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The influence of a combined elevation-vegetation site factor on the nature of the stable humus formed… Drijber, Rhae Alda 1986

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THE INFLUENCE OF A COMBINED ELEVATION-VEGETATION SITE FACTOR ON THE NATURE OF THE STABLE HUMUS FORMED IN SOILS DERIVED FROM VOLCANIC ASH By RHAE ALDA DRIJBER B.Sc. Agr. , The University of Br i t ish Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Soi l Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1986 ©Rhae Alda Drijber, 1986 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ^ jfsx^rve^ The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D a t e £ / * 7 u l 3a / 9 f t i i ABSTRACT An elevational sequence of soi ls on the volcano Iztaccihuatl was selected to study the effect of a combined elevation-vegetation site factor on the nature of the stable humus formed in soi ls developed from recent volcanic ash. One sample plot was selected in each of three vegetation zones; Abies re l igiosa , Pinus hartwegii and Zacatonal, an alpine grassland. In Part 1, non-parametric s ta t is t ics were used to evaluate several parameters measured on the Ah^ and Ah^ s o i l horizons. In Part 2, composite samples were prepared and the polymeric humic acid and fraction A were bulk isolated. The results from Part 1 indicated that the soi ls were at an early stage in their development. The degree of mineral weathering increased with elevation and with depth in the p r o f i l e . In the surface mineral horizons humus-Al complexes were dominant with insignificant formation of "amorphous" aluminosilicate clay minerals. Horizon differences in organic matter characteristics were not apparent in the Abies rel igiosa and Zacatonal zones, but were present in the Pinus hartwegii zone. The humus content differed l i t t l e among the three zones, although qualitative differences related to the vegetation site factor were apparent in the kinds of organic components in the fulv ic acid fract ion. The Ch/Cf ratio 1% was related to the regional climate. E^QQ HA indicated that the humic acids from the three vegetation zones were similar in maturity; hence, 1% zonal differences in E^QQ HA and E^/E^ HA were related to the average polymer molecular weight which increased from the Abies rel igiosa to the Zacatonal zone. i i i In Part 2, elemental and infrared analysis fai led to detect any zonal differences in the humic acid and fraction A. However, the data supported the rapid formation of a high molecular weight, highly aliphatic humic acid fraction r ich in nitrogen. Four major phenolic acids of l ignin origin were identif ied in the humic acid and fraction A hydrolysates -protocatechuic acid, p-hydroxybenzoic acid, v a n i l l i c acid and syringic acid - which established a lignin-derived component of the polymeric humus fractions. The source of vegetation for humus formation could be clearly distinguished from the ratios of the major phenolic acids ident i f ied . iv TABLE OF CONTENTS Page ABSTRACT . . . . . . . •' . i i TABLE OF CONTENTS . . . . . . . . iv LIST OF TABLES . . . . . . . v i i i LIST OF FIGURES . . . . . . . . x i LIST OF PLATES xiv ACKNOWLEDGEMENT xv 1.0 INTRODUCTION . . . . . . . . 1 2.0 LITERATURE REVIEW 3 2.1 Global Distribution of Volcanoes . . . . 3 2.2 Soils Derived from Volcanic Ash- . . . . 8 2.2.1 Distribution and Classif icat ion . . . 8 2.2.2 Profi le Characteristics . . . . 9 2.2.3 Occurrence of Andisols on the Landscape . 10 2.2.A Structure and Charge Characteristics of the Noncrystalline Clay Minerals . . . 12 2.2.5 Formation and Transformation of Clay Minerals 16 2.3 Introduction to Humus Formation . . . 19 2.4 Lignin - Its Nature and Possible Role in Humus Formation . . . . . . . . 21 2.A.1 Synthesis of Lignin in the Plant. . . 23 2.4.2 Phenol Coupling Reactions . . . . 23 2.4.3 Differentiation of Plant Classes on the Basis of Lignin Composition . . . 27 V Page 2.4.4 The Modified Lignin Theory of Soi l Humus . 28 2.5 Humus - A Microbial Product . . . . 33 2.5.1 "De Novo" Synthesis of Polyphenols by Microorganisms . . . . . 33 2.5.2 Synthesis of "Humic Acid-Like" Polymers . 37 2.5.3 Degradation of Lignin by Microorganisms . 38 2.5.4 Simple Phenolic Compounds Detected During the Decay of Plant Tissues . . . 43 2.5.5 The Mild Extraction of Phenolic Acids from Soils and Peats . . . . . 45 2.6 Phenolic Degradation Products of Humic Substances . 49 2.7 The Nature of Soi l Humic Substances and their Relationship to Genetic Soi l Types . . . 56 2.7.1 Humus Fraction Ratios . . . . 56 2.7.2 Spectrophotometric Properties of Humic Substances . . . . . . 59 3.0 STUDY LOCATION . . . . . . . 62 3.1 Iztaccihuatl Vegetation Zones . . . . 67 3.2 Site Selection and Experimental Design . . 70 3.3 Site Characteristics . . . . . . 73 4.0 METHODS . . . . . . . . 79 4.1 Literature Review . . . . . . 79 4.1.1 Methods for Estimating the Content of "Amorphous" Aluminosilicate Clays in Soils . 79 4.1.2 Extraction and Fractionation of Soi l Humus . 81 v i Page 4.1.3 Humus Degradation Methods . . . . 86 4.1.4 Detection of Phenolic Compounds . . . 88 4.2 Part 1 - S t a t i s t i c a l Study . . . . . 90 4.2.1 Sample Preparation . . . . . 90 4.2.2 Routine Chemical Analyses . . . . 90 4.2.3 Humus Fractionation Procedure and Fraction Analysis . . . . . . 91 4.3 Part 2 - Composite Study . . . . . 94 4.3.1 Composite Sample Preparation . . . 94 4.3.2 Bulk Isolation Procedure . . . . 95 4.3.3 D i a f i l t r a t i o n and Concentration of Extracts . 95 4.3.4 Humic Acid and Fraction A Analysis . . 96 5.0 RESULTS AND DISCUSSION . . . . . . 105 5.1 Part 1 - S ta t i s t i ca l Study . . . . . 105 5.1.1 Chemical Analysis of the Modal Pit Profiles . 105 5.1.2 S t a t i s t i c a l Analysis of the Ah^ and Ah^ Soi l Horizons from Iztaccihuatl Vegetation Zones . . . . . . 117 5.1.2.1 Hygroscopic Moisture (%HM), Loss on Ignition (%LI), Total Carbon (%Ct) and Total Nitrogen (%Nt) . . . . 119 5.1.2.2 Extractable Iron (%Fe), Aluminium (%A1) and Si l icon (%Si) . . . . 122 5.1.2.3 Review of the Relevant Data from the German-Mexican Project on the Soils of the Sierra Nevada de Mexico . . . 128 5.1.2.4 Humus Fraction Distribution . . . 133 v i i Page 5.1.2.5 Humus Fraction Ratios . . . . 137 5.1.2.6 Measured Properties of the Humic Acid and Fraction A Extracts . . . . 147 5.1.2.7 Optical Properties of the Humic Acid and Fraction A Extracts . . . . 152 5.2 Part 2 - Composite Study . . . . . 159 5.2.1 Bulk Isolation Recoveries and the Molecular Weight Distribution of the Humic Acid and A Fractions . . . . . . , 159 5.2.2 Elemental Analysis of the Freeze-dried Humic Acid and A Fractions . . . . 162 5.2.3 Infrared Spectra of the Humic Acid and A Fractions . . . . . . 165 5.2.3 Phenolic Acid Hydrolysis Products of the Humic Acid and A Fractions . . . . 170 6.0 CONCLUSION . . . . . . . . 198 7.0 BIBLIOGRAPHY . . . . . . . . 201 8.0 APPENDICES . . . . . . . . 218 1. Modal Pit Descriptions . . . . . 218 2. Humus Fractionation Procedure . . . . 221 3. Solution Carbon Analyser - Walkley Black Wet Oxidation Comparison for Extract Organic Carbon . . . 223 4. PVP - Column Method for Fulvic Acid Fractionation . 226 5. Individual Sample Data for S ta t i s t i ca l Analysis . 227 6. S t a t i s t i c a l Analysis of Parameter Means by AN0VA . 235 7. Correlation Matrix of Chemical Parameters . . 240 v i i i LIST OF TABLES Table Page 1 Experimental Plot Characteristics . . . . 7 5 2 HPLC Chromatographic Conditions . . . . 103 3 Chemical Analysis of the Modal Pit Soi l Horizons from Iztaccihuatl Vegetation Zones . . . . 106 4 Extractable Iron (%Fe), Aluminium (%A1) and Si l i con (%Si) from Modal Pit Soi l Horizons . . . . 113 5 Median Values and 95% Confidence Intervals for Hygroscopic Moisture (%HM), Loss on Ignition (%LI), Total Carbon (%Ct) and Total Nitrogen (%Nt) from Ah and Ah^ S o i l Horizons . . . . . . 120 6 Median Values and 95% Confidence Intervals for Acid Ammonium Oxalate (ox), Pyrophosphate (py) and Citrate-Bicarbonate-Dithionite (cbd) Extractable Aluminium (%A1) and Si l i con (%Si) from Combined Ah^ and Ah^ So i l Horizons . . . . . . . . 123 7 Median Values and 95% Confidence Intervals for Acid Ammonium Oxalate (ox), Pyrophosphate (py) and Citrate-Bicarbonate-Dithionite (cbd) Extractable Iron (%Fe) from Ah^ and Ah^ Soi l Horizons. . . . . 124 8 Relative Dissolution of Materials Containing Iron, Aluminium and Si l icon by Pyrophosphate, Acid Oxalate and Citrate-Bicarbonate-Dithionite . . . . 126 9 Distribution of Iron, Aluminium and Si l icon Containing Materials in the Recent Volcanic Ash Soils of Iztaccihuatl . . . . . . . . 127 10a Representative Profi le from Iztaccihuatl Study Area: Chemical Properties of Prof i le 04 Including Comparative Median Data from the Soils of the Three Vegetation Zones 130 i x Table Page 10b Physical Properties of Profi le 04 . . . 130 10c Particle Size Distribution of Profi le 04 Approximated to the C . S . S . C . System . . . . . . 131 lOd Mineral Distribution of the 1C Ash: 0.20-0.063 mm Particle Size Fraction. . . . 131 lOe Verif icat ion of 1C Ash Parent Material Throughout the Three Vegetation Zone Study Area . . . . 132 11 Median Values and 95% Confidence Intervals for %C in the Humus Fractions . . . . . . 134 12 Median Values and 95% Confidence Intervals for Calculated Ratios of Absolute Humus Fraction Amounts . 138 13 Median Values and 95% Confidence Intervals for the C/N Ratio and % PSS in the Humic Acid and A fractions . 138 14 Median Values and 95% Confidence Intervals for the Optical Properties of the Humic Acid Fraction . . 153 15 Median Values and 95% Confidence Intervals for the Optical Properties of Fraction A 154 16 Composite Sample Bulk Isolation Recoveries of Humic Acid and Fraction A and their Nominal Molecular Weight Distribution . . . . . . 160 17 Elemental Analysis of the Humic Acid and A Fractions . 160 18 Assignments of Infrared Absorption Bands . . . 169 19 Percent Recovery and Correction Factors for the Identified Phenolic Acids from the Successive Ether/ NaHC03 / Ether Extraction . . . . . 169 20 HPLC Analysis of the Humus Fraction Hydrolysis Products - jag Phenolic Acid / g C Humic Acid or Fraction A . . . . . . . 177 X Table Page 21 HPLC Analysis of the Humus Fraction Hydrolysis Products - Phenolic Acids as % of Total Phenolic Acids Identified . . . . . . . 189 22 HPLC Analysis of the Humus Fraction Hydrolysis Products - jag Phenolic Acid / 100 g Soi l . . . 189 23 HPLC Analysis of the Humus Fraction Hydrolysis Products - Calculated Ratios of the Major Phenolic Acids Identified . . . . . . . 195 xi LIST OF FIGURES Figure Page 1 Volcanoes of the World . . . . . . 5 2 Classif icat ion of Volcanic Materials. . . . 6 3 Volcanoes of Central Mexico . . . . . 7 4 Charge Characteristics of Variable Charge Clay Minerals . . . . . . . . 15 5 Transformation of Variable Charge Clay Minerals . . 15 6 Humus Formation Theories. . . . . . 20 7 Structural Scheme of Coniferous Lignin According to Freudenberg . . . . . . . . 22 8 Synthesis of Lignin as Outlined by Higuchi et a l . . 22 9 Formation of the Phenoxy Radical . . . . 25 10 Representative Linkages of Phenylpropane Units in Conifer Lignin. Model Compound: °< -guaiacyl-glycerol-& -coniferyl ether . . . . . . 26 11 Lignin Composition of Plants . . . . . 29 12 Transformation of Orsel l inic Acid by Fungi Imperfecti . 36 13 Transformation of p-Hydroxycinnamic Acid by Fungi Imperfecti . . . . . . . . 36 14 Intradiol Cleavage of Aromatic Rings by White-rot Fungi 41 15 Transformation of Coniferaldehyde and p-Hydroxycinnam-aldehyde by Epicoccum nigrum, Stachybotrys atra and Aspergillus sydowi. . . . . . . 41 16 Phenolic Units Obtained by Reductive Cleavage of Soi l Humic Acids . . . . . . . . 51 17 Structure of Flavonoids . . . . . . 51 18 Sequence of Strata of Soi l Forming Materials on x i i Figure Page Iztaccihuatl . . . . . . . . 66 19 Iztaccihuatl Elevation - Vegetation - Climate Zones . 68 20 Monthly Climatic Data from the Huayatlaco Climato-logical Station on Popocatepetl . . . . 69 21 Procedure for Bulk Isolation of Humic Acid and Fraction A . . . . . . . 9 7 22 U l t r a f i l t r a t i o n Procedure for Isolation of Polymeric Humic Acid and Fraction A . . . . 98 23 Humus Fraction Hydrolysis and Extraction of Phenolic Acids for HPLC Detection . . . . . 100 24 Representative Standard Chromatogram of p-Hydroxy-benzoic Acid, Protocatechuic Acid and Syringic Acid by Methanol Gradient Elution . . . . . 104 25 Distribution of Chemical Parameters within the Modal Pit P r o f i l e s . . . . . . . . 107 26 Infrared Absorption Spectra of the Humic Acid and A Fractions . . . . . . . . 166 27 HPLC Chromatograms of the Phenolic Acid Hydrolysis Products: the Humic Acid Fraction . . . . 172 28 HPLC Chromatograms of the Phenolic Acid Hydrolysis Products: the A Fraction. . . . . . 174 29 HPLC Analysis of the Humus Fraction Hydrolysis Products - Total jag Phenolic Acids / g C in the Humic Acid and A Fractions . . . . . . 178 30 HPLC Analysis of the Humus Fraction Hydrolysis Products - pg Phenolic Acid / g C Humic Acid or Fraction A . . . . . . . 179 31 HPLC Analysis of the Humus Fraction Hydrolysis x i i i Figure Page Products - Calculated Ratios of the Major Phenolic Acids Identified . . . . . . 196 xiv LIST OF PLATES Plate Page 1 The Volcanoes Iztaccihuatl and Popocatepetl . . 65 2 The Abies Religiosa Zone . . . . . 76 3 The Pinus Hartwegii Zone . . . . . 77 A The Zacatonal Zone . . . . . . 78 X V ACKNOWLEDGEMENT The author would l ike to thank Dr. Lawrence Lowe for the opportunity to v i s i t Central Mexico and study the volcanic ash soi ls of Iztaccihuatl . A special thanks to Dr. Quinones whose experience and guidance made the expedition a success, and whose his tor ical anecdotes were most entertaining. The author would also l ike to express thanks to the other members of the expedition, in particular Dr. Takaki for his outline of the elevation-vegetation zones on Iztaccihuatl, and to Rosemary Lowe for her help in plant ident i f ica t ion . Laboratory assistance and guidance of Esther Yip and Bernie Von Spindler are gratefully acknowledged. 1 1.0 INTRODUCTION During the last few decades an important focus of s o i l organic matter research has been on the elucidation of the chemical structure of s o i l humic substances and their possible relationship to genetic s o i l types. Several experimental techniques have been used to study the chemical structure of humic substances with varying degrees of success. However, the products of several chemical degradation methods seemed to indicate a relationship between some of the constituents of these complex polymers and the native vegetation of the s o i l from which they were derived. Although this did not help those studying the structure of humic compounds, i t did point to a genetic origin of humic substances in general, and has given s o i l formation theorists much to evaluate regardin the pathways of humus formation. Soi l formation, and in a sense s o i l humus formation, can be regarded as a result of the five s o i l forming factors. The general features of climate and vegetation were shown by the Russian s o i l scientists to be the dominant factors in s o i l formation; thus they developed the concept of s o i l zonality. The other factors of parent material, topography and time are also important. Local variations in topography can have a profound influence on the microclimate, and consequently the vegetation, enough to alter the pedogenic processes and resulting s o i l type. The selection of a study area to evaluate a combined elevation-vegetation site factor on the nature of the humus formed was a d i f f i c u l t task due the necessity of keeping the other s o i l forming factors constant With good fortune the author was afforded a unique opportunity to v i s i t 2 Central Mexico under the guidance of an experienced pedologist, Dr. Quinones. Through his expertise we were able to locate an area in the Sierra Nevada de Mexico which met a l l the necessary requirements. In the region south-west of the Volcano Iztaccihuatl and the Paso de Cortes an elevational sequence of soi ls developed on recent volcanic ash was selected. The sequence comprised three "climax" vegetation zones: Abies re l igiosa , Pinus hartwegii and Zacatonal, a mixed grassland dominated by Festuca species. Preliminary examination of the l i terature uncovered a joint German-Mexican project directed by Glinter Miehlich (1980) which gave an extensive s o i l and geological data base for the area. The objectives of this study were: 1. To investigate the relationship between elevation and the nature of the stable humus formed, u t i l i z i n g humus fractionation techniques and measured properties of the fractions obtained. 2. To examine the humic acid and fulv ic acid fractions for evidence of lignin-derived phenolic acids which relate to the native vegetation. 3 2.0 LITERATURE REVIEW 2.1 Global Distribution of Volcanoes The distribution of volcanoes over the globe i s determined by plate boundaries where lithosphere i s created or destroyed. There i s generally a clear association of vulcanicity with orogeny, the formation of mount-ains. At the present time there are five to six hundred active volcanoes and a greater number of r e l i c volcanoes. The chief volcanic regions are the great circum-Pacific orogenic belt , the Caribbean orogenic belt , and the Alpine-Mediterranean-North African-Himalayan belt which stretches down through Indonesia (Figure 1). Examples of volcanoes within plates are the Hawaiian in the Pacific plate and the Rift Valley regions of Africa in the African plate. Submarine volcanoes are prevalent in the former plate as well as along the Mid-Atlantic ridge (Press and Siever, 1978; Whitten and Brooks, 1972). The nature of the lava and hence the style of eruption i s dependent on the nature of the plate boundary (Press and Siever, 1978). Oceanic divergent zones and intraoceanic volcanoes produce f l u i d basaltic lava. The c o l l i s i o n of oceanic plates produces basaltic and andesitic lavas, whereas the c o l l i s i o n of an oceanic plate with a continental margin may also produce rhyol i t i c lava. The eruptive style of a volcano depends on the viscosity of the lava, being a function of i t s gas content and comp-osition (Figure 2). The more viscous lavas give explosive eruptions of fragmental or pyroclastic materials. Less viscous lavas give quiet lava flows. The viscosity increases from basaltic to andesitic to r h y o l i t i c A lavas. Volcanoes may extrude more than one type of lava in addition to pyroclastic materials during their eruptive history. Thus, volcanoes usually go through an evolutionary sequence starting with one type of lava and eruptive style and progressing through several stages u n t i l quiescent. The volcanoes of Cental Mexico are distributed along two main fractures, the Clarion and San Andreas (Figure 3). These main fractures constitute the so called "Neovolcanic Axis of Mexico" which covers an area nine hundred kilometres long, seventy to one hundred kilometres wide, and l i e s between the eighteenth and nineteenth para l le ls . This zone contains sixteen major volcanoes, including the Sierra Nevada volcanic chain, and separates the high central plateau of Mexico from the rugged and deeply dissected terrain of Meso-America (Lorenzo, 1959). Figure 1. Volcanoes of the World. F i g u r e 2. C l a s s i f i c a t i o n o f V o l c a n i c M a t e r i a l s . Decreasing V i s c o s i t y — > Decreasing Eruption Force — > TRACHYTE RHYOLITE DACITE ANDESITE BASALT 80%-APPR0XIMATE D O * • MINERAL 40% • CONTENT 20% • P o t a F B l o t . , H I K - ^ v Q u a r t z s s i u m e l d s p a r • B i o t i - x ^ ' P l a g i o c l a s e F e l d s p a r . t e , H o r n b l e n d e ^ >* ** 0 Si P y r o x e n e 1 i v i n e y*? COMPOSITION TYPE FELSIC ( S i and A l r i c h ) | INTERMEDIATE | MAFIC Figure 3. Volcanoes of Central Mexico (Lorenzo, 1959). 8 2.2 Soils Derived from Volcanic Ash 2.2.1 Distribution and Classif icat ion Soils derived from volcanic ash are intimately associated with the volcanic regions of the world (see Figure 1). These soi ls have two essential characteristics - a clay fraction dominated by amorphous material and a high accumulation of humus in the surface horizon. Several desig-nations have been given to these s o i l s , often arising from the Japanese word "Anshokudo" meaning dark (an), coloured (shoku), s o i l (do) (Leamy et a l . , 1980). In Japan the term "Ando" s o i l i s widely used as i s "Kuroboku" s o i l . More descriptive terms include black s o i l s , grassland brown earth soi ls and p r a i r i e - l i k e brown forest s o i l s , a l l of which infer s imilar i ty to Chernozemic Ah horizons (Ishizuka and Black, 1977). The FAO-Unesco World Soi l Map uses the term "Andosols" to denote soi ls formed from materials r ich in volcanic glass and commonly having a dark surface horizon (Leamy et a l . , 1980). Soi l Taxonomy does not recog-nize these so i l s at the order l e v e l , but c l a s s i f i e s them under the sub-order Andepts of the order Inceptisol . They are soi ls formed predominantly, but not exclusively, in volcanic ash. They have a low bulk density and a high allophane content, or consist mainly of pyroclastic materials (Leamy et a l . , 1980). In 1978, G.D. Smith set out a proposal for the Andisol order which was revised in 1983. The central concept of the order revolves around v i t r i c or andic s o i l properties as outlined by ICOMAND, circular letter no. 5 (Leamy, 1983): 9 V i t r i c s o i l properties The s o i l material has: 1. more than 60% by volume of the whole s o i l i s cinders, pumice or pumice-like material, or more than 40% by weight of the sand fraction (0.05-2 mm) is volcanic glass, and has: 2. either an acid-oxalate extractable aluminium value of 0.4% or more, or a 4 M K0H extractable aluminium value of 0.3% or more. Andic s o i l properties The s o i l material has: .1. a bulk density at 1/3 bar water retention of the fine earth fraction of less than 0.9 g per cubic centimetre, and has: 2. a phosphate retention value of more than 85%, and has: 3. either an acid-oxalate extractable aluminium value of 2.0% or more, or a 4 M K0H extractable aluminium value of 1.5% or more. 2.2.2 Profi le Characteristics An important feature of Andisols i s the rapid release of s i l i c a , aluminium and iron from the parent ash and the subsequent formation of humus-aluminium complexes or amorphous aluminosilicate clays. This process imparts certain characterisit ics to the p r o f i l e , namely a high accumulation of humus, often 15 to 30 % in the surface horizon, which forms a thick, dark f r iable Ah horizon with a pseudosilt fabric , a high water holding capacity, and a low bulk density (Wada, 1980; Tate and Theng, 1980; Duchaufour, 1977; Leamy et a l . , 1980). Thixotrophy, or liquefaction under pressure, i s often a characteristic of well developed Andisols containing 10 allophane (Flach et a l . , 1980). Phosphate retention, and CEC measured at pH 7 are high; however, the effective CEC i s lower due to the so i l s being acidic in reaction with a dominance of variable charge surface minerals (Parf i t t , 1980). A major contributor to the CEC would be the organic matter (Tate and Theng, 1980). Profi le development may extend from an A/C prof i le to an A/B/C profi le under intense weathering conditions and/or increasing s o i l age. B horizons are characterized by a brown to yellow colour, blocky structure, and a dominance of alumino-s i l i c a t e minerals such as allophane (Leamy et a l . , 1980). Several ash f a l l s result in s t ra t i f ied soi ls up to several metres thick with each layer displaying the characteristics of i t s pre-burial pedological development. 2.2.3 Occurrence of Andisols on the Landscape The occurrence of Andisols on the landscape i s determined by the intrazonal factors of parent material, topography and time (Duchaufour, 1977; Parf i t t and Saigusa, 1985). Climate and vegetation are thought to play a less important role . Andisols, however, are not generally formed in an arid environment or one marked by extensive dry periods (Duchaufour, 1977; Flach et a l . , 1980). Exceptions are certain v i t r i c Andisols which have been reported from an aridic moisture regime in Syria (Leamy, 1983). Soils developed from volcanic ash along a l t i tudinal climosequences seem to be rather heavily influenced by climate. On Mount Vulture in southern Italy, Spodosols tend to form at higher elevations due to a high r a i n f a l l producing a leaching environment ( L u l l i and B i d i n i , 1980). On Mount Amiata in Tuscany, L u l l i and Bidini (1980) reported Inceptisols at elevations 11 < 1400 m and Spodosols at > 1400.m. At lower elevations Vertisols may form due to the accumulation of leached bases and dissolved s i l i c a (Duchaufour, 1977). In a climosequence of soi ls in the Central Cordillera of Columbia, Cortes and Franzmeier (1972) found temperature to be inf luent ia l in Andisol B horizon development. The warmer temperatures at lower elevations resulted in increased weathering of the ash, a greater clay content, and a lower accumulation of organic matter which allowed the formation of amorphous aluminosilicate minerals such as allophane. These processes were expressed by the presence of a yellow-brown to grayish-brown B horizon. The appearance of the B horizon showed Andisol profi le d i f f e r -entiation, and was not attributed to Spodosol formation. In this s o i l sequence, although the r a i n f a l l was the highest at the lowest elevation, i t was thought that the effective precipitation increased with elevation due to a lower temperature and almost permanent cloudiness. However, the s o i l at the lowest elevation contained a placic horizon cemented by iron and aluminium which seemed to indicate the process of podzoli-zation (Cortes and Franzmeier, 1972). Parf i t t and Saigusa (1985) reported the formation of Spodosols and Andepts in the same volcanic ash beds in New Zealand. Again, this was attributed to a difference in r a i n f a l l . Spodosol formation, therefore, i s common in volcanic ash, occurring where extensive r a i n f a l l permits the removal of bases and forms an acid envir-onment. In addition, the mobilization of fulvic acid precursors, alone or in combination with iron and aluminium, results in the accumulation of sesquioxides and organic matter in the i l l u v i a l B horizon. Andisols, as indicated above, are not the exclusive soi ls formed 12 from volcanic ash, but Entisols , Mol l i so ls , Spodosols, and under extreme weathering, U l t i s o l s , A l f i s o l s and Oxisols may form (Leamy et a l . , 1980). In Japan, which i s highly volcanic, the formation of Ando soi ls i s determined primarily by pedoclimate. Thin ash deposits have a pedoclimate similar to that of the previous surface. The interaction of the ash deposit thickness and the ground water level allows three groups based on moisture status to be outlined; a dry type, a moderate type and a wet type. The dry type, which i s characterized by good drainage and a low water content, forms soi ls of the brown-forest group. Ando soi ls are produced by the moderate and wet type regimes (Ishizuka and Black, 1977). The speed at which the ash weathers w i l l determine the course of s o i l development in thick ash deposits (Ishizuka and Black, 1977). Once the weathered ash has a high enough clay content to increase i t s water holding capacity there w i l l be a succession of vegetation from dry adapted species to wet adapted species with a concomitant increase in the supply of organic matter to the s o i l . This evolution allows soi ls of the Ando group to develop (Ishizuka and Black, 1977). In summary, the essential characteristics for Andisol development are a rapid and easily weathered parent material, a moist pedoclimate without extensive dry periods, and an absence of conditions conducive to Spodosol formation such as a high r a i n f a l l and severe leaching. 2.2.4 Structure and Charge Characteristics of the Noncrystalline Clay Minerals Several chemical and physical properties of Andisols, as outlined in section 2.2.2, are related to the nature and composition of the 13 noncrystalline aluminosilicate clay minerals. Allophane and imogolite are the most widely reported of these minerals. Gibbsite, halloysite and kaolinite are found in Andisols at later stages of pedogenesis. Allophane i s a naturally occurring hydrous aluminosilicate of varying chemical composition while maintaining a predominance of S i - O - A l bonds (Wada, 1977). This i s not the case with phyllosi l icate minerals which are dominantly S i - O - S i bonded with S i - O - A l bonds l inking the tetrahedral and octahedral sheets. Allophanes are represented by a series of minerals with SiO^/Al^O^ molar ratios between 1.0 and 2.0, r^OC + V A ^ O ^ molar ratios between 2.5 and 3.0, and are characterized by a high specific surface area (Wada, 1980). Under a high resolution electron microscope allophane i s seen as a hollow sphere with a shel l likened to a "defect kaolin" structure with an A1-0,0H octahedral sheet and a Si ,Al -0 ,0H tetrahedral sheet. The linkage of the tetrahedra and octahedra and their placement in the shell are not clear; hence, they are called "short-range order" minerals (Wada, 1980). Imogolite i s also an aluminosilicate mineral of high specific surface, but i t s morphology is different from that of allophane. Imogolite has a similar composition to one end member of the allophane series, Si02/Al202 ratio of 1.0, but allophane has more structural water. This i s the result of allophane containing aluminium in tetra-hedral coordination as well as octahedral coordination (Wada, 1980). Under the electron microscope imogolite appears as smooth and curved threads varying in diameter from 10 to 30 nm, and extending several jam in length (Wada, 1980). These threads are made up of finer tube units running in p a r a l l e l . Thus, imogolite i s termed "paracrystalline" . 1A The tube units consist of an external modified gibbsite- l ike surface with aluminium in octahedral coordination. The internal surface has exposed s i lanol groups, Si-OH, arising from the isolated orthosilicate anions (Wada, 1977, 1980). The behaviour of allophane and imogolite, the noncrystalline hydroxides, organic matter, and the edge sites of phyllosi l icate minerals i s due to the variable charge nature of their surfaces and their a b i l i t y to participate in ligand exchange reactions. These surfaces have been termed "variable charge surfaces" (Theng, 1980), "constant potential surfaces" (Uehara and Gillman, 1981), "amphoteric surfaces" (Parf i t t , 1980) and "variable potential surfaces" (Bowden et a l . , 1980). The surface charge for these minerals results from the adsorption of the potential determining ions, H^0+ and 0H~ (Uehara and Gillman, 1981). It i s a direct function of pH, and other factors such as the ionic strength, temperature, valence of the counter ion and dielectr ic constant of the medium (Uehara and Gillman, 1981). This i s in direct contrast to phyllosi l icate minerals which obtain most of their charge, termed perm-anent, by atomic substitution. Specif ical ly adsorbed ions which enter the Stern layer and form coordination complexes with the surface w i l l also effect surface charge. An important example i s the specific adsorption of phosphate anions which introduce a net negative charge to the surface and increase the CEC (Uehara and Gillman, 1981). The adsorption of phosphate i s a serious f e r t i l i t y problem in Andisols. Figure A gives some examples of charge development for these clay minerals. 15 ure 4. Charge Characteristics of Variable Charge Clay Minerals (Wada, 1980). OH, OH" + H + I 2 . H + | Allophane A l (4) <• A l (4) / V ^ O H OH" /V^OH OH 0" + H + I H + | Allophane S i (A) *• S i (A) S i l a n o l group Imogolite / \ \ 0 H - / \ \ 0 C r y s t a l edges 0 0 0 0 0 H . + OH + H + 0 + HO0 | 2 H + | H + I 2 Allophane A l (6) * A l (6) * A l (6) Imogolite ' -Gibb s i t e low pH ° H U H high pH Cr y s t a l edges (AEC) ** OH- OH" + H-0 I 2 H + I 2 Al-humus A l (6) * A l (6) OH" » number i n brackets r e f e r s to coordination number of metal. ** AEC = anion exchange capacity. ure 5 . Transformation of Variable Charge Clay Minerals (Violante and Wilson, 1983). dehydration, HALLOYSITE a R 1 " R > KA0LINITE VOLCANIC ASH PR0TO-IMOG0LITE' ALLOPHANE -> r e s i l i c a t i o n d e s i l i c a t i o n M IMOGOLITE > GIBBSITE 16 2.2.5 Formation and Transformation of Clay Minerals The formation of "amorphous" aluminosilicate minerals such as allophane in Andisols i s closely related to the nature and amount of s o i l humus. Hetier claims the most important property of Andisols i s linked to the formation of a "humus-allophane" complex (Duchaufour, 1976). The abundance of humus prevents amorphous clay formation while the allophane (or active aluminium) stabil izes the humus from biodegradation. Allophane i s found only in horizons where the ratio of iron plus aluminium divided by carbon extracted by a pyrophosphate solution i s greater than 0.1 (Wada and Higashi, 1976; Parf i t t and Saigusa, 1985). Thus, allophane is only present in the early stages of s o i l formation when organic matter is very low, or, in later stages when net mineralization occurs (Aomine and Kobayashi, 1966; Duchaufour, 1976). Once conditions are favourable for neoformation, the particular suite of minerals formed w i l l depend on the stage of s o i l formation, the s o i l horizon, the petrological nature of the ash, the thickness of overburden ash deposits and the pH (Wada, 1980). In the early stages of s o i l formation, the addition of organic matter to the ash deposit surface and i t s subsequent decomposition to form s o i l humus, finds the aluminium and iron released by the weathering of - the ash largely as A l - and Fe-humus complexes (Wada and Higashi, 1976; Higashi and Wada, 1977). The s i l i c a released from the ash forms opaline s i l i c a even in heavily leached volcanic ash soi ls containing a spodic horizon (Parfit t and Saigusa, 1985). In a later stage of s o i l formation a B horizon may differentiate . This occurs when the organic matter level i s not high enough to complex a l l the released aluminium, and allophane and/ 17 or imogolite form in s i t u , possibly by coprecipitation of s i l i c a and aluminium (Wada and Higashi, 1976; Farmer et a l . , 1980; Violante and Wilson, 1983). Thus, allophane and imogolite are generally found in the B horizons of present day Andisols, and in buried A and B horizons of palaeosol Andisols where the addition of organic matter has been arrested (Higashi and Wada, 1977). Wada (1980) noted that the formation of allophane and imogolite was influenced by pH. In laboratory studies using solutions of orthosilicate anions and hydroxyaluminium cations imogolite formed at pH < 5. Allophane formed irrespective of the pH, but i t s nature depended on whether the pH was less than or greater than 5. This pH dependence was noted earlier by Yoshinaga and Aomine (1962a,b), and was used to isolate allophane and imogolite from s o i l s ; allophane dispersing in both acid and alkaline media and imogolite dispersing only in acid media. The transformation of allophane and imogolite in the s o i l seems to depend on the s o i l solution s i l i c a concentration. When the amount of s i l i c a in the s o i l solution i s high, as favoured by a thick overburden ash deposit, a stagnant water regime, a low r a i n f a l l and a s i l i c a rich parent material, halloysite would form as the result of res i l i ca t ion of allophane or imogolite. In ash derived soi ls i t i s common for allophane to decrease and halloysite to increase with depth (Violante and Wilson, 1983; Parf i t t et a l . , 1983). Conditions favouring desi l icat ion such as a higher r a i n f a l l , greater leaching, a basic parent material and a thin overburden deposit would lead to gibbsite formation possibly through imogolite (Wada, 1977). Vegetation may influence the s o i l solution s i l i c a concentration by the alteration of leaching regimes (Parfit t et a l . , 18 1983). The removal of s i l i c a by plant roots may lead to the formation of allophane or gibbsite in root channels (Parfit t et a l . , 1983). These transformations are outlined in Figure 5. In summary, the formation of allophane and imogolite i s primarily governed by the concentration of aluminium in the s o i l solution which i s intimately linked to the s o i l humus, and the s o i l pH. They are further transformed by interaction with the s o i l solution s i l i c a concentration. 19 2.3 Introduction to Humus Formation Humus is a biological product. Humification involves the biological degradation of plant and animal remains with the subsequent formation of dark-coloured, amorphous, acidic , high molecular weight polymers that are more stable than the starting materials. These are termed "humic substances" and can be operationally defined, on the basis of s o l u b i l i t y , into humic acids, fulvic acids and humins. They are distinct from "non-humic substances" which constitute the known classes of organic compounds Several theories have been put forward to describe the process whereby organic tissues introduced into the s o i l become humic substances. B r i e f l y , they stem from three possible origins; modified plant constit-uents such as l ignin (the "ligno-protein theory"); reducing sugars and amino acids produced by microorganisms as metabolic by-products (the "sugar-amine condensation theory"); and, simple polyphenols produced either by microbial synthesis from non-aromatic carbon sources or by microbial degradation of aromatic polymers such as l ignin (the "polypheno theory"). The latter two theories postulate an enzymatic decomposition of plant remains to simple monomers with subsequent polymerization of constituents with or without the participation of microbial enzymes. These theories are outlined in Figure 6. Based on the objectives of this study emphasis w i l l be placed on the role of l ignin in humus formation, and on the microbial neoformation of phenolic compounds and the trans-formation of lignin-derived phenolic compounds. 2 0 F i g u r e 6. Humus F o r m a t i o n T h e o r i e s ( S t e v e n s o n , 1982) . PLANT RESIDUES ( a n i m a l and m i c r o b i a l r e m a i n s ) * f rom n o n - a r o m a t i c c a r b o n . 21 2.4 Lignin - Its Nature and Possible Role in Humus Formation Lignin i s the second most abundant plant constituent after cellulose and makes up approximately 20 to 30 % of the dry weight of the woody tissues of plants (Gross, 1979). In the c e l l walls of plants l i g n i n i s intimately associated with the structural polysaccharides. The compos-i t i o n of l i g n i n , i t s biosynthesis and the mode of linkage of the constit-uent units within the polymer, has been the focus of l ignin research since the 1890's when Klason isolated coniferyl alcohol from the cambial sap of plants (Gottlieb and Hendricks, 1945). A l k a l i fusion of isolated l ignin produced a small amount of catechol, guaiacol and protocatechuic acid. From this and other reports available at the time Klason suggested that l ignin was a oxidation product of coniferyl alcohol or a polymerizat-ion product of coniferaldehyde (Harkin, 1967). Erdtman later claimed that the oxidation of coniferyl alcohol was really an enzymatic phenol dehydro-genation (Harkin, 1967). The greatest advances in l i g n i n chemistry were made by Freudenberg and co-workers from the late 1920's to the late 1960's. Based on their investigations they furthered the theories of Klason and Erdtman to postulate that l ignin was a condensation polymer or mixture of polymers derived from one or more simple units related to Klason's coniferyl alcohol (Gottlieb and Hendricks, 1945). Through laboratory synthesis of l i g n i n - l i k e polymers from cinnamyl alcohols, polymerized by phenol oxidases, Freudenberg arrived at his classic "spruce l i g n i n " model in the 1960's. This i s widely accepted today. A representation of conifer l ignin showing the prominant structural features i s given in Figure 7. 22 gure 7. Structural Scheme of Coniferous Lignin According to Freudenberg (Flaig et a l . , 1975). gure 8. Synthesis of Lignin as Outlined by Higuchi et a l . (Crawford, 1981). C A R B O N D I O I I D E • i S H I K I M I C A C I D I i H , - T O O H M O W ^ I O- M E T M Y T R A N S F E R A S E S - A D E N O S Y L M E T M I O N I N E C 1 P o o l O - M E T H Y T R A N S F E R A S E CH}q M O ^ C M ^ C H - C O O M C H J O * " S I K A P I C A C I D t M0-Cj>-CH.CM-CO0H CHjO S - M Y D R O X Y F E R U L I C A C I D t H0-O-CH = CH-C00H -CHjO F C R U L I C A C I D t HO-O'CM- C H - C O O M HO C A F F E I C A C I O T S I N A P T L A L C O H O L -C O N I F E R Y L A L C O H O L M O W CHj-CO - C O O H P R E P H E N I C A C I D I M O O C H v C O - COOH - » M 0 - f > C M 2 - C H - « X ) H . HO-^-CH = CH-COOH ^ - H V D R O X Y C I N N A W I C T » © - C H = C H - C O O H P•COUMARYL A L C O H O L -f H Y O R O X Y P H E N Y L P Y R U V I C L - T Y R O S I N E A C I D ^ • C H 2 - C O - C O O H • ^ > - C H J - C H - C O O H P H E N Y L P T R U V I C A C I D L - P H E N Y L A L A N I N E C I N N A W I C A C I D 23 2.4.1 Synthesis of Lignin in the Plant At the present time the mechanisms of l i g n i n formation in the plant are f a i r l y well understood. Lignin i s a natural product of enzymatic polymerization, via phenol coupling reactions, of three substituted cinnamyl alcohols: coniferyl alcohol, p-hydroxycinnamyl alcohol and sinapyl alcohol (Figure 8). Its synthesis begins with the key intermed-iate shikimic acid which i s a six carbon ring structure. It i s produced by ring closure of a seven carbon compound formed by the combination of phosphoenolpyruvic acid from glycolysis and D-erythrose-4-phosphate from the pentose phosphate pathway. Shikimic acid i s the direct precursor in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine. In grasses the enzymatic deamination of tyrosine leads directly to p-hydroxycinnamic acid (p-coumaric acid), whereas in other plant genera p-hydroxycinnamic acid i s formed by deamination of phenylalanine to give cinnamic acid which i s then hydroxylated. Further enzyme mediated hydroxy-lations lead to the other substituted cinnamic acid derivatives; caffeic acid, ferul ic acid and sinapic acid . These acids are then enzymatically reduced to form the substituted cinnamyl alcohols l i s ted above. Oxidative polymerization of the alcohols by peroxidases forms the l ignin polymer. The synthesis of l ignin i s outlined schematically by Higuchi et al.(1977) in Figure 8. 2.4.2 Phenol Coupling Reactions Phenol coupling reactions form the basis of l i g n i n biosynthesis as well as many other natural products such as tannins, lignans, pigments, ant ibiot ics , alkaloids and humic substances (Crawford, 1981). The 24 central reaction i s the formation of a phenoxy or phenolate radica l . The phenolate radical can be formed from a phenolate anion by the loss of one electron, or from a phenol by the loss of a proton in addition to an electron as indicated in Figure 9 (Musso, 1967). These radicals then couple by dimerization to give stable products. Electron density i s concentrated on the oxygen atom or the ring carbons ortho or para to the hydroxyl group. Thus, coupling only occurs at these three positions, C-C bonds being favoured over C-0 bonds unless steric factors or a lkyl substituents are present (Musso, 1967). Coupling can also occur with conjugated side chains. The most common bond in conifer l ignin i s the & -aryl-ether bond as represented by the model compound « < - g u a i a c y l -glycerol-/^ - coniferyl ether (Hurst and Burges, 1967). This linkage comprises close to 50 % of the total linkages in conifer l i g n i n (Crawford, 1981). Figure 10 i l lus t ra tes these bond types. In biological systems such as plants the formation of phenoxy radicals i s controlled by a group of enzymes known as phenol oxidases. These enzymes are divided into three classes: tyrosinases (O2 :o-diphenol oxido-reductase), laccases (O2 :p-diphenol oxidoreductase) and peroxidases ( H 2 O 2 : oxidoreductase). It i s generally agreed that peroxidases are involved in l i g n i n biosynthesis (Harkin, 1967; Crawford, 1981). The more reactive polyphenols such as the trihydroxy compounds may autooxidize without the aid of enzymes. This i s very important in the formation of humic sub-stances and w i l l be explored more f u l l y la ter . In summary, l i g n i n i s a complex, highly crosslinked, aromatic polymer of phenylpropane units . It i s formed by enzymatic dehydrogenation coupling reactions with the dominant linkage being the /S -aryl-ether bond. 25 Figure 9 . Formation of the Phenoxy Radical. phenol " o phenolate ion ortho activated para a PHENOXY RADICAL Hvdroquinone OH OH Pyrocatechol OH Cf- -H--H-OH -H« Resorcinol OH - I T H ° 2 0 < J k v ^ 0 H * k ^ 0 H 26 Figure 10. Representative Linkages of Phenylpropane Units in Conifer Lignin. Model Compound: »<.-guaiacylglycerol- /8 - coniferyl ether (Crawford, 1981; Hurst and Burges, 1967). H2C0H HC—0—(/ XVC=C—CH o0H OH oc -guaiacylglycerol - £ - coniferyl ether BOND TYPE STRUCTURE PROPORTION % Arylglycerol-/6-aryl £ ° O c ethers 9 Noncyclic benzyl aryl Jl^oO^ 6 - 8 ethers c c Biphenyl 9.5-11 c 1,2-Diarylpropane 9-0" 0" 7 9 Phenylcoumaran C--0 c —o 9-12 Diphenyl ethers c $ 3.5-4 . 9 -27 The l ignin polymer i s of high average molecular weight, polydisperse, insoluble in concentrated acids and contains few hydrolyzable bonds. Methoxyl groups are the most abundant functional group followed by phenolic hydroxyl. There i s one free phenolic hydroxyl group for every five monomer units . Alcoholic hydroxyl and carbonyl groups are present on the propane side chain (Hurst and Burges, 1967). The characteristic red colour given by the reaction of l ignin with phloroglucinol i s the result of these free coniferaldehyde groups. 2.4.3 Differentiation of Plant Classes on the Basis of Lignin Composition The early l i terature on l i g n i n dealt almost entirely with coniferous l ignin conveying the idea that l ignin was a polymer of coniferyl alcohol. In 1944 Hibbert et a l . treated the l i g n i n from hardwood trees with alkaline nitrobenzene, obtaining a mixture of v a n i l l i n and syringaldehyde as products (Gottlieb and Hendricks, 1945). This led to the belief that the nature of the aldehydes obtained from nitrobenzene oxidation of l ignin was a key feature differentiating angiosperms from gymnosperms. It was later found that angiosperms could be further differentiated on the basis of p-coumaryl residues into monocotyledons and dicotyledons, the former containing a high percentage of p-hydroxybenzaldehyde on nitrobenzene oxidation. It must be emphasized that assigning a " typical l i g n i n " to a plant group warrants caution due to considerable structural v a r i a b i l i t y among species which may affect the nature of the degradation products obtained (Gross, 1979). There i s also v a r i a b i l i t y in l i g n i n content and composition among different plant organs. Lignin i s present in the xylem ce l l s of 28 wood, the husks and shells of seeds, cones, etc . , the stalks of grasses, ferns, etc . , and plant roots (Gross, 1979). Gymnosperm l ignin contains dominantly coniferyl units with a low amount of p-coumaryl units . Syringyl content i s very low or absent. The angiosperm dicotyledons contain coniferyl (guaiacyl) and syringyl units in approximately equal amounts, p-coumaryl units being very low. The monocotyledons contain approximately equal amounts of a l l three units (Crawford, 1981; Gross, 1979). Higuchi et a l . , cited by Crawford (1981), defined three major types of l i g n i n : guaiacyl l i g n i n , found in conifers, lycopods, ferns and horsetails ; guaiacyl-syringyl l i g n i n , present in dicotyledons and a few exceptional gymnosperms; and, guaiacyl-syringyl-p-hydroxyphenyl l i g n i n , found only in the highly evolved grasses. In the grasses considerable amounts of p-coumaric acid are bound as esters to the grass l ignins and are not actually incorporated into the structure. Alder has suggested that grass l ignins should therefore be c lass i f ied as normal guaiacyl-syringyl l ignins (Crawford, 1981). The l i g n i n composition of the plant classes i s given in Figure 11. 2.4.A The Modified Lignin Theory of Soi l Humus In the early 1930's the modified l ignin theory of s o i l humus, was introduced by Waksman and others (Stevenson, 1982). The resistance of l ignin to microbial decomposition in the s o i l compared to other plant constituents suggested a possible role for l ignin as a precursor to s o i l humic acids. Evidence, which led to this conclusion, was largely presumptive and based on non-isolative experimental techniques (Bremner, 1954; Dubach and Mehta, 1963). Waksman's method of proximate analysis 29 Figure 11. Lignin Composition of Plants. OH OH OH p-Coumaryl Coniferyl Sinapyl (Guaiacyl) Grasses Conifers Hardwoods a. alcohol side chain: -CH-CH-CH„OH. 30 was classic in this respect. He termed the residue remaining after a sequential extraction of s o i l organic matter the "ligno-protein complex", which he believed were the s o i l humic acids. The protein was incorporated through the Schiff reaction: (modified lignin)-CH0 + RNH^ » (modified lignin)-C—NHR + H 20 (Stevenson, 1982) Further support of this theory was based on the similar so lubi l i ty of s o i l humic acids and l ignin in reagents such as pyridine, alcohol and a l k a l i ; both formed precipitates in acid solutions; and laboratory synthesized ligno-protein complexes had similar properties to s o i l humic acids. In addition, both l ignin and humic acids contained aromatic rings, phenolic hydroxyl groups, and methoxyl groups, the extent of which diminished with the degree of decomposition (Stevenson, 1982). Several changes have been shown to occur in l ignin during humification: 1. A significant change in the elemental composition was apparent; carbon decreased, oxygen and nitrogen increased. 2. The increase in nitrogen was largely non-hydrolyzable. 3. The increase in oxygen was reflected in an increase in carbox-yl groups and total ac idi ty . A. There was a decrease in methoxyl groups but no concomitant increase in phenolic hydroxyl groups. 5. The u l t r a v i o l e t - v i s i b l e spectra showed a loss of maxima characteristic of l i g n i n with the production of rather featureless spectra similar to those of s o i l humic acids. 31 6. Changes in the infrared spectra were especially apparent in the carbonyl region, 1700 cm *, due to the production of carboxyl groups and in the 16A0 to 1610 cm ^ region due to the incorporation of nitrogen. Changes in the 1000 to 1500 cm ^ region were primarily due to alterations in the propane side chain and loss of methoxyl groups. 7. The infrared spectra became less well defined as humification proceeded. 8. The process of l ignin transformation to humic acids was largely oxidative. The apparent absence of change in the phenolic hydroxyl content during humification was unexpected since the number of methoxyl groups decreased. Recent evidence supports a reaction of nitrogen with the released phenolic hydroxyl groups, possibly by oxidation to quinones (Flaig et a l . , 1975). It has been noted that the free radical content of l ignin increases during humification (Flaig et a l . , 1975). Experiments with model compounds indicate that demethylation of methoxyl groups i s a prerequiste to condensation with nitrogen compounds ('Haider et a l . , 1965). This further supports the quinone mechanism outlined below: (Stevenson, 1982; Flaig et a l . , 1975) demethylation 32 Due to the apparent success of degradative techniques in elucidating the chemical structure of l i g n i n , Gottlieb and Hendricks (1945) subjected s o i l organic matter to alkaline nitrobenzene oxidation and high pressure catalytic hydrogenation. They fai led to find any degradation products indicative of l ignin and concluded: 1. The material derived from plant l i g n i n in the s o i l i s drastically altered in the kind and pattern of peripheral groupings on the aromatic r ing . 2. If l i g n i n - l i k e materials are present in s o i l organic matter a large percentage of the original hydroxyl groups are absent and carbonyl groups have appeared. Subsequent use of the nitrobenzene oxidation by Morrison (1958, 1963) produced less than 1 % of the total s o i l organic carbon as lignin-derived phenolic aldehydes. This led Morrison to reject the simple ligno-protein theory of Waksman. 33 2.5 Humus - A Microbial Product Soi l microorganisms contribute to humus formation in several ways. They are the chief decomposers of macro-residues into smaller molecules for microbial metabolism or synthesis of humic polymers. Substrates include plant l ignins , flavonoids and tannins in addition to carbohydrates and proteins. Through the secondary metabolism of microorganisms several classes of organic compounds are produced. Noted are melanoidins (Bremner, 1954), pigments (Steelink and T o l l i n , 1967; Hurst, 1967), simple poly-phenols (Haider et a l . , 1972; Martin and Haider, 1971), anthraquinones (Saiz-Jimenez et a l . , 1975), perylene derivatives (Kumada and Matsui, 1970) and antibiotics (Steelink and T o l l i n , 1967). Some or a l l of these substances may serve as precursors to humic substances. Autolysis of microbial ce l l s or f rui t ing bodies may release dark-coloured, ac idic , "humic a c i d - l i k e " polymers into the s o i l environment where they may be further altered to form s o i l humus. In essence there are many aspects of microbial ac t ivi ty which could produce precursors to s o i l humic substances. The following sections focus on the "de novo" synthesis of polyphenols by microorganisms, and on the microbial degradation of l i g n i n to simple monomers. 2.5.1 "De Novo" Synthesis of Polyphenols by Microorganisms The "de novo" concept of s o i l polyphenols was credited to the Russian researchers Trusov and Williams. In the early 1900's these two scientists claimed humus formation was the result of hydrolytic decomp-osition of plant remains followed by the synthesis of aromatic compounds. 34 Oxidation to hydroxyquinones and condensation formed the dark-coloured s o i l humus (Stevenson, 1982). Kononova rejected the l i g n i n origin of s o i l humus based on histological experiments. She claimed there were two stages of decomposition: 1. Fungal attack on the simple carbohydrates and part of the protein and cellulose in the medullary rays, cambrium and cortex. 2. Further decomposition of cellulose of the xylem by aerobic myxobacteria with the formation of brown humic matter. (Bremner, 1954) During these two stages the l ignin i s hardly touched, and humic substances are produced by the myxobacteria using carbohydrate as the carbon source. Recently, polyphenols released by the decomposition of l ignin and tannin have been included in the process emphasizing the diverse origin of polyphenols for humus formation (Bremner, 1954; Stevenson, 1982). Since the pioneering work by Kononova several scientists have made important contributions to the role of microorganisms in humus formation. Noted are J .P . Martin, K. Haider, W. F l a i g , H.M. Hurst and Z. F i l i p . Several species of microscopic fungi imperfecti were shown by Martin, Haider and co-workers to be very productive in the synthesis of polyphenols from non-aromatic carbon sources. Detailed investigations were carried out with Epicoccum nigrum (Haider and Martin, 1967), Stachybotrys atra, Stachybotrys chartarum (Martin and Haider, 1969) and Hendersonula  toruloidea (Martin et a l . , 1972). H. toruloidea was found to be outstand-ing in both the number of phenols and weight of "humic a c i d - l i k e " polymer produced. 35 Secondary metabolism was induced by a nutrient shortage or by the accumulation of primary metabolic intermediates (Haider et a l . , 1972). Synthesis occurred via the shikimic acid pathway (cinnamic acid deriv-atives) and acetate-malonate pathway (orse l l in ic acid derivatives) (Haider et a t . , 1972; Haider and Martin, 1967). A key polyphenol synthesized by the studied fungi was o r s e l l i n i c acid (Haider and Martin, 1967; Martin et a l . , 1972; Haider et a l . , 1972). Oxidation, decarbox-ylation and hydroxylation reactions transformed o r s e l l i n i c acid into several other phenols, as outlined in Figure 12. A second phenolic acid produced in small quantities by several fungi imperfecti, with the exception of H. toruloidea, was p-hydroxycinnamic acid. Beta-oxidation of the propane side chain, ortho-hydroxylation and decarboxylation were the key reactions to other phenols (Figure 13). As noted in Figures 12 and 13, g a l l i c acid and pyrogallol were common to both pathways. Resorcinol was only formed from o r s e l l i n i c acid, whereas p-hydroxycinnamic acid was the parent molecule to proto-catechuic acid . Toluene derivatives were common products of orcinol , formed by decarboxylation of o r s e l l i nic acid, but were not produced from p-hydroxycinnamic acid. Ring methylation of p-hydroxycinnamic acid was not evident. Methoxyl derivatives were not produced from either acid; a key feature in distinguishing lignin-derived phenolic acids such as f e r u l i c , v a n i l l i c and syringic acids from microbially produced phenolic acids. Phloroglucinol, a 1,3,5 - trihydroxybenzene, can be formed from o r s e l l i n i c acid or cresorsel l inic acid. It has also been noted as a degradation product of flavonoids (Hurst, 1967). Substitution patterns on the benzene ring were characteristic of the parent phenol; -3,5-(meta) 36 Figure 12. Transformation of Orsel l inic Acid by Fungi Imperfecti (Haider et a l . , 1972). CH» CH» ^ \ COOH ^ \ COOH H O ^ ^ O H H O ^ / ^ O H H O ^ / ^ O H H O ^ ^ O H o r s e l l i n i c o c i d CHi orcinol OH 2,3,5-trihydroxy-toluene CHs HO' l % v / ^OH OH 3,4,5-trihydroxy-loluene 3,5-dihydroxy-benzoic acid COOH HO OH gallic ocid OH resorcinol H O ^ ^OH OH pyrogollol Figure 13. Transformation of p-Hydroxycinnamic Acid by Fungi Imperfecti (Haider et a l . , 1972). OOH COOH COOH CH f p-hydro / benzoic OH roxy-ocid OH V COOH p-hydroxy- \ ^ CH cinnomic ocid * '•OH OH coffelc acid COOH OH OH protocatechiiic ocid COOH OH gallic ocid .OH OH OH 2,3,4 -trihydroxy-benzoic acid H O ^ ^ O H H O ^ >*OH OH pyrogollol 37 substitution for orcinol derivatives and -4-(para)substitution for p-hydroxycinnamic acid derivatives. Transformation of the phenolic intermediates were oxidative in nature: oxidation of side chains and methyl groups to carboxylic acid, ring hydroxylation, decarboxylation to carbon dioxide and demethylation (important in the transformation of lignin-derived phenolic compounds). 2.5.2 Synthesis of "Humic Acid-Like" Polymers Several of the phenols noted in Figures 12 and 13 were found to be sensitive to autooxidation and polymerization reactions. This was evident from the formation of dark-coloured polymeric material in the culture solutions and ce l l s of the above fungi. As noted ear l ier , phenol coupling was the favoured mechanism in the formation of polymers from polyphenols (Section 2.4.2). Williams (1984) summarized four possible mechanisms for phenol oxidation: 1. Polyphenols are autooxidized by molecular oxygen (C^) particularly under alkaline conditions. 2. The oxidation by may be catalyzed by polyphenol oxidases from a variety of sources. 3. Peroxidases catalyze a rapid oxidation of polyphenols by- hydrogen peroxide. 4. Inorganic constituents of soi ls such as higher oxides of manganese are capable of oxidizing quinols to quinones. Mechanism 4 has been further substantiated by the work of Wang et a l . (1983a,b) and Kumada and Kato (1970) for oxides of aluminium and iron. 38 The absence of phenol oxidase enzymes in the fungal culture solutions stressed the sensit ivi ty of these phenols to autooxidation at pH values > 6 (Haider et a l . , 1972). The phenols that were particularly sensitive were the asymmetric and vic inal substituted trihydroxybenzenes, -benzoic acids and -toluenes. Their reactivity stemmed from the hydroxyhydro-quinone grouping in equilibrium with the phenoxy radical and hydroxy-quinone. Less reactive were g a l l i c acid, pyrogallol and unsubstituted hydroxyhydroquinone. Once formed these reactive phenols may combine with less reactive phenols such as resorcinol (meta substituted), amino compounds or pre-existing phenolic polymers (Haider et a l . , 1972). 2.5.3 Degradation of Lignin by Microorganisms Several classes of microorganisms have the a b i l i t y to degrade l i g n i n . These saprophytic microorganisms include the fungi (white-rot, brown-rot and soft-rot (fungi imperfecti)) , actinomycetes and several bacteria. Controversy exists over the extent of l ignin degradation by white-rot fungi. Complete degradation to carbon dioxide and c e l l constituents i s supported by Haider et a l . (1972), whereas Crawford (1981) concludes that l i g n i n in i t s natural state i s not readily u t i l izable as a carbon/energy source, but an additional more readily available carbon source such as cellulose i s required for extensive degradation. These two views are not necessarily exclusive since l i g n i n in i t s natural state i s intimately associated with the structural polysaccharides. The i n s o l u b i l i t y of the l i g n i n polymer requires the use of extra-cel lular enzymes for i t s degradation. It i s generally accepted that phenol oxidases are important for degradation (Crawford, 1981), except 39 that this does not agree with their basic function - phenol coupling. A recent review by Kirk (1975) stressed the lack of knowledge on the enzyme systems involved and the speculative nature of past l i terature . Nevertheless, this speculation led to three major conclusions regarding the degradation of l ignin by white-rot fungi: 1. The surfaces accessible to enzymes were greatly modified; carbon, hydrogen and methoxyl contents decreased,and oxygen, carbonyl and carboxyl contents increased. 2. Attack on side chains and ring carbons took place simultaneously. 3. Cleavage of the ring occurred while s t i l l in the polymer. Crawford (1981) cited two possible mechanisms for l ignin degradation by microorganisms: 1. Depolymerization of the l ignin macromolecule with the release of monomeric and dimeric fragments which are transported into the microbial ce l ls where they are degraded. 2. Dearomatization of the intact polymer by cleavage of the rings while they are s t i l l bound in the macromolecule, followed by erosion of the resulting polymeric, aliphatic network. Pathway 2 i s generally favoured; however, i t i s l i k e l y that more than one degradation mechanism may be operating at any one time. Certain preparatory steps have been implicated in the degradation process. Introduction of an a<-carbonyl group to the propane side chain may be required as a f i r s t step in i t s degradation (Kirk, 1975) or for cleavage of the aryl-ether bond between adjacent monomers (Hurst and 40 Burges, 1967). Ring cleavage involves two steps; preparation of the ring by the introduction of hydroxyl groups ortho or para to each other followed by aerobic cleavage (Crawford, 1981; Kirk, 1975). Hydroxyl groups can be introduced to the ring by monooxygenases or by demethylation of existing methoxyl groups. Protocatechuic acid, a common intermediate formed during the catabolism of l i g n i n , i s enzymatically cleaved by a dioxygenase via the "ortho-f ission" (" intradiol") pathway forming /S-ketoadipic acid (Figure 14). Degradation products of /S -ketoadipic acid are then funnelled into the tricarboxylic acid cycle. Fungi imperfecti can extensively transform lignin-derived phenolic compounds. The reaction sequence i s similar to that reported in Section 2.5.1 for p-hydroxycinnamic acid. However, an additional demethylation reaction also occurs. In model studies this reaction occurs rather late in the degradation sequence after the formation of v a n i l l i c acid by x3 -oxidation (Hurst and Burges, 1967). Earl ier demethylation may occur in the s o i l environment producing orthohydroxy monomers sensitive to oxidation and polymerization. The transformation of coniferaldehyde and p-hydroxycinnamic acid by fungi imperfecti demonstrates the nature of the reactions involved: oxidation, demethylation, /S -oxidation, decarboxy-lation and hydroxylation (Figure 15). The "humic a c i d - l i k e " polymers produced by the fungi when grown on l i g n i f i e d substrates were similar to peat and s o i l humic acids in elem-ental analysis, exchange capacity, total ac idi ty , phenols released upon sodium amalgam reduction and resistance to microbial degradation in the s o i l (Martin and Haider, 1969, 1971). Phenolic structures were thought to be major contributors to fungal humic acids. Yields of identif ied 41 Figure 14. Intradiol Cleavage of Aromatic Rings by White-rot Fungi (Kirk, 1975). OH HOOC "OH 0, HOOC 'COOH COOH Figure 15. Transformation of Coniferaldehyde and p-Hydroxycinnamaldehyde by Epicoccum nigrum, Stachybotrys atra and Aspergillus sydowi (Martin and Haider, 1971). pyrogollol 42 phenols, based on reductive degradation of the polymers, ranged from 2 to 10 % of the starting materials. Yields from s o i l and peat humic acids were 3 to 6 % (Martin et a l . , 1974). Piper and Posner (1972b) reported yields of 12 to 32 % for s o i l humic acids using the same procedure. The actual phenol content of the polymers may have been higher since reductive degradation cleaves diaryl ether bonds but not a l k y l - a r y l ether or biphenyl (C-C) structures (Piper and Posner, 1972a). Also, several model phenols were destroyed by more than 50 % during the reduction (Martin et a l . , 1974). Biphenyl structures, which were not cleaved by reduction, may contribute s ignif icantly to the humic acid polymer since phenol coupling reactions favoured C-C bond formation (Section 2.4.2). Schnitzer et a l . (1973), based on humic acid degradation with alkaline potassium permanganate, disputed the importance of phenolic structures to fungal and s o i l humic acids. They claimed that "fungal humic acids were complex organic materials containing aliphatic and aromatic structures, only some of which were phenolic". Several studies indicated a major aliphatic component in the ether-soluble sodium amalgam reduction products of s o i l humic acids (Mendez and Stevenson, 1966; Stevenson and Mendez, 1967; Tate and Goh, 1973). 14 C-labeled model phenolase polymers were used to determine the extent of biodegradation of certain phenolic carbons in the s o i l . Ring carbons were highly s tabi l ized, propane side chains were degraded to a limited extent, possibly through /3-oxidation, and carboxylic acid and methoxyl groups attached directly to the ring were readily u t i l i z e d . It was estimated that 10 to 20 % of intact p-hydroxycinnamic acids and caffeic acids were stabil ized against biodegradation. These phenolic acids were A3 more readily linked into the model phenolase polymers due to the electron-donating effect of the acryl ic side chain. Free phenolic acids were readily u t i l i z e d by the microorganisms or were linked into humic polymers (Haider and Martin, 1975). 2.5.A Simple Phenolic Compounds Detected During the Decay of Plant Tissues The production of high molecular weight substances occurs quite rapidly during the humification of plant residues. A slower, oxidative phase then follows producing material of lower average molecular weight (Swift and Posner, 1977). This rapid production of high molecular weight materials coincides with a decrease in extractable phenolic compounds. Kuwatsuka and Shindo followed the behaviour of phenolic acids during the decay of rice straw (Kuwatsuka and Shindo, 1973; Shindo and Kuwatsuka, 1975a,b, 1976). Rice straw was incubated at 50° C and 60 % water content for A5 days during which time subsamples were extracted with methanolic sodium hydroxide, a c i d i f i e d , and the phenolics extracted into ether. Gas chromatographic analysis detected several major phenolic acids; in order of abundance, p-coumaric, f e r u l i c , v a n i l l i c , p-hydroxybenzoic, s a l i c y l i c and syringic . P-coumaric and ferul ic acids were bound as esters to the grass l ignins (Kuwatsuka and Shindo, 1973). The former amounted to over 50 % of the total phenolic acids i d e n t i f i e d . Minor amounts of benzoic, g a l l i c , caf fe ic , sinapic, gentisic , protocatechuic and /Q - resorcylic acids were also detected. The highest concentrations of phenolic acids were detected at zero days incubation after which they decreased s i g n i f i -cantly. An exception was p-hydroxybenzoic acid which decreased s l i g h t l y . Myskow and Morrison (196A) found a similar trend during the incubation of 44 lupin and white melilot roots in sand. Ferulic and p-coumaric acids were detected from the lupin roots at zero days incubation. They had largely disappeared after 30 days when syringic and v a n i l l i c acids became dominant. 14 The incubation in s o i l of C-labeled oat roots demonstrated a rapid and 14 heterogeneous incorporation of the C-label into a l l organic matter fractions, coinciding with the peak of microbial ac t ivi ty (Sinha, 1972a,b). The newly formed humic substances were preferentially stabil ized into the humic acid and humin fractions, possibly through the incorporation of oligomeric l i g n i n fragments and microbial biomass. Time courses allowed Shindo and Kuwatsuka (1975a) to follow the changes in phenolic acid composition and content with temperature and moisture. At 50° C, under flooded conditions, the amount of phenolic acids peaked then declined. It seemed as i f the warmer temperatures had stimulated the production of phenolic acids, either from l ignin or through microbial synthesis, but the lack of oxygen had prevented their polymerization into humic substances, or their degradation (Sections 2.5.2 and 2.5.3). Similar behaviour was noted by Sinha (1972a). Aerobic conditions favoured the production of humic acids and higher molecular weight fulvic acids, while anaerobic conditions caused the accumulation of lower molecular weight fu lv ic acids and water-soluble compounds. The transformations of p-coumaric and ferul ic acids observed by Shindo and Kuwatsuka (1975a,b) were similar to those observed in the trans-formation of lignin-derived phenolic acids by fungi imperfecti (Section 2.5.1). The major end-product was protocatechuic acid which was readily autooxidized to form humic substances or cleaved to produce microbial substrates (Section 2.5.3). Shindo and Kuwatsuka (1975b) also cited 45 evidence for the reversible methylation of the para hydroxyl group of p-coumaric acid to form p-methoxycinnamic acid, and ferul ic acid to form 3,4-dimethoxycinnamic acid. Any further transformation was not observed. The interactions of polyphenols with s o i l constituents were invest-igated using both column and batch methods by Shindo and Kuwatsuka (1976). Adsorption processes modify the behaviour of phenolic acids in the s o i l . The extent of adsorption in subsurface horizons was related to the dominant clay mineral; allophane > montmorillonite > kaol ini te . In surface horizons adsorption was positively correlated with humus content, a reflect ion of humus s t a b i l i t y . The structure of the phenolic acid was very important; a longer side chain increased, while a methoxyl group decreased adsorption. Protocatechuic acid was strongly adsorbed, possibly due to chelation or autooxidation of the catechol moiety. The other phenolic acids, p-coumaric, p-hydroxybenzoic, <=<-resorcylic, ferul ic and v a n i l l i c , were rapidly leached from the surface mineral horizons. 2.5.5 The Mild Extraction of Phenolic Acids from Soils and Peats Phenolic acids play a central role in the humification process; consequently, they reflect the biochemical status of the s o i l (Hanninen et a l . , 1981). Several attempts have been made to extract these acids according to their form in the s o i l . Weak extractants such as water, dilute base, calcium hydroxide and hot ethyl acetate have been used to extract the "free" phenolic acids (Whitehead et a l . , 1982, 1983; 'Katase, 1981a). This form is particularly important to allelopathic studies as i t reflects the biochemical status of the s o i l solution accessible to plant roots. Free phenolic acids also serve as direct precursors for 46 humus formation. The combined forms are extracted by weak alkaline hydrolysis. An additional pool, the non-extractable phenolic acids, are obtained only by vigorous degradative techniques such as acid hydrol-ysis , oxidation or reduction. These methods w i l l be explored in Section 2.6. Katase (1981a,b,c) investigated the phenolic acids in peat and forest s o i l . The same four acids detected by Kuwatsuka and Shindo (1973) from rice straw were also found to be most abundant in forest and peat s o i l . They were p-coumaric acid, ferul ic acid, v a n i l l i c acid and p-hydroxybenzoic acid. Three forms of the acids were defined; a free form extractable with hot ethyl acetate, A, a combined form extractable with hot ethyl acetate and released by alkaline hydrolysis, B, and a combined form not extractable with organic solvent but released by alkaline hydrol-ysis , C. The distribution of these four acids within the three forms was found to differ between the peat and forest s o i l s . Total phenolic acids released were greater for the peat s o i l by an order of magnitude. The phenolic acids, ferul ic and p-coumaric, were especially abundant in form C of the peat s o i l . In contrast, v a n i l l i c acid prevailed in the forest s o i l suggesting that peat humus was transformed less than forest humus. The absence of resorcinol and phloroglucinol in the sodium amalgam reduction products of peat humus suggested that microorganisms played a less important role in peat formation than in s o i l humification (Hanninen et a l . , 1981). Insoluble l ignin macromolecules may contribute to this pool. The source of organic matter may also be important. Esters of p-coumaric and ferul ic acids were noted in graminaceous species (Whitehead 47 et a l . , 1982; Hartley and Buchan, 1979), and p-coumaric acid was espec-i a l l y abundant in sphagnum (Morrison, 1963; Morita, 1968). Coniferyl units, high in conifer l i g n i n , formed v a n i l l i c acid through microbial oxidation. The free form, A, contained the least phenolic acids reflect ing i t s dynamic nature. The transformed phenolic acids, p-hydroxybenzoic and v a n i l l i c , were the major contributors. Form B phenolic acids may be viewed as being weakly esterif ied to organic components soluble in organic solvents. This form was very low in the peat s o i l . However, in the forest s o i l form B was high in p-coumaric and ferul ic acids possibly due to a solubil izat ion of higher molecular weight l i g n i n fragments through microbial transformation. The interaction of form C with s o i l minerals was probably more important in the forest s o i l than the peat s o i l . Stabil ization of transformed phenolic acids by adsorption to mineral surfaces would f a c i l i t a t e their subsequent polymerization to form humic substances (Wang et a l . , 1983a). A similar pattern was noted in forest and peat humus by Hanninen et a l . (1981). There was a clear dist inction in the types of phenolic compounds isolated from the fulv ic acid fractions. In the forest humus the amount of v a n i l l i c acid and i t s demethylated form, protocatechuic acid, was three times that of p-coumaric acid. The opposite occurred in the peat humus. In general, the forest humus contained a greater proport-ion of benzoic acid derivatives (protocatechuic, p-hydroxybenzoic and v a n i l l i c acids) compared to cinnamic acid derivatives (p-coumaric and ferul ic acids) reflecting a greater degree of l ignin transformation. Whitehead et a l . (1982) investigated the relationship between plant 48 species and the simple phenolic acids extracted from s o i l s . Sample plots containing three different plant groups; monocotyledons (grasses), dicotyledons and pteridophytes (ferns), were sampled for soi ls and roots. The soi ls were extracted with reagents increasing in a l k a l i n i t y from water to dilute sodium hydroxide. The amount of phenolic acids extracted increased with pH. The roots contained greater amounts of extractable phenolic acids than the soi ls by at least an order of magnitude. A clear separation in the content of p-coumaric and ferul ic acids was apparent between the grass roots and the roots of other plant species. In terms of /jg phenolic ac id/ g carbon, the grasses contained the above phenolic acids in the tens of thousands while the roots of other species contained less than two thousand. This trend was reflected by ferul ic acid in the s o i l s , but was not as clear for p-coumaric acid. The plant roots showed a large var iabi l i ty in amount and composition of extracted phenolic acids which was not observed in the s o i l s . It was suggested that during humification there was a tendancy towards uniformity in composition with a marked reduction in the amount of extractable phenolic acids. 49 2.6 Phenolic Degradation Products of Humic Substances The objective of a chemical degradation technique i s to produce simple units representative of the main structural units in the polymer. This i s true whether the goal i s structural or genetic. Chemical degrad-ations can be c lass i f ied as either hydrolytic, oxidative or reductive. A severe l imitation in these techniques i s that they are either too mild, producing low yields of products, or too drastic, producing units with no relationship to the original polymer. Other limitations include a lack of bond specif i ty , uncertainty in the reaction mechanism and the production of ar t i facts through molecular rearrangements, functional group shif ts , condensations, etc. In order for degradative techniques to be used with any measure of confidence these problems must be understood and i f possible corrected. Several investigators believe that a succession of techniques with increasing strength i s required. Another approach is to hydrolyze the humic acid molecules prior to degradation to provide a more homogeneous starting material ( R i f f a l d i and Schnitzer, 1973). This latter technique may prove useful for structural studies on the humic acid "core", but losses of up to 50 % by weight of organic material could seriously obscure any genetic relationships. Despite the above l imitations, a definite l ink has been established between the phenolic degradation products of s o i l humus and the overlying vegetation. Morrison (1958, 1963) was one of the f i r s t to relate vegetat-ion to the phenolic aldehydes released during the alkaline nitrobenzene oxidation of s o i l humus. Detected in the oxidation products from soi ls and peats were the phenolic aldehydes, syringaldehyde, v a n i l l i n and 50 p-hydroxybenzaldehyde, and the related acids, syringic, v a n i l l i c and p-hydroxybenzoic. Lesser amounts of the cinnamic acid derivatives, p-coumaric and f e r u l i c , were detected. A comparison of the oxidation products of sphagnum and sphagnum peat gave corresponding results . Both were low or absent in syringyl and v a n i l l y l derivatives and dominant in p-hydroxybenzyl derivatives. In pine forest humus, v a n i l l i n was dominant followed by low amounts of p-hydroxybenzaldehyde. Syringaldehyde was very low or absent. This conformed to the known composition of gymnosperm l ignin (Section 2.4.3). A peat profi le containing birch remains gave large amounts of syringaldehyde on oxidation, as expected. A cr i t ic ism of this work was that yields were very low; 1 to 4 % for peat soi ls and 0.5 to 1 % for mineral s o i l s . Detection of phenolic compounds by paper chromatography also had i t s l imitations, notably low sensit ivi ty and poor quantitization. Nevertheless, a lignin-derived component of s o i l organic matter was established. Sodium amalgam was used'by Burges et a l . (1964) to reduce s o i l humic acids. The degradation products were divided into two categories: l i g n i n -derived units and flavonoid-derived units (Figure 16). The latter category encompassed both microbially synthesized phenols (reviewed in Section 2.5.1) and flavonoid degradation products. Lignin-derived phenolic comp-ounds and their transformed products were covered in Section 2.5.3. Flavonoids are ubiquitous in higher plants, mosses and ferns, but are absent in microorganisms and lichens (Morita, 1968; Harborne and Simmonds, 1964). Three major groups are recognized; anthocyanins, respon-sible for the colour of flowers and f r u i t s , flavonols which form the basis of condensed tannins, and flavones (Salisbury and Ross, 1978). 51 Figure 16. Phenolic Units Obtained by Reductive Cleavage of Soi l Humic Acids (Burges et a l . , 1964). C . C , Lignin-derived units C O O H O H C O O H I C H , C . C , C H , = R2 = H p-Hydroxybenzoic acid R, = H, R2 = OCH3 V a n i l l i c acid Rj = #2 = OCH^ Syringic acid R, = Rj = H, R 2 = OCH^ Guaiacylpropionic acid H, R^ = OH Protocatechuic acid Rj = R 2 = OCH^ Syringylpropionic acid O H Flavonoid-derived units K j / ^ O H O H R 1 = OH Phloroglucinol R, = H Resorcinol C . C , ' C H a O H R l " R l -R, = R 2 = OH Methylphloroglucinol H, R 2 = OH 2,6-Dihydroxytoluene OH, R 2 = H 2,4-Dihydroxytoluene Unassigned units C O O H H O \ ^ O H 3,5-Dihydroxybenzoic acid Pyrogallol H O " \ ^ O H O H Figure 17. Structure of Flavonoids (Salisbury and Ross, 1978), OH ^7 from acetate-malonate pathway 3' 5' from cinnamic acid 52 A l l have a C^C^C^ carbon structure containing a pyran ring (Figure 17). The A ring i s based on phloroglucinol formed by the acetate-malonate pathway. The B ring i s formed from cinnamic acid. Hydroxyl or methoxyl groups may be substituted at the 3' , 4' , or 5' positions of the B ring, thus bearing a resemblance to lignin-derived phenolic compounds. Reductive degradation of model flavonoid compounds confirms the origins of the flavonoid units l i s ted in Figure 16 (Burges et a l . , 1964; Tate and Goh, 1973). Burges et a l . (1964) claimed that chromatographic patterns of reduct-ion products provided a "fingerprint" technique for characterizing humic acids of different origins . The phenolic compounds that enabled this to be distinguished were derived from the l ignin of the overlying vegetation, microbially-derived phenolic compounds being common to most s o i l s . In Section 2.4.3 i t was noted that syringyl residues were present in the l ignin of deciduous hardwoods but absent in coniferous softwoods. This difference allowed the separation of humus formed under deciduous vegetat-ion from that formed under coniferous vegetation. V a n i l l i c , protocatechuic and p-hydroxybenzoic acids were ubiquitous among the soi ls investigated. An exception was moss humus which did not contain any lignin-derived phenolic compounds. Protocatechuic acid was present as such in the humic acid polymers since a l k y l - a r y l ether bonds are not cleaved by reductive degradation (Burges et a l . , 1964; Piper and Posner, 1972a). Piper and Posner (1972a) evaluated the sodium amalgam reduction method using model compounds, infrared spectroscopy and chromatography. Their results were generally favourable. Reductive degradation was found to cleave diphenyl and dibenzyl ether bonds but not diphenyl methane, 53 biphenyl, or a l k y l - a r y l ether bonds. A considerable amount of aliphatic material was produced which was not alleviated by 6 N HC1 hydrolysis prior to reduction. Up to 30 % of the original humic acid could be recovered as phenolic material provided conditions were optimized. Mendez and Stevenson (1966) had l i t t l e success with this method. They emphasized the aliphatic nature of the reduction products and stressed caution in structural studies. Other problems were related to the sensit-i v i t y of the reaction mixture to reoxidation. Schnitzer et a l . (1973) pointed out that humic acids were d i f f i c u l t to reduce as they had a natural tendency towards oxidation. The successful use of sodium amalgam reduction products of humic acids as "fingerprints" required that they be extracted from soi l s of widely different origins (Piper and Posner, 1972b; Dormaar, 1969). Piper and Posner (1972b) f e l t that reductive degradation was better suited to estimating the "degree of transformation" of s o i l humic acids. The nature and amount of the phenolic acid degradation products were related to the s o i l extractant, the molecular size of the humic acid mole-cules, the s o i l ' s clay mineral composition and particle size dis t r ibut ion. Humic acids that were extracted by sodium hydroxide following a pyrophos-phate extraction gave more phenolic compounds upon reductive degradation, primarily the less transformed v a n i l l i c , syringic and ferul ic acids. This was also true for humic acids of higher average molecular weight. Humic acids associated with the fine clay fraction and with minerals of higher specific surface area also contained less transformed phenolic acids, possibly due to a protective factor. These humic acids bore a closer resemblance to l ignin than the more oxidized, lower molecular weight 54 humic acids (Piper and Posner, 1972b). The s o i l environment also affected the nature of the phenolic degradation products. Conditions conducive to low biological act ivi ty such as gleying or cold temperatures produced greater amounts of phenolic acids, particularly v a n i l l i c acid, upon reductive degradation (Tate and Goh, 1973). Jacquin reported an inverse relationship between the amount of v a n i l l i c acid released during hydrolysis and the degree of humification (cited in Tate and Goh, 1973). The relationship between phenolic degradation products and molecular weight was further investigated by Tate and Anderson (1978). Gel chrom-atographic fractions of s o i l humic acids were subjected to acid hydrolysis with the phenolic degradation products detected by gas chromatography. Two soi ls were examined; a Bh horizon of a Podzol or iginal ly under coni-ferous vegetation (kauri forest) but presently under scrub and bracken, and an A horizon under hard beech forest. The major hydrolysis products were protocatechuic acid, p-hydroxybenzoic acid and v a n i l l i c ac id . Syringic acid was not identif ied possibly due to interference problems in gas chromatography (Tate, 1972; Stevenson and Mendez, 1967). Cinnamic acid derivatives were also not ident i f ied . Katase (1981b) noted that substituted cinnamic acid derivatives were polymerized during acid treat-ment . The gel-excluded humic acid fractions from both soi ls upon hydrolysis released mainly protocatechuic acid with lesser amounts of p-hydroxybenzoic and v a n i l l i c acids. Lower molecular weight humic acid fractions were only prevalent in the Bh horizon, releasing p-hydroxybenzoic acid in small amounts. The excluded humic acid fractions were thought to bear a closer 55 resemblance to l i g n i n . However, considerable amounts of protocatechuic acid in the hydrolysates indicated extensive demethylation of v a n i l l i c acid; either by microorganisms or during acid hydrolysis when i t was possible that some demethylation occurred, although concentrated acids and vigorous reaction conditions are usually required to cleave a l k y l -aryl bonds (Morrison and Boyd, 1973). Furthermore, Katase (1981b) found that the recovery of v a n i l l i c acid by reflux in 6 N HC1 or 2 N NaOH was independent of pH. This suggested that the demethylation of v a n i l l i c acid occurred in the s o i l environment. The hydrolysis products of the Podzol Bh horizon did not reflect the past kauri vegetation (Tate and Anderson, 1978). 56 2.7 The Nature of Soi l Humic Substances and Their Relationship to  Genetic S o i l Types Several chemical and physical techniques have been used to charact-erize humic substances, the primary goal being to uncover the nature of the high molecular weight, dark-coloured, amorphous humic acid fract ion. The early humic acid l i terature focused on a defined chemical composition (Stevenson, 1982). With the a r r i v a l of humus fractionation came the realization that humic acids were a complex mixture of substances. Studies on the fulvic acid fraction proceeded at a slower rate due to the necessity of concentrating the voluminous acid f i l t r a t e to a workable volume. Forsyth made a major breakthrough when he passed the fulvic acid fraction through activated charcoal eliminating salts , simple organic compounds, etc. in the non-adsorbed f i l t r a t e . Elution of the charcoal pad with a series of solvents released several defined fractions (Stevenson, 1982). As new techniques and more sophisticated instruments were developed i t became apparent that despite the overall s imilari ty in humic substances there were perceptible differences related to s o i l type. 2.7.1 Humus Fraction Ratios The fractionation of humus into humic acids, fulvic acids and humins provided an additional tool for studying humus formation and s o i l genesis. It had long been recognized that humus fractions differed in nature and amount among genetic s o i l types and even between horizons of the same s o i l . The humus fraction ratio Ch/Cf, defined as the ratio of carbon in the humic acid fraction to carbon in the fulvic acid fraction, had been used for 57 taxonomic purposes in Russia (Kononova, 1961), Europe (Duchaufour, 1977), Canada (Lowe, 1980) and Japan (Tokudome and Kanno, 1965a,b, 1968). It had also been used to demonstrate the nature and direction of s o i l processes (Sinha, 1972a,b; Anderson, 1979; Swift et a l . , 1970). The Ch/Cf ratio was found to vary with latitude in the zonal soi ls of Russia. The humus reserves, Ch/Cf ratio and "aromaticity" of the humic acid fraction followed a hyperbolic pathway from north to south, reaching a maximum in the Chernozemic s o i l zone (Kononova, 1961). Similar trends were reported in the Humic Allophane soi ls of Japan (Tokudome and Kanno, 1965a,b, 1968) and in the Chernozemic soi ls of the Canadian Prairies (Lowe, 1980; Anderson, 1979). The correlation among humus reserves, Ch/Cf ratio and degree of condensation of humic acids suggested that conditions which favoured humus accumulation also favoured the polymerization of humic acid precursors. The accumulation of humus in the s o i l was closely related to climate, vegetation and parent material. These s o i l forming factors determined the amount of organic matter added to the s o i l , i t s mode of decomposition and the balance between mineralization and immobilization. Conditions which favoured the accumulation of humus included an ameliorating vegetation, a neutral s o i l environment conducive to microbial act ivi ty and the poly-merization of humic acid precursors, a s tabi l iz ing parent material high in surface active clay minerals, a moderate hydrothermal regime with adequate aeration and alternating wet and dry cycles. The mode of humus deposition in a s o i l horizon can be inferred from the Ch/Cf ra t io . In surface mineral horizons, organic matter formed by in s i tu root decomposition produced a high Ch/Cf ra t io . Ch/Cf ratios 58 greater than 1.0 were typical in Ah horizons of temperate grassland soi ls (Lowe, 1980; Kononova, 1961). However, cold temperatures or anaerobic conditions enhanced the formation of fulvic acids. Low Ch/Cf ratios were found in subsurface horizons where i l l u v i a l organic matter had accumulated (Lowe, 1980). The enrichment of fulvic acids relative to humic acids was due to their greater mobility, particularly in acid media. Lowe (1975) fractionated the acid-soluble components of a Muck s o i l using polyvinylpyrrolidone (PVP). The PVP separated the fulvic acid fraction into two major components; a straw-coloured, non-adsorbed poly-saccharide-rich fraction (fraction C) and a dark-coloured, adsorbed poly-phenol-rich fraction (fraction A). The Ca/Cf ratio was defined as the ratio of the carbon in the A fraction to the carbon in the total fu lv ic acid fract ion. The Ca/Cf ratio was influenced by the source of vegetation, the s o i l acidity and the leaching regime and seemed to reflect the biolog-i c a l status of the s o i l . B r i e f l y , soi ls developed under grass tend to have low Ca/Cf ratios with the fulvic acid fraction dominated by polysac-charides. A neutral s o i l environment and large biomass caused the deplet-ion of polyphenols through polymerization or microbial degradation. The polyphenol-rich l i t t e r and low biological act ivi ty present in the organic horizons of coniferous forest soi ls (Berg et a l . , 1980) produced high Ca/Cf rat ios . The acid environment and leaching regime common to these soi ls favoured the i l l u v i a t i o n of polyphenols into the B horizon producing a Ca/Cf ratio greater than 0.5 (Lowe, 1980). The Ca/Cf ratio clearly distinguished Podzolic Bf and Luvisolic Bt i l l u v i a l horizons (Lowe, 1980). 59 2.7.2 Spectrophotometric Properties of Humic Substances Three regions of the electromagnetic spectrum are widely used to study the nature of humic substances. Absorption in the ultraviolet and vis ible regions i s due to electronic transitions within molecules. Resonances in the infrared region are due to the vibrations and rotations of atoms in functional groups giving rise to characteristic absorption bands. The absorbance of humus extracts in the ultraviolet and vis ible regions increases with decreasing wavelength, with humic acids showing a higher absorptivity per unit concentration than fulvic acids. However, the spectra are rather featureless due to the molecular complexity of the polymers. In the infrared region several broad, poorly defined peaks are evident with absorptions characteristic of COOH, C=0, C=C and C-H being most common. Conjugated multible bond chromophores (C=0, C=C) are responsible for absorption in the ultraviolet region. The absorbance at A =280 nm (E o o r i ) is widely used as an index of aromaticity. Positive correlations with the C/H ratio and negative correlations with hydrolyzable carbon and nitrogen supports this conclusion (Anderson et a l . , 1974a,b). The colour quotient, E . / E , , defined as the ratio of absorbance at 4 o A =400 or 465 nm to absorbance at X =600 or 665 nm, was thought to reflect the degree of aromatic condensation (Kononova, 1961) or maturity (Lowe and Godkin, 1975) of the humic acids. However, Anderson et a l . (1974a) found no significant correlations between the E^/E^ ratio and chemical properties related to the aromatic character of humic acids such as C/H ratios or levels of hydrolyzable carbon and nitrogen. Several investigators have shown an inverse relationship between the E , / E A ratio and polymer molecular 60 weight (Chen et a l . , 1977; Swift et a l . , 1970; Anderson et a l . , 1974a,b). According to Chen et a l . (1977) a low E^/E^ ratio reflected a large molecular size or weight, a high carbon content, and a relat ively low content of oxygen, carboxyl groups and total ac idi ty . These concepts were in harmony with the elemental and functional group compositions and molecular weights of humic and fulvic acids. Kumada (1965) considered the colour of humic acids to be their essential characteristic . Humic acids were c lass i f ied into three major types based on their u l t r a v i o l e t - v i s i b l e absorption spectra; A, B and Rp. A further type, P, was introduced when absorptions characteristic of perylene derivatives were present. Two absorption indicies were defined: the RF factor which denoted the intensity of l ight absorption at 600 nm per unit weight of humic acid, and A logK which represented the i n c l i n -ation of the absorption curve from 600 to 400 nm ( A l o g K = log E^/E^). A logK decreased and RF increased with the degree of humification. The major humic acid types were found to be characteristic of certain soi ls in Japan (Kumada, 1965, 1975; Suzuki and Kumada, 1972). Types A and B represented the mature stage of humification. Type A humic acids were found in volcanic ash s o i l s , weakly calcareous soi ls and Chernozemic s o i l s . Type B humic acids were common in so i l s of the brown forest group. Immature Rp-type humic acids were found in brown forest s o i l s , red s o i l s , organic horizons, composts, etc. and were assumed to form during the rotting of plant residues. They represented the i n i t i a l stages of humif-ication displaying characteristics similar to l ignins , tannins, etc. (Suzuki and Kumada, 1972). Detailed characterization of the humic acid types was carried out 61 by several Japanese investigators (Kuwatsuka et a l . , 1978; Tsutsuki and Kuwatsuka, 1978a,b, 1979a,b; Kumada and Matsui, 1970; Matsui and Kumada 1977a,b). Several trends were evident. The elementary compositions (Kuwatsuka et a l . , 1978), the content of oxygen-containing functional groups (Tsutsuki and Kuwatsuka, 1978a) and the levels of hydrolyzable substances (Tsutsuki and Kuwatsuka, 1979a,b) were proven by variance analysis to be s ignif icantly different among the humic acid types. The degree of humification, as defined by RF and A l o g K , decreased in the order A > B > Rp. Chemical parameters which followed this trend were % C, atomic C/H, total acidity , and carboxyl and carbonyl groups. % H, alcoholic and phenolic hydroxyl, and methoxyl groups were in the reverse order. Nitrogen behaved quite differently showing v a r i a b i l i t y in the immature Rp-type humic acids but decreasing from Rp- to B- to A-type humic acids. Two processes were evident: a rapid formation of humic substances r ich in nitrogen followed by a slower release of nitrogen with further humification. The above trends and plots of atomic H/C and 0/C ratios suggested two processes of humification. The early to middle stages of humification were in the direction of dehydrogenation or demethanation which eliminated aliphatic components. Later stages involved dehydration-condensation reactions (Kuwatsuka et a l . , 1978). These latter reactions were supported by X-ray evidence of coal bands in A-type humic acids (Matsui and Kumada, 1977a), and by the decreased susceptibil i ty of A-type humic acids to oxidative and reductive degradation (Matsui and Kumada, 1977a,b). 62 3.0 STUDY LOCATION The Sierra Nevada volcanic chain i s located in Central Mexico approximately 60 km south-east of Mexico Ci ty . The Sierra Nevada, or "snowy mountain" (Farrington, 1897), forms a north-south chain of four volcanoes linked by saddles; Tlaloc , Telapon, Iztaccihuatl and Popocatepetl (Miehlich, 1980). One of these saddles, the Paso de Cortes, allowed Hernan Cortes to march into the Aztec capi tal , Tenochtitlah, and conquer the Valley of Mexico in 1521. The Sierra Nevada separates to the east and west the high valley basins of Puebla and Mexico, respectively. The peaks of Popocatepetl and Iztaccihuatl exceed 5000 m above sea level and are glaciated even though they are located south of the Tropic of Cancer and well within the tropics. Popocatepetl, in Nahuatl, means "smoking mountain" (Farrington, 1897), and i t i s the second highest mountain in Mexico reaching an altitude of 5452 m. It has a c lass ica l cone shape often called s t ra t i f ied due to several lava flows interbedded with unconsolidatated pyroclastic materials. Popocatepetl was formed during the Pleistocene (10,000 to 2 mil l ion years before present), and was active during the Holocene (recent) (Lorenzo, 1959). The major part of the Sierra Nevada, including the flanks- of Iztaccihuatl , are covered by the recent pyroclastic materials of Popocatepetl (Miehlich, 1980). Iztaccihuatl forms a long narrow ridge cut into three well defined peaks (Plate 1) (Farrington, 1897). The peaks are equidistant from one another with the central peak reaching 5286 m making i t the third highest mountain in Mexico (Lorenzo, 1959). The snow covered silhouette of 63 Iztaccihuatl bears a str iking resemblance to a woman lying on her back. The northern, central and southern peaks form the head, breast and feet, respectively. The ridge i s a l i t t l e less than 3 km long with the feet lying approximately 16 km from Popocatepetl. Iztaccihuatl , the name given by the Aztecs, translates to "white woman". Legend claims that a goddess was executed and forever rooted to the spot where she f e l l . Popocatepetl, her lover, remained by her side venting his sorrows with heavings of ashes and floods of lava tears (Farrington, 1897). Iztaccihuatl was formed during the late Oligocene and Miocene (26 mil l ion years before present) with hornblende, trachy-andesitic lava flows forming the basal part. Volcanic act ivi ty began again in the Pliocene (2 to 12 mil l ion years before present) with porphyritic, pyroxene-andesitic lava forming the actual mountain. During the Pleistocene the summit was formed from a small cone of red basaltic-andesitic lava scoria and basalt (Lorenzo, 1959). Extensive glaciation and erosion carved the present silhouette with aeolian-relocated ash and glac iof luvia l sediments covering the lower slopes (Miehlich, 1980). The soi ls of the Sierra Nevada have developed from a series of alternating deposits of andesitic pumice and ash layers, pyroclastic materials of Popocatepetl (Miehlich, 1980). The sequence of soil, forming materials found in the sampling area, and their approximate ages are given in Figure 18. Variations of these strata form the stratigraphic s o i l units of the Sierra Nevada. The dominant s o i l units are Andosols; the the specific unit being a function of geographic location, climate, and age and type of parent material. The so i l s of the Sierra Nevada are c lass i f ied by a system introduced 64 by Miehlich (1980). Four Andosol units, using the diagnostic horizons of Soi l Taxonomy and the FAO, are defined; Andine, Andic, Andos and Thixic Andosols. B r i e f l y , the f i r s t corresponds to the FAO V i t r i c Andosol, the second and third to the FAO Humic Andosol. Thixic Andosols are found in young, l i t t l e weathered relocated ash sediments (toba sediments) and cont-ain dominantly s i l i c a in the clay fract ion. Andine Andosols are poorly developed and occur exclusively in the most recent ash of Popocatepetl (1C ash). They are limited to the climate zones between 2700 and 4200 m. Three subunits are defined; Cryandine Andosols which occur on icy-dry slopes at high elevation, Mesandine Andosols which are found on cold, humid middle slopes, and Thermandine Andosols which occur on the cool-dry lower slopes. The Mesandine Andosols cover the largest portion of the Sierra Nevada forming a ring around Popocatepetl, occurring on the Paso de Cortes and on the southwestern and eastern flanks of Iztaccihuatl . Andic and Andos Andosols show greater pedological development than Andine Andosols. A high humus content, pronounced thixotrophy and the presence of allophane in the clay fraction are their distinguishing features. Prof i le differentiation may also occur. These so i l s are found exclusively in the older ashes of Popocatepetl (2C and 3C). 6 5 Popocatepetl "Smoking Mountain" - The source of volcanic ash. 66 Figure 18. Sequence of Strata of Soi l Forming Materials on Iztaccihuatl (Miehlich, 1980). 1C Approximate Age B.P. (yr) 400 C= ash P= pumice IP 900 2C 3000-5000 2P 5000 3C 10,000 3P 10,500 67 3.1 Iztaccihuatl Vegetation Zones The vegetation zones on Iztaccihuatl form a well defined elevational sequence. The valley bottoms have been largely cleared for farming and villages with an oak forest appearing at 2000 m. A series of plant associations then follow, as diagramed in Figure 19. Beyond the forested l imi ts , alpine grasslands (zacatonal) cover extensive areas of the Paso de Cortes, Iztaccihuatl and Popocatepetl. The Paso de Cortes had been cleared of forest by the Aztecs, and wind prevented i t s reestablishment. Monthly climatic data has been recorded at the Huayatlaco Climato-logical Station on Popocatepetl, elevation 3620 m (Figure 20). Minimum temperatures are often below 0°C during the winter months, November through February. Frost i s common at higher elevations with snow being infrequent and lasting only a few days. Maximum temperatures reach the mid to late teens in the Spring just prior to the rainy season. Monthly precipitation during the rainy season, May through September, averages from 150 to over 200 mm. 68 Figure 19. Iztaccihuatl Elevation-Vegetation-Climate Zones. * Climatic data from Miehlich (1980). Figure 20. Monthly Climatic Data from the Huayatlaco Climatological Station on Popocatepetl (3620 m, North Slopes). Years averaged Temperature • • 17 Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. 70 3.2 Site Selection and Experimental Design There are two major access routes to Iztaccihuatl . The south access from Amecameca to the Paso de Cortes passes through several dist inct vegetation zones on the west face of Iztaccihuatl as outlined in Figure 19. A preliminary survey defined the nature and boundaries of the vegetation zones with respect to elevation, slope, aspect, topography, etc. It was decided to sample distinct vegetation zones rather than a continuum as the former was more s t a t i s t i c a l l y powerful for vegetation and required less sampling. Three "climax" vegetation zones were selected; Abies re l ig iosa , Pinus hartwegii and the Zacatonal. These three zones are within the elevational boundaries of the Andine Andosols developed in recent volcanic ash (Miehlich, 1980). The vegetation zones at lower elevations contained mixed species, and the soi ls were developed in different parent materials. Therefore, they were neglected in this study. Sampling was done over a three week period in January of 1982. A single experimental plot within each vegetation zone was chosen using the following c r i t e r i a : 1. The plot was representative of the zone. 2. The s o i l parent material was the most recent ash of Popocatepetl, designated 1C (Miehlich, 1980). 3. The ash layer was deep enough for adequate profi le expression. 4. The exposure was restricted to approximately a west aspect. 5. The slopes were chosen to be less than 25° with even topography. 6. The plots were located approximately mid-way between bioclimatic zone boundaries to ensure no sampling of transition zones. 71 A completely random experimental design was chosen to maximize the degrees of freedom associated with the experimental error and to minimize the complexity of s t a t i s t i c a l analysis. Within each plot one modal pit was fully described and sampled (Appendix 1 ) . Seven other pit locations were determined from random number tables. If the location f e l l on an obstacle or near the plot boundary the next random number was used. Emphasis was on recent processes; hence, surface mineral horizons were sampled at two depths corresponding to Ah^ and Ah 2 horizons. In addition the forest floor was sampled where appropriate. For s t a t i s t i c a l analysis a total of A8 samples from the three zones were used. A factorial arrangement of treatments was selected; three vegetation zones and two horizons making a total of six treatments with eight observations per treatment. St a t i s t i c a l analysis by ANOVA requires the following assumptions to be met: 1. The variance for a l l treatments must be equal. 2 . The observations within each treatment must follow a normal distribution. 3. The observations are independent. 4. The observations are selected randomly from a population' of a l l possible observations. With only eight observations per treatment i t i s virtually impossibl to verify normality or homogeneity of variance. However, the simplistic design and basic objectives of this study are easily analysed by non-parametric s t a t i s t i c s . 72 Non-parametic s ta t is t ics avoids using population parameter means which rely heavily on a normal dis t r ibut ion. Instead, observations are ranked, a s ta t is t i c i s computed and the nul l hypothesis i s accepted or rejected based on a chosen significance l e v e l . The MIDAS s t a t i s t i c a l package at the University of Br i t i sh Columbia contains both two-sample and multi-sample comparisons. The former uses the Mann-Whitney U test and the latter the Kruskal-Wallis test. The median test i s included in both. The Mann-Whitney U and the Kruskal-Wallis tests require that the random samples be independent and drawn from population's with continuous distributions. The median test only requires random samples. The nul l hypotheses tested in this study were: HO: The three zones were from a common population. HO: The two s o i l horizons were from a common population. The alternative hypotheses were: HI: At least one zone was not from the common population. HI: The two s o i l horizons were from different populations. 73 3.3 Site Characteristics Details on the experimental plots within each vegetation zone are given in Table 1. Discrepancies between the elevational boundaries as given in Figure 19 and those reported for the experimental plots were caused by variations in topography and aspect. Plots selected showed minimal disturbance, both man-made and natural. Selective logging and grazing were evident in several areas, but not within the experimental plots. One tree was logged near the boundary of the Pinus hartwegii plot , but i t s effect was negligible . Topographical i r regular i t ies , such as depressions, mounds, windthrow disturbances, etc. were also not evident. Fire scars were present in the Abies rel igiosa and Pinus hartwegii plots on veteran trees, and charcoal fragments occurred in the s o i l p r o f i l e s . Charcoal was absent in the Zacatonal plot . The experimental plot selected within the Abies rel igiosa zone seemed typical (Plate 2). A f a i r l y dense but open tree canopy allowed sufficient l ight penetration to the forest floor producing extensive herbaceous coverage. The dominant species were Pyrola secunda, Sibthorpia  pichenchensis, Senecio tolucanus, Senecio angulifolius and Alchemilla  procumbens. Scattered occurrences of bunchgrass (Festuca amplissima) and moss completed the herbaceous layer giving an estimated coverage of > 30%. Relatively thick FH layers (10 to 15 cm) were typical of the plot . Several scattered patches of Vaccinium spp. and Senecio barba-Johannis completed the shrub layer. The main canopy consisted of mature trees with circum-ferences at breast-height up to 2 m. Veteran and dominant trees were few. The limbs of some trees were thickly covered with moss, "old man's beard". 74 The Pinus hartwegii plot contained an extensive bunchgrass community, the dominant species being Festuca tolucensis (Plate 3). The tree canopy was very open allowing considerable l ight to reach the forest f loor . Herbaceous species other than grass were few, and shrubs were negligible . Moss was present at the lower end of the plot possibly due to a hydrolog-i c a l effect . There was evidence of overland water flow which occurred during the rainy season. Interspersed between bunchgrass colonies were large bare patches of s o i l with l i t t l e FH. However, pine needles were abundant even in areas distant from the tree canopy. There was consider-able variation in the circumference of mature trees, some reaching over 2 m at breast-height. The experimental plot in the Zacatonal zone was selected north of the Paso de Cortes to avoid sampling induced grassland (Plate 4). Festuca  tolucensis and Festuca amplissima were dominant with lesser amounts of Calamagrostis and Muhlenbergia spp. Growth was not as luxuriant as in the Pinus hartwegii plot, and herbaceous species were few. There were consid-erable patches of bare ground with numerous channels formed by surface-water run-off . The plot was also s l ight ly concave but of lesser slope than the Abies rel igiosa or Pinus hartwegii plots. Sampling was done while a thin patchy snow cover existed and frost penetrated to 5 cm, but this posed few problems. The 1C pumice layer (Miehlich, 1980) was not reached even though the modal pit extended to a depth greater than 1 m. Table 1. Experimental Plot Characteristics. Abies r e l i g i o s a P l o t l o c a t i o n : km from Amecameca, by road Plot s i z e : width x length (n>) Elevation: mid-plot (m) Aspect: Slope: (°) Dominant f l o r a : 15 50 x 100 3240 WNW 23 Abies r e l i g i o s a  Pyrola secunda  Sibthorpia pichenchensis  Senecio tolucanus  Senecio a n g u l i f o l i u s  Achemilla procumbens  Vaccinium spp. Senecio barba-Johannis Pinus hartwegii Zacatonal 20.8 30.5 75 x 50 50 x 75 3520 3910 SW 23 15 Pinus hartweRii Festuca t o l u c e n s i s Festuca tolucensia  Festuca amplissima Plate 2 . The Abies Religiosa Zone. The modal pit p r o f i l e . Festuca tolucensis Festuca tolucensis and Festuca amplissima 79 4.0 METHODS The investigative research for this project can be divided into two sections. The f i r s t encompasses a l l samples individually and thus gives a measure of parameter v a r i a b i l i t y within a zone or horizon. The purpose of Part 1, or the s t a t i s t i c a l study, i s to give a general overview on the nature of the soi ls and the processes occurring. The second section, designated Part 2, examines the s o i l organic matter for phenolic acids indicative of vegetation. Composite samples were prepared for each of the s o i l zones to measure a population "mean". Population var iabi l i ty i s not examined; hence, s t a t i s t i c a l l y powerful statements cannot be made. Nevertheless, general trends can be explored. 4.1 Literature Review of Methods 4.1.1 Methods for Estimating the Content of "Amorphous" Aluminosilicate Clays in Soils Semiquantitative analysis of "amorphous" aluminosilicate clays in soi ls typically involves their so lubi l i ty behaviour in various extract-ants. Allophane and imogolite can be separated by their differ ing behaviour with respect to pH. Allophane disperses in both acid and base whereas imogolite flocculates when the pH i s raised to 10.5 -11 (Ishizuka and Black, 1977). Neither are very soluble in 2% sodium carbonate ^3200^), citrate-bicarbonate-dithionite (cbd) nor pyrophosphate solutions (Parf i t t , 1980, 1983; Yoshinaga and Aomine, 1962a,b). Selective dissolution analysis coupled with infrared spectroscopy 80 was a technique developed by Wada and Greenland (1970) to semiquantitat-ively determine the amount of "amorphous" aluminosilicate clays in volcanic ash s o i l s . Ultrasonics was used to disperse the s o i l , possibly preceded by a hydrogen peroxide ( H 2 O 2 ) treatment to remove s o i l organic matter. The use of chemical reagents to remove cementing agents such as iron oxides was avoided. The clay suspensions obtained were successively treated with cbd, 2% ^200^ and 0.5 N NaOH. The weight of residue obtained after each treatment was measured to estimate the percent weight loss. Elemental analysis of the soluble fraction determined the amount of S i , Al and Fe lost during the treatment. A dif ferent ia l IR spectrum was recorded by placing the KBr disks made from the clay residues before and after treat-ment in the sample and reference c e l l s , respectively. Cbd extracts iron oxides with some co-extracted aluminium and humus complexes. 2% ^200^ dissolves "allophane-like" consituents and weakly dissolves opaline s i l i c a . Allophane-like constituents have not been isolated, but they appear to be aluminium-rich with 5102^120^ molar ratios between 0.2 and 1.4. Their IR features are similar to allophane, but the major Si(Al)0 absorption band i s sharper and occurs at a lower frequency due to a higher Al content (940-960 cm ^). The opaline, s i l i c a Si-0 band occurs at a higher frequency (1070 cm )^ with a shoulder at 1200 cm-"'". The 0.5 N NaOH treatment dissolves allophane, imogolite and opaline s i l i c a . Recall , that during isolat ion of allophane and imogolite, imogolite flocculates when the pH is raised to 10.5 - 11. This contra-dictory behaviour may stem from differences i s a l k a l i concentration; 0.5 N NaOH versus a pH of 10.5 to 11. The main absorption band shifts to 81 a higher frequency as the amount of Si increases from imogolite (925-935 cm )^ to allophane (940-1040 cm )^ to opaline s i l i c a (1070 cm Imogolite i s further differentiated by a bimodal Si(Al)0 absorption band, 980-1010 and 925-935 cm An additional peak at 348 cm * has also been used for imogolite identif icat ion by Farmer (Wada, 1980). To study humus complexes in Andosols, Wada and Higashi (1976) modified the original extraction sequence to include a 0.1 M pyrophosphate extraction at the beginning. From their results they concluded that the humus formed f i r s t in the A horizon had a very low complexing a b i l i t y for Al and Fe and was l i t t l e dissolved by pyrophosphate. With time, the humus evolved into forms capable of complexing the released Al and Fe from the ash and were dissolved by pyrophosphate. In mature, old, possibly buried horizons the reaction of humus with allophane and hydrous oxides made the humus complexes once again less soluble in pyrophosphate. Parf i t t (1983) developed a very simple method for estimating the content of allophane and other inorganic constituents in soi ls by measuring cbd, sodium pyrophosphate and acid oxalate extractable Fe, Al and S i . Allophane was determined from the acid oxalate extractable Si content. The Si content of natural s o i l allophanes was close to 14%; therefore, dividing the acid oxalate extractable Si by 0.14 gave the % allophane content of the s o i l . Humus-Al,Fe complexes and crystal l ine iron oxides were also determined using this procedure (see Section 5.1.2.2). 4.1.2 Extraction and Fractionation of S o i l Humus The study of s o i l humus involves the separation of the humus from the non-humus and mineral constituents of the s o i l . In the past, non-isolative 82 methods such as proximate analysis were used to characterize the s o i l organic matter. However, this was of limited and uncertain value, especially in well humified mineral s o i l s . Quantitative extraction of humic substances from the s o i l i s a d i f f i c u l t procedure, since the humus is closely associated with the mineral fraction including crystal l ine clay minerals, sesquioxides and b i - and tr i -valent cations. Consequently, any extraction procedure w i l l not remove a l l of the s o i l humus and tends to extract certain humus pools. The separation of humified material from non-humified or par t ia l ly humified material and microbial biomass is also less than complete due to the similar solubi l i ty of certain components in many reagents. Several factors must be taken into consideration when planning an extraction scheme. A primary goal i s to extract unaltered material in high enough yields to adequately represent the s o i l humus. Other consid-erations in selecting an extractant include the nature of the s o i l and the necessity for pretreatments. For example, soi ls high in exchangeable calcium or carbonates may require an i n i t i a l acid extraction to improve subsequent extraction in a l k a l i . Soils high in sesquioxides or humus-Al, Fe complexes require the presence of a complexing agent such as pyrophos-phate. Volcanic ash soi ls containing humus-allophane complexes are e f f i c i e n t l y extracted only with sodium hydroxide (Duchaufour, 1977). A further consideration i s the nature of subsequent analyses, and possible contamination from the extractant. For example, contamination from pyrophosphate or sulphuric acid precludes analysis for phosphorus and sulphur in the extracts. The c lass ical s o i l organic matter extraction procedure uses a l k a l i 83 (Stevenson, 1982). Generally, 0.1 to 0.5 N sodium hydroxide (NaOH) i s used, the higher concentration extracting less humus but of lower ash content (Sequi et a l . , 1975). Yields range from 30 to 90 %. Both hot and cold extractions have been used with increased extraction efficiency at elevated temperatures, but less mature, more aliphatic humic substances are obtained. Furthermore, there i s an increased risk of alkaline hydrol-ysis and oxidation at elevated temperatures. Pyrophosphate, a complexing agent, has been widely used both alone and in combination with NaOH. Pyrophosphate extracts a more oxidized humus of lower average molecular weight, and represents a more "mature" humus pool (Swift et a l . , 1970; Lowe and Godkin, 1975; Tan, 1978; Butler and Ladd, 1969). Extraction with hot 0.1 M sodium hydroxide followed by cold sodium pyrophosphate (pH 8) i s used by Kumada et a l . (1967) to obtain information on two humic and fulvic acid fractions differ ing in maturity. When combined with a l k a l i , sodium pyrophosphate provides an eff ic ient overall extractant for a wide range of soi ls without the need for an acid pretreatment (Lowe, 1980). However, in humus isolat ion studies pyrophosphate i s generally avoided due to the d i f f i c u l t y in removing i t from the extracts. A further variation on the combined NaOH-pyrophosphate extraction scheme was developed by Anderson et a l . (1974a). In this procedure a weakly-humified, high molecular weight, largely aliphatic humic acid fraction (HA-B) was obtained by sonification in water of the NaOH-pyro-phosphate extraction residue. This humus pool was thought to be associated with the clay fraction and provided a potentially labi le form of plant nutrients (Anderson, 1979). , Humus extraction i s generally followed by fractionation into humic 84 acids and fulvic acids. These fractions are operationally defined on the basis of s o l u b i l i t y . The hurain fraction i s the non-extractable organic matter and contains microbial biomass, non-humified plant remains, sequest-ered organic matter and humus strongly bound to the mineral fraction of the s o i l . The humic acid fraction (HA) is soluble in alkaline solutions but precipitates upon a c i d i f i c a t i o n . The fulvic acid fraction (FA) is soluble in both acid and base. Several methods have been presented in the literature for the further separation of the above fractions. The more elaborate schemes tend to lose sight of their objectives in the multitude of fractions obtained. Simpler schemes have been found to be the most informative. The HA fraction in earlier studies, especially in Europe, was frequently fractionated using electrolytes into gray and brown humic acids, the former flocculating due to their association with the fine clay fract ion. Alcohol was often used to separate an alcohol-soluble hymatomelanic acid fraction (Stevenson, 1982). As previously mentioned, Anderson et a l . (1974) obtained the HA-B fraction by sonif icat ion. The FA fraction has been separated by adsorption onto activated charcoal (Forsyth's method (Stevenson, 1982)) or onto polyvinylpyrrolidone (PVP) (Lowe, 1975). PVP has been used in plant biochemistry to isolate phenols from plant extracts (Anderson and Sowers, 1968). The separation i s based on the formation of hydrogen bonds between the phenolic OH group and the carbonyl group of the insoluble pyrrolidone polymer. Bond formation occurs only at acid pH, the pH of maximum retention depending on the nature of the phenol (Anderson and Sowers, 1968). Disruption of the hydrogen bond with base allows the phenols to be eluted from the PVP. 85 Lowe (1975) used this technique to separate the FA fraction into a nearly colourless, non-adsorbed polysaccharide-rich fraction C and a strongly coloured, adsorbed polyphenol-rich fraction A obtained by elution of the PVP with dilute base. Recovery of fraction A was very good (>91%) with the lowest recoveries occurring at high FA concentration (Lowe, 1975). Some coloured material remained on the PVP but this was negligible. Several experimental factors have been shown to influence the amount of extracted humus, and i t s separation into HA and FA. The soil/extractant ratio influenced both the amount of extracted humus and the Ch/Cf ratio (carbon in the HA fraction/carbon in the FA fraction) . A high ratio reduced extraction efficiency, but also increased the amount of HA relative to FA (Schuppli and McKeague, 1984). Kumada et a l . (1967) avoided this problem by adhering to a defined ratio of organic C (g) to extractant volume (ml) of < 1:300 w/v. The centrifugal force used in separating the i n i t i a l humus extract from the s o i l residue, and for separating the HA precipitate from the FA supernatant also influenced the Ch/Cf ra t io . In the f i r s t instance, lower speeds increased the HA fraction resulting in higher Ch/Cf ratios (Lowe, 1980). In the lat ter , higher speeds had l i t t l e effect on HA but decreased FA, increasing the Ch/Cf ratio (Schuppli and McKeague, 1984; Schnitzer et a l . , 1981). In the li terature centrifuge speeds of 5000 to 6000 rpm were standard (Lowe, 1980; Schnitzer et a l . , 1981). The pH of HA-FA separation also influences the Ch/Cf ra t io . The pH of KA precipitation should be <1 to minimize the effects of metals (Sequi et a l . , 1975a). However, pH's of 1 to 2 have been commonly used with l i t t l e 86 adverse effects (Lowe, 1980; Anderson, 1979; Schnitzer et a l . , 1981; Schuppli and McKeague, 1984). Precipitation of HA at pH's > 2 i s not recommended. The dependence of the Ch/Cf ratio on the experimental conditions emphasizes the necessity to standardize the humus extraction and fractionation procedure i f different soi ls are to be compared. 4.1.3 Humus Degradation Methods The chemical degradation of s o i l humic substances was introduced in Section 2.6. Oxidative, reductive and hydrolytic procedures have been widely used, the method being dependent on the study objective - structural or genetic. Structural studies on s o i l humic compounds have favoured oxidative procedures ranging in severity from mild persulphate (Martin and Gonzalez-Vila, 1984) and alkaline cupric oxide (Dormaar, 1979) oxidations to the more drastic alkaline potassium permanganate and ni t ro -benzene oxidations (Schnitzer and Khan, 1972; Ogner, 1973; Wildung et a l . , 1970; Maximov et a l . , 1977; Morrison, 1958, 1963). A severe limitation to these more drastic procedures was the deep degradation of the humic polymer with the production of units that bore l i t t l e resemblance to the starting material (Maximov et a l . , 1977). Further limitations included: 1. An increased risk of autooxidation reactions and the formation of ar t i fac ts under alkaline conditions. 2. Pre-methylation was essential to protect aromatic rings containing l a b i l e functional groups from cleavage (Maximovet a l . , 1977). 3. The production of considerable amounts of pentacarboxylic acids suggested degradation of the condensed ring system. 4. The harsh oxidative methods lacked select ivi ty in bond cleavage 87 making i t d i f f i c u l t to interpret reaction pathways. The milder degradative procedures were limited by low yields of identif iable compounds and an i n a b i l i t y to cleave covalent C-C bonds contained within the humic acid "core". Consequently, mild degradations were of l i t t l e use to structural studies unless they formed part of a degradation sequence. Nevertheless, the milder reductive and hydrolytic procedures proved invaluable to studies on the genetic origins of humic substances (Burges et a l . , 1964; Piper and Posner, 1972b, Tate and Goh, 1973; Tate, 1972; Tate and' Anderson, 1978). The sodium amalgam reductive cleavage of s o i l humic acids was int ro-duced by Burges et a l . (1964). This method stirred up much controversy in the soi ls l i terature as other researchers stressed the sensi t ivi ty of the reduction mixture to re-oxidation and the production of large amounts of aliphatic material (Mendez and Stevenson, 1966; Stevenson and Mendez, 1967; Schnitzer et a l . , 1973; Dormaar, 1969). Several investigators had l i t t l e success with the method, and low amounts of phenolic compounds were obtained (Stevenson and Mendez, 1967; Schnitzer et a l . , 1973; Dormaar, 1969). However, Martin and Haider (1969), Matsui and Kumada (1977a,b) and Piper and Posner (1972a,b) obtained good results when optimum conditions of amalgam to HA were used. Further discussion on the sodium amalgam reduction method was given in Section 2.6. Hydrolytic procedures varying in strength from mild acid or alkaline extractions (Katase, 1981a,b,c; Whitehead et a l . , 1972, 1983; Kuwatsuka and Shindo, 1973; Shindo and Kuwatsuka, 1975a,b, 1976) to moderately severe alkaline and acid hydroysis under reflux ( R i f f a l d i and Schnitzer, 88 1973; Neyroud and Schnitzer, 1975a,b; Tate and Anderson, 1978; Anderson et a l . , 1978) have been used to degrade humic substances or extract whole s o i l s . These procedures attacked the peripheral side chains of organic molecules cleaving relat ively weak linkages such as hydrogen bonds; ester, glycoside and peptide (amide) linkages; and, salt bridges. Approximately 1/3 to 1/2 of the s o i l organic matter i s dissolved represent-ing the more labi le humus components ( R i f f a l d i and Schnitzer, 1973). Hydrolysis with 6 N HC1 under reflux has been used to "clean-up" humic acid molecules by removing proteins, peptides, amino acids, sugars, uronic acids, phenols, metals, etc. Acid and alkaline hydrolysis w i l l not cleave covalent C-C bonds and generally not ether bonds, except under very vigorous conditions. An exception is the benzyl ether bond which is easily cleaved by acids. The reaction conditions during acid hydrolysis may lead to condensation and decarboxylation reactions ( R i f f a l d i and Schnitzer, 1973; Schnitzer, 1972). Katase (1981b) noted the loss of cinnamic acid derivatives via condensation reactions during acid hydrolysis. The risk of oxidative changes and the production of ar t i facts i s increased during alkaline hydrolysis. Furthermore, polymerization through phenol coupling reactions is favoured at alkaline pH. 4.1.4 Detection of Phenolic Compounds The analytical detection of phenolic compounds has centered upon chromatographic methods. Techniques ranging from crude paper and t h i n -layer chromatography (Burges et a l . , 1964; Piper and Posner, 1972a,b; Morrison, 1958, 1963) to sophisticated gas chromatography (Tate, 1972; 89 Anderson et a l . , 1978; Neyroud and Schnitzer, 1975a,b; Ogner, 1973; Kuwatsuka and Shindo, 1973; Katase, 1981a) were evident in the soi ls l i terature . Gas chromatographic methods s ignif icantly increased the r e l i a b i l i t y and speed of analysis as well as the detection l imits for phenolic compounds. Improved verif icat ion of compound identity was provided by the combination of gas chromatography with mass spectrometry. However, derivatization was essential to make the phenolic compounds v o l a t i l e . Furthermore, there was a size l imit to the molecules that could be analysed, and thermal degradation was a definite r i s k . Recent developments in l iquid chromatography column design produced columns able to withstand high pressure. This led to a new technique called high-performance l iquid chromatography (HPLC). Several advantages were evident in this technique. F i r s t , derivatization was not necessary. Secondly, thermal degradation was not a problem, and thirdly , there was no l imit to the size of molecules that could be chromatographed (Wulf and Nagel, 1976). HPLC also offered selec t ivi ty , high resolution, speed and sensit ivi ty (Wulf and Nagel, 1976; Charpentier and Cowles, 1981). Consequently, the use of this technique in the soi ls l i terature has escalated in recent years (Whitehead et a l . , 1982; Hartley and Buchan, 1979; Hanninen et a l . , 1981). 90 4.2 Part 1 - S ta t i s t i ca l Study 4.2.1 Sample Preparation The samples collected in Mexico were part ia l ly dried and vis ible roots, macrofauna, etc. were removed prior to packaging for export. A few days later at the University of Br i t i sh Columbia the samples were f u l l y a i r -dr ied and passed through a 2 mm sieve to remove small twigs, pumice, charcoal, etc. Greater than 90% of the s o i l material easily passed a 35 mesh (0.42 mm) sieve, the remainder was ground with a mortar and pestle. Subsamples were ground to 100 mesh (0.149 mm). The s o i l samples were stored in plastic containers or glass jars . The FH horizons were prepared by gently r o l l i n g the material on a 2 mm sieve, removing large fragments, twigs, charcoal, etc. Fungal mycelia and moss were removed from several Abies rel igiosa FH samples. The remaining material was ground with a mortar and pestle. 4.2.2 Routine Chemical Analyses The following analyses were carried out on 35 mesh s o i l samples. Results were reported on an oven-dry basis, except for hygroscopic moisture which was reported on an air-dry basis (Gardner, 1965). . Loss on ignit ion was done according to Hesse (1971) with a three hour ignit ion period at 450 °C. Total carbon was determined by dry combustion in a Leco Induction Furnace, Model 521 (Al l ison , 1965a). The pH was determined on the modal pits only, both in water and in 0.01 M C a C ^ . A 1:1 or 1:2 s o i l to water ratio was used for mineral horizons, which was increased to 1:5 for organic horizons. A Radiometer PHM62 Standard pH meter with a 91 calomel in glass combination electrode was used for the pH measurements. Soil samples ground to 100 mesh were used for the following analyses. Total nitrogen was determined by a semi-microkjeldahl procedure (Bremner, 1965). Extractable iron, aluminium and s i l i c o n were determined by the acid ammonium oxalate method at pH 3 (McKeague and Day, 1966) and by the sodium pyrophosphate method at pH 10 (Bascomb, 1968). Elements in the sodium pyrophosphate extracts were recorded shortly after extraction due to possible hydroxide formation. The citrate-bicarbonate-dithionite reduction method at pH 7.3 extracted an additional form of iron and aluminium (Mehra and Jackson, 1960). An atomic absorption spectroscopy unit measured the total i ron, aluminium and s i l i c o n in the extracts. A low temperature air-acetylene flame was used for iron and a high temp-erature nitrous oxide-acetylene flame for aluminium and s i l i c o n . Total sulphur was determined on the modal samples only using a Fisher Sulphur Analyser, Model 47. 4.2.2 Humus Fractionation Procedure and Fraction Analysis The humus fraction distribution was determined by a modified method of Lowe (1980) (Appendix 2). Duplicate extractions were required to isolate adequate amounts of the polyphenolic fraction A. The Ah^ and Ah^ horizon samples ground to 35 mesh from a l l three vegetation zones were analysed by this procedure. The ratio of organic carbon to extract-ant was kept below 1:300 (w/v) with a maximum of 20 g s o i l per 250 ml centrifuge bottle (Kumada et a l . , 1967). Polyvinylpyrrolidone (PVP) was used to separate the fulvic acid fraction into a polysaccharide-rich fraction (fraction C) and a poly-92 phenol-rich fraction (fraction A) (Lowe, 1975). Prior to use, the PVP was suspended in water, allowed to settle , and the fines were removed by decantation. This was repeated several times. The PVP was then washed in a Buchner funnel successively with 0.1 N NaOH, d i s t i l l e d water, 0.1 N H2S0^ and d i s t i l l e d water. After drying several hours a mortar and pestle was used to crush the PVP. The washed PVP was stored in a brown bottle for up to one week. The extract carbon contents were measured in duplicate by the Walkley-Black wet oxidation method (All ison, 1965b). Aliquots of 2 to 50 ml were evaporated to dryness on a steam bath, oxidized with 0.5 N K^C^O.^, and titrated with 0.25 N FeSO^^H^O to a greenish-brown end-point with ferroin indicator. The carbon content in several extracts was also measured on an Astro Solution Carbon Analyser, Model 1850 (Appendix 3). The percent carbon in the humus fractions were defined as follows 1. %Cf = % carbon in the fulvic acid fraction (FA). 2. %Ch = % carbon in the humic acid fraction (HA). 3. %Cc = % carbon in the polysaccharide-rich fraction C. 4. %Ca = % carbon in the polyphenol-rich fraction A = %Cf - %Cc. 5. %Ce = % extractable carbon =(%Ch + %Cf / Ct) x 100% Several analyses were made on the humic acid and fraction A extracts. These included total nitrogen, total sugars and optical density at X = 400 and 600 nm. The nitrogen and sugar contents were reported as ratios to total extract carbon and were determined in duplicate. Total nitrogen was determined by a modified semi-microkjeldahl method using a 15 ml aliquot of humic acid extract and a 25 ml aliquot of fraction A extract. 93 Digestion, d i s t i l l a t i o n and t i t ra t ion were as described by Bremner (1965). The hydrolyzable sugars in the humic acid and fraction A extracts, expressed as a glucose equivalent, were determined by hydrolysis with 1 N H^SO^ for 1 hour in an autoclave. Determination of the ppm sugar content in the hydrolysate was by a modified phenolsulphuric colorimetric method (Whistler and Wolfram, 1962). Absorbance was measured at A = 480 nm with a Bausch and Lomb Spectronic 20 using matched glass cuvettes. Glucose was used for the standard curve. A Model 550 Perkin-Elmer UV-Visible Spectrophotometer measured the absorbance of diluted extracts at X = 400 and 600 nm. Absorbance measure-ments at A = 600 nm were subject to less relative error than at 665 nm for solutions low in absorbance (Lowe and Kumada, 1984). Aliquots of 2 to 7 ml were diluted to 50 ml in a volumetric flask with 0.1 N NaOH adjusted to pH 12.00 1 0.02. Absorbances were measured within 2 hours of humic acid dissolution or elution of fraction A from PVP to avoid alkaline 1%C 1%C oxidation, and were reported on a 1% carbon basis ( E , n f ! , E , ° ). 9A A.3 Part 2 - Composite Study A.3.1 Composite Sample Preparation The results of Part 1 were used to prepare the composite samples. S ta t i s t i ca l analysis indicated that the two s o i l horizons, Ah^ and A l ^ , came from the same s o i l population in the Abies rel igiosa and Zacatonal zones, but were distinct populations in the Pinus hartwegii zone. Consequently, four composite samples were prepared; Abies re l ig iosa , Pinus hartwegii Ah^, Pinus hartwegii kh^ and Zacatonal. The composite samples were prepared from equal contributions of organic matter from each s o i l sample. The humic acids and fraction A were to be extracted; hence, the percent carbon in the humic acid fraction (%Ch) was used as a guide. Soi l samples that were very low in %Ch were neglected as they would lower the f i n a l organic matter concentration in the composite sample. Also omitted were samples extreme in more than three parameters measured in Part 1. In total six samples out of forty-eight were omitted, f ive from the Abies rel igiosa zone and one from the Pinus hartwegii zone. Sample weights were determined by %Ch. The composite samples were thoroughly mixed on a large sheet of brown paper by cornering, s p l i t t i n g , etc. and then stored in plastic bottles. A preliminary humus fractionation using 0.1 N NaOH and the method of Lowe (1980) determined the bulk i s o -lat ion s o i l extraction weights. Carbon contents in the humus fractions were determined by the Astro Solution Carbon Analyser. This data allowed estimates of bulk isolat ion recoveries. 95 4.3.2 Bulk Isolation Procedure Details of the bulk isolat ion procedure are given in Figure 21. The extractant used was 0.1 N NaOH with a ratio of s o i l to extractant of 1:10. The extraction was carried out under to avoid alkaline oxidation. Hydrochloric acid (HC1) was used instead of sulphuric acid (^SO^) for a l l acidifying steps due to the d i f f i c u l t y of removing sulphate from the extracts. Pyrophosphate was not included in the extractant for this same reason. The separation of the fulvic acid fraction into fraction A and fraction C was accomplished by a "column-PVP set-up" (Appendix 4). This procedure allowed large volumes of fulvic acid to be processed in a re la t -ively short time, and was more eff ic ient since the adsorbate was concent-rated into a small volume. Elution of the PVP with base desorbed a small volume of fraction A (< 500 ml) which was easily passed through a H + -exchange resin to remove cations. 4.3.3 D i a f i l t r a t i o n and Concentration of Extracts After preliminary t r i a l s , nominal molecular weight cut-offs of 10,000 daltons for humic acids and 1,000 daltons for fraction A were selected to remove non-polymeric materials from the extracts. I n i t i a l l y , i t was desired to study more than one molecular size range. However, very l i t t l e of the humic acid fraction from Pinus hartwegii Ah^ was retained above molecular weight 100,000, and i t was estimated that >70% f e l l between molecular weights 50,000 and 100,000. Prior to d i a f i l t r a t i o n the humic acid fraction was redissolved from an acid precipitate in dilute NaOH, and the pH was adjusted to 7 with HC1. The fraction A collected from the H+-exchange resin was also adjusted to 96 pH 7 with NaOH. Diaf i l t ra t ion and concentration were done in an Amicon Model TCF-10 N2~pressurized d i a f i l t r a t i o n c e l l using a Diaflow YM2 membrane (nominal molecular weight retention 1,000) and a Diaflow PM10 membrane (nominal molecular weight retention 10,000) (Figure 22). Fi l t rates were collected and analysed for total carbon on the Astro Solution Carbon Analyser. Humic acid and fraction A retentates were collected in salt (Na+) form. Estimates of retentate carbon were made by diluting 1 or 2 ml aliquots to 50 ml in a volumetric f lask. Carbon balances and recoveries were then calculated. The retentates were frozen and later freeze-dried. Fraction weights were recorded, and subsamples were pulverized using a mortar and pestle then stored in small v i a l s . The carbon balance was later revised after elemental analysis of the freeze-dried retentates. A.3.4 Humic Acid and Fraction A Analysis Elemental analysis of the ground freeze-dried samples was done by Canadian Microanalytical Service Limited. The ash contents were deter-mined by ignit ion at 200 °C for 1 hour followed by 2 hours at 450 °C and 3 hours at 650 °C. Vis ible inspection determined the ashing complete. Infrared spectra were recorded on a Perkin-Elmer 283 B Infrared Spectro-photometer with Infrared Data Station. Samples weighing 1.5 mg were thoroughly mixed with oven-dried KBr to a weight of 300 mg. The disk was pressed, evacuated and then immediately scanned against air in the refer-ence c e l l . A pure KBr reference disk was then made and scanned for moist-ure bands between 3300 - 3000 cm * and 1720 - 1500 cm * . These bands were negligible . 97 F i g u r e 21 . P r o c e d u r e f o r B u l k I s o l a t i o n o f Humic A c i d and F r a c t i o n A . Weigh 150 g co m p o s i t e sample i n t o 2.5 L b o t t l e . Add 1.5 L 0.1 N NaOH. D i s p l a c e a i r w i t h N2. Shake o v e r n i g h t . L e t s e t t l e . Decant or s i p h o n o f f a l k a l i n e e x t r a c t . R e - e x t r a c t r e s i d u e w i t h 0.75 L 0.1 N NaOH. N 2. Shake 1 h r . Le t s e t t l e . Decant a l k a l i n e e x t r a c t . D i s c a r d s o i l r e s i d u e . A l k a l i n e e x t r a c t A c i d i f y t o pH 1.5 w i t h 6 N HC1. Le t s e t t l e . S i p h o n o f f FA from HA p p t . HA Pour HA s l u r r y i n t o 250 ml c e n t r i f u g e b o t t l e s . C e n t r i f u g e a t 6000 rpm f o r 15 min. Decant FA co m b i n i n g w i t h p r e v i o u s FA. FA F i l t e r FA t h r o u g h Whatman #1. I s o l a t e f r a c t i o n A on "Column-PVP s e t - u p " (Appendix A ) . HA "Cl e a n - u p " D i s s o l v e HA i n t o s m a l l volume of 2N NaOH. D i v i d e among c e n t r i f u g e b o t t l e s . D i l u t e . C e n t r i f u g e a t 6000 rpm f o r 20 min. Decant HA. D i s c a r d s o l i d s . I D i l u t e HA w i t h d i s t i l l e d w a t e r . F i l t e r t h r o u g h Whatman #1, c h a n g i n g f i l t e r paper o f t e n . A c i d i f y t o pH 1.5. L e t s e t t l e o v e r n i g h t . S i p h o n o f f FA I f FA c o l o u r e d , t r e a t w i t h PVP. Repeat d i s s o l u t i o n and a c i d i f i c a t i o n o f HA u n t i l FA p a l e i n c o l o u r . F r a c t i o n A F r a c t i o n C d i s c a r d Pass f r a c t i o n A t h r o u g h H -exchange r e s i n . F r a c t i o n A-H ULTRAFILTRATION L a s t p r e c i p i t a t i o n , c e n t r i f u g e . Decant p a l e FA. D i s c a r d . S t o r e a c i d i f i e d HA p p t . i n r e f r i g e r a t o r u n t i l u l t r a f i l t r a t i o n . ULTRAFILTRATION 98 Figure 22. U l t r a f i l t r a t i o n Procedure for Isolation of Polymeric Humic Acid and Fraction A. HA D i s s o l v e HA p p t . i n NaOH. A d j u s t t o pH 7 w i t h HC1. I F i l t r a t e PM10 membrane MW c u t - o f f 10,000 d a l t o n s A c i d i f y t o pH 1.5. L e t s e t t l e . C e n t r i f u g e . HA < 10,000 FA R e t e n t a t e MW > 10,000 D i l u t e 1.00 ml a l i q u o t t o 50.00 ml. Measure %C. FREEZE-DRY RETENTATE HA D i s s o l v e i n 0.1 N NaOH. Make t o volume i n v o l u m e t r i c f l a s k . Measure XC on A s t r o S o l u t i o n Carbon A n a l y s e r . D i s c a r d HA < 10,000. T r e a t w i t h PVP, I s o l a t e f r a c t i o n A. H +-exchange r e s i n . Add t o p r e v i o u s l y i s o l a t e d f r a c t i o n A. F r a c t i o n A-H A d j u s t t o pH 7 w i t h NaOH. F i l t r a t e MW < 1,000 Make t o volume i n v o l u m e t r i c f l a s k s ( s e v e r a l l i t e r s ) . Measure XC on A s t r o S o l u t i o n Carbon A n a l y s e r . D i s c a r d f i l t r a t e . YM2 membrane MW c u t - o f f 1,000 d a l t o n s R e t e n t a t e MW > 1,000 D i l u t e 2.00 ml t o 50.00 ml i n v o l u m e t r i c f l a s k . Measure XC on A s t r o S o l u t i o n Carbon A n a l y s e r . FREEZE-DRY RETENTATE A 99 Samples weighing 0.5000 g humic acid and 0.3000 g fraction A were hydrolyzed in 6 N HC1 for 2 hours in a Model 750 Fisher S t e r i l i z e r set at 120 °C and 15 p s i . The yield of fraction A from the Zacatonal zone composite sample was quantitatively very low; hence, only 0.2000 g was hydrolyzed. The phenolic acids were recovered from the acid hydrolysate by a successive anhydrous ethyl ether/ 2% NaHCO^/ anhydrous ethyl ether extraction. Each extraction was done in t r i p l i c a t e (Kuwatsuka and Shindo, 1973a). This extraction sequence separated acidic phenols from neutral phenols, the former being isolated for analysis. After evapor-ation of the f i n a l ether extract the residue was quantitatively dissolved in 2.00 ml methanol (HPLC grade), pressed through a nucleopore f i l t e r into a small glass v i a l and capped t ight ly . Manipulations at this stage were rapid to avoid evaporation of the solvent. The methanolic solutions obtained were pale to medium yellow indicating the presence of v is ible l ight absorbing chromophores and phenolic oligomers in addition to simple phenolic acids. Details of the hydrolysis and extraction procedures are given in Figure 23. Detection of the phenolic acids was by high-performance l iquid chromatography (HPLC). A Spectra Physics HPLC System was used containing a SP 8700 Solvent Delivery System, SP 4100 Computing Integrator and a SP Variable Wavelength Detector. The phenolic acids were separated on a 25 cm RP-18 reverse phase column packed with a s i l i c a bonded non-polar 18 carbon chain stationary phase. A reverse phase column changes the elution order of sample components with the most polar compounds eluting f i r s t (Harvath, 1981). A Bioanalytical Systems LC-22 Temperature Control-ler and LC-23A Column Heating Compartment were used to maintain the column 100 Figure 23. Humus Fraction Hydrolysis and Extraction of Phenolic Acids for HPLC Detection. 0.5000 g HA (0.3000 g A ) H y d r o l y z e w i t h 7.00 ml 6N HC1 f o r 2 hours H y d r o l y s a t e I C e n t r i f u g e , 2500 rpm, 15 min. P r e c i p i t a t e dry and weigh S u p e r n a t a n t F i l t e r t h r o u g h pre-weighed Whatman #A2 f i l t e r paper i n t o 125 ml s e p a r a t o r y f u n n e l dry f i l t e r paper and weigh E x t r a c t 3x w i t h 50 ml anhyd. * e t h e r . 1 - s o l u b l e " 1 aq. phase h y d r o l y s i s p r o d u c t s d i s c a r d E x t r a c t 3x w i t h 2% NaHC0 3, pH 8 " a c i d i c " e t h e r - b i c a r b o n a t e s o l u b l e " p h e n o l i c a c i d s " e t h e r phase " n o n - a c i d i c phenols' A c i d i f y t o pH 1.5 w i t h HC1 E x t r a c t 3x w i t h anhyd. e t h e r aq. phase d i s c a r d e t h e r - b i c a r b o n a t e - e t h e r s o l u b l e " p h e n o l i c a c i d s " dry w i t h Na^O^ Ev a p o r a t e e t h e r P h e n o l i c a c i d r e s i d u e D i s s o l v e i n 2.00 ml HPLC grade MeOH F i l t e r t h rough n u c l e o p o r e . PHENOLIC ACIDS i n methanol HPLC anhydrous ethyl ether 101 temperature at 45 °C. A "fixed-loop" injection port allowed precise quantitization of injection volumes. A l l solvents were HPLC grade and passed through a nucleopore f i l t e r prior to use. Solvents were contin-ually degassed with helium by the solvent delivery system. The chromatographic conditions used are outlined in Table 2. Two gradient elution solvent systems were compared for both sample and standard peak resolution; acetonitri le - H^O - acetic acid (Charpentier and Cowles, 1981) and methanol - H 20 - acetic acid. The methanol - H^O -acetic acid system has been successively used by Hanninen et a l . (1981). A low concentration of acetic acid was included to buffer the pH and supress ionization of the carboxylic acid groups. The acetonitri le system gave a good standard chromatogram but the fast elution times of the compounds caused a crowded, less resolved sample spectrum. The v a n i l l i c acid peak often total ly obscured the syringic acid peak, especially when the latter was low in concentration. The methanol system significantly increased the component retention times leading to a better resolved sample spectrum but broader peaks. Complete resolution of the four phenolic acids (Table 2) under study was obtained, particularly syringic and v a n i l -l i c acids. Consequently, this mobile phase solvent system was chosen for data collection while the former system was used to verify peak identi ty . Further verif icat ion was done by observing the change in detector sensit-i v i t y at A = 260 nm for standard and sample chromatograms. The sample hydrolysates were found to contain v a n i l l i c acid in con-centrations greater by up to an order of magnitude compared to the other three phenolic acids; consequently, v a n i l l i c acid was determined separ-ately. A single point rather than a multilevel calibration was selected 102 based on previous findings of a linear response versus concentration curve passing through the origin for the range under investigation. Syringic, protocatechuic and p-hydroxybenzoic acids were determined together using a multilevel calibration curve. Protocatechuic acid was not linear over the range examined. Syringic acid was linear but did not pass through the or ig in . P-hyroxybenzoic acid could have been determined by single point cal ibration, but i t s concentration in the sample hydrol-ysates easily allowed i t s inclusion with the former two phenolic acids. Blank runs of both solvent systems were checked frequently for the absence of contaminating peaks. An example of a methanol gradient elution standard chromatogram is given in Figure 24. 103 Table 2. HPLC Chromatographic Conditions Detector s e n s i t i v i t y : 0.01 Wavelength : 280 nm Chart speed : 0.5 cm/min Attenuation : 8 or 16 Flow r a t e : 2 ml/min Column temperature : 45 °C C a l i b r a t i o n : e x t e r n a l standards; m u l t i l e v e l or s i n g l e point Standards : protocatechuic a c i d (3,4-dihydroxybenzoic a c i d ) , p-hydroxy-benzoic a c i d , v a n i l l i c a c i d (3-methoxy-4-hydroxybenzoic a c i d s y r i n g i c a c i d (3,4-dimethoxybenzoic a c i d ) Gradient e l u t i o n : methanol - H„0 - a c e t i c a c i d t (min) % A methanol % B 1% a c e t i c a c i d 0 11 89 5 11 89 8 14 86 14 14 86 30 11 89 * Composition change i s gradual not stepwise. Retention times of standards i n methanol gradient: RT (min) protocatechuic a c i d 4.95 p-hydroxybenzoic a c i d 8.41 v a n i l l i c a c i d 12.05 s y r i n g i c a c i d 16.05 * Retention times v a r i e d s l i g h t l y between d i f f e r e n t days but were very c o n s i s t a n t on a given day. R e c a l i b r a t i o n was necessary each day. 104 Figure 24. Representative Standard Chromatogram of p-Hydroxybenzoic Acid, Protocatechuic Acid and Syringic Acid by Methanol Gradient Elution. 0 10 20 30 min 105 5.0 RESULTS AND DISCUSSION The results and discussion is divided into Part 1 and Part 2. Part 1 i s further divided into two sections. The f i r s t explores trends in the modal pit prof i les . The second s t a t i s t i c a l l y evaluates several parameters measured on the Ah^ and Ah^ s o i l horizons and forms the basis for comp-osite sample preparation in Part 2. Included with section 2 i s the l i terature from the joint German-Mexican project by Giinter Miehlich (1980). The mineralogical data on the parent ashes presented in the above report i s used to verify a common parent material throughout the three vegetation zones. Part 2 examines the characteristics of the humic acid and poly-phenolic fu lv ic acid fraction (fraction A) obtained through bulk isolat ion of the composite samples. In particular , emphasis i s placed on the phen-ol i c acid hydrolysis products of the humic acid and fraction A as they relate to vegetation. 5.1 Part 1 - S t a t i s t i c a l Study 5.1.1 Chemical Analysis of the Modal Pit Profiles The chemical parameters measured for the modal pit profiles .are given in Tables 3 and A. Their depth distribution i s given in Figure 25. Single sample determinations were made for a l l parameters except total nitrogen (%Nt) which was the mean of duplicate determinations. Duplicates were included periodically to check for reproducibil i ty . The interpretat-ion of parameter trends within the modal pit profiles was very speculative due to only one profi le being examined, and several parameters displayed Table 3. Chemical Analysis of the Modal Pit Soi l Horizons from Iztaccihuatl Vegetation Zones. ZONE HORIZON PH 1 ( H 2 0 ) PH 1 (0.01 M C a C l 2 ) %HM % L I %Ct -oven-dry b a s i s -%Nt A b i e s r e l i g i o s a FH A h l Ah2 Ah 3 B IP pumice 6.5 6.3 6.8 6.8 6.6 6.3 5.5 6.2 6.05 0.80 0.42 0.33 0.55 1.50 32.3 3.4 1.8 1.4 2.1 4.4 16.8 1.7 0.70 0.57 0.90 1.9 0.103 0.046 0.031 0.049 0.111 P i n u s h a r t w e g i i FH Ah l Ah2 Ah3 Ah4 IP pumice 4.5 4.9 5.8 5.7 6.1 6.1 4.2 5.0 6.80 2.35 0.64 0.74 0.86 1.26 63.5 11.7 2.5 2.9 3.1 3.7 30.3 5.5 1.1 1.3 1.5 1.4 0.273 0.058 0.070 0.068 0.071 Z a c a t o n a l A h l Ah 2 Ah 3 Ah4 Ah 5 5.7 5.8 5.7 5.9 6.1 4.8 4.9 0.91 0.92 1.31 1.58 1.81 4.2 3.5 4.2 4.7 4.3 2.0 1.9 2.0 2.2 1.9 0.133 0.122 0.123 0.135 0.121 1. Mean o f d u p l i c a t e d e t e r m i n a t i o n s . 107 Figure 25. Distribution of Chemical Parameters Within the Modal Pit Prof i les . 108 109 quite wide 95% confidence intervals (Section 5.1.2). Hence, trends apparent in the modal pit profiles may not be characteristic of the zone in general. The s o i l pH in water was below neutrality for a l l zones and horizons indicating the absence of carbonates. A pH decrease ranging from 0.6 to 0.9 was observed when determined in 0.01 M C a C ^ ; hence, considerable hydrogen was present on the exchange complex. The highest pH was found in the Abies rel igiosa zone which approached neutrality in the middle of the p r o f i l e . The FH layer was not particularly acid, pH (H^O) = 6.5 , suggesting a high proportion of less humified organic matter or adequate biological ac t ivi ty which prevented the accumulation of organic acids. The high acidity typical of coniferous l i t t e r was not apparent. The high pH in the Ah^ horizon reflected minimal weathering of the profi le and a low content of well humified organic material with high exchange capacity. Although a considerable proportion of the exchange complex seemed to be dominated by H + , the near neutral pH reflected minimal leach-ing and base depletion despite heavy rains throughout several months of the year (Figure 20). Contributing factors included the young age and high porosity of the s o i l , reduced temperatures due to the alpine location, a dominance of sand-sized particles with low surface area, and evidence of overland flow which would dissipate much of the rain water. Significantly lower pH's were present in the Pinus hartwegii and Zacatonal zones, in particular the Pinus FH and Ah^ horizons. The Zacatonal modal pit varied less than half a pH unit with depth, matching the pH in the lower horizons of the Pinus hartwegii modal p i t . This suggested that the acidifying effect of the pine l i t t e r did not extend 110 into the Pinus Ah^ horizon. In the Zacatonal surface horizons the pH was up to one unit lower than in the corresponding horizon in the Abies zone. The pH was between 5.5 and 6.0, possibly reflecting active A l ^ + on the exchange s i tes . The f i r s t hydrolysis reaction for the aluminium ion (Al^ + ) has a pK of approximately 5.0. Increased levels of extract-able Al in the Zacatonal zone supported this view. However, while the levels of extractable Al increased signficantly with depth, an increase of ^ a pH unit was noted. Thus, the proportion of exchangeable A l ^ + may have decreased with depth as other amorphous forms became dominant. The levels of extractable Al in the Pinus hartwegii zone were s ignif icantly less than in the Zacatonal zone. Hence, the similar pH present in the Pinus Ah^ horizon reflected some influence of pine l i t t e r leachates, although not to the same extent as in Pinus Ah^. Simple aliphatic acids present in grass root exudates may have had some bearing on the pH's found in the Pinus hartwegii and Zacatonal zones compared to the Abies rel igiosa zone which lacked significant grass cover. The organic matter source possibly influenced the nature of the humus formed, particu-l a r l y i t s "degree of humification" and functional group content. However, as w i l l be later shown, humic acid formation was controlled by the region-a l climate, and there was no infrared or elemental evidence to support any differences in the nature of the humic acids found within the three zones. The percent hygroscopic moisture (%HM) gives an indication of the amount of adsorbed water in the air-dry state. Structural water i s not included as temperatures in excess of 150 °C are required for i t s removal. The amount of adsorbed water reflects the nature of the adsorbing surface I l l and the surface area accessible to the water molecules. Indirectly, %HM gives a statement on the "degree of transformation" and "amount" of s o i l co l lo ids , organic or mineral. The Abies and Pinus FH horizons had a high %HM due to their high organic matter content. A sudden drop was noted as the mineral s o i l was entered, which continued into the Ah^ horizons, especially in the Pinus modal p i t . There was l i t t l e further change with depth except for a modest increase in the IP pumice layers. The pumice layers, as w i l l be shown later , were s ignif icantly more weathered than the above s o i l horizons which would contribute to i t s surface adsorption capacity. In the Zacat-onal modal pit %HM increased steadily with depth from the kh^ horizon. A change in organic matter content was not responsible, but a similar depth pattern was observed for oxalate extractable Al and S i , and less so for Fe. Thus, %HM was influenced heavily by inorganic colloids at depth in the Zacatonal zone. The percent loss on ignit ion (%LI), total carbon (%Ct) and %Nt f o l -lowed similar trends within each modal pit due to their interrelationship. In the Abies modal pit a minimum was reached in the Ah^ horizon, which then increased to the IP pumice layer. In the Pinus modal pit a minimum was reached in the Ah^ horizon with a consistent, but s l ight ly higher level continuing down the p r o f i l e . In forested ecosystems the s o i l surface would be enriched with carbon in the form of a relat ively thick humus layer followed by a thin Ah horizon. Humus levels decreased s ignif icantly in the B and C horizons. The coniferous s o i l zones on Iztaccihuatl cont-ained relat ively thick Ah horizons with organic matter accumulations restricted primarily to the FH and Ah, horizons. The distribution of 112 carbon at depth in the Abies and Pinus modal pits was consistent with the distribution of roots within the profiles (Appendix 1). In the Abies Ah^ and Ah^ horizons few roots were present although rotting wood was evident. Coarse roots were plent i ful in the IP pumice layer reflect ing a zone of increased moisture which the deeper rooting species could exploit . Thus, the primary source of humus in the IP pumice layer was in si tu root decomposition. However, some i l l u v i a t i o n of organic matter possibly occurred during the rainy season and was intercepted by the more weathered lower horizons and pumice layer. The higher levels of pyro-phosphate extractable Al and Fe in the Ah^ horizon and IP pumice layer supported this conclusion. In the Pinus modal pit the amount of roots declined in the Ah^ horizon with very few roots extending to any depth in the p r o f i l e . A modest proportion of %Ct in the Abies rel igiosa and Pinus hartwegii zones was due to charcoal, possibly derived from vegetation at the time of volcanic eruption. In the Zacatonal modal pit %LI, %Ct and %Nt were relatively constant with depth reflect ing a uniform input of organic matter into the prof i le , primarily through in si tu decomposition. Extractable iron (%Fe), aluminium (%A1) and s i l i c o n (%Si) are given in Table 4. Three extractant types were used to characterize the forms of extractable elements. Acid ammonium oxalate (ox) at pH 3 extracts Al(Fe) from organic ligands, "amorphous" aluminium oxides and hydroxides and from aluminosilicates such as allophane and imogolite (Parf i t t , 1980, 1983). The extraction mechanism involves complex form-ation and not acid dissolution (McKeague and Day, 1966). Opaline s i l i c a and volcanic glasses are not affected by this treatment. Table 4. Extractable Iron (%Fe), Aluminium (%A1) and Sil icon (%Si) from Modal Pit Soi l Horizons. ZONE HORIZON Z F e ox Z A l o x % S i o X c bd % A 1 ^ c bd % F e p y Z A l p y A b i e s r e l i g i o s a All 1 0.19 0.21 0.057 0.28 0.16 0.13 0.13 Ah2 0.10 0.17 0.056 0.16 0.098 0.060 0.080 Ah 3 0.088 0.11 0.040 0.12 0.068 0.060 0.060 B 0.19 0.21 0.076 0.22 0.11 0.11 0.10 IP pumice 0.29 0.63 0.25 0.42 0.30 0.19 0.25 P i n u s h a r t w e g i i Ahl 0.17 0.29 0.029 0.29 0.26 0.16 0.25 Ah 2 0.11 0.27 0.081 0.17 0.18 0.065 0.17 Ah3 0.11 0.32 0.081 0.18 0.22 0.076 0.21 Ah4 0.15 0.37 0.12 0.21 0.22 0.11 0.21 IP pumice 0.25 0.74 0.34 0.35 0.30 0.096 0.22 Z a c a t o n a l Ahl 0.16 0.44 0.13 0.26 0.31 0.055 0.24 Ah 2 0.15 0.44 0.14 0.25 0.29 0.076 0.24 Ah3 0.17 0.56 0.18 0.27 0.32 0.11 0.29 Ah4 0.22 0.76 0.28 0.35 0.40 0.13 0.33 Ah5 0.24 0.87 0.33 0.38 0.41 0.11 0.30 oven-dry b a s i s 114 Citrate-bicarbonate-dithionite (cbd) i s primarily a reducing agent, but contains citrate to complex and retain the solubi l i ty of the released Fe and A l . Both crystal l ine and amorphous forms of Fe are attacked. Cbd extracts Fe in the fe r r i c state (Fe"^+) from crystal l ine iron oxides by reducing i t to the more soluble ferrous state (Fe^ + ) . Aluminium, primarily from humus complexes, i s usually co-extracted. Amorphous aluminosilicates are only s l ight ly attacked. Sodium pyrophosphate (py) i s a strong complexing agent for Fe and A l . The high extractant pH of 10 aids in the dispersion of organic molecules by increasing the net negative charge on the co l lo ids . The presence of sodium on the exchange sites increases the thickness of the diffuse double layer which lowers the electrostatic attraction between co l lo ids . Dispersion also increases the accessibi l i ty of the pyrophosphate ligand to the humus - Fe, Al complexes. The a b i l i t y of pyrophosphate to form a stronger complex with the Fe and Al favours their release from the humus. The distribution of organically complexed Fe and Al mimicked the distribution of %Ct in a l l three modal pit prof i les . There was no s i g n i f -icant difference in %Fe among the three modal pits except for accumulat-py ions in Abies Ah, and IP, and Pinus Ah, horizons. %A1 decreased from 1 1 py the Zacatonal zone through to the Abies zone. However, %A1 was except-p y ionally high in Pinus Ah^ due to a high %Ct and acid pH, and in Abies IP possibly due to i l l u v i a l organic matter, or the root material providing a source of complexing agents for the released Fe and A l . At depth in the profi les a relat ively constant ratio of %Fe + %A1 / %Ct = 0.20-0.23 py py was reached. This suggested that the "nature" of the humus formed at depth was similar among the three zones, emphasizing a common climate, 115 age and type of parent material. Higher ratios were present in the surface horizons due to the abundance of less decomposed organic matter with low complexing a b i l i t y . %Al ck (j and 7„hl were similar , both extracting the same pool of organically complexed A l . The highest levels of Al were extracted by ammonium oxalate. However, in the Abies and Pinus surface horizons much of this Al was organically complexed with l i t t l e "amorphous" Al being present. The amount of "amorphous" Al increased s ignif icant ly in the lower Ah^ horizons and in particular the IP pumice layers. In the Zacatonal zone s ignif icantly higher levels of ^ A 1 q x were present with "amorphous" forms becoming dominant at depth. In the Abies modal pit , organic forms of Fe were dominant over crystall ine or amorphous forms. A similar situation was apparent in the Pinus modal pit except ^Fe^ w a s less prevalent at depth. In the Zacatonal modal pit organic and inorganic forms of Fe were equal, particularly at depth in the p r o f i l e . The levels of extractable Fe were not s ignif icantly different among the surface horizons, but tended to establish a defined order of abundance in the middle of the prof i les ; Zacatonal > Pinus > Abies. These differences became less apparent at depth except for high %Fe in the Abies IP pumice layer. py Oxalate extractable s i l i c o n (%SiQx) was s ignif icantly greater in the Zacatonal modal pit exclusive of the Ah,, horizon which approached the Abies and Pinus IP pumice layers. In general, low values were present in the Abies and Pinus modal pits u n t i l the IP pumice layer was reached. The high mobility of s i l i c i c acid favoured some movement from the upper profi le into the lower profi le and ground water. However, the highly 116 weathered appearance of the IP pumice layers also supported in s i tu weathering as a dominant process. In the Zacatonal zone 7oS±^ increased steadily with depth. The behaviour of %Si closely followed that of ox J %A1 q x suggesting a relationship between the two elements. However, the very low extractable levels of %Si and %A1 made i t d i f f i c u l t to assign their identity to "amorphous" aluminosilicates such as allophane. In conclusion, the young volcanic ash soi ls of Iztaccihuatl showed limited accumulation of humus at this stage, in s i tu processes being dominant. Some evidence of i l l u v i a l organic matter was noted in the Abies rel igiosa and Pinus hartwegii zones, possibly low molecular weight fulvic acid polyphenols produced in the humus layers. Evidence of in s i tu root decomposition was apparent in the Abies IP pumice layer. The levels of extractable Al and Si seemed to be s ignif icantly influenced by elev-ation; increasing from the Abies to the Zacatonal zone. Thus, a climate or vegetation effect was present which influenced the soi ls moisture status, and hence, the degree of mineral weathering. Although maximum precipitation occurred near the boundary of the Abies and Pinus zones, the "effective precipitation" may have been greater in the Zacatonal zone due to reduced temperatures at the higher elevation (Cortes and Franzmeier, 1972). Furthermore, increased radiation intensity during the spring months (Figure 20) and the lack of an insulating tree canopy in the Zacatonal zone would increase the s o i l temperature and promote weathering of the ash. The degree of weathering also increased with depth in the p r o f i l e . The loss of moisture through evapotranspiration would be confined largely to the surface horizons, maintaining moist, but freely drained conditions at depth. In general, the profiles had been l i t t l e 117 weathered, and the low content of extractable elements precluded any defini t ive statements on the nature of the amorphous minerals present. 5.1.2 S ta t i s t i ca l Analysis of Ah^ and kh^ Soi l Horizons from Iztaccihuatl Vegetation Zones The results from the s t a t i s t i c a l study, Part 1, w i l l be presented in the following sections. For each measured parameter a total of 48 samples divided among 6 groups were analyzed. The individual sample data used in the s t a t i s t i c a l analysis i s given in Appendix 5. As indicated in Section 4.2, analysis of means by ANOVA was subject to error due to the i n a b i l i t y to confirm a normal dis t r ibut ion. Consequently, median values, 95% confidence intervals and the results from non-parametric s t a t i s t i c a l analysis were recorded in the data tables. For comparative purposes, corresponding tables containing mean data and the results from ANOVA are presented in Appendix 6. Footnotes were used to indicate where the homo-geneity of variance was not met. Non-parametric s ta t i s t i cs were used to evaluate differences among zones and horizons. The K-sample comparison used the Kruskal-Wallis and median tests, the latter being more appropriate when several t ies were present. The two null hypotheses tested were: H0:1 The three zones were from a common population. HO:2 The two s o i l horizons were from a common population. A confidence level of 95% (*) or 99% (**) was chosen for rejection of H0:1. HO:2 was rejected at the 95% confidence l e v e l . Ranking of sample medians, the value below which 50% of the data f e l l , was by two-sample comparison (95% confidence level) using the Mann-Whitney U and median 118 tests. S t a t i s t i c a l l y , this ranking procedure must be viewed with some caution since the degrees of freedom has changed from the original K-sample rejection. Nevertheless, general conclusions can be drawn. The presentation of the median data depended on the outcome of the two-sample horizon comparison. For a l l parameters measured the Ah^ and Ah^ horizons were not s ignif icantly different at the 95% confidence level in the Abies rel igiosa and Zacatonal zones, and HO:2 was accepted. However, horizon differences were noted for several parameters in the Pinus hartwegii zone. Therefore, when H0:2 was rejected the Ah^ and Ah^ s o i l horizons from a l l three zones were analyzed separately despite the two s o i l horizons forming a common population in the Abies and Zacatonal zones. When HO:2 was accepted the Ah^ and Ah^ horizons in each zone were combined and analyzed as one sample for zonal differences. The text w i l l draw attention to the data presentation used in each section. A correlation matrix was determined for a l l variables using 48 samples and 46 degrees of freedom (Appendix 7). A signficant correlation at the 95% confidence level had a correlation coefficient r > 0.29, and > 0.37 at the 99% confidence l e v e l . Verif icat ion of l ineari ty was done by plotting each significant correlation. In several instances significant correlations were highly scattered about the regression l ine making a true linear relationship doubtful. A good linear trend was found with r > 0.7; 49% of the variation in one variable could be accounted for by i t s regression on the other variable. A "true" linear relationship was apparent with r ' s > 0.9. The spread of data points along the axes may not have been wide enough to give a significant correlation, and a wider range of soi ls may be required under those circumstances. On the other 119 hand, the narrow data ranges may have caused an insignificant correlation to become s ignif icant . This emphasized the need for data plots to verify the val idi ty of regression coeff ic ients . 5.1.2.1 Hygroscopic Moisture (%HM), Loss on Ignition (%LI), Total Carbon (%Ct) and Total Nitrogen (%Nt) The median values for the above parameters are given in Table 5. Two-sample comparison of the Abies and Zacatonal Ah^ and Ah^ horizons indicated that there was no significant difference between the two horizons. However, the Ah^ and Ah^ horizons were s ignif icantly different in %HM, %LI, %Ct and %Nt in the Pinus hartwegii zone. Consequently, the horizon data was presented separately for a l l three zones. The C/N ratio was not s ignif icantly different between Pinus Ah^ and Ah^; there-fore, they were combined for analysis. %HM, %LI and %Ct were s ignif icant ly different among the three zones in the Ah^ horizons but not the Ah^ horizons. Pinus Ah^ contributed to this outcome through a high organic matter content. %Ct was s igni f -icant at the 95% confidence l e v e l ; however, two-sample analysis failed to distinguish rank among the three zones emphasizing the ambiguity of this ranking procedure. %HM was not s ignif icantly correlated with oxalate extractable Fe, Al or S i , but was correlated with citrate-bicarbonate-dithionite and pyrophosphate extractable forms. Thus, the amorphous materials in the Ah^ and Ah^ horizons were not significant enough to contribute to the pool of "active surfaces", leaving organic matter as the key source of %HM. A similar conclusion was reached in Section 5.1.1 . %Nt was not significant in the Ah, horizons. However, the same Table 5. Median Values and 95% Confidence Intervals for Hygroscopic Moisture (%HM), Loss on Ignition (%LI), Total Carbon (%Ct) and Total Nitrogen (%Nt) from Ah and Ah„ Soi l 1 Horizons. ZONE %HM % L I ZCt %Nt C/N 1 oven-dry b a s i s Ah, h o r i z o n s , n=8 Abies r e l i g i o s a 0 . 8 1 a b 3.4 a 1.7 a 0.103 16.2 b 0.63-1.93 2.8-9.3 1.2-4.8 0.080-0.238 15.2-20.2 P i n u s h a r t w e g i i 1.26 b 6.9 b 3 . 1 3 0.167 18.7 C 1.18-2.41 5.7-11.7 2.7-5.5 0.149-0.273 17.8-20.0 Z a c a t o n a l 0.91 a 4 . 2 a b 2.0 a 0.133 15.0 a 0.78-1.33 4.0-6.3 1.8-3.2 0.119-0.209 14.4-15.2 A t ^ h o r i z o n s , n=8 %HM % L I % C t % Nt* A b i e s r e l i g i o s a 0.59 2.5 1.1 0.061 3 0.42-1.50 1.6-6.7 0.68-3.3 0.40-0.222 Pinu s h a r t w e g i i 0.80 3.7 1.7 0.090 3 0.67-1.52 3.0-7.2 1.4-3.3 0.071-0.159 Z a c a t o n a l 0.93 4.3 1.9 0.133 a 0.70-1.35 3.5-6.8 1.8-3.3 0.117-0.215 1. S i g n i f i c a n c e l e v e l : 95%= *, 99%= **. A n a l y s i s by k-saraple c o m p a r i s o n . 2. The Ah and Ah h o r i z o n s were combined f o r C/N, n=16. 121 ambiguity as for %Ct was apparent during analysis of the Ah^ horizons. The 95% confidence intervals were consistently high for a l l three zones reflect ing the variable input and incorporation of N into s o i l humus. This may have contributed to the fai lure of the ranking procedure. Nevertheless, the levels of %Nt in the Ah^ horizons seemed to increase from the Abies to the Zacatonal zone. The carbon to nitrogen ratio (C/N) in the combined Ah^ and Ah 2 horizons was s ignif icantly different among the three zones. The highest C/N ratio was found in the Pinus hartwegii zone possibly due to a high input of less easily degraded polyphenolic material poor in N. Slower decomposition rates from the more acidic conditions could also be responsible. Berg et a l . (1980) claimed the l ignin level to be more important than the C/N ratio to l i t t e r decomposition rates. The C/N ratio of the Abies zone was also high indicating a similar situation, although a low pH was not responsible. The lowest C/N ratio was found in the Zacatonal zone. The presence of significant amounts of charcoal in the Abies rel igiosa and Pinus hartwegii zones may have distorted the "true" C/N ra t io . However, charcoal was absent from the Zacatonal zone which had a C/N ratio of 15. Well humified materials typically have C/N ratios close to 10, particularly in mature grassland ecosystems. This suggested that the humus of these soi ls was relat ively immature. The 95% confidence intervals of the above parameters were greatest in the Abies rel igiosa and Pinus hartwegii zones and least in the Zacatonal zone. Inputs of organic substrates in the former zones would be highly varied in nature and amount, encompassing l ignins , tannins, carbohydrates, proteins, l i p i d s , etc. The spatial v a r i a b i l i t y would be 122 great with inputs from both the humus layer and from in situ root decomposition. In the Zacatonal zone inputs would be more homogeneous, primarily through in si tu root decomposition and root exudates, with lesser amounts of highly resistant materials such as l ignins and tannins, and greater amounts of readily decomposable substrates such as carbo-hydrates and proteins. 5.1.2.2 Extractable Iron (%Fe), Aluminium (%A1) and Sil icon (%Si) The extractable %Fe, %A1 and %Si were extremely low, < 0.5%, and caution was required in data interpretation (Tables 6 and 7). However, the 95% confidence intervals about the medians were narrow allowing rel iable comparisons among zones. Extractable %A1 was not significantly different between Ah^ and kh^ horizons at the 95% confidence l e v e l ; therefore, they were combined for analysis. Mean and median values were very close suggesting l i t t l e deviation from normality (Appendix 6). However, the homogeneity of variance was not sat isfied for % S i Q x . Extractable %Fe was s ignif icant ly different between Pinus Ah^ and Ah^ horizons; consequently, they were analyzed separately. Extractable %A1 and %Si increased with elevation from the Abies to the Zacatonal zone confirming the relationship noted in Section 5.1.1 for the modal pit prof i les . A plot of %Si and %A1 showed a linear trend r r r ox ox with r = 0.73. The similari ty in extracted amount between 7akl^ anc* %A1^^^ suggested that they were extracting the same humus-Al pool. The correlation coefficient between %A1 and %A1 , , was r = 0.96, and a py cbd plot of the data verif ied a linear relationship. The highest levels of %A1 were extracted by ammonium oxalate; however, 2/3 of this Al was in 123 Table 6. Median Values and 95% Confidence Intervals for Acid Ammonium Oxalate (ox), Pyrophosphate (py) and Citrate-Bicarbonate-Dithionite (cbd) Extractable Aluminium (%A1) and Si l icon (%Si) from Combined Ah. and Ah„ Soil Horizons.^ ZONE %A1 """" %A1 %A1 ™ %Si ox py cbd ox -oven-dry basis-Ah. + Ah„ horizons, n=16 Abies religiosa 0.21 3 0.14a 0.17 a 0.0453 0.18-0.24 0.12-0.18 0.11-0.21 0.040-0.057 Pinus hartwegii 0.32b 0.25b 0.26b 0.061b 0.30-0.33 0.23-0.27 0.25-0.29 0.049-0.081 Zacatonal 0 . 3 8 ° 0.24b 0 . 2 9 ° 0.12C 0.36-0.44 0.22-0.28 0.27-0.34 0.11-0.13 1. Significance l e v e l : 95% = * , 99% = * * . Analysis by k-sample comparison. 124 Table 7. Median Values and 95% Confidence Intervals for Acid Ammonium Oxalate (ox), Pyrophosphate (py) and Citrate-Bicarbonate-Dithionite (cbd) Extractable Iron from Ah and Ah„ Soi l Horizons. ZONE %Fe %Fe """"" %Fe ox py oven-dry basis Ahj horizons, n=8 Abies religiosa 0.15 0.10 a b 0.24 0.15-0.20 0.081-0.14 0.20-0.30 Pinus hartwegii 0.14 0.12b 0.24 0.14-0.17 0.11-0.16 0.23-0.29 Zacatonal 0.15 0.0583 0.24 0.13-0.16 0.050-0.096 0.21-0.27 Ah^ horizons, n=8 •» %Fe ox %Fe py ^Fe L J cbd Abies religiosa 0.15b 0.081 0.18 0.14-0.19 0.050-0.14 0.15-0.24 Pinus hartwegii 0.13 a 0.091 0.20 0.13-0.15 0.086-0.13 0.20-0 .'24 Zacatonal 0.14 a b 0.076 0.23 0.13-0.17 0.060-0.11 0.21-0.27 1. Significance l e v e l : 95% = * , 99% = * * . Analysis by k-sample comparison. 125 humus-Al complexes. A plot of %A1 and %A1 confirmed a linear r r ox py relationship with r = 0.86. The correlation between %A1 and %A1 * ox cbd was even higher, r = 0.93, indicating some extraction of amorphous forms of Al by cbd. In general, %Fe was not s ignif icantly different among the three zones. However, %Fe was s ignif icantly greater in Pinus Ah^ than in Zacatonal Ah^. This possibly stemmed from the more acidic conditions and higher levels of organic complexing agents in the Pinus Ah^ horizons. This condition was not apparent in the A l ^ horizons suggesting a surface phenomenon in the Pinus zone. In the Ah~ horizons, Abies %Fe was v 2 ox significantly greater than Pinus H F e ^ . The narrow 95% confidence interval of Pinus Ah^ possibly contributed to this outcome. The Abies and Zacatonal zones were not s ignif icantly different in %Fe. The three extractable forms of Fe were not highly correlated, the most significant correlation being between %Fe and %Fe , , , r = 0.65. py cbd Table 8 outlines the relative dissolution of materials containing Fe, Al and Si by sodium pyrophosphate, acid ammonium oxalate and c i t rate-bicarbonate-dithionite. The method of Parf i t t (1983) is used to estimate the %Fe, %A1 and %Si in various forms. The results are speculative due to the low amounts of extractable elements present, and are recorded in Table 9. Examination of the data in Table 9 indicated that very l i t t l e Al was present in forms other than humus-Al complexes. The content of allophane was < 1% and decreased from the Zacatonal zone to the Abies zone. The distribution of Fe among the three forms was d i f f i c u l t to assess, and no patterns were evident. In conclusion, the above analysis 126 Table 8. Relative Dissolution of Materials Containing Iron, Aluminium and Sil icon by Pyrophosphate, Acid Oxalate and Citrate-Bicarbonate-Dithionite (Parf i t t , 1980, 1983). FORM PYROPHOSPHATE ACID OXALATE CITRATE-BICARBONATE-DITHIONITE Humus A l , Fe 100 100 100 Allophane (Al , Si) 5 100 30 Imogolite (Al , Si) 5 100 30 "Amorphous" Fe oxide 0 100 100 Crystalline Fe oxide 0 0 100 Opaline Si 0 0 0 Crystalline Si 0 0 0 Table 9. Distribution of Iron, Aluminium and Silicon Containing Materials in the Recent Volcanic Ash Soils of Iztaccihuatl. ZONE HUMUS Al ALLOPHANE A l a ALLOPHANE S i b ALLOPHANE A l / S i %ALLOPHANE %A1 Al - Al Si a/b x 28/27 (Si /14)100% py ox py ox ox Ah^ + Ah^ horizons Abies religiosa 0.14 0.07 0.045 1.6 0.32 Pinus hartwegii 0.25 0.07 0.061 1.2 0.44 Zacatonal 0.24 0.14 0.12 1.2 0.86 ZONE HUMUS Fe "AMORPHOUS Fe" "CRYSTALLINE Fe" Ahj Ah 2 Ah^ Ah 2 Ah^ Ah 2 Abies religiosa Pinus hartwegii Zacatonal 0.10 0.081 0.12 0.091 0.058 0.076 0.050 0.069 0.020 0.039 0.092 0.064 0.090 0.030 0.10 0.070 0.090 0.090 128 supported the hypothesis of Wada and Higashi (1976) that in the early stages of s o i l formation the Al and Fe released by weathering of the ash existed largely as A l , Fe - humus complexes. The data also confirmed the recent age and limited weathering of the ash. 5.1.2.3 Review of the Relevant Data from the German-Mexican Project on the Soils of the Sierra Nevada de Mexico To rel iably assess soil-vegetation relationships the other factors of s o i l formation; parent material, climate, age and topography, must be held relat ively constant. The data from Miehlich (1980) was therefore examined to verify the nature of the parent material throughout the three vegetation zones. As noted in Section 3.0, the Mesandine Andosol s o i l unit covered the largest portion of Popocatepetl and Iztaccihuatl and spanned the elevational ranges of the three vegetation zones. These soi ls were poorly developed and occurred exclusively in the most recent 1C ash of Popocatepetl. The stratigraphic s o i l unit mapped for the area was diagrammed in Figure 18. Based on this unit there were two possible parent materials for s o i l formation in the three vegetation zones; the 1C and 2C ashes of Popocatepetl. The most recent ash of Popocatepetl, 1C, was the most l i k e l y parent material; however, i t was necessary to verify that the 2C ash was not being exposed at the land surface in the study area. The analytical data from Miehlich (1980) clearly separated the soi ls derived from the two parent materials. In particular, the oxalate and cbd extractable Fe, Al and Si were s ignif icantly greater in the older 2C ash compared to the more recent 1C ash. Thus, the data from Miehlich and comparative elemental 129 data from the three vegetation zones were presented in Table lOe. Clearly, the extractable elements in the soi ls of the study area were far removed from those of the 2C ash, and f e l l well within the ranges reported for the 1C ash. Consequently, the 1C ash of Popocatepetl was the only possible parent material for s o i l formation in the study area. For further analytical data Profi le 04 from Miehlich was selected as being the closest in location and chemical properties to the soi ls in the study area. The chemical and physical characteristics of Profi le 04, and the mineral and particle size distributions typical of the 1C ash are presented in Tables lOa-d. The analytical results for Profi le 04 closely matched those found in the study area, particularly the soi ls sampled in the Abies rel igiosa zone. The particle size distribution suggested a very young and l i t t l e weathered s o i l (Table 10c). Very fine to fine sand was the most abundant particle size class with lesser amounts of medium sand and coarse s i l t . The clay content as well as gravel was very low. The dominance of particles with low specific surface area would seriously hinder surface adsorption phenomenon, organo-mineral complex formation, cation and anion exchange capacities and nutrient supply. Table lOd examines the mineral distribution of the 1C ash. The high content of v i t r i c material confirmed i t s volcanic o r i g i n . The absence of quartz and the identif icat ion of Ca-rich feldspars and pyroxenes c lass i f ied the ash as andesitic (Figure 2). However, Na, K - feldspars, typical of dacitic ashes, were also ident i f ied . 130 Table 10a. Representative Profile from Iztaccihuatl Study Area (Miehlich, 1980) : Chemical Properties of Profile 04 including Comparative Median Data from the Soils of the Three Vegetation Zones. PARAMETER 04 Ah, ABIES Ah, PINUS Ah, ZACATONAL Ah %Ct 1.8 1.7 3.1 2.0 %Nt 0.13 0.10 0.17 0.13 C/N 13.7 16.1 18.0 14.7 P H 1 5.4 5.5 4.2 4.8 %Fe 3.8 - -cbd 0.33 0.24 0.24 0.24 %Fe 0.090 0.15 0.14 0.15 ox %Si 29.1 _ _ _ 1 2 %Si 1 0.050 0.045 0.061 0.12 ox %A1 8.9 _ _ %A1 1 0.23 0.21 0.32 0.38 ox 1. Profile 04 pH: I N KC1; Veg etation zones pH: 0.01 M CaCl„ 2. Vegetation zones: median Ah -^  + Al^. Table 10b. Physical Properties of Profile 04 (Miehlich, 1980). Bulk density 0.9 %Total pore space 64.4 %Volume of solids 35.6 Thixotrophy weak 131 Table 10c. Particle Size Distribution of Profi le 04 Approximated to the C . S . S . C . System (Miehlich, 1980). > 2.0 mm % HUMUS FREE % HUMUS FREE Amorphous clay fraction^ Crystalline clay fraction 4.1 2.1 6.2 Fine s i l t Medium s i l t Coarse s i l t 2.9 8.3 14.6 2 5 . 8 Very fine to fine sand Medium sand Coarse sand 49.6 18.1 0.3 68.0 NaOH treatment after defferation and organic matter removal but prior to particle size separation. % amorphous clay calculated by difference to 100%.. Table lOd. Mineral Distribution of the 1C Ash: 0.20-0.063 mm Particle Size Fraction (Miehlich, 1980). Mineral % Grain counts Light to brown glasses Opaque glasses or glass encrusted particles Sanidine (Na,K - feldspar) Plagioclase (Ca.Na - feldspar) Pyroxene 74 .2 7 .3 5.5 2.5 9 . 7 Table lOe. Verification of 1C Ash Parent Material Throughout the Three Vegetation Zones. SAMPLE %A1 3 %Si %Fe %A1 . , 4 %Fe ox ox ox cbd 1C ash (400 years) 1 0.20-0.59 0.0 -0.14 0.0 -0.40 0. 10-0.59 0.11-0.50 2C ash (3000-5000 yr ) 1 0.80-2.2 0.15-0.74 0.40-0.80 0. 20-1.1 0.41-0.90 2 Abies religiosa zone 0.21 0.045 0.15 0.17 0.24 2 Pinus hartwegii zone 0.32 0.061 0.14 0.26 0.24 Zacatonal zone^ 0.38 0.12 0.15 0.29 0.24 1. Ranges based on >8 observations (Miehlich, 1980). 2. Horizon with the highest median was selected. 3. Oxalate extractable A l , Si and Fe determined by Schwertmann (1964) in Miehlich (1980). 4. Cbd extractable Al and Fe determined by Mehra and Jackson (1960) in Miehlich (1980). 133 5.1.2.A Humus Fraction Distribution The humus fraction median data i s given in Table 11. The distribution of carbon among the humus fractions separated the extractable organic carbon into dist inct groups allowing the three zones to be compared by the nature of their organic matter components. The carbon in the humic acid fraction (%Ch) and fraction A (%Ca) were s ignif icantly different at the 95% confidence l e v e l ; hence, these two parameters were l i s ted by horizon for a l l vegetation zones. The other three parameters; fulvic acid carbon (%Cf), polysaccharide-rich fraction C carbon (%Cc) and total extractable carbon (%Ce),were not s ignif icantly different between horizons and were combined for analysis. S t a t i s t i c a l analysis of the combined Ah^ and Ah^ horizons indicated that there was no significant difference among the three vegetation zones in %Cf. However, s ignif icantly less fulvic acid polysaccharides (%Cc) were present in the Abies rel igiosa zone compared to the Pinus hartwegii and Zacatonal zones. The absence of grass cover in the Abies religiosa zone resulted in a lower input of plant polysaccharides to the s o i l , and possibly affected microbial ac t ivi ty and the production of microbial polysaccharides. The amounts of humic acid carbon (%Ch) and polyphenol-rich fraction A (%Ca) were s ignif icantly different among the Ah^ horizons but not the Ah 2 horizons of the vegetation zones. Ranking of the three vegetation zones in %Ch by the two-sample comparison method was not possible; however, Pinus Ah^ tended to be higher than the other two zones. %Ca was s i g n i f -icantly greater in Pinus Ah^ than Zacatonal Ah^. Abies Ah^ was intermed-iate between the two. The 95% confidence intervals for %Ch and %Ca were T a b l e 11. Median V a l u e s and 95% C o n f i d e n c e I n t e r v a l s f o r %C i n the Humus F r a c t i o n s . ZONE %Ch %Ca %Cf -a i r - d r y basis-%Cc »# 2 ZCe Ahj horizons, n=8 Abies r e l i g i o s a 0.46° 0.30-1.5 0.23 a b 0.19-0.51 Ahj + Ah^ horizons, n=16 0.40 0.36-0.65 0.20 0.14-0.37 58 65-61 Pinus hartwegii 0.84a 0.76-1.7 0.39 0.30-0.69 0.65 0.41-0.96 0.34 0.23-0.46 53" 52-58 Zacatonal 0.55° 0.44-0.86 0.15° 0. 13-0.19 0.48 0.44-0.62 0.32 0.30-0.44 a 50' 49-53 Ah^ horizons, n=8 Abies r e l i g i o s a ZCh 0.36 0.15-1.1 %Ca 0.20 0.13-0.27 Pinus hartwegii 0.48 0.35-0.99 0.19 0.16-0.54 Zacatona1 0.52 0.44-0.95 0.14 0.13-0.21 1. Significance l e v e l : 95%= *, 99%=**. Analysis by k-saraple comparison. 2. The Ah| and Ah2 horizons were combined for %Cf, %Cc and %Ce, n=16. 135 high in the Abies and Pinus Ah^ horizons indicating considerable spatial v a r i a b i l i t y in organic matter inputs. This was not unexpected from the diversity of vegetative cover in the Abies and Pinus zones. In the Zacatonal zone inputs were more homogeneous, both compositionally and spat ial ly , as indicated by the narrow 95% confidence intervals . In the Ah^ horizons a similar situation was present except %Ca in the Abies rel igiosa zone spanned a very narrow range. Thus, the production of fulvic acid polyphenols, either through in si tu decomposition or i l l u v -iat ion from the humus layer, was quite uniform compared to the surface Ah^ horizons which showed visual evidence of faunal mixing (see also %LI for Abies FH in Table 3). The above differences in %Ch and %Ca stemmed largely from the influence of pine l i t t e r in the Pinus hartwegii zone. The large input (although uneven) of pine l i t t e r to the s o i l surface in addition to canopy drip would greatly increase the levels of acid-soluble polyphenolic mater-i a l (%Ca) in the Ah^ horizons. This polyphenolic material was undoubtedly the source of low pH found in the Pinus hartwegii zone (Section 5.1.1). However, this effect was reduced in the Ah^ horizons as indicated by a significant decrease in %Ca and a pH one unit higher. The s l ight ly higher %Ch in the Pinus Ah^ horizons may have resulted from this increased level of "humic acid precursors". The significant decrease in %Ch and %Ca from Pinus Ah^ to Ah^ suggested that the majority of the polyphenolic material remained in the surface Ah^ and FH horizons with humus formation by in si tu processes being dominant in the Ah 2 horizons. The absence of zonal differences among the Ah„ horizons supported in si tu processes in the Abies rel igiosa 136 and Zacatonal zones. I l luviat ion of acid-soluble polyphenols undoubtedly occurred during the rainy season, but the Ah^ horizons were not a major zone of accumulation. As mentioned in Section 5.1.1 some accumulation of i l luvia ted material was evident in the IP pumice layers which contained greater amounts of "surface active" materials. The percent extractable carbon (%Ce) reflected the extent of inter-action of the s o i l humus with the inorganic s o i l components. It also indicated the amount of undecomposed or part ial ly decomposed humin fract -ion. %Ce was not s ignif icantly different between Ah-^  and A l ^ horizons; hence, they were combined for analysis. %Ce decreased from the Abies to the Zacatonal zone. The levels of extractable Al (Section 5.1.2.2) increased along the above sequence suggesting some stabi l izat ion of the humus with A l . The dominant particle size class (Section 5.1.2.3) was fine sand; thus, strong clay-organic matter complexes were not major contributors to the humin pool. However, i t was possible that very large, aliphatic humic acid molecules could be stabilized to some extent by physical interaction with the s i l t and fine sand (cf. to Anderson et a l . (1974a) HA-B fraction) . The presence of charcoal in the Abies rel igiosa and Pinus hartwegii zones would lower %Ce. Thus, in the absence of charcoal %Ce would be even higher emphasizing the easily extracted, "immature" humus in the above zones. This was not unexpected considering the soi ls recent age and lack of catalytic clay-sized particles (Wang et a l . , 1983a). It was probable that the unextractable humin fraction consisted largely of large a l iphat ic , immature humic acid molecules, part ial ly decomposed plant materials such as fibrous roots, etc. which would increase with grass 137 influence, and microbial biomass. Visible roots, needles, etc. were removed during sample preparation. 5.1.2.5 Humus Fraction Ratios The interpretation of absolute humus fraction amounts (Section 5.1. 2.4) was speculative due to the absence of data on the nature, amount and spatial distribution of organic matter inputs into the s o i l prof i le , decomposition rates, levels of microbial and faunal ac t iv i ty , movement of organic components within the p r o f i l e , etc. Furthermore, there was considerable spatial var iabi l i ty within a sample plot in the humus fraction amounts as indicated by their wide 95% confidence intervals (Table 11). The calculation of humus fraction ratios removed some of this v a r i a b i l i t y by placing a l l samples in a relative frame of reference. The narrow 95% confidence intervals noted for the humus fraction ratios , especially in the Pinus hartwegii and Abies rel igiosa zones, confirmed this decrease in v a r i a b i l i t y . Consequently, the "quality" rather than the "quantity" of s o i l humus was expressed, and the process of humus formation, the d i s t -ribution of organic components among humus pools, and the nature of the s o i l environment could be more rel iably interpreted. Median humus fraction ratio data i s given in Table 12. The .calculated ratios , Ch/Cf, Ca/Cf and Ch/Ca, were not s ignif icantly different between Ah^ and Ah^ horizons and were combined for analysis. Thus, the horizon differences noted in the Pinus zone for %Ch and %Ca were not reflected in the humus fraction rat ios . This was rather surprising considering the vegetative differences among the three zones. The Abies rel igiosa zone was s t r i c t l y a coniferous system with associated shrubbery, forbs and a 138 Table 12. Median Values and 95% Confidence Intervals for Calculated Ratios of Absolute Humus Fraction Amounts.^ ZONE Ch/Cf Ch/Ca** Ca/Cf""" Ah^ + Ah^ horizons, n=16 Abies religiosa 0.97 0.73-1.2 2.0 a 1.4-2.5 0.49 0.47-0.54 Pinus hartwegii 1.1 1.0-1.2 2 . 4 ° 2.2-2.6 0.46u 0.45-0.47 Zacatonal 1.2 1.1-1.3 3.8U 3.3-4.4 0.30d 0.29-0.33 1. Significance l e v e l : 95% = 99% = * * . Analysis by k-sample comparison. Table 13. Median Values and 95% Confidence Intervals for the C/N Ratio and %PSS in the Humic Acid and A Fractions. ZONE C/N HA C/N A %PSS-C %PSS-C HA-C A-C Ah^ + Ah^ horizons, n=16 Abies rel igiosa 12.0a 26.7b 5.4 a 6.4 a 11.6-14.5 24.7-28.9 4.6-6.3 6.1-8.4 Pinus hartwegii 14.5b 25.6b 6.1 b 6.8 a 14.0-15.5 24.3-30.7 5.7-6.4 6.3-7.1 Zacatonal 13.0a 1 9 . 3 ° 6.8^ 8.3 12.9-13.4 18.3-21.4 6.5-7.7 7.5-9.4 a _ „c „ „b 1. Significance l e v e l : 95% = * , 99% = * * . Analysis by k-sample comparison. 139 relat ively thick FH humus layer. The Pinus hartwegii zone was a mixed coniferous-grassland system with l i t t l e FH but abundant pine needle l i t t e r on the bare mineral s o i l . The Zacatonal zone represented a typ-i c a l bunchgrass community with large patches of bare mineral s o i l being v i s i b l e . The poss ibi l i ty that similar processes of humus formation were occurring in both s o i l horizons, despite vegetative differences, was strongly supported by this lack of horizon different iat ion. The Ch/Cf ratio was not s ignif icantly different among the three vegetation zones indicating a regional climatic effect . A similar conclusion was reached by Lowe (1980) who reported Ch/Cf ratios greater than 1.0 (average 1.68) for Ah horizons of both grassland and forested temperate s o i l s . Several studies have reported increased Ch/Cf ratios with increased but not excessive moisture. For example, in Japan Ch/Cf ratios typical of Humic Allophane soi ls ranged from 0.9 to 1.2 in the drier north to 0.8 to 1.9 in the humid south (Tokudome and Kanno, 1965a). The Ch/Cf ratios for the young volcanic ash soi ls of Iztaccihuatl f e l l well within the ranges reported above for the more well developed Andosols. Thus, the Ch/Cf ratio may be an int r ins ic property of the s o i l from i t s youngest stage. A similar conclusion was reached by Anderson et a l . (1974a). In the Chernozemic grassland soi ls of the Canadian Prairies the Ch/Cf ratio was found to increase from the Brown s o i l zone (mean Ch/Cf = 1.49) to the Black s o i l zone (mean Ch/Cf = 2.06) (Lowe, 1980). The increase in elevation from the Abies rel igiosa zone to the Zacatonal zone of approximately 700 m was not enough to signif icantly influence the soi ls moisture status, and hence, the Ch/Cf ra t io . However, an increasing trend with elevation was apparent in the 95% confidence intervals . This 140 possibly reflected a s l ight ly more favourable environment for polymerizat-ion reactions with increasing al t i tude. Several factors could account for this observation. Grass influence, noted to favour humic acid formation, increased with elevation as did the levels of extractable %A1 (Section 5.1.2.2). Amorphous aluminosilicates have been reported to increase humic acid formation through their catalytic action on low molecular weight polyphenols (Wang et a l . , 1983a,b). The correlation coefficient between %A1 and the Ch/Cf ratio was r = 0.51. However, the data points were ox highly scattered about the regression l ine making i t s significance questionable. Nevertheless, as w i l l be discussed later , %A1 was s i e n i f -ox icantly correlated with the Ch/Ca ratio (r = 0.83). A further factor that may have increased the Ch/Cf ratio was evidence of freeze-thaw cycles at higher elevations, particularly in the Zacatonal zone. However, alpine and arctic temperate s o i l Ah horizons generally had Ch/Cf < 1.0 (mean = 0.63) (Lowe, 1980). The different sources of organic matter in the three zones seemed to have l i t t l e effect on %Ch (Table 11) or the Ch/Cf ra t io . In the Abies rel igiosa zone organic matter inputs would be highly varied, both comp-osi t ionally and spat ia l ly . This was supported by wider 95% confidence intervals for %Ch and the Ch/Cf ra t io . The 95% confidence interval for %Ch decreased s l ight ly from the Ah^ to the Ah^ horizons possibly s ignal-l ing a change in organic matter source. In the Pinus hartwegii zone the surface Ah^ horizons would receive both pine l i t t e r and grass inputs, the pine influence lessening in the Ah 2 horizons. This change was reflected in the width of the 95% confidence interval for %Ch, decreasing from 0.94 in the Ah., horizons to 0.64 in the Ah„ horizons, approaching that in the U l Zacatonal zone (0.51). This suggested that the source of organic matter for humus formation in the Pinus Ah^ horizons was primarily in si tu root decomposition. The composition of organic matter inputs into the Zacatonal zone would be highly uniform to the effective rooting depth, grass roots and exudates being the primary source of organic matter for humus formation. The a b i l i t y of grass root systems to explore the total of the s o i l volume produced a more uniform input of organic matter into the profi le compared to other plant species. This was reflected in the narrower 95% confidence intervals for Ch/Cf in both the Pinus and Zacatonal zones compared to the Abies zone. Hence, there was a possible connection between the Ch/Cf ratio var iabi l i ty and the source of organic matter for humus formation. The Ca/Cf ratio reflects the proportion of polyphenolic material in the fulvic acid fract ion. High Ca/Cf ratios are associated with conditions of low biological ac t ivi ty due to low temperatures or acid pH, and lead to the accumulation of soluble polyphenols with limited production of polysaccharides. These conditions are often met in coniferous ecosystems where acidic conditions l imit microbial act ivi ty and the forest humus layer provides a readily available source of polyphenols. The cold temp-eratures prevailing in alpine or arctic soi ls also enhances the formation of fulvic acid polyphenols (Lowe, 1980). The Ca/Cf ratio decreased s l i g h t l y , but s ignif icantly at the 99% confidence l e v e l , from the Abies to the Pinus zone. A much larger and significant decrease was noted from the Pinus to the Zacatonal zone. In the Abies zone low amounts of fulvic acid polysaccharides (%Cc) were responsible for the high Ca/Cf ra t io . The Zacatonal zone contained high 142 levels of polysaccharides but l i t t l e fulvic acid polyphenols which resulted in a low Ca/Cf ra t io . The Pinus zone was high in both fulvic acid polyphenols and polysaccharides; thus, a combined effect was apparent but favouring a high Ca/Cf ra t io . The low proportion of polyphenolic fulvic acid material in the Zacatonal zone was typical of grassland ecosystems. The Chernozemic soi ls of the Canadian Prairies have Ca/Cf ratios ranging from 0.09 to 0.25, increasing from the Brown to the Black s o i l zones. The Ca/Cf ratio in the Zacatonal zone (median = 0.30) was s l ight ly higher than the neutral Chernozemic soi ls possibly due to a more acidic pH (5-6) or the alpine location which may have affected microbial ac t ivi ty (Lowe, 1980). The environment in the Zacatonal zone favoured the elimination of fulvic acid polyphenols either through microbial degradation or polymerization reactions to form humic acids. The higher levels of s o i l polysaccharides possibly indicated enhanced microbial synthesis, although considerable polysaccharides are present in grass root exudates. The absence of a significant horizon difference in the Pinus hartwegii zone in the humus fraction ratios was unusual since %Ch and %Ca decreased considerably from the Ah^ to the Ah^ horizons, and the abundance of pine l i t t e r on the s o i l surface would provide phenolic compounds to the surface Ah^ horizons for humus formation. The extremely narrow 95% confidence interval for Ca/Cf in the Pinus hartwegii zone (0.45-0.47) suggested that contributions from the two possible sources, in si tu root decomposition and i l l u v i a t i o n from the pine l i t t e r layer, produced similar Ca/Cf ratios in the two horizons. If in si tu grass root decomposition was the major mechanism in Pinus Ah„, the Ca/Cf ratio should have approached that in the 143 Zacatonal zone. However, in si tu contributions from small, fibrous pine rootlets would also affect the f i n a l Ca/Cf ratio obtained, as well as i l luvia ted polyphenols from the pine l i t t e r . Hence, although in si tu grass root decomposition was a major source of polyphenols (and consequently humic acid) in the Ah^ and Ah^ horizons, i t was d i f f i c u l t to ascertain the importance of i l luvia ted material in the A l ^ horizons. The presence of considerable i l l u v i a t e d material would, by def ini t ion , form a B horizon, and this was definitely not observed. The absolute humus fraction amounts and their ratios were not s ignif icantly different between horizons in the Abies rel igiosa and Zacatonal zones supporting in s i tu humus formation. The lower levels of polyphenolic fulvic acid (%Ca) in the Abies Ah^ horizons suggested that the majority of the polyphenols produced in the relatively thick humus layer remained there. In a coniferous system there are two major mechanisms for organic matter input into a p r o f i l e ; i l l u v i a t i o n of water-soluble organic compounds produced primarily in the humus layer or in si tu organic matter decompos-i t i o n . Forest s o i l horizons such as Podzolic Bf have low Ch/Cf and high Ca/Cf ratios indicating a dominance of i l l u v i a l organic matter. The Ca/Cf ratios reported for the Abies and Pinus zones (0.49 and 0.46, respectively) were considerably higher than ratios reported by Lowe (1980) for temperate Ah horizons (mean = 0.25). In fact, they were closer to ratios reported for i l l u v i a l Podzolic Bf horizons (mean = 0.58), forest humus layers (> 0.40) and alpine or arctic Ah horizons (mean = 0.38) (Lowe, 1980). Consequently, the high elevation of the s o i l zones placed them in an alpine environment which favoured the accumulation of fulvic acid polyphenols. 144 Several factors are known to favour humic acid formation. These include a neutral s o i l environment with adequate base status to promote microbial ac t iv i ty , a good annual supply of organic matter, adequate aeration, a moderate hydrothermal regime, alternating wet-dry or freeze-thaw cycles, and the presence of catalytic or s tabi l iz ing clay minerals or hydrous oxides. These conditions are typically met in Chernozemic Ah horizons. Fulvic acid formation or accumulation is favoured under conditions of low biological ac t ivi ty brought about by acidic conditions, flooding or cold temperatures which prevent or slow the polymerization of low molecular weight fulvic acid polyphenols. These conditions may be met in coniferous humus layers and i l l u v i a l B horizons, gleyed s o i l s , and alpine or arctic s o i l s . The Ch/Ca ratio i s the ratio of carbon in the humic acid fraction to carbon in the polyphenolic-rich fraction A. This ratio has not been discussed in the l i terature making i t s interpretation d i f f i c u l t . Both fractions contain phenolic material and are intimately associated with each other in the s o i l environment. During the decomposition process of plants simple polyphenols, including microbial by-products, are released into the s o i l . Their fate can be summed up in the following four processes: 1. They enter the fulvic acid fraction as part of the polyphenolic fraction A, and they are oxidatively polymerized to form larger molecular weight fulvic acid polyphenols which are not highly condensed. 2. The simple polyphenols are oxidatively polymerized, but the reaction mechanisms reduce the number of functional groups 145 essential for maintaining i t s a c i d - s o l u b i l i t y , or the molecular size attained decreases the surface area to such a point that the polymer enters the "acid-insoluble" humic acid pool. 3. The polyphenols are further degraded or ut i l ized by the microbial population. 4. The simple polyphenols pass into lower horizons. Support for a l l four processes, often operating simultaneously, can be found in the l i terature . However, considerable controversy existed over whether the fulvic acid fraction was a precursor, end-product or neither to the humic acid fract ion. Studies on the decomposition process 14 have shown radioactive C to be heterogeneously incorporated into a l l humus fractions which were developed rather quickly paral lel with the i n i t i a l period of vigorous microbial ac t ivi ty (Sinha, 1972a). According to Swift and Posner (1977) the decomposition of fresh plant material produced a rapid formation of high molecular weight, acidic , humified products which was followed by a slower, prolonged, oxidative degradation to lower molecular weight materials. A similar theory was put forth by Matsui and Kumada (1977a) and Tsutsuki and Kuwatsuka (1984) where an increase in humification was associated with oxidative polymerization and intramolecular condensation leading to a darker colour, decreased molecular weight, atomic H/C and A l o g K , and increased oxygen and carbon contents of the humic acid polymers. Schnitzer (1978) extended the theory further to postulate the oxidative degradation of humic acids to form fulv ic acids, a process involving a reduction in molecular weight and carbon content (note, opposite to Japanese theory) as aliphatic components were s p l i t 146 off , and a concomitant increase in oxygen content and total ac idi ty . Anderson et a l . (1974a) equated the same process in the conversion of the immature, highly al iphatic , high molecular weight HA-B fraction to the more mature, aromatic, lower molecular weight HA-A fraction (conventional HA fract ion) . However, they claimed that the relative proportions of the humus fractions do not change, and they were a feature of humus formation from the earliest stages of decomposition. In the recent volcanic ash soi ls of Iztaccihuatl the latter process of HA —>FA conversion was not l i k e l y to be as important as the i n i t i a l build-up of a high molecular weight, a l iphat ic , immature humic acid fract ion. Hence, when interpreting the Ch/Ca ratio the fate of the simple polyphenols was the most obvious route to follow. The Ch/Ca ratio in a l l three zones was greater than 2.0 suggesting humic acid formation to be the dominant process (Process 2). The Ch/Ca ratio was not signficantly different between the Abies rel igiosa (median = 2.0) and the Pinus hart-wegii (median = 2.4) zones, but increased s ignif icantly in the Zacatonal zone (median = 3 . 8 ) . This high ratio in the Zacatonal zone was primarily due to low %Ca; the levels of %Ch being not highly different among the three zones. Therefore, conditions in the Zacatonal zone seemed to prod-uce less simple polyphenols or favoured their removal either by microbial degradation or polymerization to form humic acids. Phenol coupling reactions were favoured at pH's > 6 (Section 2.4.2); however, in the Zacatonal zone pH's.of 5.7 - 5.8 were common. Kyuma and Kuwaguchi (1964) claimed that these "polyphenols, once in the s o i l , would be adsorbed by allophane and be cata lyt ical ly oxidized to give dark coloured, highly acidic polymerized products similar to humic substances, regardless of the 147 acid environment and low base status of the s o i l " . Oxalate-extractable A l , which increased with elevation, could have had a similar affect on poly-phenols as indicated by the high correlation, r = 0.83, between Ch/Ca and %A1 ox 5.1.2.6 Measured Properties of the Humic Acid and Fraction A Extracts The C/N ratio and hydrolyzable sugar content (%PSS) of the humic acid and fraction A extracts are given in Table 13. There was no significant difference between horizons, and they were combined for analysis. Several factors can influence the cycling of nitrogen in the s o i l and the amount of nitrogen incorporated into the forming humic polymers. Important are the nitrogen supplying power of the native vegetation (including biological N fixat ion) , the act ivi ty of the s o i l microorganisms which i s largely controlled by the nature of the organic matter inputs into the s o i l and the s o i l environment, the s o i l C/N ratio as i t s reflects the balance between nitrogen mineralization and immobilization into the s o i l biomass, and the forms of nitrogen released to the s o i l solution (complex polymers versus simple amino acids, free amines and amino sugars) which determine the mode of linkage to the humic polymers. The C/N ratios of the humic acid fractions (C/N HA = 12.0 --14.5) were similar to those reported for humic acids extracted from tropical volcanic ash soi ls (C/N HA = 12.5 - 13.3) ( G r i f f i t h and Schnitzer, 1975). However, the C/N ratios of fraction A (C/N A = 19.3 - 26.7) were s i g n i f i -cantly greater than those reported for the fulvic acid fraction (C/N FA = 15.4 - 16.3) in the above report. The inclusion of the polysaccharide-rich fraction C, often abundant in bound amino acids, amino sugars, etc. 148 (Sequi et a l . , 1975b), in the fulvic acid fraction probably contributed to a lowering of the C/N ratio compared to the polyphenol-rich fraction A examined in this study. The relationship between the degree of humification and the nitrogen content of the humic acid fraction had received much attention in the l i terature . Lowe and Godkin (1975) reported lower nitrogen contents in the immature humic acids isolated from L and F forest humus layers compared to the more humified H layers. This suggested that the nitrogen content of the humic acids increased during humification. The nitrogen content of decomposing l ignin had also been shown to increase during humification (see Section 2.4.4). Conversely, Kuwatsuka et al.(1978) suggested an enrichment of nitrogen into the humic acid fraction in the early stages of humus formation which was released with further humification. However, they also noted Rp-type humic acids (the most recently formed) had n i t r o -gen contents ranging from very low to very high, possibly reflecting the source of vegetation. Anderson et a l . (1974a) could not find any rela t -ionship between "aromaticity" ( ie . maturity) and the hydrolyzable carbon and nitrogen contents of s o i l humic acids. The recent age of the soi ls in this study and their f a i r l y high nitrogen contents favoured the enrichment hypothesis of Kuwatsuka et a l . (1978), and i t was unlikely that zonal differences in C/N HA were a reflect ion of humic acid maturity. The C/N HA ratio was highest in the Pinus hartwegii zone possibly due to a high input of polyphenolic materials low in nitrogen from the pine l i t t e r , and the low pH which would decrease microbial ac t ivi ty and the cycling of nitrogen in the s o i l . A large proportion of the nitrogen would be derived from the grass vegetation; although, the high s o i l C/N 149 ratio (18.7) implied nitrogen immobilization by the s o i l biomass. Lower C/N HA ratios were found in the Abies rel igiosa and Zacatonal zones. The low C/N HA and s o i l C/N in the Zacatonal zone reflected the greater ni t ro -gen supply and increased cycling in grassland ecosystems. The Abies rel igiosa zone contained insignificant grass cover, but possibly received considerable nitrogen from the ground cover of forbs and shrubs. Also, the near neutral pH and s o i l C/N ratio (16.2) would favour microbial act ivi ty and nitrogen turnover. Hence, the C/N HA ratios were influenced by a combination of vegetation and environmental effects . The nitrogen content of the polyphenol-rich fraction A had been l i t t l e examined in the l i terature , and there were no reported theories on the significance, i f any, of i t s C/N rat io . This was partly due to the low nitrogen content of this pool and the belief that i t contributed l i t t l e to the overall nitrogen cycle. The C/N A ratio was high in the coniferous s o i l zones, but decreased s ignif icantly in the Zacatonal zone. The C/N A ratios in the coniferous zones were comparable to the > 30,000 molecular size fraction of the Lulu muck s o i l (C/N A = 28) (Lowe, 1975). Increased nitrogen and polysaccharide contents were associated with increased mole-cular weight of the Lulu muck fraction A. The lower molecular weight polyphenols had low nitrogen contents and atomic H/C ratios suggestive of aromatic materials. In this study a positive correlation was found between the C/N A ratio and the Ca/Cf rat io , r = 0.59, which formed two groups when plotted; the Zacatonal zone and the coniferous s o i l zones. A similar , but inverse correlation, was found between the C/N A ratio and the %PSS-C/total A-C, r = -0.59. However, a plot of the parameters fai led to demonstrate a grouping tendancy, and the data were quite 150 scattered. The above correlations suggested that a considerable proportion of fraction A nitrogen was present in hydrolyzable aliphatic linkages, which increased as the relative levels of polysaccharide-rich fraction C increased. The C/N HA ratio was highly correlated to the s o i l C/N rat io , empha-sizing the enrichment of nitrogen into the humic acid pool. Approximately half of the v a r i a b i l i t y in the s o i l C/N ratio could be attributed to the humic acid C/N rat io . A further source of var iabi l i ty came from the polysaccharide-rich fraction C; limited influence being associated with fraction A due to i t s high C/N ra t io . The C/N HA ratio was correlated neither to the C/N A ratio nor to %PSS-C/total HA-C suggesting that the humic polymers contained l i t t l e nitrogen in adsorbed or covalently bonded polysaccharides. However, elemental analysis of the bulk isolated humic acid fractions (Part 2) gave atomic H/C ratios suggestive of materials with a substantial aliphatic component (H/C = 1.24-1.31). It was probable that a large proportion of the humic acid nitrogen existed as adsorbed or bonded proteins, peptides and amino acids. Lesser amounts of nitrogen would be present in heterocyclic forms based on the recent ages of these humic molecules. The hydrolyzable sugar content (%PSS) of humic acid and fraction A was based on the phenol-sulphuric acid colorimetric method. The data were presented as % polysaccharide carbon to total humic acid or fraction A carbon. (%PSS-C = %Glucose-C , which i s used in the appendices). Several drawbacks were present in this method which at best only estimates the % polysaccharides. There was possible interference from other reducing substances in addition to the variation in X. max for various monosacchar-151 ides. Also, the hydrolysis conditions were a compromise between good glycosidic cleavage and monomer destruction (Stevenson, 1982). Further-more, i t cannot be assumed that the sugar monomers were covalently attached to the humic polymer since the inexact nature of the humic acid and fraction A extraction procedure allowed for the poss ibi l i ty of adsorbed polysaccharide material. The %PSS-C/HA-C, and to a lesser extent %PSS-C/A-C, reflected the increase in grass influence from the Abies to the Zacatonal zone. Increased polysaccharides were associated with fraction A compared to the humic acid fraction which may indicate contamination from fraction C. Possible sources of "glucose equivalents" included physically adsorbed ol igo- and polysaccharides, phenolic glycosides, other glycoside bonds, and ester formation with uronic acids. The fraction A %PSS-C in the Pinus hartwegii zone did not reflect the increase in grass influence noted for the humic acid fract ion. The highly polyphenolic nature of the fulvic acid fraction and the low pH found in this zone may have affected the bonding of polysaccharide materials to the phenolic polymers, ie phenolic glycoside formation. Furthermore, the high Ca/Cf ratio suggested a dilution of the polysacchar-ide material able to react with the polyphenols. A similar situation was noted for C/N A in the Abies and Pinus zones. In conclusion, the Ca/Cf ratio influenced the C/N A ratio and the %PSS content of the polyphenol-r ich fraction A. 152 5.1.2.7 Optical Properties of the Humic Acid and Fraction A Extracts The absorbance or optical density of a solution at a fixed wavelength is governed by the Beer-Lambert law: log I 0 / I = A = kcb, where I 0 = the incident l ight intensity, I = the l ight intensity after passing through the absorbing medium, k = the extinction coefficient , c = the concentration of absorbing substance and d = the path length of absorbing medium. The extinction coefficient , k, is governed by the nature of the absorbing substance and the wavelength of l i g h t , X . The extinction coefficient of humic compounds i s thought to increase with particle molecular weight, the percent carbon content, the degree of molecular condensation and the ratio of carbon in aromatic rings to carbon in aliphatic structures (Kononova, 1966. Cited in Stevenson, 1982). Fulvic acids tend to have low extinction coefficients regardless of source (Stevenson, 1982). The optical properties of the humic acid fractions are given in Table 14. Those of fraction A are given in Table 15. Extinction coefficients were based on a 1% carbon solution at pH 12.0. Significant horizon differences were noted in the Pinus hartwegii zone for E ^ Q Q HA and E^/E^ HA, a l l other parameters did not differ between the two horizons, ^^QQ' often used as an index of maturity or aromaticity (Lowe and Godkin, 1975), was not s ignif icantly different among the three zones. This suggested that the humic acids from the three vegetation zones were of similar maturity. In Part 2 atomic H/C ratios supported this conclusion. The interpretation of E ,„„ was subiect to some inaccuracy due to the low solution absorbance 600 J at this wavelength. This error could be magnified by computation of the E , / E , ratio due to the low denominator value. Nevertheless, significant U o differences at the 99% confidence level were noted in both the Ah, and Ah 0 153 Table 14. Median Values and 95% Confidence Intervals for the Optical Properties of the Humic Acid Frac t ion . 1 ZONE E ^ C HA 2 ^400 H A F 1 7 ° C HA* ^600 " h E A / E 6 HA Ah^ + Ah^ horizons, n=16 Ah^ horizons, n=£ Abies rel igiosa 217.3 187.9-235.1 46.8" 40.7-52.1 4.56 4.44-4.85 Pinus hartwegii 209.2 188.4-232.7 5 0 . l u 40.7-58.6 3.87u 3.76-4.40 Zacatonal 203.9 197.7-209.7 61.0 58.2-64.2 3.35 a 3.24-3.49 1 %C * * F HA b600 H A E . / E , HA 4 6 Ah 2 horizons, n=8 Abies rel igiosa 4 2 . 9 ° 38.3-56.4 4.63 4.38-5.18 Pinus hartwegii 66.0L 56.6-72.9 3.46u 3.35-4.25 Zacatonal 62. T 56.6-65.9 3.30 3.20-3.46 1. Significance l e v e l : 95% = * , 99% = * * . Analysis by k-sample comparison. 2. The Ah^ and Ah 2 horizons were combined for E ^ Q Q , n=16. 154 Table 15. Median Values and 95% Confidence Intervals for the Optical Properties of Fraction A."^ F 1%C * * F 1%C * ^400 600 A Ah, + Ah„ horizons, n=16 Abies rel igiosa 129.0C 9.30b 12.8b 127.6-132.9 9.00-10.5 12.6-14.5 Pinus hartwegii 123.9b 1 0 . 3 ° 12.2a 120.7-131.7 9.90-10.5 11.8-12.8 Zacatonal 107.4a 8.60a 12.5 a b 105.2-113.2 7.90-9.20 12.1-13.0 1. Significance l e v e l : 95% = * , 99% = * * . Analysis by k-sample comparison. 155 horizons for the above parameters. In the Ah, horizons E , ~ ~ HA increased significantly from the Abies 1 600 ° to the Zacatonal zone, the reverse trend occurring in the E ^ / E ^ rat ios . In the Ah^ horizons E ^ Q HA was highest in the Pinus hartwegii zone f o l -lowed by the Zacatonal zone. The significant increase in E ^ Q Q HA from Pinus Ah^ to Ah^ seemed to reflect a change in the dominant source of organic matter from pine to grass, or possibly demonstrated the invasion of native grassland by pine. The high concentration of polyphenols and low pH present in the surface Ah^ horizons possibly influenced the "nature" of the humic acids formed. This influence was less apparent in the Ah^ horizons where conditions were similar to the Zacatonal zone. To explain these observations the factors controlling absorbance in the vis ible region must be examined. Absorbance in the vis ible can stem from two origins; the presence of organically complexed transition metals and extensive conjugation of non-metallic chromophores (eg. C=0, -IM^. C=C, e tc . ) , the latter being generally more important. Humic acids are thought to consist of an aromatic "core", possibly polycyclic ring systems, with attached peripheral aliphatic side chains containing numerous funct-ional groups. The nature of this "core" reflects the degree of conden-sation or maturity of the humic acid polymers, the more mature humic acids having increased absorbance in the vis ible region and decreased E ^ / E ^ or A logK (Kononova, 1961; Kumada, 1965). Highly aliphatic humic acids with l i t t l e or no "core" material would absorb significantly in the v is ible region only i f they contained abundant conjugated chromophores; hence, increased particle molecular weight could enhance absorption. Several studies have shown an inverse relationship between the E . / E , ratio and 156 polymer molecular weight (Chen et a l . , 1977; Anderson et a l . , 1974a,b). Thus, within humic molecules of similar aromaticity, E ^ Q Q and the E ^ / E ^ ratio may reflect the polymer molecular weight. However, when comparing humic molecules extracted from widely differ ing soi ls with a range in molecular complexity, the absorbance due to aromaticity may take preced-ence and influence the E . / E , ra t io . 4 o This hypothesis favoured a similar maturity in the humic acids extr-acted from the three vegetation zones as there was no significant d i f f e r -ence in E . _ _ HA, atomic H/C ratios and the elemental content (Part 2). 400 The grassland Ah^ humic acids had increased E ^ ^ compared to the conifer-ous Ah^ humic acids indicating a higher polymer molecular weight in the former. In the Pinus Ah^ horizons reduced influence from the pine l i t t e r and a less acid pH favoured polymerization reactions producing particle molecular weights similar to those found in the Zacatonal zone. Further studies on the optical characteristics and the size fraction distributions of the humic acid fractions would be needed to verify this hypothesis. The absorbance of the A fractions in the v is ible region was lower compared to the humic acid fractions. The extremely low absorbance at h = 600 nm indicated limited conjugation within the polymers. Further-more, the low absorbance values created a greater error potential, in the E . / E , A ra t io . L i t t l e reference has been made in the soi ls l i terature on 4 6 the optical properties of the fulvic acid fraction except to note i t s lower absorptivity and higher E ^ / E ^ ratio compared to the humic acid fract ion. The high E ^ / E ^ ratio has been related to i t s lower particle molecular weight (Chen et a l . , 1977). E ^ Q Q A increased signif icantly from the Zacatonal zone to the Abies 157 zone which possibly reflected the greater input of polyphenols (%Ca) into the coniferous s o i l zones. As indicated ear l ier , most polyphenols prod-uced in the Zacatonal zone did not remain in the fulvic acid fraction but were polymerized to form humic acids or degraded by microorganisms. The high Ca/Cf ratio in the coniferous s o i l zones suggested limited biological act ivi ty which possibly enhanced the formation of higher molecular weight polyphenols due to them being in the system longer. The size fraction distribution of the bulk isolated A fractions in Part 2 supported this this theory (see Table 1 6 ) . A similar trend was evident in E ^ Q Q A except the Abies rel igiosa and Pinus hartwegii zones were reversed. The E ^ / E ^ A ratios were d i f f i c u l t to interpret as they did not totally agree with the analysis of the E ^ Q Q or E ^ Q Q A data. Nevertheless, the data seemed to support the overall low molecular weight and limited conjugation of the chromophores in the A fractions. The s t a t i s t i c a l analysis of organic matter parameters in Part 1 led to the following general conclusions: 1 . The humus in the Ah^ and A l ^ horizons of the Abies rel igiosa and Zacatonal zones represented a single population. 1% 2 . Horizon differences in %Ch, %Ca, E , ° HA and E , / E , HA were noted 6 0 0 4 6 in the Pinus hartwegii zone. Thus, two dist inct humic acid populations were evident from the optical properties. 3. Qualitative differences in the kinds of organic matter components were evident from the humus fraction ratios . These differences were related primarily to the vegetation site factor ( ie . Ca/Cf) . 4 . The alpine location of the s o i l zones favoured the production or accumulation of fulvic acid polyphenols; hence, Ca/Cf ratios were 158 higher than those reported for temperate Ah horizons (Lowe, 1980). 5. The Ch/Cf ratio was not influenced by the vegetation site factor, but reflected the overall regional climate. However, a sl ight elevational trend was apparent in the 95% confidence intervals . 1% 6. The E , ° HA optical data indicated that the humic acids from the 400 three zones were similar in "maturity". Hence, zonal differences 1% in E , ° HA and E , / E , HA were related primarily to differences in 600 A 6 polymer molecular weight which increased from the Abies to the Zacatonal zone. 1% 7. E ^ Q J HA increased significantly from Pinus Ah^ to Pinus Ah^ which suggested either a change in the dominant source of organic matter for humus formation, or a change in the s o i l environment. 8. The optical characteristics of the humic acids in the Pinus A l ^ horizons were similar to the Zacatonal zone emphasizing a vegetat-ion effect on the "nature" of the humic acid formed. 159 5.2 Part 2 - Composite Study The purpose of Part 2 i s to examine in detail the characteristics of the bulk isolated humic acid and A fractions from the three vegetation zones. As previously indicated, the results are not s t a t i s t i c a l l y based, but tend to give a general overview on the nature of the polymers, and in particular, the phenolic acid hydrolysis products as they relate to vegetation. Abbreviated designations for the composite sample humic acids and fraction A are as follows: Zone Humic acid (HA) Fraction A Abies rel igiosa AH AA Pinus hartwegii Ah^ P1H P1A Pinus hartwegii Ah 2 P2H P2A Zacatonal ZH ZA 5.2.1 Bulk Isolation Recoveries and the Molecular Weight Distribution of the Humic Acid and A Fractions The recovery of the humic acid fractions ranged from 82 to 100 % (Table 16). Fraction A recovery ranged from 70 to 83 %. Less than com-plete recovery of the humus fractions could stem from two major method-ological differences between the test extraction procedure and the bulk isolat ion procedure. In the bulk isolation procedure the alkaline humus supernatant was siphoned-off from the unextractable s o i l sol ids . In the test extraction a centrifugal separation was made which would increase the extraction eff ic iency. The other difference was in the isolation of the 160 Table 16. Composite Sample Bulk Isolation Recoveries of Humic Acid and Fraction A and their Nominal Molecular Weight Distr ibution. Sample X Recovery % D i s t r i b u t i o n of Recovered F r a c t i o n AH P1H P2H ZH 88 82 100 97 < 10,000 6.2 7.7 6.6 5.9 > 10,000 93.8 92.3 93.4 94.1 AA P1A P2A ZA 70 83 78 79 < 1,000 16.0 28.7 24.5 39.0 > 1,000 84.0 71.3 75.5 61.0 1. The recovery of HA and f r a c t i o n A was based on a te s t e x t r a c t i o n of the composite samples using 0.1 N NaOH and the method of Lowe (1980). %C i n the humus ex t r a c t s was determined on the Astro Solution Carbon Analyser. 2. The XC i n the lower molecular weight f r a c t i o n s was measured i n s o l u t i o n on the Astro Solution Carbon Analyser. The recovery of the high molecular weight f r a c t i o n s was based on the weight of the freeze-dried extract and i t s %C (uncorrected for ash) content from elemental a n a l y s i s . Table 17. Elemental Analysis of the Humic Acid and A Fractions. Sample %Ash XC %H %N %S X01 C/N H/C N/S 0/C . ash-free basis atomic r a t i o s AH 24.5 45.1 4.6 3.9 0.32 46.1 11.6 1.24 12.0 1.02 P1H 20.6 48.7 5.3 3.6 0.40 42.0 13.5 1.31 9.0 0.86 P2H 22.8 47.7 4.9 3.5 0.41 43.4 13.6 1.24 8.7 0.91 ZH 20.8 47.3 5.0 4.0 0.50 43.2 11.8 1.27 7.9 0.91 AA 27.0 49.0 4.3 1.3 0.19 45.2 37.7 1.05 7.1 0.92 P1A 29.7 51.0 4.3 1.3 0.27 43.3 39.2 1.02 4.8 0.85 P2A 29.7 48.9 4.3 1.4 0.27 45.2 34.9 1.05 5.0 0.92 ZA 27.9 47.7 4.2 1.7 0.32 46.1 28.1 1.05 5.2 0.97 1. %0 determined by 100% - £ %C + %H + %N + %S. 161 polyphenolic A fraction on PVP. In the bulk isolation procedure a column-PVP set-up was used (Appendix 4) and very concentrated fraction A solut-ions were eluted from the PVP with less eff ic ient recovery. The test extraction method dealt with much lower solution concentrations and used less PVP (1 g PVP/100 ml FA); hence, elution of fraction A from the PVP was more e f f i c i e n t . Also some loss was unavoidable during the d i a f i l t r a t -ion procedure. P1H percent recovery was quite low due to i t s high %Ch (1.21). A soi l : so lut ion ratio of 1:10 was used for a l l composite samples; hence, the extraction of humus in P1H was less e f f i c i e n t . The higher % recovery for P2H and ZH may have resulted from their lower %Ch (0.53 and 0.62 %, respectively). However, AH was similar in %Ch (0.59) but was recovered in lesser amounts. The differences in recovery of the humus fractions could also be the result of increased "operator efficiency" as the author became more adept at the bulk isolation procedure. The low % recovery for AA was due to discarding a pale yellow supernatant FA near the end of the HA-FA separation which possibly should have been kept. Nevertheless, this would not affect the analytical results on the polymeric humus fractions. The d i a f i l t r a t i o n and concentration of the bulk isolated humic acid and A extracts (Section 4.3.3 and Figure 22) was designed to separate non-polymeric and polymeric materials, and also release entrapped lower molecular weight molecules. As previously noted few humic acid molecules were greater than MW 100,000, and a large proportion seemed to f a l l bet-ween 50,000 and 100,000 (Section 4.3.3). Greater than 90% of the humic acid material was above MW 10,000, and this distribution was common for a l l composite samples. This seemed to stress a common environment for 162 humic acid formation in the three zones. Also, the conditions favoured the formation of higher molecular weight polymers since less than 10% of the humic acid molecules were below MW 10,000. The distribution of molecular weights within the A fractions differed considerably among the three zones. The relative proportion of molecules less than MW 1,000 seemed to increase from the Abies to the Zacatonal zone. Hence, polymeric fraction A molecules were more prevalent in the coniferous s o i l zones which may have related to their high Ca/Cf ratio (see Section 5.1.2.7 for previous discussion). 5.2.2 Elemental Analysis of the Freeze-dried Humic Acid and A Fractions The elemental composition and ash content of the humic acid and A fractions are given in Table 17. The ash content of the humic acid fractions ranged from 20.6 to 24.5 %, and the A fractions from 27.0 to 29.7 %. The dominant element in both fractions was sodium as the fractions were isolated in Na + -salt form. In retrospect, i t would have been advan-tageous, especially for infrared work, to have isolated the fractions in acid form. This could be accomplished by passing the diaflowed and con-centrated extracts through a column packed with H+-exchange resin, although further losses of organic matter would occur. Iron and aluminium-were present in the humic acid extracts, but were negligible in the fraction A extracts emphasizing the eff ic ient clean-up of metals by the H+-exchange resin . S i l i c a and other salts made up the balance of the ash. Reported ranges for the elementary composition of humic acids and fulvic acids are given below (Stevenson, 1982): 163 (%) dry and ash-free basis Element Fulvic acid Humic acid C 4 0 - 5 0 50 - 60 0 4 4 - 5 0 30 - 35 H 4 - 6 4 - 6 N <1 - 3 2 - 6 S 0 - 2 0 - 2 In general, humic acids are richer in carbon but poorer in oxygen than fulvic acids. Oxygen containing functional groups include carboxyl (COOH), phenolic hydroxyl, alcoholic hydroxyl, enolic hydroxyl and methoxyl. Less numerous are carbonyl groups in quinones and ketones, ethers and lactones. Fulvic acids are generally richer in carboxyl and alcoholic hydroxyl whereas humic acids contain more phenolic hydroxyl ( G r i f f i t h and Schnitzer, 1975). The oxygen not accounted for in humic acids possibly occurs as unknown ether linkages or as heterocyclic comp-ounds in the aromatic "core". Recall , the fulvic acid fraction includes the polysaccharide-rich fraction C; hence, higher carbon and lower oxygen contents would be expected for the polyphenol-rich fraction A. There were no apparent differences in the humic acids or A fractions isolated from the composite samples in %C, %H, %N and %0 among the three vegetation zones. This agreed with the basic findings of Schnitzer (1978) who was unable to detect any distinct effects of climate on the elemental compositions of the humic and fulvic acids examined. %S i n -creased s l ight ly in both the humic acid and A fractions from the Abies to the Zacatonal zone. This possibly stemmed from the release of sulphur 164 gases during volcanic eruption which would decrease with distance from the source Popocatepetl. The %C contents for the humic acids were lower than the reported ranges. Conversely, the %0 contents were higher. The %C and %0 contents were not s ignif icantly different from the polyphenolic A fractions. Thus, the humic acids were quite oxidized but not condensed, and possibly contained a high proportion of oxygen in aliphatic functional groups. The high %H and atomic H/C ratios suggested highly aliphatic humic acids. Furthermore, the humic acids were enriched with nitrogen, possibly aliphatic nitrogen. On the other hand, the atomic H/C ratios of fraction A emphasized highly aromatic materials with low nitrogen contents. A considerable proportion of the %0 must be in carboxylic acids attached to aromatic rings. The high content of hydrolyzable sugars (Section 5.1. 2.6) also indicated aliphatic hydroxyls were present. The relationship between elemental composition and the degree of humification has received much attention in the soi ls l i terature . In Japan, the %C, %0 and C/N ratio of humic acids increased with humification as the %H, %N and atomic H/C ratio decreased (Kumada, 1965; Tokudome and Kanno, 1965b; Kuwatsuka et a l . , 1978). Supporting evidence came from Anderson et a l . (1974a) who noted the HA-A fraction to have higher %C, narrower H/C ratios , lower molecular weights and increased resistance to acid hydrolysis compared to the high molecular weight, highly al iphat ic , immature HA-B fract ion. The humic acids obtained in this study were similar in elemental composition to those produced catalyt ical ly by Wang et a l . (1983a) and to the HA-B fraction of Anderson et a l . (1974a). The %C content of the cata lyt ical ly produced humic acids ranged from 47.4 to 53.0 %, with atomic 165 H/C ratios of 1.18 to 1.22. Mean HA-B elemental compositions were %C = 52.3%, %H = 5.82% and atomic H/C = 1.32. For comparison, the HA-A fraction had %C = 54.1%, %H = 4.21% and atomic H/C = 0.92. Both authors concluded that the %C content increased with further humification. In conclusion, there was a clear resemblance in elemental composition between the humic acids extracted from the three vegetation zones and the immature, highly aliphatic humic acids noted above. This lent support to the relationship between the E^/E^ ratio and particle molecular weight discussed in Section 5.1.2.7 . Thus, the low E^/E^ ratios recorded for the humic acids in this study were not a reflect ion of their maturity, but were due to their high particle molecular weight. 5.2.3 Infrared Spectra of the Humic Acid and A Fractions The infrared spectra recorded for the humic acid and A fractions are given in Figure 26. Several factors make the quantitative analysis of infrared absorption bands d i f f i c u l t . F i r s t , the bands are very broad due to the molecular complexity of the molecules. Second, low sample weights are used in the KBr pellets (1-2 mg sample / 300 mg KBr) which results in possible weighing and transfer error, and there are differences among samples in ash content which would be d i f f i c u l t to correct by adjustments in sample weight. And third , i t i s not easy to obtain uniform sample distribution within the KBr pel le t . Hence, only the general features and relative intensities of the absorption bands w i l l be discussed. The major absorption bands in the HA spectra were at 3410, 2910, 2820, 1600, 1380, 1270 and 1035 cm . A reduced number of absorption bands were present in the A fraction spectra with peaks at 3410, 1600, 1380 and 1035 166 ure 26. Infrared Absorption Spectra of the Humic Acid and A Fractions. 4000 3000 2000 1600 1400 1000 4000 3000 2000 1600 1400 1000 800 167 cm . A slight shoulder was vis ible at 2910 cm . Table 18 gives the standard assignments of the infrared absorption bands. The 3410 cm 1 band was assigned to H-bonded OH stretch, a very small amount being due to moisture absorption by the KBr disk. The aliphatic C-H stretch absorb at 2910 c m - 1 . The strong bands at 1600 and 1380 cm"1 replaced the 1720 cm 1 band usually noted for undissociated carboxyl groups. The 1600 and 1380 cm 1 bands were assigned to the carboxylate ion stretch (COO ) (Higashi and Wada, 1977), and this masked possible aromatic C=C vibrat-ions at 1610 cm 1 and the Amide I and II bands at 1620 and 1510 cm 1 , respectively. The shoulder at 1270 cm 1 in the HA spectra possibly indie ated the presence of a few undissociated carboxyl groups. The 1035 cm 1 band has been assigned to the S i - 0 - S i vibrations of s i l i c a t e impurities. This band was much more pronounced in the HA spectra compared to the fraction A spectra which was in agreement with the s i l i c a content of the two fractions. The shoulder on the 1035 cm 1 peak in the HA fractions (exclusive of AH) possibly reflected the C-0 stretch of polysaccharides which increased from the Abies to the Zacatonal zone (Section 5.1.2.6). However, this shoulder was not apparent in the fraction A spectra which had higher %PSS contents. The 1150 cm 1 shoulder in the HA spectra possibly related to ash composition since i t was not apparent in the fraction A spectra. The relative absorption band intensities were similar among the four HA spectra. A s l ight ly sharper 1035 cm 1 peak was noted in AH which was l i k e l y due to i t s higher ash content. Strong aliphatic C-H stretching was noted in a l l HA spectra which corresponded to the higher atomic H/C ratios reported in Table 17. Several authors have reported the presence 168 of this band in the higher molecular weight, more aliphatic humic acid fractions (Butler and Ladd, 1969; Tan and Giddens, 1972; Swift et a l . , 1970). The peak size and shape of the 1600 and 1380 cm 1 bands were very similar reflecting a similar content of carboxyl groups. This was also supported by elemental analysis which fai led to detect any differences in %0 content among the HA samples. The 2910 cm 1 band was reduced to a slight shoulder in the A fraction spectra which confirmed the aromatic nature of this fraction and the efficiency of the PVP separation. Fulvic acids tend to be richer in car-boxyl groups compared to humic acids (Stevenson ,1982). However, carbox-ylate absorption intensities were similar between the HA and A fraction spectra. The A fractions had a s l ight ly higher ash content which could account for this apparent s imilar i ty ; although, both fractions had similar %0 contents. The above relationship possibly included the polysaccharide fraction, r ich in uronic acids, which would explain the discrepancy. The reduced overall intensity of the AA spectrum could not be explained by variations in ash or elemental content, and a lower sample weight or uneven sample distribution in the KBr disk may have been responsible. The infrared spectra and elemental analysis data supported the f o l -lowing conclusions: 1. The humic acids differed l i t t l e in composition among the three vegetation zones. The same was true for the A fractions. 2. The humic acids were highly aliphatic based on the high atomic H/C ratios and aliphatic absorption bands in the infrared spectra. This inferred a high particle molecular weight. 3. The A fractions were dominantly aromatic with low atomic H/C 169 Table 18. Assignments of Infrared Absorption Bands (Stevenson, 1982). Frequency (cm ') Assignment 3400-•3300 0 — H stretching, N — H stretching (trace) 2940-•2900 Aliphatic C — H stretching 1725-•1720 C = 0 stretching of C O O H and ketones (trace) 1660-•1630 C = 0 stretching of amide groups (amide 1 band), quinone C = 0 and/or C = 0 of H-bonded conjugated ketones 1620--1600 Aromatic C = C , strongly H-bonded C = 0 of conjugated ketones? 1590--1517 C O O " symmetric stretching, N — H deformation + C = N stretching (amide II band) 1460--1450 Aliphatic C — H 1400--1390 OH deformation and C—O stretching of phenolic O H , C — H deformation of C H 2 and C H 3 groups, C O O " antisymmetric stretching 1280--1200 C — O stretching and O H deformation of C O O H , C—O stretching of aryl ethers 1170--950 C—O stretching of polysaccharide or polysaccharide-like substances, Si—O of silicate impurities. Table 19. Percent Recovery and Correction Factors for the Identified Phenolic Acids from the Successive Ether/ NaHCCL/ Ether 1 Extraction. Phenolic Acid X recovery SD Correction Factor Protocatechuic 64.2 0.59 1.56 p-Hydroxybenzoic 85.9 0.88 1.16 V a n i l l i c 76.7 0.94 1.30 Syringic 73.3 0.44 1.36 1. Recovery based on three extractions of solutions containing 50 ppm of the above phenolic acids. Duplicate HPLC determinations. 170 ratios and insignificant aliphatic absorption in the infrared. 4. The humic acid and A fractions seemed to differ only in aromat-i c i t y , %N content and molecular weight. They were similar in %0 content and carboxylate absorption intensity. 5. The inverse relationship between the E^/E^ ratio and polymer molecular weight was supported by the aliphatic nature of the humic acid fraction. 6. The solubi l i ty behaviour of the two humus fractions did not stem from differences in elemental content; therefore, differences related to surface area and particle molecular weight were l i k e l y responsible. 5.2.4 Phenolic Acid Hydrolysis Products of the Humic Acid and A Fractions The phenolic acid hydrolysis product data was based on a single hydrolysis and extraction of the products into ether/NaHCO^/ether. The analysis of composite samples did not allow estimates of zone v a r i a b i l i t y ; hence, one hydrolysis was considered sufficient to indicate general trends among the zones. To minimize errors associated with variations in the hydrolysis or extraction procedures the four HA or A samples were treated simultaneously. A recovery test of the four phenolic acids - protocate-chuic, p-hydroxybenzoic, v a n i l l i c and syringic - from the ether/NaHCO^/ ether extraction procedure was done to correct the sample hydrolysate data. The results are shown in Table 19. The recovery of the four phenolic acids ranged from 64.2 to 85.9 % and seemed to reflect the aqueous s o l u b i l i t i e s of the phenolic acids. The reproducibilty of the extraction sequence and the precision of the HPLC measurements was excellent. Standard deviations 171 ranged from 0.44 to 0.94 %. The sample hydrolysates were measured by HPLC under three sets of conditions with periodic replicat ion. Precision was very good, < t 5%. The lower precision for the sample hydrolysates comp-ared to the standards possibly stemmed from matrix effects and the s l ight ly elevated baseline in the former. A recovery test from the hydrolysis stage was not determined since these phenolic acids were not "acid-sensit-ive" (Katase, 1981b). The extraction sequence of ether/NaHCO^/ether served to separate acidic from neutral phenols, the latter being insoluble in aqueous bicar-bonate. Consequently, the microbially derived phenols, phloroglucinol, pyrogallol , resorcinol, etc . , as well as the l ignin derived phenyl propane alcohols and aldehydes would not be determined. Furthermore, the substit-uted cinnamic acid derivatives, p-coumaric, f e r u l i c , synapic, e tc . , would be polymerized during acid hydrolysis; hence, they would not be detected by HPLC (Katase, 1981b). The HPLC chromatograms of the HA and A fraction hydrolysis products are given in Figures 27 and 28. A methanol:H20:acetic acid gradient elut- . ion and a reverse-phase RP-18 column was used for the separation. The chromatographic conditions and phenolic acid retention times were given in Table 2. The reverse-phase column changed the normal elution order found in parti t ion chromatography, and the most polar compounds were eluted f i r s t . Hence, hydroxyl groups decreased while methoxyl groups increased the phenolic acid's retention time. The f i r s t sharp peak evident in the chromatograms was due to a refractive index change as the mobile phase reached the UV detector. The HPLC chromatograms were very simple, and only four major peaks and a number of minor peaks were detected. In 172 Figure 27. HPLC Chromatograms of the Phenolic Acid Hydrolysis Products: the Humic Acid Fraction. 0 1 8 12 T6 20 24 28 30 min Pinus hartwegii Ahl HA 173 Figure 27 Cont. -J 0 i 8 12 16 20 24 28 30 min Pinus hartwegii Ah2 HA 0 4 8 12 16 20 24 28 30 min Zacatonal HA 174 Figure 28. HPLC Chromatograms of the Phenolic Acid Hydrolysis Products: the A Fraction. I I 0 t 8 12 16 20 24 28 30 min Abies religiosa A o' t 8 12 16 20 24 28 30 min Pinus hartwegii Ahl A 175 u r e 28 C o n t . 0 4 8 12 16 20 24 28 30 min Pinus hartwegii Ah2 A 0 4 (3 12 16 20 24 28 30 min Zacatonal A 176 order of appearance, the major peaks were protocatechuic acid, p-hydroxy-benzoic acid, v a n i l l i c acid (the most prominant peak) and syringic acid. It was interesting to note that these four acids were of l ignin or ig in ; although, p-hydroxybenzoic acid and protocatechuic acid could also be of microbial or ig in . The cinnamic acid derivatives and neutral phenols of microbial or flavonoid or igin , which have longer retention times than the benzoic acid derivatives, were not detected even after one hour elution time. Apparent differences in the intensities of the minor peaks among the chromatograms was due to differences in attenuation (AT); AT = 8 for P2H, ZH, AA, and ZA, and AT = 16 for AH, P1H, P1A and P2A. Additional UV-absorbing chromophores such as acidic oligomeric polyphenols produced the s l ight ly elevated baseline. The % hydrolyzed and HPLC hydrolysis product data for the HA and A fractions are given in Table 20. Bar-graphs of the total and individual phenolic acids are given in Figures 29 and 30, respectively. The data was presented as jag phenolic acid per g HA or A carbon. The % HA hydrolyzed ranged from 24 to 31 % which was not substantial compared to the losses of up to 50% for HA's isolated from Chernozemic and volcanic ash s o i l A hor-izons ( R i f f a l d i and Schnitzer, 1973). The 20 hour refluxing in 6 N HC1 used by the above authors was considerably more drastic than the autoclave method used herein. Losses for the AA and P1A samples were similar to the HA samples, 30 and 29 %, respectively. The greatest losses occurred in the P2A (38%) and ZA (58%) samples. The high proportion of polyphenolic material removed during d i a f i l t r a t i o n less than MW 1,000 suggested that these fractions were of lower average molecular weight (Table 16), and perhaps represented a more "immature" or dynamic humus fraction. The Table 20. HPLC Analysis of the Humus Fraction Hydrolysis Products - /ig Phenolic Acid / g C Humic Acid or Fraction A. Sample % Hydrolyzed^ Proto p-Hba Van Syr T o t a l T o t a l (ash free) n fig phenolic acid / g carbon HA or A (ash free) L % HA or A (ash free) AH 25 581 288 2802 160 3831 0.17 P1H 27 736 428 1701 436 3302 0.16 P2H 24 263 329 970 182 1743 0.083 ZH 31 285 502 1254 468 2508 0.12 AA 30 1262 212 1774 170 3417 0.17 P1A 29 1581 416 2234 212 4443 0.23 P2A 38 2115 612 2025 219 4970 0.24 ZA 58 1918 905 2107 501 5431 0.26 1. The Xhydrolyzed i s only an estimate since ; a weight loss measurement on < 0.5 g has a high error p o t e n t i a l i n addition to possible ash content change: s, e t c . 2. Corrected for recovery from ether / NaHC03 / ether e x t r a c t i o n (Table 19). 178 Figure 29. HPLC Analysis of the Humus Fraction Hydrolysis Products -Total yug Phenolic Acids / g C in the Humic Acid and A Fractions, 5000 • 4000 3000 pg/gC 2000 1000 AH P1H P2H ZH PIA P2A ZA 179 Figure 30. HPLC Analysis of the Humus Fraction Hydrolysis Products ;jg Phenolic Acid / g C Humic Acid or Fraction A. 2800 2400 2000 1600 1200 800 400 Van COOH OH AH PIH P2H ZH AA PIA P2A ZA 2000 1600 1200 >"g/gC 800 Proto COOH OH OH 400 AH PIH P2H ZH AA PIA P2A ZA 180 Figure 30 Cont. 1000 p-Hba COOH 800 600 pg/gC 400 200 AH P1H P2H ZH AA P1A P2A ZA 500 400 300 . Mg/gC 200 100 Syr COOH CH30 OH AH P1H P2H ZH AA P1A P2A ZA 181 increase in % A hydrolyzed from the Abies to the Zacatonal zone possibly stemmed from differences in aliphatic content since the % glucose increased along the above sequence. However, the IR spectra and atomic H/C ratios did not support this . The total phenolic acids hydrolyzed were similar in magnitude to hydrolysates of whole mineral soi ls (Katase, 1981c; Whitehead et a l . , 1982) and to HA and FA fractions (Hartley and Buchan, 1979; Hanninen et a l . , 1981). Peat soi ls were of an order of magnitude greater (Katase, 1981a,c). The phenolic compounds readily extracted by water, dilute acid and base amounted to less than 10% (often less than 1%) of the total phenolic compounds obtained by more vigorous hydrolytic procedures (Katase, 1981a,c; Whitehead et a l . , 1982), and represented a more dynamic humus pool import-ant to allelopathic studies. The latter forms were more strongly bound to the humus fractions and constituted the largest pool. The total phen-ol i c acids identif ied on a % HA or A (ash free) basis were much less than 1%; HA = 0.083 to 0.17 % and A = 0.17 to 0.26 %. In light of the following factors this was not surprising. 1. The degradation of l i g n i n by microorganisms was thought to involve dearomatization of the l ignin macromolecule with microbial uptake of the released simple aliphatic compounds (Section 2.5-3). During this process few simple phenols would be released to the s o i l solution. This was supported by the low levels of extract-able phenolic compounds, in the order of jag/g, from plant roots and decomposing plant residues (Myskow and Morrison, 1964; White-head et a l . , 1982; Whitehead et a l . , 1983; Kuwatsuka and Shindo, 1973). Much of the easily extractable forms would exist free in 182 the plant tissues or as phenolic esters. These would be rapidly u t i l i z e d by the microbial population. 2. The phenols released would be actively transformed by the micro-bial population through -oxidation of the propane side chain, decarboxylation, demethylation, hydroxylation and ring f ission (Sections 2.5.1 and 2.5.3). 3. Reactive phenols with the ortho or para hydroxy grouping would be readily oxidized to quinones and participate in nucleophilic addition and polymerization reactions. 4. Phenols containing free positions ortho or para to the phenolic hydroxyl group would participate in phenol coupling reactions with the formation of C-C and C-0 covalent bonds stable to acid hydrolysis (Section 2.4.2). Further condensation-type reactions would lead to stable cycl ic structures forming the humic acid "core". 5. The catalytic action of clay-sized minerals and hydrous oxides, allophanes, e tc . , would favour processes 3 and 4 (Wang et a l . , 1983a,b). Furthermore, inorganic surfaces may act as "templates" to s ter ica l ly align components for polymerization reactions. On the other hand, inorganic constituents may serve to protect phenolic and other compounds from further degradation by inhib-i t t i n g extracellular enzymes (Aomine and Kobayashi, 1964, 1966). Thus, large aliphatic polymers containing attached phenolic compounds could be formed such as Anderson et a l ' s . (1974a) HA-B fract ion. 183 Thus, the concentration of hydrolyzable phenolic acids in the humus fractions would represent the balance between phenolic acid release and microbial degradation, transformation and conversion into non-hydrolyzable forms. Evidence suggests that very few simple phenolic compounds remain in the s o i l solution for long in a free state (Katase, 1981a-c; Haider and Martin, 1975; Shindo and Kuwatsuka, 1975a; Myskow and Morrison, 1964). The % phenolic acids in the HA and A fractions were equal in the Abies zone. In the Pinus and Zacatonal zones the % phenolic acids in the A fractions were considerably greater than the HA fractions. In the HA fractions the % phenolic acids decreased in the following order; AH > P1H > ZH > P2H. The distribution among the A fractions differed consider-ably from the HA fractions with the least % phenolic acids being present in AA. A significant increase was noted in P1A, P2A and ZA which were not s ignif icant ly different . The concentration of ac id- labi le phenolic acids in the humus fractions depends on a number of factors. 1. The net balance between phenolic acid production and microbial degradation or transformation. 2. The sensi t ivi ty of the individual phenolic compounds to phenol coupling reactions. The phenolic compounds which form C-C and .. C-0 covalent bonds w i l l not be released by acid hydrolysis; hence, they w i l l not be part of this pool. 3. The HA or A molecular size as i t affects surface area, accessib-i l i t y of bonding si tes , and so lu b i l i ty behaviour. 4. The rate of conversion of attached phenolic compounds into non-hydrolyzable forms. 184 5. The % distribution of the humus fractions. 6. The s o i l pH as i t affects microbial ac t iv i ty , reaction mechanisms, polymer configuration and surface area. The higher concentration of phenolic acids in the A fraction compared to the HA fractions was primarily due to their smaller molecular size, aqueous so lubi l i ty and low %Ca dis t r ibut ion. The Ch/Ca ratios ranged from 2.0 to 3.8 (Table 12); hence, when calculated on a total s o i l basis the phenolic acids associated with the HA fractions were 1.2 to 3.5 times that of the A fractions. Thus, the phenolic acids were preferentially stabilized into the HA fract ion. The % phenolic acids in the A fractions increased signif icantly with the introduction of grass vegetation. This possibly stemmed from a change in the nature of the A fractions or the s o i l environment. Previous data indicated that the polymeric nature of the A fractions decreased from the Abies to the Zacatonal zone (Sections 5.1.2.7 and 5.2.1). For instance, 1% the E ^ Q Q A optical data supported a decrease in molecular conjugation with the zonal sequence Abies to Zacatonal. However, the atomic H/C ratios did not reflect a difference in aromaticity. The fraction of molecules < MW 1,000 also increased s ignif icantly along the above zonal sequence, and the % hydrolyzed was much greater in P2A and ZA compared to AA and PIA. Consequently, the A fractions in the Pinus and Zacatonal zones seemed of lower average molecular weight and represented a more dynamic humus pool. Several authors have noted a positive relationship between HA mole-cular weight and the amount of phenolic acid degradation products (Piper and Posner, 1972b; Tate and Anderson, 1978). An inverse relationship to 185 the degree of humification has also been reported (Tate and Goh, 1973; Hanninen et a l . , 1981; Katase, 1981a-c). The HA's in this study have been shown to be rather immature, highly aliphatic and of high average molecular weight. Perceptible differences among the four HA's in the amounts of phenolic acids released during acid hydrolysis seemed not to be related either to differences in molecular weight or to the degree of humification. In fact, the E ^ / E ^ optical data indicated that the average polymer mole-cular weight increased from the Abies to the Zacatonal zone which corres-ponded to a decrease in % hydrolyzable phenolic acids. Also, there were 1% no significant differences in "aromaticity" by E ^ Q Q HA optical data or atomic H/C ratios among the HA samples. The supply of simple polyphenols to the coniferous s o i l zones would be great due to the humus layers and pine l i t t e r . In Pinus Ah^ microbial act ivi ty would be reduced by the acid pH. Less influence from the pine l i t t e r would be present in Pinus Ah^ which was supported by lower %Ca and increased pH. The higher pH would favour microbial act ivi ty with fewer simple polyphenols being released during grass root decomposition. On a total s o i l basis this fraction contained the least phenolic acids (Table 22). The similari ty in % phenolic acids between AH and P1H was due to the high %Ch in Pinus Ah^; consequently, there was a dilution of the phenolic acids. However, when calculated on a total s o i l basis the phenolic acids associated with P1H were twice that in AH (Table 22). In the Zacatonal zone the s o i l pH was favourable to microbial act ivi ty and the lower Ca/Cf ratio suggested polymerization into HA's or degradation of the released polyphenols. Thus, the concentration of phenolic acids in the HA fractions was related to the net release of polyphenols to the 186 s o i l . This was determined by the vegetation site factor. The four major phenolic acids identif ied in this study appeared to be ubiquitous among soi ls (Katase, 1981a-c; Whitehead et a l . , 1982, 1983; Hanninen et a l . , 1981). Their distribution and form in the s o i l differed with the s o i l type (forest humus versus peat (Katase, 1981a-c)), the source of vegetation (Katase, 1981a-c; Hanninen et a l . , 1981; Morrison, 1958, 1963; Burges et a l . , 1967), the degree of humification (Tate and Goh, 1973) and polymer molecular weight (Tate and Anderson, 1978). Their origins were discussed at length in the li terature review (Sections 2.4 and 2.5) but w i l l be brief ly re-capitulated here. In the l ignin macromolecule the above acids existed as cinnamyl alcohol derivatives. During oxidative degradation of the l ignin polymer a small portion of the cinnamyl alcohols, free or s t i l l attached to the macromolecule, would be oxidized to form cinnamic acid derivatives. /3 -oxidation of the propionic side chain form-ed the substituted benzoic acid derivatives l is ted above. Further react-ions included demethylation, hydroxylation and decarboxylation. These reactions occurred at a later stage in the degradation sequence (Hurst and Burges, 1967). The methoxylated phenolic acids, v a n i l l i c and syringic, have not been synthesized in vitro by microorganisms; hence, they were largely derived from l i g n i n with small contributions from the B ring of flavonoids (Burges et a l . , 1964). Precursors to v a n i l l i c acid would also include ferul ic acid bound as esters to monocotyledon l ignin (Shindo and Kuwatsuka, 1975a). Protocatechuic acid had three possible origins; de novo synthesis by microorganisms from p-hydroxycinnamic acid, demethy-lation of v a n i l l i c acid or hydroxylation of p-hydroxybenzoic acid. Caffeic acid, an intermediate in the biosynthesis of l i g n i n , may contribute in 187 minor amounts. P-hydroxybenzoic acid has been synthesized in small amounts by several fungi imperfecti (Haider et a l . , 1972), but was largely derived from p-coumaryl residues, abundant as esters in grass lignins (Shindo and Kuwatsuka, 1975a). The l ignin composition of plants was reviewed in Section 2.4.3. Gymnosperm l ignin was found to contain dominantly coniferyl (guaiacyl) units with a low amount of p-coumaryl units . Syringyl units were low or absent. Angiosperm dicotyledons (deciduous species including numerous shrubs and forbs) contained approximately equal amounts of coniferyl and syringyl units, p-coumaryl units being very low. The angiosperm monocot-yledons contained approximately equal contributions from a l l three units . However, considerable amounts of p-coumaric acid and ferul ic acid were bound as esters to the grass l ignins (Crawford, 1981; Shindo and Kuwatsuka, 1975a). These units were readily released by mild hydrolysis and would be actively u t i l i z e d by the microbial population. The phenolic acids released during acid hydrolysis would be bound to the humic polymers by " a c i d - l a b i l e " bonds such as esters, amides, phenolic glycosides and peptides. In the larger HA molecules these polyphenols would exist primarily as peripheral units. Covalent C-C bonds, ethers (except benzyl ethers) and biphenyl or cycl ic structures would not be affected. Simple phenolic acids attached by salt bridges, e tc . , or mole-cules solubilized from non-humified organic matter including phenolic acids weakly attached to the l ignin polymers, would be removed in the non-polymeric fractions during d i a f i l t r a t i o n . Polymeric l ignin fragments solubilized during alkaline extraction would precipitate with the HA fract -ion. The dominance of phenyl propane alcohols and the acid-resistant 188 nature of i t s major bond types (Figure 10) would prevent significant cont-ributions from unaltered plant l ignin to the phenolic acid hydrolysis products. Thus, the phenolic acid hydrolysis products were an integral part of the polymeric humic substances. The % distribution of the phenolic acids as a total of the phenolic acids identif ied i s given in Table 21. The weight of phenolic acids per 100 g s o i l i s given in Table 22, which corrects for variations in humus fraction amounts. Abbreviated designations of the four major phenolic acids are: Protocatechuic acid Proto p-Hydroxybenzoic acid p-Hba V a n i l l i c acid Van Syringic acid Syr V a n i l l i c acid was dominant in the HA fractions, representing between 50 and 73 % of the total phenolic acids ident i f ied . The concentration of v a n i l l i c acid in the HA fractions decreased sharply in the Pinus hartwegii and Zacatonal zones with the introduction of grass vegetation. The v a n i l l i c acid content of P1H, P2H and ZH were not s ignif icantly different; 50 to 56 % of the total phenolic acids identif ied (Table 21). When c a l -culated on a total s o i l basis (Table 22) the content of v a n i l l i c acid and i t s demethylated product, protocatechuic acid, were substantially greater in AH and P1H compared to P2H and ZH. Increased production of polyphenols from the forest l i t t e r and reduced microbial ac t ivi ty favoured the accumulation of phenolic acids in the humic acid fractions. The concentration of v a n i l l i c acid in the A fractions was high, 189 Table 21. HPLC Analysis of the Humus Fraction Hydrolysis Products -Phenolic Acids as % of Total Phenolic Acids Identified. Sample Proto p-Hba Van Syr AH 15.2 7.5 73.1 4.2 PIH 22.3 13.0 51.5 13.2 P2H 15.1 18.9 55.7 10.4 ZH 11.4 20.0 50.0 18.6 AA 36.9 6.2 51.9 5.0 PIA 35.5 9.4 50.3 4.8 P2A 42.6 12.3 40.7 4.4 ZA 35.3 16.7 38.8 9.2 Table 22. HPLC Analysis of the Humus Fraction Hydrolysis Products -/ i g Phenolic acid / 100 g s o i l . Sample %Ch or %Ca Proto p-Hba Van Syr Total AH 0.59 344 170 1659 95 2268 PIH 1.21 890 518 2058 528 3994 P2H 0.53 140 175 515 97 927 ZH 0.62 176 311 776 290 1553 AA 0.19 239 40 335 32 646 PIA 0.29 452 119 639 61 1271 P2A 0.15 317 92 304 33 746 ZA 0.11 219 103 240 57 619 1. Data from 0.1 N NaOH test extraction procedure. 190 accounting for 38 to 52 % of the total phenolic acids identif ied (Table 21). However, on a total s o i l basis the HA fractions were much richer in v a n i l l i c acid, particularly in the Abies zone and Pinus Ah^ horizons (Table 22). Extensive demethylation of v a n i l l i c acid to form protocat-echuic acid was noted in the A fractions. The Proto/Van ratio shown in Table 23 indicated that demethylation was 3 to A times greater in the A fractions compared to the HA fractions. This possibly stemmed from the lower molecular weight and greater so lubi l i ty of the A fractions which increased their accessibi l i ty to microbial enzymes. The extent of demethylation was greater in P2A and ZA which seemed to reflect their less polymeric nature, and perhaps greater level of microbial a c t i v i t y . When combined, v a n i l l i c acid and protocatechuic acid represented 61.A to 88.3 % of the total phenolic acids in the HA fractions, and 7A.1 to 88.8 % of the total phenolic acids in the A fractions (Table 21). This high contribution was not unexpected in the Abies rel igiosa zone where coniferous vegetation was the dominant source of organic matter. However, lower contributions would be expected in the Pinus hartwegii and Zacatonal zones where grass vegetation was s ignif icant . If the average composition of monocotyledon l ignin was used as a guide for the Zacatonal zone, contributions from v a n i l l i c acid and protocatechuic acid nearer to 33% would be expected. Instead, 61.4% (HA) and 74.1% (A) of the total phenolic acids were contributed by the above two acids. Therefore, these two acids were selectively enriched in the s o i l humus fractions compared to p-hydroxybenzoic acid and syringic acid. To understand this phenomenon the process of l ignin degradation and the fate of the individual monomers must be examined. 191 In the l ignin macromolecule coniferyl units are more highly condensed compared to syringyl units due to blocking with methoxyls of the positions ortho to the phenolic hydroxyl group in the la t ter . P-coumaryl units are also more condensed, but their contribution to the l ignin structure i s minimal. Consequently, during the oxidation of l ignin with nitrobenzene syringyl units are preferrentially s p l i t - o f f (Gross, 1979). A similar situation may be present in the s o i l during oxidative degradation of l i g n i n by microorganisms. Once released the syringyl units would be rapidly oxidized, decarboxylated and demethylated to form pyrogallol . The vic inal trihydroxy grouping of this molecule induces rapid polymer-ization through quinones or phenol coupling reactions. Hence, very few syringyl residues would be bound to the humus polymers in ac id- labi le form. As previously noted, p-coumaryl units actually incorporated into the l i g n i n framework were few (Section 2.4.3). The p-coumaryl residues were largely derived from p-coumaric acid which forms esters with the l ignin structure. Mild extraction of undecomposed plant material released p-coumaric and ferul ic acids in considerable amounts (Kuwatsuka and Shindo, 1973; Myskow and Morrison, 1964; Whitehead et a l . , 1982). These easily extractable forms would be readily released and degraded by the microbial population. The mild extraction of peat and forest s o i l (Katase, 1981a-c), and the FA fraction from a s o i l underlying ryegrass (Hartley and Buchan, 1979) indicated extensive beta-oxidation of the released p-coumaric and ferul ic acids to form p-hydroxybenzoic and v a n i l l i c acids. The reactivity of the individual phenolic acids w i l l also determine their susceptibil i ty to phenol coupling reactions; and hence, resistance to acid hydrolysis. The reactivity of phenolic compounds i s related to 192 their chemical structure. The rate of polymerization decreases in the following order: v ic inal trihydroxy ( ie . pyrogallol) > v ic inal dihydroxy ( ie . protocatechuic acid, catechol) > monohydroxy ( ie . benzoic acid) . Electron attracting groups such as carboxyl w i l l decrease the rate of polymerization whereas electron donating groups such as methyl or methoxyl w i l l increase the rate (Wang et a l . , 1983a). Steric factors w i l l also influence both the rate and mode of coupling. P-hydroxybenzoic acid i s not readily coupled; hence, i t s reactivity w i l l depend on i t s rate of hydroxylation to form protocatechuic acid. Protocatechuic acid, once decarboxylated to form catechol, would rapidly polymerize. Protocatechuic acid i s also a key intermediate in the cleavage of aromatic compounds by microorganisms (Section 2.5.3). Thus, the balance of the above processes w i l l determine the persistence of intact phenolic acids in the system. By and large, v a n i l l i c acid seems to be the most persistent monomer. The concentrations of p-hydroxybenzoic and syringic acids were highest in PIH and ZH. The concentrations in AH and P2H were not s t a t i s t i c a l l y different (Figure 30 and Table 20). When calculated on a total s o i l basis p-hydroxybenzoic acid and syringic acid were considerably less in the Abies zone compared to the Pinus and Zacatonal zones (Table 22). In the Abies zone forbs and shrubs would contribute largely to this pool. The % distribution of p-hydroxybenzoic acid increased s ignif icantly from PIH to P2H which was similar to ZH. Syringic acid decreased s l ight ly from PIH to P2H and was s ignif icant ly greater in ZH. The differ ing behaviour of p-hydroxybenzoic acid and syringic acid, which were derived from the grass vegetation in the Pinus zone, possibly was due to biological effects related to pH. In Pinus Ah1 the lower pH possibly reduced demethy-193 lation of syringic acid which would be more prevalent in Pinus Ah^ due to a higher pH. The lower biological act ivi ty in Pinus Ah^ may have allowed downward movement of p-hydroxybenzoic acid into Pinus Ah^. P-hydroxybenzoic acid has been observed to be rapidly leached from s o i l columns (Shindo and Kuwatsuka, 1976). In the A fractions p-hydroxyben-zoic acid increased signif icantly from AA to PIA to P2A to ZA (Table 20). The highest concentration of syringic acid was in ZA, the other three A fractions being not s ignif icant ly different . The above trends were also evident in the % distribution of syringic and p-hydroxybenzoic acids in the A fractions (Table 21). Thus, the influence of grass vegetation in the polymeric HA and A fractions was directly evident in the Pinus hartwegii and Zacatonal zones. The relationship between the nature of the vegetation and the phenolic acid hydrolysis products was particularly evident from ratios of the phenolic acids (Table 23 and Figure 31). The calculation of phenolic acid ratios eliminated variations in the total amounts of phenolic acids by putting a l l samples in a relative frame of reference. The effects of vegetation were particularly evident in the p-Hba/Van and p-Hba/Proto ratios which increased with grass influence. The above ratios in both the HA and A fractions increased considerably from A to PI to P2 to However, the p-Hba/Proto ratios were very low in the A fractions due to the high proportion of protocatechuic acid. Hence, in the A fractions this ratio was not very r e l i a b l e . The ratios of Syr/Van and Syr/Proto emphasized grass influence in the HA samples, but fai led to distinguish between the Abies and Pinus A fractions. However, higher ratios were noted for the Zacatonal zone. The ratios in the HA fractions separated the 194 three zones; Zacatonal > Pinus hartwegii > Abies re l ig iosa . The relative influence of grass in the Pinus Ah^ and Ah^ horizons was not clearly established. Nevertheless, grass influence was present in both horizons confirming the importance of in situ grass root decomposition to HA form-ation in the Pinus hartwegii zone. 195 Table 23. HPLC Analysis of the Humus Fraction Hydrolysis Products -Calculated Ratios of the Major Phenolic Acids Identified. Sample p-Hba/Van p-Hba/Proto Syr/Van Syr/Proto Proto/Van AH 0.10 0.50 0.057 0.28 0.21 P1H 0.25 0.58 0.26 0.59 0.43 P2H 0.34 1.25 0.19 0.69 0.27 ZH 0.40 1.76 0.37 1.64 0.23 AA 0.12 0.17 0.096 0.14 0.71 P1A 0.19 0.26 0.095 0.13 0.71 P2A 0.30 0.29 0.11 0.10 1.04 ZA 0.43 0.47 0.24 0.26 0.91 196 Figure 31. HPLC Analysis of the Humus Fraction Hydrolysis Products -Calculated Ratios of the Major Phenolic Acids Identif ied. p-Hba / Van 0.5 0.4 0.3 0.2 o. 1 AH PIH P2H ZH PIA P2A ZA p-Hba / Proto 2.0 1.5 • 1.0 0.5 - —, AH PIH P2H ZH AA H_JL PIA P2A ZA 197 Figure 31 Cont, 0.4 Syr / Van 0.3 0.2 o. 1 AH PIH P2H ZH AA PIA P2A ZA Syr / Proto 1.5 1.0 0.5 n n AH PIH P2H ZH AA PIA P2A ZA 198 6.0 CONCLUSION In Part 1 the most recent ash of Popocatepetl was established as the parent material for s o i l formation in the study area. The low cont-ent of organic matter and extractable Fe, Al and Si indicated the soils were at an early stage in their development. Nevertheless, the degree of mineral weathering increased with elevation and with depth in the pro-f i l e . In the surface mineral horizons humus-Al complexes were dominant supporting the theory of Wada and Highashi (1976) that in the early stages of s o i l formation the Al and Fe released by weathering of the ash existed largely as Fe-, Al-humus complexes. "Amorphous" aluminosilicate minerals such as allophane were ins ignif icant . In the Abies rel igiosa and Zacatonal zones horizon differences in organic matter characteristics were not apparent; hence, the humus in the Ahj and A l ^ horizons represented one population. However, horizon differences were noted for several parameters in the Pinus hartwegii zone which seemed to reflect the mixed coniferous-grassland ecosystem. The humus content differed l i t t l e among the three vegetation zones, although qualitative differences related to the vegetation site factor in the kinds of organic components were apparent. The distribution of polysaccharide and polyphenolic components in the fulvic acid fraction was clearly related to vegetation-type. The alpine location of the s o i l zones favoured the accumulation of fulvic acid polyphenols; hence, Ca/Cf ratios were higher than those reported for temperate Ah horizons (Lowe, 1980). The Ch/Cf ratio was not influenced by the vegetation site factor, but was related to the regional climate. The Ch/Cf ratio of these recent volcanic 199 ash soi ls was comparable to more well developed Andosols which suggested this ratio to be an i n t r i n s i c property of the s o i l from i t s youngest stage. 1% The E ^ Q Q HA optical data indicated that the humic acids from the three vegetation zones were similar in maturity; hence, zonal differences 1% in EgQQ HA and E^/E^ HA were related to the average polymer molecular weight which increased from the Abies rel igiosa to the Zacatonal zone. The optical characteristics of the humic acid fraction in the Pinus hart-wegii Ah^ and Ah^ s o i l horizons seemed to stress a difference in organic matter source for humus formation or the s o i l environment. Consequently, the humic acids in Pinus Ah^ were closer in optical characteristics to the Zacatonal zone. Fraction A had lower absorbance in the v is ible region which was consistent with i t s lower molecular weight and limited degree of conjugat-ion. However, optical characteristics suggested a more polymeric fraction A in the coniferous s o i l zones compared to the Zacatonal zone. This was substantiated in Part 2 from the molecular size distribution of the bulk isolated A fractions. Elemental and infrared analysis of the bulk isolated humic acid and fraction A, in combination with the optical properties found in Part 1, demonstrated a difference between the two humus fractions in molecular weight, aromaticity and %N content. %C, %0 and carboxylate absorption intensities were similar between the two fractions. Elemental and infrared analysis fa i led to detect any zonal differences in the humic acid and fraction A. Nevertheless, the data supported the rapid formation of a high molecular weight, highly aliphatic humic acid fraction r ich in nitrogen 200 in these recent volcanic ash s o i l s . The phenolic acid hydrolysis product data established a l i g n i n -derived component of the polymeric humic acid and fraction A. The participation of l ignin in humus formation was through microbial degrad-ation of the l ignin macromolecule and transformation of the released simple polyphenols. 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Abies rel igiosa Deoth Horizon Description 11-0 FH "Very dark grayish brown (10YR 3/2 dry)" 0-1] Ahj "Black (10YR 2/1); dark gray (10YR 4/1 dry); sandy loam; very weak granular and very fine and fine angular blocky; loose, s l ight ly sticky, s l ight ly plast ic , thixotrophy not detectable; abundant fine roots, plent i ful medium roots; abundant fine and medium pores; separation from underlying horizon gradual and wavy with tongue extending to 40 cm downward" 11-23 Ah 2 "Black (10YR 2/1); dark gray (10YR 4/1 dry); sandy loam; very weak fine subangular blocky; loose, s l ight ly sticky, s l ight ly plast ic , thixotrophy not detectable; plent iful fine and medium roots; very few coarse roots; abundant fine and medium pores; very few 1 cm charcoal fragments; separation from underlying horizon gradual and wavy" 23-43 Ah 3 "Black (10YR 2/1); dark gray (10YR 4/1 dry); loamy sand; very weak medium subangular blocky; loose, not sticky, s l ight ly plast ic , s l ight ly smeary, thixotrophy not detectable; very few fine, medium and coarse roots; abundant fine and medium pores; mottling: reddish, diffuse, rotted root part ic les ; separation from under-lying horizon gradual and f l a t " 43-63 Ah^(B) "Black (10YR 2/1); dark gray (10YR 4/1 dry); loamy sand; weak coarse subangular blocky; loose, s l ight ly sticky, s l ight ly plas t ic , smeary, thixotrophy not detectable; very few fine and medium roots, few coarse roots; abundant medium pores, plent i ful fine pores; mottling: reddish, diffuse, rotted root part ic les ; frequent small charcoal; frequent small pumice; separation from underlying horizon gradual and f l a t " 63+ IP pumice "Very dark brown (10YR 2/2); dark grayish brown (10YR 4/2 dry); loamy sand; firm medium subangular blocky; s l ightly smeary, thixotrophy not detectable; plent iful coarse roots; abundant medium pores, p lent i ful fine pores; abundant ocre coloured highly weathered pumice gravel uniformly distributed; very few small charcoal" 219 Pinus hartwegii Depth Horizon cm Description 11-0* 0-12 FH Ah 1 12-41 Ah, 41-53 Ah, 53-80 Ah, 80+ 1P pumice "Very dark grayish brown (10YR 3/2 dry)" "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loam; very weak medium subangular blocky; loose, s l ight ly sticky, s l ight ly plast ic , thixotrophy not detectable; abundant very fine and fine roots, plent-i f u l medium roots; abundant fine pores; frequent small charcoal; separation from underlying horizon abrupt and f l a t " "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loam; very weak medium angular blocky; loose, s l ight ly sticky, s l ight ly plas t ic , thixotrophy not detectable; p lent i ful very fine and fine roots; abundant fine and medium pores; separation from underlying horizon gradual and f l a t " "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loam; very weak granular and fine angular blocky; loose, s l ight ly sticky, s l ight ly plast ic , thixotrophy not detectable; very few very fine roots, p lent i ful medium roots, very few coarse roots; abundant fine and medium pores; very few subrounded weathered pumice gravel, frequent small pumice; few small charcoal; separation from underlying horizon gradual and f l a t " "Black (10YR 2/1); very dark gray (10YR 3/1 dry); sandy loam; moderate fine angular blocky; very f r iable , s l ight ly sticky, s l ight ly plast ic , thixotrophy not detectable; very few fine, medium and coarse roots; abundant fine and medium pores; frequent small pumice; frequent small charcoal; separation from underlying horizon abrupt and wavy" "Very dark grayish brown (2.5Y 3/2); grayish brown (2.5Y 5/2 dry); loam; moderate fine and medium angular blocky; very f r i a b l e , very sticky, s l ight ly plas t ic , thixotrophy not detectable; p lent i ful fine roots, very few coarse roots; abundant fine and medium pores; very few subrounded pumice gravel, abundant small pumice; very few 1 cm charcoal fragments, abundant small charcoal" The modal pit FH horizon was thicker than average for the zone. Most s o i l pits had l i t t l e FH but contained an F horizon of dominantly pine needles. 220 Zacatonal Depth Horizon Description cm  0-4 Ah "Black (10YR 2/1); very dark gray (10YR 3/1 dry); sandy loam; very weak granular and fine angular blocky; loose, s l ight ly sticky, s l ight ly plas t i c , s l ight ly smeary, thixotrophy not detectable; plent i ful fine and medium roots; abundant fine and medium pores; horizon frozen following snowfall and frost , not usually frozen; frequent small pumice; separation from underlying horizon abrupt and f l a t " 4-34 Ah 2 "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loamy sand; very weak granular and fine angular blocky; loose, s l ight ly sticky, s l ight ly plas t i c , very s l ight ly smeary, thixotrophy not detectable; plent i ful fine and medium roots; abundant fine and medium pores; very few subrounded, s l ight ly weathered yellowish pumice gravel, frequent small pumice; separation from underlying horizon gradual and f l a t " 34-47 Ah 3 "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loamy sand; very weak angular blocky; very f r i a b l e , s l ight ly sticky, s l ight ly plas t ic , smeary, thixo-trophy not detectable; few fine roots, p l e n t i f u l med-ium roots; abundant fine and medium pores; very few subrounded s l ight ly weathered yellowish pumice gravel, abundant small pumice; separation from underlying horizon gradual and f l a t " 47-64 Ah 4 "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loamy sand; very weak angular blocky; very f r i a b l e , s l ight ly sticky, s l ight ly plas t ic , smeary, thixo-trophy not detectable; few fine roots, p l e n t i f u l medium roots; abundant fine and medium pores; few subrounded weathered yellowish pumice gravel with grouping tendency, abundant small pumice; separation from underlying horizon gradual and f l a t " 64-73 Ah 5 "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loamy sand; very weak angular blocky; very f r i a b l e , s l ight ly sticky, s l ight ly plas t ic , thixotrophy not detectable; few medium roots; abundant fine and medium pores; abundant small pumice" 221 Appendix 2. Humus Fractionation Procedure. Extractant: 0.1 M sodium hydroxide - 0.1 M sodium pyrophosphate. Method: Duplicate sample extraction to isolate enough polyphenolic fraction A. 1. Weigh sample containing 0.5 g organic carbon into 250 ml centrifuge bottle. Add 150 ml extractant. Cap. Shake overnight on reciprocal shaker. 2. Centrifuge at 6000 rpm (5860 RCF) for 15 minutes. Decant alkaline extract into 1L beaker. 3. Re-extract residue with 100 ml extractant for 1 hour with reciprocal shaking. Centrifuge and decant alkaline extract as before, combining with f i r s t extract. Discard residue. 4. Acidify alkaline extract to pH 1.5 ± 0.02 with 6 N H S0^. Let stand for 30 minutes. Pour half of extract into 2-250 ml centrifuge bottles Centrifuge and decant supernatant FA into 1L beaker. Pour second half of ac idif ied alkaline extract into same centrifuge bottle. Centrifuge and decant FA as before. 5. Dissolve precipitate (HA) in centrifuge bottle with small amount of 2 N NaOH. Once dissolved, dilute with d i s t i l l e d water to —' 100 ml. 6. Repeat step 4. 7. F i l t e r FA through Whatman #1 f i l t e r paper into 1L volumetric f lask. Make to volume. 8. Re-dissolve HA precipitate in centrifuge bottle with a small amount 222 of 2 N NaOH. Make to volume depending on amount (100, 250, 500 ml) with d i s t i l l e d water. Centrifuge about 200 ml of HA at 6000 rpm for 20 minutes. Use HA to balance bottles, not d i s t i l l e d water! F i l t e r through Whatman #1 f i l t e r paper into erlenmeyer. PVP Separation of FA Fraction and Isolation of Fraction A: 1. Measure 100 ml of FA and save for carbon analysis. Pour remaining 900 ml FA into 1L erlenmeyer containing 9.0 g washed and dried PVP. Let stand 30 minutes, swirling intermittently. Let PVP set t le . F i l t e r about 250 ml supernatant through Whatman #1 and save this fraction C for carbon analysis. Place 1L beaker under funnel to catch remaining fraction C and washings. Replace f i l t e r paper with a fresh one. Poke hole in used f i l t e r paper and rinse any PVP back into the 1L erlenmeyer with dilute H2S0^. F i l t e r remaining supernatant rinsing PVP into f i l t e r paper. Wash pad of PVP several times with dilute H2S0^ to remove any remaining fraction C. Discard f i l t r a t e . 2. Desorb fraction A: Using a metal spatula scoop out PVP into a 100 ml beaker. Poke hole in f i l t e r paper and rinse paper with 0.1 N NaOH into beaker. Add 30 ml 2 N NaOH to PVP, s t i r , and let soak for 20 minutes. F i l t e r off PVP through Whatman #1 paper. Collect fraction A in a 100 ml volu-metric f lask. Rinse PVP pad with 0.1 N NaOH being careful not to go over the 100 ml mark. Make to volume. Measure carbon content of extracts by the Walkley-Black Wet Oxidation Procedure. 223 Appendix 3. S o l u t i o n Carbon Analyser — Walkley-Black Wet O x i d a t i o n The Astro S o l u t i o n Carbon Analyser uses a low temperature UV-promoted chemical o x i d a t i o n of e x t r a c t carbon with IR d e t e c t i o n of the released Q^. The oxidant used i s 1 M sodium persulphate. C a l i b r a t i o n i s based on e x t e r n a l standards with three ranges of ppm C c o n c e n t r a t i o n ; 0.1-100 ppm C, 100-500 ppm C and 500-2500 ppm C. A f i x e d i n j e c t i o n loop of the c o r r e c t volume i s determined by the range s e l e c t e d . Data output i s micro-processor c o n t r o l l e d . R e p r o d u c i b i l i t y i s ± 2% or b e t t e r . The Walkley-Black Wet Oxidation procedure uses chromic a c i d as the oxidant which i s produced by a c i d i f i c a t i o n of the dichromate species w i t h concentrated H^SO^. The oxidant l e f t unused i s determined by t i t r a t i o n with f e r r o u s sulphate, f e r r o i n being used as the i n d i c a t o r . R e p r o d u c i b i l i i s very good, 2-5 %. Two basic assumptions are used i n t h i s method. 0 4+ 1. The o x i d a t i o n s t a t e of carbon changes by +4 (C to C ), which assumes an i n i t i a l o x i d a t i o n s t a t e of zero. 2. The e f f i c i e n c y of o x i d a t i o n i s 80%. The redox r e a c t i o n f o r the o x i d a t i o n i s : Comparison f o r E x t r a c t Organic Carbon. ^ 4Cr + 3C0 2 + 8H 20 Half r e a c t i o n s : Cr 6+ + 3e ->Cr 3+ o 12 e l e c t r o n t r a n s f e r C 1 C + 4e ^•Equivalent weight of C 12 g C / mwt C = 3 g C / equiv 4 equiv C / mwt C ppm C i n E x t r a c t = (2.5 ml FeS0 4 x N FeS0 4) x 3 x 1.25 x 10 ml e x t r a c t ** 80% o x i d a t i o n e f f i c i e n c y 224 Humus fractions isolated by the method of Lowe (1980) from four Chernozemic Ah horizons and four Andosol Ah horizons (from this study) were analyzed by the two methods. The results , presented as a ratio of the ppm C determined by the Walkley-Black procedure to ppm C determined by the Astro Solution Carbon Analyser, are given below: %Cf %Ch %Cc Ch/Cf Ca/Cf X 0.92 1.17 0.88 1.29 1.09 Sx 0.029 0.059 0.019 0.052 0.053 The low standard deviations of the above ratios suggested a system-atic variation in the Walkley-Black procedure, possibly due to the assumpt ions. The Walkley-Black procedure tended to underestimate the fulvic acid and polysaccharide fraction C carbon. The humic acid carbon was overest-imated. When four Podzolic B horizons, two peats and two root mats were analyzed comparable ratios for %Cf and %Cc were obtained; however, the Walkley-Black procedure underestimated the %Ch in the Podzoic B horizons. This possibly indicated differences in the humic acids isolated from diffe ent s o i l types. Factors contributing to the underestimation of extract carbon by the Walkley-Black Wet Oxidation Method are: 1. Higher oxides of Mn, etc. which compete with the dicromate ion for oxidizable species. 2. An oxidation efficiency < 80%. 3. Loss of C during heat evaporation. Decarboxylation of uronic acids during heating. 4. An oxidation state of carbon greater than zero. Factors contributing to overestimation of extract carbon by the Walk-ley-Black Wet Oxidation Method are: 225 1. Presence of CI or reduced species such as ferrous iron in the extracts. 2. Oxidation efficiency > 80%. 3. Oxidation state of carbon less than zero. The underestimation of ppm C in the FA and fraction C could be due to an oxidation state of carbon greater than zero. It i s unlikely that the oxidation efficiency i s less than 80%. The oxidation state of C in the HA fraction may be less than zero since FA and fraction C are generally more oxidized than HA. Further research needs to be done to verify the factors contributing to the error in the Walkley-Black Wet Oxidation method. 226 Appendix A. PVP - Column Method for Fulvic Acid Fractionation, Column l e n g t h = 6-8" diameter = 2" PVP adsorbed f r a c t i o n A "orange-brown" * l 3 p a l e y e l l o w f r a c t i o n C " f i l t r a t e " F u l v i c Acid F r a c t i o n a t i o n Procedure: 1. Weigh required amount of PVP to f i l l column 1/2 to 1/3. Add d i s t i l l e d water, s t i r , l e t s e t t l e and decant f i n e s . Repeat several times. 2. Set up PVP column with suction apparatus as shown. 3. Place Whatman #1 f i l t e r paper, cut to f i t , i n t o bottom of column. Wet and seal with suction. 4. Pour P V P - d i s t i l l e d water s l u r r y evenly i n t o column. 5. Wash PVP i n column successively with; 2 column volumes of 0.1 N NaOH 2 column volumes of d i s t i l l e d water 2 column volumes of 0.1 N HC1 2 column volumes of d i s t i l l e d water 6. Pass FA f r a c t i o n through PVP pad to adsorb f r a c t i o n A. Wash column with 2-3 volumes of 0.01 N HC1 u n t i l f i l t r a t e c o l o u r l e s s . Save or discard f i l t r a t e " f r a c t i o n C". 7. Elute coloured f r a c t i o n A from PVP with 1 volume of 0.1 N NaOH followed by 0.01 N NaOH u n t i l most of the coloured material has been eluted. 8. Pass f r a c t i o n A through H +- exchange r e s i n or adjust pH to 7 and store i n r e f i g e r a t o r . 227 Appendix 5. Individual Sample Data for S t a t i s t i c a l Analysis. CD U . o. E co N O O O in N O -3 o o o O O - H -3 sr N O o o o o o o d d O N CM o d O N —I o d d r— ^ co in N O o o o o N O C O o o C O O g C N C O C N r t C N o o o o o o o o <t in N D N O —» rt rt \o <N C N oo in CN rt CN CN rt CN ON m o co CN CN O O C N C N O O N D -3 C O co o co co co co co o -3 00 CN N O rt o o o o o -~ m N D in O N O CN N O <f rt CN rt r—• rt o o o o o c N i n o o v D - a - a o » o r ^ m c o < r c o e o c o s r O O O O O O O O r ^ - c N C O c n o r ^ o o m i n N o m c o s r c o m c N o O O o o o o o o o o o o o o o o O o o o o o o CN CO CM CN m o co o 00 00 r- CN CN m - J co N O N O co m in o CN CO o CN GO oo co r-- oo -3 m in m <r in sr co in m N O c o N O c o s r O N r t O O s r O c o O N s r r - ^ i n c O N O rtOO — 0 — C N -sr oo co co in co oo O N O N rt O " en CO o o o o o * N O O N O N en O ST N O CN in CN O N m CO N O O N 00 o o -3 o N O m CN CN N O 00 o -3 CN sr co m 00 N O rt O N rt rt O N sr in CN o CN sr co m -3 co CO CN CN CN CO CN CO CN CN * O N o oo o N O r~ O N sr o r~ o -3 o o in O N oo in N O m CO CO rt co sr sr CN CN m CN o N O 00 CN rt CN O N rt rt CO O N co _ CN co CN in O N m O N m N O in N O sr N O N O CO sr m m CN N O O N 00 CO o sr s: o o o o CN 00 CO o o <—i NO oo oo rt o CO O N GO rt CN -3 oo sr r- O N N O O N -3 ST en CN rt rt rt CN O N rt CO rs o o N O o o o o o —• ~ rt CN — -* rt rt o rt rt o o rt o 00 S r t C N f O v f i n N O i ^ C L . a . a . o - 0 - a . D - a . rt CN CO ST M ts] tS] S3 N O o * 228 •£> o o o oo <r -J O O — —< co ro r~ o> co —< o o s r m o r s c o O v O o o o o o o o o sr o ~' — i s r t M — ro o o d o d o d d o o o o o o o o o o o o O —< CN O \C O \0 CN —< CN - - N N N O —i ro co 00 in CN —i —• in —H co m m C N — *-. — ro ro ro ro ro ro O ro ro o ro ro r~- CO —i ro si ro ro O O O O O O O O o o o o o o o o sr rs so cj* — —4 O m \ £ > o o s r \ O s r s r s r * o m c N s r r o s r c N O O O O O O O O O O O O O O O O O O O O O O O O n CO IN N C N in r- O in in 00 o —I CN CO O o — O — O sj CT> CO ^ CO o m m oo oo o _ H — o ST O CM vD O ro —i r-<j\ o> rs 0N O O O o o o sr m oo — o o o o o o o o o o o o o o o o O o o c N r s y o c o o o o r~ in — i c N L n c N — O O C N ~ H — co — o o CN ro sr in sr rs. sr rs ro ro cr> in 00 ro CO 00 00 00 cr-o in CN CN — CO CN — — — —c — —i ro CN — CN CN — -J o CO — — sr C N ro oo sr ro co ro ro in ro C N co oo cr* sr sr C N co O C O ro O sr in sr oo m o sr sr \o cr* m r*-o r~ rs oo vo O O — O O — ' O O o — — o o o o o ro in oo O ro C N O — — O O O O O in C N in in cr- o> o. E C N C N C N C N C N C N C N C N —* CN •< •< 00 •< C N CM C N C N C N C N 2: — C N ro sr in a. a- a. a. a. a. — CN CN CN CN CN CN CN CO m ts] Sample No. Z F e c b d Z A l ^ 7,Fepy %AJ p y AMI 0.281 0.162 0.131 0.131 A l l 0.164 0.112 0.0602 0.0904 A31 0.199 0.195 0.0806 0.171 A41 0.265 0.205 0.131 0.182 A51 0.216 0.149 0.101 0.121 A71 0.248 0.197 0.121 0.182 A81 0.299 0.246 0.143 0.234 A91 0.235 0.180 0.101 0.152 PM1 0.289 0.255 0.164 0.246 P l l 0.250 0.285 0.137 0.269 P21 0.234 0.266 0.117 0.263 P31 0.245 0.301 0.127 0.289 P41 0.222 0.320 0.106 0.304 P51 0.235 0.260 0.111 0.253 P61 0.225 0.271 0.106 0.263 P71 0.246 0.275 0.137 0.268 ZM1 0.264 0.312 0.0550 0.242 Z l l 0.266 0.351 0.0960 0.298 Z21 0.259 0.340 0.0937 0.284 Z31 0.212 0.256 0.0580 0.212 Z41 0.236 0.293 0.0504 0.222 Z51 0.229 0.282 0.0682 0.242 Z61 0.255. 0.303 0.0758 0.273 Z71 0.214 0.246 0.0453 0.201 *oven-dry basis %Cf %Ch 7Xc %Ca 0.470 0.227 0.399 0.523 0.412 0.695 1.02 0.825 1.47 1.19 0.958 0.848 0.762 0.557 0.645 0.832 0.502 0.625 0.685 0.449 0.428 0.546 0.572 0.353 0.456 0.127 0.391 0.505 0.301 0.759 1.52 0.815 1.71 1.24 0.839 0.933 0.736 0.805 0.758 0.924 0.553 0.861 0.855 0.439 0.506 0.635 0.685 0.313 0.243 0.114 0.213 0.267 0.196 0.375 0.510 0.447 0.779 0.634 0.557 0.455 0.380 0.330 0.341 0.438 0.354 0.439 0.494 0.314 0.296 0.385 0.381 0.244 0.227 0.114 0.186 0.255 0.215 0.320 0.510 0.378 0.693 0.557 0.401 0.394 M 0.381 0.228 0.304 0.394 0.147 0.186 0.191 0.134 0.132 0.162 0.191 0.110 230 sr rs \o in m cn in o o> — — C N C N — in rs C N — i C N — o o o o o o s r s r m m i n C N v O c N c n s T N O N o a o o o i n — O O O O O O O O m r~ cn o — C N o* cn O N in N O m rs sr C N in oo O N — — — — — o o o o o o o o o CO O C N C N — r~ C N O 1 C N co o i — cn n O C N — C N cn — — d o" o" d o" d d NO a* vO m — sr — in o C N C N C N O O O O O O O O sr O m r~. N O —< cn cr* o C N N O C N cn cn cn o o o o o o o — — C N sr C N sr *o sr N O C O —< — rs cn sr <6 O d o o oo o co sr cn — N O rs O N — vo sr m o sr o cn in cn O N — sr m a-C N vo m C N sr cn o sr in in N O O O O O O O O O o o o o o o o cn rs O N N O cn P ^ GO O N C N sr cn cn o o cn N O o> o o N O o — sr cn sr sr cn o> o cn — i sr r-O m N O sr cn rs o o o o o o o o o o o o cn cn sr O O C N — — cn — o v o o c n c o r s f s - s r r s — C N O N O rs N O O O — — — ' C N — o — C N C N C N C N C N C N C N sr C N C N C N sr O O C N cn cn C N C N C N C O N D r- o C N C N o o o o o o o o o o o o o o o o o o o cn C N m C N O O — — — C N C O *£>incncNCNsracsr o o — — — — o o rs ps co rs. O N rs cn C N N O m o m in in m N O *o n o co c o» co a- r s o o — — o o o o o O O — r— N O C N GO cn O N in co m m m N O rs oo in O O O O O o o o o o o o o o o o o o o o o o co o oo O N o« oo o o — cn o cn — — C N — — — in O N sr CO 00 N O — — C N C N C N cn in cn N O o ^ c~> C N C N C N C N O N oo in r- C N o C N oo sr sr N O O N r-. o C N cn cn C N C N C N cn O sr O O O O O O O O O O o sr C N N D vD cn sr — — C N cn cn o oo oo m C N — — N O N O N O m — cn — o — C N C N C N sr C N O N sr o — O N o C N C N — C N O N sr O N O C N C N C N C N C N C N C N C N o o o o o o o o o o o o o o o o o o o C N C N C N C N C N C N C N C N s — N O oo C N C N C N C N C N C N C N C N x — C N cn sr in N O r-0 . 0 . 0 . 0 . 0 . 0 . 0 . 0 . C N C N C N C N C N C N rsi CM sr M N O C O cu > o * Sample No. Ch/Cf Ch/Ca Ca/Cf %Ce C/N HA C/N A %Glucose C % Glucose C Total HA-C Total A-C AMI 0.97 2.01 A l l 0.56 1.12 A31 0.98 2.10 A41 0.97 1.98 A51 0.73 1.40 A71 1.10 2.38 A81 1.49 2.98 A91 0.99 2.16 PM1 1.16 2.47 P l l 1.04 2.22 P21 0.88 1.22 P31 1.10 2.37 P41 0.97 1.93 P51 1.45 3.54 P61 1.18 2.50 P71 1.11 2.35 ZM1 1.10 3.75 Z l l 1.38 4.63 Z21 1.25 4.49 Z31 0.98 3.28 Z41 1.18 3.83 Z51 1.16 3.93 Z61 1.20 ' 3.59 Z71 0.89 2.85 0.48 56 11.6 0.50 60 10.7 0.47 61 8.8 0.49 60 11.3 0.52 58 11.6 0.46 55 13.5 0.50 53 15.2 0.46 54 14.5 0.47 58 16.5 0.47 58 14.0 0.42 52 14.0 0.46 54 14.5 0.50 59 14.8 0.41 47 13.9 0.47 53 13.5 0.47 56 15.5 0.29 54 12.9 0.30 53 13.6 0.28 49 12.9 0.30 49 12.9 0.31 49 13.1 0.30 49 13.0 0.33 50 12.3 0.31 56 13.3 22.0 4.60 6.40 20.3 4.64 6.05 22.2 5.27 6.31 26.7 5.36 6.25 29.9 6.33 7.85 29.6 7.54 7.71 28.2 4.64 5.45 27.6 7.81 8.41 31.5 6.43 6.89 30.7 6.46 7.36 31.1 6.66 6.69 24.3 6.39 7.11 32.1 6.40 6.53 23.2 6.30 8.46 25.6 5.52 6.96 27.2 5.70 7.10 18.3 6.16 7.67 20.7 6.53 7.10 21.4 7.04 7.73 19.1 7.43 8.30 21.8 6.39 7.31 21.0 10.5 9.21 19.3 6.44 8.44 16.1 6.64 9.76 Sample No. Ch/Cf Ch/Ca Ca/Cf %Ce C/N HA C/N A %Clucose C %Glucose C Total HA-C Total A-C AM2 0.48 0.89 A12 0.69 1.20 A22 1.56 2.89 A42 0.94 1.76 A62 1.22 2.48 A72 1.68 3.95 A82 1.06 1.71 A92 0.59 1.26 PM2 0.93 2.08 P12 0.99 1.82 P22 1.08 2.63 P32 1.14 2.52 P42 1.21 2.68 P52 1.16 2.53 P62 1.02 2.21 P72 1.24 2.78 ZM2 1.17 3.79 Z12 1.34 4.59 Z22 1.26 4.36 Z32 1.01 3.09 Z42 1.19 3.99 Z52 1.13 3.46 Z62 1.19 ' 3.24 Z72 0.92 3.21 0.54 62 11.7 0.57 68 20.9 0.54 57 13.6 0.53 59 12.0 0.49 56 10.1 0.43 53 12.2 0.62 68 16.6 0.47 58 13.0 0.45 53 13.5 0.54 60 17.4 0.41 51 13.2 0.45 54 15.0 0.45 53 15.3 0.46 51 15.0 0.46 50 14.3 0.45 55 16.5 0.31 52 12.8 0.29 50 13.8 0.29 49 13.5 0.33 50 12.4 0.30 50 13.4 0.33 48 13.3 0.37 49 12.2 0.29 53 13.2 19.0 6.26 8.55 27.0 3.90 5.55 28.9 4.32 6.01 27.7 5.15 6.51 29.7 5.91 5.82 24.8 6.05 8.53 24.7 5.44 8.81 26.2 6.27 7.36 22.1 6.11 7.10 18.2 5.95 6.84 28.9 5.67 6.16 24.5 5.30 6.31 27.3 5.16 5.76 23.2 5.88 8.53 25.6 6.30 6.23 29.0 7.12 5.88 17.0 6.88 9.40 20.8 6.64 7.48 22.1 6.83 7.20 18.1 7.76 8.86 22.3 8.32 7.48 19.7 7.68 10.9 18.9 6.44 9.50 15.4 7.28 9.23 233 Sample No. EA00 C H A E 6 0 0 C H A E A/E 6 HA F 1 X 0 i LW0 A F 1 % C A ^OO fl E 4/E 6 A AMI A l l A31 A41 A51 A71 A81 A91 231.0 184.6 224.3 232.5 217.3 197.3 227.2 182.2 52.1 44.9 46.8 51.8 47.6 40.7 47.4 39.0 4.44 4.11 4.80 4.49 4.56 4.85 4.80 4.68 122.6 127.5 129.4 127.6 138.5 130.2 133.3 129.0 9.7 10.0 9.0 8.8 11.0 10.1 9.2 10.9 12.6 12.7 14.4 14.6 12.6 12.8 14.4 11.9 PM1 P l l P21 P31 P41 P51 P61 P71 178.3 174.9 179.4 193.9 188.4 231.2 209.2 210.8 40.6 40.7 45.9 51.9 50.1 58.6 54.1 55.0 4.40 4.30 3.91 3.73 3.76 3.95 3.87 3.84 131.2 133.3 124.4 123.9 125.2 131.7 118.6 133.2 11.2 9.9 10.3 9.4 9.9 10.3 9.2 10.9 11.8 13.5 12.1 13.3 12.6 12.8 13.0 12.2 ZM1 Z l l Z21 Z31 Z41 Z51 Z61 Z71 209.6 204.3 195.0 199.3 209.7 198.2 215.9 197.7 61.0 64.2 58.3 58.2 62.6 56.8 62.9 61.1 3.44 3.19 3.35 3.42 3.35 3.49 3.43 3.24 112.3 113.9 117.3 107.4 112.9 106.3 119.2 92.8 9.9 9.2 9.1 8.4 9.4 7.9 9.4 7.9 11.4 12.4 12.9 12.8 12.0 13.4 12.6 11.7 234 Sample No. E ^ C HA HA E ^ HA E ^ C A E ( . J J C A E , ^ A AM2 143.2 38.5 3.72 125.3 10.5 11.9 A12 129.6 25.5 5.09 132.9 10.7 12.5 A22 235.1 50.8 4.63 127.6 9.0 14.2 A42 247.0 56.4 4.38 128.8 8.9 14.5 A62 260.4 54.5 4.78 132.9 9.0 14.8 A72 246.0 51.6 4.77 129.6 8.8 14.8 A82 198.6 38.3 5.18 129.1 9.3 13.9 A92 187.9 42.9 4.38 122.4 9.7 12.7 PM2 232.7 68.0 3.42 116.4 10.1 11.6 P12 181.7 42.8 4.25 136.6 11.2 12.2 P22 206.3 56.6 3.64 121.3 9.7 12.5 P32 234.6 70.5 3.33 120.7 10.3 11.7 P42 221.0 63.3 3.49 118.9 9.6 12.4 P52 228.6 66.0 3.46 123.7 10.5 11.8 P62 244.5 72.9 3.35 116.6 10.3 11.3 P72 243.0 68.4 3.55 126.1 10.5 12.0 ZM2 205.3 64.2 3.20 107.0 8.9 12.0 Z12 197.5 59.2 3.30 113.2 8.9 12.8 Z22 203.9 62.7 3.25 112.7 8.6 13.0 Z32 195.8 56.5 3.46 105.2 7.9 13.3 Z42 192.3 56.6 3.40 110.6 9.0 12.3 Z52 217.3 65.9 3.30 103.4 7.9 13.2 Z62 218.0 65.7 3.32 101.9 8.2 12.5 Z72 208.5 65.4 3.19 93.3 7.7 . 12.1 1. Sample No. legend: zone / p i t no. / horizon ; A = Abies r e l i g i o s a , P= Pinus hartwegii, Z = Zacatonal ; M = modal p i t ; 1 = Ahl horizon, 2 = Ah2 horizon. ZONE %HM %LI %Ct %Nt C/N -oven-dry basis-Ahl horizons, n=8 Abies r e l i g i o s a 0.99 4.3 2.1 0.12 16.8 (0.49) (2.5) (1.3) (0.061) (2.0) Pinus hartwegii 1.4 7.5 3.5 0.19 18.0 (0.43) (2.1) (1.0) (0.046) (0.078) Zacatonal 0.95 4.8 2.2 0.15 14.9 (0.22) (1.2) (0.63) (0.040) (0.50) Ah2 horizons, n=8 %HM %LI %Ct %Nt Abies r e l i g i o s a 0.73 3.1 1.4 0.088 (0.38) (1.8) (0.92) (0.064) Pinus hartwegii 0.91 4.2 1.9 0.096 (0.28) (1.5) (0.73) (0.031) Zacatonal 0.96 4.6 2.2 0.14 (0.26) (1.3) (0.67) (0.041) 1. Signi f i c a n c e l e v e l : 95%= *, 99%= **. Analysis by ANOVA. 2. The Ahl and Ah2 horizons were combined for C/N, n=16. 3. Homogeneity of variance not met. 236 ** ** „ * * „ * * 2 ZONE ZAl %A1 ZAl . . ZSi ox py cbd ox -oven-dry basis-Ahl + Ah2 horizons, n=16 Abies r e l i g i o s a 0.21 0.15 0.17 0.049 (0.039) (0.048) (0.049) (0.0085) Pinus hartwegii 0.32 0.25 0.26 0.063 (0.022) (0.034) (0.034) (0.020) Zacatonal 0.40 0.25 0.30 0.12 (0.041) (0.035) (0.036) (0.013) 1. S i g n i f i c a n c e l e v e l : 95%= *, 99%= **. Analysis by AN0VA. 2. Homogeneity of variance not met. ZONE %Fe %Fe ZFe ox py cbd oven-dry basis Ahl horizons, n=8 Abies r e l i g i o s a 0.16 0.11 0.24 (0.025) (0.028) (0.045) Pinus hartwegii 0.15 0.13 0.24 (0.013) (0.020) (0.021) Zacatonal 0.15 0.068 0.24 (0.014) (0.019) (0.022) Ah2 horizons, n=8 %Fe %Fe %Fe t J ox py cbd Abies r e l i g i o s a 0.15 0.093 0.21 (0.025) (0.040) (0.057) Pinus hartwegii 0.13 0.095 0.20 (0.010) (0.011) (0.036) Zacatonal 0.14 0.078 0.24 (0.013) (0.018) (0.022) 1. S i g n i f i c a n c e l e v e l : 95%= *, 99%= **. Analysis by AN0VA. * * # 1 # 1 7 # 1 9 # # T ZONE %Ch %Ca J %Cf J > %Cc 5 , 1 %Ce 5 , 1 a i r - d r y basis Ahl horizons, n=8 Ahl + Ah2 horizons, n=16 Abies r e l i g i o s a 0.61 0.28 0.48 0.24 58.6 (0.43) (0.12) (0.22) (0.12) (4.6) Pinus hartwegii 0.99 0.42 0.70 0.38 54.0 (0.33) (0.15) (0.34) (0.17) (3.5) Zacatonal 0.61 0.16 0.51 0.36 50.6 (0.19) (0.031) (0.11) (0.081) (2.3) Ah2 horizons, n=8 %Ch %Ca^ Abies r e l i g i o s a 0.44 (0.33) 0.20 (0.056) Pinus hartwegii 0.54 (0.23) 0.23 (0.13) Zacatonal 0.58 (0.20) 0.15 (0.036) 1. Significance l e v e l : 95%= 2. The Ahl and Ah2 horizons 3. Homogeneity of variance *, 99%= **. were combined not met. Analysis by AN0VA. for %Cf, %Cc and %Ce, n=16. 238 ZONE Ch/Cf Ch/Ca Ca/Cf Ahl + Ah2 horizons, n=16 Abies r e l i g i o s a 1.0 (0.36) 2.0 (0.80) 0.50 (0.48) Pinus hartwegii 1.1 (0.14) 2.4 (0.50) 0.46 (0.032) Zacatonal 1.2 (0.14) 3.8 (0.56) 0.31 (0.023) 1. S i g n i f i c a n c e l e v e l : 95%= *, 99%= **. Analysis by ANOVA. 2. Homogeneity of variance not met. ZONE C/N HA C/N A %GIucose C Total HA-C *» 2 %Glucose C Total A-C Ahl + Ah2 horizons, n=16 Abies r e l i g i o s a 13.0 (2.88) 25.9 (3.44) 5.6 (1.1) 7.0 (1.2) Pinus hartwegii 14.8 (1.20) 26.5 (3.91) 6.1 (0.52) 6.9 (0.79) Zacatonal 13.0 (0.460) 19.5 (2.12) 7.2 (1.1) 8.5 (1.1) 1. S i g n i f i c a n c e l e v e l : 95%= 99%= •». Analysis by ANOVA. 2. Homogeneity of variance not met. 239 ZONE E , J J C HA E 6 ^ C HA E./E, HA Ahl + Ah2 horizons, n=16 Ah] horizons, n=8 Abies r e l i g i o s a 209.0 46.3 4.59 (36.9) (4.69) (0.247) Pinus hartwegii 209.9 49.6 3.97 (24.5) (6.65) (0.247) Zacatonal 204.3 60.6 3.36 (8.25) (2.62) i%r ** E HA L600 H A Ah2 horizons, n=8 (0.104) ** 3 E A/E 6 HA Abies r e l i g i o s a 44.8 (10.5) 4.62 (0.464) Pinus hartwegii 63.6 (9.47) 3.56 (0.269) Zacatonal 62.0 (4.02) 3.30 (0.930) ZONE E n C A** ^400 * E l X C A** ^600 A ** 3 V E 6 A Ahl + Ah2 horizons, n=16 Abies r e l i g i o s a 129.2 9.66 13.5 (4.04) (0.781) (1.08) Pinus hartwegii 125.1 10.2 12.3 (6.39) (0.586) (0.623) Zacatonal 108.1 8.64 12.5 (7.58) (0.676) (0.583) 1. Signficance l e v e l : 95%= *, 99%= •*. Analysis by ANOVA. 2. The Ahl and Ah2 horizons were combined for E^qq, n=16. 3. Homogeneity of variance not met. 240 Appendix 7 . Correlation Matrix of Chemical Parameters. CD O r~ r- to rr CM CO rr r- o »n 01 O in 01 CO CO rr to r> co o r- 01 01 •r- IB o r- rr o to T O) CB CB in r- in in oo O > rr rr O O in CN o in rr IB o O CN "J CD 01 r~ in CB 01 r~ O O • 0. r- r> r- t- IB to rr IB IB o to r~ r» in r- p~ O to O CP in ui — u. 1 1 l oo in in 01 rr CO IB in CN t> o r~ O in CO CO to in 01 O in r~ in D CB O rr r~ — in CM O r- o o 1 Ol CO 01 in CM in r> rr O rr 01 as CM CO o 00 01 CN CO 03 01 o IB IB in CO CD p-* to co CB O oc -^ O • o in in ID in IB O 01 in o — 01 T in CB — in r- t> CB O CM rr _J '' i '' — < ro in CN O r- O IB in r- O CM CO IB CN CO to •9 O CM 03 O O a co CM 01 to 01 o CO O O O r> — in t- O O CN CM O CO i> CO 0) O o to O o 01 rr IB CN oo CD in CB to 01 in CD 01 • V t- r> ID oo CN in rr O o in IB in IB r> CB <3 CB in CM 1 to ,' o CO UJ — u. CM IP CB in O uo CO f> O r- CN co 01 o CN to 01 r~ o O 01 in to co CM O CO in CO CB CM O O r- O CO CN co CB CN <3 rr 01 rr to X CD IB "3 *- rr CO O o oo in CM in r- CB o O CO r> rr O •— CN • O CN CM CM CN O in CM r- O o rr IB CO to CN o CD CD CD CO CB ^~ r^  CN — '' 1 ( 1 i i 1 l i 1 I t l 1 I i i — l/l CM CO rr in o O r> in m CN IB 01 to CD 03 in 01 in r^  Ol CN t> CO r~ co in rr O CN to O IB r~ r~ co 00 r~ IT in in rr CM co 0) X CO r- O r- O to to to O in CM CO t> O CN CN CM rr • D to co CO CO in CN O r> rr Ol CO CM co O in CO co r- O rr — < ' \ 1 ' ' ' ' 1 r> O CO rr in O o O CO IB IB CM CM to 01 co CO 01 01 CN CO o o in in r- orj in r- o rr O CN »— o CN to CO CN 01 CM CO CB r~ 00 X 0) CT1 CO IB O IB o •»- rr O CM CM in CM »3 r- r- t- CB r- rr • o CM CM CN CM CO O o CM in O rr O CN CO CN CM CN O *- •— O O UJ •' - i 1 i '' — u. r- o ro CO IB O o in in O CO CO CO IB CM in CO O 00 to 01 to CD CM i- 01 in o r- in IB O) in 01 O o O to CN CO r- \— rr 01 -•— O) O) co r~ T- o IB t- r- in IB 01 CB CN 01 to in in to in CN CM CN CN o o o CM in CM -•— CO o to CM in •9 o n in CM t> in • Z •' t i 1 '' 01 o CM O 00 o CB in rr O r- rr r- CB co CO "3 CD CM in CB 01 o ! » IB o in in CO 00 co *~ 01 co in 03 co CO t- CN CO O CN 01 ro CO rr in o O o "9 O 01 *- IB CO CB r> to CM •— O CO CB 01 01 01 01 o o CO in o CO IB IB oo 01 Ol CP CB CB in CO rr O • -^- 1 l •' 03 Z CO 0) r~ O 03 CO _ in o cn r- ^ CN co 03 CD 01 CM CB rr CM CM CO O IB 01 co in cn r- r- O) o CO CN O) CO O CD rr CO in CB CO 00 01 O in r- CB r- ~- co O vS in co in •— CP CO CM 01 rr 01 0) cn O cn CN CN to CN in IB Ol Ol Ol CO CO in to »— CN CN to 1 t '' r- cj to CM O r» O CO co CO CD CM in o co in ^ o to t- CM CD CN r- CB rr rr O CO r- r~ r> co CO CN i o 01 co O o O CB rr rr t- rr r^  r- O 01 •3 co to co ~— 01 O o cn in f> CB to CN CO ^~ CN i> CB CM 0) cn O 01 01 CN CN CO CM IB u> Ol 01 01 00 00 in CO — CM CN to • t—1 - •' 1 t CB —J 0) O CN 01 O o IB in in IB IB r- CB Ol CN O r~ r~ O 01 CN 0) O rr CM CM in r~ O CO O to CO 03 in in P~ Ol 01 CO 01 o r-CO O r- 00 to 01 0) •r- IB to 03 CN «3 CO in 01 in CB rr O) o cn 00 01 CN CN CO CM r- in r- IB Oi 01 Ol CO CO in CM O CN CN CO • u. 1 1 in cj o 01 co 00 CM r~ CN CN CO to r- 01 to r- CB CN 01 CN CO r- in 01 o 01 rr CM o rr in rT CM u> n on 01 in r- CO in CD r> CD to r^  O 01 r~ 00 to 0) 01 *- IB CN O 1 oo CM •<t CO IT) 01 in CB rr ©s O 01 cn 01 01 CN CM to CN in IB 01 o 01 03 co in CN O CM CM CO - s rr I i s« s* s« ffs Q o sp X X X CO CO > > s« u. < u. ?s s« oS o o D O o a CL Ss ss D o CJ CJ I < £ U. 1- 1- z UJ UJ U. I (J < < I I < Ui z Z I o _i u z <J li. < C/l u. < la- < CJ o o CJ CJ CJ CJ CJ CJ CJ CJ rr in IB r> co 00 o CN to rr in IB r- CO 01 O CN CO rr in CD r-CM CN CN CN CM CN CN CM VARIABLE 1G.ALPY% 17. CF7. 18. CH% 1 9 C C % 2 0 C A D % 2 1 .CAM 0/. 22 . CHCF 23. CHCA 24. CACF 25. CE% 2G.CNH 27. CNA 28. GLCHC% 29 . GLCAC% 30. E41%H 31. E61%H 32 . E4E6H 33 . E4 1%A 34. E6T/.A 35. E4E6A 1.0000 . 5596 .6195 . 7 140 . 3238 .2733 .6024 .6469 - . 5 4 5 3 - . 6 2 2 6 . 1234 - . 0 7 5 1 . 3516 . 1 170 . 1 180 .4787 - .5572 - . 28 1 1 - . 1 8 3 7 - . 1 1 5 8 16 . ALP Y% .5596 1.0000 .9345 .9386 .9301 .9129 . 3296 .0932 .0388 - .0758 . 2908 .4001 . 1 143 - . 1202 - .2142 - .2901 . 1588 . 3334 .2871 .0484 17 . CF% .6195 . 9345 1.OOOO .9090 .8355 . 8263 .6101 . 3239 - . 0 4 6 3 - . 2062 . 2691 . 3478 .0739 - . 1168 - . 0 2 1 9 - . 1 4 8 5 .1361 . 2877 . 1528 . 1726 18 . CH% .7140 .9386 .9090 1.0000 .7473 . 7528 .4186 . 3702 - . 2 9 1 0 - . 3246 . 1885 . 2033 . 3086 .0439 - .2128 - . 0 9 2 3 - . 0 9 1 9 .0685 .0884 - .0251 19 . CC% . 3238 . 9301 . 8355 . 7473 1.0000 .9587 . 1913 - . 2159 . 3870 . 1962 . 3582 . 5498 - . 1103 - . 2696 - . 1 8 5 0 - . 4 5 7 9 . 4017 .5631 .4544 . 1 186 20. CAD% . 2733 .9129 . 8263 . 7528 .9587 .0000 . 1892 1 . 2060 . 3554 . 1847 . 2982 . 6494 .0803 . 2648 . 1923 .4789 .4274 .5417 .4203 . 1356 21 . CAM% .6024 . 3296 .6101 .4186 . 1913 . 1892 .0000 .7316 . 2928 . 5024 .0682 .0728 .0704 .0070 . 5202 . 3858 .0906 .0328 .2518 . 3090 22 . CHCF .6469 .0932 . 3239 . 3702 - . 2 1 5 9 - . 2 0 6 0 .7316 1.0000 - .8248 - . 7194 - .1144 - . 4 2 0 9 .4507 .3475 . 2455 . 5781 - . 5 4 5 8 - . 5 3 9 0 - . 5 2 6 9 .0181 23. CHCA - . 5 4 5 3 .0388 - . 0 4 6 3 - . 2910 . 3870 . 3554 - . 2928 - .8248 1.OOOO . 769 1 . 2603 .5919 - . 6 2 7 3 - . 4 8 9 3 - . 0 2 3 9 - . 6 0 2 4 .7617 .7936 .5655 . 2647 24 . CACF - . 6226 - . 0 7 5 8 - . 2 0 6 2 - . 3 2 4 6 . 1962 . 1847 - . 5 0 2 4 - . 7194 .769 1 1.OOOO . 2365 . 3001 - .5345 - . 3 4 7 0 - . 3 3 5 2 - . 7 1 2 9 . 6864 . 5469 . 3691 . 2025 25 . CE% . 1234 . 2908 . 2691 . 1885 . 3582 . 2982 .0682 - . 1144 2603 . 2365 1.OOOO . 2497 - . 1 2 4 6 - . 1 7 4 8 - . 3 7 5 7 - . 2 2 2 9 .0858 . 2016 . 4446 - . 3 3 9 5 26 . CNH - . 0751 . 4001 . 3478 . 2033 .5498 . 6494 .0728 - . 4 2 0 9 .5919 . 3001 .2497 M 1.OOOO - . 2 6 8 8 - . 5935 .0864 - . 3 9 4 2 . 5608 . 7030 .4919 . 2488 27 . CNA VARIABLE 28. GLCHC% 29 . GLCAC% 30. E41%H 31 .E61°/»H 32 . E4EGH 33. E41%A 34. E6T/.A 35 . E4E6A VARIABLE 28 GLCHC% 29. GLCAC% 30. E41%H 31 . EG1%H 32. E4E6H 33. E41%A 34. EG1%A 35. E4E6A . 1256 - . 0 8 9 5 - . 0 4 7 2 - . 1 5 2 7 . 1207 . 2773 . 1884 . 1 144 4 . HM% 1.0000 .6109 - . 1467 . 2444 - . 4 4 9 5 - . 4 9 7 8 - . 3 2 2 8 - . 1 9 7 7 28. GLCHC% .1271 - . 0896 - .0444 - . 1513 . 1208 . 2767 . 1858 . 1 173 5. CF .6109 1.OOOO - . 1321 . 2095 - . 3626 - .5814 - . 4403 - . 1 4 9 7 29. GLCAC% . 1683 - . 0 7 2 5 - . 0 9 8 5 - . 1 1 4 2 .0246 .2191 . 1584 .0745 6 . LI% - . 1467 - .1321 1.OOOO .6262 - . 0398 - . 0014 - . 2 2 4 7 .3168 30. E4 1%H . 159 1 - .0634 - .0734 - . 1 2 16 .0556 . 2303 . 1461 . 1062 7 . CT% . 2444 . 2095 .6262 1 OOOO - . 7921 - .6137 - . 3769 - .2778 31 . E6 1%H . 2677 . 0693 - . 0256 - .024 1 - . 0368 .0878 - .0408 . 1761 8 . NT% - .4495 - . 3626 - .0398 - .7921 1.OOOO . 7660 .3191 .5626 32. E4E6H N= 48 DF= 46 R© .0500= 2845 R© .0100= 3683 - .3406 - . 477 1 - . 1872 - .2957 . 2759 . 5055 .6490 - .2224 9 . CN - . 3452 - .2967 .0727 - .2575 . 3695 .3122 .0225 . 3692 10. FEOX% . 4790 . 3540 .0668 .6471 - . 78 14 - .6124 - .3612 - .3054 1 1 . ALOX% .5310 . 5609 - . 0 0 7 3 .6383 - . 7 8 5 6 - . 8 6 7 3 - . 5 3 9 6 - . 3 9 8 5 12 . SIOX% . 108 1 .0190 . 284 1 . 1099 .024 1 .048 1 - . 1779 . 3031 13 . FECBD7, . 4328 . 1873 . 1050 . 5279 - .6304 - . 4 0 5 3 - . 2 9 0 5 - . 1 3 2 3 14 . A LCBD% - . 256 1 - .3666 . 2668 - . 2 193 .4348 . 6 104 . 3000 . 3930 15 . FEPY% - . 4978 - . 5 8 14 - . 0014 - . 6 1 3 7 . 7660 1.OOOO . 7207 .3171 33 . E4 1%A - . 3228 - . 4 4 0 3 - . 2 2 4 7 - . 3 7 6 9 .3191 . 7207 1.OOOO - . 4256 34. E6 1°/„A - . 1 9 7 7 - . 1 4 9 7 3168 - . 2 7 7 8 .5626 .3171 - .4256 1.OOOO 35. E4E6A K3 ro 

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