil ity of such compounds to microbial degradation is similar. The overall percentage mineral-ization of sulphur is quite high and may in part be due to the characteristic "flush of mineralization of sulphur" reported by many workers following drying and re-wetting of soils, Williams [1967], The ratios of N:S mineralized after six weeks was found to be smaller that the init ial ratios in soils and particle-size separates alike, (Table 24). Only in the <0.2um fractions were the ratios of N:S mineralized (6 weeks) similar to those in the original fractions. 156 < 2000 u 2-50 ••• I - 2 0.2-1 < 0.2 LANGLEY Ah DELTA Ap i HATZIC Ap HAZELWOOD Ah, a : 4 " 0 0 ! 1 TIME (weeks) F i g u r e 16 S u l p h u r M i n e r a l i z e d from P a r t i c l e - S i z e S e p a r a t e s D u r i n g E i g h t Weeks o f I n c u b a t i o n 157 £ Q . CL Q L U N _J < o r L U o r z> X Q_ -J ZD 0") 400-1 200H CLOVERDALE Ap • 0 . 2 - 1 LI -© 2 - 5 0 p I - 2 L l < 2000 \i 8 TIME (weeks) F i g u r e 17 S u l p h u r M i n e r a l i z e d from P a r t i c l e - S i z e S e p a r a t e s D u r i n g E i g h t Weeks o f I n c u b a t i o n 158 TABLE 24 N/S Ratio of Soils and P-S Separates Compared to Ratio of N to S Mineralized at Six Weeks N:S Ratio of Fractions N:S Mineralized After 6 Weeks LANGLEY Ah < 2000 um 5.9 3.1 2-50 10.3 3.0 1-2 9.4 2.0 0.2-1 4.7 3.6 < 0.2 7.4 6.2 HAZELW00D Ah < 2000 um 7.4 3.6 2-50 5.3 3.9 1-2 7.4 3.2 0.2-1 6.8 4.9 HATZIC Ap < 2000 um 6.3 7.9 2-50 5.1 2.5 1-2 5.4 3.8 0.2-1 3.9 2.9 < 0.2 5.1 5.9 CLOVERDALE Ap < 2000 um 5.6 3.7 2-50 7.1 2.7 1-2 8.5 3.4 0.2-1 8.9 5.3 DELTA Ap < 2000 pm 3.9 2-50 6.4 -1-2 6.0 5.9 0.2-1 6.0 3.9 < 0.2 5.3 5.3 159 These r e s u l t s s u g g e s t s u l p h u r i s m i n e r a l i z e d a t a somewhat h i g h e r r a t e than n i t r o g e n , which s u p p o r t s the f i n d i n g s o f N e l s o n [1964] who found i n l a b o r a t o r y i n c u b a t i o n t h a t the r a t i o f o r N:S m i n e r a l i z e d was a p p r e c i a b l y s m a l l e r than the N:S r a t i o i n the o r i g i n a l s o i l . In c o n t r a s t t o t h i s , however, White [1959] i n New Z e a l a n d and W i l l i a m s . [1967] i n A u s t r a l i a found t h a t the r a t i o s f o r N:S m i n e r a l i z e d g e n e r a l l y exceeded t h e c o r r e s p o n d i n g r a t i o s i n t h e o r i g i n a l s o i l s . 6.3.6 Phosphorus The m i n e r a l i z a t i o n o f phosphorus d u r i n g the c o u r s e o f the e x p e r i m e n t was somewhat e r r a t i c , however, t h e r e a r e some o b s e r v a t i o n s worthy o f n o t e . F i r s t l y , a l m o s t a l l f r a c t i o n s m i n e r a l i z e d some phosphorus d u r i n g the f i r s t two weeks o f i n c u b a t i o n , F i g u r e s 18 and 19. S e c o n d l y , the m i n e r a l i z a t i o n o f P w i t h r e s p e c t t o p a r t i c l e - s i z e i s a p p a r e n t l y the r e v e r s e o f t h a t f o r n i t r o g e n , d e c r e a s i n g w i t h p a r t i c l e - s i z e . For t h e <0.2um f r a c t i o n s , i n two o u t o f t h r e e measure-ments, t h e r e was a n e t i m m o b i l i z a t i o n o f P. A l s o , w h i l e t h e r e were many f l u c t u a t i o n s i n the amounts o f e x t r a c t a b l e phosphate w i t h t i m e , up t o s i x weeks, t h e r e was a s m a l l n e t m i n e r a l i z a t i o n o f P i n most f r a c t i o n s . I t i s d i f f i c u l t t o p r e c i s e l y i n t e r p r e t t h e o v e r a l l r e s u l t s i n view o f the d i f f e r e n c e s o b s e r v e d among s o i l s and w i t h i n i n d i v i d u a l p a r t i c l e - s i z e f r a c t i o n s . T h i s d i f f i c u l t y i s compounded by the d i f f e r e n c e s d e t e r m i n e d i n i n o r g a n i c v e r s u s o r g a n i c P w i t h i n p a r t i c l e -s i z e c l a s s e s . A l l f r a c t i o n s had m e a s u r a b l e q u a n t i t i e s o f i n o r g a n i c -P, which was perhaps i n a d e q u a t e t o meet m i c r o b i a l r e q u i r e m e n t s . D u r i n g < 2000 u 2-50 I - 2 0.2 - I < 0 .2 LANGLEY Ah CLOVERDALE Ap HATZIC Ap HAZELWOOD Ah, TIME (weeks) Figure 18 Phosphorus Mineralized from Particle-Size Separates During Eight Weeks of Incubation 161 DELTA Ap 1300-k < 0.2 u — • E 1100-C L Q . Q LU M -J < 01 700 ? 500 CO 3 Q: o x Q_ CO o X 300-< 2000 p KXH \> 0 . 2 - 1 M « 4 6 TIME (weeks) 8 F i g u r e 19 Phosphorus M i n e r a l i z e d from P a r t i c l e - S i z e S e p a r a t e s D u r i n g E i g h t Weeks o f I n c u b a t i o n 162 the f i r s t two weeks o f i n c u b a t i o n when o r g a n i c m a t t e r breakdown was v e r y r a p i d , ( a s i n d i c a t e d by the l a r g e amounts o f N and S m i n e r a l i z e d ) some P was a l s o r e l e a s e d . I t a p p e a r s l i k e l y t h a t most o f t h i s p hosphate was s u b s e q u e n t l y r e - i n c o r p o r a t e d i n t o m i c r o b i a l t i s s u e . Phosphate m i n e r a l i z e d up t o s i x weeks o f i n c u b a t i o n , e x p r e s s e d as a p e r c e n t a g e o f the t o t a l o r g a n i c - P , i s shown i n T a b l e 23. With the e x c e p t i o n o f the 2-50um and l-2um f r a c t i o n s o f the D e l t a s o i l , v e r y T i t t l e o f the o r g a n i c - P i n the v a r i o u s f r a c t i o n s was m i n e r a l i z e d . T h i s i s perhaps not too s u r p r i s i n g as i t has been no t e d by s e v e r a l w o r k e r s , [Thompson e t a l _ . , 1954; W i l l i a m s , 1950; W i l l i a m s and L i p s e t t , 1960], t h a t t he r a t e o f m i n e r a l i z a t i o n o f P i s s l o w e r than t h a t o f e i t h e r C, N, o r S, and t h a t the c o n t r i b u t i o n o f o r g a n i c - P t o p l a n t growth i n temperate s o i l s i s perhaps m i n i m a l . 6.3.7 R e l a t i o n s h i p s Between Carbon N i t r o g e n , Phosphorus and S u l p h u r  i n S o i l s and P a r t i c l e - S i z e F r a c t i o n s The r e l a t i o n s h i p s between c a r b o n , n i t r o g e n , s u l p h u r , and phosphorus (both t o t a l and o r g a n i c ) i n the u n f r a c t i o n a t e d s o i l s and p a r t i c l e - s i z e s e p a r a t e s a r e shown i n T a b l e 25. In e v e r y i n s t a n c e the c o r r e l a t i o n c o e f f i c i e n t was h i g h l y s i g n i f i c a n t , and a f f i r m s the c l o s e r e l a t i o n s h i p between t h e s e elements i n s o i l o r g a n i c m a t t e r . M u l t i p l e c o r r e l a t i o n s were a l s o made between C, N, S, and P p l u s t h e r a t i o s o f t h e s e e l e m e n t s , and the amounts o f ammonium-N, n i t r a t e - N , s u l p h a t e and phosphate m i n e r a l i z e d a t two, f o u r , and s i x weeks. The c o r r e l -a t i o n c o e f f i c i e n t s a r e p r e s e n t e d i n T a b l e 26. 163 TABLE 25 C o r r e l a t i o n C o e f f i c i e n t s ( r * ) f o r R e l a t i o n s h i p s Between C, N, P and S V a r i a b l e % %c %N %S %P % P ( 0 ) C 1.0000 N 0.9012 1.0000 S 0.8362 0.8657 1.0000 P 0.6218 0.6915 0.7784 1.0000 p ( o ) 0.7775 0.8616 0.7663 0.5604 1.0000 % 0 1 = 0.372 TABLE 26 C o r r e l a t i o n C o e f f i c i e n t s ( r ) * f o r R e l a t i o n s h i p s Between C, N, P, and S and NH^-N, NO^-N, T o t a l - N , Su phate and P h o s p h a t e M i n e r a l i z e d a t 2, 4, and 6 Weeks V a r i a b l e s %C %N %S %? % p(o) C/N N/S N / P ( 0 ) C/S c / p ( o ) NH.-N ( 2 weeks) 0.5688 0.7405 0.7277 0.6349 0.7239 -0.5537 N.S. N.S. -0 4098 N.S. NO^-N 0.5770 0.7255 0.7597 0.7226 0.7652 -0.5849 N.S. N.S. -0 4771 -0.4169 T o t a l - N " 0.5824 0.7489 0.7530 0.6787 0.7526 -0.5755 N.S. N.S. -0 4422 N.S. S u l p h a t e 0.6481 0.7508 0.7327 0.6910 0.7752 -0.6128 N.S. N.S. -0.3731 -0.4269 Phosphate " NH.-N (4 weeks) NO^-N N.S. 0.5938 N.S. 0.7510 N.S. 0.7596 -0.5028 0.7469 N.S. 0.7067 N.S. -0.5790 N.S. N.S. N.S. N.S. -0 N.S. 4351 N.S. N.S. 0.6582 0.8143 0.8122 0.7919 0.7673 -0.6232 N.S. N.S. -0. 4399 N.S. T o t a l - N 0.6284 0.7855 0.7897 0.7748 0.7400 -0.6044 N.S. N.S. -0. 4407 N.S. S u l p h a t e 0.6615 0.7613 0.7464 0.7078 0.7724 -0.6282 N.S. N.S. N.S. -0.4201 Phosphate " N.S. N.S. N.S. -0.4217 -0.3958 N.S. N.S. N.S. N.S. 0.3827 NH.-N (6 weeks) 0.6326 0.7913 0.7727 0.7307 0.7665 -0.6082 N.S. N.S. -0. 4134 N.S. NCht-N 0.6374 0.7857 0.7781 0.7742 0.7519 -0.6134 N.S. N.S. -0. 4140 N.S. T o t a l - N 0.6374 0.7916 0.7784 0.7559 0.7619 -0.6132 N.S. N.S. -0. 4152 N.S. S u l p h a t e " 0.6701 0.7561 0.7418 0.6734 0.7746 -0.6261 N.S. N.S. N.S. -0.4239 Phosphate " N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. V o l . = ° - 3 7 2 -N.S. = Not S i g n i f i c a n t . 165 O r g a n i c C, t o t a l N and t o t a l S were p o s i t i v e l y and s i g n i f i -c a n t l y c o r r e l a t e d w i t h ammonium-N, n i t r a t e - N , t o t a l m i n e r a l N, and s u l p h a t e m i n e r a l i z e d a t two, f o u r and s i x weeks o f i n c u b a t i o n , but not w i t h p hosphate. S i m i l a r l y , t o t a l - P and o r g a n i c - P were p o s i t i v e l y and s i g n i f i c a n t l y r e l a t e d t o ammonium-N, n i t r a t e - N , t o t a l m i n e r a l - N , and s u l p h a t e ; and n e g a t i v e l y c o r r e l a t e d w i t h phosphate a t f o u r weeks. T o t a l - P was a l s o n e g a t i v e l y c o r r e l a t e d w i t h phosphate a t two weeks, but n o t s i g n i f i c a n t l y c o r r e l a t e d a t s i x weeks. The C/N r a t i o was n e g a t i v e l y c o r r e l a t e d w i t h n i t r o g e n and s u l p h u r m i n e r a l i z e d but not w i t h p h osphorus, a t two, f o u r , and s i x weeks, w h i l e N/S and N / P ^ r a t i o s were not c o r r e l a t e d w i t h e i t h e r N* S, o r P m i n e r a l i z e d . The C / P ^ r a t i o was p o s i t i v e l y and s i g n i f i c a n t l y c o r r e l a t e d w i t h phosphate m i n e r a l i z e d a t f o u r weeks. From t he f o r e g o i n g d i s c u s s i o n i t i s c l e a r t h a t t h e f o r m a t i o n o f o r g a n o - m i n e r a l complexes i n s o i l s c a nnot be d i r e c t l y r e s p o n s i b l e f o r the a p p a r e n t s t a b i l i t y o f o r g a n i c m a t t e r . In f a c t t h e da t a s u g g e s t q u i t e t h e o p p o s i t e , as h i g h e r r a t e s o f m i n e r a l i z a t i o n o f N, P, and S were o b s e r v e d i n the <0.2um f r a c t i o n . A more l o g i c a l e x p l a n a t i o n t h e r e f o r e , m i ght be Edwards' [1966] m i c r o a g g r e g a t e t h e o r y whereby c l a y m i n e r a l s and o r g a n i c c o l l o i d s a r e condensed t o form p a r t i c l e s <250um i n d i a m e t e r . Such m i c r o a g g r e g a t e s were found t o be q u i t e s t a b l e and thus c o u l d e f f e c t i v e l y i m m o b i l i z e and o c c l u d e c o n s i d e r a b l e amounts o f o r g a n i c m a t t e r . T h i s t h e o r y , however, w h i l e f e a s i b l e does not seem t o be a p p l i c a b l e f o r some s o i l s as m i n e r a l i z a t i o n r a t e s o f N and P i n u n f r a c t i o n a t e d s o i l s a p p roached t h o s e o f s u b - c l a y - s i z e f r a c t i o n s m e r e l y by a d d i n g i n o c u l u m t o the s o i l . 166 Consequently, there are several implications arising from the study regarding the fate of organic chemicals added to the soil environment. Structural consideration apart, i t appears that the rate of biological degradation of such substances will be influenced by: 1. The presence of N, S, or P containing functional groups which can bond directly or via polyvalent cations to clays or hydrous oxides or iron and aluminum, or to organic colloids. 2. The ratio of C:N, C:S and C:P in the substance. 3. The presence of microorganisms in the soil capable of metabolizing the original substance as well as the intermediate products. 6.4 Conclusion A study of the particle-size separates of five gleysolic soils revealed that there was a distinct distribution pattern for C, N , P, and S; as particle-size decreased the content of these elements increased. There was also a narrowing of the C/N, C/S, and C / P ( Q ) ratios with decreasing particle-size, and this was significantly correlated with N, S, and P mineralized from these fractions. The incubation technique devised from the preceding study proved effective. The percentage organic C, N, and S were positively and significantly correlated with NH -^N, NOg-N, total -N and sulphate mineralized during the incubation but not with phosphate. Total -P and organic -P were also positively correlated with N and S mineralized 167 but n e g a t i v e l y c o r r e l a t e d w i t h phosphate. The p e r c e n t a g e o f t o t a l N, S, and P m i n e r a l i z e d a f t e r s i x weeks i n c u b a t i o n ranged from 3.6-18.6 p e r c e n t f o r N, 8.3 - 21 p e r c e n t f o r S and 0.2 - 60.8 p e r c e n t f o r P; the <0..2ym f r a c t i o n s g e n e r a l l y h a v i n g t h e h i g h e s t p e r c e n t a g e m i n e r a l i z a t i o n , e x c e p t i n the case o f P. The r a t e o f m i n e r a l i z a t i o n o f N and S was a l s o h i g h e s t i n the <0.2um f r a c t i o n s . The r a t i o s o f N:S m i n e r a l i z e d a t s i x weeks was s i g n i f i c a n t l y l e s s than t h e N:S r a t i o s o f t h e o r i g i n a l p a r t i c l e - s i z e s e p a r a t e s e x c e p t f o r the <0.2um f r a c t i o n where t h e y were a p p r o x i m a t e l y the same. The aver a g e p e r c e n t a g e S m i n e r a l i z e d was 14.1 p e r c e n t , 16.6 p e r c e n t , 13.7 p e r c e n t , 14.7 percent... and 15 p e r c e n t f o r t he <2mm, 2-50um, 1 .-2pm,. 0.2-1 ym and <0.2ym f r a c t i o n s r e s p e c t i v e l y . On t h e b a s i s o f the r a t e s and amounts o f N, P, and S m i n e r a l -i z e d from t h e p a r t i c l e - s i z e s e p a r a t e s e s p e c i a l l y the <0.2ym f r a c t i o n , i t was c o n c l u d e d t h a t the bonding o f m i n e r a l and o r g a n i c c o l l o i d s i n s o i l s i s u n l i k e l y t o be d i r e c t l y r e s p o n s i b l e f o r the b i o l o g i c a l s t a b i l -i t y o f the o r g a n i c m o i e t y , but t h a t subsequent a g g r e g a t i o n o f such complexes c o u l d o c c l u d e l a r g e amounts o f o r g a n i c m a t t e r t h e r e b y r e n d e r i n g i t i n a c c e s s i b l e t o s o i l m i c r o b e s . 168 7. GENERAL SUMMARY AND CONCLUSIONS A comprehensive review of the literature concerning the dispersion of soils for the isolation of organo-mineral complexes and the relationships between carbon, nitrogen, phosphorus, and sulphur in soils, was presented in this thesis. Special emphasis was placed on the development of ultrasonic dispersion methods for the isolation of complexes, and the apparent resistance to microbial degradation of the organic portion of such complexes. A detailed study of the characteristics of the Biosonik BP-III ultrasonic vibrator with regards to the dispersion of soils and its effects on soil constituents was undertaken. On the basis of the results obtained i t was concluded that a soil suspension (1:10 or 1:5 soil/water-ratio) not exceeding a total volume of 125 ml could be effectively dispersed by vibration for 20 minutes at a probe setting of 80. It was found that the efficiency of soil dispersion was reduced by about 30-40 percent unless there was a rest period of approximately 25-30 minutes between consecutive dispersions. The condition of the probe tip was found to exert a major influence on the effectiveness of dispersion, and the polishing of the probe tip with fine (400-grit) emery paper after each 20 minutes of operation retarded the onset of pitting. After about every 30 hours of operation it is recommended that the probe tip be machined on a lathe to restore its surface to a smooth condition. 169 The h e a t i n g e f f e c t o f u l t r a s o u n d t r e a t m e n t was found t o be a good i n d e x o f t h e o u t p u t o f u l t r a s o n i c energy from t he probe f o r a p e r i o d o f up t o 25-30 m i n u t e s . When water o r a s o i l s u s p e n s i o n was v i b r a t e d beyond 30 minutes w i t h o u t i n s u l a t i o n , t h e s u s p e n s i o n t e m p e r a t u r e l e v e l l e d o f f a t about 75-80°C i n d i c a t i n g a h i g h r a t e o f he a t l o s s t o the s u r r o u n d i n g atmosphere. S o i l s u s p e n s i o n s s h o u l d be c o o l e d d u r i n g d i s p e r s i o n t o e l i m i n a t e p o s s i b l e thermo-chemical r e a c t i o n s . P a r t i c l e - s i z e a n a l y s i s o f v i b r a t e d s o i l s u s p e n s i o n s , con-f i r m e d the f i n d i n g s o f many o t h e r workers t h a t u l t r a s o n i c v i b r a t i o n , w i t h o u t the a i d o f c h e m i c a l r e a g e n t s c o u l d e f f e c t i v e l y d i s p e r s e s o i l s . A m ajor f i n d i n g which emerged from t h i s s t u d y , however, was t h a t l a r g e amounts o f Fe, A l , S i , and C were s o l u b i l i z e d d u r i n g t h e . v i b r a t i o n o f s o i l s i n water and t h a t t h i s e f f e c t c o u l d be s u p p r e s s e d by d i s p e r s i n g s o i l s i n a d i l u t e e l e c t r o l y t e s o l u t i o n , 0.01 M C a C ^ b e i n g v e r y e f f e c t i v e . I t was noted t h a t t h e r e was a h i g h l y s i g n i f i c a n t i n v e r s e r e l a t i o n s h i p between Fe and o r g a n i c - C i n s o l u t i o n , f o l l o w i n g u l t r a s o n i c v i b r a t i o n . T h i s was unexpected and s u g g e s t s t h a t d i s s o l v e d Fe may be p r e c i p i t a t e d as an iron-humate complex. A f r a c t i o n a t i o n scheme f o r t h e b u l k i s o l a t i o n o f s o i l p a r t i c l e - s i z e f r a c t i o n s was d e v e l o p e d based on u l t r a s o n i c d i s p e r s i o n o f t h e s o i l and a system o f c o n t i n u o u s f l o w c e n t r i f u g a t i o n . I t was fo u n d t h a t c o n t i n u o u s f l o w c e n t r i f u g a t i o n r e d u c e d a l m o s t by one-h a l f t h e time i n v o l v e d i n o b t a i n i n g p a r t i c l e - s i z e s e p a r a t e s . The t e c h n i q u e gave e f f e c t i v e and q u a n t i t a t i v e s e p a r a t i o n o f the v a r i o u s s o i l s i z e f r a c t i o n s , w i t h an ave r a g e o f 98 p e r c e n t o f the s o i l m a t e r i a l r e c o v e r e d . 170 The d i s t r i b u t i o n and amounts o f C, N, P, and S i n f i v e G l e y s o l i c s o i l s and t h e i r p a r t i c l e - s i z e f r a c t i o n s (2-50um, l-2ym, 0.2-lum, and <0.2um) were d e t e r m i n e d . , I t was found t h a t as p a r t i c l e - s i z e d e c r e a s e d the c o n t e n t o f t h e s e elements i n c r e a s e d and a na r r o w i n g o f the C/N, C/S, and C / P ^ r a t i o s w i t h d e c r e a s i n g p a r t i c l e - s i z e was o b s e r v e d . An i n c u b a t i o n s t u d y was c o n d u c t e d t o d e t e r m i n e t he optimum c o m b i n a t i o n o f s e v e r a l f a c t o r s ( m o i s t u r e , sand, i n o c u l u m and n u t r i e n t s ) f o r the m i n e r a l i z a t i o n o f N, P, and S i n complexes. A e r o b i c c o n d i t i o n s were m a i n t a i n e d i n the i n c u b a t i o n v e s s e l s . by the use o f 1.5 m i l p o l y e t h y l e n e f i l m , (which a l l o w d i f f u s i o n o f oxygen and C 0 2 but r e t a r d s t h a t o f water v a p o r ) and by v e n t i n g the j a r s a t weekly i n t e r v a l s . A l s o , t h r e e d i f f e r e n t e x t r a c t a n t s were compared f o r the e x t r a c t i o n o f m i n e r a l forms o f t h e s e e l e m e n t s , v i z . 0.01 N C a C l 2 , 0.5 N NaHC0 3 pH 8.5, and 1 N KC1 pH 2.0. R e a d i l y measurable amounts o f . m i n e r a l N, P, and S were d e t e c t e d f o l l o w i n g the a e r o b i c i n c u b a t i o n o f a s o i l p a r t i c l e - s i z e s e p a r a t e . A e r o b i c c o n d i t i o n s were a p p a r e n t l y m a i n t a i n e d t h r o u g h o u t the e x p e r i m e n t a t the lower m o i s t u r e l e v e l (20 p e r c e n t by w e i g h t ) and were enhanced by m i x i n g the s o i l f r a c t i o n w i t h t h r e e times i t s we i g h t o f q u a r t z sand. The a d d i t i o n o f an i n o c u l u m appeared t o be o f c r i t i c a l i m p o r tance t o the p r o d u c t i o n o f n i t r a t e and s u l p h a t e , w h i l e the i n c l u s i o n o f s u p p l e m e n t a r y n u t r i e n t s a l s o proved t o be e f f i c a c i o u s f o r n i t r a t e and s u l p h a t e p r o d u c t i o n . The a c c u m u l a t i o n o f m i n e r a l phosphorus was f a v o u r e d by t h e h i g h e r m o i s t u r e l e v e l , (40 p e r c e n t by w e i g h t ) . On the b a s i s o f t h e s e r e s u l t s an a e r o b i c i n c u b a t i o n 171 t e c h n i q u e was d e v i s e d f o r f u r t h e r i n v e s t i g a t i o n o f the m i n e r a l i z -a t i o n o f N, P, and S from o r g a n o - m i n e r a l complexes and s o i l p a r t i c l e -s i z e s e p a r a t e s i n a l l f i v e s o i l s i n c l u d e d i n t h i s s t u d y . The i n c u b a t i o n t e c h n i q u e d e v i s e d from the p r e c e d i n g s t u d y proved v e r y e f f e c t i v e . The p e r c e n t o r g a n i c C, N, and S i n the f r a c t i o n s were p o s i t i v e l y and s i g n i f i c a n t l y c o r r e l a t e d w i t h NH^-N, NO^-N, t o t a l - N , and s u l p h a t e m i n e r a l i z e d d u r i n g t h e i n c u b a t i o n but not w i t h phosphate. T o t a l - P and o r g a n i c - P were a l s o p o s i t i v e l y c o r r e l a t e d w i t h N and S m i n e r a l i z e d but n e g a t i v e l y c o r r e l a t e d w i t h phosphate. The p e r c e n t a g e o f t o t a l N, S, and P m i n e r a l i z e d a f t e r s i x weeks i n c u b a t i o n ranged from 3.6 - 18.6 p e r c e n t f o r N, 8.3 - 21 p e r c e n t f o r S and 0.2 - 60.8 p e r c e n t f o r P; the <0.2um f r a c t i o n s g e n e r a l l y h a v i n g the h i g h e s t p e r c e n t a g e m i n e r a l i z a t i o n , e x c e p t i n the c a s e o f P. The r a t e o f m i n e r a l i z a t i o n o f N and S was a l s o h i g h e s t i n the <0.2um f r a c t i o n s . The r a t i o s o f N:S m i n e r a l i z e d a t s i x weeks were s i g n i f i c a n t l y l e s s than t h e N:S r a t i o s o f the o r i g i n a l p a r t i c l e - s i z e s e p a r a t e s e x c e p t f o r t he <0.2um f r a c t i o n where they were a p p r o x i m a t e l y the same. 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N i t r o g e n , s u l p h u r and p h o s p h o r u s , t h e i r i n t e r a c t i o n s and a v a i l a b i l i t y . I n t . Soc. S o i l S c i . T r a n s . Comm. II & IV: 93-111. S o i l C h e m i s t r y and F e r t i l i t y . A b e r d e e n , 1966, Ed. G.V. J a c k s . . 1967. Some f a c t o r s a f f e c t i n g t h e m i n e r a l i z a t i o n o f o r g a n i c s u l p h u r i n s o i l s . P l a n t & S o i l . 26: 205-221. WILLIAMS, J.D.H., J.K. SYERS, T.W. WALKER and R.W. REX. 1970. A c o m p a r i s o n o f methods f o r the d e t e r m i n a t i o n o f s o i l o r g a n i c p h o s p h o r u s . S o i l S c i . 110: 13-18. YARIV, S., J.D. RUSSEL and V.C. FARMER. 1966. I n f r a r e d s t u d y o f t h e a d s o r p t i o n o f b e n z o i c a c i d and n i t r o b e n z e n e i n m o n t m o r i l l o n i t e . I s r a e l J . Chem. 4: 201. 195 APPENDIX I A COLORIMERIC METHOD FOR THE DETERMINATION OF AMMONIUM-N IN SOIL EXTRACTS 1.1 Introduction The kjeldahl method has been widely used for the determin-ation of total and ammonium-nitrogen in soils and water samples. Other methods such as microdiffusion and nesslerization, while highly sensitive have not been as successfully applied; the former because it is less adaptable and the latter because i t is unreliable. Recently interest has been revived in the Berthelot reaction of ammonia with phenol and hypochlorite for the colorimetric determin-ation of ammonia. Van Slyke and Hiller [1933] observed that in alkaline solution, ammonia forms an intense blue dye with a phenol-sodium hypochlorite reagent, in amounts proportional to the amount of ammonia present. The minimum transmittance (maximum absorbance) was found to occur at 625 nm, [Snell and Snell, 1949]. This method has not hitherto been applied to the determination of total or ammonium-N in soils and soil extracts, however, i t appears to have several advantages over the methods currently used. Noble [1955] stated that the phenol-hypochlorite method could be recommended for its high sensitivity but that warnings usually appear concerning poor reproducibility and color instability. He (Noble) made a systematic study of the phenol-hypochlorite method 196 i n o r d e r t o d e v e l o p a p r o c e d u r e which c o u l d be used r o u t i n e l y f o r the d e t e r m i n a t i o n o f t r a c e amounts o f N i n p e t r o l e u m s t o c k s , and found t h a t t h e most c o n s i s t e n t r e s u l t s were o b t a i n e d when the phenol r e a g e n t was added f i r s t , f o l l o w e d a l m o s t i m m e d i a t e l y by the h y p o c h l o r -i t e s o l u t i o n . A f t e r t he mixing o f t h e r e a g e n t s , c o l o r was d e v e l o p e d by p l a c i n g t he r e a c t i o n tube i n a b o i l i n g w a t e r - b a t h f o r 6-8 m i n u t e s . Noble a l s o found t h a t t he s m a l l e r t he volume o f s o l u t i o n i n which c o l o r development o c c u r r e d t he g r e a t e r was the c o l o r i n t e n s i t y , thus he argued t h a t t he s e n s i t i v i t y o f the method c o u l d be i n c r e a s e d by ke e p i n g t o a minimum, t h e f i n a l volume o f t h e s o l u t i o n . B o l l e t e r , Bushman and T i d w e l l [ 1961] were among the f i r s t w o r k e r s . t o a t t e m p t t o e x p l a i n t h e r e a c t i o n mechanism, whereby the b l u e c o l o u r o f the r e a c t i o n between ammonium i o n s , phenol and hypo-c h l o r i t e i o n s was d e v e l o p e d . They proposed t h a t t he b l u e c o l o u r o f t h e s o l u t i o n was due t o t h e d i s s o c i a t i o n o f i n d o p h e n o l , i n a l k a l i n e s o l u t i o n , formed by a complex r e a c t i o n between ammonia and the r e a g e n t s . The f i r s t s t e p o f the s e r i e s o f r e a c t i o n s s u g g e s t e d by B o l l e t e r et_ al_. [ 1961] was the f o r m a t i o n o f c h l o r o a m i n e ; NHt + " O C T -> NH 0C1 + H o 0 S e c o n d l y c h l o r o a m i n e r e a c t s w i t h phenol t o form q u i n o n e c h l o r a m i n e ; 197 NhLCl + x ,;—O H -y 0 — N - CI which c o u p l e s w i t h a n o t h e r mole o f phenol t o form t h e y e l l o w a s s o c i a t e d i n d o p h e n o l ; 0 =( — N - CI + <\ V-OH •*• 0 =( )= N -A >-0H ( I n d o p h e n o l ) T h i s compound d i s s o c i a t e s i n a l k a l i n e s o l u t i o n t o g i v e a b l u e c o l o u r ; Indophenol —^ ( fW \= \\ ( B l u e complex) B o l l e t e r ejt al_. [1961] s t a t e d t h a t t h e h y p o c h l o r i t e r e a g e n t must be added t o t h e ammonia s o l u t i o n b e f o r e t h e p h e n o l , i n o r d e r t o g e t c o l o u r d e velopment, and f u r t h e r i n t i m a t e d t h a t i f phenol was added f i r s t , t h e c h l o r i n e atom becomes a t t a c h e d t o p h e n o l , t h e r e b y r e d u c i n g t h e h y p o c h l o r i t e c o n c e n t r a t i o n and b l o c k i n g t h e r e a c t i o n o f c h l o r o -amine w i t h t h e p h e n o l . These a s s e r t i o n s , however, have n o t been s u b s t a n t i a t e d by s u b s e q u e n t r e s e a r c h as Weatherburn [1967] has d e m o n s t r a t e d t h a t the b l u e c o l o r d e v e l o p s r e g a r d l e s s o f t h e sequence o f a d d i t i o n o f the r e a g e n t s . T h i s b r i n g s i n t o q u e s t i o n t h e r e a c t i o n scheme pr o p o s e d by B o l l e t e r e t a l . [ 1 9 6 1 ] , and t o d a t e t h e 198 n a t u r e o f the complex formed by t h i s r e a c t i o n i s s t i l l u n d e t e r m i n e d . Chaney and Marbach [1962] c o n f i r m e d t h a t sodium n i t r o -p r u s s i d e ( d i s o d i u m p e n t a c y a n o - n i t r o s y l o f e r r a t e I I I ) i n c r e a s e d s e v e r a l -f o l d t h e r a t e and i n t e n s i t y o f c o l o r development o f t h e ammonia-p h e n o l - h y p o c h l o r i t e complex. They p r o p o s e d t h a t t h e v a r i o u s r e a g e n t s r e q u i r e d f o r t h e development o f t h e b l u e c o l o r , be combined i n t o two r e a g e n t s , (a) phenol + n i t r o p r u s s i d e (b) NaOH + h y p o c h l o r i t e , t o s i m p l i f y t h e p r o c e d u r e . Chaney and Marbach [1962] a l s o n o t e d t h a t t h e s e r e a g e n t s were s t a b l e f o r 60 days o r more, i f k e p t i n amber b o t t l e s and r e f r i g e r a t e d . Weatherburn [1967] remarked t h a t t h e p h e n o l - h y p o c h l o r i t e method f o r t h e e s t i m a t i o n o f ammonia, "has made d e t e r m i n a t i o n s on u l t r a - m i c r o q u a n t i t i e s o f sample p o s s i b l e , " and f u r t h e r i n v e s t i g a t e d t h e method, " w i t h t h e aim o f recommending a r e l i a b l e y e t s i m p l e t e c h n i q u e f o r a busy l a b o r a t o r y . " W e atherburn, u s i n g t h e method d e s c r i b e d by Chaney and Marbach [1962] examined t h e e f f e c t s o f v a r i o u s f a c t o r s on c o l o u r development; (a) t h e sequence o f a d d i t i o n o f r e a g e n t s . (b) t h e t i m i n g o f r e a g e n t a d d i t i o n s . ( c ) v a r i a t i o n s i n c o n d i t i o n s o f t i m e and t e m p e r a t u r e . He found t h a t B e e r ' s Law was f o l l o w e d i n a c o n c e n t r a t i o n r a n g e o f 0.5 - 6 ug NH^ - N, and t h a t n i t r o p r u s s i d e i n c r e a s e d s e n s i t i v i t y more than t e n f o l d . S a t i s f a c t o r y c o l o u r development w i t h m i n i m al p r e c a u t i o n s was g i v e n by a v a r i e t y o f r e a g e n t c o n c e n t r a t i o n s and by r e a c t i o n t e m p e r a t u r e s o f 20, 25, 37, and 75°C, w i t h i n t h e pH rang e 9.9 - 12.1. 199 B eecher and W h i t t e n [1970] f o l l o w i n g c l o s e l y the p r o c e d u r e o f Weatherburn [1967] and Chaney and Marbach [1962], r e p o r t e d t h a t the p h e n o l - h y p o c h l o r i t e method was most s e n s i t i v e between the narrow pH range o f 11.7 t o 11.9 and p r o p o s e d t h e use o f a phosphate b u f f e r t o m a i n t a i n t h e r e a c t i o n pH w i t h i n t h i s narrow r a n g e . The p r o c e d u r e f o r t o t a l and NH 4 - N i n s o i l s o u t l i n e d i n t h e s u c c e e d i n g pages was based on t h e work o f t h e a u t h o r s c i t e d p r e v i o u s l y , however, a t t e n t i o n s h o u l d a l s o be drawn t o t h e work o f M i t c h e l l [1972] which a p p e a r e d i n t h e l i t e r a t u r e a f t e r t h i s work was i n i t i a t e d . M i t c h e l l [1972] d e s c r i b e d a method f o r t h e m i c r o d e t e r m i n a -t i o n o f n i t r o g e n i n p l a n t t i s s u e s based c l o s e l y on t h e method o f Beecher and W h i t t e n [ 1 9 7 0 ] , The method was t e s t e d by t h e a u t h o r and found t o be e f f e c t i v e f o r p l a n t samples and a v a r i e t y o f s o i l s , 2+ 3+ e x c e p t t h o s e r i c h i n c e r t a i n c a t i o n s , n o t a b l y Ca and Fe . 1.2 Methods and M a t e r i a l s 1.2.1 Reagents 1. Phenol S o l u t i o n -- D i s s o l v e 10 g phenol and 50 mg sodium n i t r o -p r u s s i d e i n w a ter and d i l u t e t o one l i t r e . 2. H y p o c h l o r i t e ( B u f f e r ) S o l u t i o n - - D i s s o l v e 5 g NaOH, 3.75 g * a nhydrous N a 2 H P 0 4 , 32 g N a 3 P 0 4 « 12H 20 and 10 ml 5% sodium h y p o c h l o r i t e s o l u t i o n {6% a v a i l a b l e c h l o r i n e ) i n w a t e r and d i l u t e t o one l i t r e . 3. E t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d (EDTA) — ( i ) D i s s o l v e 1 g EDTA i n 100 ml w a ter by a d d i n g 10 N. NaOH d r o p w i s e , and s t i r r i n g m a g n e t i c a l l y -- a d j u s t f i n a l pH 200 o f t h e s o l u t i o n t o pH 10, o r ( i i ) D i s s o l v e 5 g EDTA i n w a t e r as d e s c r i b e d above. 4. S t a n d a r d NH^-N S o l u t i o n -- D i s s o l v e 0.472 g ( N H 4 ) 2 S 0 4 i n w a t e r and d i l u t e t o one l i t r e . . T h i s s o l u t i o n c o n t a i n s 100 ug NH 4-N/ml. 5. NaOH -- D i s s o l v e 4.8 g NaOH i n w a t e r and d i l u t e t o one l i t r e . 1.2.2 NH 4-N i n S o i l E x t r a c t s 1.2.2.1 P r o c e d u r e P i p e t t e a s u i t a b l e sample o f t h e e x t r a c t (5 - 10 ml) o r s t a n d a r d s o l u t i o n i n t o a 50 ml v o l u m e t r i c f l a s k . Add 1 ml o f t h e one p e r c e n t EDTA s o l u t i o n w i t h a r a p i d d e l i v e r y p i p e t t e and o n e - h a l f ml o f t h e NaOH s o l u t i o n . Add 5 ml o f t h e phenol s o l u t i o n and d i l u t e t h e s o l u t i o n to a b o u t 40 ml, th e n add 5 ml o f t h e h y p o c h l o r i t e s o l u t i o n , d i l u t e t o mark and r e a d a b s o r b e n c e a t 625 nm a f t e r one hour. The r e a g e n t s c a n be c o n v e n i e n t l y s t o r e d and e a s i l y d i s p e n s e d by u s i n g O x f o r d p i p e t t o r s , m a n u f a c t u r e d by O x f o r d L a b o r a t o r i e s . A s o r b a n c e measurements f o r both s t a n d a r d s and s o i l e x t r a c t s s h o u l d be c o r r e c t e d by s u b t r a c t i n g t h e r e a d i n g s o b t a i n e d from r e a g e n t b l a n k s . B l a n k s may v a r y i n a b s o r b a n c e from 0 - 0.012 a b s o r b a n c e u n i t . I t i s recommended t h a t d e i o n i z e d w a t e r be used t h r o u g h o u t t h e p r o c e d u r e a l t h o u g h i n p r a c t i c e v e r y low b l a n k s were o b t a i n e d by u s i n g d i s t i l l e d w a t e r . The s t a n d a r d c u r v e i s l i n e a r o v e r t h e c o n c e n t r a t i o n range o f 1 - 30 ugN. 2+ C a C ^ e x t r a c t s , o r s o i l e x t r a c t s h i g h i n Ca i o n s w i l l c a u s e p r e c i p i t a t i o n o f Ca-phosphate upon t h e a d d i t i o n o f t h e 201 2+ hypochlorite-buffer solution. If the concentration of Ca is less than N/10 then 3 ml of the 5 percent EDTA solution will prevent 2+ precipitation. If, however, the Ca concentration exceeds 1/10 normal, the hypochlorite reagent should be prepared without the phos-phates and additional NaOH solution added to ensure that the pH of the final solution is about pH 11.7. There was no interference from Fe, Al , Mn, Cu, or Zn when the recommended amount of EDTA solution was used. 1.2.3 Total N 1. Catalyst 0.6 g HgO and 20 g K2S04 were ground and thoroughly mixed in a mortar; 2 g of this mixture per sample was used as the digestion catalyst. 2. Procedure Weigh 20 - 100 mg of air dried 60 mesh soil (depending on N content), then transfer the sample to a 30 ml Kjeldahl flask and add 2 g catalyst followed by 2 ml concentrated H2S04. Digest the sample for 2 hours, then transfer quantitatively to a 200 ml volumetric flask and dilute to mark. A blank should also be run concurrently with the digested samples. A 2 ml sample of the digest is pipetted into a 50 ml volumetric flask, 2 ml of the EDTA solution added followed by 4 ml of the NaOH solution. Next, add 5 ml phenol solution, dilute to about 40 ml and add 5 ml hypochlorite-buffer.solution, then dilute to mark. Read absorbance after one hour at 625 nm. 202 The proposed phenol-hypochlorite method (outlined above) for the determination of total and NH^-N in soils was compared with the standard micro-Kjeldahl method, as described in the Official methods of the Association of Official Agricultural Chemists. The method was also compared with the semi-micro Kjeldahl procedure for total N in soils described by Bremner [1965]. 1.3 Results and Discussion Tables 1 and 2 show comparisons between the proposed method and the standard micro- and semi-micro Kjeldahl methods for total -N determination. The correlation coefficient for the data in Table 1 was r = 0.97 and for Table 2 r = 0.997. It is evident from the magnitude of the correlation coefficients that the proposed method gave a good estimate of the total -N in a variety of soil materials, embracing organic layers (L, F and H), mineral so i l , and soil particle-size separates. The proposed method has the advantage over the standard methods of being quite simple and better suited to routine laboratory analysis. The phenol-hypochlorite method is also relatively free from interferences, is characterized by a high degree of precision and has considerable f lexibi l i ty for accommodating samples with widely differing nitrogen contents. Mitchell [1972] suggested that with high nitrogen substances the final reaction mixtures can be diluted at the time of reading to ensure that "absorbances are in the range where instrument settings can be read more accurately." 203 TABLE 1 Comparison o f t h e P h e n o l - H y p o c h l o r i t e Method w i t h t he M i c r o - K j e l d a h l Method f o r T o t a l - N i n S o i l s S o i l P h e n o l - H y p o c h l o r i t e Method 0/ M M i c r o - K j e l d a h l Method M 11 LANGLEY Ah 2-•50y 0. 43 0. 45 0. 46 0. 46 1-•2 y 0. 60 0. 54 0. 62 0. 50 HAZELWOOD Ah 1 2--50y 0. 23 0. 26 0. 25 0. 25 1--2 y 0. 58 0. 54 0. 62 0. 60 HATZIC 2--50y 0. 22 0. 20 0. 23 0. 18 1--2 y 0. 29 0. 27 0. 30 0. 28 C o r r e l a t i o n c o e f f i c i e n t ( r ) = 0.97 (P < 0.01). 204 TABLE 2 Comparison o f the P h e n o l - H y p o e h l o r i t e Method w i t h t he Semi-Micro K j e l d a h l Method f o r T o t a l - M i n S o i l s S o i l H o r i z o n P h e n o l - H y p o c h l o r i t e Method rt/ HI Semi-Micro K j e l d a h l Method lo N L(F)H 1. 57 •1. 61 Ae 0 20 0 22 Bfh 6J1 • 0 25 0 18 Bfh 6J2 0 29 0 24 Bfh C 0 21 . 0 16 (LF)H 1 40 1 40 Ah 0 80 0 77 AB 0 37 0 31 Bm 0 .27 0 • 25 (LF.)H 1 .58 1 .77 Average 0 694 0 691 C o r r e l a t i o n c o e f f i c i e n t ( r ) = 0.997 (P < 0.01) 205 APPENDIX 2 MANGANESE, COPPER AND ZINC EXTRACTED FROM SOIL PARTICLE SIZE SEPARATES BY 0.1 N HC1 BEFORE AND AFTER ASHING AT 550°C 2.1 I n t r o d u c t i o n T h e r e i s v e r y l i t t l e i n f o r m a t i o n c u r r e n t l y a v a i l a b l e i n t h e l i t e r a t u r e c o n c e r n i n g t h e amounts and a v a i l a b i l i t y o f m i c r o n u t r i e n t e l e m e n t s i n o r g a n o - m i n e r a l complexes and s o i l p a r t i c l e - s i z e s e p a r a t e s . Much i n f o r m a t i o n , however, has been p u b l i s h e d on t h e t o t a l amounts o f t r a c e e l e m e n t s i n s o i l s , as w e l l as, on t h e a v a i l a b l e f r a c t i o n s [Swaine, 1955]. In b o t h n u t r i t i o n a l and p e d o l o g i c a l s t u d i e s i t would be h e l p f u l t o know t h e forms i n which t r a c e e l e m e n t s o c c u r and a l s o t h e p r o p o r t i o n s and p a r t i c l e - s i z e d i s t r i b u t i o n o f t h e s e forms i n t h e s o i l . Walsh, P i e r c e and F l e m i n g [1956] and Connor, Shimp and Tedrow [1957] r e p o r t e d c o n c e n t r a t i o n s o f t r a c e e l e m e n t s i n whole s o i l s and c l a y f r a c t i o n s , and Berrow [1958] g i v e s v a l u e s f o r s o i l s and sand, s i l t and c l a y f r a c t i o n s . Le R i c h e and Weir [1973] have r e c e n t l y r e p o r t e d t h a t t h e r e was an e n r i c h m e n t o f m i c r o n u t r i e n t e l e m e n t s i n t h e c l a y f r a c t i o n s o f two b u r i e d s o i l s . M i c r o n u t r i e n t s may be bound i n t h e s o i l : (a) i n w a t e r - s o l u b l e form. (b) as e x c h a n g e a b l e c a t i o n s . ( c ) as n o n - e x c h a n g e a b l e e l e m e n t s i n an o r g a n i c complex. (d) i n a s s o c i a t i o n w i t h , Fe, A l , and sometimes manganese o x i d e s . 206 (e) as c o n s t i t u e n t s o f t h e c r y s t a l l a t t i c e s o f o t h e r s a n d - , s i l t - , and c l a y - s i z e d m i n e r a l s , [Le R i c h e and W e i r , 1963], M i c r o n u t r i e n t c a t i o n s such as Zn, Cu and Fe a r e known t o e n t e r i n t o t h e c r y s t a l l a t t i c e o f l a y e r s i l i c a t e s t h r o u g h isomorphous s u b s t i t u t i o n and may a l s o be h e l d a t t h e s i l i c a t e s u r f a c e by c a t i o n exchange f o r c e s . S o i l o r g a n i c m a t t e r forms v e r y s t a b l e complexes w i t h most o f t h e s e e l e m e n t s and M o r t e n s e n [ 1 9 6 3 ] , Hodgson [ 1 9 6 3 ] , S c h n i t z e r [1969] and S t e v e n s o n and A r d a k a n i [1972] have r e v i e w e d t h e l i t e r a t u r e p e r t a i n i n g t o t h e r e l a t i o n s h i p s between v a r i o u s m e t a l s and s o i l o r g a n i c m a t t e r as w e l l as t h e known and p r o p o s e d mechanisms by which m e t a l s a r e a b s o r b e d by o r g a n i c m a t t e r . M i c r o n u t r i e n t s may a l s o be a d s o r b e d by t h e s e s q u i o x i d e s and amorphous c o n s t i t u e n t s i n s o i l s . I t seems r e a s o n a b l e t h e r e f o r e t o s u g g e s t t h a t most o f t h e m i c r o n u t r i e n t e l e m e n t s w i l l be i n t i m a t e l y a s s o c i a t e d w i t h t h e o r g a n o -m i n e r a l complex, e i t h e r as c a t i o n b r i d g e s o r bonded t o t h e m i n e r a l o r o r g a n i c f r a c t i o n . To t e s t t h i s h y p o t h e s i s t h e amount o f Mn, Cu and Zn e x t r a c t e d by 0.1 N HC1 from a r a n g e o f p a r t i c l e - s i z e s e p a r a t e s was compared. The amounts o f t h e s e e l e m e n t s e x t r a c t e d a f t e r i g n i t i o n o f t h e s e p a r a t e s a t 550°C f o r one hour t o d e s t r o y o r g a n i c m a t t e r was a l s o d e t e r m i n e d . 207 2.2 M a t e r i a l s and Methods D u p l i c a t e 100 mg samples o f each s e p a r a t e were p l a c e d i n 50 ml c e n t r i f u g e t u b e s which had been p r e v i o u s l y washed w i t h 6N HC1. 25 ml o f 0.1 N HC1 was added t o each t u b e , which was then f i t t e d w i t h a p l a s t i c s t o p p e r and shaken f o r one hour on a r e c i p r o c a t i n g s h a k e r , a t 120 s t r o k e s / m i n u t e . The s u s p e n s i o n s were c e n t r i f u g e d a t 12,000 xg f o r 30 m i n u t e s and t h e s u p e r n a t a n t s o l u t i o n t r a n s f e r r e d t o 50 ml v o l u m e t r i c f l a s k s and d i l u t e d t o mark. Manganese, c o p p e r and z i n c i n t h e s o l u t i o n s were d e t e r m i n e d by Atomic A b s o r p t i o n S p e c t r o p h o t o m e t r y u s i n g t h e P e r k i n - E l m e r Model 306 u n i t . 2.3 R e s u l t s and D i s c u s s i o n Data a r e p r e s e n t e d i n T a b l e 1 f o r manganese, c o p p e r and z i n c e x t r a c t e d from t h e 2-50 , 1-2 , 0.2-1 and <0.2um f r a c t i o n s o f f i v e G l e y s o l i c s o i l s . I t a p p e a r s t h a t as p a r t i c l e - s i z e d e c r e a s e t h e c o n t e n t o f t h e s e e l e m e n t s i n c r e a s e , which i s n o t s u r p r i s i n g because as p r e v i o u s l y shown, t h e o r g a n i c m a t t e r c o n t e n t o f t h e s e p a r a t e s a l s o i n c r e a s e w i t h a d e c r e a s e i n p a r t i c l e s i z e , ( T a b l e 21 i n t e x t ) . 2.3.1 Manganese R e l a t i v e l y l a r g e amounts o f Mn were e x t r a c t e d f r o m t h e v a r i o u s f r a c t i o n s and f o r t h r e e o u t o f f i v e s o i l s . The maximum amount o f Mn was e x t r a c t e d from t h e 0 . 2 - l u f r a c t i o n . Manganese i s generally abundant in soils, although the concentration of Mn ions in the soil solution or occupying exchange sites on clays is usually quite small. Characteristically, most Mn occurs in soils as very insoluble oxides of tetravalent of trivalent Mn, however, Heintze [1957] suggested that the amount present in organo-metallic complexes may be considerable in soils high in organic matter. 2+ 4+ The compounds of Mn and Mn are soluble in HC1, as are some of the Mn-organic matter complexes, hence i t is not possible to pinpoint the source of the Mn found in solution. A comparison of the means for Mn extracted before and after ignition of the samples suggests that there was no significant change in extractable Mn. However, in some instances there were marked changes. For example, in the Hazelwood so i l , i t was found that after ignition of the 0.2-lu fraction, extractable Mn increased from 18.5 ppm to 274 ppm; whereas for the <0.2p fraction there was a decrease from 792 ppm to about 190 ppm. Such dramatic changes in solubility must be due to at least two separate mechanisms. Where an increase in extractable Mn was observed i t may be argued that the destruction of organic matter upon ignition of the sample resulted in the liberation of complexed Mn. Conversely, i t is possible that the oxidation of 2+ 4+ Mn and Mn to higher valence states resulted in reduced solubility of Mn in dilute HC1. 209 TABLE 1 Mn, Cu, and Zn E x t r a c t e d from S o i l P a r t i c l e - S i z e S e p a r a t e s By 0.1N HC1 B e f o r e and A f t e r A s h i n g B e f o r e A f t e r F r a c t i o n (u m) Mn Cu Zn Mn Cu Zn LANGLEY Ah 2-50 501.3 38.3 58.8 475.5 54.3 165 1-2 591.3 58.5 112.5 487 70 205 0.2-1 499 38 82 554 61 230 <0.2 620 150 425 695 310 2900 HAZELWOOD Ah! 2-50 56.5 31.5 55.5 141 38 137.5 1-2 96.5 35.5 115.3 209 37 225 0.2-1 18.5 44 66.5 274.5 68 355 <0.2 792.5 380 555 192.5 342.5 975 HATZIC Ap 2-50 121 29.5 105 210 27.3 260 1-2 152.5 33.3 72.3 276.5 37.5 307.5 0.2-1 285.5 56 90.5 447 59.5 305 <0.2 155 152.5 402.5 292.5 287.5 1350 CLOVERDALE Ap 2-50 254 21 50.5 275.5 49.5 182.5 1-2 502.5 19 50.8 595 38 275 0.2-1 747.5 43.5 94 510 60 330 <0.2 210 130 602.5 247.5 322.5 1700 DELTA Ap 2-50 42.3 21.5 35 77 25.5 235 1-2 122.5 49.8 77.5 214 38.5 220 0.2-1 326.5 61.5 174.5 247 57 410 <0.2 170 142.5 275 210 250 1300 AVERAGE 313.2 76.8 175 331.5 111.7 603.4 210 2.3.2 Copper and Z i n c Hodgson, L i n d s a y and T r i e r w e i l e r [1966] showed t h a t more th a n 98 p e r c e n t o f the Cu i n d i s p l a c e d s o i l s o l u t i o n s was i n an o r g a n i c complexed form and i t i s b e l i e v e d t h a t t o a l e s s e r e x t e n t t h e same i s t r u e f o r Zn. The d a t a i n T a b l e 1 a l s o t e n d s t o s u p p o r t t h i s c o n c l u s i o n , as t h e r e i s a marked i n c r e a s e i n e x t r a c t a b l e Cu and Zn as p a r t i c l e - s i z e d e c r e a s e d , which i s c o i n c i d e n t w i t h an i n c r e a s e i n o r g a n i c m a t t e r c o n t e n t o f t h e f r a c t i o n s . HC1 e x t r a c t s n o t o n l y f r e e Cu and Zn i o n s b u t a l s o some o f the complexed i o n s as w e l l and t h e r e i s a p p a r e n t l y more Zn tha n Cu i n the p a r t i c l e - s i z e s e p a r a t e s , t h e a v e r a g e s f o r twenty samples b e i n g 175 ppm and 76.8 ppm r e s p e c t i v e l y . However, q u i t e a p a r t from t h e t o t a l c o n c e n t r a t i o n s o f Zn and Cu i n t h e samples more Zn m i g h t be e x p e c t e d i n s o l u t i o n because Zn te n d s t o form l e s s s t a b l e complexes t h a n Cu. S c h n i t z e r and Khan [1972] have shown t h a t t h e s t a b i l i t y c o n s t a n t s o f a number o f c a t i o n - f u l v i e a c i d complexes a r e i n t h e o r d e r : F e 3 + > A l 3 + > C u 2 + > N i 2 + > C o 2 + > P b 2 + = C u 2 + > Z n 2 + > M n 2 + > M g 2 + . I t was assumed t h a t b e c a u s e Cu and Zn e x i s t e d m a i n l y as o r g a n o - m e t a l l i c complexes i n s o i l , t h e o x i d a t i o n o f o r g a n i c m a t t e r by i g n i t i o n s h o u l d r e s u l t i n a s i g n i f i c a n t i n c r e a s e i n e x t r a c t a b l e Cu and Zn. The d a t a i n t a b l e one i n d e e d c o n f i r m s t h e i n i t i a l a s s u m p t i o n as e x t r a c t a b l e Cu i n c r e a s e d from 76.8 t o 111.7 ppm and Zn from 175 t o 603.4 ppm. 211 APPENDIX I I I A D i s s o l u t i o n E f f e c t s o f S h a k i n g S o i l s i n Water 20 M i n u t e s ppm S o i l Na K Ca Mg Fe A l Mn S i C LANGLEY Ah 35 3.1 4 4.6 9 11 0 30 -Bt 44 7.8 0 2.7 36 39 0.2 140 -HAZELWOOD A h ] 12.2 5.1 23.6 7.8 9 9 0.5 40 -Bg 13.8 4.3 5.2 5.1 12 17 0.2 60 -HATZIC Ap 21.9 18.8 27.6 15.2 10 10 0.9 30 -Bg 14.3 4.3 4.0 3.3 10 14 0.1 60 -CLOVERDALE Ap 20.5 11.7 60.4 23.1 8 4 3.6 10 -Btg 71.3 3.9 0 2.2 29 33 0.2 80 -DELTA Ap 19.8 41.1 15 137.4 8 8 0 20 -Bg 157.6 23.5 1.6 5.6 14 16 0.1 60 -AVERAGE A l 21.9 16 26.1 37.6 8.8 8.4 1.0 26 -- B . 60.2 8.8 2.2 3.8 20.2 23.8 0.2 80 -APPENDIX I I I B D i s s o l u t i o n E f f e c t s o f U l t r a s o n i c V i b r a t i o n o f S o i l s i n Water ppm S o i l Na K Ca Mg Fe A l S i C LANGLEY Ah 38 1.6 1.6 5.2 28 32 120 420 Bt 54 24.6 0 16 466 404 2440 90 HAZELWOOD A h 1 18.4 4 10.8 15 28 24 140 250 Bg 20.2 7.8 0 7.8 98 n o 530 200 HATZIC Ap 19.6 8.6 50 9.0 38 38 200 170 Bg 19 9 0 8.6 182 184 820 150 CLOVERDALE Ap 40.4 5.8 22.2 18.2 24 26 70 250 Btg 77 19.6 0 15.8 562 582 2680 120 DELTA Ap 87 58.6 2.6 28 174 164 830 590 Bg 182 41 0 10.6 218 214 1390 120 213 APPENDIX I I I C D i s s o l u t i o n E f f e c t s o f U l t r a s o n i c V i b r a t i o n o f - S o i l s i n 0.01M C a C l 2 ppm S o i l Na K Mg Fe A l S i LANGLEY Ah 107 94 400 0 2 60 Bt 161 40 842 0 0 170 HAZELWOOD A h ] 48.4 16 136.2 27 6 80 Bg 48.8 10 258 0 12 100 HATZIC Ap 30.6 28 238 0 4 50 Bg 55.6 17.2 350 0 0 140 CLOVERDALE Ap 76 22 214 16 0 70 Btg 302 8.6 802 0 0 100 DELTA Ap 120 124.4 474 0 4 120 Bg 246 100.4 500 0 0 100 APPENDIX I I I D D i s s o l u t i o n E f f e c t s o f U l t r a s o n i c V i b r a t i o n o f S o i l s i n 0.01M B a C l 0 ppm S o i l Na K Ca Mg Fe A l Mn S i LANGLEY Ah 93.4 23.5 1236 420.7 0 11 29.6 100 Bt 165.6 61.4 898 1009 0 0 30.6 200 HAZELWOOD 13.8 23.5 1040 150.8 46 20 9.4 120 Bg 17.5 23.5 944 450 6 25 0 120 HATZIC Ap 21.2 68.4 1038 432 26 13.0 34.6 100 Bg 20 31.3 1440 634 0 0 584 120 CLOVERDALE Ap 21.4 25.8 1488 418 56 0 111 70 Btg 287.5 53.2 964 987 0 0 34.6 140 DELTA Ap 21.4 213.1 1170 356.3 0 0 9.8 70 Bg 239.2 143.1 458 512 25 0 0 130 215 APPENDIX I I I E D i s s o l u t i o n E f f e c t s o f U l t r a s o n i c V i b r a t i o n o f S o i l s i n 0.1M NaCl . ppm S o i l K Ca Mg Fe A l Mn S i LANGLEY Ah 32.8 682 299 0 6 16.5 150 Bt 56.2 732 687 0 0 21 200 HAZELWOOD Ah 1 36 682 112 6 11 3 130 Bg 31.2 632 199.4 0 12 1 130 HATZIC Ap 82.1 678 189.7 29 13 23.8 150 Bg 32.1 1028 277 0 0 38.6 170 CLOVERDALE Ap 36 820 194 0 25 - 70 Btg 10 620 780 69.6 0 - n o DELTA Ap 136 684 400 53.8 25 - 60 Bg 117.2 330 440 69.6 0 - n o 216 APPENDIX I I I F D i s s o l u t i o n E f f e c t s o f U l t r a s o n i c V i b r a t i o n o f S o i l s i n 0.03M NaCl _ ppm S o i l K Ca Mg Fe A l Mn S i C LANGLEY Ah 19.6 222 164.2 0 0 6.3 30 210 Bt 37.5 176 285.8 0 0 6.4 80 6 HATZIC Ap 53.2 252 110.7 3.0 3.0 7.9 50 50 Bg 24.6 326 136.2 0 0 17.3 70 36 217 APPENDIX IV A Ammonium-N E x t r a c t e d D u r i n g E i g h t Weeks o f I n c u b a t i o n ' S o i l (um) Week 0 2 4 6 8 LANGLEY Ah <2000 18.2 103.2 128.8 119.4 90 2-50 33.8 79.4 108.2 92.5 70 1-2 75 62.5 139.4 172.5 195 0.2-1 65 221.3 320.7 366.3 375 <0.2 94 713 719 750 725 HAZELWOOD A h ] <2000 20.7 151.3 212.5 215 200 2-50 55.1 93.8 158.8 124.4 105 1-2 82.5 88.8 160 146.3 125 0.2-1 133.9 435.8 553.8 580 585 HATZIC Ap <2000 23.8 220 281.9 274.4 250 2-50 80 151.3 171.3 160.7 140 1-2 90 143.2 205 193.2 190 0.2-1 150 315 365 393.2 415 <0.2 100 650 700 625 600 CLOVERDALE Ap <2000 21.9 182.5 230 257.6 270 2-50 70 163.8 189 170 150 1-2 136.9 340 395.7 422.5 445 0.2-1 177.5 620 798 781.3 765 DELTA Ap <2000 19.4 60 87 65.7 35 2-50 30.8 46.3 73.2 54.4 25 1-2 57.5 156.3 216.3 195 175 0.2-1 116.3 255 273.8 368.8 350 <0.2 100 469 719 656 620 218 APPENDIX IV B N i t r a t e - N E x t r a c t e d D u r i n g E i g h t Weeks o f I n c u b a t i o n S o i l (um) Week 0 2 4 6 8 r r LANGLEY Ah <2000 5 80 120 150 175 2-50 17 40 90 145 160 1-2 10 50 150 205 230 0.2-1 30 205 340 390 405 <0.2 25 350 675 725 775 HAZELWOOD A h ] <2000 13 137 203 254 281 2-50 12 76 132 167 178 1-2 10 75 128 162 173 0.2-1 35 298 472 515 597 HATZIC Ap <2000 12 176 198 281 285 2-50 12.5 57 93 114 127 1-2 3.5 108 145 201 216 0.2-1 16 205 309 369 397 <0.2 17 301 503 649 707 CLOVERDALE Ap <2000 6 149 230 267 288 2-50 12 70 116 152 178 1-2 20 211 321 385 426 0.2-1 18 293 601 753 821 DELTA Ap <2000 . 9 35 70 101 120 2-50 8 31 57 84 97 1-2 10 91 153 204 233 0.2-1 14 191 289 336 376 <0.2 15 305 650 720 735 219 APPENDIX IV C T o t a l - N (NH. + NCu - N) E x t r a c t e d D u r i n g E i g h t Weeks o f I n c u b a t i o n S o i l (um) Week 0 2 4 6 8 K K 1 „ .... LANGLEY Ah <2000 23 183.2 228.8 269.5 265 2-50 51 114.9 198.2 237.6 230 1-2 85 112.5 289.4 377.5 425 0.2-1 95 426.3 660.7 756.3 780 <0.2 119 1063 1394 1475 1500 HAZELWOOD A h ] <2000 33.7 288.3 415.5 469 481 2-50 67 169.6 290.8 291.4 283 1-2 92.5 163.8 288 308.2 298 0.2-1 168.9 733.8 1026 1095 1182 HATZIC Ap <2000 35.8 396 480 555.4 535 2-50 92.5 208.3 264.3 274.7 267 1-2 93.5 251.2 250 294.1 406 0.2-1 166 520 674 762.2 812 <0.2 117 951 1203 1274 1307 CLOVERDALE Ap <2000 27.9 332 460 524.6 558 2-50 82.2 233.8 304.8 322 328 1-2 156.9 551 716.7 807.5 871 0.2-1 195.6 913 1398 1534 1586 DELTA Ap <2000 28.4 95 156.9 166.7 155 2-50 38.8 77.3 130.2 138.4 122 1-2 67.6 247.2 369.3 399 408 0.2-1 130.3 446 662.8 704.8 726 <0.2 115 774 1369 1376 1355 220 APPENDIX IV D S u l p h a t e E x t r a c t e d D u r i n g E i g h t Weeks o f I n c u b a t i o n S o i l (urn) Week 0 2 4 6 8 Hr-"" LANGLEY Ah <2000 14 57 74 93 105 2-50 18 38 65 81 92 1-2 78 147 195 222 241 0.2-1 121 217 271 303 326 <0.2 148 275 343 365 376 HAZELWOOD A h ] <2000 21 77 122 143 152 2-50 10 53 71 68 60 1-2 43 74 94 n o 123 0.2-1 137 247 299 328 336 HATZIC Ap <2000 11 48 73 77 85 2-50 16 52 74 88 76 1-2 23 51 86 103 108 0.2-1 44 147 206 249 261 <0.2 87 220 273 284 288 CLOVERDALE Ap <2000 20 84 138 155 159 ,2-50 12 51 82 101 105 1-2 47 178 215 241 273 0.2-1 130 284 368 381 398 DELTA Ap <2000 5 2 3 4 4 2-50 8 3 4 2 3 1-2 49 77 99 105 111 0.2-1 68 161 217 215 201 <0.2 155 290 370 395 380 221 APPENDIX IV E Phosphate E x t r a c t e d D u r i n g E i g h t Weeks o f I n c u b a t i o n S o i l (um) Week 0 2 4 6 8 LANGLEY Ah <2000 1.0 9.2 4.0 3 1.6 2-50 0.75 6.8 5.5 2.8 6.0 1-2 0.25 4.2 2.8 5.0 4.5 0.2-1 0.25 2.4 3.0 3.0 2.6 <0.2 12.5 5.0 17.5 2.5 6.0 HAZELWOOD A h ] <2000 95.5 134.5 137.5 89.3 70 2-50 131 197.3 198 106.5 55 1-2 44.5 106.4 104.3 73.5 48 0.2-1 16.8 47 28 35.2 24 HATZIC Ap <2000 120 182.3 185.5 143.5 130 2-50 148.8 258 249 . 159.5 105 1-2 82.3 178 119 157 148 0.2-1 21.3 51 30.7 24.3 18 <0.2 45 90 50 27.5 20 CLOVERDALE Ap <2000 44 70.5 32.9 50.3 25.6 2-50 46.9 89.4 35.2 45.3 22 1-2 41.5 77.4 32.5 38.2 18.1 0.2-1 22.2 56.3 16.7 37.7 10.2 DELTA.Ap <2000 544.4 502.5 587.5 547.5 601 2-50 512.6 601.3 586.3 559.5 545.5 1-2 365 650 587.5 547.5 514.2 0.2-1 377.5 66.2 49 28.8 24.2 <0.2 1300 1162.5 1260 1258 1250 222 APPENDICES REFERENCES BEECHER, G.R. and B.K. WHITTEN. 1970. Ammonia d e t e r m i n a t i o n : Reagent m o d i f i c a t i o n and i n t e r f e r i n g compounds. A n a l y t i c a l Biochem. 36: 243-246. BOLLETER, W.T., C.J. BUSHMAN and P.W. TIDWELL. 1961. S p e c t r o -p h o t o m e t r i c d e t e r m i n a t i o n o f ammonia as i n d e p h e n o l A n a l . Chem. 33: 593. CHANEY, A.L. and E.P. MARBACH. 1962. M o d i f i e d r e a g e n t s f o r determin-a t i o n o f Urea and ammonia. C l i n . Chem. 8: 130-132. FISKELL, J.G.A. 1965. Methods o f S o i l A n a l y s i s 2. Ed. C A . B l a c k , Agron. 9: 1078-1089. GEDROITS, K.K. 1963. Chemical a n a l y s i s o f s o i l s . T r a n s l a t e d from R u s s i a n P u b l . by the N a t i o n a l S c i e n c e F o u n d a t i o n , Wash., D.C HODGSON, J.G., W.L. LINDSAY and.J.F. TRIERWEILER. 1966. M i c r o -n u t r i e n t c a t i o n c o m p l e x i n g i n s o i l s o l u t i o n I I . Complex-i n g o f z i n c and c o p p e r i n d i s p l a c e d s o l u t i o n from c a l c a r e o u s s o i l s . S o i l S c i . Soc. Amer. P r o c . 30: 723-726. — I E RICHE, H.H. and A.H. WEIR. 1963. A method o f s t u d y i n g t r a c e elements i n s o i l f r a c t i o n s . J . S o i l S c i . 14: 225-235. LE RICHE, H.H. 1973. The d i s t r i b u t i o n o f minor elements among t h e components o f a s o i l d e v e l o p e d i n l o e s s . Geoderma 9: 43-57. MITCHELL, R.L. 1964. T r a c e elements i n s o i l s . In C h e m i s t r y o f the S o i l 2nd Ed., E d i t e d F.E. Bear, R e i n h o l d P u b l . Corp. MITCHELL, H.L. 1972. M i c r o d e t e r m i n a t i o n o f n i t r o g e n i n p l a n t t i s s u e s . J . A.O.A.C. 55: 1-3. 223 NOBLE, E.D. 1955. D e t e r m i n a t i o n o f t r a c e K j e l d a h l n i t r o g e n i n . p e t r o l e u m s t o c k s . A n a l . Chem. 27: 1413-1416. SNELL, F.D. and C.T. SNELL. 1949. C o l o r i m e t r i c methods o f a n a l y s i s V o l . II 3 r d ed. P u b l . D. Van N o s t r a n d Co. I n c . STEWART, B.A. 1966. N i t r o g e n - s u l p h u r r e l a t i o n s h i p s i n p l a n t t i s s u e s , p l a n t r e s i d u e s , and s o i l o r g a n i c m a t t e r . S o i l Chem. and F e r t i l i t y . I n t . Soc. S o i l S c i . T r a n s . Commission II & IV Aberdeen, Ed. G.V. J a c k s , 131-138. VIETS, F.G. J r . AND L.C. BOAWN. 1965. Methods o f S o i l A n a l y s i s 2. Ed. C A . B l a c k , A g ron. 9: 1090-1101. WEATHERBURN, M.W. 1967. P h e n o l - H y p o c h l o r i t e r e a c t i o n f o r d e t e r m i n -a t i o n o f ammonia. A n a l . Chem. 39: 971-974. 

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