The Influence of a Combined Elevation-Vegetation Site

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 British 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 Soil Science)

We accept this thesis as conforming

to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

June 1986

©Rhae Alda Drijber, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.

Department of ^ jfsx^rve^

The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3

Date £/*7ul 3a /9ft ii

ABSTRACT

An elevational sequence of soils 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 soils developed from recent volcanic ash. One sample plot was selected in each of three vegetation zones; Abies religiosa, Pinus hartwegii and Zacatonal, an alpine grassland. In Part 1, non-parametric statistics were used to evaluate several parameters measured on the Ah^ and Ah^ soil 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 soils were at an early stage in their development. The degree of mineral weathering increased with elevation and with depth in the profile. 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 religiosa and Zacatonal zones, but were present in the Pinus hartwegii zone. The humus content differed little among the three zones, although qualitative differences related to the vegetation site factor were apparent in the kinds of organic components in the fulvic acid fraction. 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 religiosa to the

Zacatonal zone. iii

In Part 2, elemental and infrared analysis failed 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 rich in nitrogen. Four major phenolic acids of lignin origin were identified in the humic acid and fraction A hydrolysates - protocatechuic acid, p-hydroxybenzoic acid, vanillic 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 identified. iv

TABLE OF CONTENTS

Page

ABSTRACT ...... •' . ii

TABLE OF CONTENTS ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES ...... xi

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 Classification ... 8

2.2.2 Profile 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 Soil 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 Soil Humic Substances and their Relationship to Genetic Soil 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 Soil Humus . 81 vi

Page

4.1.3 Humus Degradation Methods .... 86

4.1.4 Detection of Phenolic Compounds ... 88

4.2 Part 1 - Statistical 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 Diafiltration 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 - Statistical Study ..... 105

5.1.1 Chemical Analysis of the Modal Pit Profiles . 105

5.1.2 Statistical Analysis of the Ah^ and Ah^ Soil 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 Silicon (%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 vii

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 Statistical Analysis . 227

6. Statistical Analysis of Parameter Means by AN0VA . 235

7. Correlation Matrix of Chemical Parameters . . 240 viii

LIST OF TABLES

Table Page

1 Experimental Plot Characteristics .... 75

2 HPLC Chromatographic Conditions .... 103

3 Chemical Analysis of the Modal Pit Soil Horizons from Iztaccihuatl Vegetation Zones .... 106

4 Extractable Iron (%Fe), Aluminium (%A1) and Silicon (%Si) from Modal Pit Soil 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^ Soil 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 Silicon (%Si) from Combined Ah^ and Ah^ Soil 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^ Soil Horizons. .... 124

8 Relative Dissolution of Materials Containing Iron, Aluminium and Silicon by Pyrophosphate, Acid Oxalate and Citrate-Bicarbonate-Dithionite .... 126

9 Distribution of Iron, Aluminium and Silicon Containing Materials in the Recent Volcanic Ash Soils of Iztaccihuatl...... 127

10a Representative Profile from Iztaccihuatl Study Area: Chemical Properties of Profile 04 Including Comparative Median Data from the Soils of the Three Vegetation Zones 130 ix

Table Page

10b Physical Properties of Profile 04 ... 130

10c Particle Size Distribution of Profile 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 Verification 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 Soil ... 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 Classification 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 al. . 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 Orsellinic 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 Soil

Humic Acids ...... 51

17 Structure of Flavonoids ...... 51

18 Sequence of Strata of Soil Forming Materials on xii

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 ...... 97

22 Ultrafiltration 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 Profiles...... 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 xiii

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 XV

ACKNOWLEDGEMENT

The author would like to thank Dr. Lawrence Lowe for the opportunity to visit Central Mexico and study the volcanic ash soils of Iztaccihuatl.

A special thanks to Dr. Quinones whose experience and guidance made the expedition a success, and whose historical anecdotes were most entertaining.

The author would also like 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 identification. 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 soil organic matter research has been on the elucidation of the chemical structure of soil humic substances and their possible relationship to genetic soil 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 soil from which they were derived. Although this did not help those studying the structure of humic compounds, it did point to a genetic origin of humic substances in general, and has given soil formation theorists much to evaluate regardin the pathways of humus formation.

Soil formation, and in a sense soil humus formation, can be regarded as a result of the five soil forming factors. The general features of climate and vegetation were shown by the Russian soil scientists to be the dominant factors in soil formation; thus they developed the concept of soil 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 soil type.

The selection of a study area to evaluate a combined elevation- vegetation site factor on the nature of the humus formed was a difficult task due the necessity of keeping the other soil forming factors constant

With good fortune the author was afforded a unique opportunity to visit 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 all the necessary requirements. In the region south-west of the Volcano Iztaccihuatl and the Paso de Cortes an elevational sequence of soils developed on recent volcanic ash was selected. The sequence comprised three "climax" vegetation zones: Abies religiosa, Pinus hartwegii and Zacatonal, a mixed grassland dominated by

Festuca species. Preliminary examination of the literature uncovered a joint German-Mexican project directed by Glinter Miehlich (1980) which gave an extensive soil 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, utilizing humus fractionation techniques and measured properties of the fractions obtained.

2. To examine the humic acid and fulvic 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 is determined by plate boundaries where lithosphere is created or destroyed. There is 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 relic 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 is dependent on the nature of the plate boundary (Press and Siever, 1978). Oceanic divergent zones and intraoceanic volcanoes produce fluid basaltic lava.

The collision of oceanic plates produces basaltic and andesitic lavas, whereas the collision of an oceanic plate with a continental margin may also produce rhyolitic lava. The eruptive style of a volcano depends on the viscosity of the lava, being a function of its 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 rhyolitic 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 until 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 lies between the eighteenth and nineteenth parallels. 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. Figure 2. Classification of Volcanic Materials.

Decreasing Viscosity —> TRACHYTE RHYOLITE DACITE ANDESITE BASALT Decreasing Eruption Force —>

80%- -^v Quartz APPR0XIMATE DO* • - x MINERAL Pota ssium ^' Plagioclase 40% • F eldspar Feldspar. Si CONTENT Pyroxene 20% • >* • ** Blot. ,HIK Bioti te, Hornblende ^ 0 1 i v i n e y*? COMPOSITION TYPE FELSIC (Si and Al rich) | INTERMEDIATE | MAFIC Figure 3. Volcanoes of Central Mexico (Lorenzo, 1959). 8

2.2 Soils Derived from Volcanic Ash

2.2.1 Distribution and Classification

Soils derived from volcanic ash are intimately associated with the volcanic regions of the world (see Figure 1). These soils 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 soils, often arising from the Japanese word "Anshokudo" meaning dark (an), coloured (shoku), soil (do) (Leamy et al., 1980). In Japan the term "Ando" soil is widely used as is "Kuroboku" soil. More descriptive terms include black soils, grassland brown earth soils and prairie-like brown forest soils, all of which infer similarity to Chernozemic Ah horizons (Ishizuka and Black, 1977).

The FAO-Unesco World Soil Map uses the term "Andosols" to denote soils formed from materials rich in volcanic glass and commonly having a dark surface horizon (Leamy et al., 1980). Soil Taxonomy does not recog• nize these soils at the order level, but classifies them under the sub• order Andepts of the order Inceptisol. They are soils 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 al., 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 vitric or andic soil properties as outlined by ICOMAND, circular letter no. 5 (Leamy, 1983): 9

Vitric soil properties

The soil material has:

1. more than 60% by volume of the whole soil is 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 soil properties

The soil 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 Profile Characteristics

An important feature of Andisols is the rapid release of silica,

aluminium and iron from the parent ash and the subsequent formation of

humus-aluminium complexes or amorphous aluminosilicate clays. This process

imparts certain characterisitics to the profile, namely a high accumulation

of humus, often 15 to 30 % in the surface horizon, which forms a thick,

dark friable 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 al., 1980). Thixotrophy, or liquefaction under

pressure, is often a characteristic of well developed Andisols containing 10

allophane (Flach et al., 1980). Phosphate retention, and CEC measured at pH 7 are high; however, the effective CEC is lower due to the soils being acidic in reaction with a dominance of variable charge surface minerals (Parfitt, 1980). A major contributor to the CEC would be the organic matter (Tate and Theng, 1980). Profile development may extend from an A/C profile to an A/B/C profile under intense weathering conditions and/or increasing soil age. B horizons are characterized by a brown to yellow colour, blocky structure, and a dominance of alumino- silicate minerals such as allophane (Leamy et al., 1980). Several ash falls result in stratified soils up to several metres thick with each layer displaying the characteristics of its pre-burial pedological development.

2.2.3 Occurrence of Andisols on the Landscape

The occurrence of Andisols on the landscape is determined by the intrazonal factors of parent material, topography and time (Duchaufour,

1977; Parfitt 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 al., 1980). Exceptions are certain vitric Andisols which have been reported from an aridic moisture regime in Syria (Leamy, 1983).

Soils developed from volcanic ash along altitudinal 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 rainfall producing a leaching environment (Lulli and Bidini, 1980). On Mount Amiata in Tuscany, Lulli 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 silica

(Duchaufour, 1977).

In a climosequence of soils in the Central Cordillera of Columbia,

Cortes and Franzmeier (1972) found temperature to be influential 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 profile differ- entiation, and was not attributed to Spodosol formation. In this soil sequence, although the rainfall was the highest at the lowest elevation, it was thought that the effective precipitation increased with elevation due to a lower temperature and almost permanent cloudiness. However, the soil 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). Parfitt 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 rainfall.

Spodosol formation, therefore, is common in volcanic ash, occurring where extensive rainfall 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 illuvial B horizon.

Andisols, as indicated above, are not the exclusive soils formed 12

from volcanic ash, but Entisols, Mollisols, Spodosols, and under extreme weathering, Ultisols, Alfisols and Oxisols may form (Leamy et al., 1980).

In Japan, which is highly volcanic, the formation of Ando soils is 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 is characterized by good drainage and a low water content, forms soils of the brown-forest group. Ando soils are produced by the moderate and wet type regimes (Ishizuka and Black, 1977).

The speed at which the ash weathers will determine the course of soil development in thick ash deposits (Ishizuka and Black, 1977).

Once the weathered ash has a high enough clay content to increase its water holding capacity there will 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 soil. This evolution allows soils 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 rainfall 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 is a naturally occurring hydrous aluminosilicate of varying chemical composition while maintaining a predominance of Si-O-Al bonds (Wada, 1977). This is not the case with phyllosilicate minerals which are dominantly Si-O-Si bonded with Si-O-Al bonds linking 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 + VA^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 is seen as a hollow sphere with a shell 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 is also an aluminosilicate mineral of high specific surface, but its 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 is 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 parallel. Thus, imogolite is termed "paracrystalline". 1A

The tube units consist of an external modified gibbsite-like surface with aluminium in octahedral coordination. The internal surface has exposed silanol 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 phyllosilicate minerals is due to the variable charge nature of their surfaces and their ability 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" (Parfitt, 1980) and "variable potential surfaces" (Bowden et al., 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 is a direct function of pH, and other factors such as the ionic strength, temperature, valence of the counter ion and dielectric constant of the medium (Uehara and Gillman, 1981). This is in direct contrast to phyllosilicate minerals which obtain most of their charge, termed perm• anent, by atomic substitution. Specifically adsorbed ions which enter the

Stern layer and form coordination complexes with the surface will also effect surface charge. An important example is 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 is a serious fertility 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+ 2 I . H+ | Allophane Al (4) <• Al (4)

/V^OH OH" /V^OH OH 0" + H+ I H+ | Allophane Si (A) *• Si (A) Silanol group

Imogolite /\\ 0H- /\\0 Crystal edges 0 0 0 0

+ + 0H. OH + H 0 + HO0 2 | 2 H+ | H+ I Allophane Al (6) * Al (6) * Al (6) Imogolite ' - Gibbsite low pH °H UH high pH Crystal edges (AEC) **

OH- OH" + H-0 2 2 I H+ I Al-humus Al (6) * Al (6) OH"

» number in brackets refers to coordination number of metal.

** AEC = anion exchange capacity.

ure 5. Transformation of Variable Charge Clay Minerals (Violante and Wilson, 1983).

dehydration, HALLOYSITE aR1"R > KA0LINITE

PR0TO-IMOG0LITE' resilication VOLCANIC -> ALLOPHANE ASH

desilication M IMOGOLITE > GIBBSITE 16

2.2.5 Formation and Transformation of Clay Minerals

The formation of "amorphous" aluminosilicate minerals such as allophane in Andisols is closely related to the nature and amount of

soil humus. Hetier claims the most important property of Andisols is

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) stabilizes the humus from biodegradation.

Allophane is found only in horizons where the ratio of iron plus aluminium

divided by carbon extracted by a pyrophosphate solution is greater than

0.1 (Wada and Higashi, 1976; Parfitt and Saigusa, 1985). Thus, allophane

is only present in the early stages of soil 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 will depend on

the stage of soil formation, the soil horizon, the petrological nature

of the ash, the thickness of overburden ash deposits and the pH (Wada,

1980).

In the early stages of soil formation, the addition of organic matter

to the ash deposit surface and its subsequent decomposition to form soil

humus, finds the aluminium and iron released by the weathering of- the ash

largely as Al- and Fe-humus complexes (Wada and Higashi, 1976; Higashi and

Wada, 1977). The silica released from the ash forms opaline silica even

in heavily leached volcanic ash soils containing a spodic horizon

(Parfitt and Saigusa, 1985). In a later stage of soil formation a B

horizon may differentiate. This occurs when the organic matter level is

not high enough to complex all the released aluminium, and allophane and/ 17

or imogolite form in situ, possibly by coprecipitation of silica and aluminium (Wada and Higashi, 1976; Farmer et al., 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 its 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 soils; allophane dispersing in both acid and alkaline media and imogolite dispersing only in acid media.

The transformation of allophane and imogolite in the soil seems to depend on the soil solution silica concentration. When the amount of silica in the soil solution is high, as favoured by a thick overburden ash deposit, a stagnant water regime, a low rainfall and a silica rich parent material, halloysite would form as the result of resilication of allophane or imogolite. In ash derived soils it is common for allophane to decrease and halloysite to increase with depth (Violante and Wilson,

1983; Parfitt et al., 1983). Conditions favouring desilication such as a higher rainfall, 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 soil solution silica concentration by the alteration of leaching regimes (Parfitt et al., 18

1983). The removal of silica by plant roots may lead to the formation of allophane or gibbsite in root channels (Parfitt et al., 1983). These transformations are outlined in Figure 5. In summary, the formation of allophane and imogolite is primarily governed by the concentration of aluminium in the soil solution which is intimately linked to the soil humus, and the soil pH. They are further transformed by interaction with the soil solution silica 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 solubility, 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 soil become humic substances.

Briefly, they stem from three possible origins; modified plant constit• uents such as lignin (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 lignin (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 will be placed on the role of lignin in humus formation, and on the microbial neoformation of phenolic compounds and the trans• formation of lignin-derived phenolic compounds. 20

Figure 6. Humus Formation Theories(Stevenson, 1982).

PLANT RESIDUES

(animal and microbial remains)

* from non-aromatic carbon. 21

2.4 Lignin - Its Nature and Possible Role in Humus Formation

Lignin is 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 cell walls of plants lignin is

intimately associated with the structural polysaccharides. The compos•

ition of lignin, its biosynthesis and the mode of linkage of the constit•

uent units within the polymer, has been the focus of lignin research

since the 1890's when Klason isolated coniferyl alcohol from the cambial

sap of plants (Gottlieb and Hendricks, 1945). Alkali fusion of isolated

lignin produced a small amount of catechol, guaiacol and protocatechuic

acid. From this and other reports available at the time Klason suggested

that lignin 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 lignin 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 lignin 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

lignin-like polymers from cinnamyl alcohols, polymerized by phenol oxidases,

Freudenberg arrived at his classic "spruce lignin" model in the 1960's.

This is widely accepted today. A representation of conifer lignin showing

the prominant structural features is given in Figure 7. 22

gure 7. Structural Scheme of Coniferous Lignin According to Freudenberg (Flaig et al., 1975).

gure 8. Synthesis of Lignin as Outlined by Higuchi et al. (Crawford, 1981).

CH}q SINAPTL ALCOHOL - MO^CM^CH-COOM

CHJO*"

CARBON DIOIIDE SIKAPIC ACID I • O- MET MYTRANSF ERASE t i M0-Cj>-CH.CM-CO0H S -ADENOSYLMETMIONINE C 1 Pool CHjO S - MYDROXYFERULIC ACID

SHIKIMIC ACID O-METHYTRANSFERASE t H0-O-CH = CH-C00H - CONIFERYL ALCOHOL I CHjO i FCRULIC ACID t H ,-TOOH HO-O'CM- CH- COOM MOW ^ MO W CHj-CO -COOH HO

PRE PHENIC ACID CAFFEIC ACIO I T P•COUMARYL ALCOHOL- MOOCHvCO - COOH -»M0-f>CM2-CH-«X)H . HO-^-CH = CH-COOH

HYOROXYPHENYLPYRUVIC L- TYROSINE f ^-HVDROXYCINNAWIC ACID T

^•CH2-CO - COOH • ^>-CHJ-CH-COOH » ©- CH = CH-COOH PHENYLPTRUVIC ACID CINNAWIC ACID L-PHENYLALANINE 23

2.4.1 Synthesis of Lignin in the Plant

At the present time the mechanisms of lignin formation in the plant are fairly well understood. Lignin is 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 is a six carbon ring structure. It is 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 is 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 is formed by deamination of phenylalanine to give cinnamic acid which is then hydroxylated. Further enzyme mediated hydroxy-

lations lead to the other substituted cinnamic acid derivatives; caffeic acid, ferulic acid and sinapic acid. These acids are then enzymatically reduced to form the substituted cinnamyl alcohols listed above. Oxidative polymerization of the alcohols by peroxidases forms the lignin polymer.

The synthesis of lignin is outlined schematically by Higuchi et al.(1977) in Figure 8.

2.4.2 Phenol Coupling Reactions

Phenol coupling reactions form the basis of lignin biosynthesis as well as many other natural products such as tannins, lignans, pigments, antibiotics, alkaloids and humic substances (Crawford, 1981). The 24

central reaction is the formation of a phenoxy or phenolate radical.

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 is 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 alkyl substituents are present (Musso, 1967). Coupling can also occur with conjugated side chains. The most common bond in conifer lignin is the

& -aryl-ether bond as represented by the model compound «<-guaiacyl- glycerol-/^ -coniferyl ether (Hurst and Burges, 1967). This linkage comprises close to 50 % of the total linkages in conifer lignin (Crawford,

1981). Figure 10 illustrates these bond types.

In biological systems such as plants the formation of phenoxy radicals is 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 (H2O2: oxidoreductase). It is generally agreed that peroxidases are involved in lignin biosynthesis (Harkin, 1967; Crawford, 1981). The more reactive polyphenols such as the trihydroxy compounds may autooxidize without the aid of enzymes. This is very important in the formation of humic sub• stances and will be explored more fully later.

In summary, lignin is a complex, highly crosslinked, aromatic polymer of phenylpropane units. It is 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 ortho activated para a

phenolate ion PHENOXY RADICAL

Hvdroquinone OH

-H-

OH

Pyrocatechol

OH OH Cf- -H- -H«

Resorcinol

-IT

Figure 10. Representative Linkages of Phenylpropane Units in Conifer Lignin. Model Compound: »<.-guaiacylglycerol-/8 -coniferyl ether (Crawford, 1981; Hurst and Burges, 1967).

H2C0H

X HC—0—(/ VC=C—CHo0H

OH

oc -guaiacylglycerol -£ - coniferyl ether

BOND TYPE STRUCTURE PROPORTION %

Arylglycerol-/6-aryl £ °Oc 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 9-12 Phenylcoumaran C--c —o0

Diphenyl ethers c $ 3.5-4

.9- 27

The lignin polymer is 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 is 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 lignin with phloroglucinol is the

result of these free coniferaldehyde groups.

2.4.3 Differentiation of Plant Classes on the Basis of Lignin Composition

The early literature on lignin dealt almost entirely with coniferous

lignin conveying the idea that lignin was a polymer of coniferyl alcohol.

In 1944 Hibbert et al. treated the lignin from hardwood trees with alkaline nitrobenzene, obtaining a mixture of vanillin and syringaldehyde as products (Gottlieb and Hendricks, 1945). This led to the belief that

the nature of the aldehydes obtained from nitrobenzene oxidation of lignin 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 lignin" to a plant group warrants caution due to considerable structural variability among species which may affect the nature of the degradation products obtained

(Gross, 1979). There is also variability in lignin content and composition among different plant organs. Lignin is present in the xylem cells of 28

wood, the husks and shells of seeds, cones, etc., the stalks of grasses, ferns, etc., and plant roots (Gross, 1979).

Gymnosperm lignin contains dominantly coniferyl units with a low amount of p-coumaryl units. Syringyl content is 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 all three units (Crawford, 1981; Gross, 1979). Higuchi et al., cited by Crawford

(1981), defined three major types of lignin: guaiacyl lignin, found in conifers, lycopods, ferns and horsetails; guaiacyl-syringyl lignin, present in dicotyledons and a few exceptional gymnosperms; and, guaiacyl-syringyl-p-hydroxyphenyl lignin, found only in the highly evolved grasses. In the grasses considerable amounts of p-coumaric acid are bound as esters to the grass lignins and are not actually incorporated into the structure. Alder has suggested that grass lignins should therefore be classified as normal guaiacyl-syringyl lignins (Crawford,

1981). The lignin composition of the plant classes is given in Figure 11.

2.4.A The Modified Lignin Theory of Soil Humus

In the early 1930's the modified lignin theory of soil humus, was introduced by Waksman and others (Stevenson, 1982). The resistance of lignin to microbial decomposition in the soil compared to other plant constituents suggested a possible role for lignin as a precursor to soil 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 soil organic matter the "ligno-protein complex", which he believed were the soil humic acids. The protein was incorporated through the Schiff reaction:

(modified lignin)-CH0 + RNH^ » (modified lignin)-C—NHR + H20

(Stevenson, 1982)

Further support of this theory was based on the similar solubility of soil humic acids and lignin in reagents such as pyridine, alcohol and alkali; both formed precipitates in acid solutions; and laboratory synthesized ligno-protein complexes had similar properties to soil humic acids. In addition, both lignin 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 lignin 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 acidity.

A. There was a decrease in methoxyl groups but no concomitant increase

in phenolic hydroxyl groups.

5. The ultraviolet-visible spectra showed a loss of maxima characteristic

of lignin with the production of rather featureless spectra similar

to those of soil 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 lignin transformation to humic acids was largely

oxidative.

(Stevenson, 1982; Flaig et al., 1975)

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 al., 1975). It has been noted that the free radical content of lignin increases during humification (Flaig et al., 1975).

Experiments with model compounds indicate that demethylation of methoxyl groups is a prerequiste to condensation with nitrogen compounds ('Haider et al., 1965). This further supports the quinone mechanism outlined below:

demethylation 32

Due to the apparent success of degradative techniques in elucidating

the chemical structure of lignin, Gottlieb and Hendricks (1945) subjected

soil organic matter to alkaline nitrobenzene oxidation and high pressure catalytic hydrogenation. They failed to find any degradation products

indicative of lignin and concluded:

1. The material derived from plant lignin in the soil is drastically

altered in the kind and pattern of peripheral groupings on the

aromatic ring.

2. If lignin-like materials are present in soil 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 soil 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

Soil 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 lignins, 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 Tollin, 1967; Hurst, 1967), simple poly• phenols (Haider et al., 1972; Martin and Haider, 1971), anthraquinones

(Saiz-Jimenez et al., 1975), perylene derivatives (Kumada and Matsui,

1970) and antibiotics (Steelink and Tollin, 1967). Some or all of these substances may serve as precursors to humic substances. Autolysis of microbial cells or fruiting bodies may release dark-coloured, acidic,

"humic acid-like" polymers into the soil environment where they may be further altered to form soil humus. In essence there are many aspects of microbial activity which could produce precursors to soil humic substances.

The following sections focus on the "de novo" synthesis of polyphenols by microorganisms, and on the microbial degradation of lignin to simple monomers.

2.5.1 "De Novo" Synthesis of Polyphenols by Microorganisms

The "de novo" concept of soil 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 soil humus (Stevenson, 1982). Kononova rejected the lignin origin of soil 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 lignin is hardly touched, and humic substances are produced by the myxobacteria using carbohydrate as the carbon source. Recently, polyphenols released by the decomposition of lignin 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. Flaig, H.M. Hurst and Z. Filip.

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 al., 1972). H. toruloidea was found to be outstand• ing in both the number of phenols and weight of "humic acid-like" polymer produced. 35

Secondary metabolism was induced by a nutrient shortage or by the accumulation of primary metabolic intermediates (Haider et al., 1972).

Synthesis occurred via the shikimic acid pathway (cinnamic acid deriv• atives) and acetate-malonate pathway (orsellinic acid derivatives)

(Haider et at., 1972; Haider and Martin, 1967). A key polyphenol synthesized by the studied fungi was orsellinic acid (Haider and Martin,

1967; Martin et al., 1972; Haider et al., 1972). Oxidation, decarbox• ylation and hydroxylation reactions transformed orsellinic 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, gallic acid and pyrogallol were common to both pathways. Resorcinol was only formed from orsellinic acid, whereas p-hydroxycinnamic acid was the parent molecule to proto- catechuic acid. Toluene derivatives were common products of orcinol, formed by decarboxylation of orselli 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 ferulic, vanillic and syringic acids from microbially produced phenolic acids. Phloroglucinol, a 1,3,5 - trihydroxybenzene, can be formed from orsellinic acid or cresorsellinic 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 Orsellinic Acid by Fungi Imperfecti (Haider et al., 1972).

CH» CH» COOH ^\ COOH ^\

HO^^OH HO^/^OH HO^/^OH HO^^OH

orsellinic orcinol 3,5-dihydroxy- resorcinol ocid benzoic acid

CHi CHs COOH OH

l HO' %v/^OH HO OH HO^ ^OH OH OH OH 2,3,5-trihydroxy- 3,4,5-trihydroxy- gallic ocid pyrogollol toluene loluene

Figure 13. Transformation of p-Hydroxycinnamic Acid by Fungi Imperfecti (Haider et al., 1972).

OOH COOH COOH .OH

OH OH COOH OH OH OH roxy- CH f p-hydro protocatechiiic 2,3,4 -trihydroxy- / benzoic ocid ocid benzoic acid

OH V COOH p-hydroxy- \ ^ CH cinnomic ocid * COOH

'•OH HO^ ^OH HO^ >*OH OH OH OH coffelc acid gallic ocid 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 cells of the above fungi. As noted earlier, 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 soils 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 al.

(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 sensitivity of these phenols to autooxidation at pH values

> 6 (Haider et al., 1972). The phenols that were particularly sensitive were the asymmetric and vicinal 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 gallic 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 al., 1972).

2.5.3 Degradation of Lignin by Microorganisms

Several classes of microorganisms have the ability to degrade lignin.

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 lignin degradation by white-rot fungi. Complete degradation to carbon dioxide and cell constituents is supported by Haider et al. (1972), whereas Crawford (1981) concludes that lignin in its natural state is not readily utilizable as a carbon/energy source, but an additional more readily available carbon source such as cellulose is required for extensive degradation. These two views are not necessarily exclusive since lignin in its natural state is intimately associated with the structural polysaccharides.

The insolubility of the lignin polymer requires the use of extra• cellular enzymes for its degradation. It is 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 literature.

Nevertheless, this speculation led to three major conclusions regarding the degradation of lignin 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 still in the polymer.

Crawford (1981) cited two possible mechanisms for lignin degradation by microorganisms:

1. Depolymerization of the lignin macromolecule with the release of

monomeric and dimeric fragments which are transported into the

microbial cells where they are degraded.

2. Dearomatization of the intact polymer by cleavage of the rings

while they are still bound in the macromolecule, followed by

erosion of the resulting polymeric, aliphatic network.

Pathway 2 is generally favoured; however, it is likely 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 first step in its 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 lignin, is enzymatically cleaved by a dioxygenase via the "ortho-fission" ("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 is 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 vanillic acid by x3 -oxidation (Hurst and Burges, 1967). Earlier demethylation may occur in the soil 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 acid-like" polymers produced by the fungi when grown on lignified substrates were similar to peat and soil humic acids in elem• ental analysis, exchange capacity, total acidity, phenols released upon sodium amalgam reduction and resistance to microbial degradation in the soil (Martin and Haider, 1969, 1971). Phenolic structures were thought to be major contributors to fungal humic acids. Yields of identified 41

Figure 14. Intradiol Cleavage of Aromatic Rings by White-rot Fungi (Kirk, 1975).

OH 0, 'COOH COOH HOOC "OH HOOC

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 soil and peat humic acids were 3 to 6 % (Martin et al., 1974). Piper and Posner (1972b) reported yields of 12 to 32 % for soil 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 alkyl-aryl 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 al., 1974). Biphenyl structures, which were not cleaved by reduction, may contribute significantly to the humic acid polymer since phenol coupling reactions favoured C-C bond formation (Section 2.4.2).

Schnitzer et al. (1973), based on humic acid degradation with alkaline potassium permanganate, disputed the importance of phenolic structures to fungal and soil 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 soil 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 soil. Ring carbons were highly stabilized, 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 utilized. It was estimated that 10 to 20 % of intact p-hydroxycinnamic acids and caffeic acids were stabilized against biodegradation. These phenolic acids were A3

more readily linked into the model phenolase polymers due to the electron- donating effect of the acrylic side chain. Free phenolic acids were readily utilized 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, acidified, and the phenolics extracted into ether. Gas chromatographic analysis detected several major phenolic acids; in order of abundance, p-coumaric, ferulic, vanillic, p-hydroxybenzoic, salicylic and syringic. P-coumaric and ferulic acids were bound as esters to the grass lignins (Kuwatsuka and Shindo, 1973). The former amounted to over

50 % of the total phenolic acids identified. Minor amounts of benzoic, gallic, caffeic, 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 signifi• cantly. An exception was p-hydroxybenzoic acid which decreased slightly.

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 vanillic acids became dominant. 14 The incubation in soil of C-labeled oat roots demonstrated a rapid and 14 heterogeneous incorporation of the C-label into all organic matter fractions, coinciding with the peak of microbial activity (Sinha, 1972a,b).

The newly formed humic substances were preferentially stabilized into the humic acid and humin fractions, possibly through the incorporation of oligomeric lignin 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 if the warmer temperatures had stimulated the production of phenolic acids, either from lignin 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 fulvic acids and water-soluble compounds.

The transformations of p-coumaric and ferulic 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 ferulic acid to form

3,4-dimethoxycinnamic acid. Any further transformation was not observed.

The interactions of polyphenols with soil 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 soil.

The extent of adsorption in subsurface horizons was related to the dominant clay mineral; allophane > montmorillonite > kaolinite. In surface horizons adsorption was positively correlated with humus content, a reflection of humus stability. 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, ferulic and vanillic, 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 soil (Hanninen et al., 1981). Several attempts have been made to extract these acids according to their form in the soil. Weak extractants such as water, dilute base, calcium hydroxide and hot ethyl acetate have been used to extract the "free" phenolic acids (Whitehead et al., 1982, 1983; 'Katase,

1981a). This form is particularly important to allelopathic studies as it reflects the biochemical status of the soil 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 will be explored in Section

2.6.

Katase (1981a,b,c) investigated the phenolic acids in peat and forest soil. The same four acids detected by Kuwatsuka and Shindo (1973) from rice straw were also found to be most abundant in forest and peat soil. They were p-coumaric acid, ferulic acid, vanillic 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 soils. Total phenolic acids released were greater for the peat soil by an order of magnitude. The phenolic acids, ferulic and p-coumaric, were especially abundant in form

C of the peat soil. In contrast, vanillic acid prevailed in the forest soil 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 soil humification (Hanninen et al., 1981). Insoluble lignin macromolecules may contribute to this pool. The source of organic matter may also be important. Esters of p-coumaric and ferulic acids were noted in graminaceous species (Whitehead 47

et al., 1982; Hartley and Buchan, 1979), and p-coumaric acid was espec• ially abundant in sphagnum (Morrison, 1963; Morita, 1968). Coniferyl units, high in conifer lignin, formed vanillic acid through microbial oxidation.

The free form, A, contained the least phenolic acids reflecting its dynamic nature. The transformed phenolic acids, p-hydroxybenzoic and vanillic, were the major contributors. Form B phenolic acids may be viewed as being weakly esterified to organic components soluble in organic solvents. This form was very low in the peat soil. However, in the forest soil form B was high in p-coumaric and ferulic acids possibly due to a solubilization of higher molecular weight lignin fragments through microbial transformation. The interaction of form C with soil minerals was probably more important in the forest soil than the peat soil.

Stabilization of transformed phenolic acids by adsorption to mineral surfaces would facilitate their subsequent polymerization to form humic substances (Wang et al., 1983a).

A similar pattern was noted in forest and peat humus by Hanninen et al. (1981). There was a clear distinction in the types of phenolic compounds isolated from the fulvic acid fractions. In the forest humus the amount of vanillic acid and its 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 vanillic acids) compared to cinnamic acid derivatives (p-coumaric and ferulic acids) reflecting a greater degree of lignin transformation.

Whitehead et al. (1982) investigated the relationship between plant 48

species and the simple phenolic acids extracted from soils. Sample plots containing three different plant groups; monocotyledons (grasses), dicotyledons and pteridophytes (ferns), were sampled for soils and roots.

The soils were extracted with reagents increasing in alkalinity 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 soils by at least an order of magnitude. A clear separation in the content of p-coumaric and ferulic acids was apparent between the grass roots and the roots of other plant species. In terms of

/jg phenolic acid/ 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 ferulic acid in the soils, but was not as clear for p-coumaric acid. The plant roots showed a large variability in amount and composition of extracted phenolic acids which was not observed in the soils. 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 is to produce simple units representative of the main structural units in the polymer.

This is true whether the goal is structural or genetic. Chemical degrad• ations can be classified as either hydrolytic, oxidative or reductive.

A severe limitation in these techniques is 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 specifity, uncertainty in the reaction mechanism and the production of artifacts through molecular rearrangements, functional group shifts, condensations, etc. In order for degradative techniques to be used with any measure of confidence these problems must be understood and if possible corrected. Several investigators believe that a succession of techniques with increasing strength is required. Another approach is to hydrolyze the humic acid molecules prior to degradation to provide a more homogeneous starting material (Riffaldi 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 limitations, a definite link has been established between the phenolic degradation products of soil humus and the overlying vegetation. Morrison (1958, 1963) was one of the first to relate vegetat• ion to the phenolic aldehydes released during the alkaline nitrobenzene oxidation of soil humus. Detected in the oxidation products from soils and peats were the phenolic aldehydes, syringaldehyde, vanillin and 50

p-hydroxybenzaldehyde, and the related acids, syringic, vanillic and p-hydroxybenzoic. Lesser amounts of the cinnamic acid derivatives, p-coumaric and ferulic, were detected. A comparison of the oxidation products of sphagnum and sphagnum peat gave corresponding results. Both were low or absent in syringyl and vanillyl derivatives and dominant in p-hydroxybenzyl derivatives. In pine forest humus, vanillin was dominant followed by low amounts of p-hydroxybenzaldehyde. Syringaldehyde was very low or absent. This conformed to the known composition of gymnosperm lignin (Section 2.4.3). A peat profile containing birch remains gave large amounts of syringaldehyde on oxidation, as expected. A criticism of this work was that yields were very low; 1 to 4 % for peat soils and

0.5 to 1 % for mineral soils. Detection of phenolic compounds by paper chromatography also had its limitations, notably low sensitivity and poor quantitization. Nevertheless, a lignin-derived component of soil organic matter was established.

Sodium amalgam was used'by Burges et al. (1964) to reduce soil humic acids. The degradation products were divided into two categories: lignin- 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 fruits, flavonols which form the basis of condensed tannins, and flavones (Salisbury and Ross, 1978). 51

Figure 16. Phenolic Units Obtained by Reductive Cleavage of Soil Humic Acids (Burges et al., 1964).

COOH = R2 = H p-Hydroxybenzoic acid

C.C, R, = H, R2 = OCH3 Vanillic acid

OH Rj = #2 = OCH^ Syringic acid

Lignin-derived COOH R, = H, R^ = OH Protocatechuic acid units I CH,

Rj = H, R2 = OCH^ Guaiacylpropionic acid C.C, CH,

Rj = R2 = OCH^ Syringylpropionic acid

OH

Kj/^OH R1 = OH Phloroglucinol R, = H Resorcinol Flavonoid- OH

derived R R2 = OH Methylphloroglucinol units l " C.C, R l - H, R2 = OH 2,6-Dihydroxytoluene 'CHa OH R, = OH, R2 = H 2,4-Dihydroxytoluene

COOH

3,5-Dihydroxybenzoic acid HO\^OH Unassigned units Pyrogallol HO"\^OH OH

Figure 17. Structure of Flavonoids (Salisbury and Ross, 1978),

3'

OH ^7 5'

from from acetate-malonate cinnamic acid pathway 52

All have a C^C^C^ carbon structure containing a pyran ring (Figure 17).

The A ring is based on phloroglucinol formed by the acetate-malonate pathway. The B ring is 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 listed in Figure 16 (Burges et al., 1964; Tate and

Goh, 1973).

Burges et al. (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 lignin of the overlying vegetation, microbially-derived phenolic compounds being common to most soils. In

Section 2.4.3 it was noted that syringyl residues were present in the lignin 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. Vanillic, protocatechuic and p-hydroxybenzoic acids were ubiquitous among the soils 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 alkyl-aryl ether bonds are not cleaved by reductive degradation (Burges et al., 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 alkyl-aryl 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 little 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• ivity of the reaction mixture to reoxidation. Schnitzer et al. (1973) pointed out that humic acids were difficult 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 soils of widely different origins (Piper and Posner, 1972b; Dormaar, 1969).

Piper and Posner (1972b) felt that reductive degradation was better suited to estimating the "degree of transformation" of soil humic acids.

The nature and amount of the phenolic acid degradation products were related to the soil extractant, the molecular size of the humic acid mole• cules, the soil's clay mineral composition and particle size distribution.

Humic acids that were extracted by sodium hydroxide following a pyrophos• phate extraction gave more phenolic compounds upon reductive degradation, primarily the less transformed vanillic, syringic and ferulic 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 lignin than the more oxidized, lower molecular weight 54

humic acids (Piper and Posner, 1972b).

The soil environment also affected the nature of the phenolic

degradation products. Conditions conducive to low biological activity

such as gleying or cold temperatures produced greater amounts of phenolic

acids, particularly vanillic acid, upon reductive degradation (Tate and

Goh, 1973). Jacquin reported an inverse relationship between the amount

of vanillic 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 soil humic acids were subjected to acid hydrolysis with the phenolic degradation products detected by gas chromatography.

Two soils were examined; a Bh horizon of a Podzol originally 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 vanillic acid.

Syringic acid was not identified possibly due to interference problems

in gas chromatography (Tate, 1972; Stevenson and Mendez, 1967). Cinnamic acid derivatives were also not identified. Katase (1981b) noted that

substituted cinnamic acid derivatives were polymerized during acid treat• ment .

The gel-excluded humic acid fractions from both soils upon hydrolysis

released mainly protocatechuic acid with lesser amounts of p-hydroxybenzoic

and vanillic 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 lignin. However, considerable amounts of protocatechuic acid in the hydrolysates indicated extensive demethylation of vanillic acid; either by microorganisms or during acid hydrolysis when it was possible that some demethylation occurred, although concentrated acids and vigorous reaction conditions are usually required to cleave alkyl- aryl bonds (Morrison and Boyd, 1973). Furthermore, Katase (1981b) found that the recovery of vanillic acid by reflux in 6 N HC1 or 2 N NaOH was independent of pH. This suggested that the demethylation of vanillic acid occurred in the soil 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 Soil Humic Substances and Their Relationship to Genetic Soil 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 fraction.

The early humic acid literature focused on a defined chemical composition

(Stevenson, 1982). With the arrival 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 filtrate 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 filtrate. 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 it became apparent that despite the overall similarity in humic substances there were perceptible differences related to soil 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 soil genesis.

It had long been recognized that humus fractions differed in nature and amount among genetic soil types and even between horizons of the same soil.

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 soil processes

(Sinha, 1972a,b; Anderson, 1979; Swift et al., 1970).

The Ch/Cf ratio was found to vary with latitude in the zonal soils 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 soil zone (Kononova, 1961). Similar trends were reported in the Humic Allophane soils of Japan (Tokudome and Kanno,

1965a,b, 1968) and in the Chernozemic soils 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 soil was closely related to climate, vegetation and parent material. These soil forming factors determined the amount of organic matter added to the soil, its mode of decomposition and the balance between mineralization and immobilization. Conditions which favoured the accumulation of humus included an ameliorating vegetation, a neutral soil environment conducive to microbial activity and the poly• merization of humic acid precursors, a stabilizing 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 soil horizon can be inferred from the Ch/Cf ratio. In surface mineral horizons, organic matter formed by in situ root decomposition produced a high Ch/Cf ratio. Ch/Cf ratios 58

greater than 1.0 were typical in Ah horizons of temperate grassland soils

(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 illuvial 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 soil 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 fulvic acid fraction. The Ca/Cf ratio was influenced by the source of vegetation, the soil acidity and the leaching regime and seemed to reflect the biolog• ical status of the soil. Briefly, soils developed under grass tend to have low Ca/Cf ratios with the fulvic acid fraction dominated by polysac• charides. A neutral soil environment and large biomass caused the deplet• ion of polyphenols through polymerization or microbial degradation. The polyphenol-rich litter and low biological activity present in the organic horizons of coniferous forest soils (Berg et al., 1980) produced high

Ca/Cf ratios. The acid environment and leaching regime common to these soils favoured the illuviation 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 illuvial 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 visible regions is 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 visible 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 (Eoori) 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 al., 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 al. (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,/EA ratio and polymer molecular 60

weight (Chen et al., 1977; Swift et al., 1970; Anderson et al., 1974a,b).

According to Chen et al. (1977) a low E^/E^ ratio reflected a large molecular size or weight, a high carbon content, and a relatively low content of oxygen, carboxyl groups and total acidity. 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 classified into three major types based on their ultraviolet-visible 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 light absorption at 600 nm per unit weight of humic acid, and A logK which represented the inclin• ation of the absorption curve from 600 to 400 nm ( AlogK = 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 soils 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 soils, weakly calcareous soils and Chernozemic soils. Type B humic acids were common in soils of the brown forest group.

Immature Rp-type humic acids were found in brown forest soils, red soils, organic horizons, composts, etc. and were assumed to form during the rotting of plant residues. They represented the initial stages of humif• ication displaying characteristics similar to lignins, tannins, etc.

(Suzuki and Kumada, 1972).

Detailed characterization of the humic acid types was carried out 61

by several Japanese investigators (Kuwatsuka et al., 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 al., 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 significantly different among the humic acid types. The degree of humification, as defined by RF and AlogK, 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 variability 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 rich 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 al., 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 susceptibility 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 is located in Central Mexico approximately 60 km south-east of Mexico City. 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 capital, 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 it is the second highest mountain in Mexico reaching an altitude of

5452 m. It has a classical cone shape often called stratified due to several lava flows interbedded with unconsolidatated pyroclastic materials.

Popocatepetl was formed during the Pleistocene (10,000 to 2 million 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 it the third highest mountain in Mexico (Lorenzo, 1959). The snow covered silhouette of 63

Iztaccihuatl bears a striking resemblance to a woman lying on her back.

The northern, central and southern peaks form the head, breast and feet, respectively. The ridge is a little 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 fell.

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 million years before present) with hornblende, trachy-andesitic lava flows forming the basal part. Volcanic activity began again in the Pliocene

(2 to 12 million 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 glaciofluvial sediments covering the lower slopes (Miehlich, 1980).

The soils 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 soil units of the Sierra Nevada. The dominant soil units are Andosols; the the specific unit being a function of geographic location, climate, and age and type of parent material.

The soils of the Sierra Nevada are classified by a system introduced 64

by Miehlich (1980). Four Andosol units, using the diagnostic horizons of

Soil Taxonomy and the FAO, are defined; Andine, Andic, Andos and Thixic

Andosols. Briefly, the first corresponds to the FAO Vitric Andosol, the second and third to the FAO Humic Andosol. Thixic Andosols are found in young, little weathered relocated ash sediments (toba sediments) and cont• ain dominantly silica in the clay fraction.

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. Profile differentiation may also occur. These soils 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 Soil Forming Materials on Iztaccihuatl (Miehlich, 1980).

Approximate Age B.P. C= ash (yr) P= pumice 1C 400

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 limits, 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 its 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 is 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 distinct 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 statistically powerful for vegetation and required less

sampling. Three "climax" vegetation zones were selected; Abies religiosa,

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 soils 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 criteria:

1. The plot was representative of the zone.

2. The soil parent material was the most recent ash of Popocatepetl,

designated 1C (Miehlich, 1980).

3. The ash layer was deep enough for adequate profile 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 statistical 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 fell 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 Ah2 horizons. In addition the forest floor was sampled where appropriate. For statistical 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.

Statistical analysis by ANOVA requires the following assumptions to be met:

1. The variance for all 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 all

possible observations.

With only eight observations per treatment it is 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 statistics. 72

Non-parametic statistics avoids using population parameter means which rely heavily on a normal distribution. Instead, observations are ranked, a statistic is computed and the null hypothesis is accepted or rejected based on a chosen significance level. The MIDAS statistical package at the University of British 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 is 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 null hypotheses tested in this study were:

HO: The three zones were from a common population.

HO: The two soil horizons were from a common population.

The alternative hypotheses were:

HI: At least one zone was not from the common population.

HI: The two soil 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 its effect was negligible. Topographical irregularities, such as depressions, mounds, windthrow disturbances, etc. were also not evident.

Fire scars were present in the Abies religiosa and Pinus hartwegii plots on veteran trees, and charcoal fragments occurred in the soil profiles.

Charcoal was absent in the Zacatonal plot.

The experimental plot selected within the Abies religiosa zone seemed typical (Plate 2). A fairly dense but open tree canopy allowed sufficient light 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 light to reach the forest floor.

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- ical effect. There was evidence of overland water flow which occurred during the rainy season. Interspersed between bunchgrass colonies were large bare patches of soil with little 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 slightly concave but of lesser slope than the Abies religiosa 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 religiosa Pinus hartwegii Zacatonal

Plot location: 15 20.8 30.5 km from Amecameca, by road

Plot size: 50 x 100 75 x 50 50 x 75 width x length (n>)

Elevation: 3240 3520 3910 mid-plot (m)

Aspect: WNW SW

Slope: 23 23 15 (°)

Dominant flora: Abies religiosa Pinus hartweRii Festuca tolucensia Pyrola secunda Festuca tolucensis Festuca amplissima Sibthorpia pichenchensis Senecio tolucanus Senecio angulifolius Achemilla procumbens Vaccinium spp. Senecio barba-Johannis Plate 2. The Abies Religiosa Zone.

The modal pit profile. Festuca tolucensis Festuca tolucensis and Festuca amplissima 79

4.0 METHODS

The investigative research for this project can be divided into two sections. The first encompasses all samples individually and thus gives a measure of parameter variability within a zone or horizon. The purpose of Part 1, or the statistical study, is to give a general overview on the nature of the soils and the processes occurring. The second section, designated Part 2, examines the soil organic matter for phenolic acids indicative of vegetation. Composite samples were prepared for each of the soil zones to measure a population "mean". Population variability is not examined; hence, statistically 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 soils typically involves their solubility behaviour in various extract- ants. Allophane and imogolite can be separated by their differing behaviour with respect to pH. Allophane disperses in both acid and base whereas imogolite flocculates when the pH is 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 (Parfitt,

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 soils. Ultrasonics was used to disperse the soil, possibly preceded by a hydrogen peroxide (H2O2) treatment to remove soil 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 Si, Al and Fe

lost during the treatment. A differential IR spectrum was recorded by

placing the KBr disks made from the clay residues before and after treat• ment in the sample and reference cells, respectively.

Cbd extracts iron oxides with some co-extracted aluminium and humus

complexes. 2% ^200^ dissolves "allophane-like" consituents and weakly

dissolves opaline silica. 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 is sharper and occurs at a lower

frequency due to a higher Al content (940-960 cm ^). The opaline, silica

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 silica. Recall, that during isolation of allophane and imogolite,

imogolite flocculates when the pH is raised to 10.5 - 11. This contra•

dictory behaviour may stem from differences is alkali 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 silica (1070 cm

Imogolite is 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 identification 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 first in the A horizon had a very low complexing ability for

Al and Fe and was little 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.

Parfitt (1983) developed a very simple method for estimating the content of allophane and other inorganic constituents in soils by measuring cbd, sodium pyrophosphate and acid oxalate extractable Fe, Al and Si. Allophane was determined from the acid oxalate extractable Si content. The Si content of natural soil allophanes was close to 14%; therefore, dividing the acid oxalate extractable Si by 0.14 gave the

% allophane content of the soil. Humus-Al,Fe complexes and crystalline iron oxides were also determined using this procedure (see Section 5.1.2.2).

4.1.2 Extraction and Fractionation of Soil Humus

The study of soil humus involves the separation of the humus from the non-humus and mineral constituents of the soil. In the past, non-isolative 82

methods such as proximate analysis were used to characterize the soil organic matter. However, this was of limited and uncertain value, especially in well humified mineral soils. Quantitative extraction of humic substances from the soil is a difficult procedure, since the humus is closely associated with the mineral fraction including crystalline clay minerals, sesquioxides and bi- and tri-valent cations. Consequently, any extraction procedure will not remove all of the soil humus and tends to extract certain humus pools. The separation of humified material from non-humified or partially humified material and microbial biomass is also less than complete due to the similar solubility of certain components in many reagents.

Several factors must be taken into consideration when planning an extraction scheme. A primary goal is to extract unaltered material in high enough yields to adequately represent the soil humus. Other consid• erations in selecting an extractant include the nature of the soil and the necessity for pretreatments. For example, soils high in exchangeable calcium or carbonates may require an initial acid extraction to improve subsequent extraction in alkali. Soils high in sesquioxides or humus-Al,

Fe complexes require the presence of a complexing agent such as pyrophos• phate. Volcanic ash soils containing humus-allophane complexes are efficiently extracted only with sodium hydroxide (Duchaufour, 1977).

A further consideration is 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 classical soil organic matter extraction procedure uses alkali 83

(Stevenson, 1982). Generally, 0.1 to 0.5 N sodium hydroxide (NaOH) is used, the higher concentration extracting less humus but of lower ash content (Sequi et al., 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 is 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 al., 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) is used by Kumada et al. (1967) to obtain information on two humic and fulvic acid fractions differing in maturity. When combined with alkali, sodium pyrophosphate provides an efficient overall extractant for a wide range of soils without the need for an acid pretreatment (Lowe, 1980). However, in humus isolation studies pyrophosphate is generally avoided due to the difficulty in removing it from the extracts.

A further variation on the combined NaOH-pyrophosphate extraction scheme was developed by Anderson et al. (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 labile form of plant nutrients (Anderson, 1979). ,

Humus extraction is generally followed by fractionation into humic 84

acids and fulvic acids. These fractions are operationally defined on the basis of solubility. The hurain fraction is 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 soil. The humic acid fraction (HA) is soluble in alkaline solutions but precipitates upon acidification. 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 fraction. Alcohol was often used to separate an alcohol-soluble hymatomelanic acid fraction

(Stevenson, 1982). As previously mentioned, Anderson et al. (1974) obtained the HA-B fraction by sonification.

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 is 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 its 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 al. (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 initial humus extract

from the soil residue, and for separating the HA precipitate from the FA

supernatant also influenced the Ch/Cf ratio. In the first instance, lower speeds increased the HA fraction resulting in higher Ch/Cf ratios (Lowe,

1980). In the latter, higher speeds had little effect on HA but decreased

FA, increasing the Ch/Cf ratio (Schuppli and McKeague, 1984; Schnitzer et al., 1981). In the literature centrifuge speeds of 5000 to 6000 rpm were standard (Lowe, 1980; Schnitzer et al., 1981).

The pH of HA-FA separation also influences the Ch/Cf ratio. The pH of KA precipitation should be <1 to minimize the effects of metals (Sequi et al., 1975a). However, pH's of 1 to 2 have been commonly used with little 86

adverse effects (Lowe, 1980; Anderson, 1979; Schnitzer et al., 1981;

Schuppli and McKeague, 1984). Precipitation of HA at pH's > 2 is not recommended. The dependence of the Ch/Cf ratio on the experimental conditions emphasizes the necessity to standardize the humus extraction and fractionation procedure if different soils are to be compared.

4.1.3 Humus Degradation Methods

The chemical degradation of soil 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 soil 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 nitro• benzene oxidations (Schnitzer and Khan, 1972; Ogner, 1973; Wildung et al.,

1970; Maximov et al., 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 little resemblance to the starting material (Maximov et al., 1977). Further limitations included:

1. An increased risk of autooxidation reactions and the formation of

artifacts under alkaline conditions.

2. Pre-methylation was essential to protect aromatic rings containing

labile functional groups from cleavage (Maximovet al., 1977).

3. The production of considerable amounts of pentacarboxylic acids

suggested degradation of the condensed ring system.

4. The harsh oxidative methods lacked selectivity in bond cleavage 87

making it difficult to interpret reaction pathways.

The milder degradative procedures were limited by low yields of identifiable compounds and an inability to cleave covalent C-C bonds contained within the humic acid "core". Consequently, mild degradations were of little 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 al., 1964; Piper and Posner, 1972b, Tate and Goh,

1973; Tate, 1972; Tate and' Anderson, 1978).

The sodium amalgam reductive cleavage of soil humic acids was intro• duced by Burges et al. (1964). This method stirred up much controversy in the soils literature as other researchers stressed the sensitivity 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 al., 1973; Dormaar, 1969). Several investigators had little success with the method, and low amounts of phenolic compounds were obtained (Stevenson and Mendez, 1967; Schnitzer et al., 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 al., 1972, 1983; Kuwatsuka and Shindo, 1973; Shindo and Kuwatsuka, 1975a,b, 1976) to moderately severe alkaline and acid hydroysis under reflux (Riffaldi and Schnitzer, 88

1973; Neyroud and Schnitzer, 1975a,b; Tate and Anderson, 1978; Anderson et al., 1978) have been used to degrade humic substances or extract whole soils. These procedures attacked the peripheral side chains of organic molecules cleaving relatively weak linkages such as hydrogen bonds; ester, glycoside and peptide (amide) linkages; and, salt bridges.

Approximately 1/3 to 1/2 of the soil organic matter is dissolved represent• ing the more labile humus components (Riffaldi 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 will 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 (Riffaldi 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 artifacts is 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 thin- layer chromatography (Burges et al., 1964; Piper and Posner, 1972a,b;

Morrison, 1958, 1963) to sophisticated gas chromatography (Tate, 1972; 89

Anderson et al., 1978; Neyroud and Schnitzer, 1975a,b; Ogner, 1973;

Kuwatsuka and Shindo, 1973; Katase, 1981a) were evident in the soils literature. Gas chromatographic methods significantly increased the reliability and speed of analysis as well as the detection limits for phenolic compounds. Improved verification of compound identity was provided by the combination of gas chromatography with mass spectrometry.

However, derivatization was essential to make the phenolic compounds volatile. Furthermore, there was a size limit to the molecules that could be analysed, and thermal degradation was a definite risk.

Recent developments in liquid chromatography column design produced columns able to withstand high pressure. This led to a new technique called high-performance liquid chromatography (HPLC). Several advantages were evident in this technique. First, derivatization was not necessary.

Secondly, thermal degradation was not a problem, and thirdly, there was no limit to the size of molecules that could be chromatographed (Wulf and

Nagel, 1976). HPLC also offered selectivity, high resolution, speed and sensitivity (Wulf and Nagel, 1976; Charpentier and Cowles, 1981).

Consequently, the use of this technique in the soils literature has escalated in recent years (Whitehead et al., 1982; Hartley and Buchan,

1979; Hanninen et al., 1981). 90

4.2 Part 1 - Statistical Study

4.2.1 Sample Preparation

The samples collected in Mexico were partially dried and visible

roots, macrofauna, etc. were removed prior to packaging for export.

A few days later at the University of British Columbia the samples were

fully air-dried and passed through a 2 mm sieve to remove small twigs,

pumice, charcoal, etc. Greater than 90% of the soil 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 soil

samples were stored in plastic containers or glass jars.

The FH horizons were prepared by gently rolling the material on a

2 mm sieve, removing large fragments, twigs, charcoal, etc. Fungal mycelia and moss were removed from several Abies religiosa 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 soil 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

ignition was done according to Hesse (1971) with a three hour ignition

period at 450 °C. Total carbon was determined by dry combustion in a

Leco Induction Furnace, Model 521 (Allison, 1965a). The pH was determined on the modal pits only, both in water and in 0.01 M CaC^. A 1:1 or 1:2

soil 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 silicon 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 iron, aluminium and silicon 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 silicon. 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 all 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 soil per 250 ml centrifuge bottle (Kumada et al., 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, distilled water, 0.1 N

H2S0^ and distilled 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 (Allison, 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, distillation and titration 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,nf! , 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.

Statistical analysis indicated that the two soil horizons, Ah^ and Al^,

came from the same soil population in the Abies religiosa and Zacatonal

zones, but were distinct populations in the Pinus hartwegii zone.

Consequently, four composite samples were prepared; Abies religiosa,

Pinus hartwegii Ah^, Pinus hartwegii kh^ and Zacatonal.

The composite samples were prepared from equal contributions of

organic matter from each soil 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. Soil samples that were very low in %Ch were

neglected as they would lower the final 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, five from the Abies religiosa 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, splitting,

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 iso•

lation soil extraction weights. Carbon contents in the humus fractions

were determined by the Astro Solution Carbon Analyser. This data allowed

estimates of bulk isolation recoveries. 95

4.3.2 Bulk Isolation Procedure

Details of the bulk isolation procedure are given in Figure 21.

The extractant used was 0.1 N NaOH with a ratio of soil 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 all acidifying steps due to the difficulty 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 relat• ively short time, and was more efficient 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 Diafiltration and Concentration of Extracts

After preliminary trials, 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. Initially, it was desired to study more than one molecular size range. However, very little of the humic acid fraction from Pinus hartwegii Ah^ was retained above molecular weight 100,000, and it was estimated that >70% fell between molecular weights 50,000 and 100,000.

Prior to diafiltration 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. Diafiltration and concentration were done in an Amicon

Model TCF-10 N2~pressurized diafiltration cell using a Diaflow YM2 membrane (nominal molecular weight retention 1,000) and a Diaflow PM10 membrane (nominal molecular weight retention 10,000) (Figure 22).

Filtrates 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 flask. 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 vials.

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 ignition at 200 °C for 1 hour followed by 2 hours at 450 °C and

3 hours at 650 °C. Visible 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 cell. 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

Figure 21. Procedure for Bulk Isolation of Humic Acid and Fraction A.

Weigh 150 g composite sample into 2.5 L bottle. Add 1.5 L 0.1 N NaOH. Displace air with N2. Shake overnight.

Let settle. Decant or siphon off alkaline extract.

Re-extract residue with 0.75 L

0.1 N NaOH. N2. Shake 1 hr.

Let settle. Decant alkaline extract. Discard soil residue.

Alkaline extract

Acidify to pH 1.5 with 6 N HC1. Let settle. Siphon off FA from HA ppt.

HA FA Pour HA slurry into 250 ml centrifuge bottles. Centrifuge at 6000 rpm for Filter FA through Whatman #1. Isolate 15 min. Decant FA combining with fraction A on "Column-PVP set-up" previous FA. (Appendix A).

HA "Clean-up" Fraction A Fraction C discard Dissolve HA into small volume of 2N NaOH. Divide among centrifuge bottles. Dilute. Centrifuge at 6000 rpm for 20 min. Decant HA. Discard solids. Pass fraction A through I H -exchange resin. Dilute HA with distilled water. Filter through Whatman #1, changing filter paper often.

Fraction A-H

Acidify to pH 1.5. Let settle overnight. Siphon off FA

If FA coloured, treat ULTRAFILTRATION with PVP.

Repeat dissolution and acidification of HA until FA pale in colour.

Last precipitation, centrifuge. Decant pale FA. Discard. Store acidified HA ppt. in refrigerator until ultrafiltration.

ULTRAFILTRATION 98

Figure 22. Ultrafiltration Procedure for Isolation of Polymeric Humic Acid and Fraction A.

HA Dissolve HA ppt. in NaOH. Adjust to pH 7 with HC1.

PM10 membrane MW cut-off 10,000 daltons

I Filtrate Retentate MW > 10,000 Acidify to pH 1.5. Dilute 1.00 ml aliquot to

Let settle. Centrifuge. 50.00 ml. Measure %C.

HA < 10,000 FA FREEZE-DRY RETENTATE HA

Dissolve in 0.1 N NaOH. Treat with PVP, Make to volume in volumetric Isolate fraction A. flask. H+-exchange resin. Measure XC on Astro Add to previously isolated Solution Carbon Analyser. fraction A. Discard HA < 10,000.

Fraction A-H Adjust to pH 7 with NaOH.

YM2 membrane MW cut-off 1,000 daltons

Filtrate Retentate MW < 1,000 MW > 1,000 Make to volume in volumetric Dilute 2.00 ml to 50.00 ml in flasks (several liters). volumetric flask. Measure XC on Astro Solution Measure XC on Astro Solution Carbon Analyser. Carbon Analyser. Discard filtrate. 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 Sterilizer set at 120 °C and 15 psi. 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 triplicate (Kuwatsuka and

Shindo, 1973a). This extraction sequence separated acidic phenols from neutral phenols, the former being isolated for analysis. After evapor• ation of the final ether extract the residue was quantitatively dissolved in 2.00 ml methanol (HPLC grade), pressed through a nucleopore filter into a small glass vial and capped tightly. 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 visible light 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 liquid 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 silica 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 first (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 )

Hydrolyze with 7.00 ml 6N HC1 for 2 hours

Hydrolysate I Centrifuge, 2500 rpm, 15 min.

Precipitate Supernatant dry and weigh Filter through pre-weighed Whatman #A2 filter paper into 125 ml separatory funnel dry filter paper and weigh

Extract 3x with 50 ml anhyd. * 1 ether. 1 -soluble" aq. phase hydrolysis products discard

Extract 3x with

2% NaHC03, pH 8

"acidic" ether phase ether-bicarbonate soluble "non-acidic phenols' "phenolic acids"

Acidify to pH 1.5 with HC1 Extract 3x with anhyd. ether

aq. phase ether-bicarbonate-ether soluble discard "phenolic acids"

dry with Na^O^ Evaporate ether

Phenolic acid residue

Dissolve in 2.00 ml HPLC grade MeOH Filter through nucleopore.

PHENOLIC ACIDS in methanol HPLC

anhydrous ethyl ether 101

temperature at 45 °C. A "fixed-loop" injection port allowed precise quantitization of injection volumes. All solvents were HPLC grade and passed through a nucleopore filter 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; acetonitrile - H^O - acetic acid (Charpentier

and Cowles, 1981) and methanol - H20 - acetic acid. The methanol - H^O - acetic acid system has been successively used by Hanninen et al. (1981).

A low concentration of acetic acid was included to buffer the pH and supress ionization of the carboxylic acid groups. The acetonitrile system gave a good standard chromatogram but the fast elution times of the compounds caused a crowded, less resolved sample spectrum. The vanillic acid peak often totally 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 vanil• lic acids. Consequently, this mobile phase solvent system was chosen for data collection while the former system was used to verify peak identity.

Further verification was done by observing the change in detector sensit• ivity at A = 260 nm for standard and sample chromatograms.

The sample hydrolysates were found to contain vanillic acid in con• centrations greater by up to an order of magnitude compared to the other three phenolic acids; consequently, vanillic 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 origin. P-hyroxybenzoic acid could have been determined

by single point calibration, but its concentration in the sample hydrol-

ysates easily allowed its 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 sensitivity : 0.01 Wavelength : 280 nm Chart speed : 0.5 cm/min Attenuation : 8 or 16 Flow rate : 2 ml/min Column temperature : 45 °C Calibration : external standards; multilevel or single point Standards : protocatechuic acid (3,4-dihydroxybenzoic acid), p-hydroxy• benzoic acid, vanillic acid (3-methoxy-4-hydroxybenzoic acid syringic acid (3,4-dimethoxybenzoic acid)

Gradient elution : methanol - H„0 - acetic acid

t (min) % A methanol % B 1% acetic acid

0 11 89 5 11 89 8 14 86 14 14 86 30 11 89

* Composition change is gradual not stepwise.

Retention times of standards in methanol gradient: RT (min) protocatechuic acid 4.95 p-hydroxybenzoic acid 8.41 vanillic acid 12.05 syringic acid 16.05

* Retention times varied slightly between different days but were very consistant on a given day. Recalibration was necessary each day. 104

Figure 24. Representative Standard Chromatogram of p-Hydroxybenzoic Acid, Protocatechuic Acid and Syringic Acid by Methanol Gradient Elution.

30 min 0 10 20 105

5.0 RESULTS AND DISCUSSION

The results and discussion is divided into Part 1 and Part 2. Part

1 is further divided into two sections. The first explores trends in the modal pit profiles. The second statistically evaluates several parameters measured on the Ah^ and Ah^ soil horizons and forms the basis for comp• osite sample preparation in Part 2. Included with section 2 is the literature from the joint German-Mexican project by Giinter Miehlich (1980).

The mineralogical data on the parent ashes presented in the above report is used to verify a common parent material throughout the three vegetation zones. Part 2 examines the characteristics of the humic acid and poly- phenolic fulvic acid fraction (fraction A) obtained through bulk isolation of the composite samples. In particular, emphasis is placed on the phen• olic acid hydrolysis products of the humic acid and fraction A as they relate to vegetation.

5.1 Part 1 - Statistical 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 is given in Figure 25.

Single sample determinations were made for all parameters except total nitrogen (%Nt) which was the mean of duplicate determinations. Duplicates were included periodically to check for reproducibility. The interpretat• ion of parameter trends within the modal pit profiles was very speculative due to only one profile being examined, and several parameters displayed Table 3. Chemical Analysis of the Modal Pit Soil Horizons from Iztaccihuatl Vegetation Zones.

ZONE HORIZON PH1 PH1 %HM %LI %Ct %Nt

(H20) (0.01 M CaCl2) -oven-dry basis-

Abies religiosa FH 6.5 6.05 32.3 16.8

Ahl 6.3 5.5 0.80 3.4 1.7 0.103 Ah2 6.8 6.2 0.42 1.8 0.70 0.046 Ah 3 6.8 0.33 1.4 0.57 0.031 B 6.6 0.55 2.1 0.90 0.049 IP pumice 6.3 1.50 4.4 1.9 0.111

Pinus hartwegii FH 4.5 6.80 63.5 30.3

Ahl 4.9 4.2 2.35 11.7 5.5 0.273 Ah2 5.8 5.0 0.64 2.5 1.1 0.058 Ah3 5.7 0.74 2.9 1.3 0.070 Ah4 6.1 0.86 3.1 1.5 0.068 IP pumice 6.1 1.26 3.7 1.4 0.071

Zacatonal Ahl 5.7 4.8 0.91 4.2 2.0 0.133 Ah 2 5.8 4.9 0.92 3.5 1.9 0.122 Ah 3 5.7 1.31 4.2 2.0 0.123 Ah4 5.9 1.58 4.7 2.2 0.135 Ah 5 6.1 1.81 4.3 1.9 0.121

1. Mean of duplicate determinations. 107

Figure 25. Distribution of Chemical Parameters Within the Modal Pit Profiles. 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 soil pH in water was below neutrality for all 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 CaC^; hence, considerable hydrogen was present on the exchange complex. The highest pH was found in the Abies religiosa zone which approached neutrality in the middle of the profile. The FH layer was not particularly acid, pH (H^O) = 6.5 , suggesting a high proportion of less humified organic matter or adequate biological activity which prevented the accumulation of organic acids.

The high acidity typical of coniferous litter was not apparent. The high pH in the Ah^ horizon reflected minimal weathering of the profile 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 soil, 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 pit. This

suggested that the acidifying effect of the pine litter 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 Al^+ on the exchange sites. The first 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 Al^+ may have decreased with depth as other amorphous forms became dominant.

The levels of extractable Al in the Pinus hartwegii zone were significantly less than in the Zacatonal zone. Hence, the similar pH present in the Pinus Ah^ horizon reflected some influence of pine litter 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 religiosa zone which lacked significant grass cover. The organic matter source possibly influenced the nature of the humus formed, particu• larly its "degree of humification" and functional group content. However, as will be later shown, humic acid formation was controlled by the region• al 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 is not included as temperatures in excess of 150 °C are required for its removal.

The amount of adsorbed water reflects the nature of the adsorbing surface Ill

and the surface area accessible to the water molecules. Indirectly, %HM gives a statement on the "degree of transformation" and "amount" of soil colloids, 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 soil was entered, which continued into the Ah^ horizons, especially in the Pinus modal pit. There was little further change with depth except for a modest increase in the IP pumice layers. The pumice layers, as will be shown later, were significantly more weathered than the above soil horizons which would contribute to its 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 Si, and less so for Fe. Thus, %HM was influenced heavily by inorganic colloids at depth in the Zacatonal zone.

The percent loss on ignition (%LI), total carbon (%Ct) and %Nt fol• 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 slightly higher level continuing down the profile. In forested ecosystems the soil surface would be enriched with carbon in the form of a relatively thick humus layer followed by a thin Ah horizon. Humus levels decreased significantly in the B and C horizons. The coniferous soil zones on Iztaccihuatl cont• ained relatively 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 plentiful in the IP pumice layer reflecting 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 situ root decomposition. However, some illuviation 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 profile. A modest proportion of %Ct in the Abies religiosa 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 reflecting a uniform input of organic matter into the profile, primarily through in situ decomposition.

Extractable iron (%Fe), aluminium (%A1) and silicon (%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

(Parfitt, 1980, 1983). The extraction mechanism involves complex form• ation and not acid dissolution (McKeague and Day, 1966). Opaline silica and volcanic glasses are not affected by this treatment. Table 4. Extractable Iron (%Fe), Aluminium (%A1) and Silicon (%Si) from Modal Pit Soil Horizons.

ZONE HORIZON ZFe ZAl %Si %A1 %Fe ZAl c bd c bd^ ox o x o X py py

Abies religiosa 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

Pinus hartwegii 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

Zacatonal 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 basis 114

Citrate-bicarbonate-dithionite (cbd) is primarily a reducing agent, but contains citrate to complex and retain the solubility of the released

Fe and Al. Both crystalline and amorphous forms of Fe are attacked.

Cbd extracts Fe in the ferric state (Fe"^+) from crystalline iron oxides by reducing it to the more soluble ferrous state (Fe^+). Aluminium, primarily from humus complexes, is usually co-extracted. Amorphous aluminosilicates are only slightly attacked.

Sodium pyrophosphate (py) is a strong complexing agent for Fe and Al.

The high extractant pH of 10 aids in the dispersion of organic molecules

by increasing the net negative charge on the colloids. The presence of

sodium on the exchange sites increases the thickness of the diffuse

double layer which lowers the electrostatic attraction between colloids.

Dispersion also increases the accessibility of the pyrophosphate ligand

to the humus - Fe, Al complexes. The ability 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 all three modal pit profiles. There was no signif•

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-

py

ionally high in Pinus Ah^ due to a high %Ct and acid pH, and in Abies IP

possibly due to illuvial organic matter, or the root material providing a source of complexing agents for the released Fe and Al. At depth in the

profiles a relatively 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 ability.

%Alck(j and 7„hl were similar, both extracting the same pool of organically complexed Al. 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 little "amorphous" Al being present. The amount of "amorphous" Al increased significantly in the lower Ah^ horizons and in particular the IP pumice layers. In the

Zacatonal zone significantly higher levels of ^A1qx were present with

"amorphous" forms becoming dominant at depth.

In the Abies modal pit , organic forms of Fe were dominant over crystalline or amorphous forms. A similar situation was apparent in the

Pinus modal pit except ^Fe^ was less prevalent at depth. In the Zacatonal modal pit organic and inorganic forms of Fe were equal, particularly at depth in the profile. The levels of extractable Fe were not significantly different among the surface horizons, but tended to establish a defined order of abundance in the middle of the profiles; Zacatonal > Pinus >

Abies. These differences became less apparent at depth except for high

%Fe in the Abies IP pumice layer. py

Oxalate extractable silicon (%SiQx) was significantly 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 until the IP pumice layer was reached.

The high mobility of silicic acid favoured some movement from the upper profile into the lower profile and ground water. However, the highly 116

weathered appearance of the IP pumice layers also supported in situ weathering as a dominant process. In the Zacatonal zone 7oS±^ increased steadily with depth. The behaviour of %Si closely followed that of ox J

%A1qx suggesting a relationship between the two elements. However, the very low extractable levels of %Si and %A1 made it difficult to assign their identity to "amorphous" aluminosilicates such as allophane.

In conclusion, the young volcanic ash soils of Iztaccihuatl showed limited accumulation of humus at this stage, in situ processes being dominant. Some evidence of illuvial organic matter was noted in the

Abies religiosa and Pinus hartwegii zones, possibly low molecular weight fulvic acid polyphenols produced in the humus layers. Evidence of in situ root decomposition was apparent in the Abies IP pumice layer. The levels of extractable Al and Si seemed to be significantly influenced by elev• ation; increasing from the Abies to the Zacatonal zone. Thus, a climate or vegetation effect was present which influenced the soils 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 soil temperature and promote weathering of the ash. The degree of weathering also increased with depth in the profile. 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 little 117

weathered, and the low content of extractable elements precluded any definitive statements on the nature of the amorphous minerals present.

5.1.2 Statistical Analysis of Ah^ and kh^ Soil Horizons from Iztaccihuatl Vegetation Zones

The results from the statistical study, Part 1, will 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 statistical analysis is given in Appendix 5. As indicated in

Section 4.2, analysis of means by ANOVA was subject to error due to the inability to confirm a normal distribution. Consequently, median values,

95% confidence intervals and the results from non-parametric statistical 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 statistics 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 ties were present. The two null hypotheses tested were:

H0:1 The three zones were from a common population.

HO:2 The two soil 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 level. Ranking of sample medians, the value below which 50% of the data fell, was by two-sample comparison (95% confidence level) using the Mann-Whitney U and median 118

tests. Statistically, 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 all parameters measured the Ah^ and

Ah^ horizons were not significantly different at the 95% confidence level

in the Abies religiosa 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^ soil horizons from all three zones were analyzed separately despite

the two soil 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 will draw attention to the data presentation used in each section.

A correlation matrix was determined for all 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 level. Verification of linearity was done

by plotting each significant correlation. In several instances significant

correlations were highly scattered about the regression line 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

its 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 soils may be required under those circumstances. On the other 119

hand, the narrow data ranges may have caused an insignificant correlation

to become significant. This emphasized the need for data plots to verify

the validity of regression coefficients.

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 significantly different

in %HM, %LI, %Ct and %Nt in the Pinus hartwegii zone. Consequently, the

horizon data was presented separately for all three zones. The C/N

ratio was not significantly different between Pinus Ah^ and Ah^; there•

fore, they were combined for analysis.

%HM, %LI and %Ct were significantly 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 signif•

icant at the 95% confidence level; however, two-sample analysis failed to

distinguish rank among the three zones emphasizing the ambiguity of this

ranking procedure. %HM was not significantly correlated with oxalate

extractable Fe, Al or Si, 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„ Soil 1 Horizons.

ZONE %HM %LI ZCt %Nt C/N 1

oven-dry basis

Ah, horizons, n=8

Abies religiosa 0.81ab 3.4a 1.7a 0.103 16.2b 0.63-1.93 2.8-9.3 1.2-4.8 0.080-0.238 15.2-20.2

Pinus hartwegii 1.26b 6.9b 3.13 0.167 18.7C 1.18-2.41 5.7-11.7 2.7-5.5 0.149-0.273 17.8-20.0

Zacatonal 0.91a 4.2ab 2.0a 0.133 15.0a

0.78-1.33 4.0-6.3 1.8-3.2 0.119-0.209 14.4-15.2

At^ horizons, n=8 %HM %LI %Ct %Nt*

Abies religiosa 0.59 2.5 1.1 0.0613 0.42-1.50 1.6-6.7 0.68-3.3 0.40-0.222

Pinus hartwegii 0.80 3.7 1.7 0.0903 0.67-1.52 3.0-7.2 1.4-3.3 0.071-0.159

Zacatonal 0.93 4.3 1.9 0.133a 0.70-1.35 3.5-6.8 1.8-3.3 0.117-0.215

1. Significance level: 95%= *, 99%= **. Analysis by k-saraple comparison. 2. The Ah and Ah horizons were combined for 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 all three zones reflecting the variable input and incorporation of N into soil humus.

This may have contributed to the failure 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 Ah2 horizons was significantly 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 al. (1980) claimed the lignin level to be more important than the C/N ratio to litter 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 religiosa and Pinus hartwegii zones may have distorted the

"true" C/N ratio. 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 soils was relatively immature.

The 95% confidence intervals of the above parameters were greatest in the Abies religiosa 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 lignins, tannins, carbohydrates, proteins, lipids, etc. The spatial variability 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 situ root decomposition and root exudates, with lesser amounts of highly resistant materials such as lignins 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 Silicon (%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 reliable comparisons among zones. Extractable %A1 was not significantly different between Ah^ and kh^ horizons at the 95% confidence level; therefore, they were combined for analysis. Mean and median values were very close suggesting little deviation from normality (Appendix 6).

However, the homogeneity of variance was not satisfied for %SiQx.

Extractable %Fe was significantly 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 profiles. A plot of %Si and %A1 showed a linear trend r r r ox ox

anc with r = 0.73. The similarity in extracted amount between 7akl^ *

%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 verified 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 Silicon (%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 0.213 0.14a 0.17a 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 0.38° 0.24b 0.29° 0.12C 0.36-0.44 0.22-0.28 0.27-0.34 0.11-0.13

1. Significance level: 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„ Soil Horizons.

ZONE %Fe %Fe """"" %Fe ox py oven-dry basis

Ahj horizons, n=8

Abies religiosa 0.15 0.10ab 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 %Fe ^Fe cbLdJ py ox Abies religiosa 0.15b 0.081 0.18 0.14-0.19 0.050-0.14 0.15-0.24

Pinus hartwegii 0.13a 0.091 0.20 0.13-0.15 0.086-0.13 0.20-0 .'24

Zacatonal 0.14ab 0.076 0.23 0.13-0.17 0.060-0.11 0.21-0.27

1. Significance level: 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 significantly different among the three zones. However, %Fe was significantly 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 Al^ horizons suggesting a surface phenomenon in the Pinus zone. In the Ah~ horizons, Abies %Fe was v 2 ox significantly greater than Pinus HFe^. The narrow 95% confidence interval of Pinus Ah^ possibly contributed to this outcome. The Abies and Zacatonal zones were not significantly 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 citrate- bicarbonate-dithionite. The method of Parfitt (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 little 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 difficult to assess, and no patterns were evident. In conclusion, the above analysis 126

Table 8. Relative Dissolution of Materials Containing Iron, Aluminium and Silicon by Pyrophosphate, Acid Oxalate and Citrate- Bicarbonate-Dithionite (Parfitt, 1980, 1983).

FORM PYROPHOSPHATE ACID OXALATE CITRATE-BICARBONATE- DITHIONITE

Humus Al, 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 Ala ALLOPHANE Sib ALLOPHANE Al/Si %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 Ah2 Ah^ Ah2 Ah^ Ah2

Abies religiosa 0.10 0.081 0.050 0.069 0.090 0.030

Pinus hartwegii 0.12 0.091 0.020 0.039 0.10 0.070

Zacatonal 0.058 0.076 0.092 0.064 0.090 0.090 128

supported the hypothesis of Wada and Higashi (1976) that in the early stages of soil formation the Al and Fe released by weathering of the ash existed largely as Al, 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 reliably assess soil-vegetation relationships the other factors of soil formation; parent material, climate, age and topography, must be held relatively 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 soil unit covered the largest portion of Popocatepetl and Iztaccihuatl and spanned the elevational ranges of the three vegetation zones. These soils were poorly developed and occurred exclusively in the most recent 1C ash of Popocatepetl. The stratigraphic soil unit mapped for the area was diagrammed in Figure 18. Based on this unit there were two possible parent materials for soil formation in the three vegetation zones; the 1C and 2C ashes of Popocatepetl.

The most recent ash of Popocatepetl, 1C, was the most likely parent material; however, it 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 soils derived from the two parent materials. In particular, the oxalate and cbd extractable Fe, Al and Si were significantly 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 soils of the study area were far removed from those of the 2C ash, and fell well within the ranges reported for the 1C ash. Consequently, the 1C ash of Popocatepetl was the only possible parent material for soil formation in the study area.

For further analytical data Profile 04 from Miehlich was selected as being the closest in location and chemical properties to the soils in the study area. The chemical and physical characteristics of Profile 04, and the mineral and particle size distributions typical of the 1C ash are presented in Tables lOa-d. The analytical results for Profile 04 closely matched those found in the study area, particularly the soils sampled in the Abies religiosa zone. The particle size distribution suggested a very young and little weathered soil (Table 10c). Very fine to fine sand was the most abundant particle size class with lesser amounts of medium sand and coarse silt. 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 vitric material confirmed its volcanic origin. The absence of quartz and the identification of Ca-rich feldspars and pyroxenes classified the ash as andesitic (Figure 2). However, Na, K - feldspars, typical of dacitic ashes, were also identified. 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

PH1 5.4 5.5 4.2 4.8 %Fe 3.8 - - 0.33 0.24 0.24 0.24 cbd %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: IN 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 Profile 04 Approximated to the C.S.S.C. System (Miehlich, 1980).

% HUMUS FREE % HUMUS FREE

Amorphous clay fraction^ 4.1 6.2 Crystalline clay fraction 2.1

Fine silt 2.9 Medium silt 8.3 25.8 Coarse silt 14.6

Very fine to fine sand 49.6 Medium sand 18.1 68.0 Coarse sand 0.3

> 2.0 mm

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

74.2 Light to brown glasses

7.3 Opaque glasses or glass encrusted particles 5.5 Sanidine (Na,K - feldspar) 2.5 Plagioclase (Ca.Na - feldspar) 9.7 Pyroxene 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 0.21 0.045 0.15 0.17 0.24 Abies religiosa zone 2 0.32 0.061 0.14 0.26 0.24 Pinus hartwegii zone 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 Al, 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 is given in Table 11. The distribution of carbon among the humus fractions separated the extractable organic carbon into distinct 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 significantly different at the 95% confidence level; hence, these two parameters were listed by horizon for all vegetation zones. The other three parameters; fulvic acid carbon (%Cf), polysaccharide-rich fraction C carbon (%Cc) and total extractable carbon (%Ce),were not significantly different between horizons and were combined for analysis.

Statistical analysis of the combined Ah^ and Ah^ horizons indicated that there was no significant difference among the three vegetation zones in %Cf. However, significantly less fulvic acid polysaccharides (%Cc) were present in the Abies religiosa 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 soil, and possibly affected microbial activity and the production of microbial polysaccharides.

The amounts of humic acid carbon (%Ch) and polyphenol-rich fraction

A (%Ca) were significantly different among the Ah^ horizons but not the

Ah2 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 signif• 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 Table 11. Median Values and 95% Confidence Intervals for %C in the Humus Fractions.

»# 2 ZONE %Ch %Ca %Cf %Cc ZCe -air-dry basis-

Ahj horizons, n=8 Ahj + Ah^ horizons, n=16

Abies religiosa 0.46° 0.23ab 0.40 0.20 58 0.30-1.5 0.19-0.51 0.36-0.65 0.14-0.37 65-61

Pinus hartwegii 0.84a 0.39 0.65 0.34 53" 0.76-1.7 0.30-0.69 0.41-0.96 0.23-0.46 52-58

Zacatonal 0.55° 0.15° 0.48 0.32 50'a 0.44-0.86 0. 13-0.19 0.44-0.62 0.30-0.44 49-53

Ah^ horizons, n=8 ZCh %Ca

Abies religiosa 0.36 0.20 0.15-1.1 0.13-0.27

Pinus hartwegii 0.48 0.19 0.35-0.99 0.16-0.54

Zacatona1 0.52 0.14 0.44-0.95 0.13-0.21

1. Significance level : 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 variability 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 spatially, as indicated by the narrow 95% confidence intervals. In the

Ah^ horizons a similar situation was present except %Ca in the Abies religiosa zone spanned a very narrow range. Thus, the production of fulvic acid polyphenols, either through in situ decomposition or illuv- iation 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 litter in the Pinus hartwegii zone. The large input

(although uneven) of pine litter to the soil surface in addition to canopy drip would greatly increase the levels of acid-soluble polyphenolic mater• ial (%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 slightly 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 situ processes

being dominant in the Ah2 horizons. The absence of zonal differences among the Ah„ horizons supported in situ processes in the Abies religiosa 136

and Zacatonal zones. Illuviation 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 illuviated 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 soil humus with the inorganic soil components. It also indicated the amount of undecomposed or partially decomposed humin fract• ion. %Ce was not significantly different between Ah-^ and Al^ 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 stabilization of the humus with Al. 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, it was possible that very large, aliphatic humic acid molecules could be stabilized to some extent by physical interaction with the silt and fine sand (cf. to Anderson et al.

(1974a) HA-B fraction).

The presence of charcoal in the Abies religiosa 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 soils recent age and lack of catalytic clay-sized particles (Wang et al., 1983a). It was probable that the unextractable humin fraction consisted largely of large aliphatic, immature humic acid molecules, partially 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 soil profile, decomposition rates, levels of microbial and faunal activity, movement of organic components within the profile, etc. Furthermore, there was considerable spatial variability 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 variability by placing all 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 religiosa zones, confirmed this decrease in variability. Consequently, the "quality" rather than the "quantity" of soil humus was expressed, and the process of humus formation, the dist• ribution of organic components among humus pools, and the nature of the soil environment could be more reliably interpreted.

Median humus fraction ratio data is given in Table 12. The .calculated ratios, Ch/Cf, Ca/Cf and Ch/Ca, were not significantly 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 ratios. This was rather surprising considering the vegetative differences among the three zones. The Abies religiosa zone was strictly 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 2.0a 0.49 0.73-1.2 1.4-2.5 0.47-0.54

Pinus hartwegii 1.1 2.4° 0.46u 1.0-1.2 2.2-2.6 0.45-0.47

Zacatonal 1.2 3.8U 0.30d 1.1-1.3 3.3-4.4 0.29-0.33

1. Significance level: 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 religiosa 12.0a 26.7b 5.4a 6.4a 11.6-14.5 24.7-28.9 4.6-6.3 6.1-8.4

Pinus hartwegii 14.5b 25.6b 6.1b 6.8a 14.0-15.5 24.3-30.7 5.7-6.4 6.3-7.1

a _ „c „ „b Zacatonal 13.0a 19.3° 6.8^ 8.3 12.9-13.4 18.3-21.4 6.5-7.7 7.5-9.4

1. Significance level: 95% = *, 99% = **. Analysis by k-sample comparison. 139

relatively thick FH humus layer. The Pinus hartwegii zone was a mixed coniferous-grassland system with little FH but abundant pine needle litter on the bare mineral soil. The Zacatonal zone represented a typ• ical bunchgrass community with large patches of bare mineral soil being visible. The possibility that similar processes of humus formation were occurring in both soil horizons, despite vegetative differences, was strongly supported by this lack of horizon differentiation.

The Ch/Cf ratio was not significantly 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 soils. 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 soils 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 soils of Iztaccihuatl fell well within the ranges reported above for the more well developed

Andosols. Thus, the Ch/Cf ratio may be an intrinsic property of the soil from its youngest stage. A similar conclusion was reached by Anderson et al. (1974a). In the Chernozemic grassland soils of the Canadian

Prairies the Ch/Cf ratio was found to increase from the Brown soil zone

(mean Ch/Cf = 1.49) to the Black soil zone (mean Ch/Cf = 2.06) (Lowe, 1980).

The increase in elevation from the Abies religiosa zone to the Zacatonal zone of approximately 700 m was not enough to significantly influence the soils moisture status, and hence, the Ch/Cf ratio. However, an increasing trend with elevation was apparent in the 95% confidence intervals. This 140

possibly reflected a slightly more favourable environment for polymerizat• ion reactions with increasing altitude. 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 al., 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 line making its significance questionable. Nevertheless, as will be discussed later, %A1 was sienif- 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 soil 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 little effect on %Ch (Table 11) or the Ch/Cf ratio. In the Abies religiosa zone organic matter inputs would be highly varied, both comp- ositionally and spatially. This was supported by wider 95% confidence intervals for %Ch and the Ch/Cf ratio. The 95% confidence interval for

%Ch decreased slightly from the Ah^ to the Ah^ horizons possibly signal• ling a change in organic matter source. In the Pinus hartwegii zone the surface Ah^ horizons would receive both pine litter and grass inputs, the

pine influence lessening in the Ah2 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 Ul

Zacatonal zone (0.51). This suggested that the source of organic matter

for humus formation in the Pinus Ah^ horizons was primarily in situ 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 ability of grass root systems to explore the total

of the soil volume produced a more uniform input of organic matter into

the profile 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 variability and the source of organic matter for humus formation.

The Ca/Cf ratio reflects the proportion of polyphenolic material in

the fulvic acid fraction. High Ca/Cf ratios are associated with conditions

of low biological activity 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 limit microbial activity and the forest humus

layer provides a readily available source of polyphenols. The cold temp•

eratures prevailing in alpine or arctic soils also enhances the formation

of fulvic acid polyphenols (Lowe, 1980).

The Ca/Cf ratio decreased slightly, but significantly at the 99%

confidence level, 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 ratio. The Zacatonal zone contained high 142

levels of polysaccharides but little fulvic acid polyphenols which resulted in a low Ca/Cf ratio. The Pinus zone was high in both fulvic acid polyphenols and polysaccharides; thus, a combined effect was apparent but favouring a high Ca/Cf ratio.

The low proportion of polyphenolic fulvic acid material in the

Zacatonal zone was typical of grassland ecosystems. The Chernozemic soils of the Canadian Prairies have Ca/Cf ratios ranging from 0.09 to

0.25, increasing from the Brown to the Black soil zones. The Ca/Cf ratio in the Zacatonal zone (median = 0.30) was slightly higher than the neutral Chernozemic soils possibly due to a more acidic pH (5-6) or the alpine location which may have affected microbial activity (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 soil 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 litter on the soil 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 situ root decomposition and illuviation from the pine litter layer, produced similar Ca/Cf ratios in the two horizons. If in situ 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 situ contributions from small, fibrous pine rootlets would also affect the final Ca/Cf ratio obtained, as well as illuviated polyphenols from the pine litter. Hence, although in situ grass root decomposition was a major source of polyphenols (and consequently humic acid) in the Ah^ and Ah^ horizons, it was difficult to ascertain the importance of illuviated material in the Al^ horizons. The presence of considerable illuviated material would, by definition, form a B horizon, and this was definitely not observed. The absolute humus fraction amounts and their ratios were not significantly different between horizons in the

Abies religiosa and Zacatonal zones supporting in situ 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 profile; illuviation of water-soluble organic compounds produced primarily in the humus layer or in situ organic matter decompos• ition. Forest soil horizons such as Podzolic Bf have low Ch/Cf and high

Ca/Cf ratios indicating a dominance of illuvial 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 illuvial 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 soil 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 soil environment with adequate base status to promote microbial activity, 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 stabilizing 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 activity 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 illuvial B horizons, gleyed soils,

and alpine or arctic soils.

The Ch/Ca ratio is 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 literature making its interpretation difficult. Both

fractions contain phenolic material and are intimately associated with

each other in the soil environment. During the decomposition process of

plants simple polyphenols, including microbial by-products, are released

into the soil. 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 its acid-solubility, 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 utilized by the microbial

population.

4. The simple polyphenols pass into lower horizons.

Support for all four processes, often operating simultaneously, can be found in the literature. However, considerable controversy existed over whether the fulvic acid fraction was a precursor, end-product or neither to the humic acid fraction. Studies on the decomposition process 14 have shown radioactive C to be heterogeneously incorporated into all humus fractions which were developed rather quickly parallel with the initial period of vigorous microbial activity (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 AlogK, 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 fulvic acids, a process involving a reduction in molecular weight and carbon content

(note, opposite to Japanese theory) as aliphatic components were split 146

off, and a concomitant increase in oxygen content and total acidity.

Anderson et al. (1974a) equated the same process in the conversion of the

immature, highly aliphatic, high molecular weight HA-B fraction to the more mature, aromatic, lower molecular weight HA-A fraction (conventional

HA fraction). 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 soils of Iztaccihuatl the latter process

of HA —>FA conversion was not likely to be as important as the initial

build-up of a high molecular weight, aliphatic, immature humic acid

fraction. 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 all 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 religiosa (median = 2.0) and the Pinus hart• wegii (median = 2.4) zones, but increased significantly 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 soil, would be adsorbed by allophane and be catalytically 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 soil". Oxalate-extractable Al,

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 soil and

the amount of nitrogen incorporated into the forming humic polymers.

Important are the nitrogen supplying power of the native vegetation

(including biological N fixation), the activity of the soil microorganisms

which is largely controlled by the nature of the organic matter inputs

into the soil and the soil environment, the soil C/N ratio as its reflects

the balance between nitrogen mineralization and immobilization into the

soil biomass, and the forms of nitrogen released to the soil 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 soils (C/N HA = 12.5 - 13.3) (Griffith and Schnitzer, 1975).

However, the C/N ratios of fraction A (C/N A = 19.3 - 26.7) were signifi•

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 al., 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 literature. 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 lignin 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 nitro• gen contents ranging from very low to very high, possibly reflecting the source of vegetation. Anderson et al. (1974a) could not find any relat• ionship between "aromaticity" (ie. maturity) and the hydrolyzable carbon and nitrogen contents of soil humic acids. The recent age of the soils in this study and their fairly high nitrogen contents favoured the enrichment hypothesis of Kuwatsuka et al. (1978), and it was unlikely that zonal differences in C/N HA were a reflection 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 litter, and the low pH which would decrease microbial activity and the cycling of nitrogen in the soil. A large proportion of the nitrogen would be derived from the grass vegetation; although, the high soil C/N 149

ratio (18.7) implied nitrogen immobilization by the soil biomass. Lower

C/N HA ratios were found in the Abies religiosa and Zacatonal zones. The low C/N HA and soil C/N in the Zacatonal zone reflected the greater nitro• gen supply and increased cycling in grassland ecosystems. The Abies religiosa zone contained insignificant grass cover, but possibly received considerable nitrogen from the ground cover of forbs and shrubs. Also, the near neutral pH and soil C/N ratio (16.2) would favour microbial activity 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 little examined in the literature, and there were no reported theories on the significance, if any, of its C/N ratio. This was partly due to the low nitrogen content of this pool and the belief that it contributed little to the overall nitrogen cycle. The C/N A ratio was high in the coniferous soil zones, but decreased significantly 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 soil (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 ratio, r = 0.59, which formed two groups when plotted; the Zacatonal zone and the coniferous soil 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 failed 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 soil C/N ratio, empha• sizing the enrichment of nitrogen into the humic acid pool. Approximately half of the variability in the soil C/N ratio could be attributed to the humic acid C/N ratio. A further source of variability came from the polysaccharide-rich fraction C; limited influence being associated with fraction A due to its high C/N ratio. 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 little 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 is 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, it 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 possibility 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 oligo- 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 fraction. 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- rich 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 I0/I = A = kcb, where I0 = the incident light intensity, I = the light 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 light, X . The extinction coefficient of humic compounds is 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^QQ HA and E^/E^

HA, all 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 significantly 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 Ah0 153

Table 14. Median Values and 95% Confidence Intervals for the Optical

Properties of the Humic Acid Fraction.1

C 2 17 C ZONE E ^ HA F ° HA* EA/E6 HA ^400 HA ^600 "h

Ah^ + Ah^ horizons, n=16 Ah^ horizons, n=£

Abies religiosa 217.3 46.8" 4.56 187.9-235.1 40.7-52.1 4.44-4.85

Pinus hartwegii 209.2 50.lu 3.87u 188.4-232.7 40.7-58.6 3.76-4.40

Zacatonal 203.9 61.0 3.35a 197.7-209.7 58.2-64.2 3.24-3.49

1 %C ** F HA E./E, HA b600 HA 4 6

Ah2 horizons, n=8

Abies religiosa 42.9° 4.63 38.3-56.4 4.38-5.18

Pinus hartwegii 66.0L 3.46u 56.6-72.9 3.35-4.25

Zacatonal 62. T 3.30 56.6-65.9 3.20-3.46

1. Significance level: 95% = *, 99% = **. Analysis by k-sample comparison.

2. The Ah^ and Ah2 horizons were combined for E^QQ, 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 religiosa 129.0C 9.30b 12.8b 127.6-132.9 9.00-10.5 12.6-14.5

Pinus hartwegii 123.9b 10.3° 12.2a 120.7-131.7 9.90-10.5 11.8-12.8

Zacatonal 107.4a 8.60a 12.5ab 105.2-113.2 7.90-9.20 12.1-13.0

1. Significance level: 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^ ratios.

In the Ah^ horizons E^Q HA was highest in the Pinus hartwegii zone fol•

lowed by the Zacatonal zone. The significant increase in E^QQ 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 visible region must be examined. Absorbance in the visible 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, etc.), 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 visible region and decreased E^/E^ or

A logK (Kononova, 1961; Kumada, 1965). Highly aliphatic humic acids with little or no "core" material would absorb significantly in the visible

region only if 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 al., 1977; Anderson et al., 1974a,b).

Thus, within humic molecules of similar aromaticity, E^QQ and the E^/E^ ratio may reflect the polymer molecular weight. However, when comparing humic molecules extracted from widely differing soils with a range in molecular complexity, the absorbance due to aromaticity may take preced• ence and influence the E./E, ratio. 4 o This hypothesis favoured a similar maturity in the humic acids extr• acted from the three vegetation zones as there was no significant differ• 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 litter 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 visible 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 ratio. Little reference has been made in the soils literature on 4 6 the optical properties of the fulvic acid fraction except to note its lower absorptivity and higher E^/E^ ratio compared to the humic acid fraction. The high E^/E^ ratio has been related to its lower particle molecular weight (Chen et al., 1977). E^QQ A increased significantly from the Zacatonal zone to the Abies 157

zone which possibly reflected the greater input of polyphenols (%Ca) into the coniferous soil zones. As indicated earlier, 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 soil zones suggested limited biological activity 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 16). A similar trend was evident in E^QQ A except

the Abies religiosa and Pinus hartwegii zones were reversed. The E^/E^ A

ratios were difficult to interpret as they did not totally agree with the analysis of the E^QQ or E^QQ A data. Nevertheless, the data seemed to

support the overall low molecular weight and limited conjugation of the

chromophores in the A fractions.

The statistical analysis of organic matter parameters in Part 1 led

to the following general conclusions:

1. The humus in the Ah^ and Al^ horizons of the Abies religiosa and

Zacatonal zones represented a single population. 1% 2. Horizon differences in %Ch, %Ca, E, ° HA and E,/E, HA were noted 600 4 6

in the Pinus hartwegii zone. Thus, two distinct 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 soil 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 slight

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^QJ 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 soil environment.

8. The optical characteristics of the humic acids in the Pinus Al^

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 is 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 statistically 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 religiosa AH AA

Pinus hartwegii Ah^ P1H P1A

Pinus hartwegii Ah2 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 isolation procedure. In the bulk isolation procedure the alkaline humus supernatant was siphoned-off from the unextractable soil solids. In the test extraction a centrifugal separation was made which would increase the extraction efficiency. 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 Distribution.

Sample X Recovery % Distribution of Recovered Fraction

< 10,000 > 10,000

AH 88 6.2 93.8 P1H 82 7.7 92.3 P2H 100 6.6 93.4 ZH 97 5.9 94.1

< 1,000 > 1,000

AA 70 16.0 84.0 P1A 83 28.7 71.3 P2A 78 24.5 75.5 ZA 79 39.0 61.0

1. The recovery of HA and fraction A was based on a test extraction of the composite samples using 0.1 N NaOH and the method of Lowe (1980). %C in the humus extracts was determined on the Astro Solution Carbon Analyser.

2. The XC in the lower molecular weight fractions was measured in solution on the Astro Solution Carbon Analyser. The recovery of the high molecular weight fractions was based on the weight of the freeze-dried extract and its %C (uncorrected for ash) content from elemental analysis.

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 ratios

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 efficient 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 efficient. Also some loss was unavoidable during the diafiltrat• ion procedure.

P1H percent recovery was quite low due to its high %Ch (1.21). A soil:solution ratio of 1:10 was used for all composite samples; hence, the extraction of humus in P1H was less efficient. 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 diafiltration 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 fall 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 all 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 soil 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, it 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 efficient clean-up of metals by the H+-exchange resin. Silica 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 40-50 50 - 60

0 44-50 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

(Griffith 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 in• creased slightly 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 significantly 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 soils literature. 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 al., 1978). Supporting evidence came from

Anderson et al. (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 aliphatic, immature HA-B fraction.

The humic acids obtained in this study were similar in elemental composition to those produced catalytically by Wang et al. (1983a) and to the HA-B fraction of Anderson et al. (1974a). The %C content of the catalytically 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 reflection 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 difficult. First, 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 difficult to correct by adjustments in sample weight. And third, it is not easy to obtain uniform sample distribution within the KBr pellet. Hence, only the general features and relative intensities of the absorption bands will 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 visible 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 cm-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 Si-0-Si vibrations of silicate impurities.

This band was much more pronounced in the HA spectra compared to the fraction A spectra which was in agreement with the silica 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 it was not apparent in the fraction A spectra.

The relative absorption band intensities were similar among the four

HA spectra. A slightly sharper 1035 cm 1 peak was noted in AH which was likely due to its higher ash content. Strong aliphatic C-H stretching was noted in all 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 al.,

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 failed 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 slightly higher ash content which could account for this apparent similarity; although, both fractions had similar

%0 contents. The above relationship possibly included the polysaccharide fraction, rich 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 fol• lowing conclusions:

1. The humic acids differed little 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 COOH 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 COO" 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 OH, C—H

deformation of CH2 and CH3 groups, COO" antisymmetric stretching 1280--1200 C—O stretching and OH deformation of COOH, 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

Vanillic 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-

icity, %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 solubility 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 likely

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 variability; 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, vanillic 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 solubilities 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 replication. 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 slightly 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 lignin derived phenyl propane alcohols and aldehydes would not be determined. Furthermore, the substit• uted cinnamic acid derivatives, p-coumaric, ferulic, synapic, etc., 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 partition chromatography, and the most polar compounds were eluted first. Hence, hydroxyl groups decreased while methoxyl groups increased the phenolic acid's retention time. The first 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

ure 28 Cont.

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, vanillic acid (the most prominant peak) and syringic acid.

It was interesting to note that these four acids were of lignin origin; although, p-hydroxybenzoic acid and protocatechuic acid could also be of microbial origin. The cinnamic acid derivatives and neutral phenols of microbial or flavonoid origin, 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 slightly 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 soil A hor• izons (Riffaldi 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 diafiltration 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 Total Total

(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 is only an estimate since ;a weight loss measurement on < 0.5 g has a high error potential in addition to possible ash content change:s , etc.

2. Corrected for recovery from ether / NaHC03 / ether extraction (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

Van 2400

COOH 2000

OH 1600

1200

800

400

AH PIH P2H ZH AA PIA P2A ZA

2000 Proto COOH 1600

OH OH 1200

>"g/gC

800

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 Syr COOH

400

CH30 OH

300 .

Mg/gC

200

100

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 soils (Katase, 1981c; Whitehead et al., 1982)

and to HA and FA fractions (Hartley and Buchan, 1979; Hanninen et al.,

1981). Peat soils 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 al., 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• olic acids identified 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 lignin by microorganisms was thought to involve

dearomatization of the lignin 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

soil 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 al., 1982; Whitehead et al., 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

utilized 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 fission

(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 cyclic structures forming the humic acid

"core".

5. The catalytic action of clay-sized minerals and hydrous oxides,

allophanes, etc., would favour processes 3 and 4 (Wang et al.,

1983a,b). Furthermore, inorganic surfaces may act as "templates"

to sterically align components for polymerization reactions.

On the other hand, inorganic constituents may serve to protect

phenolic and other compounds from further degradation by inhib-

itting extracellular enzymes (Aomine and Kobayashi, 1964, 1966).

Thus, large aliphatic polymers containing attached phenolic

compounds could be formed such as Anderson et al's. (1974a) HA-B

fraction. 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 soil 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 significantly different.

The concentration of acid-labile 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 sensitivity of the individual phenolic compounds to phenol

coupling reactions. The phenolic compounds which form C-C and

.. C-0 covalent bonds will not be released by acid hydrolysis;

hence, they will not be part of this pool.

3. The HA or A molecular size as it affects surface area, accessib•

ility of bonding sites, and solubility behaviour.

4. The rate of conversion of attached phenolic compounds into non-

hydrolyzable forms. 184

5. The % distribution of the humus fractions.

6. The soil pH as it affects microbial activity, 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 solubility and low %Ca distribution. The Ch/Ca ratios ranged from

2.0 to 3.8 (Table 12); hence, when calculated on a total soil 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 fraction.

The % phenolic acids in the A fractions increased significantly with the introduction of grass vegetation. This possibly stemmed from a change in the nature of the A fractions or the soil 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^QQ 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 significantly 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 al., 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^QQ HA optical data or atomic H/C ratios among the HA samples.

The supply of simple polyphenols to the coniferous soil zones would be great due to the humus layers and pine litter. In Pinus Ah^ microbial activity would be reduced by the acid pH. Less influence from the pine litter would be present in Pinus Ah^ which was supported by lower %Ca and increased pH. The higher pH would favour microbial activity with fewer simple polyphenols being released during grass root decomposition.

On a total soil basis this fraction contained the least phenolic acids

(Table 22). The similarity 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 soil basis the phenolic acids associated with P1H were twice that in AH (Table 22).

In the Zacatonal zone the soil pH was favourable to microbial activity 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

soil. This was determined by the vegetation site factor.

The four major phenolic acids identified in this study appeared to be ubiquitous among soils (Katase, 1981a-c; Whitehead et al., 1982, 1983;

Hanninen et al., 1981). Their distribution and form in the soil differed with the soil type (forest humus versus peat (Katase, 1981a-c)), the source of vegetation (Katase, 1981a-c; Hanninen et al., 1981; Morrison, 1958,

1963; Burges et al., 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 literature review (Sections 2.4 and 2.5) but will be briefly re-capitulated here. In the lignin macromolecule the above acids existed as cinnamyl alcohol derivatives. During oxidative degradation of the lignin polymer a small portion of the cinnamyl alcohols, free or still 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 listed 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, vanillic and syringic, have not been synthesized in vitro by microorganisms; hence, they were largely derived from lignin with small contributions from the B ring of flavonoids (Burges et al., 1964). Precursors to vanillic acid would also include ferulic acid bound as esters to monocotyledon lignin (Shindo and

Kuwatsuka, 1975a). Protocatechuic acid had three possible origins; de novo synthesis by microorganisms from p-hydroxycinnamic acid, demethy• lation of vanillic acid or hydroxylation of p-hydroxybenzoic acid. Caffeic acid, an intermediate in the biosynthesis of lignin, may contribute in 187

minor amounts. P-hydroxybenzoic acid has been synthesized in small amounts by several fungi imperfecti (Haider et al., 1972), but was largely derived from p-coumaryl residues, abundant as esters in grass lignins

(Shindo and Kuwatsuka, 1975a).

The lignin composition of plants was reviewed in Section 2.4.3.

Gymnosperm lignin 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 all three units.

However, considerable amounts of p-coumaric acid and ferulic acid were bound as esters to the grass lignins (Crawford, 1981; Shindo and Kuwatsuka,

1975a). These units were readily released by mild hydrolysis and would be actively utilized by the microbial population.

The phenolic acids released during acid hydrolysis would be bound to the humic polymers by "acid-labile" 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 cyclic structures would not be affected. Simple phenolic acids attached by salt bridges, etc., or mole• cules solubilized from non-humified organic matter including phenolic acids weakly attached to the lignin polymers, would be removed in the non- polymeric fractions during diafiltration. Polymeric lignin 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 its major bond types (Figure 10) would prevent significant cont• ributions from unaltered plant lignin 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 identified is given in Table 21. The weight of phenolic acids per

100 g soil is 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

Vanillic acid Van

Syringic acid Syr

Vanillic acid was dominant in the HA fractions, representing between

50 and 73 % of the total phenolic acids identified. The concentration of vanillic acid in the HA fractions decreased sharply in the Pinus hartwegii and Zacatonal zones with the introduction of grass vegetation. The vanillic acid content of P1H, P2H and ZH were not significantly different;

50 to 56 % of the total phenolic acids identified (Table 21). When cal• culated on a total soil basis (Table 22) the content of vanillic acid and its demethylated product, protocatechuic acid, were substantially greater in AH and P1H compared to P2H and ZH. Increased production of polyphenols from the forest litter and reduced microbial activity favoured the accumulation of phenolic acids in the humic acid fractions.

The concentration of vanillic 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 - /ig Phenolic acid / 100 g soil.

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 identified (Table

21). However, on a total soil basis the HA fractions were much richer in vanillic acid, particularly in the Abies zone and Pinus Ah^ horizons

(Table 22). Extensive demethylation of vanillic 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 solubility of the A fractions which

increased their accessibility 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 activity.

When combined, vanillic 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 religiosa 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 significant. If the average

composition of monocotyledon lignin was used as a guide for the Zacatonal

zone, contributions from vanillic 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 soil humus fractions compared

to p-hydroxybenzoic acid and syringic acid. To understand this phenomenon

the process of lignin degradation and the fate of the individual monomers

must be examined. 191

In the lignin 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 latter. P-coumaryl units are also more condensed, but their contribution to the lignin structure is minimal. Consequently, during the oxidation of lignin with nitrobenzene syringyl units are preferrentially split-off (Gross, 1979). A similar situation may be present in the soil during oxidative degradation of lignin by microorganisms. Once released the syringyl units would be rapidly oxidized, decarboxylated and demethylated to form pyrogallol.

The vicinal 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 acid-labile form.

As previously noted, p-coumaryl units actually incorporated into the lignin framework were few (Section 2.4.3). The p-coumaryl residues were largely derived from p-coumaric acid which forms esters with the lignin structure. Mild extraction of undecomposed plant material released p-coumaric and ferulic acids in considerable amounts (Kuwatsuka and Shindo,

1973; Myskow and Morrison, 1964; Whitehead et al., 1982). These easily extractable forms would be readily released and degraded by the microbial population. The mild extraction of peat and forest soil (Katase, 1981a-c), and the FA fraction from a soil underlying ryegrass (Hartley and Buchan,

1979) indicated extensive beta-oxidation of the released p-coumaric and ferulic acids to form p-hydroxybenzoic and vanillic acids.

The reactivity of the individual phenolic acids will also determine their susceptibility to phenol coupling reactions; and hence, resistance to acid hydrolysis. The reactivity of phenolic compounds is related to 192

their chemical structure. The rate of polymerization decreases in the following order: vicinal trihydroxy (ie. pyrogallol) > vicinal dihydroxy

(ie. protocatechuic acid, catechol) > monohydroxy (ie. benzoic acid).

Electron attracting groups such as carboxyl will decrease the rate of polymerization whereas electron donating groups such as methyl or methoxyl will increase the rate (Wang et al., 1983a). Steric factors will also

influence both the rate and mode of coupling. P-hydroxybenzoic acid is not readily coupled; hence, its reactivity will depend on its rate of hydroxylation to form protocatechuic acid. Protocatechuic acid, once

decarboxylated to form catechol, would rapidly polymerize. Protocatechuic acid is also a key intermediate in the cleavage of aromatic compounds by microorganisms (Section 2.5.3). Thus, the balance of the above processes will determine the persistence of intact phenolic acids in the system.

By and large, vanillic 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 statistically

different (Figure 30 and Table 20). When calculated on a total soil

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 significantly from

PIH to P2H which was similar to ZH. Syringic acid decreased slightly

from PIH to P2H and was significantly greater in ZH. The differing

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 activity 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 soil columns (Shindo and Kuwatsuka, 1976). In the A fractions p-hydroxyben• zoic acid increased significantly 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 significantly 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 all 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 reliable. The ratios of Syr/Van and Syr/Proto emphasized grass influence in the HA samples, but failed 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 religiosa. 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 Identified.

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 - —, H_JL AH PIH P2H ZH AA PIA P2A ZA 197

Figure 31 Cont,

Syr / Van 0.4

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 soil 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• file. In the surface mineral horizons humus-Al complexes were dominant supporting the theory of Wada and Highashi (1976) that in the early stages of soil 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 insignificant.

In the Abies religiosa and Zacatonal zones horizon differences in organic matter characteristics were not apparent; hence, the humus in the Ahj and Al^ 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 little 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 soil 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 soils was comparable to more well developed Andosols which suggested

this ratio to be an intrinsic property of the soil from its youngest

stage. 1% The E^QQ 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 religiosa to the Zacatonal zone.

The optical characteristics of the humic acid fraction in the Pinus hart• wegii Ah^ and Ah^ soil horizons seemed to stress a difference in organic matter source for humus formation or the soil environment. Consequently, the humic acids in Pinus Ah^ were closer in optical characteristics to the Zacatonal zone.

Fraction A had lower absorbance in the visible region which was consistent with its lower molecular weight and limited degree of conjugat• ion. However, optical characteristics suggested a more polymeric fraction

A in the coniferous soil 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 failed 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 rich in nitrogen 200

in these recent volcanic ash soils.

The phenolic acid hydrolysis product data established a lignin- derived component of the polymeric humic acid and fraction A. The participation of lignin in humus formation was through microbial degrad• ation of the lignin macromolecule and transformation of the released simple polyphenols. The source of vegetation for humus formation could be clearly distinguished from the ratios of the major phenolic acid hydrolysis products. 201

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8.0 APPENDICES

Appendix 1. Modal Pit Profile Descriptions.

Abies religiosa

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, slightly sticky, slightly plastic, thixotrophy not detectable; abundant fine roots, plentiful medium roots; abundant fine and medium pores; separation from underlying horizon gradual and wavy with tongue extending to 40 cm downward"

11-23 Ah2 "Black (10YR 2/1); dark gray (10YR 4/1 dry); sandy loam; very weak fine subangular blocky; loose, slightly sticky, slightly plastic, thixotrophy not detectable; plentiful 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 Ah3 "Black (10YR 2/1); dark gray (10YR 4/1 dry); loamy sand; very weak medium subangular blocky; loose, not sticky, slightly plastic, slightly smeary, thixotrophy not detectable; very few fine, medium and coarse roots; abundant fine and medium pores; mottling: reddish, diffuse, rotted root particles; separation from under• lying horizon gradual and flat"

43-63 Ah^(B) "Black (10YR 2/1); dark gray (10YR 4/1 dry); loamy sand; weak coarse subangular blocky; loose, slightly sticky, slightly plastic, smeary, thixotrophy not detectable; very few fine and medium roots, few coarse roots; abundant medium pores, plentiful fine pores; mottling: reddish, diffuse, rotted root particles; frequent small charcoal; frequent small pumice; separation from underlying horizon gradual and flat"

63+ IP pumice "Very dark brown (10YR 2/2); dark grayish brown (10YR 4/2 dry); loamy sand; firm medium subangular blocky; slightly smeary, thixotrophy not detectable; plentiful coarse roots; abundant medium pores, plentiful fine pores; abundant ocre coloured highly weathered pumice gravel uniformly distributed; very few small charcoal" 219

Pinus hartwegii

Depth Horizon Description cm

11-0* FH "Very dark grayish brown (10YR 3/2 dry)"

0-12 Ah "Black (10YR 2/1); very dark gray (10YR 3/1 dry); 1 loam; very weak medium subangular blocky; loose, slightly sticky, slightly plastic, thixotrophy not detectable; abundant very fine and fine roots, plent• iful medium roots; abundant fine pores; frequent small charcoal; separation from underlying horizon abrupt and flat"

12-41 Ah, "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loam; very weak medium angular blocky; loose, slightly sticky, slightly plastic, thixotrophy not detectable; plentiful very fine and fine roots; abundant fine and medium pores; separation from underlying horizon gradual and flat"

41-53 Ah, "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loam; very weak granular and fine angular blocky; loose, slightly sticky, slightly plastic, thixotrophy not detectable; very few very fine roots, plentiful 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 flat"

53-80 Ah, "Black (10YR 2/1); very dark gray (10YR 3/1 dry); sandy loam; moderate fine angular blocky; very friable, slightly sticky, slightly plastic, 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"

80+ 1P pumice "Very dark grayish brown (2.5Y 3/2); grayish brown (2.5Y 5/2 dry); loam; moderate fine and medium angular blocky; very friable, very sticky, slightly plastic, thixotrophy not detectable; plentiful 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 soil pits had little 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, slightly sticky, slightly plastic, slightly smeary, thixotrophy not detectable; plentiful 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 flat"

4-34 Ah2 "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loamy sand; very weak granular and fine angular blocky; loose, slightly sticky, slightly plastic, very slightly smeary, thixotrophy not detectable; plentiful fine and medium roots; abundant fine and medium pores; very few subrounded, slightly weathered yellowish pumice gravel, frequent small pumice; separation from underlying horizon gradual and flat"

34-47 Ah3 "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loamy sand; very weak angular blocky; very friable, slightly sticky, slightly plastic, smeary, thixo• trophy not detectable; few fine roots, plentiful med• ium roots; abundant fine and medium pores; very few subrounded slightly weathered yellowish pumice gravel, abundant small pumice; separation from underlying horizon gradual and flat"

47-64 Ah4 "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loamy sand; very weak angular blocky; very friable, slightly sticky, slightly plastic, smeary, thixo• trophy not detectable; few fine roots, plentiful 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 flat"

64-73 Ah5 "Black (10YR 2/1); very dark gray (10YR 3/1 dry); loamy sand; very weak angular blocky; very friable, slightly sticky, slightly plastic, 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 first 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 acidified 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 distilled water to —' 100 ml.

6. Repeat step 4.

7. Filter FA through Whatman #1 filter paper into 1L volumetric flask. 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 distilled water. Centrifuge about 200 ml of HA at 6000 rpm for 20 minutes. Use HA to balance bottles, not distilled water! Filter through Whatman #1 filter 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 settle. Filter 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 filter paper with a fresh one. Poke hole in used filter paper and rinse any PVP back into the 1L erlenmeyer with dilute H2S0^. Filter remaining supernatant rinsing PVP into filter paper. Wash pad of PVP several times with dilute H2S0^ to remove any remaining fraction C. Discard filtrate.

2. Desorb fraction A: Using a metal spatula scoop out PVP into a 100 ml beaker. Poke hole in filter paper and rinse paper with 0.1 N NaOH into beaker. Add 30 ml 2 N NaOH to PVP, stir, and let soak for 20 minutes. Filter off PVP through Whatman #1 paper. Collect fraction A in a 100 ml volu• metric flask. 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. Solution Carbon Analyser — Walkley-Black Wet Oxidation Comparison for Extract Organic Carbon.

The Astro Solution Carbon Analyser uses a low temperature UV-promoted chemical oxidation of extract carbon with IR detection of the released Q^. The oxidant used is 1 M sodium persulphate. Calibration is based on external standards with three ranges of ppm C concentration; 0.1-100 ppm C, 100-500 ppm C and 500-2500 ppm C. A fixed injection loop of the correct volume is determined by the range selected. Data output is micro• processor controlled. Reproducibility is ± 2% or better. The Walkley-Black Wet Oxidation procedure uses chromic acid as the oxidant which is produced by acidification of the dichromate species with concentrated H^SO^. The oxidant left unused is determined by titration with ferrous sulphate, ferroin being used as the indicator. Reproducibili is very good, 2-5 %. Two basic assumptions are used in this method. 0 4+ 1. The oxidation state of carbon changes by +4 (C to C ), which assumes an initial oxidation state of zero. 2. The efficiency of oxidation is 80%.

The redox reaction for the oxidation is:

^ 4Cr + 3C02 + 8H20

Half reactions: 6+ 3+ Cr + 3e ->Cr

o 12 electron transfer C 1 C + 4e

^•Equivalent weight of C 12 g C / mwt C = 3 g C / equiv 4 equiv C / mwt C

ppm C in Extract = (2.5 ml FeS04 x N FeS04) x 3 x 1.25 x 10

ml extract ** 80% oxidation efficiency 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 soil 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 is unlikely that the oxidation efficiency is 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 length = 6-8" diameter = 2"

adsorbed fraction A "orange-brown" PVP

*l3

pale yellow fraction C "filtrate"

Fulvic Acid Fractionation Procedure:

1. Weigh required amount of PVP to fill column 1/2 to 1/3. Add distilled water, stir, let settle and decant fines. Repeat several times. 2. Set up PVP column with suction apparatus as shown. 3. Place Whatman #1 filter paper, cut to fit, into bottom of column. Wet and seal with suction. 4. Pour PVP-distilled water slurry evenly into column. 5. Wash PVP in column successively with; 2 column volumes of 0.1 N NaOH 2 column volumes of distilled water 2 column volumes of 0.1 N HC1 2 column volumes of distilled water 6. Pass FA fraction through PVP pad to adsorb fraction A. Wash column with 2-3 volumes of 0.01 N HC1 until filtrate colourless. Save or discard filtrate "fraction C". 7. Elute coloured fraction A from PVP with 1 volume of 0.1 N NaOH followed by 0.01 N NaOH until most of the coloured material has been eluted. 8. Pass fraction A through H+- exchange resin or adjust pH to 7 and store in refigerator. 227

Appendix 5. Individual Sample Data for Statistical Analysis.

NO O O O O -H ON ON —I r— ^ co in NO -3 CM in NO NO CO COOgCNCOCNrtCN o o o -3 sr NO o o o o o o o o o o o o o d d odd d oooooooo

ND NO — 00 CN

cNinoovD-a-ao»o r^-cNCOcnor^oom o -~ m ND in r^mco

CN CO CM CN m o co o 00 00 r- CN CN m -J co

NO NO co m in o CO o GO oo co r-- oo -3 m in m

in m NO coNOcosrONrtOOsr sr co in OcoONsrr-^incONO oo co co oo ON ON rt CO rtOO — 0 — CN- O " en o o o o o

CN * NO ON ON en O ST NO CN in ON m CO NO ON 00 o o -3 o NO m CN CN NO 00 o -3 CN sr co m 00 NO ON ON sr in CN o CN sr co m -3 co CO CN CN CN CrtO CN CrtO rt CN CN

* ON o oo o NO r~ ON sr o r~ o -3 o o in ON oo in NO m CO CO co sr sr CN CN m CN o NO 00 CN CN ON CO ON rt co _ CN co CN in ON m ON m NO in NO sr NrtO NO CO srtr mrt m CN

NO ON 00 CO o sr o o o o CN 00 CO o o <—i NO oo oo o CO ON GO CN -3 s: oo r- ON NO ON ST en CN CN ON CO NO sr -3 rt rt rt rt rt rs ort o o o o o o —• ~ rt CN — -* rt rt o rt rt o o rt o

o. 00 SrtCNfOvfinNOi^ rt CN CO ST NO o E CL.a.a.o-0-a.D-a. M ts] tS] S3 * co 228

oo co —< •£> o -J oosrmorscoOvO sr o ~' —isrtM — ro o o O O oooooooo o o o o oooooooo oodododd

O —< CN O \C O —i ro in —H co m m O ro r~- CO O \0 CN —< CNc o 00 in CN — *-. — ro ro o —i ro ro - - N N N CN —i —• ro ro ro ro ro ro ro si ro OOOOOOOO oooooooo

sr rs so cj* — —4 Om\£>oosr\Osrsr sr*omcNsrrosrcN

OOOOOOOO OOOOOOOO OOOOOOOO

n CO IN N CN r- O 00 o in in in —I CN

sj CT> o CO O CO ^ CO o ST O CM vD o sr m m oo oo O ro —i r- m oo o — O — o _H — o rs 0N — o O O O O o o o o o o o o oooooooo

OoocNrsyocoooo CN in rs ro 00 cr- r~ in —icNLncN— o sr sr rs. sr ro cr> in ro 00 00 o in CN ro 00 CO CN OOCN~H — co — o — CO CN — — — —c — —i ro CN — CN CN —

oo in ro CN o sr co oo cr* CO sr sr CN -J — — sr CN ro ro co ro ro

co oo m o sr in CN O CO sr sr sr \o cr* m r*- ro in oo in in o O ro CN ro O in r~ rs oo vo cr- o> OO — OO—'OO o — — ooooo O — — OOOOO

CN CN CN CN CN CN CNCNCNCNCNCNCNC—* CN 00 N CN CM CN CN CN CN — CN CO m o. •< •< •< 2: — CN ro sr in ts] E a. a- a. a. a. a. Sample No. ZFecbd ZAl^ 7,Fepy %AJ py %Cf %Ch 7Xc %Ca

AMI 0.281 0.162 0.131 0.131 0.470 0.456 0.243 0.227 All 0.164 0.112 0.0602 0.0904 0.227 0.127 0.114 0.114 A31 0.199 0.195 0.0806 0.171 0.399 0.391 0.213 0.186 A41 0.265 0.205 0.131 0.182 0.523 0.505 0.267 0.255 A51 0.216 0.149 0.101 0.121 0.412 0.301 0.196 0.215 A71 0.248 0.197 0.121 0.182 0.695 0.759 0.375 0.320 A81 0.299 0.246 0.143 0.234 1.02 1.52 0.510 0.510 A91 0.235 0.180 0.101 0.152 0.825 0.815 0.447 0.378

PM1 0.289 0.255 0.164 0.246 1.47 1.71 0.779 0.693 Pll 0.250 0.285 0.137 0.269 1.19 1.24 0.634 0.557 P21 0.234 0.266 0.117 0.263 0.958 0.839 0.557 0.401 P31 0.245 0.301 0.127 0.289 0.848 0.933 0.455 0.394 M P41 0.222 0.320 0.106 0.304 0.762 0.736 0.380 0.381 P51 0.235 0.260 0.111 0.253 0.557 0.805 0.330 0.228 P61 0.225 0.271 0.106 0.263 0.645 0.758 0.341 0.304 P71 0.246 0.275 0.137 0.268 0.832 0.924 0.438 0.394

ZM1 0.264 0.312 0.0550 0.242 0.502 0.553 0.354 0.147 Zll 0.266 0.351 0.0960 0.298 0.625 0.861 0.439 0.186 Z21 0.259 0.340 0.0937 0.284 0.685 0.855 0.494 0.191 Z31 0.212 0.256 0.0580 0.212 0.449 0.439 0.314 0.134 Z41 0.236 0.293 0.0504 0.222 0.428 0.506 0.296 0.132 Z51 0.229 0.282 0.0682 0.242 0.546 0.635 0.385 0.162 Z61 0.255. 0.303 0.0758 0.273 0.572 0.685 0.381 0.191 Z71 0.214 0.246 0.0453 0.201 0.353 0.313 0.244 0.110

*oven-dry basis 230

sr rs \o in in rs srsrmminCNvOcN m r~ o* cn ON in NO m m cn in o o> CN —i cnsTNONoaoooin — cn o rs sr CN in oo ON — — CN CN — CN — — CN — — — — — o o o o o o o OOOOOOOO oooooooo

CO O CN CN — r~ CN NO a* — in sr r~. NO —< cn O1 CN co o i— cn n vO m o CN O cr* o CN NO O CN — CN cn — — — sr CN CN m CN cn cn cn d o" o" d o" d d OOOOOOOO o o o o o o o

— — CN sr CN NO rs m o cn ON CN vo m CN sr *o sr NO CO oo co sr ON — sr o — sr sr cn o cn in sr in in NO —< — rs cn sr o cn — vo sr m a- <6 O d o o OOOOOOOO o o o o o o o

cn rs ON NO o NO o> o o o> o O m NO cn P^ GO ON o NO o — sr cn —i sr cn rs CN sr cn cn cn cn sr sr cn sr r- o o o o o o o ooooo

cn cn sr O O CN — — cn — o voocn CN CN CN CO ND corsfs-srrs — CNO NO rs NO CN CN sr sr O O r- o OO — — —'CN — o — CN CN CN CN CN CN cn cn CN CN CN CN CN o o o o ooooo o o oooooooo

cn CN m CN rs ps co rs. ON rs cn r— NO CN GO cn OO — — — CNCO CN NO m o m in in m NO *o ON in co m m *£>incncNCNsracsr n o co c o» co a- rsoo m NO rs oo in oo — — — — oo — — ooooo OO —OOOO O o o oooooooo o o o o o o o

co o oo cn o — in ON sr ON oo in r- CN o CN O sr sr NO ON sr ON o« oo cn — — CO 00 NO — cn in cn NO oo r-. o o o — CN — — — CN CN CN CN cn cn CN CN CN cn o ^ c~> O O O O O O O CN CN CN CN O O O o

sr CN ND cn cn o NO NO NO m sr CN ON sr ON ON vD cn sr oo oo m — cn — o o — ON o O CNCNCNCNCNCNCNCN — — CN CN — — — CN CN CN CN CN — CN sr o o o o o o o o o o o oooooooo

cu CNCNCNCNCNCNCNCN CNCNCNCNCNCNCNCN CN CN CN CN CN CN > NO oo x — CN cn sr in NO r- NO o s — 0.0.0.0.0.0.0.0. rsi CM sr CO * M 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 0.48 56 11.6 22.0 4.60 6.40 All 0.56 1.12 0.50 60 10.7 20.3 4.64 6.05 A31 0.98 2.10 0.47 61 8.8 22.2 5.27 6.31 A41 0.97 1.98 0.49 60 11.3 26.7 5.36 6.25 A51 0.73 1.40 0.52 58 11.6 29.9 6.33 7.85 A71 1.10 2.38 0.46 55 13.5 29.6 7.54 7.71 A81 1.49 2.98 0.50 53 15.2 28.2 4.64 5.45 A91 0.99 2.16 0.46 54 14.5 27.6 7.81 8.41

PM1 1.16 2.47 0.47 58 16.5 31.5 6.43 6.89 Pll 1.04 2.22 0.47 58 14.0 30.7 6.46 7.36 P21 0.88 1.22 0.42 52 14.0 31.1 6.66 6.69 P31 1.10 2.37 0.46 54 14.5 24.3 6.39 7.11 P41 0.97 1.93 0.50 59 14.8 32.1 6.40 6.53 P51 1.45 3.54 0.41 47 13.9 23.2 6.30 8.46 P61 1.18 2.50 0.47 53 13.5 25.6 5.52 6.96 P71 1.11 2.35 0.47 56 15.5 27.2 5.70 7.10

ZM1 1.10 3.75 0.29 54 12.9 18.3 6.16 7.67 Zll 1.38 4.63 0.30 53 13.6 20.7 6.53 7.10 Z21 1.25 4.49 0.28 49 12.9 21.4 7.04 7.73 Z31 0.98 3.28 0.30 49 12.9 19.1 7.43 8.30 Z41 1.18 3.83 0.31 49 13.1 21.8 6.39 7.31 Z51 1.16 3.93 0.30 49 13.0 21.0 10.5 9.21 Z61 1.20 ' 3.59 0.33 50 12.3 19.3 6.44 8.44 Z71 0.89 2.85 0.31 56 13.3 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 0.54 62 11.7 19.0 6.26 8.55 A12 0.69 1.20 0.57 68 20.9 27.0 3.90 5.55 A22 1.56 2.89 0.54 57 13.6 28.9 4.32 6.01 A42 0.94 1.76 0.53 59 12.0 27.7 5.15 6.51 A62 1.22 2.48 0.49 56 10.1 29.7 5.91 5.82 A72 1.68 3.95 0.43 53 12.2 24.8 6.05 8.53 A82 1.06 1.71 0.62 68 16.6 24.7 5.44 8.81 A92 0.59 1.26 0.47 58 13.0 26.2 6.27 7.36

PM2 0.93 2.08 0.45 53 13.5 22.1 6.11 7.10 P12 0.99 1.82 0.54 60 17.4 18.2 5.95 6.84 P22 1.08 2.63 0.41 51 13.2 28.9 5.67 6.16 P32 1.14 2.52 0.45 54 15.0 24.5 5.30 6.31 P42 1.21 2.68 0.45 53 15.3 27.3 5.16 5.76 P52 1.16 2.53 0.46 51 15.0 23.2 5.88 8.53 P62 1.02 2.21 0.46 50 14.3 25.6 6.30 6.23 P72 1.24 2.78 0.45 55 16.5 29.0 7.12 5.88

ZM2 1.17 3.79 0.31 52 12.8 17.0 6.88 9.40 Z12 1.34 4.59 0.29 50 13.8 20.8 6.64 7.48 Z22 1.26 4.36 0.29 49 13.5 22.1 6.83 7.20 Z32 1.01 3.09 0.33 50 12.4 18.1 7.76 8.86 Z42 1.19 3.99 0.30 50 13.4 22.3 8.32 7.48 Z52 1.13 3.46 0.33 48 13.3 19.7 7.68 10.9 Z62 1.19 ' 3.24 0.37 49 12.2 18.9 6.44 9.50 Z72 0.92 3.21 0.29 53 13.2 15.4 7.28 9.23 233

F 1X0 i F 1%C A Sample No. E C HA E C HA EA/E6 HA L A E4/E6 A A00 600 W0 ^OO fl

AMI 231.0 52.1 4.44 122.6 9.7 12.6 All 184.6 44.9 4.11 127.5 10.0 12.7 A31 224.3 46.8 4.80 129.4 9.0 14.4 A41 232.5 51.8 4.49 127.6 8.8 14.6 A51 217.3 47.6 4.56 138.5 11.0 12.6 A71 197.3 40.7 4.85 130.2 10.1 12.8 A81 227.2 47.4 4.80 133.3 9.2 14.4 A91 182.2 39.0 4.68 129.0 10.9 11.9

PM1 178.3 40.6 4.40 131.2 11.2 11.8 Pll 174.9 40.7 4.30 133.3 9.9 13.5 P21 179.4 45.9 3.91 124.4 10.3 12.1 P31 193.9 51.9 3.73 123.9 9.4 13.3 P41 188.4 50.1 3.76 125.2 9.9 12.6 P51 231.2 58.6 3.95 131.7 10.3 12.8 P61 209.2 54.1 3.87 118.6 9.2 13.0 P71 210.8 55.0 3.84 133.2 10.9 12.2

ZM1 209.6 61.0 3.44 112.3 9.9 11.4 Zll 204.3 64.2 3.19 113.9 9.2 12.4 Z21 195.0 58.3 3.35 117.3 9.1 12.9 Z31 199.3 58.2 3.42 107.4 8.4 12.8 Z41 209.7 62.6 3.35 112.9 9.4 12.0 Z51 198.2 56.8 3.49 106.3 7.9 13.4 Z61 215.9 62.9 3.43 119.2 9.4 12.6 Z71 197.7 61.1 3.24 92.8 7.9 11.7 234

C C C Sample No. E^ HA HA E^ HA E^ A E(.JJ 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 / pit no. / horizon ; A = Abies religiosa, P= Pinus hartwegii, Z = Zacatonal ; M = modal pit ; 1 = Ahl horizon, 2 = Ah2 horizon. ZONE %HM %LI %Ct %Nt C/N -oven-dry basis-

Ahl horizons, n=8

Abies religiosa 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 religiosa 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. Significance level: 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 religiosa 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. Significance level: 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 religiosa 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 tJ ox py cbd Abies religiosa 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. Significance level: 95%= *, 99%= **. Analysis by AN0VA. * * # 1 #17 #19 # # T ZONE %Ch %Ca J %Cf J> %Cc 5,1 %Ce 5,1 air-dry basis

Ahl horizons, n=8 Ahl + Ah2 horizons, n=16

Abies religiosa 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 religiosa 0.44 0.20 (0.33) (0.056)

Pinus hartwegii 0.54 0.23 (0.23) (0.13)

Zacatonal 0.58 0.15 (0.20) (0.036)

1. Significance level: 95%= *, 99%= **. Analysis by AN0VA. 2. The Ahl and Ah2 horizons were combined for %Cf, %Cc and %Ce, n=16. 3. Homogeneity of variance not met. 238

ZONE Ch/Cf Ch/Ca Ca/Cf

Ahl + Ah2 horizons, n=16

Abies religiosa 1.0 2.0 0.50 (0.36) (0.80) (0.48)

Pinus hartwegii 1.1 2.4 0.46 (0.14) (0.50) (0.032)

Zacatonal 1.2 3.8 0.31 (0.14) (0.56) (0.023)

1. Significance level: 95%= *, 99%= **. Analysis by ANOVA. 2. Homogeneity of variance not met.

ZONE C/N HA C/N A %GIucose C* » 2 %Glucose C Total HA-C Total A-C

Ahl + Ah2 horizons, n=16

Abies religiosa 13.0 25.9 5.6 7.0 (2.88) (3.44) (1.1) (1.2)

Pinus hartwegii 14.8 26.5 6.1 6.9 (1.20) (3.91) (0.52) (0.79)

Zacatonal 13.0 19.5 7.2 8.5 (0.460) (2.12) (1.1) (1.1)

1. Significance level: 95%= 99%= •». Analysis by ANOVA. 2. Homogeneity of variance not met. 239

C C ZONE E,JJ HA E6^ HA E./E, HA

Ahl + Ah2 horizons, n=16 Ah] horizons, n=8

Abies religiosa 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) (0.104)

i%r ** ** 3

LE HHAA 600 EA/E6 HA

Ah2 horizons, n=8

Abies religiosa 44.8 4.62 (10.5) (0.464)

Pinus hartwegii 63.6 3.56 (9.47) (0.269)

Zacatonal 62.0 3.30 (4.02) (0.930)

nC E A** lXC ** 3 ZONE ^400 * E A*A * ^600 VE6 A

Ahl + Ah2 horizons, n=16

Abies religiosa 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 level: 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 r- rr to T O) CB CB in r- in in oo O > rr rr O O in CN ion rr IB o O CN "J CD 01 r~ in CB 01 r~ O O • 0. r- r> r- t- IB to rr o IB IB o to r~ r» in r- p~ O to O CP in ui o — 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- 1 Ol CO 01 in CM in r> rr O rr 01 as CM CO CN CO o o IB IB CO CD CB o 00 01 03 01 o — in p-* —to co O oc ^- O • o ID IB 01 01 T CB r- t> CB CM rr _J in in in O in o in in O — < 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 to o CO UJ 1 ,' — 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 oo CM r- CB CO rr •— CN O o in in o O r> O ^~ • 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 1 I t I i — l/l '' i 1 l i l 1 i

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 r~ IT in in rr CM co 0) X CO r- O r- O to to to O in CM C0O0 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 r- in IB O) in 01 O O to CN CO r- \— rr 01 -•— O) O) co r~ T- o IB t- r- in IB 0o1 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) CO CN O) CO O CD rr CO in CB CO 01 O in r- CB r- ~- co O vS ion co in •— CP CO CM 01 rr 01 00) 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 01 co O O CB rr rr t- rr ~— o CB o CN CO ^~ CN CB CM r^ r- O 01 •3 co to co 01 O o cn in f> to — i> 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 CM CM CO CO 0) O rr in r•r-~ O O to 03 in in P~ Ol 01 CO 01 o r- CO O r- 00 to 01 0) 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~ to 0) 01 *- IB CN O 1 oo CM •

ffs sp s« s* s« Q o X X X CO CO > > s« u. < u. ?s s« oS o D O a CL Ss ss D o CJ CJ I < £ U. 1- 1- z o UJ o UJ U. I (J < < I I < Ui z Z I o _i u z 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% 1.0000 .5596 .6195 .7140 . 3238 . 2733 .6024 .6469 -.5453 - . 6226 . 1234 -.0751

17. CF7. . 5596 1.0000 . 9345 .9386 . 9301 .9129 . 3296 .0932 .0388 -.0758 . 2908 . 4001

18. CH% .6195 .9345 1.OOOO .9090 . 8355 . 8263 .6101 . 3239 -.0463 -.2062 . 2691 . 3478

19CC% . 7 140 .9386 .9090 1.0000 . 7473 . 7528 .4186 . 3702 - . 2910 -.3246 . 1885 . 2033

20CAD% . 3238 .9301 .8355 .7473 1.0000 .9587 . 1913 -.2159 . 3870 . 1962 . 3582 .5498

2 1 .CAM0/. .2733 .9129 . 8263 . 7528 .9587 .0000 . 1892 -.2060 . 3554 . 1847 . 2982 . 6494

22. CHCF .6024 . 3296 .6101 .4186 . 1913 . 1892 1 .0000 .7316 - . 2928 -.5024 .0682 .0728

23. CHCA .6469 .0932 . 3239 . 3702 -.2159 . 2060 .7316 1.0000 - .8248 -.7194 -.1144 -.4209

24. CACF -.5453 .0388 -.0463 -.2910 . 3870 . 3554 . 2928 - .8248 1.OOOO .769 1 2603 .5919

25. CE% -.6226 - .0758 -.2062 -.3246 . 1962 . 1847 . 5024 -.7194 . 769 1 1.OOOO . 2365 . 3001

2G.CNH . 1234 . 2908 . 2691 . 1885 . 3582 . 2982 .0682 -.1144 . 2603 . 2365 1.OOOO .2497 M

27. CNA -.075 1 .4001 . 3478 . 2033 . 5498 . 6494 .0728 -.4209 .5919 . 3001 . 2497 1.OOOO

28. GLCHC% . 3516 . 1 143 .0739 . 3086 -.1103 .0803 .0704 .4507 -.6273 - .5345 -.1246 -.2688

29. GLCAC% . 1 170 -.1202 -.1168 .0439 -.2696 . 2648 .0070 .3475 -.4893 -.3470 -.1748 - . 5935

30. E41%H . 1 180 -.2142 -.0219 - .2128 -.1850 . 1923 . 5202 . 2455 -.0239 -.3352 -.3757 .0864

31. E61%H .4787 -.2901 -.1485 -.0923 -.4579 .4789 . 3858 . 5781 -.6024 -.7129 -.2229 -.3942

32 . E4E6H - .5572 . 1588 .1361 -.0919 . 4017 .4274 .0906 -.5458 .7617 . 6864 .0858 . 5608

33. E4 1%A - . 28 1 1 . 3334 . 2877 .0685 .5631 .5417 .0328 -.5390 .7936 . 5469 . 2016 . 7030

34. E6T/.A -.1837 .2871 . 1528 .0884 .4544 .4203 .2518 -.5269 .5655 . 3691 . 4446 .4919

35. E4E6A -.1158 .0484 . 1726 -.0251 . 1 186 . 1356 . 3090 .0181 . 2647 . 2025 -.3395 . 2488

16 . 17 . 18 . 19 . 20. 21 . 22 . 23. 24 . 25 . 26 . 27 . ALP Y% CF% CH% CC% CAD% CAM% CHCF CHCA CACF CE% CNH CNA VARIABLE

28. GLCHC% . 1256 .1271 . 1683 . 159 1 . 2677 - .3406 - . 3452 . 4790 .5310 . 108 1 . 4328 - . 256 1

29. GLCAC% -.0895 -.0896 -.0725 -.0634 . 0693 - . 477 1 - .2967 . 3540 . 5609 .0190 . 1873 - .3666

30. E41%H -.0472 -.0444 -.0985 -.0734 -.0256 - . 1872 .0727 .0668 -.0073 . 284 1 . 1050 . 2668

31 .E61°/»H -.1527 -.1513 -.1142 -.12 16 -.024 1 -.2957 - .2575 .6471 .6383 . 1099 . 5279 - . 2 193

32 . E4EGH . 1207 . 1208 .0246 .0556 -.0368 . 2759 . 3695 - . 78 14 -.7856 .024 1 -.6304 .4348

33. E41%A . 2773 . 2767 .2191 . 2303 .0878 . 5055 .3122 -.6124 -.8673 .048 1 -.4053 . 6 104

34. E6T/.A . 1884 . 1858 . 1584 . 1461 -.0408 .6490 .0225 -.3612 -.5396 - . 1779 -.2905 . 3000

35. E4E6A . 1 144 . 1 173 .0745 . 1062 . 1761 - .2224 . 3692 -.3054 -.3985 . 3031 -.1323 . 3930

4 . 5. 6 . 7 . 8 . 9 . 10. 1 1 . 12 . 13 . 14 . 15 . HM% CF LI% CT% NT% CN FEOX% ALOX% SIOX% FECBD7, A LCBD% FEPY%

VARIABLE

28 GLCHC% 1.0000 .6109 - . 1467 . 2444 - .4495 -.4978 -.3228 -.1977 K3 ro 29. GLCAC% .6109 1.OOOO -.1321 . 2095 -.3626 -.58 14 -.4403 -.1497

30. E41%H - . 1467 - . 1321 1.OOOO .6262 -.0398 -.0014 -.2247 3168

31. EG1%H . 2444 . 2095 .6262 1 OOOO -.7921 -.6137 -.3769 -.2778

32. E4E6H -.4495 -.3626 -.0398 - . 7921 1.OOOO . 7660 .3191 .5626

33. E41%A -.4978 -.5814 -.0014 - .6137 . 7660 1.OOOO . 7207 .3171

34. EG1%A -.3228 -.4403 -.2247 -.3769 .3191 . 7207 1.OOOO - .4256

35. E4E6A -.1977 -.1497 .3168 -.2778 .5626 .3171 -.4256 1.OOOO

28. 29. 30. 31 . 32. 33 . 34. 35. GLCHC% GLCAC% E4 1%H E6 1%H E4E6H E4 1%A E6 1°/„A E4E6A

N= 48 DF= 46 R© .0500= 2845 R© .0100= 3683