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Trace Elements

in the
Rhizosphere
1535/frame/FM Page 2 Tuesday, August 22, 2000 9:08 AM
Trace Elements
in the
Rhizosphere
Edited by
George R. Gobran
Walter W. Wenzel
Enzo Lombi

CRC Press
Boca Raton London New York Washington, D.C.
Library of Congress Cataloging-in-Publication Data

Trace elements in the rhizosphere / George R. Gobran, Enzo Lombi & Walter W.
Wenzel, editors.
p. cm.
Papers from a special symposium held during the Fifth International Conference on
Biogeochemistry of Trace Elements, July 1115, 1999 in Vienna, Austria.
Includes bibliographical references and index.
ISBN 0-8493-1535-2
1. SoilsTrace element contentCongresses. 2. Trace element in plant
nutritionCongresses. 3. Plant-soil relationshipsCongresses. 4.
RhizosphereCongresses. I. Gobran, George R. II. Lombi, Enzo, 1968 III. Wenzel,
Walter W. IV. International Conference on the Biogeochemistry of Trace Elements (5th :
1999 : Vienna, Austria)

S592.6.T7 T73 2000


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Preface
The term rhizosphere was introduced by Hiltner in 1904. Since then, the hidden
half of the hidden half has been investigated in its many aspects. Initially, the
interest of scientists and practitioners was directed toward plant nutrition, in partic-
ular, as to whether rhizosphere processes influence the uptake of mineral nutrients
by crop and tree species. With the general move of soil and plant scientists from
agricultural to ecological/environmental issues, the emphasis of rhizosphere research
has also increasingly shifted to studying the effect of plantmicrobial associations
on the bioavailability, uptake, and transformation of inorganic and organic contam-
inants in soils. Most recently, contaminantrhizosphere interactions have attracted
renewed attention as green plants have been proposed as being useful in the reme-
diation of contaminated soils (phytoremediation).
While potential practical applications are a driving force in rhizosphere research,
it is equally important to develop methodological tools employed to elucidate the
fundamental physical, chemical, and biological processes that create the highly
dynamic and reactive microenvironment around plant roots. Basically, two
approaches have been taken in the study of the rhizosphere: experimental investi-
gation and modeling. While a relatively large number of publications dealing with
various aspects of the rhizosphere is available, the information is still scattered and
we are far from a comprehensive understanding of the manifold interactions and
feedbacks that occur in the rhizosphere. In particular, only a few examples of studies
that link modeling to experimental work (model validation) are available, and little
is available on the fate of trace elements in the rhizosphere, even though such
information is urgently needed to improve food quality and to develop phytoreme-
diation technologies. Needless to say, the rhizosphere plays a key role in controlling
trace element contamination of the terrestrial food chain. Therefore, it is one of the
hot spots in sustaining ecosystem health.
Interest in this topic led to the organization of a special symposium entitled,
Fate of Trace Elements in the Rhizosphere, held during the Fifth International
Conference on the Biogeochemistry of Trace Elements, July 1115, 1999, in Vienna,
Austria. This special symposium provided a forum to bring together scientists
involved in various aspects of rhizosphere research. Most of the chapters in this
book comprise work that was presented by the outstanding scientists invited to this
special symposium.
The book Trace Elements in the Rhizosphere is divided in three sections. Section
I (Chapters 1 to 5) emphasizes the bioavailability of trace elements, including
radionuclides in the rhizosphere and their significance in developing management
strategies for phytoremediation. Section II (Chapters 6 to 8) deals with the complex
interactions of rootmicrobial associations, in particular mycorrhizae, and the trans-
formation of trace elements. Tools and methods available for study of the dynamics
and fate of trace elements in the rhizosphere are discussed in Section III (Chapters
9 to 12), presenting examples of experimental techniques and modeling approaches.
Chapter 1 presents a review of the role that rhizosphere processes play in the
mobilization or immobilization of trace elements and summarizes the state-of-the-
art knowledge on metal hyperaccumulators as compared to nonaccumulating plant
species. Special reference is given to the application of rhizosphere processes to
phytoremediation, and the potential of manipulating the rhizosphere to control trace
element availability for phytoremediation of crops is explored. Chapter 2 provides
a review of complementary in-depth information on trace metal bioavailability in
the rhizosphere. Chapter 3 contributes to an understanding of the specific conditions
that modify trace metal interactions in the rhizosphere. Chapter 4 extends the scope
to radiocesium. Chapter 5 presents a comprehensive review of the fate of U and Pu
radionuclides in soilplant systems. The role of rhizospheric interactions and their
application to the phytoremediation of U- and Pu-polluted soil and water environ-
ments are emphasized. Chapter 6 provides current information on exudates and
metal-organic complexes associated with rootmicrobial associations. Chapter 7
emphasizes the importance of metal speciation in symbiotic fungi (ectomycorrhiza),
presenting data generated by micro X-ray absorption spectroscopy, a promising
technique that might be more widely applied in rhizosphere research. Chapter 8
discusses bioavailability of trace elements in mycorrhizal rhizosphere systems,
hence, linking the information provided in Sections I and II. Chapter 9 explores the
application of a sequential chemical extraction technique as a means to study trace
metal fractionation in rhizosphere soils. Chapter 10 presents a technique to study
the effect of micronutrients or toxic trace elements on plant growth, using a steady-
state approach. This methodology may prove useful in rhizosphere studies, since
experimental conditions of microelement nutrition and their interactions with plant
physiological processes can modify root exudation and related processes in the
rhizosphere. Chapter 11 presents a model that describes cation exchange on plant
roots, a process that has been neglected in most models so far. Chapter 12 describes
a model of aluminum chemistry with reference to acidic forest soils.
It is hoped that this book will contribute to the body of knowledge on the secrets
of the rhizosphere. The book is suitable for advanced undergraduate and graduate
courses in soil environmental chemistry, biogeochemistry, and ecology. It should
interest professionals in those areas, as well as consulting engineers and decision
makers involved in bioremediation technology and sustaining ecosystem health.
We greatly acknowledge the authors of this book and their cooperation and
support. We also wish to extend our special thanks to many anonymous reviewers
of each chapter, especially G. gren and R. Finlay (Swedish University of Agricul-
tural Sciences, SLU, Sweden), and K. Turnau (Jagiellonian University, Krakow,
Poland). The books manuscript was carefully read by M.B. Kirkham (Kansas State
University, U.S.A.) and M.P. Huang (University of Saskatchewan, Canada), who
deserve our special thanks and appreciation. We are also grateful for the financial
support received from the Faculty of Forestry, SLU, Sweden, and the Swedish
Council for Forestry (SJFR); the Agricultural Research, IACR-Rothamsted grant-
aided support received from the Biotechnology and Biological Sciences Research
Council of the United Kingdom; and the support of the University of Agricultural
Sciences, Vienna, Fonds zur Frderung der Wissenschaftlichen Forschung, Austrian
Federal Ministry of Science and Transport.

April 2000 George R. Gobran, Walter W. Wenzel, and Enzo Lombi


Editors
George R. Gobran is Professor of Ecology-Soil Sciences with specialization in
nutrient dynamics in the rhizosphere. Since 1985, Dr. Gobran has been working at
the Department of Ecology and Environmental Research, Swedish University of
Agriculture Sciences, Uppsala, Sweden. Dr. Gobran received his Ph.D. in soil chem-
istry from the Catholic University of Louvain-la-Neuve, Belgium in 1980. He
received his M.S. in soil chemistry in 1975 and his B.S. in soil and water sciences
in 1969 from Alexandria University, Egypt. Dr. Gobran was also awarded a post-
doctoral fellowship from Texas A & M University, U.S.A. (19821984).
Professor Gobran has significant experience in research dealing with soil chem-
ical processes, with special interest in soilplant interactions and rhizospheric pro-
cesses. Currently, he focuses his research and teaching efforts on the reciprocal
effects of soilplant interactions, especially in ecosystems under environmental
stress. Dr. Gobran has written many papers and book chapters, and has participated
in several international conferences, workshops, and symposia. Additionally, he is
a reviewer for several international journals and programs and is included in the
1999 edition of Whos Who in the World. With his coeditors, Drs. Wenzel and Lombi,
Dr. Gobran organized a symposium entitled, Fate of Trace Elements in the Rhizo-
sphere, which was presented during the Fifth International Conference on the Bio-
geochemistry of Trace Elements, Vienna, July 1115, 1999.

Walter W. Wenzel is Professor of Soil Science at the University of Agricultural


Sciences in Vienna, Austria. He has been working on various fundamental aspects
of trace element biogeochemistry, with emphasis on several cationic metals and the
metalloids As and F. Currently, Professor Wenzels work is centered around exper-
imental assessment and modeling of rhizosphere processes and metal hyperaccumu-
lation. His involvement in applied research has been largely related to risk assessment
and the development of phytoremediation technologies for the cleanup of soils
contaminated with metals and organic pollutants. Professor Wenzel is coeditor of
the Journal of Environmental Quality and the Journal of Plant Nutrition and Soil
Science. He organized the Fifth International Conference on the Biogeochemistry
of Trace Elements, Vienna, July 1115, 1999. Additionally Professor Wenzel is one
of the initiators and founders of the International Society for Trace Element Bio-
geochemistry, for which he currently serves on the board of directors.

Enzo Lombi was born in Piacenza, Italy, 1968. After graduation in 1993, he earned
a Ph.D. in agricultural chemistry and conducted his research in Italy, Austria, and
the United States. During 19971998 he was Scientific Researcher at the Institute
of Soil Science, University of Agricultural Sciences, Vienna, and at the Austrian
Research Centre, Seibersdorf. His activities focused on the development and appli-
cation of low-input technologies to remediate polluted soils. At present, Dr. Lombi
is Research Scientist at the Soil Science Department of IACR-Rothamsted, UK. His
research includes various aspects of phytoremediation using hyperaccumulator
plants and chemical-assisted phytoextraction. He is interested in physiological
aspects of tolerance, metal transport, and accumulation and compartmentation in
hyperaccumulator plants. Moreover, Dr. Lombis work includes investigations con-
cerning rhizosphere processes involved in metal mobilization and uptake by plants.
A second area of investigation is the in situ fixation of heavy metals and metalloids
using industrial byproducts.
Contributors
Domy C. Adriano Salvatore Deiana
Savannah River Ecology Lab DI.S.A.A.B.A.
University of Georgia Universita degli Studi di Sassari
Drawer E Viale Italia 39, 07100 Sassari
Aiken, South Carolina 29802 Italy
U.S.A.
Bruno Delvaux
Naomi Assadian Unit Sciences du Sol
Texas Agricultural Experiment Universit Catholique de Louvain
Station Place Croix du Sud, 2/10
Texas A & M University 1348 Louvain-la-Neuve
1380 A & M Circle Belgique
El Paso, Texas 79927
U.S.A. Joseph E. Dufey
Unit Sciences du Sol
Universit Catholique de Louvain
Bjrn O. Berthelsen
Place Croix du Sud, 2/10
Norwegian University of Science and
1348 Louvain-la-Neuve
Technology
Belgique
Faculty of Chemistry and Biology
Department of Chemistry
Alain Dufresne
N-7491 Trondheim
Les Laboratoires Savoie-Dufresne Inc.
Norway
C.P. 48808, C.S.P. Outremont
Montral (Qubec), H2V 4V1
Henri Calba Canada
AMIS Programme Agronomie
Centre de Coopration Internationale Lloyd B. Fenn
en Recherche Agronomique Texas Agricultural Experiment Station
pour le Dveloppement Texas A & M University
2477 Avenue du Val de Montferrand 1380 A & M Circle
F-34032 Montpellier Cedex 1 El Paso, Texas 79927
France U.S.A.

Franois Courchesne Jos G. Genon


Dpartement de Gographie Unit Sciences du Sol
Universit de Montral, C.P. 6128 Universit Catholique de Louvain
Succursale Centre-Ville Place Croix du Sud, 2/10
Montral (Qubec), H3C 3J7 1348 Louvain-la-Neuve
Canada Belgique
Carlo Gessa Erik J. Joner
Istituto Chimica Agraria Centre de Pdologie Biologique
Universit degli Studi di Bologna CNRS-UPR6831
Viale Berti Pichat 10 17, rue Notre Dame des Pauvres
40127 Bologna B.P.5, F-54501
Italy Vandoeuvre-les-Nancy Cedex
France
George R. Gobran
Department of Ecology and
Environmental Research Nathalie Kruyts
Faculty of Forestry Unit Sciences du Sol
Swedish University of Agriculture Universit Catholique de Louvain
Sciences Place Croix du Sud, 2/10
Box 7042 1348 Louvain-la-Neuve
S-750 07 Uppsala Belgique
Sweden
Geraldine M. Lamble
Anders Gransson MS-80-101, Earth Sciences Division
Department for Production Ecology Lawrence Berkeley National
Faculty of Forestry Laboratory
Swedish University of Agriculture 1 Cyclotron Road
Sciences Berkeley, California 94720
Box 7042 U.S.A.
S-750 07 Uppsala
Sweden
Jin-Ho Lee
Philippe Hinsinger Soil and Crop Sciences Department
INRA, UFR de Science du Sol Texas A & M University
Place Viala College Station, Texas 77843
F-34060 Montpellier Cedex 1 U.S.A.
France
Corinne Leyval
Lloyd R. Hossner
Centre de Pdologie Biologique
Soil and Crop Sciences Department
CNRS-UPR6831
Texas A & M University
17, rue Notre Dame des Pauvres
College Station, Texas 77843
B.P.5, F-54501
U.S.A.
Vandoeuvre-les-Nancy Cedex
France
Benot Jaillard
Laboratoire de Science du Sol
Institut National de la Richard H. Loeppert
Recherche Agronomique Soil and Crop Sciences Department
Place Pierre Viala Texas A & M University
F-34060 Montpellier Cedex 2 College Station, Texas 77843
France U.S.A.
Enzo Lombi Alessandra Premoli
Soil Science Department DI.S.A.A.B.A.
IACR-Rothamsted Universita degli Studi di Sassari
AL5 2JQ Harpenden, Hertfordshire Viale Italia 39, 07100 Sassari
U.K. Italy

Alastair A. MacDowell Gervais Rufyikiri


MS-80-101, Earth Sciences Division Unit Sciences du Sol
Lawrence Berkeley National Universit Catholique de Louvain
Laboratory Place Croix du Sud, 2/10
1 Cyclotron Road 1348 Louvain-la-Neuve
Berkeley, California 94720 Belgique
U.S.A.
Vronique Sguin
Emmanuel Maes Dpartement de Gographie
Unit Sciences du Sol Universit de Montral, C.P. 6128
Universit Catholique de Louvain Succursale Centre-Ville
Place Croix du Sud, 2/10 Montral (Qubec), H3C 3J7
1348 Louvain-la-Neuve Canada
Belgique
Hamid Shahandeh
Bruno Manunza
Soil and Crop Sciences Department
DI.S.A.A.B.A.
Texas A & M University
Universita degli Studi di Sassari
College Station, Texas 77843
Viale Italia 39, 07100 Sassari
U.S.A.
Italy

David G. Nicholson Erik Smolders


Norwegian University of Science and Laboratory of Soil Fertility and Soil
Technology Biology
Faculty of Chemistry and Biology Katholieke Universiteit Leuven
Department of Chemistry K. Mercierlaan 92, 3001 Heverlee
N-7491 Trondheim Belgium
Norway
Walter W. Wenzel
Heino W. F. Nietfeld Institute of Soil Science
Institute of Soil Science and Forest University of Agricultural Sciences
Nutrition Gregor Mendel Strasse 33
Buesgenweg 2, D-37077 Goettingen A-1180 Vienna
Germany Austria

Amedeo Palma
DI.S.A.A.B.A.
Universita degli Studi di Sassari
Viale Italia 39, 07100 Sassari
Italy
Contents
SECTION I PHYTOAVAILABILITY OF TRACE ELEMENTS AND
RADIONUCLIDES IN THE RHIZOSPHERE AND
APPLICATION TO PHYTOREMEDIATION

Chapter 1
Dependency of Phytoavailability of Metals on Indigenous and Induced
Rhizosphere Processes: A Review ............................................................................3
Enzo Lombi, Walter W. Wenzel, George R. Gobran, and Domy C. Adriano

Chapter 2
Bioavailability of Trace Elements as Related to Root-Induced Chemical
Changes in the Rhizosphere ....................................................................................25
Philippe Hinsinger

Chapter 3
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption by Plants
Growing in Calcareous Soils...................................................................................43
Naomi Assadian and Lloyd B. Fenn

Chapter 4
Fate of Radiocesium in Soil and Rhizosphere........................................................61
Bruno Delvaux, Nathalie Kruyts, Emmanuel Maes, and Erik Smolders

Chapter 5
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems
and the Potential of Phytoremediation....................................................................93
Hamid Shahandeh, Jin-Ho Lee, Lloyd R. Hossner, and Richard H. Loeppert

SECTION II INTERACTIONS OF TRACE ELEMENTS WITH ROOT


MICROBIAL ASSOCIATIONS

Chapter 6
Interactions and Mobilization of Metal Ions at the SoilRoot Interface .............127
Salvatore Deiana, Bruno Manunza, Amedeo Palma, Alessandra Premoli,
and Carlo Gessa
Chapter 7
Analysis of Metal Speciation and Distribution in Symbiotic Fungi (Ectomycorrhiza)
Studied by Micro X-ray Absorption Spectroscopy and X-ray Fluorescence
Microscopy ............................................................................................................149
Bjrn O. Berthelsen, Geraldine M. Lamble, Alastair A. MacDowell,
and David G. Nicholson

Chapter 8
Bioavailability of Heavy Metals in the Mycorrhizosphere...................................165
Corinne Leyval and Erik J. Joner

SECTION III EXPERIMENTAL TECHNIQUES AND MODELING


APPROACHES TO STUDY THE FATE OF TRACE
ELEMENTS IN THE RHIZOSPHERE

Chapter 9
Solid Phase Fractionation of Metals in the Rhizosphere of Forest Soils ............189
Franois Courchesne, Vronique Sguin, and Alain Dufresne

Chapter 10
A Technique for Quantitative Trace Element and Micronutrient Studies
in Plants .................................................................................................................207
Anders Gransson

Chapter 11
Cation Exchange on Plant Roots Involving Aluminium: Experimental
Data and Modeling ................................................................................................227
Joseph E. Dufey, Jos G. Genon, Benot Jaillard, Henri Calba, Gervais Rufyikiri,
and Bruno Delvaux

Chapter 12
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry in Acid
Forest Soils ............................................................................................................253
Heino W. F. Nietfeld

Index......................................................................................................................309
Section I
Phytoavailability of Trace
Elements and Radionuclides in
the Rhizosphere and Application
to Phytoremediation
1 Dependency of
Phytoavailability of
Metals on Indigenous
and Induced
Rhizosphere Processes:
A Review
Enzo Lombi, Walter W. Wenzel,
George R. Gobran, and Domy C. Adriano

CONTENTS

I. Introduction.......................................................................................................4
II. PlantSoilMicrobial Interactions: The Rhizosphere ......................................5
III. Phytoremediation ..............................................................................................5
IV. Biogeochemistry of the Metals in the Rhizosphere.........................................7
A. Rhizosphere Effect on pH ....................................................................7
B. Mineralogy and Soil Composition in the Rhizosphere .......................8
C. Root Growth and Effects on Soil Physical Parameters .......................8
D. Root Exudation and Metal Mobilization in the Rhizosphere..............9
E. Precipitation Phenomena in the Rhizosphere ....................................12
F. RootMicroorganisms Interactions in the Rhizosphere.....................13
G. Volatilization in the Rhizosphere .......................................................13
V. Microtools and Special Setup to Study RhizosphereContaminant
Interactions......................................................................................................14
VI. Future Research ..............................................................................................15
VII. Summary and Conclusions.............................................................................16
Acknowledgments....................................................................................................17
References................................................................................................................17

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 3
4 Trace Elements in the Rhizosphere

I. INTRODUCTION
Several metals and metalloids such as Mn, Cu, Zn, Ni, Mo, and B play a dualistic
role in the soil-plant system. They are essential for plant growth but when present
at high concentrations, can result phytotoxic. Their bioavailability is therefore of
fundamental importance. In case of deficiency, an increase in metal bioavailability
can be beneficial for plant growth. On the other hand, in case of pollution, a large
available amount of these elements and other similar nonessential elements (such
as Pb, Cr, As, etc.) can be detrimental to plant growth. Cleanup technologies are
often limited in their efficiency by the mobility of the pollutants. This chapter
will review the rhizosphere aspects related to availability of metals and their role
in plant nutrition and emerging remediation technologies (in particular phytore-
mediation).
At the beginning of this century, several works by Arnon and Stout1,2 and other
authors demonstrated that plants need a number of micronutrients at very low
concentration. One of the first examples of increased plant production as a conse-
quence of micronutrient application is reported by Anderson3 who observed a tenfold
increase in clover production after addition of Mo to an ironstone soil in Australia.
Since then the discovery of the essentiality of other micronutrients has given an
explanation to low plant productions in many areas all around the world. The
bioavailability of micronutrients in the rhizosphere is a key factor controlling plant
nutritional status and therefore plant production.
Soil contamination by heavy metals and metalloids is a phenomenon that has
accompanied human kind throughout its history.4 Examples of contaminated sites
due to past mining activities are common throughout the world. The number of
contaminated sites already identified represents a serious problem and the list of
sites to be treated is most likely to increase along with their remediation costs.5
Soil pollution by trace elements represents a large proportion of the problem.
For instance, trace elements account for 65% of the contaminated Superfund sites
for which the U.S. EPA has signed Records of Decisions.6 The most common
inorganic contaminant is Pb (47% of sites) followed by As (41%), Cr (37%), Cd
(32%), Ni (29%), and Zn (29%).6
The need to treat contaminated soils has created a large market for remediation.
Many technologies have been developed to remediate contaminated soils.4,7-9 The
EPAs Vendor Information System for Innovative Treatment Technologies (VISITT)
lists 66 technologies to treat metal-contaminated soils. Only 9 of the 66 technologies
listed are in situ soil or groundwater treatments.6
Recently, phytoremediation has been thought to provide an alternative, environ-
mentally friendly remediation technology for the treatment of polluted soils. Phy-
toremediation refers to the use of higher plants to remove pollutants from the soil
or to reduce the hazard associated with their presence in the environment.10-12 The
market for phytoremediation in the U.S. is between $1 and 2 million. Glass13
estimated that this market might grow to $4080 million by 2005.
The interactions between soil, metals, and the root system represent key factors
controlling the success of plant production (in case of micronutrient deficiency) and
phytoremediation. Therefore, focal importance can be attributed to the part of the
Dependency of Phytoavailability of Metals 5

soil where these interactions take place: the rhizosphere. Due to the presence of the
root system, the characteristics of the rhizospheric soil differ largely from the bulk
soil.14,15 As a consequence, the biogeochemical processes in the rhizosphere will
play an important role in regulating the mobility and bioavailability of the metals
and, in turn, on its uptake by plants, volatilization, or plant-induced precipitation.
This chapter reviews the role of the rhizosphere in metal uptake, tolerance, and
volatilization. Emphasis is given to the biogeochemical processes in the rhizosphere
and their role in phytoremediation technology and plant nutrition.

II. PLANTSOILMICROBIAL INTERACTIONS: THE RHIZOSPHERE


Since the first definition of the rhizosphere given by Hiltner in 1904,16 our under-
standing of this part of the soil has significantly improved. The rhizosphere is
spatially defined as the few millimeters of soil surrounding the plant roots and
influenced by their activity. The boundary between the rhizosphere and bulk soil is
therefore difficult to individuate. Soil characteristics (texture, density, etc.), proper-
ties of the root system, nutritional status of plants, and climatic condition (humidity,
temperature, etc.) can be considered as factors able to influence the dimension of
the rhizosphere. Moreover, practical difficulties exist in sampling rhizospheric soils.
In fact, most of the methods are based on empirical approaches that may lead to
marked differences between sampling.
The rhizosphere can be functionally defined as a highly dynamic, solar/plant-
driven micro-environment that is characterized by feedback loops of interactions
between root processes, soil characteristics, and the dynamics of the associated
microbial population.17 A graphic representation of the plant-soil-microbial interac-
tions at the rhizosphere level is shown in Figure 1.1.
Soil characteristics such as pH and redox potential, nutrient status, the presence
of contaminants, and physical properties all influence root growth and the dynamics
of microbial populations. On the other hand, soil characteristics are modified by
plants by means of root exudation, uptake of nutrients and contaminants, and root
growth. The energy that drives these changes is provided by sunlight through pho-
tosynthesis. A key component of this system is represented by root exudates. It has
been calculated that between 10 and 40% of the total net C assimilated by crops is
released in the form of soluble root exudates, and insoluble materials such as cell
walls and mucilage.18-24 The microbial population of the soil is influenced in its
dimension, composition, and diversity by both soil characteristics and plant activities
(in particular release of organic C) in a feedback cycle of interactions.
In conclusion, the resulting picture of the rhizosphere shows a very dynamic
microenvironment governed by iterative reaction between its major components:
soil, plant, and microorganisms.

III. PHYTOREMEDIATION
In recent years environmental legislation has dealt with the problem of contaminated
or polluted soil and its remediation. The need for soil remediation depends primarily
6 Trace Elements in the Rhizosphere

FIGURE 1.1 Plantsoilmicrobial interactions in the rhizosphere.

on the nature of the contaminant (toxicology and ecotoxicology) and the land use.
Moreover, public perception, social costs, and political considerations play decisive
roles in the decision-making process of soil remediation.4 Traditional solutions such
as landfill of contaminated soils, that at the moment cover a large part of the
remediation market, will probably loose their economical and public acceptance.
New technologies based on environmentally friendly and low cost processes are
urgently required.
Phytoremediation refers to the use of higher plants to remove soil pollutants or
to diminish the risk associated with their presence in the environment.11,12,25-27 This
technology meets several of the desirable characteristics of an ideal remediation
process: phytoremediation reduces the hazard associated with the presence of pol-
lutants in the environment, improves soil quality and restores its functionality, and
is a low-input technology. Moreover, its public acceptance is expected to be great
and the cost should be highly competitive ($25100 t1).13 On the other hand, several
years are needed to clean up a contaminated site with the plant material available
at the moment. McGrath et al.28 calculated that it would take 9 years to reduce Zn
concentration in soil from 440 g g 1 to 300 g g 1 using Thlaspi caerulescens.
Phytoremediation can be divided into containment and removal technologies.
Containment processes such as phytoimmobilization and phytostabilization aim to
reduce the mobility, bioavailability, and/or toxicity of the pollutant in the environ-
ment. Removal processes (phytoextraction and phytovolatilization) are technologies
for cleaning up the soil.
Dependency of Phytoavailability of Metals 7

Phytoimmobilization refers to the ability of certain plants to reduce pollutant


mobility in the soil through, for example, enhancement of its precipitation in the
rhizosphere. This is a modified view of the definition of phytostabilization as
proposed by Raskin et al.27 and Cunningham and Ow.29 Phytostabilization, as
defined here, uses pollutant-tolerant plants to mechanically stabilize polluted soils
and to prevent bulk erosion and airborne transport to other environments. In addi-
tion, leachability of pollutants may be reduced due to higher evapotranspiration
rates relative to bare soils. This technology is often associated with soil amendment
treatments (in situ fixation and revegetation). Phytovolatilization processes involve
specialized enzymes that can transform and volatilize contaminants in the
plantmicrobesoil system.30,31 Phytoextraction processes extract both metallic and
organic constituents from soil by direct uptake into plants and translocation to
aboveground biomass.32 Salt et al.25 have divided phytoextraction into induced and
continuous phytoextraction. Induced phytoextraction refers to the use of chemical
compounds (in particular chelating agents) to enhance metal accumulation in high
biomass plants. This process can also be defined as chemically induced hyperac-
cumulation. Continuous phytoextraction uses hyperaccumulator plants to clean up
the soil.

IV. BIOGEOCHEMISTRY OF THE METALS IN THE RHIZOSPHERE


Heavy metals and metalloids present in the soil have different origins. These ele-
ments are generally ubiquitous in nature, but they may concentrate in the soil due
to anthropogenic and geogenic processes. Among the geogenic processes, the weath-
ering of metal-rich parent material is responsible for large concentrations of metals
such as those in calamine or serpentine soils. Other natural inputs are represented
by volcanic activities, wood/plant burning, and marine aerosols. With the only
exception of naturally mineralized soils, human activities are the primary source for
soil pollution. Anthropogenic sources of metal and metalloids such as fuel combus-
tion, waste disposal and incineration, agricultural, and industrial and mining activ-
ities have greatly accelerated the natural fluxes of trace elements.
In the soil, trace elements undergo a series of reactions that can increase or
decrease their mobility, availability, and toxicity. These reactions are influenced by
root activities (i.e., release of exudates, changes in pH, etc.) and may proceed
differently in the bulk soil in comparison with the rhizosphere. The root activities
and their influences on trace element mobility, plant nutrition, and phytoremediation
processes are reviewed in the following sections.

A. RHIZOSPHERE EFFECT ON PH

Adsorption mechanisms on soil particles are primarily controlled by pH and the


type of minerals present. Clay minerals have permanent negative charges due to
isomorphic substitution of Al and Fe and pH-dependent charges at the edge of the
crystals. Similarly, pH-dependent charges are associated with other minerals such
as oxy/hydroxides, carbonates, and organic matter. Root activities are one of the
causes of soil pH heterogeneity.33 The pH in the rhizosphere may be very different
8 Trace Elements in the Rhizosphere

from the pH of the bulk soil.34 The principal reason for these differences is thought
to be the unequal uptake of cations and anions by plant roots.35,36 A dominant factor
in this process is the form of N that is taken up by plant roots: when N is absorbed
in the form of NH4+ the plants release H+ to maintain electrical neutrality and
therefore the pH in the rhizosphere decreases. On the contrary, when NO3 is taken
up, a net efflux of HCO3 by the roots will cause the pH to increase.34,37,38 Smiley39
reported differences in the rhizosphere pH of wheat up to 2.2 units when different
sources of N where used. The large difference that may exist between rhizosphere
and bulk soil pH can cause processes of adsorption or desorption, precipitation, or
solubilization of trace elements to occur. Heavy metals become more mobile in
acidic conditions whereas the mobility of metalloids follows an opposite trend.

B. MINERALOGY AND SOIL COMPOSITION IN THE RHIZOSPHERE


Other soil characteristics involved in trace element adsorption such as soil mineral
composition and soil organic matter can differ between bulk and rhizosphere soil.
Transformation of minerals in the rhizosphere has been demonstrated by Hinsingers
group,40-42 who reported rapid vermiculization of phlogopite in the rhizosphere of
Lolium multiflorum and Brassica napus. Courchesne and Gobran43 found a signifi-
cantly lower amount of amphiboles and interlayered minerals in the rhizosphere of
Norway spruce compared to the bulk soil. These processes can be related to enhanced
weathering due to the release of H+ and organic compounds in the rhizosphere.44-46
The organic matter contents in the rhizosphere can differ from that of the bulk soil.
Plant roots release a considerable amount of organic materials, including water
soluble exudates (sugars, organic acids, and amino acids) and water insoluble mate-
rials such as cell walls, sloughed materials, and mucilage into the soil.47 Many of
these materials are readily degraded by soil microorganisms, but their continuous
release maintains the organic matter concentration at higher levels than in bulk soil.
Moreover, degradation of organic compounds in natural condition could be retarded
by the existence of a spatial separation between sites of exudate release and microbial
activity at the rhizoplane and in the rhizosphere.48

C. ROOT GROWTH AND EFFECTS ON SOIL PHYSICAL PARAMETERS


Root development of the Cd and Zn hyperaccumulator Thlaspi caerulescens is
influenced by the presence of Zn. Schwartz et al.49 have demonstrated that there is
a preferential growth of the roots in Zn-rich patches. This behavior is similar to the
positive response in terms of root branching as a response to macronutrients.50
Plant roots stabilize the soil, mechanically binding together soil particles. Root
exudates, such as mucigel and sugars, increase the soil structure and its stability.
Finally, mycorrhizal fungi associated with the roots of certain plants can improve
soil stability and structure.51,52 All these processes are effective in reducing soil
erosion and preventing the resuspension of pollutants from contaminated soils to
the adjacent areas. All these mechanisms, related to the root system and the rhizo-
sphere, are key factors in phytostabilization technology. The application of soil
amendments to contaminated land in order to reduce metal toxicity represents a
Dependency of Phytoavailability of Metals 9

FIGURE 1.2 Main factors controlling metal uptake.

complementary tool able to enhance plant growth and therefore improve phytosta-
bilization (for review see Vangronsveld et al.).53

D. ROOT EXUDATION AND METAL MOBILIZATION IN THE


RHIZOSPHERE
Bioavailability of metals in soil is a major factor that regulates metal uptake by
plants. The availability of metals to plants is controlled by the chemical fate of the
element considered, rhizosphere soil characteristics, and specific characteristics of
the plant at the level of species, cultivars, or populations. The main factors controlling
metal uptake are reported in Figure 1.2.
Among all these factors, the plants can play a decisive role in controlling metal
bioavailability through specific activity causing metal mobilization such as the
release of organic acids and phytosiderophores.
Plants growing in conditions where Fe and/or Zn are deficient can actively
increase the availability of these metals, releasing specific organic compounds.
Different strategies have been identified. Strategy I is active in dicotyledonous and
monocotyledonous species with the exception of graminaceous species. Strategy I
is based on a three-component system constituting a plasma membrane-bound induc-
ible reductase, an enhanced excretion of protons, and the release of reducing and
chelating agents.54 Strategy II, active in Graminaceous plants, is based on the release
of phytosiderophores in the rhizosphere and specific uptake systems on the root
10 Trace Elements in the Rhizosphere

FIGURE 1.3 Models of Strategy I and II plants: P.M. = plasma membrane; R = inducible
reductase; T1 = transporter for Fe2+; St = stimulated H+ pump for ATPase; X = increased
production/release of chelators/reductants; E = enhanced synthesis and secretion of phytosi-
derophores; PS = phytosiderophores; T2 = translocator for Fe3+ phytosiderophores in the
plasma membrane. (Modified from Marschner, H. et al.140 and Rmheld, V.141 With permis-
sion.)

surface. The chemical nature and the rate of release of phytosiderophores differ
among plant species and even cultivars.46,54-56 Phytosiderophores form chelates not
only with Fe but also with Zn, Cu, and Mn and are therefore able to mobilize other
micronutrients in case of deficiency.57,58 Uptake of ferrated phytosiderophores is
generally considered highly selective.48 More recently, Wiren et al.59 found evidence
that Zn can be taken up in grasses in the form of nondissociated Zn-phytosidero-
phores. The mechanisms of Strategies I and II, effective on Fe oxides or other soil
components may, as a side effect, mobilize metals adsorbed on minerals. Represen-
tations of Strategies I and II are given in Figure 1.3. A third strategy may be identified
in the capacity of microorganisms to release siderophores and the possibility for the
plants to take up these compounds.60 The existence of a microbial siderophore Fe
transport system has been proposed in oat61,62 and maize.63
Exudation of organic acids has been observed in P-deficient plants by Hoffland
et al.64 This mechanism, also reported by other authors,65,66 may have implications
for metal mobility. In fact, mineral weathering and dissolution of P-containing
minerals can increase the mobility of metals.
Plant uptake of trace elements can be enhanced through changes in metal avail-
ability in the rhizosphere. In particular, exudation of phytosiderophores and organic
acids, and changes in pH and redox potential are considered key factors controlling
metal mobility in the rhizosphere.
The ability of root exudates, collected from different plants, to mobilize Cd was
investigated by Mench and Martin.67 They found that root exudates of Nicotiana
tabacum were able to extract more Cd from soil than those of Nicotiana rustica and
Dependency of Phytoavailability of Metals 11

Zea mays. These results are in agreement with the fact that tobacco plants are well
known to accumulate fairly large amounts of Cd in their leaves.
Fenn and Assadian68 reported that Cynodon dactylon was able to mobilize Pb,
Cu, and Mn in the rhizosphere and accumulate them in the leaves. This process was
probably related to changes in rhizosphere pH and the dissolution of carbonates.
Other authors69-71 found that the mobility of Cd, Zn, and Cu increased in the soil
surrounding the roots due to acidification of the rhizosphere. The acidification
process was related to the form of nitrogen taken up by the plants. There is evidence
that the reduction mechanism present in Strategy I plants to mobilize Fe could also
be able to increase Cu and Mn uptake (Welch et al.).72
So far, only a few studies have been conducted to investigate the rhizosphere
of hyperaccumulator species. Redox potential and pH changes in the rhizosphere
of the Ni hyperaccumulator Alyssum murale and the nonhyperaccumulator Rapha-
nus sativus were compared by Bernal and McGrath73 and Bernal et al.74 The results
suggest that the source of N was the main factor responsible for pH changes and
the crop plants were able to reduce the system more effectively than the hyperac-
cumulator. These findings indicate that hyperaccumulation mechanisms may be
related to other rhizosphere processes such as the release of chelating agents and/or
to differences in the number and affinity of metal root transporters. McGrath et
al.75 and Knight et al.76 studied the chemical and physical characteristics of the
rhizosphere of Thlaspi caerulescens. They concluded that the decrease in the
mobile Zn fraction could explain only less than 10% of the total Zn uptake by the
plants. T. caerulescens was able to deplete the mobile fraction of Zn more than
the closely related, but nonaccumulator T. ochroleucum. No significant differences
in pH were observed in the rhizosphere of T. caerulescens. Knight et al.76 proposed
that two possible mechanisms could explain these results: either T. caerulescens
is able to mobilize Zn from the soil or the soil studied had a large capacity to
buffer the concentration of Zn in soil solution. Recently, Hamon and McLaughlin77
used an isotopic dilution method to determine pools of Cd and Zn in contaminated
soils that can be accessed and removed from soil by T. caerulescens and by wheat.
Their results show no difference in specific activity of Cd or Zn taken up by T.
caerulescens or wheat. This indicates that the hyperaccumulator plant was able to
access the same pools of metals that were available to the wheat plants. However,
it has to be pointed out that the Zn added in biosolids was highly labile, and the
population of T. caerulescens used in this experiment acts more as a Cd-tolerant
species than as a hyperaccumulator for Cd. These partly contradictory results
suggest that hyperaccumulator plants seem to take up from the same phytoavailable
metal pools that are accessible to other plants where this pool is large enough.
However, there may exist mechanisms in the rhizosphere of hyperaccumulators,
such as root exudation, that can support metal uptake from less accessible pools.
In field conditions, where metals are returned to the soil via senescence, the easily
available metal pools may typically be large enough to explain the amounts accu-
mulated in the plants.78
Currently, there is not enough information to understand whether mobilization
of metals in the rhizosphere constitutes one of the mechanisms responsible for
12 Trace Elements in the Rhizosphere

hyperaccumulation or not. The extraordinary metal uptake of hyperaccumulator


species could be simply related to a larger number of highly specific metal trans-
porters in the roots as reported for T. caerulescens by Lasat et al.79 If this is the
case, metal uptake by hyperaccumulator plants would be limited to decreasing the
fraction of metals bioavailable. This remediation strategy has been termed Bio-
available Contaminant Stripping (BCS).77 This strategy could be used to remove
larger amounts of the contaminant if its mobility is enhanced through rhizosphere
processes.
Another approach to mobilize metals in the soil is the application of chelating
agents to the soil. This process enhances metal accumulation in high biomass plants
and is defined as chemically assisted phytoextraction.25,80-82 Synthetic chelating
agents such as EDTA and HEDTA are effective in mobilizing metals in the soil and
increasing their accumulation in plants. An enhanced root production and release of
chelating agents could also increase accumulation processes in plants and therefore
enhance phytoextraction processes.12
Compared to most root exudates, EDTA seems to be fairly biostable and efficient
in chelating and mobilizing metals from soils.83 Another interesting aspect of the
utilization of soil additives is that their transport should primarily occur from the
bulk soil to the rhizosphere and finally to the rhizoplane, while chelating agents
exuded by the plant roots will move in the opposite direction. This should have
consequences for the efficiency of naturally exuded vs. added chelating agents,
depending on whether metals accumulate or are depleted close to the rhizoplane.
Independently, driven by the transpiration stream, convective transport of chelating
agents is likely to occur primarily toward the rhizoplane. Thus, this limits the extent
to which root exudates may penetrate toward the outer limit of the rhizosphere and
thus their capacity to solubilize metals at distance from the root.

E. PRECIPITATION PHENOMENA IN THE RHIZOSPHERE


In the rhizosphere, mass and diffusive transport and plant uptake mechanisms can
induce accumulation or depletion of ions. This fact, together with changes in pH
and rhizosphere conditions, can lead to precipitation of metal complexes. This
process can be positive in the case of pollution (reducing mobility and availability),
but can cause deficiency if the availability of micronutrients is drastically reduced.
Plant roots can release H+ or HCO3 in the rhizosphere to maintain their electrical
neutrality.34 Moreover, the redox potential can change in the rhizosphere as a con-
sequence of the release of reducing agents or microbial activity. Acidification or
alkalinization and changes in redox potential can lead to precipitation of heavy
metals or metalloids. Reduction in Cd uptake and bioavailability due to the use of
nitrate fertilizer has been reported by several authors.71,84 Salt et al.32 found reduction
of Cr6+ to Cr3+ in the rhizosphere of Brassica juncea. This process is very important
in the case of Cr, with its oxidized species being more toxic and mobile than Cr3+.
In wetland environments, Doyle and Otte85 found accumulation of As and Zn in the
rhizosphere of aquatic plants. This accumulation was related to the formation of Fe
plaques in the oxidized soil that surrounds roots. Decomposition of root material
Dependency of Phytoavailability of Metals 13

can increase soil organic matter, which is very effective in immobilizing toxic
elements such as Cu. Fairly insoluble compounds of Zn and Pb can form in the
rhizosphere for reaction with P. Pyromorphyte [Pb5(PO4)3Cl] has been reported to
form in the rhizosphere of Agrostis capillaris.86 This mineral has a very low solubility
(Ksp = 1084.4) and is thought to maintain Pb at very low concentrations in the surface
environment.87 Cotter-Howells et al. (1999) have localized pyromorphyte granules
in the outer cell walls of the epidermis of Agrostis capillaris.88 Associations between
Cd and polyphosphate granules were found in the mycorrhized roots of Pteridium
aquilinum.89 These precipitation processes may represent active tolerance mecha-
nisms that plants use to reduce heavy metal toxicity.88

F. ROOTMICROORGANISMS INTERACTIONS IN THE RHIZOSPHERE


Rootmicroorganisms interactions in the rhizosphere play a decisive role in the
bioavailability of micronutrients. For instance, the role of Mn-oxidizing bacteria has
been known for long time: in 1946 Timonin reported a large number of these bacteria
in the rhizosphere of oat cultivars sensitive to Mn deficiency.90 It is possible that
root exudates can regulate the balance between Mn-oxidizing and Mn-reducing
bacteria. It seems that Mn-tolerant cultivars can enhance the number of Mn-reducing
bacteria in the rhizosphere.91,92
A large number of interactions between roots, microorganisms, and trace ele-
ments exist in the rhizosphere and cannot be discussed in detail in this chapter. More
information, especially on the bioavailability of heavy metals as influenced by
mycorrhiza, is reported in Chapter 8 of this text.

G. VOLATILIZATION IN THE RHIZOSPHERE


Volatilization is based on different processes, including reduction to volatile ele-
mental forms and synthesis of methylated compounds of some metals and metal-
loids.12 These processes can take place in the plant or in the rhizosphere. In the latter
case, this process can be better described as plant-assisted microbial volatilization.
Phytovolatilization may be viable to remediate soil polluted by As, Hg, and Se.
Microbial methylation of As has been known for a long time and is common
to both bacteria and fungi. Bacterial methylation seems to be favored by anaerobic
conditions and may be employed only in ex situ bioreactor systems.93 Fungal
methylation seems to be important in the volatilization of As compounds used in
agriculture.94,95 The microbial production of arsines from arsenicals in soil seems
to contribute only marginally to the loss of As. Gao and Burau96 reported a loss of
volatile arsine in the range of only 0.0010.4% of the As applied to the soil as Na
cacodylate or methanearsonic acid. Processes based on volatilization of As have not
been implemented yet as they have for Hg and Se. This, as pointed out by McGrath,97
is strange considering the similarities between the pathways of biovolatilization of
these elements. Microorganisms in the rhizosphere have access to reducing power
released by plants in the rhizosphere through root exudates. This reducing power
could be used to remove As by the production of volatile reduced compounds.97
14 Trace Elements in the Rhizosphere

Unlike methylated As and Se compounds, methylated forms of Hg are extremely


toxic. Therefore, the only acceptable volatilization of Hg is through reduction of the
ion Hg2+ to gaseous elemental Hg. Rugh et al.98 have inserted the bacterial merA
genes into Arabidopsis thaliana. The transgenic plants acquired Hg resistance that
was associated with enhanced reduction and volatilization of Hg.99 In this case, Hg
volatilization can be enhanced only by increasing the fraction of Hg that can be
taken up by plants. On the other hand, reduction of Hg to elemental form is also
carried out by soil microbes.100 Enhancement of this process in the rhizosphere may
be possible, but more information is needed.
Selenium volatilization in the plantsoilmicrobes system is the best under-
stood biovolatilization process. Selenium is volatilized mainly as dimethylselenide,
a compund that is 500 to 700 times less toxic than inorganic forms of Se.101 Many
plant species have been screened for their efficiency in accumulating and volatil-
izing Se.102-104 One of the best candidates for Se phytoremediation is Brassica
juncea.103,105 Recently, de Souza et al.106 have been able to explain several aspects
of Se accumulation and volatilization by this species. Experiments with the anti-
biotic ampicillin demonstrated that rhizosphere bacteria facilitated 35% of plant
Se volatilization and 70% of Se accumulation in plants. In a second experiment
with plants grown in axenic condition or inoculated with rhizosphere bacteria, the
authors showed that inoculation increased Se concentration in the plants and Se
volatilization by a factor of five- and fourfold, respectively. The bacteria may
facilitate the maintenance of a high Se concentration in the roots, a factor that has
been indicated as the limiting factor in the process of volatilization.107 A heat-labile
compound produced by the plantbacteria interaction is probably responsible for
enhanced Se accumulation and volatilization. This compound has not yet been
identified. It can be produced by either the plant or bacteria and may act by
stimulating selenate transporters or by converting selenite to SeMet (a compound
that is readily taken up by plants).108 Different bacterial strains showed differential
abilities to enhance Se accumulation in plants. The use of seeds coated with selected
strains of bacteria could enhance Se volatilization and thereby improve phytovol-
atilization technologies.106

V. MICROTOOLS AND SPECIAL SETUP TO STUDY


RHIZOSPHERECONTAMINANT INTERACTIONS
Rhizosphere research has been carried out in field conditions as well as in the
laboratory. In field studies the separation between rhizosphere and bulk soil has been
achieved simply by gentle shaking,109-111 or brushing of roots,110,112 and in these cases
only the adhering soil has been considered as rhizospheric. Another approach is the
rhizocylinder method,113-115 in which, besides rhizosphere and bulk soil, plant roots
and adjacent soil particles are examined as a separate fraction (rhizocylinder).116
Porous membranes have been employed in field experiments to separate roots from
soil. Through this technique, a clean and intact root system is obtained as is the
passage of soil solution.117 In another study a porous membrane was used to separate
the root system of tree crops from a soil-filled container.118 At the end of the
Dependency of Phytoavailability of Metals 15

experiment, the container was removed and the soil sliced with a piston microtome
to obtain soil samples at defined distances from the rhizoplane.
In laboratory research more complicated techniques, other than the ones
described above, have been used. Different experimental setups (i.e., rhizobox, soil-
packets system, rhizosphere study container) have employed porous membranes to
separate soil at different distances from the roots.69,70,75,119-122 Other experimental
designs have been developed to control plant nutritional conditions and rhizosphere
pH,66,123 or soil redox potential.124 Root-induced changes of pH have been studied
directly in soil using glass microelectrodes,125-127 or by employing agar with a pH
indicator as substrate.128,129 In this latter case, qualitative changes in pH can be
visualized by color changes in the indicator added, and quantitative measurements
are obtained using microelectrodes,33,130,131 spectrodensiometry,132 or a method based
on the buffer curve of the gel used.133
Root growth dynamics and morphology have been studied using rhizotrons or
minirhizotrons.134-136

VI. FUTURE RESEARCH


Rhizosphere soil is very different from bulk soil in terms of pH, redox potential,
mineral composition, and organic matter cycle and composition. These parameters
also largely control the mobility of trace elements.137 As a consequence, the bio-
geochemistry of trace elements in the rhizosphere may be very different from bulk soil.
Future research should be directed to enhance several aspects of the root and
rhizosphere systems and to improve our knowledge of basic mechanisms controlling
the behavior of trace elements in the rhizosphere. In particular, future efforts should
focus on the following aspects:

1. Root biomass, morphology, and the physiology of absorption.


2. Rootmicrobial associations.
3. Mobilization of metals through exudation of natural chelating agents by
plant roots and associated microbes.
4. Modeling and experimental assessment of pathways and degradation prod-
ucts of root exudates in the rhizosphere as compared to added synthetic
agents as a predictive tool of the efficiency of plantsoil management in
phytoremediation.
5. Immobilization of metals through precipitation in the rhizosphere.
6. A better control of the rhizosphere system can increase plant uptake of
micronutrients and therefore improve plant growth. A better understanding
and control of the rhizosphere processes can be very important in the
frame of sustainable agricultural practices and organic farming.

Phytostabilization can be enhanced by the selection of tolerant species with large


and deep root systems. Genetic engineering and the use of particular microorganisms
such as Agrobacterium rhizogenes represent other powerful tools to enhance root
development. Stomp et al.138 used a wild type of A. rhizogenes to obtain trees with
16 Trace Elements in the Rhizosphere

larger root biomass. An increased soil-binding capacity of the root system can also
be achieved through enhancement of mycorrhizal associations.
Phytoimmobilization processes in the rhizosphere are caused by changes in pH
and redox potential, and by the exudation of inorganic or organic compounds such
as phosphate or phytate. Before we proceed to modify plants to improve phytoim-
mobilization processes, the mechanisms of metal precipitation should be better
understood at the rhizosphere level.
Phytoextraction represents the most studied phytoremediation technology and
many efforts have been directed at the understanding of metal accumulation in plants.
On the other hand, information on rhizosphere processes involved in metal uptake
by hyperaccumulator species is still very poor. It has not been proven that hyperac-
cumulator plants have a need to,78 and would be able to mobilize metals in the
rhizosphere; recent studies rather seem to indicate the opposite.77 However, these
plants have a very high requirement for necessary metals such as Zn and it cannot
be excluded that they have evolved mechanisms to mobilize metals in the rhizo-
sphere. In any case, an improved understanding of these mechanisms will be useful
in both phytoremediation technologies and plant nutrition research. Still the complex
interaction between plant and microorganisms and their potential role in metal
mobilization and plant uptake is largely unknown. The understanding of plantbac-
teria interactions to enhance metal uptake, such as the one recently reported for
Se,106 can represent a major step toward enhancing phytoextraction technologies.
Phytovolatilization of Se has been studied extensively and the increased under-
standing of basic mechanisms in the rhizosphere will, most likely, largely improve
this technology. Further information is required to enhance phytovolatilization of
other elements such as Hg and As.

VII. SUMMARY AND CONCLUSIONS


The role and importance of the rhizosphere for phytoremediation has been fully
recognized in the case of organic pollutants. It is generally accepted that biodegra-
dation of organic compounds is a plant-assisted process that takes place primarily
in the rhizosphere.139 At present, the possibility of enhancing phytoremediation of
soils polluted by trace elements through stimulation of rhizosphere processes is not
completely exploited. Similarly, the processes that take place in the rhizosphere can
be important to enhance uptake of micronutrients by plants. The exploitation of these
natural processes can be economical, convenient, sustainable, and acceptable in
organic farming practices.
Recent results show that plantsoilmicroorganism interactions play a key role
in phytoimmobilization and phytovolatilization processes. The metal fate in the
rhizosphere of hyperaccumulator plants is still largely unknown. So far, only a few
studies have been aimed at the comparison of metal dynamics in the soilplant
interface of hyperaccumulator vs. nonaccumulator plants.
More research is required to increase our understanding of basic rhizosphere
processes involving trace elements (both pollutants and micronutrients). Owing to
the complexity of the rhizosphere, a multidisciplinary approach is required using
tools specifically designed to study this microenvironment. Experimental approaches
Dependency of Phytoavailability of Metals 17

should be combined with modeling exercises, involving theoretical modelers and


scientists from various disciplines assessing model parameters and validating model
outputs in experiments and finally in the field.

ACKNOWLEDGMENTS
This study was supported by the University of Agricultural Sciences Vienna, Priority
Research Area Project No. 16 (Rhizosphere processes: Modeling and experimental
assessment of metal interactions with organic ligands exuded by plant roots); the
Fonds zur Frderung der Wissenschaftlichen Forschung (FWF, Project No. P13454-
BIO); and the Austrian Federal Ministry of Science and Transport. It also is part of
an ongoing project that is financed by the Swedish Council for Forestry and Agri-
cultural Research (SFJR No. 23 .0173/98).

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Technology, American Chemical Society, Washington, D.C.
140. Marschner H., Rmheld V., and Kissel M. (1986), Different strategies in higher plants
in mobilization and uptake of iron, J. Plant Nutr., 9, 695713.
141. Rmheld V. (1987), Existence of two different strategies in the acquisition of iron in
higher plants, in S.V. Winkelmann, ed., Iron Transport in Microbes, Plant and Ani-
mals, Verlag Weinheim, GE, 353374.
2 Bioavailability of Trace
Elements as Related to
Root-Induced Chemical
Changes in the
Rhizosphere
Philippe Hinsinger

CONTENTS

I. Introduction.....................................................................................................25
II. Bioavailability of Trace Elements as Related to Root-Induced
Accumulation/Depletion of Ionic Species in the Rhizosphere......................26
III. Bioavailability of Trace Elements as Related to Root-Induced
Acidification/Alkalinization of the Rhizosphere............................................30
IV. Bioavailability of Trace Elements as Related to Root-Induced
Oxidation/Reduction in the Rhizosphere .......................................................32
V. Bioavailability of Trace Elements as Related to Root-Induced
Complexation/Chelation in the Rhizosphere .................................................33
VI. Conclusions.....................................................................................................36
References................................................................................................................37

I. INTRODUCTION
Predicting the bioavailability of trace elements to plants is a major agricultural and
environmental issue. Indeed, plants are the prime entry point for trace elements in
the food chain. On the one hand, for trace elements that are essential micronutrients
for plants, animals, and human beings, the need arises for ensuring adequate levels
in agricultural products. Indeed, deficiencies of micronutrients are fairly widespread
in various types of soils worldwide. On the other hand, as a consequence of human
activities, concentrations of potentially toxic trace elements tend to build up in some
environments and ultimately cause contamination of the food chain. Similarly as
described for major nutrients by researchers such as Clarkson1 and Hinsinger,2 a
range of factors and mechanisms are implied in the transfer of trace elements from

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 25
26 Trace Elements in the Rhizosphere

the soil into plant roots.3 In addition to the uptake per se, the acquisition of trace
elements encompasses those chemical processes that occur at the soilroot interface
and that can influence the dynamics of trace elements in the so-called rhizosphere.1,2
Understanding all the mechanisms implied in the process of acquisition of trace
elements by plant roots is thus a prerequisite for assessing their bioavailability.
The rhizosphere effect was first defined by Hiltner4 to account for the increased
microbial biomass and activity that occurs in the immediate vicinity of plant roots.
This phenomenon is largely due to the large flux of C originating from root exudation.
Root exudates include a wide spectrum of organic substances. Some are very reactive
compounds that can directly affect the chemical properties of the rhizosphere and,
in particular, those properties that determine the speciation and ultimately the bio-
availability of trace elements. In addition to root exudation, research conducted in
the past decades has provided ample evidence that plant roots can drastically alter
the physical,5 and chemical properties of the rhizosphere through several other
processes.2, 3, 6-8 These processes have been reported as major steps in the process of
acquisition by plants of major nutrients such as N, P, and K in particular, and of
micronutrients such as Fe and Zn, among others. They can also have a major
influence on the dynamics of trace elements in the soil surrounding roots and are
likely to ultimately determine the actual rates of contamination of plant tissues by
trace elements that are considered as micropollutants.
The aim of this chapter is to give an overview of the possible chemical processes
involved in the rhizosphere that can affect the speciation of trace elements in the
soil solid phase and their bioavailability to plants (i.e., phytoavailability). Although
rhizosphere microorganisms are known to potentially play a role in that respect as
reviewed for mycorrhizal fungi, for instance,9,10 this chapter only addresses the
possible direct effects of plant roots.

II. BIOAVAILABILITY OF TRACE ELEMENTS AS RELATED


TO ROOT-INDUCED ACCUMULATION/DEPLETION OF IONIC
SPECIES IN THE RHIZOSPHERE
Plant roots draw water from the surrounding soil, which results in a convective
transfer of solutes toward the roots. Therefore, depending on how this flux of solutes
matches with its uptake by the roots from the soil solution, solutes will either
accumulate or be depleted in the rhizosphere.2,11-13 Ions that are present in large
concentrations, which is common for Ca and Mg in many soil environments, tend
to accumulate in the rhizosphere.11 Lorenz et al.14 showed, for instance, that the
amount of Ca and Mg transferred toward the roots by mass-flow amounted to three-
to sixfold the actual rates of uptake of these nutrients by radish roots, depending on
the method used for measuring soil solution concentrations (Table 2.1). The con-
centration of Ca and Mg would then build up in the vicinity of the roots, as shown
by Youssef and Chino.15
Conversely, for those elements that occur at low concentrations in the soil
solution, mass-flow will represent only a portion of the actual flux taken up by plant
roots. This is typically the case for P and K among major nutrients,2 and of trace
Bioavailability of Trace Elements 27

TABLE 2.1
Ratio (%) of Mass-Flow to Actual Uptake into
Leaves and Tubers of Radish (Raphanus sativa L.)
for Major Cations and Two Trace Metals in a Sandy
Loam (with Total Cd and Zn Concentrations of
2.8 and 152.5 g g1, respectively).a
Ratio (%) Ca Mg K Zn Cd

Displacement 679 579 22 39 42


Centrifugation 380 334 43 63 58
a The mass-flow was deduced from the measurements of the water
transpiration and absorption rates and the concentrations of ele-
ments in bulk soil solution, as obtained by a displacement method
or by centrifugation. The ratios are expressed as a percentage (%).

Source: Adapted from Lorenz, S. E. et al.14 With permission.

elements such as Zn and Cd, (Table 2.1). As a result of this, these elements are
expected to be depleted from the rhizosphere, as reviewed for nutrients by Jungk12
and Hinsinger.2 However, little experimental evidence exists for such a depletion
of trace elements in the rhizosphere, possibly because of the technical difficulty of
accessing measurable, reliable concentrations of trace elements in the soil solution
at this scale. Calculations have alternatively provided a means to support this
hypothesis, as shown for instance for Mn.16 This work reported good agreement
between predicted and observed Mn uptake by wheat, maize, and sugar beet, which
suggests that the calculated values of Mn depletion in the rhizosphere were realistic.
According to this, Mn concentration decreased from 0.20 M in the bulk soil to
0.16, 0.13, and 0.11 M in the rhizosphere of wheat, maize, and sugar beet,
respectively.
Whenever a decrease in the concentration of a trace element occurs, the direct
consequence will be a diffusion of the trace element toward the root along the created
gradient. According to Le Chateliers principle (mass action law), a further conse-
quence will be the shift in the various reaction equilibria involving this trace element,
in order to replenish the soil solution in the rhizosphere. For those trace elements
that are present in the soil as exchangeable cations such as Zn and Cd, their depletion
from the rhizosphere (see Table 2.1) is likely to result in a shift in the adsorption/des-
orption (cation exchange) equilibrium toward an enhanced desorption (toward the
right in Equation (1) that stands for the divalent metal M):

exchanger-M + Ca2+ exchanger-Ca + M2+ (1)

For a range of species grown on a soil spiked with 65Zn and 109Cd, similar specific
activities of Zn were found in the shoots by Hamon et al.,17 demonstrating that none
of the studied species had access to nonisotopically exchangeable pools of soil Zn.
28 Trace Elements in the Rhizosphere

In comparison, a lower specific activity of Cd was found in all species relative to


the oilseed rape, indicating that those other species had access to a nonexchangeable
pool of soil Cd. This suggests that the mobilization of Zn by plants is largely relying
on simple cation exchange equilibria as described in Equation (1). In this case,
isotopically exchangeable Zn can thus be seen as a powerful indicator for predicting
the phytoavailability of soil Zn.17, 18 McGrath et al.19 found for three soils contami-
nated by a Zn smelter or sewage sludge application that NH4NO3-extractable Zn,
i.e., essentially exchangeable Zn, was significantly depleted in the rhizosphere of
hyperaccumulator plants of the Thlaspi genus. They found, however, that more than
90% of the Zn taken up by the hyperaccumulator plants was originating from the
nonexchangeable pool of soil Zn. Other processes than cation exchange were thus
likely to be implied in the mobilization of Zn from the rhizosphere of those plants.
In addition, according to the exchange equilibrium described in Equation (1),
the concomitant increase in divalent, competing cations such as Ca and Mg in the
rhizosphere (as described above) would be expected to shift this equilibrium further
toward an enhanced desorption of the metal [toward the right in Equation (1)].
However, the increase in Ca and Mg ions in the soil solution around roots may
conversely decrease the uptake of trace elements such as divalent metals because of
increased competition for the uptake across the plasmalemma. Indeed, to date it is
acknowledged that specific uptake systems for each of these metals are unlikely to
exist. Divalent metals are thus likely to compete with each other and with alkaline
earth cations such as Mg at this level, as demonstrated in yeast for trace metals such
as Mn, Zn, and Co.20
Similarly, competition between cations has been shown to take place for alkaline
metals such as K (major cation) and 137Cs (radioactive, trace cation). Smolders et
al.21 have clearly shown that less contamination of plants by 137Cs occurred with
increasing K concentrations in the nutrient solution. A possible direct application
of such results in the field might be to use large rates of K fertilizers in 137Cs-
contaminated sites, as a possible remediation technique for agricultural land.21 How-
ever, the peculiar conditions of the rhizosphere also need to be taken into account
in that respect. Potassium concentrations can indeed be much lower than in the bulk
soil as a consequence of the high demand of the plant for K compared with its
concentration in the soil.22-24 The difference can reach up to several orders of mag-
nitude, shifting from several hundreds of M in the bulk soil solution down to 2 to
3 M in the vicinity of maize roots.22 Such a depletion of K in the rhizosphere can
then enhance the uptake of 137Cs because of the reduced competition for the uptake
at the soil solution root interface. Smolders et al.21 have indeed shown that the
measured contamination of wheat by 137Cs was much better predicted when taking
into account the rhizosphere K concentration than when based on K concentration
in the bulk soil.
The depletion of K in the rhizosphere has been shown to be responsible for the
release of nonexchangeable K in soils,24, 25 and of interlayer K in micaceous miner-
als.23 In a similar manner to K (or Rb and ammonium), 137Cs is known to have a
high affinity for those interlayer sites.26 Evidence suggests that 137Cs is preferentially
sorbed on the frayed edge sites that correspond to the terminal interlayer sites in
micaceous clay minerals27 or to the wedge sites in mica-derived vermiculites.28 It is
Bioavailability of Trace Elements 29

FIGURE 2.1 Values of the Cs adsorption distribution coefficient (Kd) of each rhizosphere
soil relative to that of the bulk (uncropped) soil as a function of the final solution Cs
concentration for a sandy (Soil S) and a clayey (Soil C) soil. The ratios of Kd values obtained
for trace concentrations of Cs are plotted against the geometric average of the Cs solution
concentrations in the rhizosphere and bulk soils. (Adapted from Guivarch, A. et al.31 With
permission.)

thus possible that the root-induced decrease of solution K concentration, which is


a critical factor governing the expansion of those clay minerals and concomitant
release of interlayer cations,29 will ultimately lead to the release of 137Cs from those
sites in the same way as occurs for interlayer K.23 The depletion of ammonium in
the rhizosphere, as shown by Scherer and Ahrens30 might further induce such a
release of 137Cs in the soil solution. Guivarch et al.31 showed that the Kd (distribution
coefficient, that is, the ratio of concentration of Cs in the adsorbed and solution
phases) of Cs adsorption increased in the rhizosphere compared with the bulk
(nonrhizosphere) soil, the biggest effect being obtained for trace concentrations of
Cs (Figure 2.1). This experiment was conducted when adding Cs to an uncontami-
nated soil. A possible explanation proposed by the authors was that the release of
nonexchangeable K (and possibly ammonium, too) induced by plant roots as a
consequence of the uptake of K (and ammonium) resulted in an increase in the
number of high affinity sites available for Cs adsorption in the rhizosphere. Further
discussion on how rhizosphere processes can affect the bioavailability of 137Cs can
be found in Chapter 4 of this volume.32
In addition to the depletion of trace elements in the rhizosphere, which has itself
a direct impact on adsorption/desorption and precipitation/dissolution equilibria, the
accumulation and depletion of major elements are also likely to interfere with the
30 Trace Elements in the Rhizosphere

dynamics of trace elements and their bioavailability to plants. These competing


effects, which would need to be better accounted for in the future, will also take
place in reactions involving chelating or complexing agents (see below). Also note
that a given element can either be depleted or accumulated, depending on how the
mass-flow will match with the plants demand, which may vary with the environ-
mental conditions and the plant species. For instance, Clegg and Gobran13 showed
for forested sites that K (and P) concentrations could increase in the rhizosphere
soil of forest trees, especially when their growth is poor (that is, when the demand
for nutrients is restricted, because of drought, for example). For trace metals as well,
rhizosphere depletion does not systematically occur. In a similar manner as some-
times occurs for major nutrients such as K and P,13 accumulation can also take place
for trace elements, whenever the plant restricts its uptake and/or increases its mobil-
ity (through the range of processes listed below). This is exemplified in this book
for Mn, Pb, Zn, and Cu, reported to accumulate in the rhizosphere of perennial, wild
plants growing in calcareous soils contaminated by smelter emissions.33

III. BIOAVAILABILITY OF TRACE ELEMENTS AS RELATED


TO ROOT-INDUCED ACIDIFICATION/ALKALINIZATION
OF THE RHIZOSPHERE
Rhizosphere acidification can take place as a direct consequence of root respiration
and the resulting buildup of pCO2, especially in compacted soil conditions.34,35 It is
often considered that the exudation of so-called organic acids by plant roots can
also contribute some proportion of rhizosphere acidification. However, as pointed
out by several authors who considered the pK values of common organic acids found
in root exudates, these compounds are released as organic anions (conjugate base)
and could not therefore be considered responsible per se for an acidification of the
rhizosphere.35-37 More generally, pH changes in the rhizosphere take place notably
as a result of the differential rates of uptake of cations and anions by plant roots.35,36
To compensate for an excess of positive charges when larger net influxes of cations
than anions are occurring, the roots release protons.36 They release hydroxyl or
bicarbonate ions in the reverse case.36 These processes result in severe pH changes
in the rhizosphere,38 which are directly involved in the dissolution of minerals,2 such
as silicates,39 carbonates,40 and phosphates in the rhizosphere.41 Some of these
minerals, such as carbonates and phosphates in particular, are known for their
potential role in the immobilization of trace elements, such as metals in the soil.42,43
Therefore, if plants can induce the release of Ca from Ca-carbonates and -phosphates
as related to the release of protons by their roots (as demonstrated by Bertrand et
al.40 and Hinsinger and Gilkes),41 such a process is also likely to induce a release
of accompanying trace metals. For instance, Hinsinger and Gilkes41 showed an
increase in Ca and P concentrations in the rhizosphere of ryegrass and subclover as
a consequence of proton release and subsequent dissolution of an apatite-type phos-
phate rock. Phosphorus fertilizers are known as a major source of input of Cd in
agricultural soils due to frequent substitutions of Cd that occur in phosphate rocks
used for manufacturing P fertilizers.44 Although Cd was not measured in the previous
Bioavailability of Trace Elements 31

work, it is likely that an increase in Cd concentration occurred concomitantly in the


rhizosphere, as a consequence of its root-induced acidification.
More generally, pH is a critical factor in the dissolution of most soil minerals,45
especially metal carbonates and metal oxides. This is illustrated by the following
equation that stands for goethite, one of the most ubiquitous Fe oxides:

FeOOH + 3 H+ Fe3+ + 2 H2O (2)

Considering this simple equation, proton release by plant roots is thus expected
to result in an enhanced dissolution of such minerals, in application of Le Chateliers
principle. It is noteworthy that most plant species (all but grasses) have been
described as Strategy I plants that respond to Fe deficiency by an increased acidifying
and reducing capacity of their roots.46 Bertrand and Hinsinger47 indeed showed that
goethite could be dissolved significantly in the rhizosphere of Strategy I plants such
as pea, white lupin, and oilseed rape. Their results, however, showed that proton
excretion by plant roots could not account on its own for the measured dissolution,
suggesting that it operated in conjunction with other rhizosphere processes such as
reduction and possibly complexation, too (see below). Many metals occur in trace
amounts in Fe oxides and will thus be released similarly as Fe in Equation (2). In
addition, the process described above for Fe will also apply similarly for other metal
oxides, Mn oxides in particular.48 The following example addresses the case of a
sewage sludge for which a substantial proportion of the Zn was likely to occur as
Zn carbonate (smithsonite).49 This sludge was mixed with quartz sand, with or
without addition of calcite, and cropped with two plant species in a cropping device
designed by Guivarch et al.31 This device enabled them to measure the total amounts
of metal taken up by the plants and to easily collect the rhizosphere material for its
chemical analysis.50 Pecqueux et al.50 showed that the uptake of Zn was by far the
biggest for rape when no calcite was added to the substrate, that is to say in the sole
treatment where a significant decrease in rhizosphere pH was recorded (Table 2.2).

TABLE 2.2
Amounts of Zn Taken up by Plants from a Sewage Sludge and
Changes in Rhizosphere pHa
pH g per pot)
Zn uptake (
Treatment Initial Ryegrass Rape Ryegrass Rape

CaCO3 6.28 0.17 6.25 0.18 6.00 0.20* 8.6 04 24.1 2.7
+CaCO3 6.81 0.08 6.81 0.19 6.88 0.14 6.8 0.9 11.5 1.5
a The sludge, which contained 2.28 g Zn kg1, was mixed with pure quartz sand at a rate

of 20 g sludge kg1. In the +CaCO3 treatment, calcite was added at a rate of 50 g kg1.
The * indicates a significant (at p < 0.05) pH change in the rhizosphere relative to the
initial pH of the substrate.

Source: Data from Pecqueux, H. et al.50


32 Trace Elements in the Rhizosphere

In comparison, the addition of calcite had little effect on the uptake of Zn by ryegrass,
which did not show any pH change in its rhizosphere. The release of protons by
rape roots and subsequent dissolution of Zn carbonate in the sludge might thus
explain the larger uptake of Zn that this species achieved.
In addition to being involved in the dissolution/precipitation equilibria of metal-
bearing minerals, pH is also considered as a key factor in sorption reactions of trace
metals with soil constituents. This is exemplified in the following equations that
describe the adsorption/desorption of a divalent metal M and concomitant deproto-
nation/protonation of goethite:51

Fe-OM+ + H+ -Fe-OH + M2+ or

Fe-O
M + 2 H+ 2 (-Fe-OH) + M2+ (3)
Fe-O

Finally, the pH is also a determinant of the speciation of metals in the soil


solution, i.e., the relative abundance of the various species of a given metal that
result from the hydrolysis and complexation reactions of the metal with the various
organic and inorganic ligands that are present.48,52 By altering soil pH in their
rhizosphere, plant species can, therefore, affect the chemistry and bioavailability of
trace elements such as metals through a wide range of chemical processes that may
be difficult to distinguish from one another using an experimental point of view.

IV. BIOAVAILABILITY OF TRACE ELEMENTS AS RELATED


TO ROOT-INDUCED OXIDATION/REDUCTION
IN THE RHIZOSPHERE
The oxidation of the rhizosphere is a well-known phenomenon that has been exten-
sively described for lowland rice among agricultural plants. In waterlogged or
submerged soils, the leakage of O2 from the roots, as a consequence of its transfer
from the shoots through the aerenchyma, provides an adequate supply of O2 for rice
root respiration. It also enables rice plants to alleviate metal toxicities that can occur
due to the ambient reducing conditions of the bulk soil and much increased solubility
of metal (Fe and Mn, in particular) oxides under such circumstances.48 This leakage
of O2 leads to a substantial reoxidation of the rhizosphere,53,54 which can ultimately
result in a precipitation of Fe and Mn oxides at the root surface or even in the root
cell walls.55 This process will thus affect the bioavailability of these metals, but also
possibly of trace metals that might coprecipitate or be adsorbed.
In comparison, reduction processes occurring in the rhizosphere are much less
documented. A decrease in the redox potential might, however, occur as a direct
consequence of the decrease in pO2 that can take place because of the consumption
of O2 by rhizosphere organisms (root and associated microbe respiration). In addi-
Bioavailability of Trace Elements 33

tion, the reduction of Fe is a major mechanism involved in the acquisition of Fe by


most plants, as pointed out previously.46,56 Strategy I species in particular have been
defined as plants exhibiting an enhanced reduction activity as a response to Fe
deficiency.46 The exudation by roots of reductant compounds such as malic or phe-
nolic (e.g., caffeic) acids has been reported,57 but Bienfait et al.58 showed that their
contribution to the reducing activity of plant roots was minor. It is now considered
that the reducing activity of plant roots takes place mostly at the root surface, as
related to the occurrence of reductase enzymes in the root cell membranes.58 The
resulting root-induced reduction of Fe is expected to shift the dissolution/precipita-
tion equilibria of Fe oxides and promote their dissolution. This should also lead to
the concomitant release of trace metals commonly associated (sorbed or included)
with these minerals in soil environments.
These redox processes are also expected to play a major role in the mobiliza-
tion/immobilization processes involving those trace elements that can occur under
various oxidation states in soil conditions, such as Mn,59,60 but also Cr, As, and Se.61
For Mn especially, various authors have provided direct, visual evidence of the
reduction of brownish Mn oxides by the roots of plants, as revealed by the root-
print-like discoloration of Mn oxide impregnated filter paper.59,62 Uren59 compared
axenically and nonaxenically grown plants and showed that little difference exists
indicating that the reduction of Mn oxide was directly induced by the reducing
activity of the roots and that the contribution of rhizosphere microflora was negli-
gible. Therefore, root-induced changes of redox conditions can influence the speci-
ation and, hence the bioavailability, of trace metals to plants and need to be studied
further in that respect.

V. BIOAVAILABILITY OF TRACE ELEMENTS AS RELATED


TO ROOT-INDUCED COMPLEXATION/CHELATION
IN THE RHIZOSPHERE
Among root exudates that are released in the rhizosphere, some compounds can
form strong complexes or chelates with a range of metals. This is particularly the
case of aliphatic and phenolic acids on the one hand, and of phytosiderophores on
the other hand. These exudates will then affect the speciation of metals in the soil
solution of the rhizosphere as shown for instance by Hamon et al.63 These authors
showed that although almost 100% of total Zn and Cd were present as free metal
ions in the soil solution at the start of their experiment, once radish had grown and
produced complexing exudates, 50 to 90% of Cd and 80 to 100% of Zn present in
the soil solution were complexed. Similarly, Merckx et al.64 showed that the pro-
portion of complexed radioactive metals, 57Co, 65Zn, and 54Mn, added to a sandy
soil increased, respectively, from 6.4, 1.9, and 0.2% under fallow to 60.6, 15.8, and
5.9% after 6 weeks of maize growth, as a consequence of root exudation. Those
complexing or chelating exudates can thus lead to an enhanced mobilization (i.e.,
bioavailability) of both micronutrients65 and undesirable trace metals. They are
characterized by very different patterns (amount and composition) among plant
species. Mench and Martin66 showed, for instance, that tobacco exudates mobilized
34 Trace Elements in the Rhizosphere

FIGURE 2.2 Amounts of trace metals extracted by the root exudates of Zea mays, Nicotiana
rustica, and Nicotiana tabacum and synthetic solutions of glucose, citric acid, and glycine
or deioinized water. Except for controls treated with water, all the exudate and synthetic
solutions contained an equivalent amount of total C, yielding a final concentration of 150 g
C g1 soil. The soil used for the experiment was an acid soil that had been earlier contaminated
with Cd(NO3)2 and contained 3.6, 11, and 485 g g1 of total Cd, Cu, and Mn, respectively.
For each metal, treatments with a different letter were significantly different at p < 0.05.
(Adapted from Mench, M. and Martin, E.66 With permission.)

substantial amounts of Cd and Mn whereas those of maize rather mobilized Cu


(Figure 2.2) and Ni.
Among the carboxylic ligands that have been reported to occur in the root
exudates, oxalate and citrate exhibit the strongest stability constants (complex for-
mation constants) with metals such as Fe and Cu, for instance.67 Organic ligands
such as oxalate and citrate can promote the dissolution of metal oxides such as Fe
oxides, according to the following reactions, which involve ligand (L) adsorption,
FeIII complexation and, ultimately, the detachment of the FeIII complex :68

FeIII-OH + L + H+ FeIIIL + H2O FeIIILaq + H2O (4)

Several authors60,69,70 have shown the exudation of citrate by the proteoid roots
of white lupin and its possible implication in the mobilization of metals such as Fe,
Mn, or Zn. Dinkelaker et al.69 showed that, when grown in a calcareous soil, citrate
was exuded at such high rates around proteoid roots that it precipitated as discrete
crystals of Ca citrate. They also reported a significant increase in DTPA-extractable
Mn and Zn. The bioavailability of these metals in such conditions as DTPA has been
proposed as a method for predicting the availability of metals in calcareous soils by
Lindsay and Norvell.71 The increase in DTPA-extractable Mn and Zn in this exper-
iment was possibly the consequence of an increased amount of total soluble Mn and
Zn in the soil solution as related to their complexation by citrate ions. It could,
however, also be attributed to the acidification of the rhizosphere that concurrently
Bioavailability of Trace Elements 35

TABLE 2.3
Stability Constants of Complexes Formed between
Fe or Divalent Metals and Two Phytosiderophores
Isolated and Purified from Root Washings of Barley
(Hordeum vulgare L.): Mugineic Acid (MA) and
Deoxymugineic Acid (DMA)
Ca2+ Mn2+ Fe2+ Zn2+ Ni2+ Fe3+ Cu2+

MA 3.81 8.30 10.14 12.69 14.92 17.71 18.10


DMA 3.34 8.29 10.45 12.84 14.78 18.38 18.70

Source: Data from Murakami, T. et al.80 with permission.

occurred as a consequence of proton release by the proteoid roots of white lupin.


In addition, other commonly found root exudates such as mucilage72 and phenolics
also have been reported as potential chelators for metals.73 They may thus play a
substantial role in the mobilization of those trace elements by plant roots.
One of the most remarkable groups of root exudates are the so-called phytosi-
derophores that are excreted by grasses.74 As their release is enhanced under Fe
deficiency, this phenomenon has been described as the Strategy II for Fe acquisi-
tion.46,75 It has, however, been shown that their release is also enhanced under Zn
deficiency,76-78 and possibly also as a response to Cu deficiency.79 Indeed these
chelators not only form strong chelates with Fe, but also with many other metals
such as Cu and Zn.80 The stability constant of the chelate formed with Cu is even
slightly larger than that formed with ferric Fe (Table 2.3). Treeby et al.81 showed
that Zn, Cu, and Mn concentrations significantly increased in the shoots of barley
as a consequence of the increased release of phytosiderophores in response to Fe
deficiency (Table 2.4). These results suggest that, although the mobility of many

TABLE 2.4
Biomass and Micronutrient Concentrations in the Shoots of Barley
(Hordeum vulgare L.) Grown in a Calcareous Luvisol and Release
of Fe Solubilizing Exudates by Fe-Sufficient (+Fe) or Fe-Deficient
(Fe) Seedlings
(mg kg1 shoot dry weight) Exudate release
Shoot dry weight (nmol Fe solubilized g1
(mg plant1) Fe Mn Cu Zn root DW per 4 hours)

+Fe 29 234 94 13 43 3.0


Fe 22a 55a 165a 39a 180a 22.5a
a Indicates a significant difference (at p < 0.05) between the two Fe treatments.

Source: Adapted from Treeby, M. et al.81 With permission.


36 Trace Elements in the Rhizosphere

metals is restricted in calcareous soils because of the high pH of the bulk soil, the
bioavailability of metals to plants such as grasses might be larger than expected.
This is because of the enhanced production of phytosiderophores that occur in
response to the low levels of available Fe in the soil solution.
When considering the effects of any organic exudate in the soil, the possible
competition between the various metals, including alkaline earth metals, needs to be
accounted for, as suggested by Jones and Darrah37 for alkaline soils containing large
concentrations of Ca and Mg. Hamon et al.63 showed, for instance, that when increas-
ing Ca concentration from 42 to 1042 mg dm3 in the soil solution, the proportion
of Zn and Cd present as free metal ions increased from 8 to 14% to 83 to 100%, as
a consequence of Ca forming complexes or chelates instead of Zn and Cd. The
competition between various trace metals and Fe for chelate formation has also been
reported to occur for phytosiderophores.81 Owing to the respective stability constants
of the complex that they can form with phytosiderophores (Table 2.3), the competition
between divalent, trace metals and Fe will decrease in the following order: Cu > Ni
> Zn > Mn. In addition, the ecological significance of the impact of the various
exudates on complexation or chelation reaction with various trace metals is still a
question for debate in real soil conditions, as they are likely to be rapidly metabolized
by soil microorganisms that are particularly abundant in the rhizosphere.7

VI. CONCLUSIONS
As drawn schematically in Figure 2.3, a range of mechanisms exists by which plant
roots can alter the chemical conditions in the soil solution in their immediate vicinity.

FIGURE 2.3 Schematic representation of the chemical interactions between plant roots, soil
solution and soil solid constituents that take place in the rhizosphere as a direct consequence
of the biological activity of the root. This diagram depicts how processes occurring at the
solution/root interface can affect (i) a range of soil solution parameters and (ii) thereby the
chemical reactions occurring at the soil solution/soil (solid) interface that ultimately determine
the bioavailability of trace elements for the plant.
Bioavailability of Trace Elements 37

Those changes in ionic and ligand concentrations, pH, and pe can have dramatic
effects on the chemical reactions that occur at the soil solution/soil (solid) interface
and that ultimately determine the bioavailability of trace elements to plants. The
mechanisms involved in these root-induced changes and in the acquisition of trace
elements can vary widely among plant species, and in response to environmental
conditions, too. This diversity needs to be further studied and used in the future,
although it definitively complicates the reliable prediction of the bioavailability
through a single, simple, universal, chemical soil test.
A substantial proportion of the ideas reported herein have been derived from
model experiments, numerical models, or the application of general concepts of soil
chemistry applied to the peculiar chemical conditions of the rhizosphere. Obviously
it is necessary to acquire additional, experimental proofs supporting these ideas and
hypotheses. On the one hand, the refinement of numerical codes by geochemists
and the development of adequate tools and analytical methods by chemists will
certainly provide great help to move forward. On the other hand, the use of new
tools such as those provided by molecular biology will help testing mechanisms
implied at the root-soil interface. This is a challenge for the future as it will help us
improve our capacity to predict and possibly to restrict the contamination of plants
by trace elements.

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40 Trace Elements in the Rhizosphere

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3 Rhizosphere Chemical
Changes Enhance Heavy
Metal Absorption by
Plants Growing in
Calcareous Soils
Naomi Assadian and Lloyd B. Fenn

CONTENTS

I. Introduction.....................................................................................................43
II. Materials and Methods ...................................................................................45
A. Site Description ..................................................................................45
B. General Survey ...................................................................................45
C. Sequential Extractions ........................................................................47
D. Statistics ..............................................................................................48
III. Results and Discussion...................................................................................48
A. General Survey ...................................................................................48
B. Vegetation ...........................................................................................49
C. The Rhizosphere .................................................................................51
IV. Conclusions.....................................................................................................56
V. Future Research ..............................................................................................58
References................................................................................................................59

I. INTRODUCTION
Lead (Pb), cadmium (Cd), chromium (Cr), nickel (Ni), zinc (Zn), copper (Cu), and
cobalt (Co) naturally occur in many soils at concentrations less than 300 mg kg1
from weathering of parent materials.1 Elevated metal concentrations often result
from industrial activity. Atmospheric deposition of Pb, Zn, Cd, and Cu from a west
Texas smelter2 operating since the late 1800s was substantial prior to mandatory
environmental laws. For example, airborne emissions of these metals between 1969
and 1971 exceeded 1000, 500, 10, and 1 metric tons for Pb, Zn, Cd, and As,
respectively.3 Adjoining soils contained Pb concentrations exceeding 3000 mg kg1

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 43
44 Trace Elements in the Rhizosphere

causing children living near the smelter to experience lead poisoning.3 After stack
emission regulations were in effect, metals were still being emitted. Worthington4
found that native lichens and mosses had been eradicated from the hills surrounding
the smelter. The diversity of winter annual plants had also diminished.
Despite the disappearance of many plants, many have flourished along the banks
of the Rio Grande River, particularly introduced species. It is difficult to generalize
plant responses to heavy metal contaminations.1 Plants are often broadly classified
as metal accumulators, indicators, or excluders.5 Metals accumulate either by foliar
or root absorption. Foliar absorption of metals occurs through leaf cuticles or sto-
mata, an important pathway when atmospheric deposition is high and/or calcareous
soils exist. Foliar absorption may be a contributing factor, but not likely the major
pathway of metal accumulation. More likely, the root and its environment (the
rhizosphere) have the major impact on heavy metal absorption even in a calcareous
environment. Metal mobility and eventual bioavailability in an alkaline calcareous
soil may be dependent on rhizosphere processes.
Nutrient (NO3, K+, and Ca2+) mobility into the rhizosphere is partially by mass
flow and a diffusion gradient established by plant water consumption.6 The presence
of excess nutrients brought into the rhizosphere by mass flow can be demonstrated
by surplus calcium (Ca) surrounding plant roots.6 Soil solution supersaturated with
Ca eventually precipitates forming CaCO3.7,8 Increased CO2 (H2CO3) in organic rich
zones, areas close to the roots for example, leads to the supersaturation of Ca in the
soil solution.9 This increased soil solution Ca concentration is governed by the
solubility product of CaCO3. The mass flow of the Ca containing water to the plant
roots and the water use by transpiration continually concentrate the Ca, leading to
continued CaCO3 precipitation. In a desert environment, considerable amounts of
CaCO3 are deposited around active roots and hyphae forming partially cemented
root channels.10 Heavy metals in a calcareous environment coprecipitate with
CaCO3,11 especially those that have ionic sizes similar to Ca, such as Zn, Mg, and
Mn.12 Metals may also adsorb on clay particles or react with hydroxide compounds
at a high pH.13,14 Precipitated and insoluble metals would control phytotoxicity.14,15
Within the rhizosphere, Gobran and Clegg16 recently published the protocols of
examining plant-available nutrients and trace elements. This conceptual model
assumes that plant roots exist in an equilibrium between the bulk soil (no roots),
rhizosphere (roots), and the soilroot interface (soil in contact with roots). The bulk
soil is the principal reservoir of nutrients. Biological activity in the rhizosphere and
soilroot interface contribute to the concentration of organic acids and other organic
compounds. Greater organic matter, weathering, changes in pH, microorganism-
mediated transformations, and chelation may contribute to a larger fraction of plant-
available metals in these soilroot fractions. Those plant species that have the
potential for significant acid production such as grasses and legumes could further
increase the availability of metals in the soilroot interface (the region nearest the
plant root) resulting in greater plant absorption even in calcareous soils.13 Root
exudates increased the solubilities of Mn and Cu in two acid soils, whereas the
solubilities of Ni and Zn were increased only in the least acidic of the two studied
soils.17 It is suggested that the soilroot interface is the most predictive location for
potential ion absorption by the plant.16
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption 45

The bioavailability of metals in a contaminated environment is frequently char-


acterized by sequential extraction protocols.18 The procedure partitions the total
metal pool into forms that are progressively less bioavailable (exchangeable most
available, and residual forms least available). Unfortunately, the utility of this
procedure may be impaired by the presence of carbonates exceeding 10% by
weight.19 Metals coprecipitated with dolomite (MgCO3 CaCO3) and calcite (CaCO3)
crystals in the presence of high CaCO3 concentrations are often released in subse-
quent extractions masking the true chemical form of the specific extraction. Despite
the limitations of this procedure in a calcareous environment, it appears to be a
method with some potential to quantify the impact of rhizosphere processes on the
bioavailability of metals.
Our first objective was to survey the presence of metals in river sediments and
desert soil adjacent to the smelter. The next objective was to evaluate the possible
metal accumulation in riparian vegetation. Our last objective was to describe the
metal chemical states in the soil environment and the possible impact of its rhizo-
sphere position on potential metal transformations to more soluble forms.

II. MATERIALS AND METHODS


A. SITE DESCRIPTION
A 100-year smelting operation in El Paso, Texas,2 was examined for the plantsoil
impact of its copper, zinc, and lead emissions. The smelter is located near the southern
end of the Franklin Mountains next to the Rio Grande River. The environment is
arid (Chihuahuan Desert) and the elevation is 1225 m. Alluvial soils above the flood
plain of the Rio Grande River and adjacent to the smelter primarily belong to the
Delnorte-Canutio Association. These are shallow, undulating soils, underlain by
caliche, found frequently in flooded arroyos or alluvial fans at the base of the Franklin
Mountains. These soils are loamy-skeletal, mixed, thermic, shallow, Typic Paleothids.
A second site was located about 11 km west of the smelter along the Rio Grande
River near the small town of Canutillo, Texas. Soils in this area belong to the
Glendale-Harkey Association. These are level, deep, brown soils that developed in
loamy, friable sediments and recently deposited on the flood plain of the Rio Grande
River. These soils are coarse-silty, mixed, calcareous, thermic, Typic Torrifluvents.

B. GENERAL SURVEY
River sediments, soils, and plant tissue were collected at the smelter and Canutillo
locations in April and July, 1993. Within 1 km of the smelter, river sediments were
collected from the channel in the lower flood plain of the river to an approximate
depth of 0.3 m. Surface soil (0 to 15 cm) and subsurface soil (15 to 30 cm) samples
were collected from the shoulder terrain along the river with five replications. All
sediment and soil collection sites had no plants growing in the immediate area.
Aboveground plant tissue (leaves and nonbarky stems) from perennial species was
also collected from a 30-m area along the riverbanks, but not necessarily near
sediment or soil sampling areas. Duplicate samples for each plant species were
46 Trace Elements in the Rhizosphere

TABLE 3.1
Identification and Grouping of Riparian Vegetation Collected at Two Sites
along the Rio Grande River, 1993
Riparian Vegetation Collection
Plant Species Common Name Sitea Plant Type Pubescence Microhabitat

Melilotus alba Clover Sm, Ca Dicot Hairy High bank


Tamarix gallica Salt Cedar Sm, Ca Dicot Smooth Low bank
Cynodon dactylon Bermuda grass Sm, Ca Monocot Smooth High bank
Lepidium Sp. Mustard Sm Dicot Hairy High bank
Salix nigra Willow Ca Dicot Smooth High bank
Carex emoryi Sedge Ca Monocot Smooth Low bank
Rume crispusx Curly dock Ca Dicot Smooth Low bank
Equisetum Horsetail Ca Monocotb Smooth Low bank
kansanum
Not identifiedc Clump grass Sm Monocot Smooth High bank
Not identified grass species Ca Monocot Smooth High bank
a Sm = Smelter; Ca = Canutillo.
b Horsetail is not monocot, but categorized as such for descriptive statistics.
c Reproductive structures not collected for definitive identification. Assuming a perennial.

collected from ten different perennial plants (Table 3.1). An identical sampling
procedure was followed at the Canutillo site. Plant species were identified using
plant flora publications by Parker20 and Warnock.21
River sediments and surface and subsurface soils were transported and stored
in plastic bags. Sample preservation and preparation included forced air-drying at
105C for 24 hours, sieving with a 2-mm screen, and pulverizing with a ball mill.
An acid-digestion with nitric acid (HNO3) and hydrogen peroxide (H2O2) following
EPA 3050A protocols was used to determine heavy metal concentrations of Cd,
Cu, Pb, Zn, Cr, Ni, Co, V, and Mn, using inductively coupled plasma spectroscopy
(EPA 200.7). Plant tissue samples were transported and stored in plastic bags. Half
of each plant sample was washed twice with deionized water. The other portion
was left unwashed. Washed and unwashed plant tissues were dried in a forced-air
dryer at 65C for at least 72 hours, ground, and passed through a 40-mesh screen.
Samples were acid-digested with nitric and perchloric acids using EPA 200.2
protocols for heavy metal determinations, using inductively coupled plasma spec-
troscopy (EPA 200.7).
Both sites were revisited in late June, 1994. Plant tissue samples of salt cedar
(Tamarix gallica) and Bermuda grass (Cynodon dactylon L.) were recollected to
verify that preparation methods were not affecting analytical outcomes. Plant tissue
samples were either left unwashed, washed with deionized water, or washed with
0.1 N HCL to remove leaf surface contamination prior to drying for heavy metal
analyses (using EPA 200.2 and 200.7 protocols).
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption 47

C. SEQUENTIAL EXTRACTIONS
In 1997, common Bermuda grass and salt cedar from the smelter site were selected
for further evaluation. Both plant species have been introduced to this area and have
aggressively grown on the banks of the Rio Grande River. A soilroot sampling
scheme was selected that follows protocols by Gobran and Clegg.16 Soil samples
were collected from three soil fractions:

1. Bulk soil (BS) soil between plants where few or no roots were expected;
2. Rhizosphere soil (RS) soil that was removed from the roots with gentle
shaking for a minute;
3. Soilroot interface soil (SRI) soil that was strongly adhering to plant
roots.

Soil samples were collected in triplicate for each plant species. Plant tissue was
also collected from each soil-sampling site. All samples were transported in a cold
chest and kept frozen until chemical analysis. Field-moist soil samples were used
for sequential metal extractions using protocols presented by Kersten and Frstner.18
About a 5-g sample was weighed into centrifuge tubes and extracted by the steps
listed in Table 3.2. The samples were done in triplicate at 25C. All samples were
analyzed for Zn, Mn, Pb, Cu, and Ca by inductively coupled plasma spectroscopy.
The exchangeable form (No. 1, Table 3.2) is usually examined by extraction
with NaCl, NH4-acetate, NH4NO3 or KCl. This form is the most available to plants.
The carbonate form of heavy metals (No. 2, Table 3.2) is usually extracted with a
weak acid such as acetic acid and NH4-acetate at a pH value of 5. The carbonate
metals are less available to the plant than the exchangeable metals. The subsequent
extractant category is easily reducible Mn-oxides or metals associated with the
amorphous oxides (No. 3, Table 3.2). These compounds are extracted with a 0.1 M

TABLE 3.2
Sequential Extraction Scheme for Soil from the Root-Zone of Bermuda Grass
and Salt Cedar
Extraction Shaking
Step Source Metals Extraction Chemical Volume Time

1 Exchangeable 1M NH4NO3 25 ml 20 min


2 Carbonate associated 1M NH4Oac, pH = 5 + acetic acid 25 ml 20 min
3 Easily reducible Mn-oxides 0.1 M NH4OH-HCl + 0.01 M 25 ml 20 min
HNO3, pH ~ 2
4 Moderately reducible 0.1M NH4 Oxalate + acetic acid, 25 ml 20 min
amorphous Fe-oxides pH = 3
5 Sulfidic and organic materials 30% H2O2 + 0.01 M HCl, pH = 2 25 ml 20 min
6 Residuals Concentrated HNO3 heated to 25 ml 60 min
80 120C

Note: Extraction method largely that of Kersten and Frstner.18


48 Trace Elements in the Rhizosphere

NH2OH-HCl in 0.01 M HNO3 extractant and are even less soluble than the carbon-
ates. If soil carbonates exceed 10 to 20%, some CaCO3 residual dissolution occurs
in this step.19 The moderately reducible Fe-oxides (No. 4, Table 3.2) constitute an
amorphous material of some significant extent, but contain metals of usually low
availability to plants. This class is extracted with NH4-oxalate + acetic acid at a pH
value of 3. The metal-associated oxides would become significant if the soil system
is acidic or anaerobic. The organic-sulfidic class (No. 5, Table 3.2) contains the
organic materials consisting largely of humic and fulvic acids that are very insoluble
and only slightly biodegradable. The extractant is H2O2 at acid pH of 2 that destroys
all organic matter and characterizes the strongly held chelated heavy metals. The
last metal fraction in the sequence is the residual fraction, extracted by concentrated
HNO3 heated to 100C (No. 6, Table 3.2).
Samples from all three fractions were also analyzed for total C (carbon), organic
C, soil pH, and electrical conductivity (EC). Representative subsamples from each
soil sample were dried, pulverized, and analyzed for total carbon using a Carlo Erba
NCHS-O analyzer using standard protocols. Subsamples pretreated with a 10% HCl
solution until bubbling (evolution of CO2) ceased were used to determine inorganic
C concentrations. Inorganic C concentration was determined indirectly by subtract-
ing the organic fraction from the total carbon fraction. Saturation extracts were made
from BS and RS samples to determine soil pH and electrical conductivity following
EPA 4500H+B and EPA 2520B protocols, respectively.

D. STATISTICS
Descriptive statistics, including means, correlations, t-tests, and analysis of variance
(ANOVA) for split plot analyses, were used to summarize chemical analyses for the
general survey of riparian vegetation, river sediments, and desert soil samples col-
lected in 1993 and 1994. For soil samples collected in 1997, ANOVA using SAS22
was used to delineate soil differences associated with plant species (Bermuda grass
and salt cedar), and among soil fractions (BS, RS, and SRI). Statistics were based
on a split plot design with plant species as main parameters and soil fraction as sub-
parameters with three replications. Additional ANOVA analyses were used to delin-
eate which heavy metal form dominated the soil rhizospheres between plant species.
Significant differences were delineated at the 0.10, 0.05, and 0.01 level of probability.

III. RESULTS AND DISCUSSION


A. GENERAL SURVEY
River sediments at both the smelter (contaminated) and Canutillo (uncontaminated)
sampling sites showed lower concentrations of all metals than soils (p > 0.05,
statistics not shown) (Table 3.3). Although conventional concentration ranges for
heavy metals in sediments are unknown, sediment concentrations of metals at both
sites were well within ranges for most uncontaminated soils.
High soil levels of Cd, Cu, Pb, and Zn were closely related to the metal emissions
from smelter stacks reported in 1975.3 Average concentrations of sediment Cu, Pb,
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption 49

TABLE 3.3
Mean Heavy Metal Concentrations in Sediment, Surface Soil, and Subsurface
Soil at Two Sites along the Rio Grande River, 1993

Conventional
Heavy Soil Sediment Surface Soila Subsurface Soilb
Metal Concentrations Smelter Canutillo Smelter Canutillo Smelter Canutillo

Cd 0.311 0.3 a3 0.0 b 6.2 a 1.2 b 2.6 a 2.1 a


Cu 550 34.3 a 4.6 b 284.1 a 8.4 b 90.8 a 11.6 b
Pb 2200 27.1 a 2.1 b 169.7 a 7.7 b 43.8 a 9.5 b
Zn 10300 34.7 a 14.6 b 108.6 a 16.7 b 42.0 a 21.4 b
Cr 0100 3.6 a 3.6 a 4.6 a 3.7 a 4.1 a 5.8 a
Ni 1 0.7 a 0.1 b 3.4 a 2.7 b 3.1 b 5.3 a
Co 14 1.5 a 1.4 a 2.0 a 1.7 a 1.9 a 2.5 a
Mn 2003000 176.4 a 180.3 a 136.6 a 128.1 b 125.7 b 191.2 a
a Soil collected at a depth of 00.15 m.
b Soil collected at a depth of 00.30 m.
c Lower case letters following numbers in each paired column are significantly different at the 0.05 level

of probability.

and Zn were relatively high (34, 27, and 34 mg kg1, respectively) near the smelter
and greater than those at the Canutillo site (Table 3.3). The lack of greater metal
deposition in sediments at the source was most likely due to a combination of factors.
Metal deposition in the river is diluted and transported in river sediment flow. Settling
of particles is dependent on particle size and water velocity according to Stokes
Law. Floating particles may take several days or weeks to settle. There is also greater
sediment disturbance near a riverbank, which would also account for such low metal
concentrations.
Conversely, surface soils near the smelter showed elevated concentrations of
284, 169, and 108 mg kg1 for Cu, Pb, and Zn, respectively (Table 3.3). To a lesser
extent, surface soils also showed Ni contamination. Contamination of these metals
was also found in subsurface soil samples, but at lower concentrations. High metal
levels would be expected in surface soils. Subsurface soil contamination could be
due to vertical movement (e.g., river flooding) over time or possibly to river levee
disturbance in reshaping the river channel and the roads parallel to the river. Soil
metal levels at the contaminated smelter site fell within the high end of conventional
ranges for uncontaminated soils (Table 3.3).

B. VEGETATION
Three plant species, clover, salt cedar, and Bermuda grass, were collected at both
sites (Table 3.4). Metal concentrations based on a water-wash preparation were
greater in leaf tissue from the smelter site than those from the Canutillo site. Cadmium
and Pb levels were at least two times greater in clover and salt cedar leaf tissue
collected from the smelter site than those from the Canutillo site. All other metal
50 Trace Elements in the Rhizosphere

TABLE 3.4
Heavy Metal Concentrations of Water-Washed Plant Tissue Collected at Two
Sites along the Rio Grande River, 1993
Clover Salt Cedar Bermuda Grass
Plant Smelter Canutillo Smelter Canutillo Smelter Canutillo
Metal Concentrations mg kg1

Cu 220 29 aa 14 b 30 NS 34 NS 127 a 16 b
Cd 0.117.6 1a 0b 2a 1b 4a 1b
Pb 0.50 11 a 4b 10 a 51 b 53 a 11 b
Zn 21120 49 a 37 b 68 a 39 b 120 a 31 b
Cr 10% of soil level 2 NS 8 NS 2 NS 1 NS 2 NS 3 NS
Ni 0.15 2 NS 2 NS 2 NS 1 NS 1 NS 1 NS
Co 0.020.5 0 NS 0 NS 0 NS 0 NS 1 NS 1 NS
Mn 1540 26 a 23 b 24 a 16 b 45 a 141 b

Note: NS = not significant.


a Different lower case letters following numbers in paired columns indicate significant differences at the
0.05 level of probability.

concentrations in clover and salt cedar tissue from the smelter site were either similar
to or greater than those metal concentrations from plant tissue collected at Canutillo.
Plant tissue concentrations of Cu, Pb, and Cr exceeded conventional ranges in all
three plant species. In addition to these three metals, Zn and Mn concentrations were
also high in Bermuda grass. Copper, Pb, and Cr concentrations in clover and salt
cedar tissue ranged from 6 to 45% of that found in contaminated surface soil at the
smelter. Metal concentrations in Bermuda grass tissue were 31 to >100% of that of
contaminated soil. Unlike the other two plant species, Bermuda grass appeared to
be an indiscriminate metal accumulator with high tissue Cu, Pb, and Zn concentra-
tions. More importantly, this indicated that riparian vegetation was either preferen-
tially absorbing soil metals or that the soil samples we collected did not reflect the
actual metal concentrations to which the plant roots are exposed.
Plants were also grouped by morphology, location, or tissue preparation cate-
gories for better interpretation of chemical results (Table 3.5). When grouped by
location, plants absorbed the greatest concentrations of metals where the greatest
concentrations were present in the soil in that environment. Plants near the smelter
showed high levels of Cd, Cu, Pb, and Zn and those at Canutillo showed higher
levels of Cr, Mn, Ni, and Co. Neither plant type nor pubescence appeared to affect
metal uptake. However, monocots tended to absorb more Cu, Pb, and Mn relative
to dicots. Plants with hairy leaves tended to accumulate more Pb. Microhabitat
seemed to be the single most critical factor affecting plant uptake of metals. Plant
species living along the river in high, dry areas had higher metal concentrations than
those rooted in low, wet areas. The flow of the river had minimized the effects of
metal deposition at the source and transported contaminated sediments downstream.
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption 51

TABLE 3.5
Average Heavy Metal Concentrations in Water-Washed
Plant Tissue Categorized by Location, Plant Type,
Morphology, Microhabitat, and Sample Preparation at
El Paso, 1993
Cd Cu Pb Zn Mn
Comparisons mg kg1

Location
Smelter 4.0 ab 87 a 26 a 76 a 35 b
Canutillo 0.5 b 20 b 12 b 56 b 142 a

Plant Type
Dicot 2.0 NS 25 NS 16 NS 60 NS 82 b
Monocota 1.7 70 19 68 123 a

Pubescence
Hairy 2.7 NS 26 NS 12 NS 44 NS 26 b
Smooth 1.6 52 19 70 123 a

Habitat
High Dry Bank 2.5 NS 59 NS 22 a 70 NS 122 NS
Low Wet Bank 0.8 24 10 b 54 67

Plant Tissue Preparation


Double water wash 3.2 NS 59 NS 12 NS 85 NS 39 NS
Acid wash 3.3 45 8 86 32

Note: NS = not significant.


a Includes Equisetum.
b Different lower case letters following numbers within each subcolumn
are significantly different at the 0.10 level of probability.

An acid-wash preparation decreased detectable metal concentrations by about


15% in comparison with unwashed tissue, particularly for Bermuda grass leaf
blades. These differences were not statistically significant (Table 3.5). Bermuda
grass has a prostrate growth habit and debris and fine dust can be easily lodged
between blades. In general, metal concentrations in plant tissue collected in 1994
were lower than those from 1993 except for increased Cd and Cu concentrations
in salt cedar tissue (data not shown). Despite fluctuations in plant tissue metals with
regard to either washing method or year, Cu, Pb, Zn, and Mn exceeded conventional
plant concentrations.

C. THE RHIZOSPHERE
Average calcium carbonate concentrations of 44.1 and 56.1 g kg1 found in the BS
fraction for Bermuda grass and salt cedar, respectively, significantly increased almost
52 Trace Elements in the Rhizosphere

TABLE 3.6
Determined Soil CaCO3 Concentration, Organic
Carbon Percentage, pH, and EC of Soil Fraction
Surrounding Bermuda Grass and Salt Cedar Roots
Organic
CaCO3 Carbon EC pH
Plant/Soil Fraction g kg1 % dS m1 Units

Bermuda Grass
Bulk 44.1 ba 0.56 b 7.35 NS 7.94 NS
Rhizosphere 35.9 b 1.32 b 11.63 7.80
Soilroot interface 80.3 a 3.80 a
Mean 53.4 NS 1.89 Bb 9.49 NS 7.87 NS

Salt Cedar
Bulk 56.1 b 0.41 b 4.86 7.82
Rhizosphere 60.9 b 1.25 b
Soilroot interface 109.6 a 7.40 a
Mean 75.5 NS 3.02 A

Note: = insufficient sample; NS = not significant.


a Different lower case letters following numbers within each subcol-
umn are significantly different at the 0.10 level of probability.
b Different upper case letters following means in each column are

significantly different at the 0.10 level of probability.

twofold to 80 and 110 g kg1, respectively in the SRI fraction (Table 3.6). An
increasing CaCO3 gradient among soil fractions was more evident for salt cedar than
for Bermuda grass. Salt cedars are high water-consuming trees with deep, woody
roots. The fibrous root system of Bermuda grass can be quite extensive, but typically
remains at shallow depths. Calcium carbonate concentrations were much greater
around salt cedar roots. Limited evaluation of electrical conductivity of the saturation
extract showed similar increases to that of CaCO3 for Bermuda grass. Organic C
also increased from BS to RS and from RS to SRI. The increase from BS to SRI
was greater than sixfold for Bermuda grass and about 18-fold for salt cedar. There
were no apparent changes in pH between BS and RS fractions. It was also observed,
but not measured, that the soil texture of the SRI was of a finer composition than
the texture of the other two soil fractions.
Heavy metal concentrations in the BS fraction were similar to metal concen-
trations of the general soil survey of the smelter site (Tables 3.3 and 3.7). However,
metal concentrations were lowest in the BS fraction and increased in the order BS
< RS < SRI (Figure 3.1). Despite increased amounts of CaCO3 in the SRI of salt
cedar, total metal concentrations were greater in the root rhizosphere of Bermuda
grass rather than of salt cedar. ANOVA analyses showed statistical differences in
metal accumulation in the rhizosphere between plant species for the most soluble
metal fractions of exchangeable and carbonate association (data not shown). Low
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption 53

TABLE 3.7
Averaged Bermuda Grass and Salt Cedar Soil Extraction Concentrations of
Heavy Metal Forms According to Soil Fraction
Soil Fraction Cu Pb Zn Mn Ca
Extractant g kg1 mg kg-1

Exchangeable BS 4.2 aa 0b b 0.02 b 1.3 b 1.2 b


RS 5.9 a 0.02 ab 0.11 b 2.5 b 1.4 b
SRI 8.2 a 0.1 b 0.44 a 1.3 a 2.8 b
Carbonate Associated BS 18.8 b 12.4 a 4.9 b 20.3 a 3.3 ab
RS 25.4 ab 13.4 a 8.0 b 22.4 b 3.0 b
SRI 38.2 a 20.1 a 14.5 a 37.8 a 4.2 a
Easily Reducible BS 5.2 a 3.8 a 2.2 b 53.0 a 4.2 ab
Mn-Oxides RS 6.1 a 3.1 a 2.5 b 31.9 a 3.0 b
SRI 8.4 a 4.4 a 6.4 a 42.7 a 5.0 a
Moderately Reducible BS 60 b 0.10 a 15.4 b 6.4 a 0.0
Fe-oxides RS 162 b 0a 15.5 b 8.9 a 0.0
SRI 337 a 0.12 a 29.0 a 9.5 a 0.0
Organic Fraction BS 59 b 2.1 a 12.7 a 7.3 a 0.2 a
RS 100 b 3.2 a 21.1 a 9.8 a 0.3 a
SRI 296 a 5.5 a 50.4 a 14.8 a 0.4 a
Residual BS 49 b 72.0 a 36.7 b 39.6 a 3.7 a
RS 102 b 109.9 a 54.4 ab 46.3 a 4.7 a
SRI 297 a 196.4 a 102.4 a 53.7 a 5.3 a
Total BS 196 90 71 127 12
RS 401 129 101 121 12
SRI 984 226 203 159 17
a Different letters after numbers in each subcolumn are significantly different at the 0.05 level of prob-
ability.
b Zero represents below detection limit.

replication likely masked more frequent significant differences between soil fraction
and plant species. It appeared that plant species not only accumulated metals
differently, but also affected the types of metal species within the RS and SRI
(Figure 3.2).
From sequential extraction of root zone soils, we found exchangeable Cu to
reach 8.2 mg kg1 in SRI soil, a value probably in excess of 100 times that needed
for plant nutrition (Table 3.7). Other than Ca and Cu, no other metal was found in
excessive amounts in the exchangeable form. Copper also dominated carbonate-
associated, easily reducible Mn-oxides, moderately reducible Fe-oxides, and the
sulfidic-organic forms. Lead species were mainly associated with carbonate and
residual forms (p > 0.01). Zinc was primarily associated with insoluble forms such
as moderately reducible Fe-oxides, organic, and residual fractions (p > 0.01).
In comparison to bulk soils, metal forms varied in the RS and SRI (Figure 3.2).
With increasing proximity to root, Cu was increasingly associated with Fe-oxides,
organic, and residual forms. Soluble Cu forms increased in the salt cedar rhizosphere,
54 Trace Elements in the Rhizosphere

FIGURE 3.1 Total concentrations of Cu, Pb, Zn, and Mn in the soil from the BS (bulk soil),
RS (rhizosphere soil), and SRI (soilroot interface) of Bermuda grass and salt cedar growing
next to a west Texas smelter site.

but not for Bermuda grass. Similarly, the increased Pb and Zn within the Bermuda
grass rhizosphere were associated with insoluble metal forms (organic and residual
forms), whereas increased soluble metal forms (moderately reducible Fe-oxide and
organic fractions) were found within the salt cedar rhizosphere.
Calcium release at the sequential step for easily reducible Mn-oxides suggested
that not all CaCO3 was dissolved in the carbonate-associated fraction as reported by
Prez-Sirvent et al.19 As a consequence, greater metal quantities may be associated
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption
FIGURE 3.2 Relative change in Cu, Pb, and Zn concentration from bulk soil, rhizosphere soil, and soilroot interface as influenced by Bermuda grass
and salt cedar growth.

55
56 Trace Elements in the Rhizosphere

with carbonates than the data might indicate. In the moderately reducible Fe-oxide
phase, large amounts of Cu and Zn were found, but no Pb or Ca (Table 3.7). Since
the extractant here was NH4-oxalate, the Ca and Pb were evidently precipitated as
oxalates. The organic fraction was high in Zn and Cu concentrations. The highest
concentrations of metals were found in the residual fraction.

IV. CONCLUSIONS
Our results reveal that heavy metals have accumulated in an alkaline desert soil, and
to a lesser degree in river sediment, at the source of smelter contamination. A general
survey of plant vegetation also showed that some plants, in particular Bermuda grass,
might accumulate metals growing on the high dry banks of the Rio Grande River.
From the data, we concluded that metal accumulation in a contaminated desert soil
was not uniform. Both the macro- and microenvironments (those minerals nearest
the root) impacted plant absorption of heavy metals. Heavy metals, Ca content, and
organic C in the soilroot interface were significantly greater than those concentra-
tions in bulk soil. Gobran et al.23 stated that mineral weathering is probably most
severe next to the root, as this is a plants mechanism to obtain required nutrients.
The question is how these metals concentrated in the plant rhizosphere.
It appears that additional abiotic processes such as wind erosion and rain splash
may also contribute to localized metal accumulation in the rhizosphere of desert
plants. Schlesinger and Pilmanis24 found soils N, P, and K were associated with the
presence of shrubs in desert habitats. Intershrub spaces had lower fertility. They
suggested that finer particles were wind-deposited on shrub mounds. Improved soil
stability, soil structure, and water infiltration allowed for greater vertical movement
of nutrients into soil. Banin et al.25 also found that plants act as biofilters of metal-
laden dust. It is possible that metal mobility in this situation was the movement of
precipitated insoluble forms from surface or bulk soils moving into the rhizosphere
as small amorphous particles rather than as ionized forms via mass flow. However,
these high soil concentrations are not excessively absorbed by the biotic species in
the arid-calcareous system (Table 3.4). It is accepted that calcite presence reduced
the potential toxicity of most heavy metals.26
Within the soil profile, the concentration of soil metals increased from bulk soil
to soil in intimate contact with soil roots (the soil rhizosphere and soilroot interface).
The development of variable metal and CaCO3 partitioning among these soil frac-
tions in the desert ecosystem is difficult to answer. Mass flow would seem to be the
more obvious answer.6 Surplus Ca in a CO2-rich environment would cause CaCO3
precipitation as plants reduce the water content of the soil.7 Salt cedar trees have a
greater water consumption potential than Bermuda grass and would account for
greater Ca as CaCO3 deposition within the salt cedar rhizosphere. Suarez et al.9 also
found a two- to threefold increase (a supersaturated Ca solution) in Ca concentration
in solution from an organic-matter-rich zone. The increased CaCO3 in the SRI could
be the result of large increases in unused Ca in the soil solution. In the presence of
high pCO2 we have a reduced precipitation potential, but yet the largest precipitated
fraction in the soilroot system. The observation of CaCO3 shells around old root
channels is frequent in arid regions.10
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption 57

In acid soils, Gobran and Clegg16 found increased nutrients (Ca) and decreased
metals (Al) next to the roots (SRI) suggesting completely different growing condi-
tions than thought from analyses of the bulk soil. In these arid zone soils, proximity
to the roots resulted in up to a threefold increase in all metals (Table 3.7). Increased
organic matter closer to the roots may decrease the tendency for CaCO3 precipita-
tion,9 but yet the largest CaCO3 accumulations were found next to the roots. Bio-
logically active soil organic matter (a heterogeneous mix of living and organic
materials that are readily circulated through biological pools) may also have con-
tributed to metal availability by increasing the acidity of soil microsites. Indirect
evidence of increased exchangeable Mn from our results (Table 3.7) suggested that
Mn released from oxides must have occurred at acidified microsites. Factors affecting
dissolution or preventing precipitation of CaCO3 would affect metal bioavailability.
Boundaries between soil BS, RS, and SRI were diffuse rather than abrupt.
Methodology to partition these soil fractions may have contributed to inaccuracy.
Sand particles were readily separated from the SRI fraction when plant roots were
gently shaken. Only fine, aggregated particles adhered to root hairs (SRI). By
definition of the SRI,16 we likely segregated the most biologically interactive and
root-influenced zone. As a consequence, metal concentrations were elevated. Using
the sequential extraction data to determine bioavailability of metals, we also noted
that much of the Cu, Zn, Pb, and Mn were associated with the CaCO3 phase. In the
easily reducible Mn-oxides fraction, a solubilization of precipitated Ca suggested
that the CaCO3 was solubilized in even greater quantities, a problem in the proper
use of these data (Table 3.7). One cannot be certain of how much of the heavy metals
(Cu, Pb, and Zn) would be coprecipitated with the manganese precipitates. Appar-
ently, a large quantity of the metals must have been surface precipitated on the calcite
crystals as much less was solubilized in the Mn-oxide extraction with even a larger
amount of solubilized Ca (residual CaCO3) than in the carbonate category. Since
this is an equilibrium state, all soil extract values would have some value as potential
reserves. These reserves would be available with localized or general soil pH value
decreases in the SRI.
Concentrations of Cd, Cu, Pb, Zn, and Mn were often many times greater in
Bermuda grass tissue than in salt cedar tissue. Although metal concentrations in
leaf tissue were not proven to be statistically different, we feel that there are real
differences between plant species in metal absorption potential that is possibly
linked to differences in the rhizosphere. The fibrous, shallow root system of Bermuda
grass creates a continuous shallow root mass where metals have accumulated from
atmospheric fallout. Conversely, salt cedar roots are typical of dicotyledonous trees
with deep woody roots with fragmented zones of active root growth. Differences
in root morphology between monocots and dicots, and the shallow soil concentration
of root mass and active absorption sites may affect differences in plant uptake of
heavy metals.
Despite the limitations of soil fractionation and sequential extraction to deter-
mine metal forms in soils, the results demonstrated that not all metals are uniformly
distributed and immobilized in a calcareous desert soil. Calcium carbonate may not
always be a plant barrier to metal uptake in a calcareous soil. Entry of metals into
the food chain is possible from wildlife grazing on grass.
58 Trace Elements in the Rhizosphere

V. FUTURE RESEARCH
This study illuminated a need to examine dominant native flora on the high lands
and plains of the Chihuahua Desert. Dicots such as mesquite (Prosopis glandulosa,
P. julifera, and P. pubescens), acacia (Acacia greggii and A. constricta), four-winged
saltbush (Atriplex canescens), and creosote bush (Larrea tridentata), and monocots
such as yucca (Yucca baccata, Y. elata, Y. torreyi) and Spanish dagger (Agave
lecheguilla) are widespread and commonly grazed by cattle and wildlife. Metal
accumulation patterns in the rhizosphere and in plant tissue needs documentation
to compare with those of introduced species such as Bermuda grass. A broader plant
survey will reveal possible mechanisms of metal mobility into the rhizosphere and
patterns of accumulation in desert environments. It seems likely that our hypothesis
of mass flow of precipitated, clay-sized particles laden with metals could effectively
occur in sandy desert soils, but it is questionable if mass flow would be a primary
metal carrier in soil textures with finer particles commonly found in cultivated lands.
The concept of soil fractionation of the root zone applied to crop fertility needs
reexamination, particularly for irrigated agriculture in arid and semiarid regions.
Pecan, cotton, corn, and wheat might benefit from root-zone nutrient studies. It is
known that Ca in an irrigated arid zone accumulates around the roots. Information
on the movement, deposition, and bioavailability of P and K needs examination.
Our understanding could be expanded by these characterizations. Barber and
coworkers6 began rhizosphere studies many years ago. However, rhizosphere data
were primarily intended for seasonal fertility recommendations. This type of research
might provide information, hopefully, to establish nutrient indices between bulk and
rhizosphere soils or information for improved cultivation techniques for future years.
We also questioned whether our methodology in physical partitioning of root
zone soil samples affected soil particle size fractionation. Did larger particles drop
out of the root zone because of size and weight, leaving small-sized particles
organically cemented to the roots? Or, were fine soil particles interfacing with
roots the result of in situ weathering processes and mass flow. As the roots grow
through the soil, they may proliferate around these smaller-sized particles, but also
be exposed to sand and silt constituents. We hypothesize that a chemical marker
such as CaCO3 in calcareous soils may be used to delineate rhizosphere boundaries
in conjunction with physical methods.
Lastly, the use of sequential extraction protocols in this study suggested that
metal accumulation was greatest in the soilroot interface at root absorption sites.
Yet, metal absorption was low in comparison to soil metal concentrations. The
novelty of insoluble metal compounds being concentrated at the root absorption site,
yet not being absorbed at high rates, leaves many questions unanswered. The phys-
ical, chemical, and biological interactions affecting metal solubilities and precipita-
tion products needs to be more closely examined.
Rhizosphere Chemical Changes Enhance Heavy Metal Absorption 59

REFERENCES
1. Fergusson, J. E., The Heavy Elements, Chemistry, Environmental Impact, and Healthy
Effects, Pergamon Press, Oxford, England, 1970, 614.
2. Woodside, T. J. and Roberts, B., The El Paso smelter, in Nelson, L. A. and Haighs,
B. R., eds, West Texas Geological Society Guide Book, 1958 Field Trip, Franklin and
Hueco Mountain, Texas, The West Texas Geological Society, Midland, TX, 1958,
7577.
3. Landrigan, P. J., Gehlbach, S. H., Rosenblum, B. F., Shoults, J. M., Candelaria, R. M.,
Barthel, W. F., Liddle, J. A., Smrek, N. W., Staehling, N. W., and Sanders, J. F.,
Epidemic lead absorption near an ore smelter. The role of particulate lead, New
England J. Med., 292, 123129, 1975.
4. Worthington, R. D., Effects of El Paso pollutants on the lichen, moss, and winter
annual flora on andesite rock formations, in Ganster, P. and Walter, H., eds., Envi-
ronmental Hazards and Bioresource Management in the United States-Mexico Bor-
derlands, UCLA Latin America Center Publication, University of California, Los
Angeles, CA, 1990, 105115.
5. Whiting, S. N., Leake, J. R., Baker, A. J. M., and McGrath, S. P., Changes in the
phytoavailability of zinc to plants sharing a rhizosphere with the zinc hyperaccumu-
lator Thlaspi caerulescens J. and C. Presi, in Iskandar, I. K., Hardy, S. E., Chang, A.
C., and Pierzynski, G. M., eds., Proceedings of the Fourth International Conference
on the Biogeochemistry of Trace Elements, June 2326, Clark Kerr Campus, Berkeley,
CA, 1997, 469.
6. Barber, S. A. and Ozanne, P. G., Autoradiographic evidence for the differential effect
of four plant species in altering the calcium content of the rhizosphere soil, Soil Sci.
Soc. Am. Proc., 34, 635637, 1970.
7. Miyamoto, S. and Pingitore, N. E., Predicting calcium and magnesium precipitation
in saline solution following evaporation, Soil Sci. Soc. Am. J., 56, 17671775, 1992.
8. Feagley, S. E. and Hossner, L. R., Ammonia volatilization reaction mechanism
between ammonium sulfate and carbonate systems, Soil Sci. Soc. Am. J., 42, 364367,
1977.
9. Suarez, D. L., Wood, J. D., and Ibrahim, I., Re-evaluation of calcite supersaturation
in soils, Soil Sci. Soc. Am. J., 56, 17761784, 1992.
10. Monger, H. C., Daugherty, L .A., and Lindemann, W. C., Microbial precipitation of
pedogenic cacite, Geology, 19, 9971000, 1991.
11. McBride, M. B., Chemisorption of Cd2+ on calcite surfaces, Soil Sci. Soc. Am. J.,
4, 2628, 1980.
12. Xiang, H. F. and Banin, A., Solid-phase manganese fractionation changes in saturated
arid-zone soils: Pathways and kinetics, Soil Sci. Soc. Am. J., 60, 10721080, 1996.
13. Mengel, K. and Kirby, E., Principles of Plant Nutrition, International Potash Institute.
Worblaufen-Bern/Switzerland, 1982, 655.
14. Harter, R. D., Effect of soil pH on adsorption of lead, copper, zinc, and nickel, Soil
Sci. Soc. Am. J., 47, 4751, 1983.
15. Basta, N. T., Pantone, D. J., and Tabatabai, M. A., Path analysis of heavy metal
absorption by soil, Agron J., 85, 10541057, 1993.
16. Gobran, G. R. and Clegg, S., A conceptual model for nutrient availability in the
mineral soil-root system, Can. J. Soil Sci., 76, 125131, 1996.
17. Mench, M. and Martin, E., Mobilization of cadmium and other metals from two soils
by root exudates of Zea mays L., Nicotiana tobacum L. and Nicotiana rustica L.,
Plant Soil., 132, 187196, 1991.
60 Trace Elements in the Rhizosphere

18. Kersten, M. and Frstner, U., Chemical fractionation of heavy metals in anoxic
estuarine and coastal sediments, Wat. Sci. Tech., 15, 121130, 1986.
19. Prez-Sirvent, C., Martinez-Sanchez, J., and Garcia-Rizo, C., Lead mobilization in
calcareous agricultural soils, in Selim, H. M. and Iskander, I. K., eds., Fate and
Transport of Heavy Metals in the Vadose Zone, Ann Arbor Press, Ann Arbor, MI,
1999, 328.
20. Parker, K. F., An Illustrated Guide to Arizona Weeds, The University of Arizona Press,
Tucson, AZ, 1982, 338.
21. Warnock, B. H., Wildflowers of the Guadalupe Mountains and the Sand Dune Country,
Texas, Sul Ross State University, Alpine, TX, 1974, 176.
22. Statistical Analysis System (SAS) Institute, Inc. SAS Users Guide: Statistics, Version
6.03, Cary, NC, 1988.
23. Gobran, G. R., Clegg, S., and Courchesne, F., Rhizospheric processes influencing the
biochemistry of forest ecosystems, Biogeochemistry, 42, 107120, 1998.
24. Schlesinger, W. H., and Pilmanis, S. M., Plant-soil interactions in deserts, Bio-
geochemistry, 42, 169187, 1998.
25. Banin, A., Navrot, J., and Perl, A., Thin horizon sampling reveals highly localised
concentrations of atmophile heavy metals in a forest soil, The Sci. Total Environ., 61,
145152, 1987.
26. Shuman, L. M., Effect of liming on the distribution of manganese, copper, iron and
zinc among soil fractions, Soil Sci. Soc. Am. J., 50, 12361240, 1986.
4 Fate of Radiocesium in
Soil and Rhizosphere
Bruno Delvaux, Nathalie Kruyts, Emmanuel
Maes, and Erik Smolders

CONTENTS

I. Introduction.....................................................................................................61
II. Environmental Fate of Radiocesium ..............................................................63
A. Atmospheric Deposition of Radiocesium ..........................................63
B. Target Ecosystems ..............................................................................63
C. Primary Interception by the Canopy ..................................................64
D. Foliar Uptake, Translocation, and Primary Contamination ...............64
E. Radiocesium Incorporation in the Biogeochemical Cycle ................64
F. Summary .............................................................................................65
III. Retention of Radiocesium in Soil ..................................................................67
A. Introduction.........................................................................................67
B. Mechanisms ........................................................................................67
1. Intrinsic Properties...............................................................69
2. Surrounding Conditions.......................................................73
C. Quantitative Analysis of Radiocesium Retention in Soils.................74
D. Radiocesium Retention and Soil Components...................................76
IV. Root Uptake and Soil-to-Plant Transfer.........................................................78
A. Root Uptake ........................................................................................78
B. Soil-to-Plant Transfer .........................................................................78
V. Mobilization of Radiocesium in the Rhizosphere .........................................79
VI. Conclusion ......................................................................................................82
References................................................................................................................83

I. INTRODUCTION
Since 1945 the radioisotopes 134Cs and 137Cs have been released in the environment
by three main routes: nuclear weapons testing, controlled discharge of waste effluents
from nuclear plants, and accidental release.1 After deposition, much of the radioce-
sium becomes disseminated in aquatic environments, but large quantities remain in
terrestrial ecosystems. From the extensive research on the environmental behavior
of radiocesium in weapons and accidental fallout, a general consensus emerged that
radiocesium exhibits a biogeochemical behavior rather similar to that of potassium,

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 61
62 Trace Elements in the Rhizosphere

a major plant nutrient. In this respect, radiocesium displays a relative mobility in


various ecosystems through uptake by plants and terrestrial and aquatic organisms.2
The Chernobyl nuclear accident has renewed interest in the environmental fate
of radiocesium because radioactive air plumes reached several European countries
and contaminated large territories, particularly forested areas.3 The isotope 137Cs was
one of the major radionuclides involved in that contamination. It poses considerable
environmental problems because of its relatively long half-life (30.17 years). Around
6% of the European territory exhibited a contamination level of Chernobyl-deposited
137Cs above 20 kBq/m2; that level was above 40 and 1480 kBq/m2 in 2.0 and 0.03%

of the European territory, respectively.4 A wide variety of ecosystems were contam-


inated, including agricultural, seminatural, and natural environments. The available
estimations in these ecosystems all show that soil acts as a major sink-source
compartment in 137Cs fluxes through plant and food chains.5-8 In fact, 137Cs-soil
contamination generates a long-term burden on food chains. For instance, food intake
in contaminated regions can contribute to 60% of the total radiation dose in man.9
A number of in situ measurements after the Chernobyl accident showed that nutrient
cycling and storage led to a much longer persistence of radiocesium in seminatural
ecosystems in comparison with agricultural cropping systems.10-13 That persistence
is crucial in managing contaminated lands.
As for other radiopollutants, the assessment of radiocesium contamination of
the above standing vegetation has long been based on the determination of Transfer
Factor (TF) or aggregated Transfer Factor (Tagg) coefficients. The former expresses
the ratio of radionuclide concentration in two compartments (soil and plant), while
the latter describes the transfer via a complete chain of parameters (e.g., from the
soil surface to the plant). Combining these transfer parameters and 137Cs, a solid-
liquid distribution coefficient (Kd) was used to assess the phytoavailability of radi-
ocesium in soil.3 Computational procedures for TF, Tagg, and Kd are described below:

concentration in plant Bq kg 1 Bq kg 1
TF = 1
or 3
(1)
concentration in soil Bq kg Bq dm

concentration in plant Bq kg 1
Tagg = (2)
concentration in soil Bq m 2

concentration in soil Bq kg 1
Kd = (3)
concentration in solution Bq l 1

Transfer factors and Kd consist of bulk measurements that do not accurately


predict the distinctive contributions of various soils to the 137Cs-plant contamination.
Generally speaking, trends in TF indicate that 137Cs soil-to-plant transfer can largely
vary between ecosystems and soils as well as in distinct horizons in a given soil.3
In this respect, two factors are of major importance in 137Cs soil-to-plant tranfer: the
type of vegetation and the soil properties.
Fate of Radiocesium in Soil and Rhizosphere 63

The uptake of radiocesium by plant roots and associated microflora is the first
step in plant, and hence food chain contamination. The chemical uptake of trace as
well as major elements is regulated by active and passive mechanisms occurring in
the rhizosphere, at the soil solutionroot interface. These processes and their inter-
action with the soil matrix, were recently reviewed in a state-of-the-art study.14 They
take place in physicochemical conditions that largely depend on ion exchange
reactions between soil components, soil solution, and plant root. The aim of this
chapter is fourfold. First, it will briefly review the environmental fate of radiocesium
and identify the major sinks and pathways for trace Cs. Second, it will focus on the
quantitative analysis and mechanisms of trace Cs retention in soil. Third, it will
briefly review the effect of ionic environment on root uptake of radiocesium as well
as recent approaches modeling the 137Cs soil-to-plant transfer. Fourth, it will consider
the mobilization of radiocesium in the rhizosphere and identify the key factors and
processes in this microenvironment.

II. ENVIRONMENTAL FATE OF RADIOCESIUM

A. ATMOSPHERIC DEPOSITION OF RADIOCESIUM


The atmospheric wet and/or dry deposits of radiocesium from nuclear weapon tests
in the 1950s and 1960s were estimated up to 1.5 1018 Bq, whereas that of the
Chernobyl accident (1986) amounted to 7.8 1016 Bq.4 The former deposits rep-
resent dispersed, relatively low level but chronic inputs, whereas the latter was a
pulse input involving a relatively high contamination level. As demonstrated after
the Chernobyl fallout, rainfall patterns have a decisive influence on the 137Cs con-
tamination of lands.15,16 The Chernobyl 137Cs far-fallout occurred in a soluble form
in western Europe,17 whereas the 137Cs near-fallout consisted of particle forms of
uranate and/or ferrite type.18 The very low solubility of these particles delays the
mobilization of radiocesium by the above standing vegetation.19,20

B. TARGET ECOSYSTEMS
The contamination of lands with radiocesium rapidly leads to its accumulation in
soil, in both agricultural areas as well as in natural and seminatural environments.
Radionuclide contamination of agricultural lands is obviously highly sensitive,
because of the direct burden on the food chain. Seminatural areas are recognized as
environments showing little interference with man (forests, upland pastures, moor-
land, marshland, peatland, alpine meadows, unimproved meadows, and tundras).
According to Poldini,21 seminatural can be best defined versus natural areas. In the
latter, the flora and fauna are original and spontaneous and the vegetation has not
undergone structural modifications by man. In the seminatural countryside the flora
and the fauna are still mainly native but the vegetation is greatly modified by human
intervention; the relationships between biological forms have been modified. The
dominant biological form has often been replaced and the derived vegetation no
longer belongs to the same formation as the original vegation.21 The seminatural
64 Trace Elements in the Rhizosphere

environments produce human food stuffs as well as various materials. The use of
these ecosystems is important for some population groups.22,23
After the Chernobyl accident, it was observed that the vegetation in natural and
seminatural environments was more highly contaminated with 137Cs than crops from
agricultural lands.10,11,24 Higher 137Cs biological half-lifes were measured in these
environments versus agricultural areas, demonstrating a longer persistence in the
natural and seminatural ecosystems.25 Consequently, one can expect in the forth-
coming years an important increase of radiocesium doses to man from the contri-
bution of these highly complex ecosystems.9,22,26 Among these environments, forest
ecosystems are particularly complex as they involve several vegetative strata as well
as diverse plant communities.

C. PRIMARY INTERCEPTION BY THE CANOPY


In forested areas, the tree canopy is recognized as an efficient interceptor for aerosols,
which can delay 137Cs accumulation in soil. Linkov27 reports interception rates
ranging between 20 and 100% of the initial 137Cs deposition versus 20 to 25% in
agricultural lands. In this respect, forested areas consist of target ecosystems with
respect to atmospheric radioactive depositions. The interception of radiocesium by
the forest canopy depends on various parameters related to the density of vegetation
cover, plant species, and development stage.3,28 Though located in the understory
vegetation, lichens and mosses play a significant sink role in the interception of
radiocesium because of their high water absorption capacity.29

D. FOLIAR UPTAKE, TRANSLOCATION, AND PRIMARY


CONTAMINATION
Part of the intercepted radiocesium is absorbed by leaves and translocated within
the plant,30,31 following the analogous element of potassium, a major plant nutrient.32
The rate of direct 137Cs absorption by leaf or needle depends to a large extent on
both the species characteristics and growth stage.3 The foliar uptake of radiocesium
directly after deposition can represent the primary source of plant contamination.33
The radiocesium that is not absorbed by leaf or needle is transferred to the soil by
rainfall through stemflow and wind as well as by the shedding of leaves and nee-
dles.28,34-36 Less than two years after the fallout event, the unabsorbed radiocesium
ends up at the surface of the soil.35 In contaminated forests in Germany, the radio-
active fallout resulted in the rapid accumulation of radiocesium in the surface
horizons of mineral soils as more than 90% of the total deposited radiocesium was
measured in the top 4 to 6 cm in less than 2 years.37 In a coniferous stand in the
Chernobyl area, Thiry et al.31 have shown that 89% of fallout radiocesium was
located in the surface horizon 3 years after the accidental pulse.

E. RADIOCESIUM INCORPORATION IN THE BIOGEOCHEMICAL CYCLE


Once in the surface horizons of the soil, the fate of radiocesium can follow three
main routes: strong retention by soil particles, migration with percolating soil solu-
tion, and uptake by plant roots and microorganisms. As radiocesium is strongly
Fate of Radiocesium in Soil and Rhizosphere 65

retained in mineral, trace Cs migration rates are quite small and generally less than
2 cm yr1.35,38,39 The major pathway for plant contamination is root uptake. Tradi-
tionally, the assessment of the contamination of above standing vegetation with
radiocesium is commonly based on the determination of Transfer Factor (TF) or
aggregated Transfer Factor (Tagg) coefficients expressing the ratio of radionuclide
concentration in the plant to that in soil. A number of in situ measurements have
been accumulated in several studies, particularly after the Chernobyl accident.
Reported TFs exhibit large seasonal as well as yearly variations in the same eco-
system.40-44 Also, they vary largely between ecosystems, soils, plant, and fungal
species. Figure 4.1 gives a global picture of reported 137Cs Tagg values measured in
agricultural as well as seminatural environments in some case studies.
The range of the orders of magnitude of the 137Cs Tagg mean values is 102 to
10 m2 kg1 in seminatural environments and 104 to 101 m2 kg1 in agricultural
1

areas. In the latter areas, some Tagg values can be as low as 5 106 m2 kg1. Noteworthy
is the fact that the 137Cs soil-to-plant transfer factor is higher in seminatural envi-
ronments than in agricultural lands. Such high transfer contributes to a much longer
persistence of radiocesium in the seminatural ecosystems, as illustrated in a number
of papers.10,12,13,47,48 In contaminated seminatural environments, mushrooms are well-
known accumulators of radiocesium (Figure 4.1).24,49,50 On a dry weight basis, fungi
accumulate between 10 and 150 times more radiocesium from soil than vascular
plants, as recently reported in the extensive reviews by Avery1 and Nimis.3 Among
the higher plants, heather, Calluna vulgaris, accumulates more radiocesium than
other species.42 The uptake of radiocesium by the understory vegetation largely
contributes to the contamination of the forest phytocenosis.3 Though not illustrated
in Figure 4.1, variations in TF or Tagg values can be related to soil properties in
seminatural environments, as reviewed by Nimis.3 In this respect, vegetation growing
in organic soils appear to be more susceptible to radiocesium contamination than
that growing in clay soils.51 Such a relationship can also appear in agricultural areas,
as a trend in decreasing Tagg values can be observed from peaty to sandy and clayey
soils (Figure 4.1). However, so far, trace Cs TF or Tagg values measured in situ have
never been significantly related to any commonly measurable soil property such as
pH, CEC, organic matter or clay content, or K level and saturation, etc. in a
mechanistic way. Even in controlled pot experiments involving agricultural soils of
widely varying textural classes, 137Cs+ TF is unrelated to clay content.50 Relatively
high radiocesium soil-to-plant transfer has been generally associated with peaty,
organic-rich soils as well as podzols and some sandy soils.51,53-58 In contrast, clay-
rich soils have been generally associated with relatively low 137Cs TF.3

F. SUMMARY
A very high variability is thus generally observed in 137Cs soil-to-plant transfer
patterns, particularly in seminatural environments. That variability requires a com-
prehensive understanding of the microbiogeochemical processes involved in the fate
of radiocesium in soils. Indeed, three main processes will govern the fate of radio-
cesium once accumulated in soil: uptake by living organisms, migration with the
soil solution, and retention on soil particles. A dynamic integration exists between
66
Trace Elements in the Rhizosphere
FIGURE 4.1 Aggregated transfer coefficients for 137Cs+ to food products from seminatural ecosystems and agricultural areas. The solid points represent
the expected values and the vertical lines represent the range of the values. (References: no asterisk:I.A.E.A.45; *Horrill et al.46; **Salt and Mayes41;
***Salt and Mayes42.)
Fate of Radiocesium in Soil and Rhizosphere 67

these processes in soil. More particularly, these processes and their interactions take
place with the highest intensity in the rhizosphere, i.e., the soil volume influenced
by root activity.14 The uptake of major and trace elements takes place in a physico-
chemical environment that depends to a large extent on ion exchange reactions
between the soil matrix, the liquid phase, and the plant root with associated micro-
flora.14,59 In this respect, soil particles and hence soil minerals are key components
of the rhizosphere as they closely interact with biota.14 The next two sections will
be devoted to i) the behavior of radiocesium in soil at the interface solid-solution
and its relationship with soil components, and ii) the uptake of radiocesium by plant
roots at the interface solution-root. The fifth section will involve the mobilization
of radiocesium in the rhizosphere, in which plant roots strongly interact with soil
minerals. Rhizospheric processes are likely to be of major importance in seminatural
ecosystems, because the soil is not fertilized.

III. RETENTION OF RADIOCESIUM IN SOIL


A. INTRODUCTION
Soil colloids can control the concentration of trace elements in the liquid phase and
hence their transfer to plants through root uptake. Sorption/desorption experiments
have been commonly used to characterize trace Cs interactions with soil colloids.
Sorption yield is measured at the end of the sorption process: here, it is the proportion
of sorbed Cs of the initial Cs contamination. Cs retention is measured at the end of
the desorption process, characterized by a plateau: it is the proportion of retained
Cs of the initial Cs contamination. Among soil colloids, organic matter weakly
retains trace Cs in soil. Indeed, the retention of radiocesium by soil organic matter
is reported to involve chiefly electrostatic binding, even if chelating agents are
present.60,61 To the contrary, radiocesium is specifically retained by micaceous clay
minerals, as documented by literature.62

B. MECHANISMS
The sorption of trace cesium by soils, sediments, and clay minerals has been widely
studied, as recently reviewed by Cornell.62 It is now generally recognized that
specific adsorption of radiocesium is associated with the presence, even in very
small quantity, of micaceous clay minerals. In particular, the interlayer space of
these phyllosilicates exhibits areas formed by the juxtaposition of nonexpandible
(1.0 nm) and hydrated (1.4 nm) zones. Such areas are called wedge zones and their
associated sites are the frayed edge sites (FES).63-65 The FESs contribute to less than
2% of the overall CEC.65,66 They selectively sorb poorly hydrated alkaline cations
K+, Rb+, Cs+ as well as NH4+ ion. Because they easily loose their hydration water,
these cations are specifically sorbed in the wedge zones. If the concentration of
such cations is sufficient, their specific sorption in the wedge zones induces the
collapse of the interlayer space, and hence their fixation by the layer silicate. This
process is illustrated in Figure 4.2 in which three steps of the collapse are schemat-
ically represented.
68 Trace Elements in the Rhizosphere

FIGURE 4.2 Schematic illustration of the collapsing process of the interlayer space of 2:1
micaceous phyllosilicates with increasing Cs+ saturation of interlayer sites (respective layer
and interlayer thickness are not at scale).

The collapse forms new wedge zones, which are closer to the border of the
particle. The hydrated cations such as Na+, Ca2+, and Mg2+ maintain expansion of
the interlayer space, but are not sorbed in the wedge zones. Thus, the low hydration
energy is the major factor explaining the specific sorption of trace Cs+ by micaceous
minerals.64 For that reason, the decrease in cation selective adsorption by the FESs
follows the increase of the hydration energy (Cs+ > Rb+ > NH4+ > K+). Similarly, the
relatively low hydration energy of Cs+ explains why hydrated cations can readily
induce the expansion of the interlayer space of a micaceous clay mineral exchanged
with K+, but not with Cs+.64
It is generally accepted that the adsorption of radiocesium by micaceous phyl-
losilicates can be described in terms of at least three kinds of sites exhibiting a
decreasing selectivity for poorly hydrated cations.64-68 The location of the three kinds
of sites in a micaceous clay mineral is schematically illustrated in Figure 4.3.
Fate of Radiocesium in Soil and Rhizosphere 69

FIGURE 4.3 Schematic view of the various kinds of sites in an idealized structure of 2:1
micaceous phyllosilicates (interlayer distances are purposely overmagnified for clarity).

First, the frayed edge sites (FES) are located in the wedge zones of the mineral,
as described above, and specifically sorb the poorly hydrated alkaline cations and
NH4+ ion. Second, the hydrated interlayer sites are located in the expanded interlayer
zones, in which the hydrated cations can be exchanged with poorly hydrated cations.
This ion exchange process can, however, induce the collapse of the interlayer space.
Third, the regular exchange sites (RES) are planar sites located on the external
surfaces of the phyllosilicate and regular ion exchange processes occur on such sites.
These processes involve exchange surfaces that are freely accessible to all ions,
hence ion exchange will be governed by valence and hydration effects. Valcke69 as
well as Hird et al.70 have measured a decreasing selectivity for trace Cs+ ions in the
FESs with increasing distance from the collapsed interlayer sites (Figure 4.3), i.e.,
the mica core (1.0 nm), toward the expanded hydrated zone (1.4 nm). Such decreas-
ing selectivity for Cs+ sorption is due to increasing accessibility of competing cations,
which are more hydrated than Cs+. In fact, defining various kinds of sites, each
exhibiting its own and precise selectivity for trace Cs+, is likely a theoretical view
that gives some useful description, but oversimplifies the reality.
The selective adsorption of poorly hydrated cations and their retention by 2:1
phyllosilicates both depend on a number of factors that can be classified in terms
of intrinsic properties of the mineral itself and external conditions related to the
environment of the mineral particles.

1. Intrinsic Properties

The intrinsic properties of the 2:1 phyllosilicates that play a key role in the Cs+
selective sorption are: the layer charge, the octahedral occupancy, the extension
of the wedge zones, the interlayer occupancy, the particle size, and the layer
stacking order.
70 Trace Elements in the Rhizosphere

The selective sorption of Cs+ ions is obviously promoted by a high layer charge
of the 2:1 phyllosilicate. In this respect, the selectivity for Cs+ would decrease in the
order mica (illite) vermiculite smectite.62,63,71 The location of the negative
charge, i.e., its distribution in the octahedral and/or tetrahedral sheet, has often been
evoked as a factor influencing the selective sorption of poorly hydrated cations. The
tetrahedral charge would exert a more direct influence on layer collapse than the
octahedral charge would, because of the proximity of the tetrahedral sheet relative
to the interlayer space.72 In this respect, a larger Cs concentration is required to induce
the collapse of smectites with dominant octahedral charges versus smectites with
dominant tetrahedral charges.73 However, Kodama et al.74 have shown in phlogopite
that the location of the charge was less a determinant than the octahedral occupancy
for the release or extractability of interlayer potassium. These authors suggested that
the location of the charge would play more of a key role on the swelling behavior of
the mineral after K extraction than on the K extractability sensu stricto.
The di- or trioctahedral character of the phyllosilicate measures the octahedral
occupancy of the mineral. It is well known that K+ ions are retained much more in
dioctahedral micaceous phyllosilicates than in the trioctahedral ones.75 The orienta-
tion of the OH dipole has long been considered as the major factor in this differ-
ence.75-77 The orientation of the OH group is quasi-perpendicular in trioctahedral
minerals, whereas it shifts toward the octahedral vacancy in the dioctahedral min-
erals, making an angle of 16 with the (a,b) plane of the layer silicate. In the latter
case, a larger distance between interlayer K+ and the proton of the OH group involves
a lower cation-to-cation repulsion and hence a larger stability in the dioctahedral
minerals versus the trioctahedral ones.75 In fluoride-rich micas, however, the K+H+
repulsion does not occur as the F ion replaces the OH dipole.78 The value of the
rotation angle of the tetrahedrons is another factor influencing the difference in
stability between the di- and trioctahedral minerals because the tetrahedron rotation
is larger in the former minerals.79
The extension of the wedge zones, i.e., the number of specific Cs+ sorbing
sites,71 increases in the order mica (illite) vermiculite.62,71 Using Rb+Ca2+ ion
exchange on a progressive K-depleted biotite (1.0 nm), Le Roux and Rich80 dem-
onstrated that, with increasing K depletion, the selectivity for Cs sorption first
increased, reached a maximum, and further decreased at the full expanded stage of
vermiculite (1.4 nm). Such variation is caused by the extension of the wedge zones
during the process of vermiculitization. The wedge zones are initiated at the border
of the mineral particles and multiplied during the early stage of the transformation
of mica. Their number can be regarded as being steady in the intergrade structure
mica-vermiculite. A further expansion leads to the coalescence of the hydrated
interlayer sites (Figure 4.3), the progressive disappearance of the wedge zones, and
hence the decrease in selectivity for Rb+ ions. Similarly, larger Cs sorption yields
have been measured after K removal from a biotite.81,82
The extension of both the wedge zones and their associated hydrated interlayer
surfaces obviously depends on the interlayer occupancy (Figures 4.2 and 4.3). In
this respect, a full occupancy of the interlayer space by K in mica reduces the
sorption yield of Cs+ ions in a large range of Cs concentration in the equilibrium
solution.81,83 Similarly, aluminium interlayering of vermiculites reduces the CEC of
Fate of Radiocesium in Soil and Rhizosphere 71

the mineral as well as K fixation84 and Cs sorption.82,85 Hydroxy-Al interlayer groups


are positively charged, but nonexchangeable polycations act as props that maintain
the basal distance at 1.4 nm.86 As such, they inhibit the collapse of the 2:1 layers 86
(Figure 4.2) and, therefore, strongly reduce Cs retention.82
Literature data thus converge to show that several intrinsic properties of the
mineral promote the selective sorption of poorly hydrated cations. These properties
are: the dioctahedral character, a high layer charge, a large number of wedge zones,
and a low degree of occupancy of the interlayers by nonexchangeable cations. In a
recent study, Maes et al.82 demonstrated that the fate of trace radiocesium at the
solid-liquid interface strongly depends on these properties, which are affected by
the weathering of mica. These authors measured the 137Cs retention at each of the
seven stages of a laboratory weathering sequence biotite (B) trioctahedral ver-
miculite (Vt) oxidized vermiculite (Vo, partly dioctahedral) Al-hydroxy inter-
layered vermiculite (HIV, both trioctahedral -HIVt- and oxidized -HIVo-). The HIVs
were placed in an Al-depleted environment using citrate (HIVt-ci and HIVo-ci). Their
results are summarized in Figure 4.4.
The parent biotite and its weathering products exhibit similar trace Cs sorption
yields. However, the 137Cs net retention values largely differed between the various
minerals. As illustrated in Figure 4.4, the interlayer occupancy by either K in biotite
or Al-polymers in HIV strongly reduces the retention of trace Cs. The transformation
mica vermiculite largely increases the 137Cs net retention. Though both vermic-
ulites (Vt, Vo) retain similar Cs amounts in a K+-Ca2+ scenario [total chloride con-
centration of 103 M and a potassium adsorption ratio (PAR) of 0.057 mM0.5; PAR
is defined as the aK/ aCa where a refers to ionic activity], the oxidized vermiculite
retains much more trace 137Cs in acid conditions (pH 3, desorption with protonated
resin), because of its dioctahedral character. Noteworthy is the fact that extracting
Al-polymers leads to the recovery of the expanded vermiculite, and hence to the
high retention of radiocesium.
The available data thus converge to show that intergrade structures such as mica-
vermiculite, i.e., weathered mica with dioctahedral domain or partly collapsed ver-
miculite, offer the mineralogical properties that strongly promote Cs+ fixation. In
other words, strong Cs+ fixation occurs on vermiculitic sites associated with mica-
ceous wedge zones.
The particle size influences the weathering of mica.75 Layer weathering occurs
in clay-sized particles (< 2 m): some interlayer spaces are completely opened from
one particle edge to the other whereas remnants of collapsed interlayers are stabilized
likely because of a change in the orientation of the octahedral OH dipole.87 Edge
weathering occurs in larger particles (> 2 m): a number of edges open simulta-
neously and weathering progresses from the edge to the mica core, producing a large
number of wedge zones.75 The ratio [surface of collapsed interlayer space (1.0 nm)
: surface of FES] is larger in clay-sized particles versus larger particles; consequently,
the small particles retain much more Cs than the large ones.64, 88
The layer stacking order in the mineral can have major incidence on the
retention of poorly hydrated cations. Gaultier and Mamy89 have shown that wetting-
drying cycles applied on a K+-exchanged smectite lead to an increase in the layer-
to-layer stacking order in the phyllosilicate, and hence to the illitization of smectite.
72 Trace Elements in the Rhizosphere

FIGURE 4.4 Schematic illustration of the variation in 137Cs+ sorption and retention (after
sequential desorption using first a K+-Ca++-saturated resin and then an H+-resin) of a laboratory
weathering sequence mica vermiculite hydroxy interlayered vermiculite (HIV)
vermiculite. (Adapted from Maes, E. et al.82)
Fate of Radiocesium in Soil and Rhizosphere 73

Similarly, Maes et al.90 demonstrated that increasing the stacking order could form
highly selective Cs-exchange sites in montmorillonites and increase Cs retention by
the clay minerals. Prolonged wetting-drying cycles were shown to largely increase
the retention of radiocesium by soils of widely varying properties, despite their
initially low retention.91

2. Surrounding Conditions

These conditions involve the composition of the solution surrounding the sorbing
sites (concentration of Cs+ and other ions, ionic strength), the solid to solution ratio
as well as the time of contact between the Cs+ sorbing particles and the solution.
Traditionally, the behavior of radiocesium at the solidsolution interface has
long been described in terms of Cs+ solid-liquid distribution coefficient (Kd). The
effect of Cs concentration on Kd has been well studied.62 At Cs concentration less
than 108 M, it seems that Kd is independent of Cs concentration in the equilibrium
solution. At larger concentration, Kd decreases with increasing Cs concentration and
consequently, the Cs sorption is nonlinear.66,92,93 Such nonlinearity is generally inter-
preted as being the expression of the heterogeneity of the Cs sorbing sites.64,90,91
With increasing Cs concentration, the FESs are progressively more and more satu-
rated with Cs+ ions. Consequently, further sorption of Cs+ ions involves exchange
sites with decreasing selectivity (hydrated interlayer sites, RES); as a result, the
mean affinity of the exchanger for Cs decreases, hence Kd diminishes. The Cs
concentration also influences the reversibility of the Cs sorption process. A very
elegant study by Hird et al.70 shows that the KCl-extractable Cs increases with
increasing Cs saturation of the FESs, but abruptly drops at a sorbed Cs level of 10
mmol kg1 in podzolic soils. That drop is attributed to the layer collapse induced by
the increasing Cs loading on the FESs (Figure 4.2).
The electrolyte background of the equilibrium solution is of major importance
in Cs sorption, because of the presence of both competing ions (K+, NH4+) and other
ions (Ca2+). Poorly hydrated cations such as K+ and NH4+ influence the Cs sorption
in two respects: i) they compete with Cs+ ions for sorption on the same exchange
sites66,67,94, and ii) they can induce layer collapse, and hence Cs trapping (Figure
4.2).88,95 Potassium and ammonium salts have long been used to extract exchangeable
Cs+ ions in soils and clays.93,96,97 Increasing concentrations of K+ and NH4+ ions in
the equilibrium solution decreases the Cs sorption yield in micaceous phyllosilicates
and soils.83,92,98-100 As K+ and NH4+ are collapse inducing cations, they can provoke
Cs trapping in micaceous phyllosilicates. The entrapment of Cs is caused by simul-
taneous introduction of Cs+, K+, and NH4+ ions in the surrounding solution,88 whereas
the edge interlayer trapping of radiocesium occurs when K+ and NH4+ ions follow
the Cs+ contamination.95 The K+ and NH4+ ions can therefore promote either the
release of trace Cs or its fixation through the trapping of the trace element. The
predominance of the ion competition effect or the Cs trapping effect over each other
will chiefly depend on the concentration of competing ions.88,95,101,102 Such predom-
inance depends also on the location and extension of the wedge zones95 as well as
on the particle size.88 With respect to the concentration, Absalom et al.102 demon-
strated that trace Cs is reversibly sorbed at PAR < 101.5 mM0.5, whereas it is
74 Trace Elements in the Rhizosphere

entrapped at PAR > 101.5 mM0.5. In other words, with increasing K concentration,
K+ ions first exchange with Cs+ ions to displace them from exchange sites and further
cause Cs trapping through layer collapse. Regarding the extension of the wedge
zones, it can be expected that K+ and NH4+ ions will act as Cs+-competitive ions if
that extension is limited (mica, weakly weathered or not) and as trapping ions if
that extension is large (strongly weathered mica, mica-vermiculite).
Cs sorption in micaceous phyllosilicates pre-equilibrated with Ca2+ ion is larger
and more rapid than that measured in the corresponding K+-saturated clay miner-
als.98,103 In addition, the Ca2+ ion seems to promote the slow migration of Cs in
deeper wedge zones of the mineral.103
Increasing the ionic strength of the equilibrium solution generally decreases
KdCs, as recently reviewed by Cornell,62 likely because of the contraction of the
diffuse double layer. The increase in the ionic strength leads to increasing values of
the Gaines-Thomas selectivity coefficient Kc(Cs+/Mn+) on illite (Mn+ = K+, Na+:
Brouwer et al. 66) and montmorillonite (Mn+ = K+, Na+, Ca2+: Staunton and Roubaud
104); such increase has been attributed to, respectively, the hydration properties of

exchanged cations and, partly, the increase of the size of the clay tactoids.
As KdCs depends on Cs+ concentration below a certain level (108 M), KdCs
decreases with decreasing solid to solution ratio in a nonlinear adsorption sce-
nario.62,105 The contact time between solid and solution seems to play a major role
on trace Cs sorption yield as well as on the reversibility of the sorption process.
Comans et al.103 observed a rapid Cs sorption on illite followed by a slow sorption
phase, the extent of which can last several weeks. Increasing sorption time leads to
an apparent decrease in the reversibility of the Cs sorption process, attributed by
these authors to slow migration of Cs into deeper wedge zones and likely to sorption
onto FESs with very high Cs specificity. Using a multidesorption technique shows
that the effect of sorption time is particularly important in mica-derived smectite,
whereas it is very small on illite and biotite (unpublished data). Wauters et al.106 and
Absalom et al.102 have also observed the time-dependent decrease of release of
radiocesium in the solution. As suggested by Comans et al.,103 strong Cs retention
is thought to occur as radiocesium slowly migrates in the collapsed interlayers of
micaceous clay minerals.95,102 This aging effect seems to reduce the phytoavailability
of radiocesium in agricultural soils.25, 107, 108

C. QUANTITATIVE ANALYSIS OF RADIOCESIUM RETENTION IN SOILS


Several approaches have been developed to quantify the retention of radiocesium in
soils. The traditional one is based on the use of Kd, which is not an intrinsic Cs+-
binding property. Comparison of Kd values has long been problematic, because
measurements were not made in similar electrolyte background conditions. Simi-
larly, Cs+ sorption-desorption data can hardly be used on a general basis, but such
data can be useful to compare soil or clays in a common experimental framework.82
Cs+ sorption has been described in a satisfactory way by using the Freundlich
isotherm.93,104,109,110
A major breakthrough in the quantitative analysis of radiocesium retention was
achieved with the RIP theory, which is briefly described here. Cremers et al.65
Fate of Radiocesium in Soil and Rhizosphere 75

quantified the number of FESs in soils by using the silver thiourea (AgTU) complex
to mask the RESs. They measured numbers of FESs contributing 0.01 to 2% of the
overall CEC and quantified the trace Cs+ to K+ selectivity of the FESs (mean
KcFES(137Cs+/K+) 103).65 The same research group57,94,111,112 further developed this
quantitative analysis and demonstrated the following equation:

RIP = KcFES(137Cs/K) [FES] = KDCs mK (mol kg1) (4)

where RIP, KcFES(137Cs/K), [FES], mK, and KDCs refer, respectively, to the radioce-
sium interception potential, the trace 137Cs to K selectivity coefficient, the number
of FESs, the fixed potassium molar concentration in the equilibrium solution,112 and
the 137Cs solid-liquid distribution coefficient measured in an electrolyte background
confining both Cs+ and K+ ions onto the FESs using AgTU94 or a fixed K+-Ca2+
scenario.112 In a very large majority of soils, the mean value of KcFES(137Cs/K)
amounts to 103.57,61,65,69,94,112 However, some of the FESs can exhibit exceedingly
high Cs to K selectivity (KcFES(137Cs/K) ~ 2 103).65 They are likely the vermiculitic
sites associated with micaceous wedge zones, as suggested by Maes et al.82,113 In
comparison with such high Kc values, the trace Cs to K selectivity in the RES pool
is quite small. Indeed, KcRES(137Cs/K) ranges between 1.0 for humic compounds114
to 5.0 in clay minerals.115 Noteworthy is the fact that lnKcRES(137Cs/K) is about 6.92
0.56 in a wide variety of soils and sediments.112 Such a trend shows that the same
type of Cs specific site is operating in various soils and sediments. Consequently,
the RIP, which combines Cs specific site selectivity and capacity, is an extremely
important property of the system.57,94 That property integrates the quantity-intensity
(Q-I) relationship.116 As such, RIP could be a useful tool to predict the potential
phytoavailability of radiocesium in soils.57
RIP values are presented in Tables 4.1 and 4.2 to illustrate their range in soils
and sediments, respectively.

TABLE 4.1
Ranges in Organic Matter, Clay Contents, and RIP
Values Measured in Various Soil Collections
Number of O.M.a Clay RIPKb RIPNc
Source Samples % % mol g1 mol g1

[57] 8 2388 323 652450 16450


[94] 12 0.58.6 0.533 674890 19882
[69] 33 797 79199 21482
[117] 30 228 0.536 545861
[118] 88 228 0.540 5011200
[121] 47 0.596 0.766 134861
a Organic matter.
b RIPK = KDCsmK+.
c RIPN = KDCsmNH4+ [see Equation (1)].
76 Trace Elements in the Rhizosphere

TABLE 4.2
Ranges of RIP Values Measured in Various Types of
Sediments and Montana Illite; Mean Values of the NH4+
to K+ Selectivity Coefficient of the Frayed Edge Sites
Sediment Type Number of RIPKa RIPNb KCFES (NH4+-K+)
or Mineral Samples mol g1 mol g1 (Mean Value)

Lacustrine 23 4588 101150 5.45


Estuarine 7 16008.6 3401010 5.71
Riverine 33 14597 12555 4.79

Montana illite 12600 2420 5.2


a RIPK = KDCsmK+.
b RIPN = KDCsmNH4+ [see Equation (1)].

Source: Adapted from Wauters, J.61

The reported values of RIPK [RIPK = KDCs mK: see Equation (1)] cover four
orders of magnitude (10 104 mol g1). Such a range illustrates fairly well the huge
variability of Cs-binding properties in soils and sediments. The range in RIPK values
are quite the same in soils and sediments, providing strong indication that basic
mechanisms in trace Cs retention are similar in both soil and sediment environments.
Table 4.1 also shows that soils rich in organic matter can exhibit substantial RIP, and
hence genuine FESs,69,70 borne by a very small amount of clay particles present in
these soils, as Cs retention by organic matter involves only electrostatic binding.114
As inferred from both Tables 4.1 and 4.2, the values of RIPN (KDCs mNH4+) are
invariably lower than those of RIPK. Such a difference confirms the preference of NH4+
over K+ ions for the sorption on the FESs. The mean NH4+ to K+ selectivity coefficient
KcFES(NH4+/K+), deduced from the ratio [RIPN:RIPK], is about 5 in sediments (Table
4.2). In fact, it generally ranges between 4 and 6 in various soils and sediments.57,94,112
Such a narrow range is an additional fingerprint of the specific action of the FESs and
the common mechanisms of trace Cs retention in soils and sediments.94,112

D. RADIOCESIUM RETENTION AND SOIL COMPONENTS


In agricultural soils, several attempts to understand the determinism of trace Cs
retention in soil have shown that this parameter is unrelated to any usual soil
property.52,119
Radioecologists have long debated about the respective roles of clay minerals
and organic compounds on the retention of radiocesium, because several observations
have suggested contrasting statements, particularly in forest soils. In particular, the
marked differences in Cs retention and soil-to-plant TFs between soil horizons gen-
erally pointed to strong retention and low TFs in organic rich horizons such as OAh
and Ah. Recent studies113,120 have supported the validity of the laboratory weathering
model,82 illustrated in Figure 4.4, in some forest acid soils. Radiocesium is specifically
Fate of Radiocesium in Soil and Rhizosphere 77

FIGURE 4.5 Log-log plot of the estimated FES capacity against the number of vermiculitic
sites. Adapted from Maes et al.113 and Delvaux et al.121 (see Table 4.1). Open symbols refer
to the weathering sequence acid brown soil podzol.

retained on vermiculitic sites neighboring micaceous wedge zones. In acid soils, Al-
interlayering of vermiculite leads to the formation of HIV minerals (hydroxy inter-
layered vermiculite), which weakly retain radiocesium. Organic matter can therefore
influence the retention of trace Cs through Al-complexation. Indeed, this process
induces Al-depletion in the soil solution and hence impedes Al-interlayering and
maintains the interlayer sites accessible for Cs retention. In this respect, the role of
soil organic matter on trace Cs+ retention is obviously indirect. In a weathering
sequence acid brown earth podzol (see Figure 4.5), Maes et al.113 further showed
that Cs retention was directly related to the soil vermiculite content, which reflects
to some extent the weathering of mica in soils because vermiculite content was
strongly horizon and depth dependent. These findings largely contribute to under-
standing the marked differences of 137Cs retention properties between the various
horizons in forest soils. Such differences were long not understood in a mechanistic
way.3 The retention of radiocesium can indeed greatly vary within very short vertical
distances in forest acid soils, particularly in those exhibiting thick humus layers.48
The RIP as measured according to Wauters et al.112 in the soil weathering sequence
78 Trace Elements in the Rhizosphere

studied by Maes et al.113 as well as in other soils121 (Table 4.1) leads to computed
[FES] capacity using KcFES(137Cs/K) 103 in equation (1), which correlates fairly well
with the number of vermiculitic sites (r = 0.75, n = 43) (Figure 4.5).
Figure 4.5 shows that the samples representing the weathering sequence brown
soil podzol113 are fairly well distributed over a large range of both estimated FES
capacity and vermiculite content, as do the samples representing a larger collection
of soils with widely varying properties121 (Table 4.1). This observation is consistent
with the conclusion that vermiculitic sites associated with micaceous wedge zones
control, to a very large extent, the retention of radiocesium in soils. In particular,
soil horizons in which the weathering of mica is active would strongly retain
radiocesium in soils.
The available data from clay, soil, and sediment studies indeed converge to show
that weathered mica and/or mica-vermiculite intergrade appear to be the mineralog-
ical structures that best fix radiocesium. The immediate structural environment of
fixed Cs in the frayed edge site exhibiting exceedingly high trace Cs to K selectivity
is, however, not yet fully characterized.

IV. ROOT UPTAKE AND SOIL-TO-PLANT TRANSFER


A. ROOT UPTAKE
The uptake of radiocesium is proportional to its concentration in the solution around
plant roots. However, root uptake of 137Cs is significantly reduced with increasing
K concentration in soil solution, at K concentration below 1 mM.117,122-125 Above 1
mM K, varying K concentration has no significant effect on trace Cs uptake.52,123 In
a nutrient-solution experiment, Smolders et al.124 have shown that the uptake of 137Cs
was most sensitively affected by K supply between 50 and 250 M K. They also
showed that K was from 3.9 to 25 times more selectively taken up than 137Cs.124
Root uptake of radiocesium is slightly reduced by increasing NH4+ concentrations
and unaffected by stable Cs concentration between 0 and 10 M of potassium,123
whereas it decreases with increasing Ca and Mg concentrations.52,126 The large
increase in trace Cs uptake rate at K concentration below 1 mM is, however, strongly
dependent on plant species.127

B. SOIL-TO-PLANT TRANSFER
Many reports demonstrate that the 137Cs soil-to-plant transfer largely varies between
soils (see above). Though extensive knowledge has been gathered on the separate
soil and plant factors likely to control 137Cs availability in soil, few studies have
analyzed the soil-to-plant transfer of radiocesium in a mechanistic
way.57,117,124,125,128,129 In meadow upland areas, the RES pool [KcRES(137Cs+/K+)1] con-
trols the concentration of radiocesium in the soil solution in organic-rich soils,
whereas the FES [mean KcFES(137Cs+/K+)103] pool does it in mineral soils.57 Conse-
quently, the 137Cs TF values are much lower in the latter soils than in the former ones.57
In a pot experiment study involving 30 agricultural soils with a wide range of
properties, Smolders et al.117 have shown that the 137Cs soil-to-plant transfer was very
Fate of Radiocesium in Soil and Rhizosphere 79

well predicted by the 137Cs and K concentrations in the soil solution. In this respect,
the bioavailability of radiocesium varies extensively between soils because of differ-
ences i) in 137Cs retention, affecting 137Cs concentration in solution, and hence its
supply to plant roots, and ii) in K availability, affecting the 137Cs root uptake process.
Based on these concepts, a soilplant 137Cs transfer model was developed by Absalom
et al.119 The soil solution concentrations of 137Cs and K were estimated from two soil
characteristics, the percent clay and the exchangeable K content, and from the total
137Cs content in soil and the time after the contamination. The model successfully

predicted soilplant transfer of 137Cs for a wide range of arable crops.


Recently, Sanchez et al.130 conducted a pot experiment study involving 23
organic-rich soils from seminatural environments. They demonstrated that high
transfer of radiocesium in these soils was related to both the high Cs mobility and
low K availability. High Cs mobility was related to a low clay content and a high
NH4+ concentration in the soil solution. This means that low RIP and more effective
NH4+-competition for mineral sorption sites (see Section III, D) enhanced trace Cs
mobility in these soils.130
Unpublished data by Thiry et al., based on a five-month pot experiment, show
that the 137Cs contamination of young spruce plants [Picea abies L. (Karst.)] from
a forest soil described by Maes et al.120 originated almost exclusively from the top
organic layer Of. In fact, the global contamination of the spruce plantlets was related
i) positively to the organic matter content and ii) negatively to the vermiculitic
character of the soil clay fraction. These authors also showed that the respective
horizon contributions to the total 137Cs soil-to-plant transfer were 92.71% in Of,
0.18% in OAh, 3.28% in Ah, and 3.83% in Bw. They suggested that the 137Cs TF
was controlled by the RES pool in the organic horizon Of and by the FESs in the
organo-mineral horizons OAh and Ah. Such a difference in radiocesium availability
for root uptake in distinct soil horizons of the forest soil was due to contrasting clay
mineralogy between horizons. Such a difference in radiocesium availability in forest
soils was clearly demonstrated for fungi.131 The largest contamination of fungi was
associated with the mycelia exploring specifically the top organic layers. In contrast,
lowest fungal contamination by radiocesium was associated with mycelia specifically
exploring deeper organo-mineral and mineral horizons.131

V. MOBILIZATION OF RADIOCESIUM IN THE RHIZOSPHERE


Though soilplant transfer is the first step by which radiocesium enters the food
chain, it has been scarcely studied in the rhizosphere, i.e., the soil volume influenced
by root activity. The rhizosphere composition differs from the soil further away from
roots because of differences in pH, ionic strength, redox potential, the presence of
root exudates, and the presence of specific rhizosphere microflora. All these factors
affect the physicochemical processes controlling trace element mobility and avail-
ability, making the rhizosphere a unique environment where plant roots and soil
minerals strongly interact.132
There are no direct observations on the 137Cs chemistry in the rhizosphere, and
most data are obtained indirectly from modeling.124,128 The rhizosphere gradients in
80 Trace Elements in the Rhizosphere

TABLE 4.3
Predicted and Observed 137Cs Activity in Wheat Shoots Grown in a
Contaminated Soil at Different K Supply
Bulk Soil Solution Predicted Soil-Root Interface Predicted Observed
K Application K 137
Cs Shoot 137Cs K 137
Cs Shoot 137Cs Shoot 137Cs
mmol pot1 M cpm mL1 cpm g1 (M) cpm mL1 cpm g1 cpm g1

0 170 4.7 345 44 2.8 4444 6192


2 810 7.8 244 776 7.7 249 755

Note: The predictions are based on soil solution composition of the bulk soil or rhizosphere soil and on
137Cs uptake characteristics measured in solution culture. The soil solution composition in the rhizosphere

was calculated with a solute transport model.

Source: Modified from Smolders, E. et al.124 and McLaughlin, M. J. M. et al.132

pH and exudate concentrations are unlikely to affect 137Cs mobility in the rhizosphere
as both factors are known not to alter the solid to liquid distribution in a significant
way. Gradients in the concentrations of cations, mainly K, or 137Cs itself, are likely
to play a significant role on plant availability of 137Cs. This is illustrated in Table
4.3. The 137Cs uptake by young wheat plants was predicted from soil solution
concentrations of 137Cs and K, either those in bulk soil solution data or those in the
rhizosphere. The predictions based on rhizosphere composition were significantly
better than those based on the bulk soil solution composition. The rhizosphere
concentrations were calculated by a solute transfer model and the uptake was pre-
dicted from soil solution composition (137Cs and K concentrations) and data on 137Cs
uptake in wheat in solution culture.124,125
The data in Table 4.3 show that K is more depleted away from the bulk soil than
137Cs. This is remarkable since K is more mobile than 137Cs in soil. This can be

explained by the fact that 137Cs is absorbed less efficiently than K by plant roots.
Indeed, these two cations have been demonstrated to be 4 to 25 times more efficient
in uptake in solution culture studies125 (see also Section IV, A). This uptake efficiency
is expressed relative to solution concentrations and is therefore related to the rate
by which these cations are removed from the soil solution. The root uptake efficiency
becomes high at low K supply, resulting in the well-known K depletion in the
rhizosphere at low K supply of the soil. In contrast, reducing 137Cs concentrations
in the rhizosphere of actively absorbing roots does not induce a stronger sink for
137Cs itself (Cs is not a plant nutrient and its uptake rate is not controlled by nutritional

demand). It should be stressed, however, that a K depletion in the rhizosphere results


in increasing plant uptake of 137Cs because of the strong effect of the K concentration
on 137Cs uptake (see Section IV, A). This effect is known as the rhizosphere mobi-
lization of radiocesium, which can be induced by K stress. Kruyts et al. (unpublished
data) have recently characterized this rhizospheric mobilization of 137Cs in a forest
soil described by Maes et al.120 They used an experimental cropping device adapted
from earlier studies132,133 and inducing K stress. In this device, the soil samples were
previously made homoionic with Ca2+ ions, whereas the plantlets previously germi-
Fate of Radiocesium in Soil and Rhizosphere 81

nated in a K-free nutrient medium. Briefly, the experimental procedure consists of


placing the 137Cs contaminated Ca2+-exchanged soil into close contact with an active
root mat of young ryegrass (Lolium multiflorum) seedlings and measuring the 137Cs
concentration in the plant after a standardized period of contact. The close contact
between soil and root consists of a macroscopic rhizosphere intensifying the
soilroot interaction. These experimental conditions enhance K stress because K
source in the device originates only from native K in potassium bearing soil minerals
as well as original K contained in the seeds. Using such an experimental design,
Kruyts et al. (unpublished data) measured a readily effective uptake of radiocesium
by plant roots as the 137Cs rhizospheric mobilization (RM) amounted to 0.07 to 23.42
% of the initial 137Cs soil contamination. They showed that the respective horizon
contributions to the total 137Cs soil-to-plant transfer were 96.7% in Of, 0.13% in
OAh, 1.34% in Ah, and 1.84% in Bw. Such contributions are quite similar to those
measured by Thiry et al. in the same forest soil, but using a pot experiment approach
involving young spruce plants (unpublished data, see Section IV, B). That similarity
suggests that short experimental measurement of the 137Cs RM can attractively
compete with time-consuming pot experiments involving longer experimental peri-
ods as well as heavier infrastructure.
In a recent study involving a wide variety of soils from seminatural
environments121 (Table 4.1), we quantified the 137Cs RM and compared it with the
137Cs extractability by sodium tetraphenylboron (NaTPB). NaTPB specifically

extracts interlayer K in micaceous minerals135 and the precipitation of low-solubility


K-tetraphenylboron136 maintains a very low K concentration in the liquid phase,
simulating a K-depleted environment. Our results are illustrated in Figure 4.6.
Figure 4.6a shows that the 137Cs RM is essentially governed by RIP, i.e., a
common trace Cs binding characteristic that is readily measurable in soil.112 Large
137Cs RM is here associated with low RIP soils, i.e., peaty soils, podzolic soils, and

sandy soils. Small 137Cs RM is associated with high RIP soils, i.e., brown soils,
whatever their clay and organic matter content. Noteworthy are the facts that i) the
RIP values cover four orders of magnitude (see also Table 4.1: unpublished data)

FIGURE 4.6 Plot of the 137Cs+ rhizospheric mobilization against RIP (a) and NaTPB-extract-
able 137Cs+ (b) in a collection of soils of widely varying properties. (Adapted from Delvaux,
B. et al.121; see Table 4.1.)
82 Trace Elements in the Rhizosphere

and ii) the 137Cs RM exhibits a very large range as it varies between 1 and 69% of
the initial 137Cs+ soil contamination. The relationship illustrated in Figure 4.6 suggests
that RIP could serve as a useful tool to predict potential 137Cs+ soil-to-plant transfer
patterns, likely because the RIP concept integrates the quantityintensity relationship
[Equation (1)]. These results further support that the FES pool controls the concen-
tration of radiocesium in the soil solution, and hence its mobility in the vicinity of
active plant roots. However, the absorption of radiocesium by the roots is also likely
driven by K depletion in this experimental device because of K stress (Ca2+ exchanged
soils, K-deficient plant). The selective extraction of 137Cs by NaTPB simulates the
Cs root-induced sink effect due to K depletion135 enhanced by rapid precipitation of
KTPB and CsTPB.136 Indeed, the correlation between 137Cs RM and NaTPB-extract-
able 137Cs (Figure 4.6b: r = 0.94, n = 47) strongly supports that K depletion in the
rhizosphere is a major driving force in the 137Cs uptake by plant roots.
Many soils in seminatural environments are expected to exhibit low RIP because
of their peaty, acidic, sandy, and/or podzolized character. The rhizospheric mobili-
zation of radiocesium can therefore be large in such environments, particularly if K
supply is low. In this respect, intense mobilization of radiocesium in the rhizosphere
can be a key process in the relatively large biorecycling of radiocesium in such areas
(Figure 4.1). This statement is supported by the results reported by Sanchez et al.130
on organic-rich soils from such areas (see Section IV, B). Small RIP, low K avail-
ability, and high NH4+ concentration in the soil solution all contribute to increased
Cs soil-to-plant transfer in these soils.
The relationships described here between soil matrix, soil solution, and plant
roots consider only the chemical interactions involving radiocesium in the rhizo-
sphere. Such chemical interactions should not occult the biological interactions
occurring as well, particularly in the uppermost soil horizons.137 A large part of
radiocesium can be immobilized by the microflora, particularly in fungal mycelia.138
Some major groups of microorganisms living in the rhizosphere are root symbionts,
such as N-fixing bacteria and mycorrhizal fungi. These fungi have a considerable
impact on the accumulation of radiocesium in seminatural environments; they
occupy a central position in the biogeochemical cycle of radiocesium in such envi-
ronments.139,140 However, very few studies have analyzed the role of fungi in the
transfer of radiocesium from soil to plant in a mechanistic way. Some studies have
emerged in the last 5 years, but they exhibit somewhat contrasting conclusions,
partly due to the fact that published results refer to mycorrhizal symbioses involving
distinct structures and species. Entry et al.141,142 observed higher contamination with
radiocesium in plants previously infected with arbuscular mycorrhizal fungi (Glomus
spp.). They suggested that relatively high 137Cs transfer to mycorrhized plants could
be very attractive in phytoremediation. Other studies reported that the infection of
roots of Picea abies by ectomycorrhizal fungi (Hebeloma crustuliniforme) decreases
the plant contamination with radiocesium.143,144

VI. CONCLUSION
The data presented in this chapter show that both clay minerals and plant roots act
as mutual competitive sinks for radiocesium in the rhizosphere. The micaceous 2:1
Fate of Radiocesium in Soil and Rhizosphere 83

clay minerals, which bear selective Cs+ sorbing frayed edge sites, control the con-
centration of radiocesium in the soil solution, and hence its uptake by plant roots.
The strongest trace Cs retention occurs on vermiculitic sites associated with mica-
ceous wedge zones; these sites can be readily quantified. As such, weathered mica
and/or mica-vermiculite intergrade appear to be the mineralogical structures that
best fix radiocesium. The intimate structural environment of the frayed edge site
exhibiting exceedingly high trace Cs to K selectivity is, however, not yet fully
characterized and merits further investigation.
The concentration of K+ ions in the soil solution plays a key role in the root
uptake of radiocesium as K depletion (K concentration < 1mM) increases the trace
Cs uptake rate, and thereby is a principal driving force in the uptake of radiocesium
by plant roots. Because K depletion promotes the weathering of micaceous phyllo-
silicates, one can expect that K depletion may promote, to some extent, trace Cs
retention on newly accessible frayed edge sites with a very high Cs+ specificity.
Such possible interaction needs further investigation.
The soilroot interaction has been presented here in terms of chemical processes
occurring in the rhizosphere. The role of fungi, particularly the mycorrhizal fungal
species in this interaction is largely unknown, from a mechanistic point of view.
Basic research in this topic is required to achieve a fuller understanding of the
bioavailability of radiocesium in soils from natural and seminatural environments.
It is also a prerequisite to lay out the scientific foundations about the use of mycor-
rhizal fungi in the phytoremediation or phytostabilization of contaminated areas.

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from agricultural and semi-natural environments after nuclear fallout, Bodenkultur,
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108. Paasikallio, A., Rantavaara, A., and Sippola, J., The transfer of cesium-137 and
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109. Hsu, C.-N. and Chang, K.-P., Sorption and desorption behavior of cesium on soil
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111. De Preter, P., Van Loon, L., Maes, A., and Cremers, A., Solid-liquid distribution of
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112. Wauters, J., Elsen, A., Cremers, A., Konoplev, A. V., Bulgakov, A. A., and Comans,
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116. Becket, Ph., The immediate Q/I relations of labile potassium in the soil, J. Soil
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117. Smolders, E, Van den Brande, K., and Merckx, R., The concentrations of 137Cs and
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119. Absalom, J. P., Young, S. D., Crout, N. M. J., Nisbet, A. F., Woodman, R. F. M.,
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soil, Eur. J. Soil Sci., 49, 133, 1998.
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different plant species, Soil Sci., 108, 64, 1969.
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H., Dodd, B. A., Singleton, D. L., and Barnett, C. L., High plant uptake of radiocesium
from organic soils due to Cs mobility and low soil K content, Environ. Sci. Technol.,
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131. Ruhm, W., Steiner, M., Kammerer, L., Hiersche, L., and Wirth, E., Estimating future
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135. Scott, A. D., Hunzinker, R. R., and Hanway, J. J., Chemical extraction of potassium
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136. Geilmann, W. and Gebauhr, W., Precipitation of alkali metals as tetraphenylboron


compounds, Z. Anal. Chem. 139, 161, 1953.
137. Kiefer, P., Prohl, G., Muller, H., Lindner, G., Drissner, J., and Zibold, G., Factors
affecting the transfer of radiocesium from soil to roe deer in forest ecosystems of
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138. Guillite, O., Melin, J., and Wallberg, L., Biological pathways of radionuclides orig-
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157, 207, 1994.
139. Heinrich, G., Uptake and transfer factors of 137Cs by mushrooms, Radiat. Environ.
Biophys., 31, 39, 1992.
140. Kammerer L., Hiersche, L., and Wirth, E., Uptake of radiocesium by different species
of mushrooms, J. Environ. Radioact., 23, 135, 1994.
141. Entry, J. A., Vance, N. C., Hamilton, M. A., Zabowski, D., Watrud, L. S., and Adriano,
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contaminated soil by three grass species inoculated with mycorrhizal fungi, Environ.
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cesium/potassium ratio on uptake and localization of cesium in Norway spruce
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and Sr-85 uptake into Picea abies seedlings, Environ. Pollution, 93, 1, 1996.
5 Bioavailability of
Uranium and Plutonium
to Plants in SoilWater
Systems and the
Potential of
Phytoremediation
Hamid Shahandeh, Jin-Ho Lee, Lloyd R. Hossner,
and Richard H. Loeppert

CONTENTS

I. Introduction.....................................................................................................94
II. Plants Growing at Heavy-Metal-Rich Sites ...................................................95
III. Strategies of Metal Tolerance.........................................................................96
IV. Phytoremediation ............................................................................................97
V. Uranium in Soils and Plants...........................................................................98
A. Uranium and the Environment ...........................................................98
B. Uranium Behavior in Soils.................................................................99
C. Uranium Behavior in Plants .............................................................101
D. Influence of Rhizosphere Processes on Uranium Absorption
by Plants ...........................................................................................102
VI. Plutonium in Plants and Soils ......................................................................103
A. Plutonium and the Environment.......................................................103
B. Plutonium Behavior in Soils ............................................................104
C. Plutonium Behavior in Plants...........................................................106
VII. U Phytoextraction .........................................................................................108
VIII. U Rhizofiltration ...........................................................................................111
IX. Enhancement of U and Pu Uptake by Plants ..............................................112
A. Effect of Synthetic Chelates and Organic Acids on U Uptake .......112
B. Effects of Synthetic Chelates and Organic Acids on Pu
Uptake ...............................................................................................115
X. Conclusions...................................................................................................116
References..............................................................................................................116

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 93
94 Trace Elements in the Rhizosphere

I. INTRODUCTION
Large areas of land have been contaminated by radionuclides and fission by-products
from nuclear weapons facilities, aboveground nuclear testing, nuclear reactor oper-
ations, and nuclear accidents.1 The principal contaminants of concern are the radi-
onuclides U, Th, Pu, 60Co, 90Sr, 135Cs, and 99Tc, and toxic metals Ba, Cd, Cr, Ni,
Pb, and Zn. Radionuclides, unlike organic pollutants, cannot be degraded. Radionu-
clides can move to the food chain, and exposure through inhalation and ingestion
of food can result in detrimental health effects, including cancer and genetic muta-
tions. The radionuclides U and Pu and their decay products result in two problems,
specifically radiation dose hazards and chemical toxicity. The radiation hazard intro-
duces the need for special precautions in reclamation beyond that associated with
nonradioactive metals.
Radioactive contamination of the environment that surrounds facilities where U
and Pu have been mined and processed for nuclear weapons and nuclear power has
occurred in many countries. Uranium is reported to be the most frequent radionuclide
contaminant in groundwater and surface soils of United States Department of Energy
(USDOE) facilities.2 One of these facilities, which is now a superfund site, is the
former Feed Materials Production Center at Fernland, OH. The soils around the
Fernland processing plant have become contaminated with U after decades of U
extraction and purification. Efforts are now underway to remediate the site. Various
technologies for removal of U have been evaluated at this site.3-6 Another well-
researched site is Rocky Flats, located north of Golden, CO, where Pu contamination
originated from leakage of drums used to store Pu.7 Plutonium particles entrapped
in the fine fraction of the surface soils have been subsequently transported by winds
and water and deposited across the soils of the site.
The remediation of these and similar areas contaminated with radionuclides
represents a worldwide problem that will require considerable economic resources
of industry and government. For the most part, contaminated soils are remediated
using engineering approaches including thermal, chemical, and physical techniques
(e.g., in situ mobilization, immobilization, degradation, and burial; or removal and
reburial, vitrification, vacuum extraction, steam flooding, pumping and leaching,
electroosmosis, and electroacoustic extraction). Various soil-washing techniques
have been developed for decontamination of radionuclides using organic and inor-
ganic acids, bases, salts, and chelating agents. The merits of these techniques have
been discussed by Francis and Dodge.5 Many of the inorganic chemicals used are
corrosive and alter or damage the soil. Synthetic organic chelating agents used in
decontamination sometimes persist in the environment, causing the radionuclides to
migrate from the disposal site.
In addition to these physical and chemical treatment methods, microbial reme-
diation (bioremediation) has become more common in the last two decades. However,
the success of microbial bioremediation has been largely limited to the degradation
of organic compounds, with less emphasis on metal and radionuclide contaminations.
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 95

Furthermore, bioremediation techniques were developed for small, heavily contam-


inated sites and were often not cost effective or risk reducing when applied to larger
sites where pollutants are widely dispersed and present at low concentration.
The removal of U and other radioisotopes from soils is theoretically simple to
achieve. Often soil is moved offsite for leaching/chelation treatments, and then
returned to its previous location.1 However, in practice, the movement of large
quantities of soil for decontamination is environmentally destructive and costly due
to transportation. Transportation also increases the risk of releasing potentially
harmful radionuclides. Some industries have opted simply to pile the contaminated
soil for later remedial treatment.8 Transport and treatment of contaminated soil
requires heavy equipment and chemicals that might adversely affect soil physical
and chemical properties for subsequent vegetative establishment. For remediating
toxic metal and radionuclide-contaminated soils by soil removal and replacement,
or excavation and burial at a hazardous waste site, the average cost is approximately
$250 per m3 of soil.8 The cost of radionuclide clean up in the U.S. alone is considered
to be more than $200 to 300 billion. The fact that remediation is so expensive is the
driving force behind the search for new technologies.9-12 In situ techniques for
decontamination of soils seem to be more desirable.

II. PLANTS GROWING AT HEAVY-METAL-RICH SITES


Recently, researchers have become interested in the use of plants to remediate soils
contaminated with organic and inorganic pollutants. Plants have been used to
prospect for heavy metals and have the potential to be used for clean up of
contamination caused by mining and/or ore processing in mining and metallurgical
industries. Research on the possible use of plants to remediate heavy metal con-
taminated sites was initiated as a result of early literature on the biogeochemical
prospecting for ores.13,14
Plants have been utilized for centuries for metal prospecting, and for centuries,
prospectors of ores have been aware that a relationship exists between vegetation and
its soil and geologic substrate. The significance of vegetative cover has been illustrated
mostly in studies of the nature and distribution of plant communities and individual
indicator plants. Indicator plant species are individual species that are confined to a
specific substrate, which may be a rock type or specific type of mineral. Indicator
plants include numerous plant species with a reputation for indicating metals. Almost
without exception, indicator plants are herbs or shrubs rather than trees. Most of these
plant species belong to three families: Leguminosae, Caryophyllaceae, and Labiatae.
In many cases, indicator plants tolerate mineral-rich soil by restricting uptake of the
normally toxic element. In other cases, indicator plants accumulate a large concen-
tration of the potentially toxic element,13 in which case the plant must also possess
an internal defense mechanism against the potential toxicity.
One of the most interesting cases of plants in mineral prospecting is the use of
serpentine flora for locating nickel (Ni) and chromite (Cr III) deposits. Also, the
discovery of selenium-rich flora in the western United States, Columbia, Canada,
and Australia resulted in the discovery of selenium (Se) and U deposits. Selenium
96 Trace Elements in the Rhizosphere

indicator plants include species of the genera Astragalus. Astragalus species cover
large areas of the western United States and were used by Cannon15 to detect U
mineralization because of the geochemical association of Se and U in the carnotite
ores of the region. Cannon mapped the distribution of Astragalus plants and was
thereby able to pinpoint several economic-grade U deposits. This discovery of U in
the western United States is an example of the successful application of the geobo-
tanical method for metal prospecting.13 Similarly, Dunn16 reported that the twigs of
black spruce (Picea mariana) contained U concentrations >100 mg kg1 ash weight,
in comparison with background levels of <1 to 60 mg U kg1. This relationship was
used to locate a massive U biogeochemical anomaly near Wollaston Lake in Canada.
Plant species that are naturally high in heavy metals have developed a defense
mechanism to tolerate absorption of the metal and, as a result, accumulate a high
concentration of metal in the plant tissue. Since heavy metals are damaging to most
plants at relatively low concentrations, the accumulation strategy requires mecha-
nisms to detoxify the metal.

III. STRATEGIES OF METAL TOLERANCE


The uptake of essential metals by plants occurs predominantly by way of channels
and transporters in the root plasma membrane. Metals are required for a variety of
metabolic processes in all organisms. However, because many metals can be toxic,
plants have evolved systems to regulate the uptake and distribution of metals. Plants
characteristically exhibit a remarkable capacity to absorb what they need and exclude
what they do not need. But most vascular plants absorb toxic and heavy metals
through their roots to some extent, from negligible to substantial. Sometimes absorp-
tion occurs because of the chemical similarity between essential and toxic metals.
For plants, the absorption of metals occurs primarily through the roots, so this is
the site for regulating their accumulation. Some plants utilize exclusion mechanisms,
where there is a reduced uptake by the roots or a restricted transport of the metal
from root to shoot. At the other extreme, hyperaccumulator plants absorb and
concentrate metals in both roots and shoots.17 Some plant species endemic to met-
alliferous soils accumulate metals in percent concentrations in the leaf dry matter.18
Once metals have crossed the root membrane, there are a variety of mechanisms
to prevent metal toxicity, including compartmentation and binding to intracellular
ligands. It is widely accepted that detoxification of metal ions within plant tissues
usually depends on chelation by appropriate ligands. Researchers have considerable
interest in determining the chemical nature of these ligands. The anionic species of
organic acids, such as citrate, malate, and malonate, are commonly found in high
concentrations in the leaves of Alyssum spp. Reeves19 pointed out that these anions
tend to be present constitutively in these plants in substantial amounts and cannot
account for the metal specificity or species variability of Ni hyperaccumulators.
Kramer et al.20 suggested that the Ni hyperaccumulation trait in Alyssum is associated
with the ability of the root system to produce substantial amounts of histidine, an
Ni-complexing ligand.
In addition to the organic acids, plants are equipped with at least two ligands,
phytochelatins (PCs) and metallothioneins (Mts), that are able to bind heavy metals
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 97

such as Cu, Zn, and Cd. Phytochelatins are a family of peptides with the general
structure [-GluCys]n-Gly, where n > 1.21 Metallothioneins are similar to PCs in
being Cys-rich, metal-complexing ligands; however, metallothioneins are proteins
synthesized by mRNA translation.

IV. PHYTOREMEDIATION
The term hyperaccumulator was introduced by Brooks et al.18 for plants growing
on serpentine sites that are capable of concentrating nickel (Ni) to more than 1000
g g1 in their leaves on a dry matter basis. A concentration of 1000 g g1 has also
been used to delineate the exceptional uptake of copper (Cu), cobalt (Co), and lead
(Pb). The delineation level is raised to 10,000 g g1 for zinc (Zn) and manganese
(Mn), because of the greater background concentrations of these metals in soil.
The concept of using hyperaccumulator plants to extract and remediate metal-
contaminated sites started in the 1970s,22 and the term phytoremediation emerged.
This term consists of the Greek prefix phyto (plant) and Latin root remedium
(to correct or remove an evil) and is defined as the use of green plants and associated
microbiota, soil amendments, and agronomic techniques to remove or render envi-
ronmental contaminants harmless.23 Chaney et al.10 defined phytoremediation as
using plants to make soil contaminants nontoxic. Green remediation, bioremediation,
and botanical-bioremediation are other names used for the process of using plants
to remediate the environment.24
Phytoremediation requires that the target metal must be sorbed by the roots and
either (1) translocated from the root to the shoot for subsequent removal of the
biomass, or (2) rendered harmless by biochemical or chemical processes associated
with the plant. In the case of U and Pu, the metals cannot be rendered harmless,
hence phytoremediation must involve absorption, translocation, and subsequent
removal. The metal is removed from the site by harvesting the plant material. After
harvesting, the biomass is processed to either recover the metal or further concentrate
the metal to facilitate disposal.
Phytoremediation has been categorized into five techniques: (1) phytoextraction
(use of plants to remove contaminants, e.g., Zn and Cd from soils);25,26 (2) phyto-
volatilization (use of plants to volatilize soil elements like Hg and Se);27-29 (3)
rhizofiltration (use of plant roots to remove contaminants like U and Pb from flowing
water);30-32 (4) phytodegradation of organic compounds by rhizosphere biodegrada-
tion;33,34 and (5) phytostabilization (use of plants to transform soil metals to less
toxic forms, e.g., to reduce Cr(VI) to Cr(III) but not to remove the metal from the
soil).10,35
Some of the successful pilot projects in phytoremediation have been: (1) Se and
boron (B) removal from saline soils in California using Brassica juncea and Festuca
arundinacea,36 (2) Zn removal from metal-contaminated soils using hyperaccumu-
lator plants,37,10 (3) lead (Pb) cleanup from a contaminated industrial site in New
Jersey using Brassica juncea,38 and (4) removal of 137Cs and 90Sr from contaminated
soils at Chernobyl.39,40
Fundamental to the environmental and economic success of phytoremediation
of heavy metals and radionuclides is the existence of high biomass plant genotypes
98 Trace Elements in the Rhizosphere

that accumulate metals or radionuclides. The effectiveness of the phytoremediation


process is summarized in the following equation:

total metal uptake or metal harvest =


(plant metal concentration) (plant biomass) (1)

Therefore, both absorption potential and biomass must be considered in the


optimization of strategies for metal removal. Unfortunately, often the metal hyper-
accumulators have low plant biomass. To maximize metal concentration in the
biomass, it is necessary to use good soil management practices (e.g., optimized soil
pH and mineral nutrition, minimum concentrations of interfering elements, and the
introduction of agents that will increase the concentration and diffusion of dissolved
metals in the soil); plant genotypes with optimized metal uptake, translocation, and
tolerance; and plants with a high biomass potential. Individualized practices must
be developed for specific sites.
An understanding of soilplant interactions and the processes involved in the
solubilization, absorption, transport, and accumulation of U and Pu contaminants
by plants in affected soils is essential for the optimization of phytoremedial tech-
niques. Increased uptake of contaminants by plants will lead to more efficient
amelioration of affected soils.
Research emphasis on the uptake of heavy metals by selected plant species is
growing. For some heavy metals (Ni, Cd, Zn), current research in the field of
phytoremediation has provided much information. Unfortunately, such fundamental
information is lacking for U and Pu.

V. URANIUM IN SOILS AND PLANTS


A. URANIUM AND THE ENVIRONMENT
There is widespread U contamination of soils at levels above regulatory standards.
For example, the Rocky Flats Plant, near Golden, CO, has soils that are contaminated
with U due to improper waste-storage practices.41 Near surface disposal of spent
fuel and other low level radioactive wastes from nuclear-power generating plants is
accomplished by solidifying the liquid wastes.42 Leachates from these systems have
presented problems. Also, the mining and milling of U results in large quantities of
waste material called tailings. These tailings consist of overburden from strip and
open pit mines as well as the by-products from ore processed in milling facilities.
These materials still contain sufficient amounts of radionuclides to warrant concern
over environmental health.43,44
Natural U exists as three predominant isotopes, 234U, 235U, and 238U, with a
relative abundance of 0.0055, 0.720, and 99.27%, respectively.45 In many instances,
the natural abundance has been altered either due to anthropogenic U enrichment
or geological processes. Uranium is primarily an alpha () emitter. Due to the
relatively high mass of the particle versus beta () and gamma () particles and
its lower velocity, the primary mode of exposure of particles is through direct
contact with the emitter. The dangers associated with exposure are mainly through
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 99

ingestion by inhalation or drinking contaminated water. The toxic effects of U are


similar to those of other heavy metals, and its physiological toxicities, other than
damage from ionizing radiation, mimic those of Pb. Uranium is toxic to kidneys,
and insoluble U compounds are carcinogenic.46 Although U and its daughter elements
have not been shown to be essential or beneficial to either plants or animals, many
plant species will absorb U and incorporate it into their biomass along with other
heavy metals.47-52 This observation suggests the possibility for remediation of U-
contaminated soils by plant uptake.
The major prior emphasis in the remediation of contaminated soils has been
chemical extraction. Chemical extraction of U has been accomplished with either
strongly acidic or strongly alkaline solutions.6 The acid leaching process, using
sulfuric acid, is the most common technology, but for carbonate-bearing soils, the
only feasible process is the alkaline leach. The extraction of soil with carbonate or
bicarbonate is highly selective for U, by the formation of the soluble uranyl tricar-
bonate complex:

UO 2 2 + + 2(HCO 3 ) + (CO 3 2 ) = UO 2 (CO 3 )3 4 + 2 H + (2)

U removals of 75 to 90% were achieved by a column leaching process in the


laboratory, using 0.5 M sodium bicarbonate as the dominant reagent.6
The Francis and Dodge5 technique consists of three steps: extraction, biodegra-
dation, and photodegradation, in which U was removed from contaminated soils and
wastes by extracting it with citric acid. Citrate complexes were readily biodegraded
by Pseudomonas fluorescens, resulting in the bioprecipitation and recovery of the
metals, whereas uranyl citrate, which is recalcitrant to biodegradation, was photode-
graded upon exposure to light, with the precipitation of U as UO3 xH2O. Francis and
Dodge5 were able to remove >85% of U from contaminated soils and sludge. Bio-
degradation followed by photodegradation of the citric acid complex resulted in >99%
recovery of the extracted U. Buck et al.3 suggested that an additional physical extrac-
tion is needed to remove U that is not removed by chemical remediation technologies.
A treatment approach that provides an oxidizing, alkaline environment rich in
carbonate, has been proposed for remediation of U-contaminated soils.53 An on-site
pilot plant in which the treatment strategy was based on this recommendation (i.e.,
sodium carbonate-bicarbonate extraction in the presence of potassium permangan-
ate) has achieved a U-removal efficiency of 83 to 92% from U-contaminated soils.54

B. URANIUM BEHAVIOR IN SOILS


Uranium is present in the soil primarily (80 to 90%) in the +6 oxidation state as the
uranyl (UO22+) cation.4,45,52 Under acidic conditions, UO22+ is the predominant U
species in the soil. Under strongly reducing conditions (E 0.2 V) and low total
carbonate concentration, the tetravalent hydroxide U(OH)4 and possibly U(OH)5
are dominant, but the equilibrium total dissolved concentration is less than 1010 M
(less than 0.025 gUL1). Under mildly reducing conditions (E > 0.2 to 0.4 V) the
hexavalent state dominates in solution, even at low carbonate concentrations.45
100 Trace Elements in the Rhizosphere

FIGURE 5.1 Species distribution for U(VI) hydrolysis and carbonate species at 2.38 mg
U(VI) L1 and 31.1 total inorganic C L1. (From Duff, M. C. and Amrhein, C.57 With
permission.)

Uranium (VI) is the most mobile form of U.55 It exists in solution predominantly
as the stable linear ion UO22+ and as soluble carbonate complexes, (UO2)2CO3(OH)3,
UO2CO3, UO2(CO3)22, UO2(CO3)34, and possibly (UO2)3(CO3)66.56-59 Within the
pH range of 4.0 to 7.5 and in the absence of dissolved inorganic ligands (carbonate,
fluoride, sulfate, and phosphate), the hydrolysis species UO2OH+, UO2(OH)2o, and
(UO2)2(OH)22+ dominate U(VI) speciation.60 The equilibrium distribution of U(VI)
hydrolysis and carbonate species is shown in Figure 5.1. Uranium in soil and water
forms complexes with sulfate and phosphate as well as with carbonate and hydroxide,
and these complexes increase the total solubility of U.
Bunzl et al.61 found that a significant portion of U contaminant in soils exposed
to vented air from U ore mine shafts was associated with soil clay minerals. The
least amount of U was found in the exchangeable and soluble forms; therefore, the
amount of U available for plant uptake and subsequent phytoremediation was limited.
In another study, it was observed that in soils spiked with U(VI) as UO2(NO3)2
6H2O and analyzed shortly afterwards, the dominant proportion of U(VI) was found
in association with the Fe-Mn oxide portion of the soil.62,63 Sorption of U(VI) onto
soil surfaces tends to increase with increasing pH (up to pH 7) and is readily
reversible by decreasing the pH.64
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 101

C. URANIUM BEHAVIOR IN PLANTS


Most of the information on the accumulation of U in plants is related to the use of
native plant species in the biological exploration of metals. There are few studies
related to the mechanisms by which plants absorb and accumulate U.65-72 In these
studies, it was generally observed that plant species differ in U accumulation. Several
studies of the relationships between plants and soils relevant to radionuclide accu-
mulation by plants have been conducted.52,50,47 Plants remove nutrients from the
readily exchangeable and soluble fractions of the soil, but negligible amounts of
U(VI) remain in the soluble and exchangeable forms for a significant period of
time.61 In one study it was found that the rate of desorption of U(VI) did increase
with time.63 In this study, it was also observed that the insoluble organic matter
fraction of the soil formed strong complexes with U(VI). Uranium accumulates
mainly in the roots.4,30,67 Depth of U in the soil profile and soil properties influences
absorption by plants.52
Contradictory information on the phytotoxicity of soil U to plants exists.72 Levels
as low as 1 mg kg1 in soil, well within the normal background range, have been
cited as toxic. Gulati et al.74 reported that wheat and tomato yield decreased contin-
uously with increasing U level in soil from 1 to 6 mg kg1. Other studies have
reported no toxicity at soil U concentrations 100 to 1000 times higher. For example,
Brassica rapa produced seed and high biomass yields at U levels of 10,000 mg kg1
in soil.73 To identify the toxic threshold of soil U, Sheppard et al.73 tested nine levels
of U in eleven soils with five plant species. They found no detrimental effects below
300 mg U kg1 in soil.
Many of the studies have used the concentration ratio (CR) to determine the
ability of various plant species to absorb radionuclides from soils or other substrates.
This value is defined as the concentration ratio of a given element (radionuclide) in
the aboveground plant tissue compared to that in the soil:

concentration of radionuclide in plant tissue


CR = (3)
concentration of radionuclide in soil

Plantsoil concentration ratios have been used in predicting the transport of


radionuclides and other elements of interest through the food chain as well as in
biogeochemical studies of radionuclides. Substantial variability in CR values have
been reported, depending on soil properties and crop species.52
Hossner et al.77 screened high biomass and rapidly growing agricultural crops
for their ability to accumulate U. Agriculture crops, such as switchgrass (Panicum
virgatum), sunflower (Helianthus annuus), and Indian mustard (Brassica juncea)
were determined to be potential metal accumulators that produce high biomass and
tolerate and accumulate heavy metals. Nanda Kumar et al.78 reported that Indian
mustard (Brassica juncea) cultivar 426308, grown in a sandPerlite mixture con-
taining 625 g Pb g1, was the most efficient accumulator of shoot Pb, with 35,000
g Pb g1 of biomass and a phytoextraction coefficient of 55.2 (the ratio between
mg of metal/g dry weight of tissue and mg of metal/g dry weight of substrate). In
addition, Indian mustard can produce 18 t biomass ha1 78 and can be easily adapted
102 Trace Elements in the Rhizosphere

to various climatic conditions and agricultural practices. Sunflower produces about


the same biomass as Brassica juncea and can readily absorb U and other metals
from its rhizosphere.78

D. INFLUENCE OF RHIZOSPHERE PROCESSES ON URANIUM


ABSORPTION BY PLANTS
The soil rhizosphere, or soil environment influenced by plant roots, has significantly
different properties than the bulk soil. Plant exudates of soluble organic and inorganic
compounds provide substrate for microbiological growth and can interact directly
with soil solids. Increased density and activity of microorganisms near plant roots
provide for the possibility of accelerated degradation of organic matter in the rhizo-
sphere. Organic carbon, pH, biological activity, and the solubility of inorganic
constituents are significantly altered in the rhizosphere. The net effect is that micro-
bial population and activities are as much as 100 times that in bulk soil.
One of the reasons for the increased interest in phytoremediation is that evapo-
transpiration by plants can help to bring contaminants to the rhizosphere. For exam-
ple, phreattophytic trees such as poplar, willow, and cottonwood, are very efficient
natural pumps. A single willow tree uses and transpires as much as 22,500 liters of
water in a single day. The water evaporates through plant transpiration, and at the
same time, the heavy metals can be absorbed by the fine root system. The plants
use of solar energy and evapotranspiration by plants can be considered a solar-
powered pump-and-treat system that helps bring contaminants to the rhizosphere
for bioremediation and containment.22
Rhizosphere degradation has been used mostly for remediation of organic con-
taminants. Unlike organic compounds that can be degraded or mineralized, the
remediation of most metals and radionuclides requires removal of the contaminant.
Plants might extract inorganic species, but effective phytoextraction requires plants
that produce high biomass, grow rapidly, and possess high-capacity uptake of inor-
ganic substrate. Interest in the use of genetically modified plants to extract heavy
metals is increasing. Rhizosphere processes can play an important role in the effec-
tiveness of these plants. For example, some plants and certain microorganisms can
chelate and concentrate metals from their surroundings. Ow79 states that plant nutri-
ent uptake is intrinsically linked to the association with rhizospheric fungi, so
engineering of rhizospheric microbes could also assist in metal phytoextraction.
Interest in adsorption and precipitation of organic and inorganic wastes on the
plant root surface is also on the rise. Adsorption to the surfaces of roots may be an
important sink for organic compounds in soils and the first step in phytoremediation.
Plants might also be able to precipitate metals such as lead by exuding phosphate
into the rhizosphere that can form insoluble lead phosphate.22 Little information is
currently available on the influence of rhizosphere environments on radionuclides.
Although no studies of the direct influence of the rhizosphere on absorption of
U by plants can be found, it is widely accepted that rhizosphere processes play a
significant role. For example, the higher CO2 partial pressure resulting from root
and microbial respiration in the rhizosphere would influence solubility of U because
of the stability of U(VI)-carbonate complexes.60 This influence of CO2 would be
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 103

greater in alkaline and calcareous soil because of the equilibrium relationship


between pH and CO32 concentration.80
Some plants and microorganisms can exude considerable concentrations of
organic metal-complexing agents. Under phosphate-deficiency stress conditions,
some plants, e.g., pigeon pea (Cajanus cajan L.), radish (Raphanus sativus L.),
rape/black mustard (Brassica nigra L.), and red clover (Trifolium pratense L. cv.
Hamidori), release organic complexing agents, such as oxalate, citrate, malate,
malonate, succinate, and tartarate, as a specific stress response.81-83 It is likely that
many other plant species exhibit a similar phosphate-deficiency stress response. The
quantity of organic-acid exudation is highly variable between plant species and
cultivars within a species. Under Fe-deficiency conditions, graminae produce a class
of nonproteinaceous amino acid (phytosiderophore),84-85 though the influence of
these compounds on U solubility has not been reported. Microorganisms also exude
a range of Fe-complexing agents, e.g., the hydroxymate siderophores,86-87 that prob-
ably contribute to U solubility. The products of organic matter decomposition in the
rhizosphere also contribute to U complexation and solubility.
Some plants will moderate the pH of the rhizosphere by the exudation of H+,
either to provide charge balance during K+ or NH4+ absorption or as a specific plant
adaptation.88 Dicots commonly exude H+ as a specific response to Fe deficiency.88-89
Even though soil composition has a strong effect on U solubility, the concen-
tration of U in the bulk soil is probably not representative of rhizosphere concen-
trations. In most natural soil situations it is likely that soluble U concentration is
higher in the rhizosphere than in the bulk soil due to the processes summarized
above. But still, manipulation of the bulk soil, e.g., by pH adjustment or addition
of complexing agents, can have a significant influence on U solubility and absorp-
tion by plants.

VI. PLUTONIUM IN PLANTS AND SOILS


A. PLUTONIUM AND THE ENVIRONMENT
Plutonium (Pu) is not a natural nuclide. The only naturally occurring nuclide that
is fissile by thermal neutrons is uranium-235 (235U), which constitutes 72% of
naturally occurring U. The other important fissile nuclide, 239Pu, is produced from
238U by neutron capture, followed by successive decays. The isotopes neptunium-

239 and -238 (239Np and 238Np) are produced by reaction on 238U and decay by
emission to plutonium. The neutron arises from fission of 235U:79,80

( n, ) 239 239
238
U 239 U Np Pu (4)

As long as nuclear reactors are in existence, whether for power generation or


scientific purposes, Pu will be produced along with a number of other radionuclides,
and certain amounts of these radionuclides might be released into the environment.
Plutonium has been used predominantly in weapons manufacture. It can be found
in minute quantities globally, as it is a fallout material from nuclear weapons testing.
104 Trace Elements in the Rhizosphere

Previous work on Pu has focused primarily on migration and transport mechanisms,


with little emphasis on the mechanisms of uptake and accumulation by plants.
Primary interest has been in determining mechanisms by which food crops might
become contaminated and the risks of bioaccumulation, with little interest in deter-
mining species of plants that might be suitable for phytoremediation. The danger
inherent in working with above background quantities of Pu has prevented scientists
from studying it very closely to determine the mechanisms of plant uptake and the
factors affecting its availability to plants. The soil chemistry of Pu is not as well
known as that of most other metals. Strict regulations for the procurement of special
nuclear materials (SNM) have severely limited the amount of Pu available for study.
Soils can be contaminated with Pu by global fallout from nuclear weapon tests
or from controlled or accidental discharges associated with the nuclear power
industry. Plutonium has been considered, because of its half-life (t1/2 = 2.41 104
y for 239Pu), to be one of the most radiologically and biologically toxic radioele-
ments. Therefore, detailed information on the fate of Pu in soils, the transfer of Pu
from soil to plants, and the translocation of Pu in plants are of considerable envi-
ronmental significance.
The mobility, solubility, and extractability of Pu in soil are mostly affected by
pH, oxidation state, organic matter, CaCO3, and hydrous oxides of aluminum (Al),
iron (Fe), and manganese (Mn).

B. PLUTONIUM BEHAVIOR IN SOILS


Plutonium ions in soil solution can exist in several oxidation states, predominantly
as Pu3+, Pu4+, PuO2+, and PuO22+. Because of the equilibrium relationships between
the various oxidation states and the relatively slow kinetics of conversion from one
state to another, it is possible for all four of these oxidation states to coexist in
appreciable concentrations in the same system.90
The downward leaching of Pu in the soil profile is relatively slow.91-93 In long-
term field studies, 238Pu and 239+240Pu concentrations in a soil remained constant with
depth over years, an indication of little vertical leaching through the soil profile.
Plutonium probably undergoes reactions within the soil shortly after deposition,
which make it less available for absorption by the plant. Plutonium is more mobile
in coarse-textured soils and less so in peats and mucks.94,95
Results from Pu-contaminated soils and plants at the Rocky Flats site96,97 suggest
that the movement of actinides was restricted to the top 20 cm regardless of soil
type, distance, or direction from the contamination source. These findings strongly
suggest that Pu mobility is limited.
In the surface horizons, 239Pu and 240Pu were primarily associated with organic
C (45 to 65%), sesquioxides (20 to 40%), and the residual fraction (10 to 15%).
Liator and Ibrahim98 found that a small portion of 239 + 240Pu was associated with
soluble (0.09 to 0.22%), exchangeable (0.04 to 0.08%), and carbonate (0.57 to
7.0%) phases.
Nishita and Haug99 studied the effects of three different clay minerals (mont-
morillonite, kaolinite, and illite) on the extractability of 239Pu (IV) as a function of
pH using an HNO3-NaOH extracting system. They prepared the clay suspensions
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 105

on the basis of equal weight or equal CEC. Plutonium extractability from montmo-
rillonite was markedly higher than that from kaolinite and illite between pH 4.0 and
10.5 on both an equal weight and CEC basis. However, on an equal weight basis,
the Pu extractability from illite was slightly greater than that from kaolinite below
pH 2.8 and between pH 7.3 and 11.6, but it was lower than that of kaolinite between
pH 2.8 and 7.3. On an equal CEC basis, the Pu extractability curves for kaolinite
and illite were reversed. Rhodes100,101 investigated Pu adsorption on a calcareous soil
containing mainly montmorillonite and kaolinite and little or no organic matter.
Strong adsorption of Pu (>98%) began at pH 2 and extended to pH 8.5. In the region
between pH 8.5 and 12.5, adsorption was lowered to a maximum of about 80%.
The ability of humic compounds (humic and fulvic acids), present in soils and
natural waters, to enhance the solubilities of metal ions is well known.102 Cleveland
and Rees103 investigated the solubilization of Pu and Am by humic compounds in
contaminated soil. They reported that the solubilization of Pu and Am by fulvic acid
was slight, though some of the radionuclide was associated with the aqueous phase
as a result of surface complexation with suspended colloids. They also found that
the resulting suspension was unstable, and over a period of a few days most of the
radionuclide was flocculated either as a result of colloid coagulation or hydrolysis.
Chelation by naturally occurring soil organic constituents was proposed as the
most likely mechanism of mobility of Pu and subsequent uptake by plants.104,105
Assumptions were made that Pu associated with low molecular-weight molecules
was more mobile and thus readily available for plant uptake.106 Plutonium associated
with higher molecular-weight molecules was assumed to be nonmobile and not
plant available.
Nishita and Haug107 studied the effects of organic (humic and fulvic acids) and
inorganic (free iron oxides, free silica, and free alumina) fractions of soil on the
sorption and extractability of 239Pu(IV) as a function of pH. They found that in
mixtures of pure humic and fulvic acids with soil inorganic fractions, the extract-
ability of Pu was substantially influenced by the organic-inorganic interaction. The
Pu(IV) was strongly sorbed on the inorganic fraction of the soil under soil pH
conditions normally encountered in natural environments. Also, they reported that
the influence of the inorganic fractions was most evident in the lower pH range,
whereas that of the organic materials was most evident in the higher pH range, i.e.,
above pH 6. The mobility of Pu in soil under the leaching action of water depended
on the type of organic matter present. If both soluble and insoluble forms of organic
matter are present in the soil under either alkaline or acidic conditions, Pu is likely
to show relatively high mobility, due to soluble complexes or chelated forms of Pu,
respectively. Nishita and Hamilton108 investigated the influence of CaCO3, organic
matter, and the inorganic fraction of a calcareous soil on the chemical extractability
of 239Pu(IV). In the system containing humic acid with CaCO3, the extractability of
Pu ranged between 0.1 and 1.3%. Free CaCO3 in all treatments drastically reduced
the Pu extractability.
Nishita et al.109 investigated the effect of 5 organic (Na-formate, -acetate, -tar-
trate, -citrate, and -oxalate) and 21 inorganic (Na2SO4, NaNO3, Na2Cr2O7, NaI,
Na2HPO4, Na4P2O7, NaOH, NH4OH, etc.) compounds on the extractability of 239Pu
from a contaminated soil. They reported that among the organic compounds, the
106 Trace Elements in the Rhizosphere

order of 239Pu extractability was Na-acetate < Na-formate < Na-oxalate < Na-tartrate
< Na-citrate. Also, among the 21 inorganic compounds, soil treated with Na2HPO4
had the least 239Pu extractability (0.07% of the dose), and soil treated with Na4P2O7
had the greatest extractability (28.6% of the dose). Other compounds that had
relatively high 239Pu extractability included NaF, Na2B4O7, NaCO3, Na2O2, NaOH,
and NH4OH.
Jacobson and Overstreet,110 who worked with an aqueous suspension of elec-
trodialized Wyoming bentonite adjusted to pH 5.5 with Ca(OH)2, reported that the
order of adsorption was Pu(III) > Pu(IV) > Pu(VI). Murray and Fukai111 also showed
that the adsorption of Pu was Pu(III) > Pu(IV) > Pu(VI) at pH 3 in sediments of
the Var River, but at pH 8, the adsorption was Pu(VI) > Pu(III) > Pu(IV). Prout112
found that Pu adsorption by a Savannah River facility soil containing mainly kaolin-
ite was >90% from solutions of Pu(III) and Pu(IV) between pH 1.2 and 2.5.
However, from solutions of Pu(VI), more than 90% adsorption was not attained
unless the pH values were >pH 6. Bondietti et al.104 found that the adsorption of
239Pu by the clay fraction of a Miami silt loam was >99.4% regardless of initial

valence, Pu(IV) or Pu(VI). The adsorption of Pu(IV) to montmorillonite was around


98%, whereas that of Pu(VI) was appreciably lower (37%). The high adsorption of
Pu(VI) on soil clay was considered to be due to the reduction of Pu(VI) to Pu(IV),
which was strongly adsorbed.
The chemistry of Pu in soil is influenced by pH, organic matter content, and
mineralogy. It has been shown that the solubility and extractability of Pu(IV) is
affected by carbonate concentration of the solution.108 In systems containing no
carbonate, an increase in the quantity of extractable Pu corresponded to an increase
of pH to 8 to 10. The explanation offered was that the dissolution of alkali-soluble
portions of soil organic matter, and possibly the dissolution of hydrous sulfide that
existed in association with Pu, were responsible for the increased amount of Pu
extracted. The percent of Pu recovered from the organic matter fraction was reduced
from 94% in systems containing no carbonate to 0.1 to 1.3% recovery in systems
where carbonate was present. Free CaCO3 in all treatments drastically reduced the
extractability of Pu, indicating the possible association with calcite. Under acidic
(pH < 3.0) or alkaline (pH > 7.0) conditions and in the presence of a high percentage
of organic matter, Pu becomes more mobile in a kaolinitic soil. This phenomenon
is likely due to the dissolution of acid and alkali soluble organic matter, respectively.
Above pH 6.0, little Pu was extracted from kaolinitic soils with small amounts of
organic matter, but extractable Pu increased with decreasing pH.113 Little (3%) of
the Pu present in the soil was readily exchangeable. The amount of Pu in association
with the organic fraction of soil was variable (2 to 13%, depending on pH) with the
majority (73 to 88%) associated with the reductant soluble fraction (Fe-Mn oxides)
and the remainder (19%) residing within the mineral lattice.93

C. PLUTONIUM BEHAVIOR IN PLANTS


Many researchers have evaluated Pu uptake and translocation in plants. A range of
concentrations of Pu present in plant tissue, relative to that in the soils in which the
plants were grown, has been reported. Plutonium concentration ratios in soil-culture
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 107

systems (CRS; concentration in plant/concentration in soil) ranged between 1010


and 103.114-123 These results indicate that Pu is not readily absorbed by plants.
Possible causes for the wide range of reported CRS values include soil characteris-
tics, particle size of Pu minerals, composition of the source material, chelation
effects, and concentration of Pu in the soil. Pimpl and Schttelkopf122 reported that
transfer coefficients (concentration ratios) are especially dependent on cation-
exchange capacity (CEC), the amount of organic material, pH, and the type of
contamination in soil. Wildung and Garland124 also offered that the soil concentra-
tions of plant-available forms of the individual actinide elements are related to a
range of complex soil processes, including microbial metabolism, ion exchange, ion
precipitation, complexation, mineral formation, and mineral dissolution.
The amount of Pu assimilated by tumbleweed (Sasola sp.) increased in Pu-
contaminated soils treated with DTPA, with up to a 1000-fold increase in uptake
reported.125 Vyas and Mistry126 found significant increases in plant uptake in both an
oxisol and a vertisol when Pu was supplied as Pu-DTPA compared to Pu-EDTA or
Pu nitrate. Vyas and Mistry126,127 also found decreases in the quantities of Pu-DTPA,
Pu-EDTA, and Pu nitrate assimilated by plants grown in nutrient solution compared
to soil. This phenomenon could be an indication of competitive absorption by another
cation that was limited in the soil, but not in the nutrient solution. Eolian transport
and subsequent deposition have been identified as the major cause of Pu movement
in agricultural ecosystems as well as the major source of shoot contamination.94,128-131
Plant root absorption can be distinguished from adsorption by soil colloids by
comparing Pu uptake by plants grown in hydroponic solution, where interactions
with the exchange complex of soil and other soil constituents are absent, with uptake
from soil.119,127 Vyas and Mistry127 reported that the Pu concentration ratio in hydro-
ponics (CRH; concentration in plant/concentration in nutrient solution) ranged
between 103 and 101.
Pimpl and Schttelkopf122 performed a literature search to evaluate the magni-
tude of transfer factors (concentration ratios) for Pu. They found transfer factors in
the range of 109 to 103. The transfer factor was dependent on the cation-exchange
capacity and pH of the soil. Transfer factors or concentration ratios (CR) were found
to be higher in the rice grain than in the shoots.117 Wildung and Garland114 reported
that the concentration of Pu in roots exceeded that in the shoots by factors of 3 to
8, depending on soil Pu concentration. Plutonium was also shown to be translocated
downward to the roots after removal from the soil. Complexing agents increased the
transfer coefficients by up to 1300-fold.122 Lipton and Goldin132 reported that
increases in plant uptake of Pu due to chelation were on the order of 1000-fold.
Regardless of the form of 238Pu contaminant used, it has been shown that the
assimilation rate by the plant is relatively constant.133 In winter wheat (Triticum
aestivum), it has been established that up to 70% of the contamination of grain by
Pu is due directly to the redeposition of dust during harvesting.130 McLeod et al.121
found that in different cropping rotations (winter wheat to soybean, fallow to corn,
continuous Bahia grass, and continuous white clover) on the same contaminated
soils, liming decreased the assimilation of Pu by all crops at all soil concentrations
of Pu. It was also determined that the assimilation rate by the plant was independent
of the concentration of Pu within the soil.121
108 Trace Elements in the Rhizosphere

Vyas and Mistry134 studied plant uptake of 239Pu and Americium-241 (241Am)
from different clay mineral (montmorillonite and kaolinite)-organic matter-sand
mixtures simulating contrasting soil types. They reported a marked reduction in
uptake of both 239Pu and 241Am with an increase in organic matter as well as clay
content of the mixtures. Reduction in the uptake of 241Am with increasing clay
content was more pronounced in the montmorillonite-organic matter mixtures com-
pared to the kaolinite-organic matter mixtures. They also observed that increasing
organic-matter content and the interaction of organic matter with montmorillonite
and kaolinite reduced the uptake of 239Pu and 241Am by plants.
In general, the information available on this topic has been from a dosimetry
standpoint, with little emphasis on developing an understanding of the mechanisms
involved in the uptake of Pu by plants. Agricultural plants were investigated for their
ability to accumulate Pu and the potential migration of Pu into humans due to
ingestion of agricultural products. More Pu was found in the seeds of soybean
(Glycine max)135 than would normally be expected. This was likely due to harvesting
practices in which the suspension of airborne particles and subsequent deposition
onto the seed contributed to the quantity of Pu present.
As with U, it is likely that rhizosphere processes could strongly influence
solubility of Pu. The predominant factors that might be expected to influence Pu
absorption are plant and microbially induced pH changes that occur in the rhizo-
sphere and the release of organic metal-complexing agents by plant roots and
microbes. It is very unlikely that specific channels or transporters exist in the root
plasma membrane for the absorption of U or Pu. Absorption likely occurs by mass
flow through nonspecific channels or plasma membrane defects. Therefore, rhizo-
sphere processes that influence root plasma membrane structure and selectivity (e.g.,
chemical or enzymatic damage to the membrane) might also contribute to the
absorption of U or Pu.

VII. U PHYTOEXTRACTION
Recently, a few U phytoextraction investigations have been reported.4,75,76,31 Some
reports on the high bioavailability of U to agriculture plants are also available.75
Results of U accumulation by some of the plant species used in the experiment of
Hossner et al.77 are shown in Figure 5.3. Uranium uptake and accumulation by plant
species grown in a soil contaminated with 100 mg U(VI) kg1 differed significantly
(Figure 5.2). Uranium concentrations in plant shoots ranged from 3.5 to 23 mg U
kg1. Indian mustard and sunflower had the highest U concentrations in the shoot,
while wheat had the lowest U concentration (3.5 mg U kg1). Sheppard et al.70 found
that U concentration of alfalfa and chard ranged from 0.0 to 27.6 mg kg1 when 50
and 100 mg U kg1 were applied to the soil as uranyl nitrate. Saric et al.67 also found
a large variation in U uptake among plants and concluded that sunflower and leafy
vegetables accumulated more U than other plants. Shoot concentrations of U in plant
species from the Brassica family were higher than from grasses77 (Figure 5.2).
Mustard green, turnip, and Chinese cabbage from the Brassica family and Swiss
chard (Beta vulgaris), a vegetable root crop, were better U accumulators than grain
crops. Sheppard et al.70 reported that Swiss chard U concentration ranged from 0.0
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems
FIGURE 5.2 Uranium concentration in shoots and roots in plant species grown in a Weswood soil (pH 7.55, CaCO3 equivalent 2.45%; fine-silty, mixed,
thermic Fluventic Ustochrept) contaminated with 100 mg U kg1 as uranyl nitrate.

109
110 Trace Elements in the Rhizosphere

FIGURE 5.3 Uranium concentration in shoots and roots of sunflower (H. annuus) grown on
soils contaminated with 300 mg U(VI) kg. Chelates were applied as solutions to soils after
plant emergence. Control denotes U concentration plants grown in soils in the absence of
chelates.

to 27.6 mg kg1 with soil contamination with uranyl nitrate at levels of 50 to 100
mg U kg1.
Uranium accumulates mainly in the roots. Uranium concentrations in roots of
different plant species were markedly different and varied from 69 to 789 mg U kg1
of root dry weight (Figure 5.2). Indian mustard and sunflower had the highest root
U concentration, and wheat and rye the lowest concentration. Sunflower and Indian
mustard are reported accumulators of U.30,38
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 111

Meyer and McLendon136 and Meyer et al.137 investigated phytotoxicity of


depleted uranium (DU) and its influence on soil microbial function. The DU was in
the chemical form of schoepite (UO2(OH)2 H2O). Soil respiration and organic
matter decomposition was significantly decreased at 500 mg DU kg1 and 25,000
mg DU kg1, respectively. Biomass, propagation, and long-term survivability of three
native grasses (buffalo grass (Buchloe dactyloides), little bluestem (Schizachyrium
scoparium), and purple threeawn (Aristida purpurea) at the Yuma Proving Grounds
in Arizona decreased only at the highest level (25,000 mg DU kg1).

VIII. U RHIZOFILTRATION
Radionuclide contamination in water is usually removed by ion exchange, reverse
osmosis, microfiltration, precipitation, or flocculation. These methods are of limited
use for large water volumes, low metal concentration, and saline water, and are
expensive. Recently, Gu et al.138 used Fe fillings as reductants in removing U from
contaminated water. Results indicated that Fe fillings were much more effective
than adsorbents such as peat, iron oxide, or organic cation exchangers in removing
uranyl (UO22+) from aqueous solution. Nearly 100% of U was removed by reaction
with Fe. This is an inexpensive and effective method, though reduced U(IV) pre-
cipitated on the Fe surface could be reoxidized to U(VI) and remobilized.
Rhizofiltration is an alternative technology of remediation in aqueous systems
that might provide a cost-effective method to treat contaminants at very low con-
centrations.31,32,139 Rhizofiltration is a technique in which plants are grown in polluted
water for subsequent sorption of the target element(s) by the live roots. Sunflower
has been shown to have some potential for rhizofiltration.30 The high root biomass
has been effective in removing heavy metals from polluted water. Sunflower grown
hydroponically was able to drastically reduce the concentration of Cr and U(VI) to
low levels within 24 hours.32 In tests carried out in Ashtabula, OH, sunflower roots
were submerged in water contaminated with 100 to 400 g U L1.140 The U content
was decreased to below the EPA standard of 20 g U L1. Sunflower was shown to
be very effective in recovering U from contaminated water.1 Uranium was accumu-
lated mainly in the roots with a final concentration 5,000 to 10,000 times greater
than the concentration in water. Both sunflower and Indian mustard tend to concen-
trate metals in the roots and translocate only a small portion of the metal to the shoots.
Scientists used sunflower plants to clean a pond at Chernobyl contaminated with
cesium and strontium, the most common radionuclides left from the fallout of the
1986 nuclear reactor disaster, which polluted more than 4.6 million hectares.141,142
Dushenkov et al.31 tested the ability of two sunflower cultivars, bean and Indian
mustard to remove U from an Ashtabula, OH, site with a U concentration of 56 g
L1. Sunflower cultivars removed more than 95% of the U from solution in 24 hours.
Almost all of the U removed from water in a laboratory experiment was concentrated
in the roots. The bioaccumulation coefficients based on the ratio of U concentration
in the roots to U concentration in the aqueous phase reached 30,000. Shoot U
concentration was <5 g g1 and root concentration was >15,000 g g1. The best
environment for U removal using rhizofiltration was a moderately acidic pH (approx-
imately 5.5). Dushenkov et al.31 concluded that root U absorption is a combination
112 Trace Elements in the Rhizosphere

of physical and chemical processes such as precipitation, chelation, and ion


exchange. Dushenkov et al.s results suggest the potential feasibility of rhizofiltration
for U removal from waste streams; however, for development of a full scale system
to treat a total volume of 2,000 million liters of U-contaminated water, the rhizofil-
tration system might have limitations.
Ebbs et al.4 found that in a hydroponic system at pH 5.0 the uranyl (UO22+)
cation was more readily absorbed and translocated by pea than the hydoxyl and
carbonate U complexes present in the solution at pH 6.0 and 8.0, respectively. The
following plant species were screened in soil and hydroponics for the ability to
accumulate U: pea, Indian mustard, turnip, red beet, alfalfa, common crown and
hairy vetch, tepary bean, oats, and corn. Of these species, tepary bean and red beet
had the greatest accumulation of U. HEDTA and citric acid were evaluated for their
influence on U uptake by red beet. Citric acid was the only amendment that signif-
icantly increased U uptake, but the increase was primarily attributed to the decrease
in soil pH from 6.8 to 5.0.
Rhizofiltration might be cost effective for groundwater treatment. Ensley143 of
Phytotech (Reston, VA) indicated that plants could be used to clean up U-contam-
inated ground water at 60 DOE sites at the cost of $2 to 6 per thousand gallons of
water treated, including capital and waste-disposal costs. In comparison, a recently
developed precipitation and microfiltration process to remove radionuclides is pro-
jected to cost $80 per thousand gallons of water.

IX. ENHANCEMENT OF U AND PU UPTAKE BY PLANTS


A. EFFECT OF SYNTHETIC CHELATES AND ORGANIC ACIDS ON
U UPTAKE
One of the limitations of phytoremediation is the lack of solubility and bioavailability
of target metals in soil. These metals are often present in the soil as insoluble forms
or as species that are strongly bound to soil particles. A solution to this problem
was reported by Huang and Cunningham144 in which nonaccumulator plants with
high biomass were grown in Pb-contaminated soil, and just before maximum growth,
a chelating agent, N-2-hydroxyethylenediaminetetraacetic acid (HEDTA), was added
to the soil. The accumulated Pb is toxic to the plants, but the plants are harvested
before death.
Chelates increase the concentration of total dissolved metal in the soil aqueous
phase and have been shown to make insoluble cations available to plants. Chelates
increase the mobility of cations to roots by replenishing those absorbed by plants.
Chelating agents, including diethylenetriaminepentaacetic acid (DTPA), ethylenedi-
aminetetraacetic acid (EDTA), ethylenediamine di-o-hydroxy-phenylacetic acid
(EDDHA), and nitrilotriacetic acid (NTA), have been used extensively as means of
supplying micronutrients to plants in intensive agriculture and in terrestrial and
aquatic ecosystems.126,145
Acidification or the application of chelates are chemical treatments commonly
used to bring sorbed metals into solution. The use of chelating agents is the most
practical way to solubilize metals.146 Dushenkov et al.11 found that EDTA was the
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 113

best chelator for the leaching of Pb from the soil and enhancing Pb uptake by plants.
In the soil, the applied chelate acts first to complex the soluble metal in the soil
solution. As the free-metal activity decreases, dissolution of bound or precipitated
metal ions begins to compensate for the shift in equilibrium. This process continues
until the equilibrium saturation of the chelator ligand with the metal is achieved, the
supply of metal from solid phases is exhausted, or the equilibrium solubility of the
metal ion with respect to the solid phase is achieved. The uptake of metal by the
plant must then involve either: (1) the release of the metal from the chelate and
absorption of free metal by the plant, (2) the absorption of intact chelate-metal
complex, or (3) the exchange of metal between the chelate ligand and a plant
metabolic ligand. In the case of U and Pu, these processes are not well understood.
The use of chelates in soils to increase metal uptake by plants has a substantial
impact on the phytoextraction of heavy metals. Chelates have been used extensively
to enhance plant nutrient uptake and the uptake of insoluble elements by plants.147-
149 Recently Huang and Cunningham144 and Huang et al.150 have shown that the

complexation of Pb with EDTA resulted in a Pb concentration of 1% in corn plants.


In this experiment, EDTA added to soil at the rates of 0.5, 1.0, and 2.0 g kg1
increased soluble Pb content of the soil solution from 0 to 4000 g mL1 within 24
hours. The Pb content of pea (Pisum sativum) reached 6000 g g1 with the addition
of 0.5 g EDTA kg1 soil. EDTA was by far the most efficient chelate used compared
to HEDTA, DTPA, EGTA (ethylenebis(oxyethlenenitrilo)tetraacetic acid), and
EDDHA. EDTA also has the advantage of being the least expensive of the chelating
agents. Marten and Hammond151 found that EDTA application to Pb-contaminated
soils increased the Pb content of biomass from 5 to 35 mg g1.
Lead translocation from roots to shoots is limited, and only a small proportion
of absorbed Pb is usually translocated from the roots to the shoots.152 The application
of EDTA to Pb-contaminated soils significantly increased Pb transport in the xylem
of plant roots,152 and sap Pb concentration was positively correlated to the increase
of Pb concentration in xylem with the increase in Pb translocation from roots to
shoots. Compared to the control, added EDTA (1.0 g kg1) increased Pb translocation
from roots to shoots 120-fold within 24 hours.
Huang et al.75 tested the effects of synthetic chelates, inorganic acids, and organic
acids on soil U desorption following addition to U-contaminated soil. Application
of citric acid (20 mmol kg1) increased the U concentration in soil solutions from
1.2 to 240 mg L1, representing a 200-fold increase. The driving force for the citric-
acid enhanced soil-U desorption was probably the complexation of U by citric acid.
Also, the low pH of the citric acid treatment resulted in the removal of coatings of
amorphous iron and aluminum sesquioxide from solid-phase U particles, which
enhanced the dissolution and transfer of U from soil to solution. Huang et al.75 also
examined 30 plant species for U uptake and translocation in U-contaminated soils
with and without citric acid treatment. Four plant species, Brassica juncea, B.
chinensis, B. narnosa, and amaranth (Amaranthus retroflexus), demonstrated signif-
icant potential for U accumulation in shoots. U concentration in the shoots of these
four plant species increased more than 1000 times in response to the application of
citric acid to the contaminated soil. There are several advantages of using citric acid
as a soil amendment for U phytoextraction. Citric acid is biodegradeable,76 which
114 Trace Elements in the Rhizosphere

helps to reduce the possible movement of U-citric acid complexes within the soil
profile. Citric-acid triggered U accumulation in plants is very rapid, which helps
reduce the risk of having plants with high U levels in the field for a long period of
time. Another advantage of using citric acid is its relatively low cost.
Hossner et al.77 studied the effect of different chelators and rates of application
on U accumulation by two potential U accumulators grown in four different soils.
They found that the degree of U uptake and accumulation among soils was different
even before chelate addition (Figure 5.3). Uranium concentrations in sunflower
shoots of the control treatment of soils contaminated with 300 mg U(VI) kg1 and
without chelate addition varied between 21 and 165 mg U kg1. Sunflower grown
in calcareous Weswood soil (pH = 7.6) had the highest U uptake, and that grown in
very acid Crowley soil (pH = 4.85) had the lowest U uptake. Chelate addition
enhanced U accumulation in both shoots and roots of sunflower in each of the four
soils (Figure 5.3). However, the increase was not as dramatic as reported by Huang
et al.,76 except in sunflower plant roots grown in Crockett and Weswood soils.
Addition of EDTA, citric acid, or oxalic acid to the acid soils, Labelle and Crowley,
did not influence the U concentration in sunflower roots (Figure 5.3). Chelator
addition increased shoot U concentrations two- to fivefold to values no greater than
500 g g1. Citric acid was the most effective of the chelates applied to Crockett
and Weswood soils. In Crowley and Labelle soils, EDTA was the most effective
chelate. The highest U accumulation was in plants growing in soils with CaCO3 or
high pH. Uranium concentration in the roots of sunflowers grown in Crockett soil
(pH = 7.1) in the presence of oxalate was more than 5500 mg kg1. The sunflower-
root U concentration of the control treatment of Crockett soil [(300 mg U(VI) kg1
of soil with no chelate addition)] was also very high (about 2700 mg U kg1). The
source of U contamination in soils in this study was uranyl nitrate which is soluble
in water at the concentrations used. Uranium in its oxidized form, U(VI), is more
soluble than the reduced U form; however, oxidized U(VI) can vary in solubility
depending on the physicochemical properties of the soil.59 In acid soils and in the
absence of complexing ligands, the uranyl cation (UO2)2+ would be readily adsorbed
onto exchange sites of clay minerals. However, in alkaline soils containing carbonate
minerals, the uranyl ion is complexed with the carbonate ion, forming highly mobile,
anionic complexes [i.e., (UO2)(CO3)22, (UO2)(CO3)34]. The processes of uranium
adsorption and complexation might have influenced the uptake and accumulation of
U in acid Labelle and Crowley soils and the calcareous Weswood and limed Crockett
soils. Uranium (VI) carbonate complexes would dominate the U(VI) speciation in
Crockett and Weswood soils. These complexes apparently did not translocate to the
shoots but seemingly precipitated either inside or outside of the roots. Uranium
solubility and mobility were limited in Labelle and Crowley soils due to the presence
of highly adsorptive Fe and Mn oxides.
In growth chamber experiments conducted by Dushenkov et al.31 more than 99%
of the U was found in the roots. The concentration of U in sunflower roots in the
rhizofiltration system was more than 10,000 g g1. Dushenkov et al.31 concluded
that the ability of roots to remove U from solution was plant species dependent.
Results of the experiments of Hossner et al.77 show that U transport from roots to
the shoots of sunflower is limited (Figures 5.2 and 5.3). Addition of synthetic chelate
Bioavailability of Uranium and Plutonium to Plants in SoilWater Systems 115

or organic complexing agents increased U concentration in the plant root but did
not contribute significantly to U accumulation in the plant tops.

B. EFFECTS OF SYNTHETIC CHELATES AND ORGANIC ACIDS ON PU


UPTAKE
The chelate complexes formed in soils serve to increase the concentration of dis-
solved elements in the root zone. Under normal conditions these elements are often
subjected to hydrolysis and sorption on soil particles. Although plant uptake of Pu
from soils is limited, the presence of chelating ligands in soils and the possible
formation of soluble complexes of Pu with these ligands could enhance the solubility
of Pu in soils. Vyas and Mistry153 investigated the influence of DTPA and EDTA
chelating agents on plant availability of Pu from soils. A large fraction, ranging from
60 to 90% of the soluble Pu added as nitrate underwent rapid conversion to precip-
itated compounds of hydrous oxides and hydroxides, as determined by selective
extraction. At 3 hours after contamination, the ion-exchangeable fraction (0.1 M
MgCl2 extracts) contained low levels of added Pu (0.7 to 19.1%). These levels
decreased further with increasing incubation time of up to 400 days. In contrast,
upon addition of chelated forms of radionuclides (Pu-DTPA and Pu-EDTA), up to
85% of the added Pu was associated with the 0.1 M MgCl2 extracts 3 hours post-
contamination, and significant amounts were extractable following incubation for
extended periods of up to 400 days. Hale and Wallace154 compared the effects of
the chelating agents DTPA and EDDHA on the uptake of heavy metal radionuclides
from soil by bush bean. DTPA increased Am uptake nearly 1000 times for 15-day-
old plants. Wallace155 also noted that DTPA promoted uptake of Am by soybean.
There was a 200-fold increase in Am uptake when the iron chelate, Fe-DTPA, was
applied to soil. It was also noted that Am was readily translocated within the plant
following initial uptake, for instance, from old leaves to newly formed leaves or to
roots, but DTPA had little effect on retranslocation.
Lipton and Goldin132 found an approximate 1000-fold increase in Pu concentra-
tion in pea plants when DTPA was added to Pu contaminated soils. Also, the DTPA
effect on Pu uptake by tumbleweed (Atriplex spp) amounted to about a 1000-fold
increase in the plant-to-soil ratio.125 A marked enhancement in the mobility of Pu
and Am through three contrasting Indian soil types was observed upon leaching with
a dilute chelating solution.156 Vyas and Mistry126 investigated the uptake of Pu from
two soils, an oxisol and a vertisol, and from nutrient solution. They found a signif-
icant increase in Pu uptake by plants from both soil types when Pu was added as
Pu-DTPA or Pu-EDTA compared to Pu-nitrate, and also found a marked enhance-
ment in uptake as well as translocation of Pu in plants due to the supply of chelated
Pu in the hydroponic system. They also observed that the Pu-EDTA complex was
less effective than the corresponding DTPA complex in enhancing the uptake of Pu
by plants. Garland et al.119 investigated whether Pu-DTPA supplied to plant roots is
absorbed and transported intact to shoots or whether it undergoes recomplexation.
In an experiment using hydroponically grown 18-day-old soybean (Glycine max)
plants placed in Pu-DTPA solution containing 48 ng of Pu mL1, the concentration
of Pu in shoot tissues and xylem exudates was determined over a 24-hour period.
116 Trace Elements in the Rhizosphere

The concentration ratio for tissues increased from 6 103 after 1 hour of uptake
to 0.3 after 24 hours. The concentration of Pu in xylem exudates increased from
about 0.03 ng Pu mL1 of exudate at 1 hour to 1.6 ng Pu mL1 at 24 hours. When
Pu was supplied as Pu(NO3)4 under these experimental conditions, less than 1 pg
Pu mL1 was found in exudates at 24 hours.

X. CONCLUSIONS
Most experimental results have shown that U and Pu accumulate mainly in the roots
of plant species. No plant species have been identified as hyperaccumulators of U
or Pu, however, there are differences in U and Pu accumulation in plants. U(VI)
uptake and translocation, and shoot and root concentrations among plant species,
vary by more than 100-fold.
It is unlikely that a phytoremediation strategy using hyperaccumulating plants
will be feasible for U and Pu remediation. However, there are plant species that can
accumulate substantial amounts of metals by changing their root environment and
altering metal availability. It has been suggested that hyperaccumulation may not be
necessary for remediation of U contaminated soils in the U.S.157
The effectiveness of remediation of soil is strongly influenced by soil type. The
use of plants to extract U and Pu from contaminated soils will require the identifi-
cation of high biomass U and Pu accumulating plant species. Two plant species,
sunflower and Indian mustard, have been identified as potential accumulators of Pu
and U. They may be especially effective in the decontamination of radionuclides in
aqueous systems, where contaminants can be accumulated in the roots using hydro-
ponic techniques. For the successful phytoremediation of soil, U and Pu must be
available to plant roots, absorbed by plant roots, and translocated from roots to
shoots. Further research to maximize both the absorption and translocation of radi-
onuclides in plants is needed.

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Section II
Interactions of Trace Elements
with Root Microbial Associations
6 Interactions and
Mobilization of Metal
Ions at the SoilRoot
Interface
Salvatore Deiana, Bruno Manunza, Amedeo
Palma, Alessandra Premoli, and Carlo Gessa

CONTENTS

I. Introduction...................................................................................................127
II. Role of the SoilRoot Interface in Uptake Processes .................................129
A. Interaction Reactions ........................................................................129
B. Mobilization......................................................................................135
C. Degradation Products and Their Activity in the Rhizosphere.........143
III. Conclusions...................................................................................................144
Acknowledgment ...................................................................................................145
References..............................................................................................................145

I. INTRODUCTION
The rhizosphere can be considered as an accumulation phase of protons, reductants,
and organic ligands produced by biological activity.1-5 Redox and complexation
reactions that take place in the rhizosphere can appreciably increase the solubility
of the soil mineral components, which leads to an increase of the macro- and
micronutrient concentration in the soil solution.6-8 During their life cycles, plants
release root exudates, which affect the mobility and uptake of nutrients as well as
the physical, chemical, and biological properties of the rhizosphere. The release of
the root exudates takes place mainly at the proximal region of the root hairs, which
are also responsible for the uptake of nutrients.
The continuous exchange of material between the soil and the root is essential
for plant nutrition, soil fertility, and environmental protection.9 The soil particles
and the root surface are joined by a mucilaginous interface, mostly made up of acid
polysaccharides.10-13 Figure 6.1 shows a transmission electron micrograph of an ultra
thin section of garlic root. The cell wall consists of two layers: the outer layer (the
soilroot interface), which is tenuous and largely mucilaginous and is characterized

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 127
128 Trace Elements in the Rhizosphere

FIGURE 6.1 Transmission electron micrograph of an ultra thin section of a garlic root. C =
cuticle. Magnification 20,000. (From Gessa, C. and Deiana, S.14 With permission.)

by a network of electrodense zones; and the inner layer, which is compact and mainly
made up of cellulose.14 The mucilaginous layer is discharged into the rhizosphere,
but is continuously regenerated by the Golgi apparatus.
The soilroot interface, which is generally 1 to 10 m thick,15 is characterized
by interlaced fibrils that define a porous system, known as free space volume. The
interface is highly hydrated and, acting as a selective accumulator for nutrients and
as a filter for chemicals, regulates their movement through and out of the cell. It
may support nitrogen-fixing bacteria as well as microorganisms, which produce
substances that antagonize the root pathogens.16
The absorption of nutrients by plants involves their transfer from the root surfaces
to the cell wall, hence to the plasmalemma, and finally to the cytoplasm. The soilroot
interface is involved in cation exchange reactions and often contains precipitates of
iron, manganese, and other elements. In fact, ion adsorption, precipitation, chelation,
reduction, and other reactions occur within the free space.17-21
Plant species differ widely in their ability to mobilize the sparingly soluble soil
compounds. As an example, iron efficient plants have adopted both morphological
modifications and chemical mechanisms for the mobilization and uptake of iron
from the rhizosphere. These include acidification of the rhizosphere, formation of
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 129

rhizodermal transfer cells, release of reducing and complexing components, and


activation of a redox pump at the plasma membrane.22-26
The concentration of the metal ions, such as iron, chromium, copper, vanadium,
and manganese at the soilroot interface, depends mainly on their oxidation state,
which determines their speciation and solubility. As a consequence, the concentration
of the metal complexed also varies. Such concentration can also be influenced by
competition among ligands. The competition for Fe(III) among caffeic acid, other
substances of low molecular weight, and the galacturonic units of the pectic sub-
stances are good examples.27
A schematic representation of the mucilage as an interface between soil and
roots, as well as some of the reactions that take place there, is presented in Figure 6.2.
Although many papers have been published concerning the role of chelating and
reducing agents in plant nutrition, the form by which metal ions are bound at the
root surface as well as the mechanisms that regulate the nutrient uptake are still
open questions.28
Root exudates are organic compounds of a different chemical nature, such as
low and high molecular weight compounds including phenolic and organic acids,
sugars, and aminoacids.29,30 The extent to which these compounds and other com-
ponents of the rhizosphere and of the cell wall matrix play a role in mediating the
metal ion solubility is still partially unknown.
Here we report on the more recent results concerning i) the interaction of the
soilroot interface with metal ions, ii) the ability of complexing and reducing
chemical species to mobilize metal ions from the interface, and iii) the effect of
such reactions and of the metal ions on the physical properties of the interface.

II. ROLE OF THE SOILROOT INTERFACE IN UPTAKE PROCESSES

A. INTERACTION REACTIONS
A Calcium-polygalacturonate (Ca-PGA) network, which resembles the natural root
mucilages and thus constitutes a good model of natural systems, was used to study
the interaction with metals.14,31 The micrograph (Figure 6.3) shows the fibrillar
structure of the Ca-PGA network. From a molecular point of view the fibrillar
arrangement of the PGA chains seems to result from the dissociation of the COOH
groups and the formation of ionic bridges. The carboxylic groups are arranged as
far apart as possible so that monomers are positioned at an angle of 120 degrees
(Figure 6.4). Interchain interaction is assured by Ca(II) bridges, each formed of two
COO groups. This structure is supposed to be stabilized by H-bonds formed
between the -OH groups of the polymers and the first layer of the water molecules.
Molecular Dynamics (MD) experiments confirm that the aggregation of the PGA
polymers is mainly due to interchain ionic bridges32 as evidenced in Figure 6.5 where
four MD cells of the Ca-PGA system, each with only two chains, are represented.
On the other hand, experiments carried out on a system formed by three PGA
chains in equilibrium with 16 Ca(II) ions, 20 Na+ ions, 12 Cl, and 1024 H2O
molecules demonstrated that calcium has a stronger affinity toward the polymer
130 Trace Elements in the Rhizosphere

FIGURE 6.2 Schematic representation of the root mucilage. SP: free space, S: soil particle,
L: ligand, ML: complex, PS: phytosiderophores, NFA: fulvic acids, NHA: humic acids, TR:
transport of Fe(II), TL: translocator for metal complexes, F: fibrils of Ca-polygalacturonate,
PM: plasma membrane.

matrix compared to Na+ ions. Calcium ions are complexed in the reticulating sites
forming interchain bridges, while Na+ ions diffuse in the free space (Figure 6.6).33
The porous system, which results from the fibrillar arrangement of the polymers,
defines the free space volume that mainly depends on the type of reticulating ion.
Calcium is an excellent reticulating agent as it induces high hydration of the network
and stabilizes the fibrillar arrangement of the system over a wide pH range.34 One
gram of Ca-PGA retains about 250 g of water; this amount increases as the Ca(II)
concentration in solution increases. On the contrary, Cu(II) destabilizes the porous
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 131

FIGURE 6.3 Scanning electron micrograph showing the fibrils and the free-space volume of
the Ca-PGA network. Magnification 100,000. (From Gessa, C. and Deiana, S.14 With
permission.)

system, as will be shown later, notably reducing the degree of hydration and then
the free-space volume. One gram of Cu-PGA retains 100 g of water and decreases
as the Cu(II) concentration increases.34
Ionic competition experiments35 between Ca(II) and Cu(II) ions indicate that:

1. The affinity of Cu(II) for the organic matrix is so high that at very low
concentrations of copper, the Cu(II) ions are nearly all complexed by the
polymer. This condition characterizes the soil liquid phase where the
calcium concentration is very high with respect to that of Cu(II).
2. The free-space volume of the Cu-Ca network decreases slowly as the
amount of Cu(II) increases up to 40%. Then a dramatic reduction occurs,
suggesting that the fibrillar system could be deeply modified if reticulated
Ca is lower than about 60%.

Scanning electron micrographs confirm the collapse of the fibrillar structure in


the Cu-Ca network. The porous system, which is well developed in the Ca-network,
is reduced in the system with 20% of reticulated copper and is critically damaged
in the system with 45% of Cu(II), as will be shown later. Subsequent treatments
with CaCl2 do not restore the fibrillar arrangement.
As the transport of a metal ion and its interaction with the organic components
of the soilroot interface can be affected by the presence of other heavy metals,
experiments on the interaction between Cu(II), Pb(II), Cd(II), and Zn(II) ions and
an artificial polygalacturonate network were carried out.
Absorption kinetics of Cu(II) (600 mM) and Pb(II) (600 mM) by the Ca(II)-
PGA network were performed at pH 3.5 and 5.5 in the presence of calcium acetate
132 Trace Elements in the Rhizosphere

FIGURE 6.4 Schematic representation of the fibril structure: conformation of two polyga-
lacturonate chains projected on the xy plane (A); the three different positions of the carboxylic
groups () refer to the molecular structure created by three rotations of 120 degrees around
the molecular axis; a, a, b, b, and c, c represent the carboxylate groups inside, above, and
below the plane, respectively. Structure of the polygalacturonic acid (B).
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 133

FIGURE 6.5 Four MD cells of the Ca-PGA system, with only two chains represented. (From
Manunza, B. et al.32 With permission.)

(6.0 mM). They indicate that Pb(II) has a slightly higher affinity toward the functional
groups of the network fibrils than Cu(II). The absorption of Pb(II) and Cu(II) at pH
3.5 were equal to 52 and 43 moles /180 moles of COOH groups, respectively,
whereas at pH 5.5 they were equal to 49 and 41 moles, respectively. The absorption
kinetics performed at the same pH values in the presence of both ions indicate that
the amount of Pb(II) absorbed does not vary considerably, whereas that of Cu(II)
decreases to 25 and 15 moles at pH 3.5 and pH 5.5, respectively (Figure 6.7). This
behavior can be attributed to both hydrolysis reactions of the metal ions and the
structural modifications of the network.
The same systems studied in the presence of Cd(II) and Zn(II) show that these
ions compete with Pb(II) and Cu(II) for the same sites.
The collapse of the fibrillar structure of the network induced by the Cu(II) ions
is characterized by the formation of microspheres, which are easily distinguishable
in the SEM microphotographs (Figures 6.8a and 6.8b), with a high Cu(II)
concentration as shown by X-ray mapping (Figures 6.9a and 6.9b). The formation
of microspheres does not occur when the collapse of the fibrillar structure is induced
by Pb(II) ions (Figures 6.10a and 6.10b). The IR spectra of the Pb(II)-PGA and
Cu(II)-PGA complexes suggest that the carboxylate groups coordinate the ion
through a sole oxygen atom. In contrast, the spectra of the Cd(II)-PGA and Zn(II)-
PGA complexes, similar to those of the Ca-polygalacturonates, indicate a bidentate
structure of the carboxylate groups in these complexes. The electron spin resonance
measurements at room temperature of the Cu(II)-PGA network show that the Cu(II)
absorption is accompanied by the formation of an inner-sphere complex. These
findings suggest that a purely electrostatic interaction between the Ca(II), Mg(II),
Mn(II), Zn(II), and Cd(II) ions and the carboxylate groups occurs (i.e., outer-sphere
complexes). On the contrary, the Cu(II), Fe(III), Cr(III), Al(III), VO(IV), and rea-
sonably Pb(II) ions form inner-sphere complexes with the carboxylate groups (Figure
134 Trace Elements in the Rhizosphere

FIGURE 6.6 Snapshots of the polygalacturonate chains from the end of the equilibrated MD
trajectory. The Ca(II) (large) and Na(I) (small) ions are represented as hard spheres. Water
molecules are omitted to make vision easier. (From Manunza, B. et al.32 With permission.)
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 135

FIGURE 6.7 Absorption kinetics at pH 3.5 and 5.5 of Pb(II) and Cu(II) by the Ca-network
in the presence of calcium acetate 6.0 mM. The initial metal concentrations were both 0.6 mM.

6.11). This justifies the markedly higher affinity of these latter ions for the polysac-
charidic matrix and the important role of the mucilages as an accumulation phase
of these metals.36,37
The results of this study have a great biochemical significance in the transfer of
heavy metals through the soilroot interface. Covalently bound ions are very dan-
gerous as they tend to induce the collapse of the fibrils of the root mucilage thus
destroying its porous structure.
The functionality of the PGA matrix depends on the metal concentration. In
fact, it acts as an accumulator of metals until their concentration is under a threshold
that is a property of the metal considered. Above this threshold the porous structure
can be seriously damaged so that it gives up acting as a selective filter, and toxic
ions can easily reach and injure the cellular biological structures. Probably, the ability
of several plants to tolerate the presence of toxic metals can be related to their
capacity to regenerate the mucigel at a high rate, allowing it to maintain its ability
to act as a filter.
The interaction studies of PGA with Fe(III), V(V), and Cr(VI) have shown that
redox reactions also occur in these systems.38 Mssbauer and Elecronic Spin Res-
onance (ESR) measurements evidenced the presence of Fe(II), VO(IV), Cr(V), and
Cr(III) ions. The redox reaction for Fe(III) and VO(V) leads to the formation of
formic acid as a decomposition product of the polymer (Figure 6.12).

B. MOBILIZATION
The metal complexed on the reticulating sites of the mucilage can be mobilized
by the root exudates. An important example of this process is represented by the
136 Trace Elements in the Rhizosphere

FIGURE 6.8 Scanning electron micrograph of the Ca-PGA network containing 10% (a) and
50% (b) Cu(II).
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 137

FIGURE 6.9 X-ray mapping of the Ca-PGA network containing 10% (a) and 50% (b) Cu(II).
Magnification 6,000.
138 Trace Elements in the Rhizosphere

FIGURE 6.10 Scanning electron micrograph of the Ca-Pb-PGA network (a) and its X-ray
mapping (b). Magnification 6,000.
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 139

FIGURE 6.11 Schematic representation of outer-sphere (a) and inner-sphere complexes (b).

FIGURE 6.12 Oxidation of the monomeric terminal unit of PGA by V(V).

mobilization of iron, which can occur through both ligand exchange or redox
reactions.
In the first case, complexing agents such as mugineic acid or compounds
synthesized by microorganisms such as DFOB (desferrioxamine-B) can free Fe(III)
reticulated on the polymer.39 Desorption kinetics carried out with CaCl2 solutions
show that this treatment does not remove the metal from the reticulating sites,
whereas DFOB, which had little effect on the amorphous iron hydroxide, forms
140 Trace Elements in the Rhizosphere

FIGURE 6.13 Iron(III) reduction process by CAF.

very stable chelates with Fe(III).40 Caffeic acid (CAF) is particularly active in the
redox processes.41-44
What happens when a reductant such as caffeic acid exuded by roots runs through
the interface? In aqueous phase, the redox reaction is pH-dependent and, at pH <
3.5, it proceeds through two steps: the first is faster and involves five electrons, while
the second involves four electrons.41 The redox reaction is characterized by the
formation of compounds able to reduce Fe(III). At pH > 3.5, the redox activity
dramatically decreases. Hydrolyzed Fe(III) species interact with CAF giving rise to
the formation of soluble complexes that successively transform into insoluble iron-
CAF polymers (Figure 6.13). We wish to point out the great chemical flexibility of
CAF, which can activate redox, complexation, polymeriation, and adsorption reac-
tions depending on the environmental conditions.41
Caffeic acid also shows a strong redox activity toward the Fe(III)-PGA com-
plexes, but the mobilization of the metal undergoes a different mechanism.27 In fact,
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 141

the kind of iron coordination in the complex varies with pH so that a different redox
activity of CAF is observed. At acid pH values from 2.4 to 4.0, the metal coordinates
five or four water molecules and one or two oxygen atoms of the COOH group,
which prevents iron from undergoing hydrolysis and precipitation processes. At pH
values from 4.7 to 7.0, where the coordinated water molecules start to deprotonate
and form nonreducible hydroxylated species, the redox reaction is less effective so
that the Fe(II) yield decreases as pH increases. As the hydrolysis of chelated iron
starts at pH values higher than that of Fe(H2O)63+, the amount of reducible iron
bound to the mucilage is much higher in the pH range of 4 to 7.
The Fe(II) produced can be separated into two fractions: diffusible and exchange-
able with CaCl2 6.0 mM. The Fe(II) mobility, in addition to electrostatic effects that
are established between Fe(II) and the PGA carboxylic groups, can be notably
conditioned by the interaction of the CAF oxidation products with the metallic center
in the reticulation sites. The fibrillary structure that characterizes the calcium network
is altered following the substitution of Ca(II) with Fe(III) ions and is restored by
the reduction of Fe(III) by CAF at pH 4.0. This arises from two distinct interaction
mechanisms, which are established between Fe(III), or Fe(II), and the carboxylic
groups of the fibrils. Iron(II), like Ca(II), forms outer sphere complexes, whereas
Fe(III) forms more stable complexes. This has important implications in plant nutri-
tion because the porous structure of the interface favors the transfer of nutrients to
the plasmalemma.
The CAF oxidation products with high molecular weight are hypothesized to
be trapped in the fibrils, whereas those with low molecular weight, such as esculetin
(6,7-dihydroxycoumarin, ESC), can diffuse in the free space (Figure 6.14).44-46 These
products were found to possess reducing and complexing activity and, therefore,
can be considered active in the mobilization of nutrients in the rhizosphere. In

CH CH COOH

OH
Free space OH
Fibril

Free space
C
C C

O O
O O O O
2+ +
Fe(II)-,Fe(III)-CAF Polymers Fe(III) FeOH Fe(OH)2
O O O O
O O

C C C

Free Space

Oxidation products of low molecular weight

FIGURE 6.14 Schematic representation of Fe(III) reduction by CAF inside the fibrils.
142 Trace Elements in the Rhizosphere

FIGURE 6.15 Structure and probable formation mechanism of products following the reduc-
tion of Fe(III) by esculetin. Fe(III)/ESC molar ratios < 3 (A) and > 3 (B).

particular, esculetin is able to reduce Fe(III) to Fe(II) in the pH range 3.0 to 4.0
through the formation of two main products whose structure and probable formation
mechanisms are reported in Figure 6.15. Their formation can be explained by
considering that the phenoxylic radicals (ArO.), well represented in the systems with
an Fe(III)/ESC molar ratio < 3, can couple or react with other radicals to form
esculetin oligomers, whereas when the Fe(III)/ESC molar ratio is > 3, esculetin is
oxidized to organic acids. At pH > 4 the reducing activity decreases and the formation
of Fe(III)-ESC complexes, which tend to precipitate, is observed. Organic acids
present in the root exudates, such as malic, pyruvic, citric, and oxalacetic acid did
not affect the redox reaction but kept these complexes in solution. This is important
as Fe(III)-ESC complexes can thus reach the plasmalemma where they can be
absorbed or subjected to Fe(III)-chelate reduction. The same molecule is able to
buffer the toxic effects of Cu(II) by reducing this ion to Cu(I), which, in the presence
of chloride ions, tends to precipitate as sparingly soluble CuCl. Other CAF oxidation
products obtained by electrochemical methods or by reactions in an alkaline medium
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 143

with molecular oxygen were found to possess both reducing and complexing activity
toward Fe(III) and a marked complexing activity toward Fe(II).43

C. DEGRADATION PRODUCTS AND THEIR ACTIVITY IN THE


RHIZOSPHERE
The uptake of metal ions depends strongly on the presence of complexing agents at
the soilroot interface and in the rhizosphere. Redox processes are often combined
with these complexation reactions. In this context, uronic and phenolic acids present
in both monomeric and polymeric forms on the root surfaces (mucigel) and the cell
wall (apoplast) play a fundamental role. The root mucilages released into the rhizo-
sphere are degraded to uronic acids, which constitute a pabulum for microorganisms
or activate redox and complexation reactions.
Iron mobilization and detoxification processes that occur in the rhizosphere
constitute a topic of particular importance in plant mineral nutrition. Studies on the
Fe(III)-D-galacturonic acid system showed that the organic molecule decomposes
to organic acids of low molecular weight, whereas the metal is reduced.47,48 In the
first reaction step, an Fe(III)-galacturonate complex was formed; the iron interacted
with both the carboxylate groups and the ring oxygen of the sugar, producing a
reducing species with a free aldheydic group, which constitutes the redox site for
the reaction (Figure 6.16). It is evident that metals capable of forming this kind of
coordination with the galacturonate anion are potential reductants and that the redox
activity of the complex would increase with its stability. Metal ions such as Cu(II),
Cr(III), Pb(II), and UO2(VI), able to form reducing complexes with D-galacturonic
acid, strongly enhance the yield in Fe(II), indicating the formation of particularly
powerful reductants.49-51 On the contrary, Ni(II), Cd(II), Ca(II), Fe(II), Mn(II)-galac-

O O O O O O
C M C M C
M

C O C OH C O
OH O -H+ O
M + GAL C C C C C C
H H
C C C C C C

OX

Fe2+; Cr3+; VO2+, FORMIC ACID AND OTHER ORGANIC ACIDS

M = Fe3+; Cr3+; Cu2+

OX = Fe3+; Cr2O7=; VO2+

FIGURE 6.16 Mechanism of the D-galacturonic acid oxidation by Fe(III), Cr(VI), and V(V).
144 Trace Elements in the Rhizosphere

turonate complexes do not induce a significant change in the Fe(II) yield with respect
to the simple Fe(III)-D-galacturonate system.
At the soilroot interface, the D-galacturonic acid can also reduce toxic-heavy
metals such as Cr(VI).52 This redox reaction can be considered a Cr(VI) detoxifica-
tion mechanism because CrO42-, a very dangerous and mobile anion in the soil-plant
system, transforms into the Cr(III) cation, which tends to precipitate as Cr(OH)3, to
form organic complexes or to be immobilized on particle surfaces. The reduction
of Cr(VI) is slow even at low pH values, but proceeds much faster in the presence
of Cr(III) or other metals which, through their reaction with D-galacturonic acid,
form reducing metal complexes. It is important to note again the particular behavior
of the redox system, which can be activated by the same reduction product.

III. CONCLUSIONS
The soilroot interface, as well as the apoplast, can be represented as an interlacing
of channels that controls the flow of water, ions, organic molecules, and complexes
with different charges through and out of the cells. This system of channels is
stabilized by ionic bridges, mainly composed of Ca-complexes. A substantial sub-
stitution of Ca(II) with ions such as Al(III), Fe(III), Cr(III), Cu(II), VO(II), and Pb(II)
in the reticulation sites can damage the interface, thus exposing the absorbing cells
to the action of dangerous environmental agents. Nutrient mobility inside the inter-
face can be notably different depending on the affinity for biopolymers. In this sense,
it is possible to distinguish:

1. Chemical species with low affinity like potassium, sodium, and several
anionic and molecular species that flow rapidly through the interface;
2. Chemical species such as Ca(II), Mg(II), Fe(II), Mn(II), Zn(II), and Cd(II)
that interact electrostatically in the reticulation sites forming outer-sphere
complexes and can be mobilized by ionic exchange;
3. Chemical species with a high affinity that interact in the reticulating sites
as inner-sphere complexes such as Al(III), Fe(III), Cr(III), Cu(II), and
Pb(II). They show low solubility and are accumulated at the interface and
apoplast from which they can be released following redox and/or com-
plexation reactions. These ions can be very dangerous for plants because
they can damage the root surface. For example, in acid soils, aluminum
can present serious problems for several crops;
4. Chemical species unable to cross the free-space system due to their high
molecular size.

The uptake of a chemical element is strongly influenced and conditioned by the


composition of the soilroot interface. Thus, pH, chemical composition, redox poten-
tial, and ionic strength play important roles in determining the chemical form of the
element and its affinity for any ligand agent.
There are questions that are still to be probed and future surveys are encouraged
concerning:
Interactions and Mobilization of Metal Ions at the SoilRoot Interface 145

the role of the chelating and reducing agents in plant nutrition,


the way in which ions are bound at the root surface,
the nature of the bond that is established between the metal ion and the
components of the root surface,
the mechanisms that determine the mobilization of these nutrients.

Therefore, our future work will focus on improving the model of the soilroot
interface by using other components present in the mucigel and extending the study
in vivo of this matrix in the plant mineral nutrition.

ACKNOWLEDGMENT
The financial support of 40% from Ministero dellUniversit e della Ricerca Scien-
tifica e Tecnologica (MURST) is gratefully acknowledged.

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7 Analysis of Metal
Speciation and
Distribution in Symbiotic
Fungi (Ectomycorrhiza)
Studied by Micro X-ray
Absorption Spectroscopy
and X-ray Fluorescence
Microscopy
Bjrn O. Berthelsen, Geraldine M. Lamble,
Alastair A. MacDowell, and David G. Nicholson

CONTENTS

I. Introduction...................................................................................................150
II. Ectomycorrhiza Structure and Function .................................................150
III. Heavy Metal Accumulation and Binding in Mycorrhizal Biomass ............152
A. Uptake and Accumulation of Heavy Metals ....................................152
B. Metal Localization ............................................................................152
C. Binding Mechanisms Speciation of Metals ................................153
IV. Techniques and Approaches for Studying Metal Speciation in Fungal
Biomass.........................................................................................................154
A. Available Approaches and Techniques.............................................154
B. X-ray Absorption Spectroscopy (XAS) ...........................................155
V. Zn Speciation in Ectomycorrhiza from Contaminated Forest Soils
Studied by Micro-XAFS ..............................................................................156
A. Materials and Methods .....................................................................156
1. Sampling and Sample Pretreatment ..................................156
2. Instrumentation and Analyses ...........................................157
B. Results and Discussion.....................................................................158
VI. Conclusions...................................................................................................161
References..............................................................................................................162

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 149
150 Trace Elements in the Rhizosphere

I. INTRODUCTION
This chapter mainly focuses on a new technique for examining metal binding and
speciation in microscopic structures, micro X-ray Absorption Fine-Structure Spec-
troscopy (mXFAS), a technique based on conventional X-ray Spectroscopy (XAS).
mXFAS may prove to be useful for analysis with respect to trace element binding
and speciation in several types of microorganisms or specific parts of biological
structures other than ectomycorrhiza, which is addressed in this chapter.
Through the years international literature has provided overwhelming documen-
tation on the ability of a great many types of microorganisms, including fungal
species with a wide variety of growth strategies, to accumulate large amounts of
trace metals. Soil fungal biomass, in addition to its numerous and important roles
in most ecosystems, makes up a significant part of the organic matter in surface
soils, with biomass values that may exceed 50 g kg1 as measured by the ergosterol
method.1 According to Bth et al.,2 FDA-active fungal length in forest humus may
exceed 400 m g1 dry soil. Thus soil fungal biomass may constitute an important
metal reservoir in surface soils. In addition to measurements of total metal content
in soil fungal biomass, knowledge on mechanisms of metal binding and speciation
is a key factor in fully assessing the role of fungal biomass in the biogeochemical
cycling of trace metals.
This chapter reviews ectomycorrhizal structures and functions with emphasis on
metal accumulation, localization, and binding mechanisms, followed by a detailed
description of the background and approaches provided by the mXFAS technique.
Other techniques for studying metal localization, distribution, and binding at a small
spatial scale are also summarized. Finally, the application of mXFAS to metal
localization and speciation in biological tissues is demonstrated in a case study on
Zn binding in ectomycorrhizal structures.

II. ECTOMYCORRHIZA STRUCTURE AND FUNCTION


Ectomycorrhiza constitutes one of seven kinds of mycorrhiza, representing a symbi-
osis between soil-borne fungal biomass and fine roots, mainly from trees.3 This
symbiosis provides several benefits to both the host and the fungus. The absorbing
ability and activities of the ectomycorrhizal and extraradical hyphae respond to
important metabolic needs of the host. The fungal hyphae within the root are, on the
other hand, protected from competition with other soil microorganisms, and are
preferential users of the host photoassimilates.4 In addition, the symbiosis is likely a
result of morphogenetic and physiological changes, altogether forming a permanent
and efficient structure.4 The fungal part of the symbiosis originates from many families
of Basidiomycotina, some of the Ascomycotina, and a few of the Zygomycotina.3
Figure 7.1 shows a cross-section of an ectomycorrhizal association between
Pisolithus and Eucalyptus displaying important parts of the symbiotic structure. The
outer part of the structure consists of the fungal sheath or mantle (Figure 7.1), which
encloses the root in fungal tissue. The fungal sheath is most often 30 to 40 m thick
and comprises 25 to 40% of the dry weight of the whole structure.3 The sheath may
Analysis of Metal Speciation and Distribution in Symbiotic Fungi 151

FIGURE 7.1 Cross-section of ectomycorrhiza of Pisolithus in association with Eucalyptus.


fs fungal sheath, hn Hartig net, epi epidermis, end endodermis, oc outer
cortex, ic inner cortex. (Photo by Treena Davies and Bernie Dell, Murdoch University.
Reprinted with permission.)

be monolayered, constructed of coherent hyphae, or structured pseudoparenchyma-


tously throughout, often consisting of two layers.3 There are types of ectomycorrhiza
that contain extramatrical mycelium directly connected to the sheath (typical for a
majority of ectomycorrhiza forming fungi) and types where no, or only sparse,
mycelium is seen on the surface of the sheath, which is typical for mycorrhizas of
Russula and several species of Lactarius. To compensate for the lack of a large
network of extraradical hyphae, the latter types typically produce a vast number of
mycorrhizal structures to explore the largest possible soil volume. From the sheath,
hyphal connections grow between epidermal and cortical cells of the host root
forming the Hartig net (Figure 7.1).3 In senescent parts of the mycorrhiza, hyphal
cells also colonize the cortex.3 Formation of a Hartig net does not, however, affect
the symplastic pathways through the cortex.5
Generally the fungal symbiont is not able to decompose complex carbon com-
pounds, but instead obtains easily degradable carbohydrates from the host.6 In return,
the mycobiont supplies the tree with nutrients such as phosphorus, nitrogen, sulphur,
zinc, and copper.7 Mycorrhizae are particularly important in phosphorus nutrition,
because the bioavailability of this element in soils is typically very low. The enhanced
nutrient uptake of mycorrhizal plants is mainly a function of the greatly increased
soil volume explored and exploited.7
152 Trace Elements in the Rhizosphere

III. HEAVY METAL ACCUMULATION AND BINDING


IN MYCORRHIZAL BIOMASS
A. UPTAKE AND ACCUMULATION OF HEAVY METALS
Fungi are able to accumulate heavy metals of environmental concern from soils
exhibiting low metal concentrations. Several fungal genera with a mycorrhizal
growth strategy are able to do so including Amanita (Cd, Cu, Hg), Boletus (Cu, Hg),
Cortinarius (Cd, Cu, Hg), Laccaria (Cd), Lactarius (Hg), and Russula (Cd, Cu,
Hg).8 Species differences and soil conditions, e.g., pH, may influence metal uptake.
According to Gast et al.9 differences among species are more important regulators
of metal accumulation in mycorrhiza than soil conditions. Krantz-Rlcker et al.,10
on the other hand, found that accumulation of Zn, Cd, and Hg in mycelia of
nonmycorrhizal fungi increased with decreasing pH. The density of extramatrical
mycelium may also influence metal retention, with a dense mycelium being observed
to decrease the potential for Cd retention.11
Most studies of heavy metal uptake and the varying ability among fungal species
to accumulate heavy metals are based on in vitro experiments where the fungal
biomass is exposed to variable, often high concentrations of heavy metals. Mycelium
of Suillus granulatus and Lactarius deliciosus may accumulate large amounts of
Cu.12 Denny and Wilkins13 observed high concentrations of Zn in Paxillus involutus,
where concentrations in extramatrical hyphae were roughly 4 times higher compared
to mantle hyphae. Interesting findings have also emerged from studies of Zn and
Cd uptake by tolerant and low-tolerant strains of Suillus bovinus14,15 showing strongly
increased uptake with increasing metal concentrations in the medium. The uptake
efficiency, however, decreased with increasing metal exposure. Colpaert and van
Assche15 also found that Zn and Cd uptake was lower in the metal tolerant than in
the intolerant strain, and that increasing Zn concentrations in the medium decreased
Cd concentrations in mycelium from both strains of Suillus bovinus.
Heavy metal accumulation in mycorrhizal biomass under field conditions is
studied to a much lesser extent. Based on a study of 11 different morphological
types of ectomycorrhiza, Berthelsen et al.1 observed the highest uptake of Zn and
Cu in Cortinarius semisanguinea. The highest concentrations of Cd were generally
found in Suillus spp. The uptake of Pb into ectomycorrhiza, however, was very
low.1 Considering the large concentrations of Pb in organic surface soils in the
study area, Berthelsen et al.1 concluded that Pb virtually seems to be excluded from
ectomycorrhizas.

B. METAL LOCALIZATION
Metal-binding sites and distribution in mycorrhizal biomass have been extensively
studied for several years. However, caution is needed in such studies since microbial
structures are fragile and may be subject to structural damages upon sample prep-
aration and analysis. This may cause both a redistribution of metals as well as
changes in the metal speciation within the fungal structures. For instance, observa-
tions on metal association with polyphosphate granules as observed by Turnau et
Analysis of Metal Speciation and Distribution in Symbiotic Fungi 153

al.16 have been considered an artifact of specimen preparation. A study by Berthelsen


et al.17 showed dramatic changes in Zn and Cu concentrations in ectomycorrhiza
depending on storage conditions and pretreatment of samples prior to metal analysis.
Both drying followed by rewetting and freezing/thawing of soil samples before
dissection of mycorrhizas for metal analysis lead to large decreases in the mycor-
rhizal concentrations of Zn and Cu compared to mycorrhizas from field moist soils.
Caution is thus needed concerning sample preparation as well as data interpretation
in studies of metalmicroorganism relationships.
Several studies have shown that large fractions of the metals are located in fungal
compartments of fine roots, as well as in the extramatrical mycelium.13,18 The fungal
sheath is identified in several studies as a major metal reservoir in mycorrhiza, e.g.,
for Zn in beech mycorrhiza,19,20 Ni and Fe in oak mycorrhiza,21 and Cd and Al in
Rhizopogon roseolus.16 More rare and special observations in this context include
large accumulations of Zn in extrahyphal, polysaccharide slime in Paxillus involutus
as observed by Denny and Wilkins.13
More detailed localization studies have revealed that Zn may be accumulated
within the cytoplasm of the mantle in beech mycorrhiza, with a gradient across the
mantle indicating physiological differentiation.20 According to Turnau et al.,16 accu-
mulation of both Cd and Al may be observed in several parts of the fungal sheath,
including electronopaque deposition in vacuoles, periplasmic spaces, and extracel-
lular material. Turnau et al.16 also found relatively large amounts of Cd and Al in
phenolic material of dead cortical cells of the host, Pinus sylvestris.

C. BINDING MECHANISMS SPECIATION OF METALS


Heavy metals are bound in fungal biomass in several ways. Metal-binding mecha-
nisms may depend on both fungal species differences and type of metal. What makes
this picture even more complicated is the fact that fungi have developed two basically
different metal uptake mechanisms: one operating at low external concentrations,
which is mainly metal specific, and one relatively nonspecific mechanism dominating
at larger metal concentrations in the substrate.22
Binding mechanisms for heavy metals in ectomycorrhizal biomass reported in
the literature are primarily associated with cystein-containing proteins, metallothio-
neins. Associations of heavy metals with metallothionein or metallothionein-like
proteins have been observed in the case of Cd and Al in Rhizopogon roseolus,16 Cd
(Zn) for different species of Glomus,23 and Cu in sporophores of Laccaria laccata
and Paxillus involutus.24 According to Morselt et al.,25 protein-bound disulphides
and metal-thiolate clusters may be formed in metal tolerant Pisolithus tinctorius.
Other studies have concluded that heavy metal exposure of fungal biomass does not
induce production of metallothioneins, which underlines the complexity and heter-
ogeneity of fungi and fungalmetal relationships. Galli et al.26 observed neither
formation of phytochelatins nor metallothioneins in a study on Laccaria laccata
exposed to Cd. This sharply contrasts with the findings by Howe et al.24 Galli et
al.26 indicated that Cd is bound to glutathione in Laccaria laccata. The potential
role of glutathione in metal detoxification has been addressed in detail by Mehra
154 Trace Elements in the Rhizosphere

and Winge.27 who stated with respect to fungi: Glutathione appears to provide the
first line of defense and may be the only detoxifying molecule under certain condi-
tions. Esser and Brunnert28 studied Cd speciation in fruiting bodies of Agaricus
bisporus. Even though they were studying individuals exhibiting high Cd concen-
trations (>70 ppm dry weight), they detected no presence of metallothioneins.
Through advanced Sephadex and Sephacel chromatography they did, however, find
three Cd-binding complexes, but these were not subject to further characterization.
By a combination of EDAX analyses and capillary electrophoresis, Turnau et
al.29 indicated that heavy metal binding to inorganic constituents may take place in
ectomycorrhiza. They concluded that oxalate and/or carbonate may be involved in
complexation of Cd and Pb in mycorrhizal structures.

IV. TECHNIQUES AND APPROACHES FOR STUDYING METAL


SPECIATION IN FUNGAL BIOMASS
A. AVAILABLE APPROACHES AND TECHNIQUES
The objective of this section is to summarize different techniques for metal analyses
on a small spatial scale (mainly mycorrhizal structures), not to provide a detailed
comparison of advantages and disadvantages of the different methods.
The most easily available methods for studying metal binding and speciation
employ indirect approaches or require comparison with complementary observa-
tions. Turnau et al.16 employed Electron Energy Loss Spectroscopy (EELS) to study
the localization and distribution of metals within the mycorrhizal structure. These
results were compared with data from the Gomori-Swift reaction (cystein-rich pro-
teins) and PATAg tests (presence of polysaccharides) to identify areas rich in metals,
polysaccharides, and/or cystein-rich proteins.
Metal speciation in terms of protein binding may be studied by different kinds of
column chromatography and gel electrophoresis,23,24,27,28 which can be accompanied
by amino acid assays to detect specific amino acids.23 Column fractions containing
metallothioneins may also be analyzed for their metal concentrations. Such methods
are more direct in terms of metal speciation, although restricted to protein binding.
Interesting results on metal speciation in mycorrhizal structures have also been
obtained using histochemical staining.25 Again, this approach is restricted to studies
of metal binding to proteins.
EDAX (Energy-Dispersive X-ray Analysis) is a multielement technique that has
been used for several years for trace element analysis in microbial structures. Studies
by Heyser and Donner,19 Wassermann et al.,21 and Turnau et al.29 have also proven
the feasibility of this technique for studies on heavy metal localization and accumu-
lation in ectomycorrhizas. Exact binding mechanisms are not obtained by this tech-
nique. Structural information on precipitates associated with the metal accumulation
may be extracted when combining EDAX and capillary electrophoresis, as demon-
strated by Turnau et al.29
The PIXE (Proton Induced X-ray Emission) technique, which had already
emerged before 1970,30 is nondestructive and may be used for multielement, quan-
titative analysis with respect to elements above atomic number 11.31 When connected
Analysis of Metal Speciation and Distribution in Symbiotic Fungi 155

to a nuclear microprobe it can be used for analysis of heavy metal concentrations


on a 10 m scale. As such, PIXE is comparable to EDAX, although PIXE provides
simultaneous determinations of typically more than 20 elements, of which several
are of environmental concern (for instance Al, As, Cr, Cu, Pb, and Zn).31 Thus PIXE
may provide a rather detailed picture of the inorganic composition of tiny structures
or areas of particular interest in microbial structures.
Other techniques and applications for metal analyses on a small spatial scale
are available, including Electron Spectroscopic Imaging (ESI). Kottke and Martin,32
for instance, used an analytical transmission electron microscope to identify phos-
phorus and aluminium in mycelium from Laccaria amethystea.
The summary of studies on metal localization, distribution, and binding mech-
anisms in microbial structures (see Chapter 3, this volume) as well as different
techniques for analysis of metal concentrations, localization, and binding mecha-
nisms on a tiny spatial scale raises some important issues. Metal localization, distri-
bution, binding mechanisms, and concentrations may vary over a broad range depend-
ing on type of organism, level of exposure, elemental differences, and so on. Most
available methods and techniques for such studies are rather target specific, especially
in terms of binding mechanisms. To obtain a more detailed metal speciation in
microbial structures, several techniques or a combination of different techniques have
to be applied. Microbial structures are fragile and may suffer from structural changes
or may more or less collapse during storage, sample preparation, and analysis.

B. X-RAY ABSORPTION SPECTROSCOPY (XAS)


During the past two decades, Extended X-ray Absorption Fine-Structure (EXAFS)
spectroscopy has emerged as an increasingly important methodology for studies on
local atomic environments. The main reasons for this are that EXAFS spectroscopy
is atom specific, is nondestructive, and can be applied to solids, liquids, and gases;
and may yield data on interatomic distances, bond angles, and types and numbers
of neighboring atoms.33
Generally the method is based on the degree and manner of absorption of
electromagnetic radiation in the X-ray region. When the photon energy derived from
an X-ray source reaches a certain level, an inner shell electron is liberated and gives
rise to a secondary radiation that may be absorbed by the absorber/sample (absorp-
tion edge).33 Figure 7.2 shows a simplified EXAFS spectrum identifying important
parts of the spectrum. The photoelectrons liberated by the primary absorber (sample)
may be scattered back to the absorber by a neighboring atom, an absorption phe-
nomenon typical of the EXAFS region (Figure 7.2).33,34 Information emerging from
this part of the absorption spectrum comprises numbers and types of neighboring
atoms. More complex events, e.g., multiple scattering, may also occur, causing the
XANES region to extend some 50 eV above the absorption edge. This part of the
absorption spectrum can yield knowledge on, for instance, bond angles.33,34
When X-ray radiation in the intermediate energy range (2 to 15 keV) is channeled
through a scanning electron microscope fitted with appropriate X-ray detectors, it
is possible to attain structural information on a high spatial resolution scale. X-ray
Fluorescence Microscopy (XRFM) and micro X-ray Absorption Fine-Structure
156 Trace Elements in the Rhizosphere

FIGURE 7.2 Simplified EXAFS spectrum, displaying the four distinct regions pre-edge,
edge, XANES region, and EXAFS region.

Spectroscopy (mXAFS) provide characterization probes on the micron scale,35 facil-


itating the study of highly heterogeneous systems. These probes are ideally suited
to the study of a variety of scientific problems in the earth and environmental sciences
that have previously not been possible to measure or understand. In addition, the
appropriate control of time and experimental conditions will provide mechanistic
information and new insight into the interaction of such complex systems. Research
areas that could directly benefit from such methodological approaches include trans-
port of nutrients and contaminants in plants and symbiotic systems; phytoremedia-
tion and phytostabilization mechanisms; mobilization of nutrients or contaminants
by colloids; mechanism of formation and functioning of biominerals in plants and
animals; information about diagenesis, climate change, and life forms by investiga-
tion of the latter. Very little is known in detail about many of the above systems.
This is largely due to the lack of available tools to investigate the systems in their
natural environment or using samples that are not sufficiently small to investigate
the changes on the scale at which they are occurring.

V. ZN SPECIATION IN ECTOMYCORRHIZA FROM CONTAMINATED


FOREST SOILS STUDIED BY MICRO-XAFS
A. MATERIALS AND METHODS
1. Sampling and Sample Pretreatment

Organic surface soil for dissection of mycorrhizae was sampled in southernmost


Norway. Organic surface soils in this part of Norway are enriched with heavy metals
such as Pb, Cd, and Zn36,37 due to long-term airborne deposition of metals, mainly
from anthropogenic sources.38,39
Ectomycorrhizas of Cortinarius sp. were carefully dissected from moist field
forest soil, washed clean of any organic or mineral debris using deionized water,
and inspected for contamination under a stereo microscope. Both fresh and air-dried
mycorrhizal materials were used for mXAFS analyses. Fresh, intact mycorrhizal
Analysis of Metal Speciation and Distribution in Symbiotic Fungi 157

tips were stored between humidified tree leaves surrounded by soil material in closed
plastic cups, and then cleaned once more prior to analysis. The intact and still living
mycorrhizal tips were then encapsulated in Kapton tape (used for mounting samples
because of its low absorptivity for high-energy radiation). Cross-sections of ecto-
mycorrhiza, approximately 1 mm thick, were prepared with a scalpel and air-dried
on Kapton tape before being covered with tape.

2. Instrumentation and Analyses

Data were collected using the X-ray microprobe on the bending magnet beamline
10.3.2 at the Advanced Light Source, Lawrence Berkeley National Laboratory,
U.S.A. A Si (III) four-bounce monochromator was used to monochromatize and
scan the synchrotron X-ray energy for the XANES spectra. Following the mono-
chromator are two grazing incidence mirrors in the Kirk-Patrick Baez arrangement
that image the synchrotron source with high demagnification to an X-ray spot
typically of size 1.5 2 micrometers. High-order harmonics are not present due to
the low energy of the ring (1.9 GeV) and the grazing incidence cutoff energy set by
the grazing incidence mirrors. XANES data were collected at the zinc K-edge
(lambda = 1.28330, energy = 9 661 eV). The beam currents ranged from 300 to
400 mA at 1.9 GeV. The energy resolution of the Si (III) monochromator was
approximately 1.4 10-4 (E/E).
The spectral energy calibration was checked by measuring the transmission
spectrum of a Zn-foil (thickness 5 micrometers; the energy of the first inflection
point being defined as the edge 9661 eV).
The focused dimensions of the beam were checked by carrying out a knife edge
test.
The intensity of the incident X-rays was measured by an ionization chamber.
Excited fluorescence X-rays from the sample were recorded using a solid state SiLi
detector (effective area 55 mm2) placed in the electron orbit plane of the storage
ring and mounted 90 degrees to the incident X-rays to minimize background caused
by scattering.
A two-dimensional elemental map for Zn and Cu was produced by scanning the
sample through the microbeam and recording the emission of K-alpha fluorescence
X-rays. From this elemental map, points of interest were chosen and the microbeam
positioned at these points. XANES spectra were collected at the chosen points by
scanning the incident X-ray energy from ca. 50 eV below the edge to ca. 300 eV
above the edge (i.e., into the EXAFS region) and measuring the K-alpha fluorescence
X-rays from the sample at each energy step. The step size of the data points was
0.5 eV/step. The XANES spectra were calculated by normalizing the intensities of
the fluorescent X-rays to that of the incident X-rays, and plotting against the X-ray
energy. Several spectra were collected and summed.
X-ray fluorescence mapping with respect to Zn and Cu was carried out on fresh,
intact mycorrhiza to identify possible metal hot spots or anomalies for speciation
studies, and on dried cross-sections to study the metal distribution within the
mycorrhiza.
158 Trace Elements in the Rhizosphere

Several XANES spectra extending into the EXAFS region with respect to Zn
were collected from a 5 m zinc particle in the mantle of the Cortinarius mycorrhiza.
Similar spectra were collected from a Zn-oxalate standard.

B. RESULTS AND DISCUSSION


The results presented here focus on Zn speciation and distribution in Cortinarius
sp., although Cu is also discussed briefly. Cortinarius spp., from the area sampled
for this case study, exhibit Zn concentrations between 250 and 900 g g1 and Cu
concentrations between 180 and 600 g g1 dry weight mycorrhiza.1
Figure 7.3 shows the results of X-ray fluorescence mapping of dried cross-
sections of Cortinarius sp. with respect to Zn (left) and Cu. The mapping was
performed at 10 m increments. X- and y-axis labels in the figure show the distance
in m. The more metals the brighter the rectangles. Despite their hazy appearance,
these figures show that the largest Zn and Cu anomalies are found in the outer, fungal
part of the symbiosis (mantle and extraradical hyphae). This was previously shown
by Denny and Wilkins13 using X-ray microanalysis (EDAX) and by Electron Energy
Loss Spectroscopy (EELS).16 The Cu mapping suffers from substantial noise, shown
by several signals outside the cross-section. Parts of this noise may be removed using
multifiltering techniques, but the main cause for the noise is the low concentrations
of Cu in the mycorrhiza compared to Zn (Figure 7.3). Rather large concentration
differences between metals make it difficult to compare spatial distribution of dif-
ferent metals through XRFM analyses. Such mappings are, however, important
prerequisites in metal binding studies using mXAFS and provide a picture of the
metal distribution within the structure. As shown in Figure 7.3, there are areas high
in Zn inside the mantle. These may be areas of Zn accumulation within the Hartig
net, with an obvious heterogenous distribution of the metal within the fine root.

FIGURE 7.3 Distribution of Zn (left) and Cu (right) in dried cross-sections of Cortinarius


sp. obtained by XRFM. High concentrations are indicated by white color. Labels indicate
distance in micrometers.
Analysis of Metal Speciation and Distribution in Symbiotic Fungi 159

FIGURE 7.4 Optical micrograph of intact Cortinarius sp. (above) and XRFM of a 400
400 micrometer part of the mycorrhiza (below). Values are shown on the legend bar as counts
per 0.5 seconds.

Figure 7.4 displays an X-ray fluorescence mapping, showing Zn distribution in


an intact mycorrhizal tip of Cortinarius sp., with the 400 400 micron area mapped
indicated on the optical micrograph of the mycorrhiza (above). Figure 7.4 shows a
rather uneven distribution of Zn within the mycorrhiza, and variable Zn accumulation
within very small regions at dimensions of only a few microns.
Based on the findings shown in Figure 7.4, the monochromatic microbeam was
directed on the area of the mycorrhiza with the largest Zn concentration, and several
scans were performed over the Zn k-edge (9659 eV). Similar scans were performed
on a Zn-oxalate standard. The use of oxalate as model compound was motivated by
several factors: (1) metal anomalies within the mycorrhizal mantle have been
observed in the same areas where large deposits of oxalate crystals are found,16,40
(2) considerable amounts of oxalate are produced in the fungal part of the symbiosis
via the tricarboxylic and glyoxylate cycles,41 and (3) several metal-oxalates, includ-
ing that of Zn, are highly insoluble.
The micro-XAFS scans with respect to Zn for both the Cortinarius sp. and oxalate
are shown in Figure 7.5. A preliminary analysis of the short extended region is
consistent with the observation by simple comparison with the very distinctive XAFS
spectrum of the Zn-oxalate standard, supporting that Zn binding and precipitation as
oxalate are of importance in the mycorrhizal mantle of Cortinarius sp. In addition,
160 Trace Elements in the Rhizosphere

eV
FIGURE 7.5 Micro-XAFS spectra extending into the EXAFS region of a 5 m Zn particle
in Cortinarius sp. and a Zn-oxalate standard.

our conclusion supports that of Sarret et al.42 who showed, using conventional XAFS,
that for Zn sequestered by lichens under contaminant exposure, the dominant product
is zinc-oxalate. Some uncertainty may be raised concerning such an interpretation,
because Zn binding to acetate produces a similar spectrum.43 Zn-acetate is, however,
highly soluble while Zn-oxalate has a very low solubility. This fact, in addition to
the consistency of the obtained XAFS spectra, thus points to binding of Zn as oxalate
as an important means of Zn immobilization in Cortinarius mycorrhiza.
Several types of fungi produce oxalate, including Basidiomycetes,44,45 mycor-
rhiza of Monotropa uniflora,46 and mycelium of Mucorales.7 Oxalate, among several
other important features,41 may precipitate calcium and thus may reduce calcium
toxicity in calcium-rich soils. The fact that Cortinarius mycorrhiza binds Zn as
oxalate may imply that oxalate precipitation is an important means for the fungus
to buffer and reduce excessive Zn uptake into the host. Based on EELS analyses
Turnau et al.16 observed a gradual decrease of Cd and Al in Rhizopogon roseolus
mycorrhiza from the mantle, along the Hartig net toward the inner region of the
hosts roots, suggesting that the fungal symbiont may act as a filtering system for
toxic elements. Cd and Al, as well as other toxic metals such as Ni and Hg (in the
+I state), form sparingly soluble oxalates. Our findings, together with the findings
of Turnau et al.16 with respect to Zn, suggest that precipitation of oxalate salts may
be involved in metal detoxification in mycorrhizal systems, and perhaps in other
organisms that produce oxalate.
Analysis of Metal Speciation and Distribution in Symbiotic Fungi 161

Zn is one of the nutrients that is absorbed and translocated to the host by


mycorrhizal fungi.7 In this context, it cannot be excluded that the fungus uses oxalate
excretion as an important means for extracting Zn from the surrounding soil.
The signal-to-noise ratio for the scans with respect to Cortinarius sp. was large
enough to scan into the Extended XAFS (EXAFS) region (extending above 10,000
eV), indicating the potential of this technique for obtaining structural data for Zn.
Similar scans with respect to Cu failed to provide significant and distinct results.
Compared to Zn, scanning with respect to Cu resulted in more noise in the EXAFS
region, strongly reducing the possibilities of extracting structural data on Cu binding
in the mycorrhiza. Even more important, a comparison of the scans for Cu in
mycorrhiza and Cu-oxalate revealed visible differences in the XANES region. Either
the Cu-oxalate model we used exhibited different structural properties than the
dominating oxalate form in the mycorrhiza, Cu is bound by other means than Zn,
or Cu binding in mycorrhiza may occur by several means, which may or may not
include oxalate precipitation. A higher solubility of Cu-oxalate compared to Zn-
oxalate (approximately three times higher solubility in cold water) may be an impor-
tant factor involved here. On the other hand, Cu is known to form much more stable
compounds, complexes, and chelates with a variety of organic compounds than does
Zn. Due to its two oxidation states (+I and +II) Cu also participates in several energy
transforming and other redox processes in organisms. In any of these cases, other
binding mechanisms for Cu probably compete effectively with oxalate precipitation.

VI. CONCLUSIONS
The case study presented here clearly demonstrates the capability of direct chemical
speciation at microscales. Micro X-ray spectroscopy has proven to be a feasible
method for studying Zn binding in ectomycorrhiza. There are, however, some factors
restricting the feasibility of this method:

1. The sample needs to contain high levels of the metal studied, or anomalies
within the structure that are sufficiently rich in the metals of interest.
Small metal concentration can cause a decrease in the signal-to-noise
ratio, which means that structural information that otherwise may be
retrieved from the EXAFS region cannot be obtained.
2. If a metal is bound by several mechanisms and no specific binding form
is dominant, there may be difficulties in interpreting the resulting data.
3. An appropriate model compound is needed, especially at low concentra-
tions of the investigated metal, from which extraction of useful structural
data from the EXAFS region is limited.
4. At present only a few synchrotrons fitted with micro X-ray facilities are
available. Accordingly, a shortage of beam-time, which affects planning
and implementation of research projects, is prevalent. In addition, there
is the problem of conservation and storage of sample material.
5. Some metals, e.g., Cd, which are important in terms of environmental
concern, are difficult to study using XAS.
162 Trace Elements in the Rhizosphere

XAS/XANES/EXAFS and mXAFS are nonetheless methods that provide infor-


mation on speciation and structure of chemical compounds, which, at present, cannot
be obtained by any other technique. To obtain a broader and more complete metal
speciation, a larger regime of applications and techniques is required.
Our case study on metal speciation in ectomycorrhizae showed that Zn is pre-
dominantly bound as sparingly soluble Zn-oxalate in the mycorrhizal mantle of
Cortinarius sp. It is suggested that oxalate excretion by the fungus is involved in
the process of providing the host with necessary Zn. Also, oxalate precipitation may
be an important means for the fungus to regulate Zn translocation to the host.
Even though our studies did not shed light on binding mechanisms with respect
to Cu in mycorrhiza, our data suggest that oxalate precipitation is not dominant, and
may even be of minor importance.

REFERENCES
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2. Bth, E., Lundgren, B. and Sderstrm, B., Fungal populations in podzolic soils
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6. Harley, J. L., The Biology of Mycorrhiza, Leonard Hill, London, 1969, Chap. III, IV.
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9. Gast, C. H., Janson, E., Bierling, J. and Hannstra, L., Heavy metals in mushrooms
and their relationship with soil characteristics, Chemosphere, 17, 789799, 1988.
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common soil fungi comparison and assessment of importance for metal distribution
in natural soil systems, Soil Biol. Biochem., 28, 967975, 1996.
11. Colpaert, J. V. and van Assche, J. A., The effects of cadmium on ectomycorrhizal
Pinus sylvestris L., New Phytol., 123, 325333, 1993.
12. Poitou, N. and Olivier, J. M., Effect of copper on mycelium of three edible ectomy-
corrhizal fungi, Agric. Ecosyst. Environ., 28, 403408, 1989.
13. Denny, H. J. and Wilkins, D. A., Zinc tolerance in Betula spp. IV. The mechanism
of ectomycorrhizal amelioration of zinc toxicity, New Phytol., 106, 545553, 1987.
14. Colpaert, J. V. and van Assche, J. A., Zinc toxicity in ectomycorrhizal Pinus sylvestris,
Plant Soil, 143, 201211, 1992.
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15. Colpaert, J. V. and van Assche, J. A., The effects of cadmium and the cadmium-zinc
interaction on the axenic growth of ectomycorrhizal fungi, Plant Soil, 145, 237243,
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16. Turnau, K., Kottke, I. and Dexheimer, J., Toxic element filtering in Rhizopogon
roseolus/Pinus sylvestris mycorrhizas collected from calamine dumps, Mycol. Res,
100, 1622, 1996.
17. Berthelsen, B. O., Olsen, R. A. and Steinnes, E., Metal concentrations in ectomyc-
orrhiza as affected by sample pre-treatment and storage conditions, Soil Biol. Bio-
chem., in press.
18. Wilkins, D. A., The influence of sheathing (ecto-) mycorrhizas of trees on the uptake
and toxicity of metals, Agric. Ecosyst., Environ., 35, 245260, 1991.
19. Heyser, W. and Donner, B., X-ray microanalytical studies on element uptake and
deposition indifferent tissues of beech mycorrhizae, Agric. Ecosyst. Environ., 28,
175179, 1989.
20. Kumpfer, W. and Heyser, W., Zinc accumulation in beech mycorrhizae a mecha-
nism of zinc tolerance, Agric., Ecosyst., Environ., 28, 279283, 1989.
21. Wasserman, J. L., Mineo, L. and Majumdar, S. K., Detection of heavy metals in oak
mycorrhizae of northeastern Pennsylvania forests, using X-ray microanalysis, Can.
J. Bot., 65, 26222627, 1987.
22. Leyval, C., Turnau, K. and Haselwandter, K., Effect of heavy metal pollution on
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23. Dehn, B. and Schepp, H., Influence of VA mycorrhizae on the uptake and redistri-
bution of heavy metals in plants, Agric. Ecosyst. Environ., 29, 7983, 1989.
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corrhizal fungi, New Phytol., 135, 123131, 1997.
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of heavy metal tolerance in ectomycorrhizal fungi, Plant Soil, 96, 417420, 1986.
26. Galli, U., Meier, M. and Brunold, C., Effects of cadmium on non-mycorrhizal and
mycorrhizal Norway spruce seedlings [Picea abies (L.) Karst.] and its ectomycor-
rhizal fungus Laccaria laccata (Scop. Ex. Fr.) Bk. & Br.: Sulphate reduction, thiols
and distribution of the heavy metal, New Phytol., 125, 837843, 1993.
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164 Trace Elements in the Rhizosphere

34. Koningsberger, D. C. and Prins, R., Eds., X-ray absorption: Principles, Applications,
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S., Padmore, H. A., Patel, J. R. and Sandler, R., Progress towards sub-micron X-ray
imaging using elliptically bent mirrors, Proc. SPIE, 3152, 126135, 1997.
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natural soil. Identification of contributions from different sources, Analyst, 120,
14791483, 1995.
37. Steinnes, E., Allen, R. O., Petersen, H. M., Rambaek, J. P. and Varskog, P., Evidence
of large scale heavy metal contamination of natural soils in Norway from long-range
atmospheric transport, Sci. Tot. Environ., 205, 255266, 1997.
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and ecology in the soil environment, Can. J. Microbiol., 42, 881895, 1996.
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80, 707715, 1988.
8 Bioavailability of
Heavy Metals in the
Mycorrhizosphere
Corinne Leyval and Erik J. Joner

CONTENTS

I. Introduction...................................................................................................165
II. Effects of Heavy Metal Pollution on Mycorrhizae......................................167
A. Field Observations ............................................................................167
B. Use of Mycorrhizal Fungi for the Diagnosis of Metal
Toxicity in Soil .................................................................................169
C. Effect of Heavy Metals on the Diversity of AM Fungi ..................169
D. Metal Tolerance of Mycorrhizal Fungi and Mycorrhizal
Plants.................................................................................................170
III. Effects of Mycorrhizal Fungi on the Bioavailability of Heavy
Metals and Their Transfer to Plants.............................................................171
A. Sorption of Metals by Mycorrhizal Fungi .......................................172
B. Accumulation of Metals in Fungal Structures .................................173
C. Solubilization of Minerals Containing Metals.................................174
D. Transport of Metals to Plants by Mycorrhizal Fungi ......................175
E. Application for Land Remediation...................................................179
References..............................................................................................................179

I. INTRODUCTION
Mycorrhizae are symbiotic interfaces between soil fungi and plant roots that permit
plants to acquire nutrients from soil with a far lower carbon expenditure than would
be necessary if all nutrients were to be taken up by the roots alone. Mycorrhizae
may also represent other advantages to plants, such as improved water relations,
enhanced resistance against certain pathogens, and enhanced tolerance to salt and
metal stress.1,2 The majority of both wild and cultured plants form mycorrhizae of
one type or another. In most cases mycorrhizae can determine plant successions and
botanical compositions of terrestrial ecosystems and are crucial for their normal
functioning.2 Evidence of their importance is also provided by their possible role

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 165
166 Trace Elements in the Rhizosphere

during plant colonization of land 420 million years ago and their persistence during
plant evolution through the present time.3
The different types of mycorrhizae have quite different host plant ranges and
are thus largely confined to different terrestrial ecosystems. A crude and general
classification4 distinguishes between ericoid mycorrhizae found in heathland and
alpine vegetation (often in high latitudes on highly organic and acid soils), ectomy-
corrhizae (ECM) found in most boreal to subarctic forest trees (intermediate acidity
and organic matter content), and arbuscular mycorrhizae (AM) that are formed by
most herbaceous plants (including agricultural plants) and with the majority of trees
in mediterranean and tropical ecosystems (near neutral soil pH and low organic
matter content).
A common feature to all types of mycorrhizae is the production of extraradical
hyphae, which explore the soil outside the root depletion zone, up to 10 cm from
the root in AM fungi.5 The biomass of mycorrhizal fungi in soil varies greatly with
mycorrhizal type, and may be in the range of 12 to 138 mg g1 soil (organic layer
of forest soil) for ECM6 and 0.1 to 1 mg g1 for AM (mineral soil).7,8 ECM often
form aggregated fungal structures (rhizomorphs, mantles, etc.), whereas AM and
ericoid mycorrhizal fungi form simple, branched hyphae in soil. Extraradical hyphal
biomass or lengths of ericoid mycorrhizae have, to our knowledge, not been reported,
but they are likely to be in the same range as AM when calculated on a soil volume
basis, i.e., around 20 m cm3. The external hyphae of mycorrhizal fungi are respon-
sible for the improved nutrient uptake in mycorrhizal plants, which is greater for
elements with narrow diffusion zones around plant roots such as P, NH4, and most
heavy metals.9
The mycorrhizosphere may be defined as the rhizosphere of mycorrhizal plants.
As most plants are mycorrhizal (unless one deals with plants from the families
Chenopodiaceae, Carophyllaceae, Brassicaceae, and Urticaceae, or plants in ster-
ilized or heavily P-fertilized soil), rhizosphere and mycorrhizosphere are thus syn-
onymous, but the latter emphasizes that the presence of mycorrhizae has been
verified. The rhizosphere is modified in several respects depending on the pres-
ence/absence of mycorrhizae. Thus, microbial communities10 and activity11 are
altered; soil pH, exchangeable acidity, and quantity of exchangeable cations may be
influenced;12 root exudation is commonly reduced;13 and root architecture, morphol-
ogy, and longevity are modified.14 Such observations, along with the often dramatic
effects of mycorrhizae on growth and nutrient uptake of plants and their ubiquitous
distribution, has led mycorrhizal researchers to state that the study of plants without
mycorrhizae is a study of artifacts.15
Heavy metal concentrations in soil can reach toxic concentrations and pose a
major environmental and health problem, mainly due to man-made pollution through
the burning of fossil fuels, mining and smelting of metalliferous ores, municipal
wastes, sewage sludge, and the use of pesticides and fertilizer amendments.
The term heavy metal strictly refers to metallic elements with a specific mass
>5 g cm3 able to form sulfides,16 but is commonly used for some metals that do
not fit this definition. Trace metals would be a more correct term to use since the
concentrations are <0.1% in soil or 100 mg kg1 in dry matter of biological samples.
Bioavailability of Heavy Metals in the Mycorrhizosphere 167

However, the term heavy metal is generally used and accepted in environmental
studies, and it will be used here.
Metals in soil are present as free metal ions, soluble metal complexes (seques-
tered to ligands), exchangeable metal ions, organically bound metals, and precipi-
tated or insoluble compounds such as oxides, carbonates, and hydroxides, or they
may be part of the structure of silicate minerals (indigenous soil content). Some of
the metals are essential for plant growth, such as Zn, Cu, Mn, Ni, and Co,17 while
others have no apparent function such as Cd, Pb, and Hg. However, all exhibit
toxicity above a certain concentration, which depends on the metal, the organism,
and the physicochemical soil properties.
Toxicity of metals in soil depends on their bioavailability, defined as their ability
to be transferred from a soil compartment to a living organism.18 This is a function
not only of their total concentration, but also of physicochemical (pH, Eh, organic
matter, clay content, etc.) and biological factors (e.g., biosorption, bioaccumulation,
and solubilization).19 As pointed out by Giller et al.,20 bioavailability cannot be
measured because it can only be assessed by the growth of an organism of interest
and an evaluation of uptake and toxicity of a metal after the fact.
Soil microorganisms including mycorrhizal fungi are affected by the presence
of high metal concentrations in soil (see reviews by Gadd21 and Giller et al.20). But
the organisms in turn influence the availability of metals in soil either directly,
through alterations of pH, Eh, biosorption, or uptake, or indirectly in the rhizosphere
through their effect on plant growth, root exudation, and resulting rhizosphere
chemistry. A unique trait of mycorrhizal fungi is that they form a direct link between
the soil and the root, providing a route for movement of elements in the rhizosphere
(Figure 8.1). These aspects of the interactions between mycorrhizae and heavy metals
in soil are the topic of the present nonexhaustive review. For full coverage, the reader
should also consult other recent reviews.22-26

II. EFFECTS OF HEAVY METAL POLLUTION ON MYCORRHIZAE

A. FIELD OBSERVATIONS
Field observations have shown that AM as well as ECM fungi and ericoid fungi
occur on heavy-metal-polluted sites. For ectomycorrhizal fungi, such studies have
focused mainly on fruiting bodies and shown a strong decrease in fruit produced
and of fruiting species with increasing pollution along a complex heavy metal
pollution gradient.27 Such studies have also shown a decrease in the abundance of
fruit for some species and an increase for others, which sometimes is rare in
unpolluted ecosystems.27
The presence of AM fungi in metal-contaminated soils has been observed by
counting spore density, estimating root colonization, or determining mycorrhizal
infectivity potential (MPN counts). Results are not always consistent, probably due
to different metal concentrations and availability in the soils. In agricultural soils
contaminated with metals through atmospheric deposition from a smelter28,29 and
sludge amendments,28 low to high levels of mycorrhizal colonization and spore
168 Trace Elements in the Rhizosphere

FIGURE 8.1 Interactions between heavy metals and the mycorrhizosphere.

numbers were observed. These effects were attributed not only to metal concentra-
tions, but also to soil pH, P content, and organic matter content, factors that influence
metal availability and mycorrhizal colonization. The effect of heavy metals on
mycorrhizal occurrence and infectivity seemed to be more a function of availability
rather than total soil concentrations.29
Mycorrhizal fungi have also been found in many mine spoils heavily polluted
with metals, where plant establishment was greatly improved by the presence of
AM and ECM.26,30 Indeed, the absence of AM colonization in some mine spoils has
been attributed to poor or absent mycorrhizal inoculum.31
AM spore numbers, root colonization (estimated by nonvital staining), and the
abundance of ECM fruiting bodies may not necessarily reflect the actual activity of
these organisms in the soil system, or even their functional structure. Mycorrhizal
infectivity, i.e., the number of infective propagules (including viable spores and other
infective units) has also been estimated in polluted soils. For example, along a
gradient of heavy metals on a metallicole site in northern France, where only
metallophytes and pseudometallophytes were able to grow, mycorrhizal infectivity
was reduced with increasing metal concentrations compared to control uncontami-
nated soil, but it was never completely suppressed. The vitality of hyphae in soil,
as evaluated by enzyme staining techniques (vital staining using alkaline phosphatase
or succinate dehydrogenase), is another way to estimate toxicity of metals to myc-
orrhizal fungi. For AM fungi, this parameter is reported to be negatively affected
by metals.32 However, the growth of such hyphae seemed to be less sensitive to
metals than plant roots, entering soil volumes amended with up to 100 mg Cd kg1.33
Most of the reports cited above do not contain estimates of metal availability,
or differences in extraction methodology have made it difficult to compare the results
Bioavailability of Heavy Metals in the Mycorrhizosphere 169

from different studies. Still, the presence of mycorrhizae in many soils polluted with
metals suggests metal tolerance of mycorrhizal fungi (see below).

B. USE OF MYCORRHIZAL FUNGI FOR THE DIAGNOSIS OF METAL


TOXICITY IN SOIL
Different techniques are used to estimate the bioavailability of metals in soil, includ-
ing chemical extractions34 and biological tests using plants or microorganisms.35 The
relevance of mycorrhizal fungi in this context is their central function in the plant
soil system, as well as their responsiveness to metals within a biologically relevant
concentration range. The effect of heavy metals on AM fungi has been estimated
by using a spore germination test, which was proposed as a bioassay for monitoring
soil metal pollution or other types of soil disturbance.36 Spores of a nonmetal-tolerant
AM fungus (Glomus mosseae, BEG 12, Banque Europenne des Glomales) were
placed between membrane filters in a simple sandwich device,36 which was buried
in soils with differing heavy metal concentrations, exposing spores to soil solution
imbibing the filters. Subsequent measurements of spore germination showed that
there was a correlation between chemically extractable Cd (Ca(NO3)2, NH4NO3) and
spore germination percentage, but that edaphic factors other than heavy metal content
also influence germination.29,36 Nutrient uptake by intact mycorrhizal Pinus sylvestris
seedlings was also proposed as a diagnostic tool for copper toxicity.37
These ecotoxicological laboratory studies measure responses to acute toxicity,
but not long-term chronic toxicity.20 Monitoring long-term field experiments where
heavy metals accumulate gradually is an alternative to short-term studies to estimate
critical metal concentrations or loading rates for biological activity in soil.

C. EFFECT OF HEAVY METALS ON THE DIVERSITY OF AM FUNGI


Most studies are concerned with the presence or absence of mycorrhizal fungi in
metal-polluted soils, but diversity of mycorrhizal fungi in metal-polluted soils has
not received much attention. In general, the diversity of AM fungi in soils, including
their spatial heterogeneity, is unknown. del Val et al.38 and Vandenkoorhuyse39
recently estimated the diversity of AM fungal spores in different plots of a long-
term, sewage-sludge-amended experimental site at two pH levels. Since mycorrhizal
spores directly extracted from soil may not be viable, trap cultures with host plants
were made and the spores extracted from the trap cultures. Both studies showed a
limited number of AM fungal species (six) in all the tested plots, including the
control plots. The number of AM fungal species was not affected by the highest
level of sewage sludge amendments, but the total number of spores decreased.
However, species richness and diversity increased at moderate levels of sludge
amendments.38 An increased diversity at moderate metal concentrations was also
found for Rhizobium leguminosarum bv. trifolii plasmids at the same experimental
site.20 Studying the AM fungi actually colonizing the roots in situ, which requires
molecular techniques to characterize the fungi, would be a complementary approach
to estimate their diversity, because the fungi-colonizing plants may not form spores
in the soil at all, or not in sufficient numbers to be detectable.
170 Trace Elements in the Rhizosphere

D. METAL TOLERANCE OF MYCORRHIZAL FUNGI AND


MYCORRHIZAL PLANTS

The ability of microorganisms to survive metal toxicity has been defined as resis-
tance or tolerance21 depending on the mechanism involved. Metal resistance
corresponds to an induced mechanism in response to heavy metals, such as the
production of metallothioneins. Metal tolerance is due to intrinsic properties and/or
modification of the environment to reduce toxicity, including binding or precipitation
via, for example, extracellular polysaccharide secretion. Other authors distinguish
between heavy metal avoidance, where the organism is able to restrict metal uptake
(reduced uptake or increased efflux, formation of complexes outside cells, organic
acid release, etc.), and tolerance, where the organism survives in the presence of
high internal metal concentrations (intracellular chelation by synthesis of ligands
such as metallothioneins, polyphosphates, and/or compartmentation within vacu-
oles).40,41 The ability of an organism to survive heavy metal toxicity may involve
more than one mechanism.
Ericoid mycorrhizal fungi grow without any sign of toxicity at 50 g Cu ml1
or 100 g Zn ml1 in liquid medium.42 Higher concentrations lead to no growth in
the case of Cu (100 g ml1) and a 60 to 80% reduction in growth with Zn (at 500
g Zn ml1). For ECM fungi, metal tolerance has been tested by their growth response
in axenic cultures exposed to increasing metal concentrations, and showed large
differences between species. Fifty percent inhibition of growth occurred at Cu and
Zn concentrations ranging from 10 to 200 g ml1, and Ni and Cd concentrations
from 1 to 10 and 0.1 to 10 g ml1, respectively.43 Among the five fungi tested, there
were no interfungal differences regarding tolerance to Pb, Hg, and Cr. For the other
metals, the most tolerant fungi were Pisolithus tinctorius and Hymenogaster sp. In
another study, isolates from metal-contaminated sites were more tolerant than cor-
responding fungi from uncontaminated sites.44 However, metal tolerant fungi have
also been isolated from nonmetal polluted soils, and a wide genetic variation exists
for metal tolerance in ECM fungi.45
Different mechanisms have been suggested for metal tolerance in ECM fungi
involving adsorption or accumulation of metals due to ion exchange, formation of
complexes, precipitation, or crystallization.46 Specific metal binding proteins were
involved in the accumulation of Cd, Cs, and Th in different ECM fungi.47,48 Copper-
binding proteins were detected in the culture media of tolerant isolates of Paxillus
involutus, but not in other fungi also regarded as tolerant, nor in any of the less
tolerant P. involutus isolates.49 Galli et al.50 did not detect metallothioneins in the
mycelium of Laccaria laccata, although Cd induced an increase in sulfate assimi-
lation and cysteine formation.
Components of fungal cell walls, such as chitin and melanins, and bind metals,
have been suggested as the main barrier against uptake of toxic metals.21,23 Denny
and Wilkins51 suggested that Zn was adsorbed to electronegative sites in the hyphal
cell wall and extrahyphal polysaccharide slime of P. involutus. Using electronic
microscopy and associated methods such as energy loss spectroscopy (EELS) and
electron spectroscopy imaging (ESI) microscopy, heavy metals were found to be
Bioavailability of Heavy Metals in the Mycorrhizosphere 171

localized in crystals within ECM mycelium,52 and in P-rich material in the vacuoles,26
suggesting immobilization mechanisms involving sequestration/compartmentation
and precipitation/complexation.
AM fungi cannot be grown without a host plant. Their tolerance to metals has
thus been tested regarding spore germination53,54 and root colonization. One isolate
of a very common AM fungal species, G. mosseae P2 (BEG 69), isolated from a
metal-contaminated soil was more tolerant to heavy metals than another isolate of
the same species (G. mosseae Gm, BEG 12) with no history of exposure to metals.53
AM isolates are often regarded as tolerant, because they are collected from metal-
contaminated soils without checking their effective tolerance. Furthermore, very few
isolates from uncontaminated soils have been checked for metal tolerance.
The mechanisms of heavy metal tolerance in AM fungi have not been studied
yet, nor has the stability of the tolerance of either AM or ECM fungi. Metal tolerance
of mycorrhizal fungi in axenic culture may be different from the tolerance of the
fungi in symbiotic conditions.55 The mechanisms of plant tolerance17 are comparable
to microbial mechanisms and differ between plants as well. Metal tolerance of
mycorrhizal plants may depend on the host plant, on fungal tolerance, and on the
compatibility between them.51 In most studies concerning metal tolerance of mycor-
rhizal plants, only the effect of the fungi has been considered, but possible plant
protective effects against heavy metal toxicity to the fungus have not been studied.
The protective effect of ectomycorrhizal fungi against heavy metal toxicity to
plants has mainly been attributed to the extramatrical mycelia binding metals and
excluding metals from the host,23,56,57 to a filtering effect of the fungal mantle,58,59
and to the whole fungal biomass reducing heavy metal availability to the host plant.56
Binding of the heavy metals by the ericoid mycorrhizal fungi60 and production
of extrahyphal slime61 explained the improved tolerance of ericoid mycorrhizae to
heavy metal toxicity.
In soils heavily contaminated with heavy metals and nutrient deficient, where
nonmycorrhizal plants died, a Glomus isolated from a metal tolerant plant, Viola
calaminaria, allowed plants like maize, barley, and alfalfa to grow.62 Using micro-
beam analysis, it was shown that heavy metals were immobilized in root tissues
containing fungal structures.63 Using compartment devices and a metal tolerant G.
mosseae, fungal immobilization of 109Cd within AM roots and reduced translocation
to the shoots were observed.33
A possible indirect effect of mycorrhizal fungi on plant metal tolerance via the
modification of the rhizosphere, suggested by Dixon and Buschena,58 was never
investigated.

III. EFFECTS OF MYCORRHIZAL FUNGI ON THE BIOAVAILABILITY


OF HEAVY METALS AND THEIR TRANSFER TO PLANTS
Mycorrhizal fungi can affect the bioavailability of heavy metals by 1) a direct effect
on the free metals in the soil solution (immobilization by adsorption, absorption,
accumulation), 2) by affecting solubilization of metal bearing minerals, and 3)
indirectly by modifying root exudation.
172 Trace Elements in the Rhizosphere

The fungi forming the three types of mycorrhizae have a capacity to protect
their host plants against excessive metal uptake that generally decreases from ericoid,
to ECM and AM fungi. This seems to be the result of adaptation to soil pH and thus
metal availability in their native environments, but it is also related to the morphology
and biomass of the fungal structures characteristic of each group of mycorrhizae.
The ectomycorrhizae are exceptional in this respect as they form an often dense and
thick sheet of fungal tissue (the mantle) that covers the root surface completely so
that any compound entering the root has to pass through the fungal mantle. This
gives ectomycorrhizae the unique ability to filter ions that enter the plant, which
the other types of mycorrhizae do not have.

A. SORPTION OF METALS BY MYCORRHIZAL FUNGI


Biosorption of heavy metals has received much interest in connection with cleaning
industrial effluents or other liquid wastes, and for the recovery of precious metals
and radionuclides (see, e.g., Gadd64). This passive mechanism of ion immobilization
on the surface of microbial cells includes processes like adsorption, ion-exchange,
complexation, precipitation, and crystallization on and within what may often be a
multilaminate, microfibrillar cell wall rich in negatively charged ligands such as
phosphoryl, carboxyl, sulfhydryl, hydroxyl, and phenolic groups.65 In addition, extra-
cellular polysaccharide slime or mucilage is commonly produced by microorganisms
and roots, forming a coat around them that is capable of considerable metal immo-
bilization.61,66,67 Soils contain adsorptive constituents other than microbial biomass
that are quantitatively by far the most important for metal immobilization, with
resulting low concentrations of metals remaining in soil solution.
The rhizosphere is, however, particularly rich in microorganisms and their asso-
ciated immobilizing matrices which, due to their location at the soilroot interface,
may contribute to reduced metal availability to plants.
Mycorrhizal fungi are part of the rhizosphere biomass, and their metal sorption
capacity is thus of interest for investigations on the fate of metals in the rhizosphere.
The first interaction in mycorrhizal metal transport is the contact between metal ions
and the hyphae in soil, and the processes that take place at the hyphal surface are
the first to influence the fate of any ion in question. Sorption of metals on mycorrhizal
mycelium may be extensive and limit the amount of metals taken up by the fungi.
Reported data specifically considering metal sorption by mycorrhizal fungi are
scarce, but have been reported for Pb on ECM68 and for Cd and Zn on AM.7 The
former ranged from 200 to 1200 mol Pb g1 at equilibrium, depending on fungal
species and the form of N in the growth medium. In the latter report, Cd and Zn
sorption was in the range 380 to 2000 mol Cd g1 for four fungi and 300 mol Zn
g1 (Figure 8.2) at similar solution concentrations (10 mg l1) as the report on Pb in
ECM. Electron-dense lead deposits on the surface of the ectomycorrhizal fungus,
with the highest Pb sorption capacity, contained molar equivalents of S and half the
molar equivalents of P,68 a phenomenon that is known from plant roots exposed to
Pb.69 This shows that surface binding of heavy metals on fungi does not follow a
monolayer Langmuir adsorption model when incubated in solutions with high con-
centrations of metals. A further complicating factor is that the sorption characteristics
Bioavailability of Heavy Metals in the Mycorrhizosphere 173

FIGURE 8.2 Sorption of Cd and Zn on arbuscular mycorrhizal hyphae of four fungi from
solutions containing 0.089 mM Cd(NO3)2 or 0.15 mM Zn(NO3)2. Bars represent S.E.M. (n = 3).

of a fungus may differ between fungi grown in vitro and in symbiosis with a plant,
with higher sorption having been found in the former.68 Though the metal sorption
capacity of AM fungi is unusually high7 and may exceed that of ECM fungi, the
total immobilization capacity of ECM would still be 1 to 2 orders of magnitude
higher than that of AM due to differences in biomass. Lower soil pH and thus higher
metal solubility does, however, potentially impose higher quantities of metals on
the former (see above).
The outcome of sorption studies like those above depends greatly on ion con-
centrations in the incubation solution and the presence of competing cations. In an
experiment on Cd sorption of AM hyphae the presence of 50 g Ca ml1 or 100 g
Zn ml1 in a solution containing 1 g Cd ml1 reduced fungal Cd sorption by 87 and
92%, respectively.7 Such considerations show that the impressive values cited above
are disproportionately high considering the low ion concentrations commonly found
in the soil solution. However, these investigations do serve to illustrate the important
capacity of fungal biosorption, and further show that at ion concentrations < 1M
more than 93% of Cd2+ may be removed from solution by these fungi.7

B. ACCUMULATION OF METALS IN FUNGAL STRUCTURES


Since ectomycorrhizal fungi can be grown without the host plant, heavy metal uptake
and accumulation by ectomycorrhizal fungi has also been shown using axenic
cultures, where the metals were added as soluble salts.44 Under these conditions, the
fungalsoil concentration ratios were around 200 and 80 for Cd, and 40 and 30 for
Zn for nontolerant and metal-tolerant isolates of Suillus bovinus, respectively.
174 Trace Elements in the Rhizosphere

Field observations showed accumulation of heavy metals in fruiting bodies of


mushrooms including ectomycorrhizae,47,48,70-72 with concentrations exceeding those
in vascular plants.73 These observations suggest that the metals are effectively taken
up by the mycelium (since they are transported to the fruiting bodies). Large differ-
ences between metals have been found70 with very high accumulation for Cd,
exclusion for Pb, and a narrower range of concentrations for Zn and Cu, suggesting
a regulation of uptake for essential elements. The concentration ratio (metal con-
centration in fungal biomass divided by soil concentration) varied with the metals
and the fungal species, with high values for Amanita muscaria. On the basis of
studies where soil factors were considered, Gast70 concluded that species differences,
and not soil factors, are the primary determinants of metal levels in fungi.

C. SOLUBILIZATION OF MINERALS CONTAINING METALS


Mycorrhizal fungi, especially ectomycorrhizal fungi, can increase the bioavailability
of heavy metals in the rhizosphere by solubilizing minerals containing metals such
as rock phosphates.74 A great number of bacteria and fungi including ectomycorrhizal
fungi have been reported as solubilizers of water-insoluble inorganic phosphates75,76
and contribute to the weathering of minerals.77 This has been mainly attributed to
the acidification of their environment and to the release of organic acids in the
rhizosphere.78 AM fungal colonization has been shown to increase the mobilization
of elements from biotite,79 but no organic acids were detected in the root exudates
of mycorrhizal plants80 and the mechanisms involved were not clear.
The availability of Cd from rock phosphates has been studied in laboratory
experiments with ectomycorrhizal fungi and three rock phosphates of different origin
and with different chemical compositions and surface (North Carolina, West Africa
A and B, containing 37, 94, and 54 mg kg1 Cd, respectively) (Leyval et al., unpub-
lished data). Growing the fungi in flasks containing nutrient medium and rock
phosphates in dialysis bags to avoid direct contact with the microorganisms showed
an increase with time of Cd concentration in the solution (Figure 8.3). The pH
decreased in the medium, and organic acids such as tartaric acid were released, which
could explain the partial solubilization of the rock phosphates. Suillus granulatus
was more efficient than Pisolithus tinctorius in promoting the release of Cd from the
North Carolina rock phosphate, with 8.6 and 3.4% of the Cd added as rock phosphate
being released from the mineral, respectively. Comparing these ectomycorrhizal fungi
with a phosphate-solubilizing rhizobacterium, Agrobacterium sp., showed that all the
Cd released from the minerals remained in solution with the bacteria, but with the
fungi, a large part of the Cd was bound to, or accumulated by, the fungi. This
suggested that both microorganisms contributed to the mobilization of Cd from rock
phosphates by solubilization, but that the ectomycorrhizal fungi were able to further
immobilize the metal. With S. granulatus, no Cd could be detected in the solution,
suggesting that all solubilized Cd was accumulated in the fungal biomass. In these
experiments absorption and adsorption were not distinguished.
When associated with plant roots in symbiosis, mycorrhizal fungi affect plant
root exudation quantitatively and modify the composition of root exudates containing
carbohydrates, amino acids, and aliphatic acids.80 Such modifications influence the
Bioavailability of Heavy Metals in the Mycorrhizosphere 175

Cd (g/flask) P (mg/flask)
5 0,5

P
4 Cd 0,4

3 0,3

2 0,2

1 0,1

0 0
0 5 10 20 30 days

FIGURE 8.3 Growth, acidification of the medium, and mobilization of cadmium and phos-
phorus from North Carolina rock phosphate by Suillus granulatus in liquid culture.

bioweathering of minerals in the rhizosphere81 and the availability of metals in the


mycorrhizosphere.

D. TRANSPORT OF METALS TO PLANTS BY MYCORRHIZAL FUNGI


Transport of elements by mycorrhizae is defined as the movement of an ion from
soil solution into the plant through the fungus, and consists of three separate steps
or processes (sensu Cooper and Tinker82): 1) Uptake (from soil solution to fungal
cytoplasm, commonly an energy-dependent active process against an ion concen-
tration gradient); 2) translocation (cytoplasmic streaming within the fungus from
the site of uptake to the organ with an interface toward the host plant, a process that
appears to depend on concentration gradients between the two sites); and 3) transfer
(between the fungus and the plant through the inter- or intracellular hyphae colo-
nizing the root cortex, a process that may be linked to symbiotic exchange of C
compounds in the opposite direction). Each of these steps has been optimized for
transport of essential nutrients that are immobile in soil (P, NH4, Zn, Cu, and others)
during mycorrhizal evolution, and may also constitute a transport route for trace
elements that are either nonessential or essential, but in excess of plant needs. To
the extent that the symbiotic partners may distinguish between nutrients and non-
essential trace elements or between adequate and excess amounts of an element,
each step may also become a barrier against harmful element uptake. There are
indications that both ectomycorrhizae and AM restrict metal transport through fungal
sequestration before transfer to the plant. This may be seen in experiments where
176 Trace Elements in the Rhizosphere

enhanced metal concentrations in roots and reduced concentrations in shoots are


due to mycorrhizal presence. Enhanced root sequestration of Ni, Zn, and Cd has
been observed with the ectomycorrhizal symbioses of birch (Betula papyrifera/Scle-
roderma flavidum for Ni)83 and oak (Quercus rubra/Suillus luteus for Zn and Cd),84
as well as for Cd in the AM symbioses of clover33 and lettuce,85 for Zn in Andropogon
geraldii30 and Cu in Agrostis capillaris.86
Mycorrhizal effects on plant uptake of metals are known to vary depending on
certain factors: The type of mycorrhizae, the fungal genotype, the individual metal
in question, and concentrations of available metals in soil. Additional factors that
may have an influence on mycorrhizal metal accumulation are related to experimen-
tal conditions (e.g., light intensity, plant growth stage, soil pH, available soil N and
P, and root densities in soil, etc.), plant species, and plant size. The latter two are
common confounding factors in mycorrhizal experiments as mycorrhizal plants
commonly grow bigger than nonmycorrhizal plants, with resulting (at times dubious)
big plantsmall plant comparisons or dilution effects (see George et al.24). Matching
plant growth by adding P to nonmycorrhizal plants may overcome this (as well as
effects contributed to internal metal sequestration by complexation with enhanced
amounts of intracellular P), but requires additional experiments to establish a proper
fertilization level.
The most pronounced overall effects of mycorrhizae on plant uptake of heavy
metals are those reported by Bradley et al.42,60 where ericoid mycorrhizae reduced
Zn and Cu uptake in three plant species by up to 65 and 80%, respectively. The
reduced metal uptake was attributed to exclusion of metals after adsorption on fungal
walls within the root cortex, as a result of which the plants could continue their
assimilatory activity as opposed to nonmycorrhizal plants that were unable to grow
when transplanted into the metal-enriched medium.
In ectomycorrhizae there are numerous examples of reduced metal uptake rel-
ative to nonmycorrhizal controls,51,55,56,84,87-89 though there are cases where no ame-
lioration occurs.55,83,90,91 Again fungal sorption seems to be a major mechanism, but
complexation by elements (P and S) enriched in or on mycorrhizal structures68 or
specific proteins,49 and a dilution effect due to enhanced nutrition and plant growth
in mycorrhizal plants84 may also explain some of the differences.
The AM have so far shown variable effects on metal exclusion/accumulation in
plants. Results may even differ between experiments with the same soil, and have
thus shown either reduced uptake of metals like Cd, Cu, and Zn in AM plants or no
effects, depending on experimental conditions other than fungi and plant species.92
The outcome has in many cases differed depending on the amount of added metals
or resulting concentrations in plants. Thus, Ietswaart et al.93 found some cases of
reduced uptake of Zn, Cu, and Pb in plants with enhanced AM colonization by
indigenous AM fungi on two soils with elevated metal contents using Agrostis
capillaris as a host. Heggo et al.94 found enhanced heavy metal uptake in mycorrhizal
soybean at resulting low leaf metal concentrations, but reduced uptake of Zn, Cu,
and Cd at high leaf concentrations (>10 mg Cd kg1, >20 mg Cu kg1, and >400 mg
Zn kg1). Leyval et al.,95 using maize, found consistently lower Pb concentrations
in shoots of mycorrhizal plants, but Zn and Cu shoot concentrations were lower in
mycorrhizal plants only at the highest soil metal concentration. A consistent exclu-
Bioavailability of Heavy Metals in the Mycorrhizosphere 177

sion of Cd was demonstrated with five varieties of lettuce and four of maize, whereas
the same plants showed variable effects of AM on uptake of Zn.96 The consistent
exclusion of Cd and Zn at high soil Zn levels were later verified for lettuce in
symbiosis with three different AM fungi in two soils with contrasting metal con-
tents.85 Contrary to these reports, Killham and Firestone97 found mainly enhanced
uptake of Cu, Ni, and in some cases Pb and Zn in mycorrhizal bunchgrass at three
metal loads and three pH levels. Cd uptake was either not affected or enhanced in
mycorrhizal plants in an experiment with maize and two AM fungi in sand culture.98
The differences in metal uptake were, however, correlated to differences in plant
size in the two latter reports, which complicates the interpretation.
Mycorrhizal transport of some heavy metals has been demonstrated for AM
using compartmented pots. In these pots, roots are restricted to one part of the pot
without metals, whereas external hyphae cross a fine mesh and grow into a root-
free soil volume where metals (sometimes as radioisotopes) have been placed. Thus,
the transport of Zn,99,100 Cu,100,101 and Cd33,100 has been demonstrated, and it has been
established that Ni is not transported by AM hyphae.100 These experiments have not
used more than one fungus each. Thus interspecific variation, which may be con-
siderable with respect to P transport,102,103 remains unresolved regarding AM trans-
port of metals.
AM effects on nutrient uptake in plants depend on root length densities in soil.104
This is because mycorrhizal functioning is based on fungal uptake of nutrients from
outside the rhizosphere and their transport back to the plant, rather than an enhanced
affinity for nutrients in soil already exploited by roots. Elements found in the
rhizosphere are taken up by the root itself and root densities high enough to make
rhizospheres overlap preclude any additional uptake by the fungi. Pot experiments
with AM and heavy metals have differed largely in pot volumes and plant growth
periods, resulting in different root length densities (data for the latter are rarely
reported, but they may be deduced from root weight data). In our laboratory, we
found different effects of the same mycorrhizal fungus on metal uptake by maize
from the same soil at contrasting root length densities. In two experiments with
similar pot size, but with plant size ten times larger in one than in the other, no
effect of AM was found in the experiment with the larger plants. The smaller plants,
however, showed reduced accumulation of Cd.92 In a recent experiment, we com-
pared metal uptake in plants as affected by pot size and AM, using an industrially
polluted soil. The results show that at high root length densities (>25 cm cm3) the
effect of mycorrhizae in reducing plant metal uptake is lower than in plants that had
three times larger soil volume available (Figure 8.4). With these results in mind we
reexamined some of the reports on AM effects on metal uptake, and found that those
reporting no difference or enhanced metal uptake by AM plants seem to have used
higher plant densities, longer growth periods and/or smaller pot volume than those
where the presence of mycorrhizae resulted in decreased metal uptake in plants. For
example, Dueck et al.105 reporting reduced uptake of Zn, used one plant per pot (1 L)
grown for 7 weeks; Heggo et al.,94 who reported reduced Zn, Cd, and Mn in AM
plants, used one plant per pot (2 L) grown for 6 weeks, and Killham and Firestone,97
who reported enhanced uptake of Cu, Ni, Pb, and Zn in AM plants, used 10 plants
per pot (1 L) and a 14-week growth period.
178 Trace Elements in the Rhizosphere

FIGURE 8.4 Zn concentration in shoots of maize grown in an industrially polluted soil (see
Weissenhorn92 for details on soil) as affected by pot size and presence (Myc) or absence (NM)
of an arbuscular mycorrhizal fungus (Glomus mosseae P2). Bars represent S.E.M. (n = 3).

Roots are commonly capable of depleting soil of immobile nutrients like P


located 1 to 2 mm from the root surface; thus root length densities are 6 to
20 cm cm3, assuming a root diameter of 0.5 mm and a 1 to 2 mm depletion
radius,106-108 they render the entire soil volume exploited by roots alone. Root length
densities of 5 cm cm3 are reached after 3 weeks in maize and 6 weeks in clover,
and 20 cm cm3 after 6 and 9 weeks, respectively, using 1 liter pots and low to
moderate fertilization and light intensity (Joner and Leyval, unpublished results).
Future experiments concerning AM effects on plant metal uptake need to take into
account, and report data on, root densities to see whether or not this aspect is among
the crucial parameters determining exclusion and/or accumulation of metals.
When heavy metals are taken up by mycorrhizae as essential micronutrients,
threshold values may exist for individual metals and plant/fungus combinations that
represent turning points in a sufficiencytoxicity continuum at which mycorrhizae
may start to interfere and restrict additional uptake. Some reports on AM have
demonstrated higher mycorrhizal metal uptake at low concentrations of metals in
shoots, but a reduction in metal uptake in mycorrhizal plants at high metal concen-
trations. Using Zn as an example, the interval at which a change from enhanced
plant uptake (or no effect) to reduced uptake took place was 300 to 400 mg kg1
with Agrostis capillaris,93 >60 mg kg1 with maize,95 and 400 to 500 mg kg1 with
soybean.94 In a report by Lambert et al.,9 Zn concentrations were consistently higher
in mycorrhizal maize and soybean, but had maximum Zn concentrations of
<30 mg kg1 for soybean and 65 mg kg1 for maize, thus possibly staying below a
threshold where mycorrhizae may reduce uptake. The relationship outlined above
Bioavailability of Heavy Metals in the Mycorrhizosphere 179

may be valid only under certain conditions. For example, Killham and Firestone97
demonstrated that reduced pH enhanced metal uptake in AM plants, with no effect
of AM on uptake of Cu, Ni, Pb, and Zn by Erharta calycina at pH 5.6, but with a
considerably increased mycorrhizal uptake at pH 3.0.

E. APPLICATION FOR LAND REMEDIATION


Since heavy metals cannot be degraded, remediation of heavy-metal-polluted soils
is limited to metal immobilization or extraction. The use of plants for bioremediation
of polluted soils, including phytoextraction and phytostabilization, has recently
attracted much attention109 (see also Lombi et al., Chapter 1 of this book). The
objective of phytoextraction is to grow plants with the ability to concentrate metals
in shoots, so that harvesting the plants would progressively decrease metal content
in soil. Most of the hyperaccumulative plants, such as Thlaspi caerulescens, which
are potential candidates for phytoextraction, belong to the family Brassicaceae,
which is a nonmycorrhizal family. However, these plants often produce little biom-
ass, and the use of other plants of mycorrhizal families, which accumulate small
amounts of metals but produce more biomass, have also been considered.110
Phytostabilization is the use of plants to reduce or eliminate the bioavailability
of metals, minimize erosion, improve soil quality (organic matter content in partic-
ular), and reduce leaching of metals. Metal-tolerant plant species, such as Agrostis
tenuis and Festuca rubra, have been used together with different amendments.111,112
In this context, mycorrhizal fungi could be considered as helpful for plant establish-
ment and maintenance,113 thus promoting better plant nutrition, ameliorating phyto-
toxicity, and revegetating polluted land and mine spoils.
A better understanding of the protection mechanisms against metal toxicity, and
also of the diversity and function of AM fungi in connection with their metal tolerance
is, however, still required for utilization of mycorrhizal fungi in phytoremediation.

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Section III
Experimental Techniques and
Modeling Approaches to Study
the Fate of Trace Elements in
the Rhizosphere
9 Solid Phase Fractionation
of Metals in the
Rhizosphere of Forest
Soils
Franois Courchesne, Vronique Sguin,
and Alain Dufresne

CONTENTS

I. Introduction...................................................................................................189
II. Materials and Methods .................................................................................191
A. Study Sites ........................................................................................191
B. Separation of Soil Components........................................................192
C. Soil Chemistry and Metal Fractionation ..........................................192
III. Results and Discussion.................................................................................193
A. Soil pH and Organic Carbon............................................................194
1. pH in H2O ..........................................................................194
2. Organic Carbon..................................................................194
B. Metal Fractionation...........................................................................196
1. BaCl2 Extraction ................................................................196
2. Pyrophosphate and Oxalate Extractions............................201
3. Acid Extraction..................................................................202
IV. Conclusions...................................................................................................203
References..............................................................................................................204

I. INTRODUCTION
The fractionation of metals in the solid phase is controlled by a series of environ-
mental factors such as the pH, redox potential, mineralogy, organic matter, clay, and
oxide content of a soil. Sequential extraction procedures have been used extensively
to estimate the distribution of metals among different chemical forms (e.g.,
exchangeable, organically complexed, oxide bound) in soils.1 One of the overriding
aims of these fractionation studies is to assess the bioavailability of metals in order
to evaluate their potential limiting effect on crops or their harmful impacts on biota
and humans.2 In this context, empirical relationships between metal forms in soils

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2001 by CRC Press LLC 189
190 Trace Elements in the Rhizosphere

and uptake by plants have been developed in agriculture and forestry as a tool to
predict the response of terrestrial ecosystems to environmental changes (e.g., acid-
ification, metal contamination) or management practices (e.g., fertilization, flood-
ing). These relationships are based on the premises that the fractionation of metals
in the bulk soil provides a realistic indication of the chemical conditions faced by
plant roots and of the pool of bioavailable metals. This seems to be the case for a
range of environments where strong relationships between metal fractions in the
bulk soil and metal content in plant parts have been established.3,4 However, this
type of link is either weak or nonexistent in many other soilplant systems.5-8
Although numerous reasons could be invoked to explain these discrepancies, we
believe that the microscale heterogeneity of soil materials needs to be considered to
better assess the relationships between the components of the soilplant system.
The soil is an extremely heterogeneous system and the chemistry of metals has
been shown to vary considerably between horizons and from one microsite to another
within a given horizon. For example, Wilcke and Kaupenjohann9 observed that
greater proportions of total metal (Cd, Ni, Zn, Cu) were found in the bioavailable
fractions at the surface than in the core of soil aggregates. Recent work on the
rhizosphere, the soil microvolume surrounding living plant roots, further documented
the microscale heterogeneity of dissolved metals in soils. Soil solution displacement
studies showed that Zn and Cd were depleted from the rhizosphere of radish (Rapha-
nus sativus cv. Crystal Ball) compared to nonrhizosphere soil.10 The results sug-
gested that Zn and Cd were taken up by plants at a higher rate than they were
supplied by mass flow. A chemical speciation analysis indicated that dissolved Zn
and Cd were mostly found in the uncomplexed ionic form (Zn2+, Cd2+) in nonrhizo-
sphere soil.11 The proportion of free Zn and Cd ions decreased in the rhizosphere
solution in response to an increase in the concentration of dissolved organic sub-
stances produced by the growth of radish. Working with heavy-metal-contaminated
soils, Lorenz et al.12 further demonstrated that the Cd concentrations in plant parts
were more closely correlated to the total or free ion concentrations in the rhizosphere
solutions than with the dissolved Cd concentrations in the nonrhizosphere soil.
However, they found no such relationship for Zn.
Microscale analyses of the solid phase of soils also emphasized the heteroge-
neous spatial distribution of metals. In a red spruce (Picea rubens Sarg.) forest, total
Al concentrations exhibited a strongly descending gradient from the bulk soil to the
root surface, while total Zn followed the inverse trend.13 Total Fe was invariant across
the rhizosphere. Energy dispersive X-ray microanalyses indicated that Al accumu-
lates in the soil material of the root surface zone of oilseed rape (Brassica napus
cv. Rafal) relative to the whole soil.14 Much of the Al taken up by plants was further
retained in the root cell walls. Using acid ammonium oxalate, Chung and Zasoski15
and Courchesne and Gobran16 observed that extractable Al and Fe were systemati-
cally higher in the rhizosphere than in the bulk fraction of agricultural and forest
soils. Gobran and Clegg17 demonstrated that the amount of NH4Cl-exchangeable Al
was higher in the rhizosphere than in the bulk fraction of a Spodosol developed
under Norway spruce (Picea abies (L.) Karst). Yet, Ruark et al.18 found no difference
Solid Phase Fractionation of Metals in the Rhizosphere of Forest Soils 191

between rhizosphere and bulk soils in terms of KCl-exchangeable Al under loblolly


pine (Pinus taeda L.).
A growing body of evidence suggests that root exudates and organic compounds
released by rhizosphere organisms increase the solubility of metals in the rhizosphere
and have an important role in elemental acquisition by plants.19 Root exudates from
tobacco (Nicotiana tabacum L.) and corn (Zea mays L.) were shown to increase the
solubility of Mn, Cu, and Cd, although the solubilities of other metals (Ni and Zn)
were not affected.20 Marschner and Rmheld8 further demonstrated that the bioavail-
ability of Fe, and possibly Cu, Mn, and Zn, was increased in the rhizosphere of
graminaceous species (Avena sativa L., Hordeum vulgare L., Triticum aestivum L.)
through the release of strong organic chelators, like phytosiderophores, by roots.
However, Jones and Brassington21 observed that root-derived, short-chain organic
acids (citric, malic, oxalic) were rapidly sorbed by Fe and Al oxides in acidic forest
soils and by ferrihydrite. The authors hypothesized that this sorption could diminish
their effectiveness in mobilizing nutrients and metals in the rhizosphere.
Whereas numerous studies characterized the fractionation of metals in bulk
agricultural and contaminated soils,22-23 similar data are scarce for the rhizospheric
component of soils, especially for uncontaminated forested ecosystems.24 This study
targets forest soils from areas situated far from metal smelters and that were not
contaminated by heavy metal loadings. Yet, some of these soils might have been
subjected to the atmospheric deposition of metals from distant industrial sources.
The objective is to test the hypothesis that the fractionation of metals between
chemical forms is different in the rhizosphere than in the bulk component of soils
and, as a corollary, that metals are accumulated preferentially in the rhizosphere.
Therefore, the approach is to compare the solid phase fractionation of metals (Al,
Fe, Mn, Cu, Zn) between the bulk and rhizosphere components of a series of soils.
In this case, we elected to study four Podzols supporting a coniferous forest. Homo-
geneous stands were selected to assure that all the roots acting on the rhizospheric
materials at a given site had the same floristic origin.

II. MATERIALS AND METHODS


A. STUDY SITES
Soils were sampled at four locations in southern Qubec (see Table 9.1) and classified
according to the Canadian system of soil classification.25 The Coniferous (CON;
Orthic Ferro-Humic Podzol), Lac Croche (CRO; Orthic Humo-Ferric Podzol), and
LaTuque (LAT; Orthic Ferro-Humic Podzol) sites are all colonized by balsam fir
(Abies balsamea). The Parc des Grands-Jardins (PGJ; Eluviated Dystric Brunisol)
site is covered by an open black spruce (Picea mariana (Mill.) B.S.P.) forest. Soils
from CON and CRO developed in a shallow (<2 m) sandy anorthositic till, while
at LAT and PGJ the soil parent material is a thick (5 to 10 m) sandy granitic
glaciofluvial outwash. All soils were well drained as evidenced by the absence of
mottling suggesting periodic or permanent reducing conditions.
192 Trace Elements in the Rhizosphere

TABLE 9.1
Selected Characteristics of the Study Sites
Parent
Site Horizona Soil Vegetation Material Mineralogyb

Coniferous (CON) BC Podzol Abies balsamea Glacial till Qz, Pla, K-f, Amp
Lac Croche (CRO) Bf, BC Podzol Abies balsamea Glacial till Qz, Pla, K-f, Amp
LaTuque (LAT) Bf, BC Podzol Abies balsamea Glaciofluvial Qz, Chl, K-f, Amp
Parc des Grands-Jardins Bf, C Brunisol Picea mariana Glaciofluvial Qz, K-f, Pla, Int
(PGJ)
a Horizon designations and soil orders according to Soil Classification Working Group.25
b Minerals in the clay fraction (< 2 m) are in order of relative abundance where: Qz = quartz, K-f = K-
feldspar, Pla = plagioclase, Int = expandable interstratified minerals, Chl = chlorite, Amp = amphibole.

B. SEPARATION OF SOIL COMPONENTS


At each site, a pedon was dug at the base of a balsam fir (CON, CRO, LAT) or a
black spruce (PGJ) tree with a 15 to 25 cm diameter at breast height. Two profile
faces presenting a similar horizon succession were then selected for sampling. On
these two faces, bulk and rhizosphere soil materials were collected at two depths:
in the upper (Bf horizon) and the lower (BC or C horizons) portions of the mineral
soil. Materials from the two faces were bulked on a horizon basis prior to chemical
analyses to yield two composite samples per profile. At CON, only the lower part
of the profile could be sampled.
The separation of soil samples into bulk and rhizosphere components was con-
ducted in the field.16,17 In each horizon, approximately 0.05 m3 ( 50 kg) of soil
material was cut and extracted from the profile. Living roots smaller than 2 mm
were then carefully removed by hand, lightly shaken in a plastic container, and stored
at 4C. All the soil adhering to the roots after they had been shaken was considered
as rhizosphere soil. This corresponded to a layer of soil about 3 mm thick that was
intimately attached to root surfaces and to the associated microflora. The remaining
material was regarded as the bulk soil and was collected 1 to 5 cm away from
individual roots. In the laboratory, the rhizosphere soil was brushed and shaken away
from roots and air-dried. The bulk soil was air-dried and freed from rootlet fragments
using plastic tweezers. All samples were gently crushed and passed through a plastic
2-mm sieve.

C. SOIL CHEMISTRY AND METAL FRACTIONATION


Soil pH in H2O was measured with a 1:2 soilsolution ratio.26 Dissolved organic
carbon (DOC) was analyzed in 1:2 water extracts with a Shimadzu TOC Analyser
(Kyoto, Japan). The organic C content was determined by combustion with a LECO
total C analyzer (St. Joseph, MI). The particle-size distribution of the bulk soil was
evaluated by the hydrometer technique in the presence of 5 g L1 Na-metaphosphate,
Solid Phase Fractionation of Metals in the Rhizosphere of Forest Soils 193

after treatment with dithionite-citrate-bicarbonate (DCB) and H2O2 to remove Fe


oxides and organic matter.27 All analyses were duplicated.
Chemical extractions with BaCl2, Na-pyrophosphate, acid ammonium oxalate,
and HNO3HCl (acid) were used to define the solid phase chemistry of Al, Fe, Mn,
Cu, and Zn in bulk and rhizosphere materials. This fractionation scheme was com-
pleted by X-ray fluorescence (XRF) analyses performed on the bulk component of
each horizon; the amount of rhizosphere material available was too small for XRF
analyses. Although we recognize that no extraction procedure can unequivocally
discriminate the various metal forms in the solid phase, we propose the following
operational assignment of extractants to chemical forms: 1) BaCl2 is considered to
displace water soluble and exchangeable metal,28 2) Na-pyrophosphate mostly dis-
solves organically complexed metals, 3) acid ammonium oxalate extracts organic
complexes and metals bound in amorphous and poorly crystalline oxides,29 4) the
acid extraction [2:1 HNO3 (50%) HCl (50%) mixture] has an effect similar to that
of oxalate, although it is thought to be more efficient in solubilizing metals bound
in inorganic crystalline oxides30 while, 5) the XRF analysis yields the total metal
content of the sample. Separate rather than sequential batch extractions were con-
ducted. We did so to avoid the cumulative loss of reactive clay particles and organic
matter during the phase separation and washing steps of the sequential procedure.
The heterogeneity of the subsamples was reduced by mixing the soil in a plastic
container after the retrieval of organic matter fragments. The individual metal frac-
tions were determined by difference. For example, the amorphous inorganic metal
content was calculated by subtracting the amount dissolved with Na-pyrophosphate
from the oxalate extract. Similarly, the residual metal fraction was estimated as the
difference between the total metal content (XRF) and the acid extraction. When
negative values were obtained in this way, the concentration of the associated form
was considered to be zero. Metals in duplicate extracts were determined by induc-
tively coupled plasma-atomic emission spectrometry (ICP-AES, Perkin-Elmer) or
graphite furnace atomic absorption spectrophotometry (GFAAS).
The comparison of chemical properties between bulk and rhizosphere components
was assessed using the Wilcoxon signed rank test in the nonparametric module of the
SYSTAT statistical package.31 A nonparametric test was selected because the number
of pairs of data was such (n = 7 horizons) that the basic assumptions of parametric
tests (e.g., equality of variances, normality of the distributions) could not be met.

III. RESULTS AND DISCUSSION


The seven horizons have a loamy sand to sandy loam texture and contain less than
12% clay-size materials. The bulk component of all horizons share a similar min-
eralogy dominated by quartz, plagioclases, and K-feldspars with minor amounts of
amphiboles.32,33 These minerals are inherited from the granite (LAT, PGJ) and
anorthosite (CON, CRO) bedrock from which the parent materials are derived. The
clay fraction of the LAT and PGJ soils also contains phyllosilicates and interstratified
expandable minerals (Table 9.1). Total chemical analyses (XRF) indicate that the
elemental composition of the bulk soil is dominated by O (40 to 48% weight), Si
194 Trace Elements in the Rhizosphere

TABLE 9.2
Total Metal Content of the Bulk
Component of the Seven Soil Horizons
Site Horizon Al Fe Mn Cu Zn
g kg1 mg kg1

CON BC 69.9 29.0 426 23 47


CRO Bf 89.0 36.4 434 18 50
BC 87.3 34.7 472 8 47
LAT Bf 88.8 46.4 534 15 59
BC 81.9 45.1 666 29 59
PGJ Bf 78.8 40.7 531 14 68
C 76.5 38.2 608 11 73

(24 to 32% weight), Al, and Fe (Table 9.2). For a given profile, the observed increase
in total Al and Fe from the BC or C to the Bf horizon reflects the accumulation of
sesquioxides in the upper portion of these podzolic soils. The total Mn, Cu, and Zn
contents are much lower than those of Al and Fe and show no systematic pattern,
although Mn tends to increase with depth in the profile.

A. SOIL PH AND ORGANIC CARBON


1. pH in H2O

The pH in H2O of soil materials is significantly ( < 0.05) lower in the rhizosphere
(mean pH = 4.84) than in the bulk (mean pH = 5.01) component of six of the seven
horizons (missing data for the LAT BC). The difference for individual horizons ranges
from 0.05 to 0.50 pH units and is somewhat more pronounced under black spruce
(PGJ) than balsam fir (Figure 9.1). Changes in pH close to roots are documented for
a variety of agricultural and forest soils and include both pH increases and decreases
with respect to the bulk soil.34,35 The main factors responsible for the change in
rhizosphere pH are the exudation and secretion of organic acids by roots and by
rhizosphere microorganisms and the relative contribution of H+ and HCO3 efflux to
the soil following the uptake of nutrients by plants.8 Hedley et al.36 demonstrated
that a greater uptake of cations than anions by plants, with the associated excess H+
release, was closely related to the decrease in rhizosphere pH under rape (Brassica
napus var. Emerald). Petersen and Bttger 37 subsequently showed that the acidi-
fication of the rhizosphere of maize (Zea mays L. cv. Goldprinz) was predominantly
controlled by H+ efflux from roots, not by the secretion of organic acids.

2. Organic Carbon

The amount of organic C is systematically higher ( < 0.05, missing data for the
LAT BC horizon) close to the root surface (mean = 20.8 g kg1) than in the bulk
soil (mean = 5.0 g kg1). The organic C content of the soils covers a wide spectrum
of values and so does the difference between rhizosphere and bulk materials (0.5 to
Solid Phase Fractionation of Metals in the Rhizosphere of Forest Soils 195

FIGURE 9.1 Soil pH in H2O for the rhizosphere and bulk components of seven forest soil
horizons. Error bars are standard deviations of duplicates. The legend for soil names is: PGJ
= Parc des Grands-Jardins, LAT = LaTuque, CRO = Lac Croche, and CON = Coniferous.
The horizons are designated according to Soil Classification Working Group.25

70 g kg1) for a given horizon (Figure 9.2). The average concentration of dissolved
organic C (DOC), as estimated by water extracts, is also significantly higher ( <
0.05) in the rhizospheric environment (28.6 mg L1) than in soil materials further
from the rootsoil interface (7.2 mg L1). This accumulation of organic matter in
the rhizosphere may result from root exudation and decay, microbial activity, and
preferential water flow along root channels and has been observed in a number of
studies.17,38 On a horizon basis, the extent of organic C enrichment in the rhizosphere
is not significantly correlated to the decrease in soil pH, suggesting that the accu-
mulation of organic substances is probably not the main acidifying mechanism in
the rhizosphere of these forest soils.
The growth and activity of roots create a microenvironment that is both acidified
and enriched in organic substances relative to the bulk soil. Moreover, the abundant
C supply of the rhizosphere increases the establishment of microbial colonies (fungi,
bacteria), which in turn, act as a source of organic matter to the rhizosphere.19,39
These specific properties of the rhizosphere are known to accelerate the weathering
of minerals at the rootsoil interface.40,41 Courchesne and Gobran16 showed that the
abundance of weatherable minerals was consistently lower in the rhizosphere than
in the bulk soil and that the magnitude of the mineralogical differences was related
to the resistance to weathering of soil minerals. For example, the amount of amphib-
oles was found to be significantly lower ( < 0.10) near the roots of Norway spruce
[Picea abies (L.) Karst] than in the bulk materials of six horizons collected from
196 Trace Elements in the Rhizosphere

FIGURE 9.2 Organic C content (log scale) in the rhizosphere and bulk components of seven
forest soil horizons. Error bars are standard deviations of duplicates. The legend for soil names
and horizon designations is the same as in Figure 9.1.

two podzolic profiles (Figure 9.3). Systematically lower amounts of plagioclases


were also measured in the rhizosphere samples, but the differences were not signif-
icant. However, no rhizosphere effect was detected using X-ray diffraction tech-
niques for the more resistant K-feldspars.

B. METAL FRACTIONATION
1. BaCl2 Extraction

Table 9.3 presents both the average amounts of metals dissolved by the various
extractants and the results of the Wilcoxon signed rank test. For all metals (Al, Fe,
Mn, Cu, and Zn) the amounts extracted by the BaCl2 solution are significantly higher
( < 0.05) in the rhizosphere than in the bulk soil as shown in Figure 9.4 for Zn.
The Wilcoxon test uses the size of rank order differences within pairs of data
(rhizosphere versus bulk) and does not rely on the average values. Therefore, the
averages are presented in Table 9.3 to illustrate the extent of differences between
rhizosphere and bulk materials, although they are not the information used to estab-
lish whether the properties of the two components are statistically different. The
mean metal concentrations in the BaCl2 extracts lie within the range for salt extrac-
tions of uncontaminated forest soils.2,42 It should be noted that the standard deviation
values in Table 9.3 reflect the high heterogeneity between soil samples rather than
analytical variation. On a profile basis, the amounts of Al and Fe in the rhizosphere
Solid Phase Fractionation of Metals in the Rhizosphere of Forest Soils 197

FIGURE 9.3 Abundance of amphiboles [expressed as a ratio between the intensity of the
amphibole peak (I) and the intensity of the 100 peak for quartz (IQZ)] in the rhizosphere and
bulk components of six horizons collected from two Podzols (P1 and P2) formed under
Norway spruce [Picea abies (L.) Karst.]. The legend for horizon names is: A = 010 cm, B1
= 1025 cm, and B2 = 2550 cm.

TABLE 9.3
Average Metal Concentrations in the Rhizosphere (R) and Bulk (B)
Components of Seven Forest Soil Horizons as Measured by Four Chemical
Extractions
Extraction
Metal Component BaCl2 Pyrophosphate Oxalate Acid

Al (g kg1) R 0.16 (0.08)a 5.54 (4.75) 16.13 ( 7.98) 31.64 (18.84)


B 0.06 (0.03) 4.67 (3.68) 15.45 ( 8.19) 28.94 (19.09)
Fe (g kg1) R 0.02 (0.02)a 1.95 (1.74) 10.20 ( 5.75) 17.58 ( 6.62)
B 0.01 (0.01) 1.45 (1.41) 9.27 ( 5.56) 15.15 ( 5.53)
Mn (mg kg1) R 2.63 (2.41)a 4.43 (4.93)a 16.94 (14.13) 61.00 (67.58)
B 0.67 (0.33) 1.43 (1.99) 19.51 (16.58) 73.14 (67.48)
Cu (mg kg1) R 4.04 (3.35)a 4.71 (3.68)a 9.01 ( 3.88)a 10.71 ( 3.74)a
B 1.71 (1.04) 0.86 (0.69) 4.81 ( 2.22) 6.79 ( 1.89)
Zn (mg kg1) R 1.96 (1.38)a 4.26 (2.73)a 5.49 ( 2.84)a 28.39 (15.19)
B 0.40 (0.26) 1.43 (1.40) 3.89 ( 2.13) 19.29 ( 9.92)
a Metal concentrations in the rhizosphere are significantly different from values measured in the bulk
at the < 0.05 level (Wilcoxon signed rank test).
Note: Standard deviation values are in parentheses.
198 Trace Elements in the Rhizosphere

FIGURE 9.4 BaCl2-extractable Zn in the rhizosphere and bulk components of seven forest
soil horizons. Error bars are standard deviations of duplicates. The legend for soil names and
horizon designations is the same as in Figure 9.1.

and bulk materials decrease with depth and follow the trend observed for organic C
(Figure 9.2) while the other three metals, Mn, Cu, and Zn (Figure 9.3) do not.
The BaCl2 salt is thought to displace the cations that are adsorbed, in an
exchangeable fashion, on the negative charges of the soil.26 In acidic forest soils,
the cation exchange capacity (CEC) increases with organic matter content and is
pH-dependent, decreasing with soil pH.43 The exchangeable metal cations are loosely
held at the soilsolution interface and can easily be replaced by other cations with
their subsequent displacement toward the soil solution. In this context, the exchange-
able form represents one of the fractions, among all the metal fractions on the soils
solid phase, that is the most readily available for uptake by the plants and biota.3
The analysis of the rhizosphere thus shows that the roots are exposed to a larger
pool (up to fivefold) of available, potentially toxic metals than what would have
been estimated from bulk soil analysis. For the seven horizons, the significant
accumulation of BaCl2 exchangeable metals in the rhizosphere parallels the observed
enrichment in organic C (Figure 9.2). Also, because the mineral horizons studied
contain very little phyllosilicate minerals, their CEC is mostly controlled by organic
matter content.44 This suggests that the larger pool of organic matter found in the
rhizosphere, through its known positive effect on the CEC of rhizospheric materi-
als,17,24 favors the retention of exchangeable metals at the rootsoil interface. The
reaction occurs even though the pH of the rhizosphere is lowered by root activity
Solid Phase Fractionation of Metals in the Rhizosphere of Forest Soils 199

FIGURE 9.5 Cumulative amounts of Cu, Zn, Fe, Mn, and Al dissolved (log scale) by the
four chemical extractions for the rhizosphere (R) and bulk (B) components of the Parc des
Grands-Jardins C horizon. Metal contents for each extractant are expressed as percentages of
the total metal content measured by X-ray fluorescence (XRF) in the bulk soil. Error bars are
standard deviations of duplicates for the acid extraction.

(Figure 9.1), a condition that should diminish the CEC of the rhizosphere and the
amount of exchangeable metals.
The exchangeable metal pool in the rhizosphere and bulk components generally
represent less than one percent of the total metal content in the soil as illustrated in
Figure 9.5 for the PGJ C horizon. The exchangeable fraction is known to constitute
a small proportion of total metals in soils.4 However, the amount of BaCl2-exchange-
able Cu reaches 13% of total Cu in the PGJ C horizon and varies from 10 to 24%
when all horizons and components are considered. Exchangeable Zn also accounts
for 2 to 8% of total Zn in six of the seven rhizosphere materials, although its
contribution remains below 2% in the bulk component. These exchangeable Zn
percentages are similar to those reported by Johnson and Petras42 for a Spodosol
and by Ma and Rao45 for contaminated soils. Up to 40% of total Zn was associated
with the exchangeable fraction of Spodosols under citrus production.46 As for Cu,
the high proportion measured in the exchangeable form is in contrast with data for
coarse-textured soils where the bulk of soil Cu is in unavailable forms, either in the
residual fraction or bound to organic matter.47 We suggest that the BaCl2 salt was
effective in displacing some of the Cu present in the weaker Cu-organic matter
complexes found at these low soil pH values.48 Under such conditions, a fraction of
the Cu that would otherwise be strongly bound to organic compounds in a less acidic
environment behaves more like an exchangeable metal.
200 Trace Elements in the Rhizosphere

TABLE 9.4
Average Metal/Organic C Molar Ratio
in the Rhizosphere (R) and Bulk (B)
Components of Seven Forest Soil
Horizons
Metal Rhizosphere Bulk

Molar Ratioa
Al 130.0 (103) 192.0 (321)
Fe 8.2 (9.0) 10.9 (21.2)
Mn 0.9 (0.9) 0.8 ( 1.1)
Cu 1.8 (2.4) 2.3 ( 3.1)
Zn 0.6 (0.6) 0.5 ( 0.6)
a [Amount of metal in the BaCl2 extraction
(M)/amount of organic C in the horizon (M)] 104.
Note: Standard deviation values are in parentheses.

The ratio of the amount of metal displaced by the BaCl2 extraction to the amount
of organic C is presented in Table 9.4. This molar ratio was calculated (conversion
of concentration units from g kg1 to M kg1) for each horizon and metal and averaged
through horizons for a given metal. The computation of this simple ratio is based
on the assumptions that organic matter constitutes the dominant source of cation
exchange sites in these soils and that organic C content represents a good analogue
of the relative abundance of such sites.17,49 The results indicate that the amounts of
exchangeable metal do not differ between the rhizosphere and bulk soil when the
metal concentration is normalized with respect to the amount of organic C (Table
9.4), even though the absolute exchangeable metal concentrations are much higher
in the rhizosphere (Table 9.3). If we accept the assumptions of the molar ratio, then
the data suggest that the proportion of exchange sites occupied by metals (per unit
mass of soil) at the surface of organic substances is virtually equal in both compo-
nents. Only the total number of organic C containing exchange sites differs, being
higher in the rhizosphere.
When interpreting the molar ratio values, consideration should be given to the
fact that the concentration of negative charges per unit mole of organic C probably
differs between the rhizosphere and the bulk soil. Because the charges carried by
the organic matter are pH-dependent, the charge density of organic substances should
be lower in the rhizosphere where the pH is lower. Also, differences in the compo-
sition of organic matter between the two soil components19,21,38 will reflect on the
nature and abundance of the functional groups capable of attracting cations. None-
theless, the observations provided by the molar ratio draw attention to the dynamic
role of organic matter enrichment in the rhizosphere on the accumulation of
exchangeable metals at the rootsoil interface. However, further work is needed on
the composition, properties, and reactivity of the organic substances present in the
rhizosphere of forest soils before their reaction with metals is fully understood.
Solid Phase Fractionation of Metals in the Rhizosphere of Forest Soils 201

FIGURE 9.6 Na-pyrophosphate-extractable Fe in the rhizosphere and bulk components of


seven forest soil horizons. Error bars are standard deviations of duplicates. The legend for
soil names and horizon designations is the same as in Figure 9.1.

2. Pyrophosphate and Oxalate Extractions

Significant differences between rhizosphere and bulk materials are limited to Cu


and Zn when both the pyrophosphate and the oxalate extractions are considered
(Table 9.3). The amounts of extractable Al and Fe are generally higher in the
rhizosphere, but a clear statistical separation of the components is lacking. For
example, Figure 9.6 shows that pyrophosphate-extractable Fe is more abundant at
the rootsoil interface in five of the seven horizons. Similar, yet inconclusive results
are obtained for Al in pyrophosphate extracts and for Al and Fe in oxalate. These
observations are comparable to the data of Courchesne and Gobran16 who showed
a systematic, but not always significant increase in oxalate Al and Fe in the rhizo-
sphere. At the scale of a given profile, the amounts of Al and Fe recovered by the
two extractions sharply decrease with depth as does organic C content (Figure 9.6).
The behavior of Mn varies as a function of the extracting solution. While pyrophos-
phate-extractable Mn accumulates in the rhizosphere, the amounts dissolved by the
oxalate solution do not differ between the two components (Table 9.3). The frac-
tionation of Mn indicates that a larger portion of total Mn is associated with amor-
phous inorganic solids in the bulk soil than in the rhizosphere.
In podzolic soil environments, Na-pyrophosphate and ammonium oxalate are
commonly used to extract the organic and the inorganic amorphous fractions, respec-
tively.50,51 Although limitations exist with regard to their specificity, the metals
recovered by these extractants are generally considered to be bound to organic
202 Trace Elements in the Rhizosphere

FIGURE 9.7 Acid ammonium oxalate-extractable Cu in the rhizosphere and bulk compo-
nents of seven forest soil horizons. Error bars are standard deviations of duplicates. The legend
for soil names and horizon designations is the same as in Figure 9.1.

substances (pyrophosphate) or associated with inorganic amorphous oxides


(oxalate). The formation of mixed solids containing organic compounds and amor-
phous and crystalline Fe and Al oxides is a typical feature of Podzol development
under forest covers.52 They accumulate as coatings on mineral grains or on aggregates
in the B and BC horizons. During the genesis of these pedogenetically derived solids,
metals like Cu, Zn, and Mn can be incorporated into their structure through a variety
of reactions, including adsorption, chelation, coprecipitation, and cation substitu-
tion.47 Changes in soil properties occurring during pedogenesis and through the
activity of the biota can favor the concentration of such chemically reactive solids,
which can, in turn, alter the distribution of metal pools. The rhizosphere is a dynamic
soil environment characterized not only by an enrichment in organic substances
(Figure 9.2) but also by the accumulation of Al and Fe oxides, at least in a majority
of horizons (Figure 9.6). The combined effect of these two processes confers a higher
metal retention capacity to rhizospheric materials than to the bulk soil. It also
represents a plausible explanation for the high pyrophosphate and oxalate-extractable
metal accumulations observed in the rhizosphere of all horizons, as shown in Figure
9.7 for Cu in the oxalate extracts.

3. Acid Extraction

Only Cu concentrations were found to differ significantly between the bulk and
rhizosphere components following the HNO3HCl extraction (Table 9.3). The results
of the acid extraction emphasize the fact that, in these forest soils, the rhizosphere
Solid Phase Fractionation of Metals in the Rhizosphere of Forest Soils 203

effect is probably confined to the smallest, most available metal pools, like the BaCl2-
exchangeable and pyrophosphate-extractable fractions (Table 9.3). It seems that the
metal contents estimated for the larger acid-soluble (Figure 9.5) and residual (average
of 63, 58, 87, 48, and 59% of the total metal content for Al, Fe, Mn, Cu, and Zn,
respectively) fractions are too extensive to be significantly affected by the develop-
ment of the root network in soils. This conclusion somewhat parallels observations
concerning the mineralogical composition of rhizospheric and bulk materials where
only the most weatherable minerals of the assemblage (e.g., amphiboles, phlogopite)
were measurably affected by the increased weathering activity generated by root
growth.53,54 Also, the component separation procedure used in this study is macro-
scopic in nature and does not assure the collection of pure rhizosphere, because
the contamination of the rhizosphere by materials from the bulk soil can occur during
manipulations. This fact may limit the detection of changes or gradients in an
environment as small as the rhizosphere. Techniques allowing the in-place charac-
terization of the chemical composition of the rootsoil interface need to be developed
to circumvent this problem.

IV. CONCLUSIONS
The major findings provided by the study of the rhizosphere and bulk components
of uncontaminated forest soils are as follows:

1. The rhizosphere is enriched in organic C and in DOC and has a lower pH


in H2O than the bulk soil. These conditions contribute to the development
of a corrosive microenvironment where the alteration of the most weath-
erable soil minerals is increased.
2. The pool of BaCl2-exchangeable metals (Al, Fe, Mn, Cu, and Zn)
increases close to roots. This fraction represents one of the most bioavail-
able metal pools in soils. The results also underline the effect of organic
matter enrichment in the rhizosphere on its capacity to accumulate
exchangeable metals. However, when the exchangeable pool is normalized
with respect to the amount of organic C (Me/C ratio), the density of cation
exchange sites occupied by metals is equivalent in both components.
3. Pyrophosphate and oxalate-extractable Cu, Zn, and to a lesser extent Mn,
are higher in the rhizosphere, while extractable Al and Fe are not signif-
icantly different. This emphasizes the contribution of organic matter accu-
mulation and sesquioxide formation on metal retention in the rhizosphere,
although the metals bound to these solids are not as available as the
exchangeable fraction.
4. Except for Cu, no difference exists for the acid (HCl-HNO3) extract,
suggesting that the detection of the rhizosphere effect is limited to the
smaller metal pools in these forest soils.

Evidence presented here from forest soils shows that the analysis of the rhizo-
spheric component of soils yields a more complete picture of the microenvironment
created and faced by the roots of coniferous trees. Indeed, the impact of the rhizo-
204 Trace Elements in the Rhizosphere

sphere on elemental availability and cycling appears to be disproportionately large


when compared to the small volume it occupies in the total mineral soil. In this
context, it is worth exploring an approach that explicitly includes the rhizospheric
environment in empirical soilplant relationships, both to improve their predictive
capacity and to better account for the functioning of soilplant systems. Furthermore,
because elemental uptake by plants is a critical component of the overall flux of
matter in a terrestrial ecosystem and because these fluxes control the health status
of a forest, we believe that the rhizosphere could provide indicators of the response
of forested ecosystems to environmental changes such as metal contamination, soil
acidification, and fluctuations in climatic conditions.

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34. Conkling, B. L., Blanchar, R. W., and Niblack, T. L., Effects of foliar and soil acidity
on the rhizosphere pH of alfalfa, corn and soybean, J. Environ. Qual., 20, 381, 1991.
35. Clegg, S., Gobran, G. R., and Guan, X., Rhizosphere chemistry in an ammonium
sulfate and water manipulated Norway spruce [Picea abies (L.) Karst.] forest, Can.
J. Soil Sci., 77, 515, 1997.
206 Trace Elements in the Rhizosphere

36. Hedley, M. J., Nye, P. H., and White, R. E., Plant-induced changes in the rhizosphere
of rape (Brassica napus var. Emerald) seedlings, New Phytol., 91, 31, 1982.
37. Petersen, W., and Bttger, M., Contribution of organic acids to the acidification of
the rhizosphere of maize seedlings, Plant Soil, 132, 159, 1991.
38. Szmigielska, A. M., Van Rees, K. C. J., Cieslinski, G., and Huang, P. M., Low
molecular weight dicarboxylic acids in rhizosphere soil of durum wheat, J. Agric.
Food Chem., 44, 1036, 1996.
39. Sguin, V., Courchesne, F., and Dufresne, A., Spciation des mtaux dans la
rhizosphre de sols forestiers et de sols contamins, in Comptes-Rendus du 3e Con-
cours tudiants du RESOL sur le sol contamin, Elektorowicz, M., and Ebadi, T.,
eds., Concordia University, Montral, 1998, 55.
40. Jongmans, A., van Breemen, N., Lundstrom, U., van Hees, P., Finlay, A. W., Srini-
vasan, R. D., Unestam, M., Giesler, R., Melkerud, P. A., and Olsson, M., Rock-eating
fungi, Nature, 389, 682, 1997.
41. Arocena, J. M., Glowa, K. R., Massicotte, H. B., and Lavkulich, L., Chemical and
mineral composition of ectomycorrhizosphere soils of subalpine fir [Abies lasiocarpa
(Hook.) Nutt.] in the Ae horizon of a Luvisol, Can. J. Soil Sci., 79, 25, 1999.
42. Johnson, C. E., and Petras, R., Distribution of zinc and lead fractions within a forest
Spodosol, Soil Sci. Soc. Am. J., 62, 782, 1998.
43. Sposito, G., The Chemistry of Soils, Oxford University Press, New York, 1989, 171.
44. Courchesne, F., Savoie, S., and Dufresne, A., Effects of air-drying on the measurement
of soil pH in acidic forest soils of Quebec, Canada, Soil Sci., 160, 56, 1995.
45. Ma, L. Q., and Rao, G. N., Chemical fractionation of cadmium, copper, nickel, and
zinc in contaminated soils, J. Environ. Qual., 26, 259, 1997.
46. Zhang, M., Alva, A. K., Li, Y. C., and Calvert, D. V., Chemical association of Cu,
Zn, Mn, and Pb in selected sandy citrus soils, Soil Sci., 162, 181, 1997.
47. Shuman, L. M., Chemical forms of micronutrients in soils, in Micronutrients in
Agriculture, Mortvedt, J. J., Cox, F. R., Shuman, L. M., and Welch, R. M., eds., Soil
Science Society of America, Madison, WI, 1991, 113.
48. Norvell, W. A., Reactions of metal chelates in soils and nutrient solutions, in Micro-
nutrients in Agriculture, Mortvedt, J. J., Cox, F. R., Shuman, L. M., and Welch, R. M.,
eds., Soil Science Society of America, Madison, WI, 1991, 187.
49. Santore, R. C., Driscoll, C. T., and Aloi, M., A model of soil organic matter and its
function in temperate forest soil development, in Carbon Forms and Functions in
Forest Soils, McFee, W. W., and Kelly, J. M., eds., Soil Science Society of America,
Madison, WI, 1995, 275.
50. McKeague, J. A., and Day, J. H., Dithionite and oxalate-extractable Fe and Al as aids
in differentiating various classes of soils, Can. J. Soil Sci., 46, 13, 1966.
51. Bascomb, C. L., Distribution of pyrophosphate extractable iron and organic carbon
in soils of various groups, J. Soil Sci., 19, 251, 1968.
52. Courchesne, F., and Hendershot, W. H., La gense des Podzols, Gog. Physique Quat.,
51, 235, 1997.
53. April, R., and Keller, D., Mineralogy of the rhizosphere in forest soils of the eastern
United States, Biogeochemistry, 9, 1, 1990.
54. Hinsinger, P., Elsass, F., Jaillard, B., and Robert, M., Root-induced irreversible trans-
formation of a trioctahedral mica in the rhizosphere of rape, J. Soil Sci., 44, 535, 1993.
10 A Technique for
Quantitative Trace
Element and
Micronutrient Studies
in Plants
Anders Gransson

CONTENTS

I. Introduction...................................................................................................207
A. Historical Rsum.............................................................................207
B. Nutrient Supply in Plant Nutrition Research ...................................211
II. Microelement Nutrition ................................................................................214
III. Toxicity of Elements.....................................................................................218
IV. The Root SubstrateRoot Interface..............................................................220
V. Concluding Remarks ....................................................................................222
References..............................................................................................................222

I. INTRODUCTION
A. HISTORICAL RSUM
Different ways of increasing harvest yield have been discussed for as long as crops
have been cultivated. For example, the peoples of ancient Egypt and China were
aware of the beneficial aspects of flooding on crop yield and used the approach
extensively. Among the first documented philosophers of plant nutrition, Aristotle
(384322 BC) and Theophrastos (371286 BC) recognized the importance of soil
as a source of materials from which plants are made.1 The influence of Aristotle on
the science of plant nutrition persisted, as evidenced by Linn2 (1751) who, in his
Philosophia Botanica, quoted Aristotles philosophy that the soil is the stomach
of plants: plantarum ventriculus est terra .
Two centuries ago, the possibility that plant growth was in any way dependent
on mineral nutrients was still being debated.3 One of the first mineral elements to
be recognized as a plant nutrient was nitrogen. This was demonstrated in 1804 by

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 207
208 Trace Elements in the Rhizosphere

TABLE 10.1
Knops culture solution, developed in 1868.4
Note the inaccurate specification, especially of the
Fe-salt, of the amounts to be added per liter of
distilled water for healthy-looking plants.
Chemical Salt Amount (g)

Ca(NO3)2 4 H2O 0.8


KNO3 0.2
KH2PO4 0.2
MgSO4 7 H2O 0.2
FePO4 trace

the Swiss scientist T. de Saussure.4 The science of mineral nutrition took advantage
of developments in chemistry, and as purer chemicals were developed, the essential
nature of other mineral elements was discovered. For example, the Frenchman E.
Gris5,6 documented the effects of iron deficiency on plant appearance, although it is
J. Sachs who is normally credited with having, in 1860, conclusively proven the
essential nature of iron.3 By the end of the 19th century, ten mineral elements were
known to be essential to plant growth and culture solutions were being used for
experimental purposes. An example of one of these early solutions is shown in Table
10.1. In 1915, Maz was one of the first to seriously challenge the belief that only
those elements listed in Table 10.1 were required by plants.7 It had become clear
that trace amounts of different elements were being added as impurities in experi-
ments with solution cultures and that some of these might well be important to plant
growth. In 1905, the French scientist G. Bertrand, recognized manganese as being
fundamental to plant growth4 and, in 1922, J.S. McHague showed that manganese
was essential to the metabolism of higher plants.3 Manganese was found in much
smaller quantities than the other elements, and was the first element to be defined
as a micronutrient, in contrast to the other macronutrients.8 By the mid-19th
century, it was clear that plants required several different elements in balanced
proportions to maintain healthy growth, and today it is general practice to list six
mineral nutrient elements that are required for plant growth as macronutrients, and
six trace elements as micronutrients (Table 10.2). Other elements are often consid-
ered as beneficial to plant growth (Table 10.3), but their true importance is debated.
At the same time other elements were recognized as being potentially growth-
retarding or toxic for several plant species when present in higher concentrations in
the rooting substrate (Tables 10.4 and 10.5). During evolution, some plants species
have developed the ability to accumulate large amounts of metals without showing
toxic responses (Tables 10.4 and 10.5). These species are often referred to as
chemoecotypes, and have developed a genetically based heavy metal resistance,
which may be modified by adaptation. Despite the current level of knowledge
regarding which elements are essential to plant growth, the quantitative requirements
and proportions of these different nutrients are much less well appreciated. For
Quantitative Trace Element and Micronutrient Studies in Plants 209

TABLE 10.2
Essential plant macro- and micronutrient
mineral concentrations considered to be
adequate for most higher plants81 and for
Betula,36 Pinus, and Picea species34 at free
access to all nutrients.
Concentration in Dry Matter
Element Higher Plants Betula Pinus Picea

g g1 dm)
Macronutrients (
N 15 40 26 22
K 10 26 12 12
P 2 5 5 4
Ca 5 2.8 3 3
Mg 2 3.5 2 1
S 1 3.6

g g1 dm)
Micronutrients (
Fe 100 280 180 160
Mn 50 160 100 90
B 20 80 50 45
Zn 20 25 15 15
Cu 6 10 10 10
Mo 0.1 3 2 2
Cl 10 10 10

example, it may be noted that, in recent times, iron has still been listed as a
macronutrient,4,9 although the proportion of iron required for healthy growth may
not be much greater than that required for many micronutrients. The distinction
between macro- and micronutrients on the basis of amounts found in plant tissues
can be misleading in a functional context. From a physiological point of view, other
definitions may be proposed, according to the function of the element in the plant
(Tables 10.6 and 10.7).

TABLE 10.3
Some elements regarded as beneficial for
higher plants.3
Element Example of Function in Higher Plants

Cl Photosynthesis and stomatal regulation


Na Essential for water regulation in halophytes
Si Biomass production
Co N2-fixation in, e.g., Leguminosae
Se Prevention of excessive phosphate uptake
210 Trace Elements in the Rhizosphere

TABLE 10.4
Examples of elements and concentrations regarded as toxic
for higher plants. Note that some of the micronutrient
elements are very toxic in slightly raised concentrations.82, 83
Concentration
Element Typical Response or Effect g g1)
(

Al Effects on cation uptake, cytological changes 500


Cd Decrease in photosynthesis and transpiration rates 1050
Cu Disturbance of carbohydrate and N-metabolism 1050
Pb Growth retardation, effects on enzymes 50100
Zn Changes in enzyme activity, cytological changes 200300

TABLE 10.5
Examples of concentrations in some chemoecotypes.
Typical Concentrations
Species Accumulated Element g g1)
(

Astragalus preussi U 70
Astragalus racemosus Se 15.000
Jasione montana As 31.000
Minuartia verna Cu 1.0501.850
Minuartia verna Pb 11.40026.300
Minuartia verna Cd 350400
Thlaspi caerulescens Zn 11.30025.000

TABLE 10.6
Classification of essential mineral nutrients according to photosynthetic
function.3 Parentheses denote that function is unclear or mainly based on
indirect evidence.
Constituents of Activators of Enzymes,
Elements Process Organic Structure Osmoregulation

Chloroplast formation Protein synthesis N, S Mg, Zn, Fe, K, (Mn)


Chlorophyll synthesis N, Mg Fe
Electron transport chain PSI + II,
Photophosphorylation Mg, Fe, Cu, S, P Mg, Mn, (K)
CO2-fixation Mg, (K, Zn)
Stomatal movement K, (Cl)
Starch synthesis, sugar P Mg, P, (K)
transport
Quantitative Trace Element and Micronutrient Studies in Plants 211

TABLE 10.7
Classification of essential mineral nutrients according to the general
function.84 Parentheses denote that function is unclear or mainly based
on indirect evidence.
Group Function Elements

1 Major constituents of organic material, essential for enzymatic N, S


processes
2 Esterification with alcholol groups in plants, energy transfer P, B, (Si)
3 Balancing anions, controlling membrane permeability, enzyme K, (Na), Mg, Ca, Mn, (Cl)
activation, osmotic potentials
4 Electron transport by valence change Fe, Cu, Zn, Mo

B. NUTRIENT SUPPLY IN PLANT NUTRITION RESEARCH


Different experimental approaches to plant culture have been used in studies of plant
nutrition. In general, there are three different ways of adding nutrients. First, the
arbitrary addition of nutrients to the rooting substrate on one or more occasions
during the study often occurs. In many cases, the amounts added and the frequency
of addition are poorly specified. The intention is normally to eliminate the occurrence
of nutrient deficiency but, because the amounts and frequency of addition are not
related to the potential for growth, unintentional deficiency can occur. This can lead
to very misleading interpretations of growth response.10-13
Secondly, in experiments where external variables, such as temperature, light
intensities, light duration, and relative humidity, often are constant or varied within
narrow ranges, there is the maintenance of a constant nutrient concentration in the
rooting substrate.14 Technically, this may feature more- or less-sophisticated systems
of flowing culture solution,15 where external nutrient concentrations are held constant
by balancing the rate of addition against the rate of nutrient removal by the plants.
By introducing flowing solutions, the demand of external concentration for ade-
quate growth decreased by orders of magnitude.16,17 At extremely low nutrient
concentrations, plant growth can be controlled by the external concentration. This
has, for example, been demonstrated with respect to nitrate supply.18 The mainte-
nance of such low concentrations at the root surface may be problematic, as rapid
flow or stirring of the culture solution is essential. In order to maintain low concen-
trations in culture solutions, nutrients must be supplied in increasingly large amounts
to compensate for the rapid withdrawal of nutrients by the plants. The disadvantage
of the approaches described above is that there are great difficulties in controlling
plant growth and thus, other plant characteristics connected with different growth
rates. To control plant growth in practice, the use of external nutrient concentration
as the driving variable has shown to be adequate only when plants are growing at,
or close to, their potential relative growth rate. It is, however, important to conclude
that external nutrient concentration is the result of two time-dependent variables,
addition rate and uptake rate. The use of chelators to buffer low concentrations of
metals has recently been used in hydroponics.19,20 The advantage of this approach
212 Trace Elements in the Rhizosphere

FIGURE 10.1 The dependence of relative addition rate of nutrients, RA (day1) and photo-
synthetic active radiation (PAR) on the relative growth rate, RM (day1), of plants. The equation
of the fitted line is y = 0.003 + 0.997 with r2 = 0.945. The symbols represent; = Betula
pendula, PAR 450 M s1 m2;  = B. pendula, PAR 250 M s1 m2;  = B. pendula,
PAR 50 M s1 m2;  = Pinus sylvestris, PAR 250 M s1 m2;  = Pinus contorta, PAR
250 M s1 m2;  = Picea abies, PAR 250 M s1 m2.

is to reduce contamination by unwanted elements and problems of controlling the


addition of diminutive amounts of micronutrients. Chelates can, however, form very
strong bindings with trace metals and will affect the speciation of several di- or
trivalent metals.19,20
The third approach eliminates the requirement of maintaining constant external
concentrations. In small plants, the amount of added nutrients per unit time must
increase in an exponential manner not to be growth-limiting. By using the relative
addition rate, RA day1, of nutrients as the growth-controlling variable, it has been
shown that plant uptake of nutrients and growth can be strictly controlled during
the exponential phase of growth (Figure 10.1).10,21-25 The relative addition of nutrients
to the roots may be defined as the addition rate, dn/dt (g day1), with respect to the
amount of nutrient, n (g), in the plant:

1 dn
RA = (1)
n dt
Quantitative Trace Element and Micronutrient Studies in Plants 213

When using RA to control the relative uptake rate of nutrients, stable relative
growth rates, RM, may be attained. Thus, the relative growth rate equals the relative
addition rate of nutrients, which may be defined as:

1 dM 1 dn
RM = = = RA (2)
M dt n dt

where M (g) is the plant mass. This is the steady-state approach and has been
used extensively in studies of plant nutrition during recent years.26 At steady-state
growth, the plant nutrient concentration, n/M, remains constant for a given genotype,
growth environment, and the chosen value of RA.27

n
d
M
=0 (3)
dt

When nutrients are added at different RA, any submaximal or maximal RM will
be in a one-to-one relationship with the corresponding RA regardless of plant species
or external environmental conditions.21,28-37 The conductivity of the culture solution
is usually very low, less than 50 S cm1 at submaximum supply rate as RA = RM.
Solution conductivity at maximum RM may be kept at about 50 S cm1, but is
normally about 100 to 200 S cm1, to ensure free access to all nutrients. The RA
of nutrient elements controls plant characters other than growth, e.g., biomass par-
titioning to above- and belowground plant parts (Figure 10.2), leaf or needle area
development, and the uptake rate of nutrient elements. Uptake rates of different
elements at the growing root tip may thus be calculated as nutrient uptake rate per
unit root growth rate, defined as dn/dMr (mol g1 root DM):

dn n M
= (4)
dMr M Mr

where n is the nutrient amount in the plant, and M and Mr are plant and root dry
matter, respectively. (Related theoretical considerations concerning the technique
have been discussed elsewhere).10,27,37,38 The use of nutrient addition rate as a treat-
ment variable has proved to be a useful tool for a number of research areas related
to plant growth and soil acidification and pollution.13,39-46
The fertility of a soil depends highly on weathering and mineralization of
inorganic nutrient elements. It is possible, by using the steady-state technique, to
mimic low concentrated fluxes of nutrients, which may occur in the rhizosphere.
Consequently, by using the technique as a tool to mimic nutrient fluxes in the
rhizosphere in field trials, soil fertility and the growth of Pinus sylvestris and Picea
abies increased with only small nutrient leakage to the soil solution.47-48
214 Trace Elements in the Rhizosphere

FIGURE 10.2 Root mass ratio (percent root DM of plant DM) at different relative addition
rates of Cu, Fe, Mn, and Zn (day1). FA denotes free access to all nutrient elements. The root
mass ratio of N at different RA (day1) is included as reference.

II. MICROELEMENT NUTRITION


It is commonly known that high growth rates of plants may be attained at low external
concentrations of nutrients. However, in order to maintain these low concentrations
in culture solutions, nutrients must be supplied in increasingly large amounts to
compensate for the rapid withdrawal of nutrients by the plants. In small plants, the
amount of added nutrients per unit time must increase in an exponential manner, so
as not to be growth-limiting. If this requirement is not taken into account, the use of
low, so called ecologically relevant nutrient concentrations may give rise to nutrient
limitation caused by insufficient supply. For convenience, however, large applications
of nutrients are often used in nutritional experiments. These may cause chemical
precipitation between nutrient elements and a toxic substance, e.g., P and Al. Poor
control of plant nutrition may thus obscure effects on plant growth, and causal
connections between plant reactions and mineral elements may be difficult to identify.
The mineral nutritional status of plants is a critical factor when interpreting plant
growth. Biomass partitioning, gas exchange, photosynthesis, water relations, sym-
biosis, senescence, and aging are some examples of plant processes that can be
altered by the nutrient status of the plant. Regardless of the process under investi-
Quantitative Trace Element and Micronutrient Studies in Plants 215

FIGURE 10.3 Calculated increase of internal zinc amount in Betula pendula plants growing
at a maximum RM of 0.25 day1 () and at a submaximum RM of 0.10 day1 (). The addition
of a constant external concentration of 5 M day1 is included (dotted line). The latter results
in growth decline and decrease in internal concentrations of nutrients within a few days. The
calculations are based on a seedling dry mass of 1 g at time = 0 and an internal plant [Zn]
of 25 g g1 DM.

gation, it is important to maintain a consistent program of nutrient supply. The


importance of the steady-state approach and the use of RA is clear when compared
to the use of constant external concentration of nutrients as the growth-driving
variable. The latter results in growth decline and decrease in internal concentrations
of nutrients within a short time because of insufficient nutrient supply. An example
of this is shown in Figure 10.3. If the supply rate is high, plant growth may be
controlled at extremely low nutrient concentrations,18 but the maintenance of such
low concentrations may be problematic, as rapid flow or stirring of the culture
solution is essential. In investigations where the RA of different micronutrients (Cu,
Fe, Mn, and Zn) was the growth-determining variable, the external concentration in
the spray-nutrient solution was below or close to the detection limit of the analytical
equipment, regardless of RA and micronutrient element.24,25,31,32 Thus, in practice, it
is extremely difficult to use external concentration of micronutrients as the driving
growth variable to achieve stable relative growth rates and stable plant properties.
The quantitative requirements for micronutrients in plant growth are less well
defined than those for macronutrients, and concepts of critical concentration and
216 Trace Elements in the Rhizosphere

FIGURE 10.4 Traditional view of the relationship between growth of plants and nutrient
status. Region I denotes severe limitation and Region II mild limitation. It is in these two
regions that the Piper-Steenbjerg effect occurs. Region III consists of more or less healthy-
looking plants. In Region IV, luxury consumption of nutrients occurs, and in Region V, toxicity
is encountered. Redrawn from Bouma.49

luxury consumption are often used in the explanation of growth response.49 At


limiting availability, especially of micronutrients, a negative relationship between
yield and plant nutrient concentration, the Piper-Steenbjerg effect (Figure 10.4), has
often been reported.50-52 The effect has been attributed to variation in tissue age53
and translocation within the plant.54 There is, however, no consistent physiological
explanation as to why a small addition of a limiting element, at extreme limitation,
should be followed by a decrease in internal concentration. The choice of plant part
to be analyzed, the age of the part, and sampling occasion may all complicate the
interpretation of the nutritional-growth relationship. The results of investigations
where the RA of different micronutrients (Cu, Fe, Mn, and Zn) was the growth-
determining variable, indicated a linear relationship between relative growth rate
and internal micronutrient concentration (Figure 10.5).24,25,31,32 These observations
are not supportive of the Piper-Steenbjerg effect. Several attempts to explain the
effect are available.53-56 The most consistent explanation may, however, be the one
proposed by Wikstrm,57 who demonstrated that the effect might be the result of
dynamic changes of plant nutrient concentration. Thus, it is probably attributable to
methodology, resulting in non-steady-state growth, where the quantification of the
added nutrient amount over time is often overlooked.23,38 One of the most important
conclusions drawn from the steady-state approach is that plant properties are constant
and time-independent as long as the plants are at steady-state growth.
Quantitative Trace Element and Micronutrient Studies in Plants 217

FIGURE 10.5 The relationship between the relative growth rate, RM day1, and plant con-
centrations of iron, manganese, copper, and zinc in small Betula pendula seedlings. The
concentrations are given as g g1 DM. Open symbols are for controlled exponential supply
rates; closed symbols are for free access to all nutrients.

It is apparent that plant RM can be controlled by regulating the relative supply


rate of different micronutrients.24,25,31,32 This was only possible by growing the plants
in growth units free from contamination by unwanted sources of the micronutrients
and by eliminating any initial amount in the plants. This was achieved after a
thorough cleaning procedure in which acid, alkali, and ethanol solutions were cir-
culated in the growth units for ca. 24 hours each. Flushing with distilled water took
place for 24 hours between each of these solutions. After the ethanol had been
circulated, flushing with distilled water continued, but the distilled water was
218 Trace Elements in the Rhizosphere

changed at intervals of 5 to 10 hours to dilute remaining impurities. Small plants


were then grown in the units in a culture solution (made of Suprapure salts) in which
no micronutrients had been added, for approximately 1 week. This last step in the
cleaning procedure was found to be crucial in effectively avoiding subsequent con-
tamination by unwanted elements. These plants were discarded and the growth unit
was rinsed with distilled water before the experiments began.
In order to control plant growth rate, it is necessary to rapidly exhaust the reserves
of the micronutrient in the seed. This meant that the seedlings had to be grown
somewhat differently in the pretreatments than those grown by Ingestad and cowork-
ers.37 In pretreatments for micronutrient experiments, small plants were weighed in
groups and cultivated in a complete culture solution with balanced nutrient propor-
tions up to a fresh weight of approximately 50 mg per plant. Analyses from such
plants, growing at free access to all nutrients, revealed internal concentrations higher
than those needed for maximum RM. The internal concentration of the element under
investigation, for constant maximum RM, was estimated from other pilot studies. This
information was later used in the experiments where the RA of the different elements
was the growth-controlling variable. The plants that had been growing at free access
to all nutrients were transferred to a complete culture solution (except the element
under investigation) used for conductivity- and pH-titrations until their mass had
increased by a factor of approximately three. The mass increase of the plants was
checked by consecutive harvests of plant groups and the internal micronutrient con-
centration in the plants was analyzed. The concentrations in the plants grown in the
solution were thus diluted during this phase. When the expected internal micronutrient
concentration for a specific RM was achieved, the plants were carefully reweighed,
after which the element was added at RAs of 0.05, 0.10, 0.15, and 0.20 day1.
Additional experiments were conducted where all nutrients were added in free access.
It is evident from the results of such investigations that in experiments where
nutrients have been added at a constant rate, plants quickly become nutrient-limited
because of insufficient nutrition. Rapid morphological and physiological changes
may occur when plant nutrition is not controlled. This method has been used to
make models for nutrient uptake in plants.58,59 To evaluate correctly and interpret
results from plant experiments, it is therefore important to control nutrition. The
results from the micronutrient studies have been used in the field trials at Asa in the
province of Smland and at Flakaliden in the province of Vsterbotten, Sweden.
Aims of the trials were to maximize biomass production without leakage of nutrients
and to study nutrient turnover in a forest ecosystem. All results found in the labo-
ratory have been shown to be relevant for the field trials.48

III. TOXICITY OF ELEMENTS


It is a well-known fact that the aluminum, calcium, and magnesium concentration in
soil leachates has been increasing as a result of increased anthropogenic soil-acidifi-
cation.60,61 Damage to conifers was attributed to adverse effects, specifically of Al3+
on tree nutrition and growth.62,63 Several investigations have been carried out to
describe aluminum toxicity in forest trees.64 Some of these have been made at high
nutrient concentrations, while others have been performed at low or so called eco-
Quantitative Trace Element and Micronutrient Studies in Plants 219

TABLE 10.8
Growth response of Picea abies (L.) Karst. to
different aluminum concentrations in culture solution
experiments.
[Al], (mM) Plant Growth Reaction Reference

0.04 Negative Rost-Siebert85


0.2 None Gransson and Eldhuset44
0.7 None Abrahamsen86
1.1 None Evers87
1 to 6 Positive Wilkins and Hodson88

logically relevant concentrations of nutrients. This makes an objective comparison


between experiments difficult and a number of investigations concerning aluminum
toxicity of tree seedlings have resulted in conflicting results (Table 10.8). By using
the steady-state approach and comparing the stability of RM before aluminum addition
with that after, it has been shown that several boreal tree species were rather insensitive
to Al3+ at a pH of 4.0 to 4.2 in the rooting substrate.43-44 Decrease in growth rate was
found at aluminum concentrations that are high compared to those found in the
rhizosphere of acid boreal forest soils.61 Other tree species have been found to be
more sensitive to high aluminum concentrations and low pH.41 By controlling the RA
of nutrients it was possible to compare growth response to elevated aluminum con-
centrations between tree seedlings growing at near-maximum RM (0.22 day1) with
those that were nutrient limited (RM = 0. 10 day1).43 Small differences were found,
although plants that are nutrient optimized seem to be more sensitive to high alumi-
num concentrations. Such a comparison would have been practically impossible with
traditional growth methods, as the requirement of nutrients change with time.
The largest effect of aluminum on nutrient uptake was a decrease in the internal
Ca- and Mg-concentrations. The nutritional disorders apparently had no effect on
plant growth at low aluminum concentrations. Decrease in Ca and Mg uptake in the
presence of aluminum is commonly reported and interpreted as detrimental for plant
growth. However, a decrease of nutrients, often occurring in excess of the quantitative
requirement, should not per se imply growth disturbances or growth decrease. It is
therefore reasonable to assume that a decrease in internal Ca and Mg concentrations
is not quantitatively large enough to influence growth at aluminum concentrations,
which are ecologically relevant in the rhizosphere of acid boreal forest soils. How-
ever, plant growth and nutrition may be further affected by aluminum because of its
antagonistic effect on nitrate65 or base cation uptake13,66 when these are close to
being, or are, growth limiting, but also because the pattern of biomass partitioning
to roots may change, especially at K- and Mg-limitation.29,30 Calcium not only acts
as an antagonist to aluminum, but also to magnesium and potassium.3,67 Such antag-
onistic effects, which may be caused by liming, were investigated by adding Mg-
or Ca- at a submaximal RA of 0.15 day1 and by keeping a concentration of Ca2+ in
the Mg-limited treatments at 0.01, 0.1, or 1 mM.40 Magnesium was given in equal
molarities in the Ca-limited treatments, and Al was added to a concentration of 0.5
220 Trace Elements in the Rhizosphere

mM in all treatments. The RM of the plants was close to RMg or RCa and was not
affected by the concentration of Mg2+, Ca2+, or Al3+ in the solution. Uptake of Ca,
at Ca-limitation, increased significantly in the roots after addition of Al, and
decreased in all plant parts when the Mg concentration of the culture solution was
raised. Fewer clear effects were found on Mg uptake at Mg-limitation. Thus it seems
as if Al interferes with the uptake of Ca and Mg in excess of growth requirements
and to a much lesser extent with the uptake of these elements when they are in low
supply. Van Oene46 developed a mechanistic model for the study of interactions
between Ca, Mg, and Al. The model is based on surface complexation of the
membrane with both specific adsorption reactions and exchange reactions included.
The behavior of the model agreed with reported inhibiting effects of Al, and showed
that effects of Al might be reversed by increased nutrient solution concentrations of
cations. However, antagonism between Ca and Mg is a well-known phenomenon in
plants3 and was clearly seen in this investigation.
The balance between Ca and Al in the root substrate is regarded as playing an
important part in the growth of trees.60,63,68 A similar approach has been advocated
by Sverdrup et al.69,70 who used molar ratios of (Ca + Mg + K/Al) as a diagnostic
tool when examining literature data about growth responses to Al. Sverdrup et al.
proposed that a molar ratio above 1 is required for normal growth of several tree
species. Such ratios do not offer a deeper physiological understanding of the
observed nutrient imbalances in forest trees. The molar ratios in the experiments
were all far below the critical value of 1 (often 0.1) in all investigations performed
with the steady-state approach, except in the highest Ca treatment in the Mg-limited
experiment.13,40,43-44 Consequently, Al-induced inhibition of cation uptake cannot
simply be explained from the balance between these elements in the soil solution.
According to Van Oene,46 the ratio is rather an expression for a mixture of pH and
Ca effects.

IV. THE ROOT SUBSTRATEROOT INTERFACE


The rhizosphere conditions differ in many ways from those more distant from the
root. Although the chemical properties of soils are important for mineral nutrient
availability and plant growth, the rhizosphere conditions and the extent to which
roots can change them are decisive for mineral nutrient uptake. Commonly, it is held
that roots act as a sink for mineral nutrients transported to the root surface by mass
flow and diffusion. The contribution of nutrient transport by mass flow to the roots
compared with nutrient uptake during the growing season varies greatly, and depends
on soil type, nutrient element, and plant species.3 In contrast to mass flow, diffusion
is regarded as an important factor of ion movement in the close vicinity of the root
surface, and is thus linked with plant properties such as root growth and correspond-
ing surface area. Among the first to emphasize the importance of root growth was
Barber.71 Clarkson72 reviewed factors affecting mineral nutrient acquisition by plants,
and concluded that the P-uptake, at moderate limitation, was correlated with the rate
of root elongation. The same correlation, but for all levels of limitation, has been
found for other mineral nutrient elements by using the concept of relative addition
rate as the driving growth variable. However, the relative root mass (and root
Quantitative Trace Element and Micronutrient Studies in Plants 221

elongation) increase for N, P, S, and Fe but decrease for K, Mn, and Cu.10,24,28,29,31,32,73
Similar results have been found with respect to Cu-limitation in Hordelymus,20 but
with no control of the stability of biomass partitioning.
The nature of a hydro- or aeroponic culture differs widely from a soil culture.
Nevertheless, the regulation of nutrient intake in the roots is the same regardless of
rooting substrate. The use of hydro- or aeroponic systems may thus, because of their
relative simplicity, be advantageous for the study of root morphology or nutrient
uptake. As the nutrients are delivered to the roots, the plant response may be
interpreted as an inherent plant property. By controlling the RA of nutrient elements,
plant characters such as biomass partitioning to the roots may be controlled and kept
stable during an experiment. Plant responses, which are treatment specific and thus
not time dependent, may then be studied more thoroughly. In experiments with the
micronutrient elements Cu, Fe, Mn, and Zn, biomass partitioning to the roots was
found to be highly dependent on the chosen value of RA and the micronutrient
element in question.24,25,31,32 The relative proportion of root biomass was lower at
severe Cu-, Mn- and Zn-limitation than at moderate or no limitation, and the ability
of plant roots to penetrate a new soil volume becomes less evident at severe Cu-,
Mn-, and Zn-limitation. By cultivating the birch plants at steady-state nutrition,
it was possible to calculate uptake rates of the micronutrient elements with respect
to the growth rates of the roots.24,25,31,32 The range in dn/dMr [Equation (4)] (excluding
FA) was much less than the range of plant RM, which implies that partitioning to
roots, or changes in root RM, and not necessarily changes in specific uptake rate, are
important components for the further acquisition of these nutrient elements. Under
field conditions, root growth can be one of the most important factors in acquisition
of nutrients,72 and apparently there is no evidence that growth response at Cu-, Mn-,
or Zn-limitation will result in a relatively improved acquisition of these elements,
because the partitioning pattern does not favor root growth. It is evident, from the
examples given above, that the steady-state technique may be a useful tool to model
and predict processes in the rhizosphere under a variety of different environmental
conditions. Studies of the rhizosphere are important for the understanding and
modeling of nutrient and trace element acquisition in forest ecosystems. Because of
the use of bulk soil chemistry and culture solution input data in computer models,
it has been questioned whether they predict the nutritional conditions of the soil
surrounding the root.47,74,75 Mass flow and diffusion are often assumed to be the most
important ion transport processes in supplying roots with nutrients,76,77 although the
magnitude of these are difficult to quantify and vary in different ecosystems.78 Rapid
plant growth can deplete nutrients from the rhizosphere, particularly if additional
nutrient supply is mediated by weathering.79 It may therefore be argued that these
variables in controlling P and K availability have been overemphasized and that
other processes, such as nutrient flux density, control nutrient availability in the
rhizosphere.47,80 Ingestad37 and coworkers12,22,24,25,28-30,31,32 showed that it was possible
to grow plants at stable and high growth rates, and at extremely low external nutrient
concentrations, by using the steady-state technique as a tool to mimic rhizospheric
inorganic nutrient fluxes. Thus, the nutrient acquisition of the plants is not depending
on diffusion and mass flow at the root surface, but on nutrient fluxes and root growth
rate. The fertility of a soil depends highly on weathering and mineralization of
222 Trace Elements in the Rhizosphere

nutrient elements. Consequently, by using the technique as a tool to mimic such


fluxes, both in the laboratory and in the field, it has been possible to incorporate
functional aspects of the rhizospheric environment into the ecosystem investigated.

V. CONCLUDING REMARKS
It has been demonstrated that steady-state nutrition can be achieved where the supply
of a micronutrient is regulated in an appropriate manner. In such studies, it is essential
that contamination by micronutrient impurities is avoided, and appropriate measures
for achieving this have been outlined. Over the range of supply for which either Cu,
Fe, Mn, or Zn was limiting, plant growth rate was positively correlated with the
amount of limiting nutrient in the plant. Because the plants were grown at steady-
state nutrition, it was possible to discuss growth response in terms of structural and
functional aspects of root and shoot growth, which may be considered to characterize
the response to micronutrient limitation.
It may be concluded from the small, direct effects of aluminum concentration
on tree seedling growth rate, that this variable (at the concentrations normally
encountered in the fine-root zone) is unlikely to account for the extent of forest
dieback that has been observed on many sites. The measured effects of aluminum
concentration on the uptake of calcium and magnesium did not result in growth-
limiting proportions of these nutrients in plant tissues. However, the proportion of
magnesium was approaching a critical level in spruce.
The findings have illustrated the feasibility and usefulness of the steady-state
approach in plant nutrition studies of potentially toxic substances and growth
response to micronutrients. As the steady-state technique has been used as a tool to
mimic rhizospheric inorganic nutrient fluxes, it has been found that nutrient acqui-
sition by plants is not dependent on diffusion and mass flow at the root surface, but
on nutrient fluxes and root growth rate.

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11 Cation Exchange on
Plant Roots Involving
Aluminium:
Experimental Data
and Modeling
Joseph E. Dufey, Jos G. Genon,
Benot Jaillard, Henri Calba, Gervais Rufyikiri,
and Bruno Delvaux

CONTENTS

I. Introduction...................................................................................................228
II. Origin, Measurement, Variability, and Implications of the
Cation Exchange Capacity of Roots (CECR)..............................................228
A. Origin ................................................................................................228
B. Measurement.....................................................................................229
C. Variability..........................................................................................229
D. Implications ......................................................................................230
III. Aluminium Exchange on Roots ...................................................................231
IV. Modeling Cation Exchange on Roots ..........................................................235
A. Review of Existing Models ..............................................................235
B. Description of the Model .................................................................237
1. Chemical Speciation ..........................................................237
2. Ion Exchange .....................................................................238
a. General Equation for Ion Exchange................239
b. The Donnan Volume ........................................241
C. Estimation of the Model Parameters and Simulations ....................242
1. Calcium-Aluminium Exchange for Clover and
Ryegrass Roots ..................................................................242
2. Cation Exchange Data for Banana and Maize
Roots ..................................................................................246
V. Conclusion ....................................................................................................247
Acknowledgments..................................................................................................247
References..............................................................................................................248

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 227
228 Trace Elements in the Rhizosphere

I. INTRODUCTION

The uptake of major and trace elements by plants is regulated both by active and
passive mechanisms at the soilroot interface. Among the active mechanisms, the
release of protons and organic substances in the rhizosphere is extensively studied
at present as they play key roles in the mobilization of nutrients and toxic elements.
A state-of-the-art paper on these processes was recently published by Hinsinger.1
These active mechanisms take place in a physicochemical environment, which
depends to a large extent on ion exchange reactions between soil matrix, solution
phase, and root. The interface between the living part of root cells (the cytosol
delimited by the plasmalemma) and the soil solution is made up of a macroreticulated
network of cell walls, which reduces solute mobility by both mechanical friction
and electrostatic interactions. The composition of the solution close to the plasma-
lemma and the functioning of proteins and enzymes included in the cell walls are
deeply influenced by ion exchange processes.
The aim of this chapter is to briefly recall the origin, sources of variability, and
practical implications of root exchange capacity, to present results of exchange
experiments involving aluminium as a model trace element, and to show how a
mathematical model can be used to simulate ion fixation on root exchange sites.

II. ORIGIN, MEASUREMENT, VARIABILITY, AND


IMPLICATIONS OF THE CATION EXCHANGE CAPACITY
OF ROOTS (CECR)

A. ORIGIN
Although exchange equilibria evidently take place inside any plant cell, the
exchange sites that can react directly with the soil solution are essentially located
in the apoplast. Carboxylic groups of uronic acids from pectin and hemicellulose
in cell walls, and proteins to a lower extent, are mostly responsible for the exchange
properties of roots.2,3 The accessibility of the exchange sites for cations from the
soil solution can be limited to the cortex when the endodermis is suberized;
exchangeable trace cations are sometimes found only in peripherical cell layers,4,5
likely because thermodynamic equilibrium throughout the apoplast is slow to
become established when the concentrations in solution of high affinity cations
are low. Also direct observation of element localization in roots (e.g., using
microprobes) does not distinguish between exchangeable cations and other forms
of accumulation.
Phospholipid head groups from plasma membranes, which are in close contact
with cell walls, and membrane proteins also exhibit cation exchange properties that
influence transport processes from apoplast to symplast,6,7 but their contribution to
total root exchange capacity is expected to be low when considering the relative
weights of plasma membranes and cell walls. Ishikawa and Wagatsuma8 reported a
strong positive correlation between CECR (i.e., including membranes) and the cation
exchange capacity of cell walls among four plant species.
Cation Exchange on Plant Roots Involving Aluminium 229

TABLE 11.1
Comparison of CECR Values (cmolc kg1) Measured by
Different Methods on Clover and Ryegrass Roots
Titration in the Presence of
Cu Fixation Ca + Mg + Na Na Mg Ca

Ryegrass 24.3 24.8 20.0 23.5 25.0


Clover 42.4 42.6 37.0 45.5 47.5

B. MEASUREMENT
The cation exchange capacity of roots can be measured in different ways, which
can influence the results and consequently the interpretation of data. Basically, as
CECR mainly originates from carboxylic groups, parameters such as pH, reference
cation, and ionic strength, used in the measurement procedures should be controlled
and mentioned. A method based on Cu adsorption followed by acid extraction was
proposed as a reference procedure by Dufey and Braun.9 Because copper is a trace
element in roots, Cu content after extensive leaching with CuSO4 can be attributed
to Cu binding with exchange sites. In addition, carboxylic groups have a very high
affinity for Cu ions, so that most native exchangeable cations are easily displaced
by CuSO4 leaching. Table 11.1 compares CECR values estimated by Cu fixation to
the sum of base cations displaced by Cu (clover and ryegrass roots were first
equilibrated with mixed CaCl2-MgCl2-NaCl solutions). Titration of acidified roots
can also provide CECR values; the data reported in Table 11.1 were obtained by
titrating protonated roots with NaOH in the presence of 0.1M CaCl2, MgCl2, or NaCl
up to pH 6, i.e., a pH value at which all carboxylic groups should be deprotonated.
These data support the validity of the Cu method for CECR measurements. This
method was recently used by Pintro et al.10 and Calba et al.11 to speciate Al forms
in maize roots.
Strontium or Ba were used as extracting cations by other authors;12-14 like Cu,
they have high affinity for root exchange sites, so they can leach the native exchange-
able cations.

C. VARIABILITY
As the uronic acid content in roots is first related to the plant species, CECR also
depends first on plant species and even on varieties or cultivars of the same species.
Wacquant15 listed CECR for a few hundred herbaceous species: monocots usually
have low CECR in the range 10 to 20 cmolc kg1 (dry matter basis), whereas most
dicots have CECR in the range 20 to 50 cmolc kg1. Experiments on forest tree
rootlets showed that coniferous trees had higher CECR than deciduous trees;16 CECR
values (titration method, pH 6) of 13.5 and 14.0 cmolc kg1 were found for beech
and oak, respectively, whereas CECR of 18.0, 20.5, 23.0, 23.5, and 26.0 cmolc kg1
were found for larch, nobel fir, pine, spruce, and douglas fir, respectively.
230 Trace Elements in the Rhizosphere

cmolc kg-1

FIGURE 11.1 CECR of pineapple roots as a function of distance from apex.

The age of roots or root parts also influences CECR.17-18 Indeed, secondary cell
walls very poor in pectin are formed in older root parts so that the increase of dry
weight is not accompanied by a proportional increase of exchange sites. For pine-
apple roots collected in the field or grown in nutrient solution (Figure 11.1, data
from F. Ikans thesis, UCL Louvain-la-Neuve, not published), we observed CECR
values ranging from 25 cmolc kg1 near the apex down to less than 10 cmolc kg1 at
20 cm from the apex.
We also noticed that CECR changes according to root order. For example, in
banana roots, we measured CECR 23 cmolc kg1 for main roots and CECR 34
cmolc kg1 for lateral roots (mean values of five cultivars, data not published).
The nutritional status of roots or the presence of toxic elements were also
reported to influence CECR. This might result from changing the average age of
the root system. Indeed toxic elements inhibit root growth and, therefore, increase
the average root age, whereas adequate nutrient supply results in healthy root systems
with well-developed young roots.

D. IMPLICATIONS
Many authors attempted to find relationships between CECR and various plant
characteristics such as adaptation to different stresses (salinity, Al toxicity, low or
high soil pH, etc.). These studies are not easy to generalize, because they deal almost
always with very limited series of plant species or plant varieties. For example, it
seems that halophytes have high CECR, which can keep enough Ca on cell walls
in Na rich soils.19 Woodward et al.20 observed that in natural plant communities, the
CECR of monocots was correlated to the cation exchange capacity of soil. According
to the authors, this might result from a competitive adaptation for the uptake of
Cation Exchange on Plant Roots Involving Aluminium 231

cation nutrients. Plants with low CECR should be handicapped for cation uptake in
soils with high CEC. Solubilization of sparingly soluble phosphorus (AlPO4, FePO4)
was hypothesized by Ae et al.21 to be related to the chelating ability of pectic
substances, i.e., to CECR; however, by comparing different plant species, these
authors demonstrated that the relationship between CECR and phosphorus solubi-
lization was not straightforward.
Tolerance to aluminium was tentatively related by many authors to CECR. Plants
with high CECR should be more sensitive to Al, because Al has high affinity for
exchange sites and impairs physical properties of roots.3,12,22 However, other mech-
anisms of Al toxicity, e.g., alteration of plasma membrane properties can also take
place,8,23-25 so that the relationship between CECR and Al tolerance is not obvious.26,27
Nevertheless, in a series of five banana cultivars (recent data, not published), we
observed that the most Al-sensitive one had CECR 30 cmolc kg1 compared to
CECR 19 to 23 cmolc kg1 for the other four cultivars. Recent studies on maize
showed that the CECR values of Al-sensitive and Al-tolerant cultivars were not
significantly different, although the total amounts of Al accumulated in roots were
greater for Al-sensitive than for Al-tolerant cultivars.11,28

III. ALUMINIUM EXCHANGE ON ROOTS


Alumino-silicates dominate the constitution of most soils so that aluminium is a
major element in the root environment. However, the solubility of alumino-silicates
is low and decreases with increasing pH, so that the ionic Al concentration in soil
solutions is negligible for pH greater than about 5. In acid soils, assuming that Al
activity should be controlled by model minerals such as gibbsite or kaolinite, one
can calculate that micromolar monomeric Al3+ concentration should be reached at
pH about 4.5, and increases 1000-fold when pH drops by one unit.29 Many mea-
surements of Al concentration in acid soil solutions are reported in the literature,
however, the question that always remains is what chemical speciation of Al has
formed, which can be answered either through analytical techniques or through
calculation. The lack of reliable methods for determining Al species in soil solutions
makes it difficult to assess the phytotoxicity of different Al forms. Monomeric as
well as polymeric Al (Al13) were often reported to be more toxic for plants than
inorganic ion pairs and, more evidently, than organic Al-complexes. Assessing actual
Al concentrations in soil solution is also a challenging task, because of the kinetic
aspects of the regulating processes that involve a number of solid phases.30 The
chemical changes induced by roots in the rhizosphere (ion depletion, release of
protons and organic acids) also have obvious effects on the concentration and forms
of Al at the soilroot interface. In any case, Al can be regarded as a trace element
in soil solutions except at very low pH in mineral soils. Therefore, the experimental
results presented below and the mathematical model developed in the next section
can be considered as a reference research approach for other trace elements that
have increasing solubility with decreasing pH.
Measurements were performed on roots equilibrated either with solutions com-
ing from acid soils of different types and different pH or from synthetic solutions.16
It was confirmed that the Al quantities adsorbed on roots are first related to CECR,
232 Trace Elements in the Rhizosphere

Exchangeable Al, cmolc kg-1

FIGURE 11.2 Relationships between exchangeable Al on roots equilibrated with soil at pH


4 and CECR values at pH 4 and 6, with regression lines forced through the origin (plant
species from top: clover, douglas fir, spruce, ryegrass, nobel fire, larch, beech, oak).

and consequently to plant species. Figure 11.2 shows results obtained on roots
equilibrated in an acid soil suspension prepared by repeated washing of a mineral
soil sample with CaCl2-HCl at pH 4. Exchangeable Al expressed in moles of charge
(extracted using CuSO4) is higher than the CECR values at pH 4 (measured by
titration in 0.1 M CaCl2) and close to the CECR values measured at pH 6. At least
three reasons can explain this observation: (1) the affinity of Al for exchange sites
is higher than the affinity of Ca, and consequently the fixation of Al induces more
deprotonation of carboxylic groups than the fixation of Ca; (2) Al might be fixed
on exchange sites not only as monomeric Al3+, but also as less charged ionic forms
such as Al(OH)2+, Al(OH)2+; (3) Al extracted by CuSO4 might come not only from
exchange sites, but also from other forms such as Al-precipitates.11,31
Other experiments were done by equilibrating roots with solutions from differ-
ent soil horizons and different soil types. Furthermore, measurements of root
exchangeable cations were carried out after adding acid or lime to soil samples.
Figure 11.3 shows typical results obtained for three horizons of a forest acid soil
from the Belgian Ardens.
The fraction of sites occupied by Al depends on the activity and on the fraction
of free Al in solution. Roots equilibrated with solutions coming from organic soil
horizons do not fix much Al even at low pH. The reason is that Al activity in solutions
from organic horizons is not controlled by Al-bearing minerals, but by exchange
and chelating processes with organic compounds. In such conditions, the root
exchange sites compete with the exchange sites of organic matter for available
Al, and the solution is not replenished with Al coming from mineral dissolution as
Cation Exchange on Plant Roots Involving Aluminium 233

cmolc kg-1
cmolc kg-1
cmolc kg-1

FIGURE 11.3 Exchangeable cations of roots equilibrated with soil solutions from three
horizons (O, A, B, from top to bottom) of a forest acid soil (Vielsalm, Belgium).

it occurs in deeper mineral horizons. Indeed, in B horizons, Al activity in solution


is not controlled by root fixation, because alumino-silicates can be considered as an
infinite Al source that replenishes the solution according to pH, silicate activity, etc.
When lime is added, pH and Ca activity increase, and Al activity decreases, so that
Ca becomes the dominant cation on root exchange sites.
Another experiment was recently conducted on bananas (data not published) in
order to compare cation loading of root exchange sites from plants grown in nutrient
solution with Al and from plants cultivated in fields. Figure 11.4 shows mean values
of Al, Ca, and Mg extracted with CuSO4 for five banana cultivars. The nutrient
234 Trace Elements in the Rhizosphere

cmolc kg-1

FIGURE 11.4 Exchangeable cations on banana roots in nutrient solution and in two soils
from Cameroon.

solution supplied by a continuous flow technique contained 78.5 M Al, 1 mM Ca,


and 0.1 mM Mg; pH at input was 4.2. Banana root samples were collected in soils
from Cameroon, an Andosol with pH-water around 6, and an acid soil with pH
around 4.3. To some extent, the composition of root exchange sites can be considered
as a probe for the chemical environment. At a given pH, the total loading of exchange
sites depends on ionic strength and on the type of cations. As the nutrient solution
and the acid soil had similar pH values, one can guess that the nutrient solution was
more concentrated than the acid soil solution, and that our nutrient solution was
relatively poor in Mg. Very low Mg content in plant tissues and leaf toxicity/defi-
ciency symptoms corroborated this observation on root exchange sites.32 On the
other hand, as the Andosol had higher pH, deprotonation of root exchange sites was
more complete. Nevertheless, the exchangeable Al loading of roots from this soil
was not negligible; the reason is likely that nutrient uptake resulted in proton release
and consequent Al solubilization in the rhizosphere even at bulk soil pH 6 (no
exchangeable Al was measured in this soil). Bananas were demonstrated to have a
high capacity of releasing protons due to their high cation requirement,32 and allo-
phanes in Andosols are important potential sources of Al. Exchangeable Mg was
especially high in roots from the Andosol, which may be related to the exchangeable
Mg content in soils; values of 2.0 and 0.8 cmolc kg1 were measured for the Andosol
and the acid soil, respectively.
Calba et al.11 recently reported Al exchange experiments on fresh maize roots
and on isolated root cell walls at different pH values ranging from 4 to 4.8. As in
Figure 11.5, at fixed Al concentration in solution (15 M), a maximum of Al fixation
Cation Exchange on Plant Roots Involving Aluminium 235

Exchangeable Al, cmolc kg-1

FIGURE 11.5 Exchangeable Al on maize roots and isolated cell walls as a function of pH.

(as assessed by CuSO4 extraction) was observed at pH 4.4. Below that value, Al
fixation decreased due to H-Al competition. This behavior is valid only for fixed Al
concentration in solution, keeping in mind that in mineral soils a decrease in pH is
normally accompanied by an increase in Al3+ activity.
Exchange experiments were also conducted in well-controlled synthetic solu-
tions in order to produce reference data that can be used to calibrate models. Roots
of clover and ryegrass were equilibrated with solutions at 2 Ca concentrations (0.5
and 5 mM), 2 pH values (3.5 and 4.0), and 4 Al concentrations (0, 25, 50, 100 M).
The experimental data were reported by Dufey et al.,16 and a comparison with
simulated values is presented in the next section of this chapter. Another series of
data was recently produced with banana roots (not published). Root equilibration
was carried out at 3 Ca concentrations (0.2, 1, and 5 mM), 3 Mg concentrations
(0.02, 0.1, and 0.5 mM), 3 K concentrations (0.2, 1, and 5 mM), 5 Al concentrations
(0, 10, 25, 50, and 100 M), and at 2 pHs (3.75 and 4.25). The model parameters
obtained from this very extended range of solution composition are given below.

IV. MODELING CATION EXCHANGE ON ROOTS


A. REVIEW OF EXISTING MODELS
Ion exchange has been one of the most widely treated subjects in soil chemistry.
Modeling exchange processes can rely on well-established fundamental concepts,
although application of models in true soil conditions is still a challenging task,
236 Trace Elements in the Rhizosphere

because of the variety of exchangers among soil constituents (including roots) and
because of the multiplicity of processes that influence soil solution composition.
Two approaches can be distinguished when dealing with ion exchange models: either
a macroscopic approach formally expressed as mass-action laws, or a molecular
approach expressed by double diffuse layer formulations.
The first mathematical formulation as mass-action law was issued in 1913 and
developed by Kerr in 1928.33,34 Simultaneously, Vanselow35 and Gapon,36 and later
Gaines and Thomas,37 proposed various expressions of the exchange constant for
binary cation exchange. If such thermodynamic treatment of cation exchange has
proven helpful in interpreting the exchange processes,38-40 it has unfortunately no
general predictive power. The cause for this is the lack of a general formulation for
the surface activity coefficients like the Debye-Hckel or Davies formulae for solu-
tion activity coefficients.34,41
Up to the beginning of the 1980s, few applications were made beyond binary
exchange.42-50 In many studies, multication exchange is treated as simultaneous
binary exchange,51-53 and most of the time, the studies were limited to model com-
pounds like smectitic clays or zeolites, but not often extended to soils.
Many authors were in search of constants for characterizing cation
exchange.32,34,54,55 But it was soon recognized that the exchange constants generally
vary with ionic strength and pH, and with the composition of the surface
phase.34,36,37,49,50,56,57 Indeed even a given clay mineral may have distinct exchange
sites and cannot be considered as a homogeneous exchanger.38,58-59 A fortiori a soil
system is a more complex mixture of various exchangers that show a different affinity
for ions.36,60-63 Furthermore, the charge itself of a series of exchangers varies with
pH, edge sites of clay minerals, oxihydroxides of Al, Fe, etc., and organic exchangers,
including root cell walls. The mass-action law alone is not sufficient to describe the
behavior of such variably charged materials.
Cation exchange may also be described in terms of statistical mechanics as
Helmholtz and Gou and Chapman did with the diffuse double layer (DDL) model.
A Stern layer may be added to the DDL model for taking into account the real size
of ions and possible specific interactions with the exchanger surface. The DDL
approach as been widely developed and applied to clay minerals,64-67 and also to
biomembranes.23,68-70 In particular, Kinraide et al. (1992) clearly demonstrated that
phytotoxicity is not related to concentrations, but to ion activity close to the plasma
membranes computed according to the DDL model.23
When dealing with exchangers such as soil organic matter or roots, a major
limitation of this model resides in the fact that DDL equations are derived for well-
defined geometry; planar surfaces received more attention than cylindrical or spher-
ical geometry, because the reference exchangers in soils have been clay minerals in
most studies. Exchange sites in roots are not confined in a simple geometrical frame,
a difficulty that is avoided by Donnan-type models. Indeed, the Donnan theory
considers mean electrostatic effects in a phase or a compartment that includes a
charged substance that cannot diffuse throughout the system due to the existence of
a barrier, be it physical (semipermeable membrane) or energetic (charged surface).
Furthermore Donnan-based models, like DDL models, can include, in addition to
electrostatic effects, specific interactions between the fixed charge and the counte-
Cation Exchange on Plant Roots Involving Aluminium 237

rions. These interactions can be thermodynamically described by intrinsic dissoci-


ation constants between the ions specifically bound to the exchanger, and the ions
of the Donnan phase, the activity of which depends on the Donnan potential and on
the activities in the ions in free solution.
With that approach, Miyajima et al.71,72 have described heavy metal (Ag, Zn,
Cu) adsorption on linear and planar organic polymers. The intrinsic constants/Don-
nan phase approach was also developed by Sentenac and Grignon73 for describing
Ca-Mg-K-H relationships in root cell walls. Models including the same principle
were used by Kinniburgh et al.,74 and Benedetti et al.75 for describing proton and
metal ion binding by humic and fulvic materials, and by Plette et al.76 for studying
the pH dependent charging behavior of bacterial cell walls. Tipping and Hurley,77
Tipping and Woof,78,79 Tipping,80 and Tipping et al.81,82 have applied similar concepts
in the CHAOS and WHAM models for describing cation (H, Al, Ca, Mg, K, Eu,
and actinides, Cu) interactions with soluble organic anions or humic materials.
Marinsky83 published a review on that subject.
In all these studies, the intrinsic dissociation constants proved to be largely
independent of experimental conditions such as pH or ionic strength. As such, this
approach appears promising, because it could allow for the description of ion
exchange based on properties of the system components, and not on experimental
conditions. Therefore, it might prove adequate for describing ion exchange on roots.
We describe our model below in detail, which is fundamentally an extension of the
Sentenac and Grignon73 model.

B. DESCRIPTION OF THE MODEL


The exchange procedure as described by Sentenac and Grignon73 has been coupled
with a chemical speciation procedure; an additional submodel describes the Donnan
volume as a function of the ionic strength and electrical potential. The formation of
charged surface complexes has been taken into account, as well as equilibration with
more than one exchanger with the same bulk solution, which is always the case in
soilroot systems.

1. Chemical Speciation

A chemical speciation involves a detailed computation of all chemical species in a


solution. These consist of free ions (K+, Ca++, NO3-, SO4=, etc.) and ion pairs or
complexes (CaNO3+, CaSO40, organo-metallics, etc.), so that the total concentration
of an element does not always correctly represent its actual form in the solution.
The speciation must be taken into account when dealing with ion exchange. Indeed
some species of a given element do adsorb whereas others do not on charged surfaces,
be they clay minerals,59,84 soils,85 dissolved organics,63 or roots.16 Whatever the
answer to that question, in the present model we compute the chemical speciation
of the bulk solution prior to equilibrating with the exchanger phase.
From a mathematical point of view, a solution may be described by a system
of chemical equilibrium equations between free ions and complexes, and of mass-
balance equations for each element. A chemical speciation program solves such a
238 Trace Elements in the Rhizosphere

system of equations. In this chapter we will not develop the equations used for
solving the chemical speciation, because this part of the model is well covered in
other papers.86

2. Ion Exchange

Without loss of generality, we shall consider a cation exchanger in the following


development, knowing that a similar approach can be used for anion exchangers.
The Donnan phase consists of the exchanger surface and the surface-bound cations,
and the Donnan free space (Figure 11.6). Part of the exchanger charge is compensated
by various cations bound by covalence or by any other specific interaction. An
electrical potential then develops around the residual charge (i.e., the surface charge
not compensated by tightly bound cations), and ion distribution in this surface-
unbound phase (or Donnan free space) is regulated according to the Maxwell-
Boltzmann equation. It should be noted that the Donnan phase is treated as a solution.

FIGURE 11.6 Diagram of the ion exchange model.


Cation Exchange on Plant Roots Involving Aluminium 239

Thus exchangeable ions, either bound or in the free space, are not expressed on the
basis of exchanger weight as molc kg1, but on the basis of the Donnan phase volume
as molc dm3.
a. General Equation for Ion Exchange
Definitions:

R one mole-charge of dissociated exchanger,


X.i species i (if required, C.i if a cation, A.i if an anion),
D the mean electrical potential in the Donnan phase (volt),
zi the charge of ion i,
Rzi-Xi a surface bound species,
F the Faraday constant (C molc1),
R the ideal gas constant (J K1 mol1),
T the temperature (K),
() denotes ion activity (dimensionless),
[] denotes ion concentration (mol dm3).

The subscript D refers to the Donnan free space, whereas no subscript refers to
the bulk solution, i.e., the solution that remains unaffected by the electrical potential.
The relationship between the activity of any ion in the Donnan free space, (XDi),
and its activity in the bulk solution, (Xi), is given by the Maxwell-Boltzmann
equation:

z F
( X Di ) = ( X i ) exp i D (1)
RT

or, for the sake of simplification, if we define a new variable, r, function of the
Donnan potential D as follows

F D
r = exp (2)
RT

then the Maxwell-Boltzmann equation becomes

( X Di ) = ( X i )r Z i (3)

The relationship between surface-bound ions and the Donnan free space is
expressed by dissociation reactions between the cations and multivalent exchange
sites, R(zi), of the same valence as the cations considered:

R zi X i R (zzi i ) + X D
240 Trace Elements in the Rhizosphere

All the exchange sites are supposed to be able to cluster, with the result that the
concentration in such multivalent sites is equal to the CECR divided by cation
valence. Therefore, the concentration in dissociated multivalent groups is calculated
as follows:

[R ]
[R (zzi i ) ] = (4)
zi

so that the intrinsic dissociation constant can be written:

[R ]( X Di )
K dint,i = (5)
z i [ R zi X i ]

Equations (3) and (5) are the basic equations of the Sentenac and Grignon73
model for cell walls.
Equation (5) implies that only neutral surface complexes are formed. Here we
introduce an extension to the original model. Charged surface complexes occur when
all the charges of a multivalent ion do not bind to an equivalent number of reactive
groups. This has been reported by Baes and Bloom87 for peat, and by Miyajima et
al.71 in linear and cross-linked polymers. Tipping63 also considers monodentate
complexes with polyvalent cations, and bidentate complexes with trivalent cations.
The formation of charged surface complexes is illustrated by the following reaction
with aluminium, where only two of the three positive charges are bound to negatively
charged surface groups:

R 2 + Al 3+ {R = Al}

Let li be the number of negatively charged surface groups R bound to a cation,


with 1 li zi. (e.g., in the above reaction, zi = 3 and li = 2). The general expression
for the intrinsic dissociation constant then becomes

[R ]( X Di )
K dint,i = (6)
l i [R li X i ]

The mass balance of exchange sites is expressed by

Cations

[R]Tot = [R ] + l [R i li Xi ] (7)

The charge balance of the Donnan phase requires


Cation Exchange on Plant Roots Involving Aluminium 241

Anions

[R ] + | z | [A i Di ]=

(8)
Cations Cations

(z l )[R
zi li
i i li Xi ] + z [C i Di ]

By grouping the surface charges on the left-hand side and the charges in the Donnan
solution on the right-hand side, Equation (8) may be condensed to

Cations Ions

[R ] (z l )[R
zi li
i i li Xi ] = z [X i Di ] (9)

Combining Equations (6) and (9), factoring by [R], and introducing the expression
of [R] in Equation (7) results in

Cations


(C Di )
1 +
Ions K dint,i
[R]Tot =
z i [X Di ] Cations

(10)

1 (C )
(z i l i ) Did
l i K int,i
zi li

b. The Donnan Volume


As the Donnan phase is treated as a solution, the Donnan volume VD of the exchanger
is an important feature of the model. The Donnan volume is a characteristic of the
exchanger, and in some cases it may be deduced from the structural properties of
the exchanger materials.73 Benedetti et al.75 observed that the Donnan volume of
humic and fulvic acid gels increased with decreasing ionic strength. In a study of
metal binding to humic acids, Kinniburgh et al.74 proposed an empirical formulation
of VD depending on ionic strength only and based on data from Benedetti et al.,75
for humic acids:

log VD = b (1 log I) 1 (11)

where I is the ionic strength and b 0.43 for humic acid. In their study of bacterial
cell walls, Plette et al.76 also used an inverse relationship between the Donnan volume
and ionic strength and found good agreement with other experimental data. The
most elaborate model for the Donnan volume is probably found in the WHAM
computer code for fulvic and humic acid particles.63 Briefly, the Donnan volume,
dependent on ionic strength, is computed from a spherical geometry with Debyes
length as radius. The volume is then restricted by a Michaelis-Menten-type function
of the dissociated charge of the organic particle. Only tentative values of the param-
eters have been obtained up to now.
242 Trace Elements in the Rhizosphere

In the present work, the Donnan volume was estimated for each set of data as
outlined below. Typical values reported for the Donnan volume of ion exchangers
are mentioned in Section C.1 and compared with the values found in our own
experiments.

C. ESTIMATION OF THE MODEL PARAMETERS AND SIMULATIONS


The parameters of our model were estimated with the nonlinear least square fitting
procedure NLIN of the statistical package SAS.88 The previously described ion
exchange procedure was translated into SAS language and imbedded into the built-
in NLIN procedure that yields the analysis of variance, the estimates of the model
parameters, their standard error, and 95% confidence interval. We also performed a
sensitivity analysis in order to assess the effect of a variation of the model parameters
on the model response. The sensitivity to a variation of a parameter will be expressed
as the mean square of the deviation from the best-fit response.

1. Calcium-Aluminium Exchange for Clover and Ryegrass Roots


The input dataset taken from Dufey et al.16 consisted of the equilibrium solution
concentrations of H, Ca, Al, and NO3, and of the exchangeable Ca and Al on clover
and ryegrass roots. Exchangeable H was not available from the original data. The
dissociation constants for H+, Ca2+, Al3+, and AlOH2+ were estimated. Various sce-
narios were tested, where either the same89 or different constants were assigned to
each plant species. The root cation exchange capacity (CECR) was also estimated
from the data, given the wide range of values proposed in the literature and their
dependency on experimental conditions. The Donnan volume was estimated for each
combination of plant x (pH, Ca).
Table 11.2 presents the analysis of variance from the NLIN fitting procedure,
with the same intrinsic dissociation constants assigned to both plants. Tables 11.3
and 11.4 show the same results with intrinsic dissociation constants depending on
the plant species. The plant-independent-constant model may be considered as a
restriction of the full plant-dependent-constant model. We may compare the good-
ness of fit of these two models with an extra sum of squares test.90 The results of
the test indicate that the RSS of the full model is significantly smaller (p > 0.99),

TABLE 11.2
Analysis of Variance, Plant-Independent
Dissociation Constants
Source DFa Sum of Squares Mean Squares

Regression 11 10292.81 935.71


Residual 45 53.66 1.19
Total 56 10346.47
a DF: degrees of freedom.
Cation Exchange on Plant Roots Involving Aluminium 243

TABLE 11.3
Analysis of Variance, Plant-Dependent Dissociation Constants
for Clover
Source DFa Sum of Squares Mean Squares

Regression 7 7824.70 1117.81


Residual 21 21.53 1.03
Total 28 7846.23

95% C.I.a
Parameter Estimate Standard Error Lower Upper

log Kdint, H 3.42 0.19 3.82 3.03


log Kdint, Ca 0.84 0.43 1.75 0.06
log Kdint, Al 2.41 0.61 3.67 1.14
log REC 0.25 0.03 0.30 0.19
log VD1b 0.39 0.34 0.31 1.09
log VD2b 0.17 0.38 0.61 0.96
log VD3b 0.73 0.35 1.46 0.00
CECR (cmolc.kg1) 57 50 6
VD1 (cm3 g-1) 2.47 0.49 12.3
VD2 (cm3 g1) 1.49 0.24 9.1
VD3 (cm3 g1) 0.18 0.03 0.9
a DF: degrees of freedom; C.I.: confidence interval.
b 1: pH 4.0 0.5 mM Ca; 2: pH 3.5 0.5 mM Ca; 3: pH 4.0 5 mM Ca.

i.e., that the fit is better with plant-dependent dissociation constants. Figure 11.7
shows the whole set of experimental data and the fitted curves, with plant-dependent
dissociation constants.
The parameter estimates, standard errors, and 95% confidence intervals are given
in Tables 11.3 and 11.4. The estimated intrinsic dissociation constants for H are
close to the 103.36 deprotonation constant of galacturonic acid, a main component
of pectin, used by Sentenac and Grignon,73 and also close to the constants of some
humic acids sites of the WHAM model.63
The intrinsic dissociation constants for Ca are greater than the values estimated
by Sentenac and Grignon73 and used by Amory and Dufey.89 Furthermore, the
constant for ryegrass is greater than for clover. Thus, according to the statistical
estimation, the association between Ca ions and the root exchange sites is looser in
ryegrass than in clover. Indeed Whitehead et al.91 reported a higher proportion of
pectin in clover roots and of hemicellulose in ryegrass. Pectin consists of longer,
more organized chains of uronic acids than hemicellulose, and this may allow easier
and closer association with cations.
For Al, the most coherent results were obtained, for both plant species, with
surface complexes of the form R3-Al0 and no association of AlOH++ with the
exchanger. The intrinsic dissociation constant for Al+++ is 102.41 for clover, with a
244 Trace Elements in the Rhizosphere

TABLE 11.4
Analysis of Variance, Plant-Dependent Dissociation Constants
for Ryegrass
Source DFa Sum of Squares Mean Squares

Regression 7 2488.80 355.54


Residual 21 11.44 0.54
Total 28 2500.24

95% C.I.a
Parameter Estimate Standard Error Lower Upper

log Kdint, H 3.22 0.22 3.67 2.76


log Kdint, Ca 0.14 0.57 1.05 1.32
log Kdint, Al 1.95 0.70 3.40 0.51
log CECR 0.49 0.03 0.55 0.42
log VD1b 0.27 0.39 1.08 0.54
log VD2b 0.35 0.43 1.24 0.54
log VD3b 1.23 0.41 2.07 0.38
CECR (cmolc kg1) 33 28 3
VD1 (cm3 g1) 0.54 0.08 3.4
VD2 (cm3 g1) 0.45 0.06 3.4
VD3 (cm3 g1) 0.06 0.01 0.4
a DF: degrees of freedom; C.I.: confidence interval.
b 1: pH 4.0 0.5 mM Ca; 2: pH 3.5 0.5 mM Ca; 3: pH 4.0 5 mM Ca.

95% confidence interval ranging from 103.67 to 101.14, and 101.95 for ryegrass, with
a 95% confidence interval ranging from 103.40 to 100.51.
The estimated CECR is 57 cmol kg1 for clover and 33 cmol kg1 for ryegrass.
These values are 25 to 60% higher than the CECR measured by Dufey and Braun9
from titration with Na, Ca, and Mg, and from copper adsorption, but they are very
close to the 63 and 31 cmol kg1 observed by Amory and Dufey89 from Ca adsorption
at pH 5.2.
The estimated Donnan volumes are listed in Table 11.3. The volumes are 4 to
5 times smaller for ryegrass than for clover. This is probably related to differences
in the cell wall structure of the plant species. For both plants the volume decreases
with increasing ionic strength. The smaller volume at pH 3.5, Ca 0.5 mM, as
compared to pH 4.0, Ca 0.5 mM, may be attributed to contracted Donnan space
resulting from higher protonation, hence lower electrical potential. A slightly smaller
potential (modulus) is indeed observed at pH 3.5 (not shown). As expected the
volumes are the smallest at the highest ionic strength, i.e., in the Ca 5 mM treatment.
The Donnan volumes estimated for the 0.5 mM Ca treatments (lower ionic
strength) range from 0.5 to 2.5 cm3g1. These values are in agreement with the
Donnan volumes reported by Sentenac and Grignon73 for root cell walls, by Benedetti
et al.75 for humic acid gels, or by Plette et al.76 for bacterial cell walls. Values even
Cation Exchange on Plant Roots Involving Aluminium 245

FIGURE 11.7 Exchangeable Ca and Al on ryegrass and clover roots as a function of Al


concentration in three types of solutions (triangles: pH 4.0, 0.5 mM Ca; circles: pH 3.5, 0.5
mM Ca; squares: pH 4.0, 5 mM Ca). Full lines are best-fit from the model.

as high as the upper confidence limits for clover (9.1 and 12.4 cm3g1) have been
reported by Ritchie and Larkum92 for algal cell walls. Benedetti et al.75 obtained
even higher values for fulvic acid gels, from 10 to 80 cm3g1 at similar ionic strength
ranging from 0.010 to 0.001, but, according to the authors, the highest values might
be suspect. On the contrary, the volumes estimated at the higher ionic strength (5
mM Ca treatments) are very small, but nonetheless close to the 0.15 cm3g1 reported
by Benedetti et al.75 for humic acids, even if the latter was established at a far higher
ionic strength.
Table 11.5 presents the sensitivity of the model response to a variation of 20%
of each parameter. For clover, the sensitivity is the highest for the dissociation constant
for the proton, then for Al, and finally Ca, 4 times lower than that for the proton. The
sensitivity to Donnan volume is of the same order of magnitude as to the dissociation
246 Trace Elements in the Rhizosphere

TABLE 11.5
Sensitivity of the Model Response to
a 20% Variation of the Parameters
Mean Square Difference
Parameter Clover Ryegrass

Kdint, H 6.7 2.0


Kdint, Ca 1.5 0.1
Kdint, Al 3.0 0.8
VD1 (cm3 g1)a 2.2 0.6
VD2 (cm3 g1) 0.9 0.3
VD3 (cm3 g1) 1.3 0.8
CECR 27.00 9
a1: pH 4.0 0.5 mM Ca; 2: pH 3.5 0.5 mM
Ca; 3: pH 4.0 5 mM Ca.

constant of calcium. The dissociation constants for ryegrass rank in the same order
as for clover, but their influence is 3 times smaller for H and Al, and about 15 times
smaller for Ca. The model is rather insensitive to the dissociation constant of Ca for
ryegrass. The sensitivity to Donnan volume for ryegrass is smaller than for clover,
and more comparable to the sensitivity to the dissociation constants. The model is
most sensitive to the root cation exchange capacity, especially for clover. The root
cation exchange capacity is the most easily measured parameter of the model.

2. Cation Exchange Data for Banana and Maize Roots

Estimation of the model parameters was also carried out for banana roots and for
maize root cell walls. The experimental conditions used for the measurements are
described briefly in Section III above. Table 11.6 presents the estimates of the
parameters from our work and from Sentenac and Grignon73 for horse beans and
lupin cell walls. In these studies, good agreement was found with experimental
data using high values of dissociation constants for monovalent ions, which shows
that the complexation of root exchange sites with these ions can be neglected in
modeling. They are confined mostly in the Donnan free phase. As for ryegrass and
clover, the intrinsic dissociation contants of protons found for banana and maize
are close to the value reported for horse beans and lupin, and all these values are
comparable with the deprotonation constant of galacturonic acid. The dissociation
constants estimated for Ca cover 2 orders of magnitude (100 to 102), which can
possibly reflect structural differences in root cell walls of the studied species. As
far as Al is concerned, satisfactory fits were obtained for clover, ryegrass, and
banana assuming only surface complexes with Al3+. However, no definitive conclu-
sion can be drawn concerning the existence of complexes with hydroxy-Al species,
because models are very poor tools for establishing detailed physicochemical mech-
anisms. Indeed, Calba et al.11 found that by considering surface complexes with
Cation Exchange on Plant Roots Involving Aluminium 247

TABLE 11.6
Intrinsic Dissociation Constants Estimated for Roots or Cell
Walls of Different Plant Species (log10 of values)
Horse Bean and Clover Ryegrass Maize Cell Banana
Complexes Lupin Cell Wallsa Rootsb Rootsb Wallsc Rootsb

R-H 3.20 3.42 3.22 3.60 3.25


R2-Ca 1.12 0.84 0.14 2.20 1.12
R3-Al 2.41 1.95 2.85 3.78
R-Al(OH)2 1.85
a Sentenac and Grignon.73
b This chapter.
c Calba et al.11

Al(OH)2+, they were able to improve the agreement of model data with measured
exchangeable Al.

V. CONCLUSION
The data reported in this chapter show that exchange properties of roots must be
considered in studies dealing with cations that have high affinity for root cell walls.
Indeed, drastic changes in the chemical properties of the apoplast can result from
changes in the composition of root exchange sites. It is also shown that a model based
on intrinsic characteristics of rootion interactions can account for this composition
in a wide range of solution compositions. Further data are needed to improve the
reliability of the model parameters, although those available already allow one to
make reasonable simulations. The theoretical approach developed in this model is
immediately applicable to other trace elements without any supplementary algorithmic
work, because the number of ionic species can take any value in the computer code.
Understanding the functioning of the rhizosphere is not an easy task, mainly
because of considerable technical difficulties. However, in acid conditions, the
increasing solubility of Al and of most trace elements strengthens the need for
mechanistic approaches at the rhizosphere scale. Only limited studies have been
reported so far,93,94 and the development of numerical tools including kinetic aspects
is a promising research field.11,95 We might expect that such studies will lead to better
understanding of plant adaptation in problem soils and will suggest appropriate
cultural practices. Implementation of adequate countermeasures against toxic trace
elements and phytoremediation should also derive benefit from numerical tools
describing the soilroot interface.

ACKNOWLEDGMENTS
We thank the European Commission for supporting part of this work through the
project REPITISC contract EV5V-CT93, and the project INCO-DC contract CEE
CT 970208.
248 Trace Elements in the Rhizosphere

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95. Jones, D.L. and L.V. Kochian, 1996, Aluminium-organic acid interactions in acid
soils. I. Effect of root-derived organic acids on the kinetics of Al dissolution, Plant
and Soil, 182, 221228.
12 Modeling the Dynamics
of the Rhizosphere
Aluminum Chemistry in
Acid Forest Soils
Heino W. F. Nietfeld

CONTENTS

I. Introduction...................................................................................................254
II. Development of the Model Approach..........................................................256
A. Model Assumptions and Hypotheses of Rhizosphere Processes.....257
B. Mathematical Model.........................................................................258
1. Ionic Flux Equations .........................................................259
2. Cation Exchange................................................................262
3. Transpiration-Induced Water Flux.....................................263
4. System of Mass Balance Equations ..................................263
5. Root Ion Uptake and H/OH Excretion..............................265
6. Boundary and Initial Conditions .......................................265
III. Materials and Methods .................................................................................266
A. Spectrum of Parameter Values Used in Model Calculations...........266
B. Numerical Solution Procedure .........................................................269
IV. Results...........................................................................................................270
A. Influence of Soil Conditions: Low NO3 Concentration...................270
1. Influence of Daily-Patterned Water Flux ..........................270
2. pH Changes at Various Bulk pH and Mb Cation
Concentrations ...................................................................273
3. Al3+ Concentration Changes at Low Bulk pH ..................277
4. Al3+ and A1-Hydroxo Complexes Concentration
Changes at High Bulk pH and Various Mb Cation
Concentrations ...................................................................278
B. Influence of Soil Conditions: Increasing NO3 Concentrations .......281
1. H+ and Aluminum Concentration at Fluctuating
Daily-Patterned Water Fluxes............................................281
2. pH Changes at Various Mb and Ma Cation
Concentrations and Bulk pH Values .................................286
3. Aluminum Concentration at Various Mb and Ma Cation
Concentrations and Different Bulk pH Values..................287

0-8493-1535-2/01/$0.00+$.50
2001 by CRC Press LLC 253
254 Trace Elements in the Rhizosphere

C. Influence of Soil Conditions: Soil Water Content ...........................290


D. Influence of Root Characteristics: Nutrient Uptake Rates
and Root Diameter............................................................................292
V. Discussion and Conclusions.........................................................................294
Acknowledgments..................................................................................................301
References..............................................................................................................301

I. INTRODUCTION
Solution chemistry of acid soils of forest ecosystems with high concentrations of
acid salts (Ma-cations: Al3+, Mn2+, Fe3+) and protons, and low concentrations of
neutral salts (Mb-cations: Na+, K+, Ca2+, Mg2+)119 has been regarded as a stress
potential for forest tree development.82,120 A wide number of recent experimental
studies have demonstrated that aluminum (Al) has adverse effects on the growth,
root structure,20,115 and nutrient uptake31,61 of forest trees. But transferring the results
of experiments with Al-enriched solution cultures to field observations is problem-
atic, although root toxicity symptoms discovered in field observations are quite
similar to those examined in solution cultures.
First of all, solution culture experiments have shown that Al generates root
damage and plant growth reduction only at high Al concentrations. In many inves-
tigations, a maximum Al concentration threshold of about 500.0 mol L1 has been
found to be tolerable for growth of European beech and spruce.22,33,116 However, this
threshold is not actually reached in soil solutions or if so, only temporarily. These
results contrast with the results of field observations and growth chamber experiments
in which much lower Al solution concentrations caused root damages.70,120 Generally,
it can be established, that the distribution patterns of the degree of forest tree vitality
within forest stands deduced from crown damages are very heterogeneous and dam-
aged trees occur in the direct neighborhood to symptom-free trees.93 These distur-
bance patterns may be considered as evidence that stress-related and stress-free
chemical conditions can occur in close proximity to one another and may be
explained, among other things, by the spatial and temporal heterogeneity of the soil
chemistry98 and physiological and morphological differences of the fine root system.
Indeed, with about the same ionic soil solution composition, Al rhizotoxicity
must be assessed differently for mycorrhizal and nonmycorrhizal plants. Nonmyc-
orrhizal trees seem to be more susceptible to unfavorable acid soil conditions.
Cumming and Weinstein17 demonstrated clearly that nonmycorrhizal pitch pine
seedlings react more sensitively to Al (50.0 to 200.0 mol L1) than mycorrhizal
ones. But the risk of Al toxicity to mycorrhizal plants cannot be clearly determined,
because reports on the role of ectomycorrhizaes in the response of Al treatment have
often been contradictory. On the one hand, depression of the mycorrhizal develop-
ment was limited to declining trees.78 These observations are supported by results
of solution culture experiments showing a potential risk of toxicity to mycorrhizae,
e.g., high Al concentrations also caused root damage and reduced root growth of
mycorrhizal spruce seedlings.55 On the other hand, the higher tolerance of
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 255

mycorrhizal trees as demonstrated by the experiments of Cumming and Weinstein17


correlated with the field observations of Rudawska et al.,104 showing that greater
soil acidity is not accompanied by a significant change in mycorrhizal status. There-
fore, the possible filter function of the mycorrhizal fungi protecting the true root
against acid soil solution has been discussed for some years.77
The effects of soil chemical heterogeneity are already demonstrated by the clear
chemical gradients between the soil layers7 producing different disturbance patterns
of the fine root system. The more harmful conditions in the lower soil layers have
caused a considerable increase in the fraction of damaged fine roots80 and a retreat
of the fine root system from the lower layer to the upper soil layer where more
favorable chemical conditions exist.120 Furthermore, after extended summer droughts
the mineralization and nitrification processes in forest ecosystems intensify during
later rewetting periods,8,10 which may change the soil solution chemistry. During and
after rewetting there is a surplus production of nitrate in the soil, and it has been
hypothesized that the soil solution temporarily acidifies due to the formation of nitric
acid.118 Decoupling of N mineralization and N uptake and a fast leaching of NO3
may lead to soil acidification and a subsequent increase in Al concentration near the
cited concentration threshold of tolerance.121 But most important for the conditions
of mineral nutrition and the risk of toxicity to the tree roots is the chemical compo-
sition of the soil pore space preferentially occupied by roots.40,50 The fine roots of
beech and spruce are predominantly found in the macropores, mostly on aggregate
surfaces,3,43 which may be characterized by a delay of Mb cations49 and higher
concentrations of Ma cations.50
However, not only are the temporal and spatial differences in soil chemical
conditions responsible for different risks of toxicity, but also the various chemical
bonding forms of Al represent different toxicity potentials.
In acid soil solutions, Al3+ may react with F, phosphate, silicate, SO42, and a
large number of organic ligands. Depending on pH, Al3+ may form hydroxo-Al
species (e.g., in solution, the portion of Al3+ is 90% at pH 4.0 where at pH 5.0 the
total Al consists of one third each Al3+, AlOH2+, and Al(OH)2+) and polynuclear
hydroxo-Al species and may possibly precipitate to form new solid phases.9
The toxicity of Al3+ has been detected for a long period; recent experimental
studies have revealed a quite different picture of the rhizotoxicity of the Al com-
plexes. The results of these investigations appear to establish the nontoxicity of Al-
sulfate59,60 and at least some organic complexes72 and the uncertainty of the toxicity
of mononuclear Al species.20
However, the direct transfer of these findings from solution cultures investiga-
tions to investigations of soil grown tree species leave possible root-induced changes
in the chemical conditions at the soil root interface (rhizosphere) completely out of
the reckoning. From recent studies it is clear that depending on the selectivity of
nutrient uptake, there are depletion and accumulation gradients around the root if
transport rate and uptake rate do not correspond.4,87 Therefore, in order to explain
tree decline symptoms through the soil chemical environment, it is necessary to
know about the dynamics of the Al chemistry at the soilroot interface, knowledge
of which is still incomplete.53
256 Trace Elements in the Rhizosphere

Accumulations of Al3+ and SO42 around roots have been detected;24 consequently
the concentration of Al-sulfate complexes in the rhizosphere may be higher as in the
bulk soil. It is well known that due to the H+/OH root efflux, which is mainly
determined by imbalance of cation/anion uptake of all major nutrients,71 the rhizo-
sphere pH may change considerably.74 Root-induced pH increases in acid soils caused
by anion uptake excess, which is usually due to high NO3 uptake, may increase the
fraction of Al hydroxocomplexes59 in the total Al concentration. In the opposite case
at a higher bulk pH, a pH decrease may cause a transformation of Al hydroxocom-
plexes in the bulk soil solution to the Al3+ ions. Furthermore, the H+/OH root efflux
rate may change with time because the root surface concentration of cations and
anions involve changes19 and therefore, the equilibrium between hydroxo-Al species
may shift. These chemical transformations are also affected by the transport charac-
teristics of the ions involved. Al complexes have their own self-diffusion velocities,
which are different from those of the underlying dissociated ions.2 Moreover, the
cation exchange between soil solution and soil exchanger may considerably modify
the chemical reactions in soil solution. Root-induced soil solution depletions can
cause a release of nutrients from the soil exchanger58 or, more generally, root-induced
decreases of cations and shifts of proton concentrations may lead to a new equilibrium
between soil solution and exchanger composition. This may lead to an additional
buffering or a release of Al3+ ions from soil exchanger sites.97
The few direct measurements of Al in the rhizosphere also show depletions and
accumulations of Al,24 which may reflect the briefly outlined possible dynamics of
the Al chemistry in the rhizosphere. It may be concluded that in the rhizosphere
there may exist a dynamic equilibrium between the various Al complexes of different
risks of toxicity. In order to evaluate risks of Al rhizotoxicity originating from
complex, interrelated mechanisms, the coupled processes of the Al chemistry in the
rhizosphere must be described by appropriate modeling approaches.
It is obvious that this can only be achieved by an extension of the known one-
component rhizosphere models18,21 to a multicomponent model approach linking
transport mechanisms and simultaneous chemical reactions.12
In this theoretical study a multicomponent model is presented that includes non-
Fickian diffusion transport and cation adsorption as the competition process for the
exchanger sites. It is the purpose of this study to use model calculations to demon-
strate various scenarios of the dynamics of the Al species in the rhizosphere as
affected by root uptake of water and nutrients and the physicochemical conditions
of the bulk soil. In detail, model calculations are introduced based on various initial
concentrations of Mb cations, NO3, and Al at various bulk pH values, soil water
conditions, and different water and nutrient uptake rates. Taking into account the
heterogeneity of the absorbing root system,113 the calculations distinguish between
different uptake patterns and root diameters of the apical and basal zones of usually
nonmycorrhizal long roots26 and mycorrhizal short roots.63

II. DEVELOPMENT OF THE MODEL APPROACH


The current model starts out from the following series of hypotheses, assumptions,
and simplifications of the physicochemical processes in the rhizosphere.
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 257

A. MODEL ASSUMPTIONS AND HYPOTHESES OF RHIZOSPHERE


PROCESSES
The rhizosphere soil is assumed to be physically and chemically homo-
geneous. The determination of the species sets is mainly constrained by
the buffer range of the soil.117,120 Next to Al3+- and H+-ions, the species
set includes the mineral nutrients relevant for the cation-anion uptake
balance, hence the rhizosphere soil solution includes = cation U anion
U complex = {H+, Al3+, Mn2+, Fe3+, Ca2+, Mg2+, K+, Na+, NH4+, H2PO4,
NO3, SO42, HCO3, OH, Cl AlSO4+, Al(OH)2+, Al(OH)2+}. The species
set may be separated into the subsets consisting of basic cations and anions
and ion complexes. It is assumed that all cations additionally exist in an
adsorbed form and the complexes are treated as equilibrium reactions.
Like the previous models, this model is based on self-diffusion and con-
vection. In addition, the diffusion potential is taken into account.84 During
ionic movement in electrolyte systems an electrical potential rises due to
the Columb interactions between all ions involved. This electrical poten-
tial, termed diffusion potential, is caused by the different self diffusion
velocities of the various ions. The driving force of the diffusion potential
gradient has been emphasized by Nye and Tinker (p. 166),87 and the need
to adequately treat this gradient as a transport-influencing factor is sup-
ported by the results of the experiments by Rhue.98 This is made especially
necessary by the fact that, owing to the high concentration of protons and
Al ions in acid soils, there are two chemical species with clear differences
in self-diffusion mobility.68
In accordance with the previous models, the steady-state condition of water
flux in the rhizosphere seems to be valid.90 Furthermore, the model proceeds
on the option of a diurnal cycle of transpiration114 because there is evidence
that the daily-patterned, transpiration-induced water flux immediately
extends to the rhizosphere soil.91 The deducible water flux velocities46,73
imply that the effects of soil solute dispersion may be neglected.
Exchange reactions between soil solution and soil exchanger involve all
cations of . The exchanger has a fixed exchange capacity although an
increase of the exchanger capacity in the rhizosphere has been observed
due to the enrichment of organic matter. Considering the velocities of
exchange reactions and transport fluxes, it is justified to consider cation
exchange to be a sufficiently fast, reversible reaction.103,122 Investigations
of proton buffering in several soils show that the short-term buffering
of pH is traceable to the exchange of protons against cations.106,107 Hence,
a slow buffer reaction as realized in Nyes85 model is not considered.
Following Prenzel and Schulte-Bisping,92 the exchange reactions may
be represented by algebraic equilibrium expressions. The adsorption of
ion pairs is not considered.
The processes considered in the model may be valid for physical condi-
tions determined by water suctions of up to 100 kPa. These water suctions
may be characterized by an appropriate diameter of water-filled pores and
258 Trace Elements in the Rhizosphere

water films of approximately 23 m. Therefore, the influence of elec-


trokinetic phenomena (e.g., streaming potential) on the ion distribution
patterns of the rhizosphere soil may be ignored. Water-solute-particle
surface interactions gain in importance at higher water-potential values
with equivalent water films of up to about 15 nm or to 1020 water
molecules in thickness (recalculated from83 with a diameter value of a
water molecule of about 200.0 pm). The water velocity distribution within
the soil pores is characterized by a relative maximum in the center of the
pores and minimum values at the pore walls. The approximate values of
the velocity distribution can be calculated by an equivalent step function
separating mobile and immobile water.64 Given the assumed physical
conditions, the spatial extent of the stagnant water portion within the water
films is considerably larger than the thickness of the diffuse layer.11
The root ion uptake is assumed to obey a model formulated according to
the widely accepted MichaelisMenten kinetics. In this study the uptake
rates reported in the literature are regarded as maximum influx rates
(potential uptake rates). However, the kinetic parameters (maximum influx
rates, MichaelisMenten constant) determined by measured influx rates
are not considered to be constant plant properties, but variable entities
describing root influx rates under the present growth conditions.109 Also
preferential and restricted ion uptake (e.g., reduction of NO3 uptake at
high NH4+ availability or a parameterization of the MichaelisMenten
constant by the Al root surface concentration)31 is not considered here.
The study proceeds on the assumption of zero flux of Al ions at the root
surface. It is known that Al easily and rapidly accesses the root apoplast,
and Al uptake by the root symplast is associated with its chemical binding
form.28 The root plasma membrane is a barrier for polyvalent ions such
as Al3+, but, e.g., neutral Al ligands can cross the plasma lemma. Therefore,
the zero flux assumption seems to be justificable for Al3+, Al(OH)2+,
Al(OH)2+, and AlSO4+ ions.
In contrast to Nyes model,85 the efflux rates of H+ or OH are calculated
implicitly via electroneutrality (see Nye and Tinker, p. 167).87 The com-
pensation of excess cation uptake by H+ and OH in the case of excess
anion uptake is evident. The excretion is also stoichimetrically equivalent
to the charge imbalance.48

B. MATHEMATICAL MODEL
It is assumed that each root, of radius r0, exerts its influence on a cylindrical volume
of soil (rhizosphere) starting from root surface with a radius of r1, i.e., the rhizosphere
is a hollow cylinder with a radius of r1 r0. The following equations are formulated
for a general set of species, = {M1, , Mn}, which includes m + 1 dissociated
(basic) cations and anions (species subsets cation and anion) that belong to 1, and
n-(m + 1) ion complexes that belong to the subset complex. Table 12.8 (Appendix)
shows a summary of the model parameters and variables.
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 259

1. Ionic Flux Equations

The flux of each ion Mi is described by the Nernst Planck equation38 that is
extended by a water flux term and is defined by

c F
J i = Di i + zc q r ci [1]
r RT i i r

where Ji is the flux of Mi, Di = f1 Di0, its soil diffusion coefficient, Di0 its self
diffusion coefficient, ci its solution concentration, zi its charge, is the volumetric
water content, and f1 is the soil-impedance factor, which accounts for the tortuosity
of the diffusion path. is the diffusion potential, T is the absolute temperature, R
is the ideal gases constant, F is Faradays constant, qr is the transpiration-induced
water velocity toward the root, and r is the radial position in the rhizosphere.
Starting out from the assumption that no net flux of electrical charge (condition
of current density) occurs in the system, that is

z J
i =1
i i =0 [2]

we can determine Equation [1]. If [1] is inserted for Ji it is possible to deduce an


expression for the unknown diffusion potential gradient,

n n
F c i

z c
ziDi qr
= [3a]
RT r n
r n i i
i =1



j=1
z j D jc j
2


z
j=1
j
2
D jc j i =1

According to the electroneutrality condition in electrolyte systems, ni=1 zici = 0, the


second term on the right-hand side of Equation [3a] disappears. The resulting
expression may be used to eliminate the unknown diffusion potential in flux Equation
[1]. This yields to

n
c c k
z D
Dizici
J i = Di i + q r ci ; 1 i n [3b]
r n k k
r
j=1
z j2 c jD j k =1

Note that the second term in Equation [3b] represents a correction term due to the
existing Columb forces between the ions. Furthermore, the electroneutrality condition
allows one to express the concentration of, e.g., Mn as cn = n1
i=1 (z i /z n)c i ; extracting
cn/r from Equation [3b] and replacing it by the derivative of the expression above
yields, for each Mi \{Mn} in the modified flux expression
260 Trace Elements in the Rhizosphere

n 1
c j
Ji = D*
j=1
ij
r
q r ci ; 1 i n 1 [4a]

where

D ij* = ij D i fij [4b]

and

zici Di
fij = n (D jz j D n z j ) [4c]
z
k =1
k
2
Dkck

and ij is the Kronecker symbol. Equations [4a], [4b], and [4c] show that the flux
of each ion is coupled with the concentration gradients of all ions.
For each ion complex Mi complex there exist ions M1, Mk 1 with
a il M l zl + a ik M k z k M i zi . The reaction is characterized by an equilibrium constant
Ki, which may be written according to the law of mass action

c Mi
Ki = [5a]
c Ml a il
c M k a ik

In this equation c Ml , c M k are the concentrations of the basic ions, c Mi the concen-
tration of the ion complex, and ail, aik are the stoechiometric coefficients.
A possibility for treating chemical ion complex reactions is by differentiating
[5a] with respect to spatial coordinates, by solving the equations thus obtained, and
by substituting the expression for these derivatives into the flux equations. The result
is what Rubin103 called the the partial differential equations option; e.g., using
Equation [5a] the differentiation of an ion complex Mi complex yields

c Mi c M k c Ml
= K i a ik c Ml a il c M k a ik 1 + a il c M k a ik c Ml a il 1 [5b]
r r r

Thus, numbering the n 1 ions from 1 to m for the independent basic ions and from
m + 1 to n 1 for the dependent ion complexes the flux Equation [4a] may be
expressed by

n 1
c c j c j
m

J i = Dc i i +
r j=1
fij
r
+
j= m +1
fij
r
q r ci

[5c]
m
c j
= D* r q c ;
j=1
ij r i 1 i n 1
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 261

which is forced by the weighted gradients of the independent basic ions as well as
by the ion pairs, where

n 1
c
D ij *: = ij D i fij +


k = m +1
fik k
c j
[5d]

for i = 1, , m and

n 1
c i c
D ij *: = D i fij +
c j
k = m +1
fik k
c j
[5e]

for i = m + 1, , n 1 and ij the Kronecker symbol. The Dij* in Equation [5b]


also include the coefficients resulting from Equation [5b]. The convective term for
ion complexes may be split into its basic ions according to [5a] (see Equation [10f]).
This flux equation includes concentration gradients of the bicarbonate and
hydroxyl ions that have to be eliminated with the aid of known chemical reaction
laws. The self-ionization of water, H+ + OH H2O, is characterized by the
expression

K W = c H c OH [6a]

where pKw = 14.069 and which may be termed here as conditional ion product because
its definition is based on concentrations instead of activities. The protolysis of H2CO3,
expressed by the equilibrium reaction H+ + HCO3 H2CO3, is according to
Equation [5a]

c H2CO3
KS = [6b]
c H c HCO3

where pKS = 6.46.112 The equilibrium of vapor CO2 and H2CO3* = CO2(aq) + H2CO3
in the soil solution, CO2(g) + H2O H2CO3*, is described by

pCO 2 K H = c H2CO3 [6c]

where KH = 3.47 107 MPa1 is the Henry constant and pCO2 is the CO2 partial
pressure.112
By setting pCO2 equal to a constant, the relation between H+ and HCO3 is
given by

1
c H c HCO3 = K = K H pCO 2 [6d]
KS
262 Trace Elements in the Rhizosphere

Hence c HCO3 and cOH are considered as a function of cH. The relations [6a] and [6d]
are used to eliminate all concentration gradients of HCO3 and OH ions in the flux
Equations [5c]. This is realized by differentiation [6a] and [6d] with respect to spatial
coordinates and by substituting the expression for these derivatives into the flux
equations, e.g., cOH/r = (cOH/cH) cH/r.
Therefore, the elimination of the concentration gradients of the hydroxyl ion
and bicarbonate ion in flux Equation [5c] (assume M1 = H+, Mm1 = HCO3 and Mm
= OH) yields

c m 1 c c c c
D i*,m 1 + D i*,m m = D i*,m 1 m 1 + D i*,m m 1 [7a]
r r c1 c1 r

where i = 1, 2, , n 1. This is possible because c1 0 is valid for all r. By


insertion, modified flux equations Ji of each ion Mi can be calculated

m 2
c j
Ji = D
i =1
ij
r
q r ci [7b]

where i = 1, , n 1 and

c m 1 c
D *ij D i*,m 1 + D i*,m m for j = 1
D ij = c1 c1 [7c]
D ij* elsewhere

2. CATION EXCHANGE
In the model the stoichiometric cation exchange reactions between the soil solution
and the soil exchange phase may be presented in accordance with thermodynamic
convention. If there are two cations Mi and Mj with charges zi and zj, the exchange
reactions can be described by

1 1 1 1
M + zi + M j X z j M j + z j + M i X zi [8a]
zi i zj zj zi

where X denotes one unit of charge of the cation exchanger with a fixed exchange
capacity CT.92 In analogy to the law of mass action one defines the selectivity
coefficient for the equation above11 as

c i1/ zi m j1/ z j
K Mseli / M j = [8b]
c j1/ z j m i1/ zi
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 263

where ci, cj are the soil solution concentrations of Mi, Mj; m k = c k / cT , k = i, j is


the equivalent fraction of Mk on the sorbed phase, c k is its exchanger concentration,
and K Mseli / M j is the selectivity coefficient. Generally, it is sufficient for a system of n1
cations in 1 to specify n1 1 equilibrium relations of type [8b] for different pairs
of cations and the additional condition that the equivalent concentrations of cations
sum up to the cation exchange capacity, which means

n1

m
i =1
i =1 [8c]

3. TRANSPIRATION-INDUCED WATER FLUX


According to the assumed steady state of the water flux, the corresponding water
flux term in Equations [1] and [7b], respectively, may be expressed by

r0
qr = q [9a]
r 0

where q0 is the constant water uptake rate of the root. Alternatively, according to
the diurnal cycle of transpiration, a cyclic water uptake rate may be defined,

q max sin ( t 8); 8 t 24a 24


q 0 (t ) = 0 16 [9b]
0; elsewhere

where a = 0, 24, 48, and q0max is the maximum water uptake rate and t is the time
in hours. The time-dependent mass flow term defines a water flow period of 16 hours
and a subsequent 8-hour diffusion period.

4. SYSTEM OF MASS BALANCE EQUATIONS


The mass balance equation for each species Mi \{Mn} is

m 2
c i 1 c j c
t
=
r r j=1
r D ij
r
+ q 0 r0 i ;
r
1 i n 1 [10a]

where ci is the concentration of the ion Mi in a unit of soil volume, i.e., for basic
cations ci = ci + ci and for both basic anions and ion complexes ci = ci, where
ci is the soil exchanger concentration, is the soil bulk density and is the volumetric
water content, and the other parameters are the same as in the equations above.
The mass balance of an ion complex, Mi, is expressed in gradients of the basic
ions (see Equation [5b]).
264 Trace Elements in the Rhizosphere

c i c c c c k
= i l + i =
t
tc t c k t
[10b]
m 2
1 c j c c c c k

r r j=1
r D ij
r
+ q 0 r0 i l + i ;
c l r c k r
m +1 i n 1

For consideration of n1 cations, the n1 1 chemical equilibrium, Equations [8b] and


[8c] can be combined to yield the n1 multicomponent isotherms:

ci = Fi (c1 , c 2 , , c n1 ); i = 1, 2, , n1 [10c]

Applications of [10a] under consideration of Equation [10c] gives the mass balance
equation for basic cations

n1
c i c c j
t
= i +
t g j=1
ij
t
=

[10d]
m 2
1 c j

r r j=1
r D ij
r
+ q 0 r0 c i ;

1 i n1

where

ci Fi
g ij = = [10e]
c j c j

The system of mass balance equations is based on the total concentration of a


chemical component, e.g., it includes all chemical bonding forms of the chemical
species considered, i.e., AlTotal = Al3+ + AlSO4+ + Al(OH)2+ + Al(OH)2+. The total
concentration of protons, HTotal, represents components that bind protons (e.g., car-
bonic acid H2CO3), i.e., it includes the protonated forms of all weak bases. But in
the particular case of protons, the self-ionization of water (Equation [6a]) and the
dissociation of H2CO3 (Equation [6d]) cause that a concentration change of H+ ions
can never be distinguished from an appropriate change of the OH and HCO3
concentration in the system. As in the case of protons the total concentration of
hydroxide ions, OHTotal also includes here the Al hydroxocomplexes; i.e., OHTotal =
OH + Al(OH)2+ + Al(OH)2+. The adequate resulting H+ concentration is therefore
the difference of the total concentrations of H and OH. For Al hydroxocomplexes,
the spatial and temporal gradients of the OH ion are transformed into H ion gradients
with the aid of the ion product of water.
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 265

5. ROOT ION UPTAKE AND H/OH EXCRETION


The flux of ions, except for protons, is defined by a parameter expression analogous
to the MichaelisMenten kinetics

c( t, r0 ) c min
J = J max [11a]
K + (c( t, r0 ) c min )

where Jimax is the maximum influx rate, ci(r0,t) the solution concentration at the root
surface, cmin the minimum concentration below which no influx occurs, and K the
MichaelisMenten affinity constant. For Al ions, it is JAl = 0.
Based on these definitions, a cation-anion balance of the uptake rates of various
ions can be calculated. Let C = and A =
M i cation \{H}z i J i M i anion \{OH , HCO3} | z i | J i
be the sum of cation and anion fluxes across the root surface. As a result, the fluxes
of the H+ or OH-ions at the root surface are defined as

(C A), if C A > 0
J H =
0 elsewhere
[11b]
( A C), if A C > 0
J OH =
0 elsewhere

6. BOUNDARY AND INITIAL CONDITIONS


The initial conditions for r0 r r1 are

c Mi ( r, t ) = c i ; 1 i n; t = 0
[12a]
cMi ( r, t ) = c i ; 1 i n1 ; t = 0

where c i and c i are the initial concentrations in soil solution and soil exchanger,
which are constant for the entire rhizosphere soil. These initial concentrations are
consistent with the equilibrium relations [5a], [6a], [6d], and [8b].
If no competition between neighboring roots is assumed the concentration at the
outer boundary remains on initial concentration, i.e.,

c M i ( r, t ) = c i ; i = 1, , n [12b]
r = r1

At the root surface, the flux equation is defined as

Ji r = r0
= J i ; i = 1, , n [12c]
266 Trace Elements in the Rhizosphere

where Ji is the parameter expression defined according to [11a] and [11b]. The
defined boundary conditions guarantee the electroneutrality.

III. MATERIALS AND METHODS


A. SPECTRUM OF PARAMETER VALUES USED IN MODEL CALCULATIONS
The calculation examples are based on given values of initial concentrations, which
are distributed uniformly in the bulk of soil. The various cases of soil chemical
conditions are characterized by differences in Mb (cases I, III, and IV in Tables 12.2
and 12.3), Ma cation concentrations (case II in Tables 12.2 and 12.3), and various
NO3 concentrations; these values are differentiated for various bulk pH (pHb) values
and consequently for various bonding forms of aluminum (subcases A (pHb 4.0)
and B (pHb 4.9)).
The self-diffusion coefficients of the ions involved are listed in Table 12.1. The
diffusion coefficients of the ion complexes are determined according to their charge,66
i.e., univalent and bivalent ions are assumed to have a diffusion coefficient of 1.5
109m2s1 and 1.0 109m2s1, respectively.
The equilibrium constants of the ion complexes involved are listed in Table 12.2.
The initial ion concentrations in the soil solution and on the soil exchanger are
presented in Tables 12.3 and 12.4, respectively. The values are based on the soil
chemical conditions found in most forest study plots.7,75
The concentration values in both soil phases are consistent with the correspond-
ing sets of cation selectivity coefficients in Table 12.5 (see Equation [8b]). The
values of the soil exchanger capacity are 95.84 mmolc kg1 (case I), 101.95 mmolc
kg1 (case II), 104.93 mmolc kg1 (case III), and 138.83 mmolc kg1 (case IV). The

TABLE 12.1
Self Diffusion Coefficients
D0 D0
Ion [10 m2s1]
9 Ion [10 m2s1]
9

Cl 2.03 b H + 9.34b
NO3 1.92b Na+ 1.33b
SO42 1.07b K+ 1.98b
OH 5.5a Mg2+ 0.705b
H2PO4 0.846a Ca2+ 0.793b
Al3+ 0.559a NH4+ 1.98b
Fe3+ 0.846a Mn2+ 0.688a
AlSO4+ 1.5c Al(OH)2+ 1.0c
Al(OH)2+ 1.5c HCO3 1.33a
a Li and Gregory.68
b Nye.86
c According to charge (cf Lasaga66).
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 267

TABLE 12.2
Equilibrium Constants of Ion Complexes
Ion Complex pK

H2CO3 6.45
AlSO4+ 3.2
AlOH2+ 5.02
Al(OH)2+ 9.3

pKsel values used in the calculations correspond to the range of pK values determined
by Prenzel and Schulte-Bisping.92
The assumed range of volumetric soil water content values () of 0.225 to 0.4
corresponds to a range of water suction values of 100 kPa to 10 kPa (Beese, p.
13 ff).6 The calculations use values of 0.225, 0.3, and 0.4 that imply soil impedance
factors (f1) of 0.0325, 0.08, and 0.22, respectively, (for calculations see Beese, p.
36 ff).6 The soil density () is assumed to be 1.19 Mg m3.
Table 12.6 shows assumed diameter values of the various root zones.

TABLE 12.3
Initial Concentrations in Soil Solution
Concentration in Soil Solution
[mmol L1]
Case I Case II Case III Case IV
Ion A B A B A B

H+(pH) 4.01 4.91 3.75 4.0 4.94 4.0 4.91


Al3+ 0.188 0.042 0.249 0.177 0.039 0.176 0.042
AlSO4+ a 0.003 0.001 0.004 0.003 0.001 0.003 0.001
Al(OH)2+ a 0.020 0.035 0.015 0.018 0.05 0.017 0.035
Al(OH)2+ a 0.001 0.134 0.004 0.009 0.207 0.008 0.136
Mn2+ 0.024 0.017 0.027 0.025 0.013 0.048 0.019
Fe3+ 0.005 0.004 0.012 0.005 0.002 0.021 0.005
Ca2+ 0.092 0.088 0.085 0.145 0.116 0.217 0.296
Mg2+ 0.041 0.040 0.040 0.050 0.037 0.051 0.071
K+ 0.045 0.047 0.040 0.062 0.024 0.060 0.049
Na+ 0.087 0.089 0.090 0.094 0.057 0.144 0.091
NH4+ 0.022 0.023 0.020 0.027 0.017 0.023 0.014
H2PO4 0.005 0.005 0.005 0.005 0.005 0.005 0.005
NO3 0.0251.25 0.025 0.025/1.4 0.025/1.15 0.025 0.025/1.25 0.025
SO42 0.025 0.025 0.025 0.025 0.025 0.025 0.025
Clb 1.120.8 0.70 0.025/1.6 0.164/1.24 0.775 0.3/1.52 1.18
a Determined from chemical equilibrium expression.
b Computed to obtain electroneutrality.
268 Trace Elements in the Rhizosphere

TABLE 12.4
Initial Concentrations on Soil Exchanger
Concentrations on Soil Exchanger
[mmolc kg1]
Case I Case II Case III Case IV
Ion A B A B A B

H+ 3.10 3.11 15.0 3.10 3.14 2.09 2.10


Al3+ 75.84 84.81 80.04 75.84 75.72 75.83 75.81
Mn2+ 2.02 2.04 2.02 3.0 3.0 4.0 4.0
Fe3+ 0.54 0.54 0.54 1.5 1.5 4.5 4.47
Ca2+ 1.24 1.24 1.0 15.0 15.02 40 40.0
Mg2+ 0.53 0.53 0.5 3.0 3.0 6.0 6.0
K+ 1.42 1.43 1.25 1.5 1.51 3.5 3.5
Na+ 0.89 0.89 0.9 1.0 1.0 1.86 1.88
NH4+ 0.79 0.79 0.7 1.0 1.0 1.0 1.02

TABLE 12.5
Selectivity Coefficients
Soil Chemical Cases

sel
Case I Case II Case III Case IV
pK M / M
i j A B A B A B

pKH/Al 1.3 1.98 1.76 1.27 2.0 1.0 1.72


pKMn/Al 0.25 0.10 0.31 0.33 0.22 0.22 0.22
pKFe/Al 0.19 0.40 0.18 0.053 0.10 0.10 0.10
pKCa/Al 0.15 0.36 0.14 0.29 0.12 0.40 0.13
pKMg/Al 0.16 0.37 0.12 0.17 0.023 0.30 0.023
pKK/Al 1.29 1.06 1.32 1.15 1.34 1.46 1.37
pKNa/Al 0.80 0.59 0.82 0.8 0.80 0.80 0.80
pK NH / Al 1.34 1.11 1.37 1.34 1.34 1.34 1.34
4

TABLE 12.6
Diameter Values of Root Absorption Zones
Root Diameter Values [mm]
Range of Values Used
Root Absorption Zone Measurements in Simulations

Apical zone 0.5a 0.5; Range: 0.250.75


Basal zone (with root hairs) 0.51.0 (0.521.3)a 0.9
Mycorrhizal roots 0.20.3b 0.25
a Murach, pers. comm., average root hair length: 100150 m (Merk, pers. comm.).
b Clowes16; Kottge.63
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 269

TABLE 12.7
Potential Uptake Rates (J max-Values)
Jmax-Values
[105 mmol m2s1]
Ion Apical Zone Basal Zone Mycorrhizae

Na+ 0.0035 0.0035 0.0035


K+ 0.25a 0.65a 0.65
Ca2+ 6.6a 0.7a 0.25e
Mg2+ 1.9a 0.3a 0.05e
Fe3+ 0.0075 0.0075 0.0075
Mn2+ 0.1 0.1 0.1
Alf 0.0 0.0 0.0
SO42 0.035c 0.035c 0.035e
H2PO4 1.0d 1.0d 2.5c
NH4+ 4.0b 4.0b 0.65c
NO3 14.0b 2.0b 0.25c
Cl 0.01b 0.01 0.01
a Hussling et al.46
b Marschner et al.73
c Harley and Smith.42
d Barber.4
e Rapp.95
f Includes Al3+ and Al-complexes.

Potential uptake rates ( Jmax-values) of the three different root absorption zones
have to be established (Table 12.7). The MichaelisMenten constant is 103 mmol
L1 and the cmin value is set to 2.0 103 mmol L1; both values are assumed to be
the same for all ions.
Calculations with diurnal-patterned water flux (Equation [9b]) use values for the
maximum water uptake rate, q0max, of up to 7.5 105 L m2 s1 (apical zone of long
roots). For the subsequent basal zone a lower value of 2.5 105 L m2 s1 is assumed.73
For mycorrhizal root zones the model calculations assume much lower values of 1.0
105 L m2 s1. For calculations with constant water flux, unchanged during the
whole simulation period (Equation [9a]), q0 is defined as 2.5 105 L m2 s1.

B. NUMERICAL SOLUTION PROCEDURE


The numerical procedure has to consist of a strategy that efficiently couples two
different processes, namely chemistry and ion transport. Each of these processes is
characterized mathematically by fundamentally different equation forms. These
forms are in the present case algebraic relationships that define equilibrium-con-
trolled ion exchange and complex reactions and partial differential equations that
describe the movement of ions through the solution of the rhizosphere soil. A well-
known solution approach involves incorporating the chemical reaction equations
directly into the partial differential equations that describe ionic mass balances. An
270 Trace Elements in the Rhizosphere

alternative approach, split operator technique,79 is used here, which keeps the alge-
braic equations that define the chemistry separate from the differential equations.
Therefore, a solution is obtained by iterating between the two sets of equations:
solving in turn the physical transport and chemical reactions until the two results
are consistent. The Galerkin method has been applied for solving the transport
equations using the piece-wise linear hat-functions. The resulted set of ordinary
differential equations is converted into a set of nonlinear algebraic equations with
the aid of the Crank-Nicolson approximations for the unknown variables and their
derivatives.1 A linearization of the algebraic equation systems has been achieved by
means of the predictor-corrector technique. Based on the total concentrations, the
concentrations of the individual species are calculated separately by a Newton
iteration algorithm.111 A final solution at each time step is obtained by iterations
between the two sets of equations until the results are consistent.

IV. RESULTS
The following scenarios have been introduced based on various soil physical (vari-
ation of soil water content) and various soil chemical conditions (various bulk
concentrations of Mb and Ma cations and NO3 and different pHb values) for different
transport conditions (e.g., for diffusion transport only, sinusoidal water fluxes (Equa-
tion [9b]) or constant water flux unchanged during the whole simulation period
(Equation [9a])) and for various root absorption rates of nutrients. The distribution
dynamics of H+ and Al ions concentrations are presented as radial concentration
patterns in the rhizosphere soil and, alternatively, temporarily as concentration devel-
opment on the root surface. If not explicitly stated, the calculation examples are
based on the potential ion uptake rates of the apical root zone, its diameter value r0
= 0.5 min, and a soil water content of = 0.3.

A. INFLUENCE OF SOIL CONDITIONS: LOW NO3 CONCENTRATION


The following calculations have been introduced with initial concentrations of NO3
of 0.025 mmol L1 (Table 12.3).

1. Influence of Daily-Patterned Water Flux

This calculation example shows the concentration changes of Al3+ and H+ ions based
on the soil chemical case I-A as influenced by daily-patterned water fluxes defined
according to Equation [9b]. The intensity of the maximum water flux rate, q0max,
determines the accumulation of Al3+ at the root surface. Figure 12.1A shows that,
depending on the particular determined maximum water flux, the Al3+ concentrations
reach an accumulation level between 0.4 mmol L1 (q0max = 2.5 105 Lm2s1) and
1.4 mmol L1 (q0max = 7.5 105 Lm2s1); also a slight increase to 0.22 mmol L1 is
calculated for diffusion transport only. Increases and decreases of Al3+ soil solution
concentration on the root surface are calculated according to the fluctuating water
fluxes (Figure 12.1B). Parallel to an increase in the soil solution, an increase in the
Al3+ equivalent fraction on the soil exchanger is observed (Figure 12.1C) with
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 271

FIGURE 12.1A Distribution of Al3+ ions depending on intensity of daily-patterned water flux
rates toward the root (soil chemical case I-A, t = 4.66d).

FIGURE 12.1B Temporal dynamics of Al3+ root surface concentration depending on intensity
of water flux rates (soil chemical case I-A).
272 Trace Elements in the Rhizosphere

FIGURE 12.1C Distribution of equivalent fraction of Al3+ ions on the soil exchanger depend-
ing on intensity of daily-patterned water flux rates (soil chemical case I-A, t = 4.66d).

increasing water flux rates. Cations that are taken up in high quantities by the root
(Ca2+, Mg2+, NH4+) leave the soil exchanger sites. A new equilibrium between soil
solution and soil exchanger is established, so that vacant soil exchanger sites are
occupied by cations that have an accumulation or low depletion in soil solution
(Al3+, Mn2+, Fe3+, K+, Na+) caused by little or no root uptake, or in the case of H+,
by root efflux (Figure 12.2B). The soil exchanger sites previously covered by nutrient
cations are occupied mainly by Al3+ ions, for the most part near the root surface
because of the high affinity of Al3+ ions for the soil exchanger sites.
For diffusion transport alone there is already a steep pH decrease; calculations
with cyclic water flux show that H+ ions additionally accumulate on the root surface
during the water flux periods, and particularly encompass a larger spatial area around
the root (Figure 12.2A). This is caused, among other things, by the root H+ excretion
rates. Figure 12.2B shows the temporal development of the root H+ excretion rates,
depending on the water fluxes. The course of the curves reflects the actual transport-
influenced ion uptake rates. First, in all cases the H+ excretion rates decrease with
increasing depletion of highly absorbed cations. Second, rising water fluxes cause,
in this case, rising cation uptake rates and consequently an excess of H+ ions.
Together with the convective H+ ion input, a cyclic pH pattern is created (not
demonstrated; compare Figure 12.1B), which is characterized by additional pH
decreases during water flux periods and increases (with respect to the previous pH
level) during the subsequent diffusion periods. Increases in soil solution correspond
to decreases in the H+ exchanger concentration and vice versa. The occupation of
soil exchanger sites by H+ ions is highest with diffusion transport only (Figure
12.2C). With water transport competing cations, particularly Al3+ ions (Figure
12.1C), acting in the simulation course previously adsorbed protons to be desorbed
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 273

FIGURE 12.2A Distribution of pH depending on intensity of daily-patterned water flux rates


toward the root (soil chemical case I-A, t = 4.66d).

again during the water flux periods. Hence, for water flux periods an input of protons
into the rhizosphere soil solution consists of the influxes caused by root excretion
and convective flux and of H+ desorption from the soil exchanger.
The following calculations have been introduced with a constant water flux rate
unchanged during the whole simulation period (q0 = 2.5 105 L m2 s1). The
simulation results represent the distribution of H+ ions and Al components at various
soil chemical bulk conditions (soil chemical cases I, III, and IV) at low and high
pHb (subclasses A and B).

2. PHCHANGES AT VARIOUS BULK PH AND MB CATION


CONCENTRATIONS
The root excretion of H+ ions and the influx of H+ ions via water influx, produce
H+ accumulations in the rhizosphere soil whose spatial-temporal distribution within
the rhizosphere soil is influenced by its chemical conditions.
Figure 12.3A demonstrates larger pH decreases with increasing Mb cation avail-
ability in the rhizosphere soil at each of the bulk pH values of about 4.0 and 4.9,
respectively. In all these chemical cases, based on soil chemical situations with low
NO3 concentration, the root uptake activity nearly causes a one-to-one exchange of
cations taken up and excreted H+ ions. But H+ ions and nutrient cations differ
considerably with regard to diffusion transport and soil chemical reactions, especially
concerning the adsorption on the soil exchanger. This is demonstrated by the distri-
bution differences of the accumulating H+ ions in soil solution (Figure 12.3A) and
274 Trace Elements in the Rhizosphere

FIGURE 12.2B Temporal development of the H+ root excretion rates depending on intensity
of water flux rates (soil chemical case I-A).

FIGURE 12.2C Distribution of the equivalent fraction of H+ ions on the soil exchanger
depending on intensity of water flux rates (soil chemical case I-A, t = 4.66d).
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 275

High pHb

Low pHb

FIGURE 12.3A Distribution of pH depending on various Mb cation concentrations and dif-


ferent bulk pH values (soil chemical cases I, III, and IV differentiated for the subclasses A
(pHb 4.0) and B (pHb 4.9); t = 4.66d; q0 = 2.5 105 Lm2s1).

on the soil exchanger (Figures 12.3B and 12.3C). At pHb 4.0 the accumulated H+
ions on the soil exchanger distribute over a large space (Figure 12.3B) while at pHb
4.9 the soil exchanger just around the root, to a maximum of 2.0 mm distance from
the root surface, is covered though with higher amounts of H+ ions (Figure 12.3C).

FIGURE 12.3B Distribution of equivalent fraction of H+ ions on soil exchanger at various


Mb cation concentrations and low bulk pH (pHb 4.0; t = 4.66d; soil chemical cases I-A, III-
A, and IV-A; q0 = 2.5 105 Lm2s1).
276 Trace Elements in the Rhizosphere

FIGURE 12.3C Distribution of equivalent fraction of H+ ions on soil exchanger at various


Mb cation concentrations and high bulk pH (pHb 4.9; t = 4.66d; q0 = 2.5 105 Lm2s1; soil
chemical cases I-B, III-B, and IV-B).

Differences of pH and mH accumulation levels between subcases I, III, and IV


of the particular soil chemical cases A and B are due to different influx rates of H+
ions into the rhizosphere soil. The differences of the H+ root excretion rates dem-
onstrated in Figure 12.3D correspond to differences in nutritional cation availabili-
ties. In particular the high concentrations of nutrients in case IV-B produce high
concentration increases of protons in solution and exchanger of the rhizosphere soil.
On the other hand, for the B-subcases the influx rates of H+ ions via water flux are
lower and proton concentrations are reduced caused by the dissociation of hydroxo-
Al complexes. For the cases I-B and III-B the root excretion rates (not demonstrated)
are similar to the calculated rates in the cases I-A and III-A; hence the calculated
spatial distribution differences of nearly identical amounts of feeded H+ ions are
clearly attributed to differences in the soil chemical conditions.
The different distributions of H+ ions on the soil exchanger are due to the com-
petition of H+ ions and the other cations, in particular Al3+ ions, for the exchanger
sites. The higher H+ occupation just around the root in subcases B compared with
subcases A is generally due to the higher affinity of protons for the soil exchanger
sites which is expressed by the higher pKH/Al sel values (and, e.g., pKsel sel
H/Ca and pKH/Mg; it is
pK H / Ca = pK H / Al + pK Al / Ca and pK Al / Ca = K Ca / Al ) in the B-cases. This is also dem-
sel sel sel sel sel

onstrated by the clear differences of the Al 3+ soil exchanger accumulation levels in


soil subcases A and B (Figure 12.4C, Figure 12.5C). The strong sorption of H + ions
reduces their transport velocity and therefore leads to the calculated high accumulation
in the soil solution in a smaller space.
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 277

FIGURE 12.3D Temporal development of the H+ root excretion rates depending on Mb cation
concentrations and initial pH values (soil chemical cases I-A (low Mb cation concentration,
pHb = 4.01); III-A (medium Mb cation concentration, pHb = 4.0); IV-A (high Mb cation
concentration, pHb = 4.0); III-B (high Mb cation concentration, pHb = 4.91); q0 = 2.5 105
Lm2s1).

3. Al3+ Concentration Changes at Low Bulk pH

In the soil cases III-A and IV-A the Al3+ soil exchanger accumulation is higher just
around the root compared with the cases III-B and IV-B but distribute over a smaller
space; in cases III-B and IV-B the Al3+ soil exchanger increases cover a larger space
(Figure 12.4C and Figure 12.5C).
Figure 12.4A shows that the water-flux induced Al3+ accumulation is modified
by the availability of Mb cations. High uptake rates of Mb cations produce depletions
in soil solutions as well as on the soil exchanger. Consequently, high bulk concen-
trations of Mb cations on the soil exchanger cause high amounts of free exchanger
sites. These exchanger sites are occupied, among other cations, by Al3+ ions producing
a lower concentration increase of Al3+ ions in soil solution. In contrast, small amounts
of Mb cations on the soil exchanger produce only small free exchanger sites during
the root uptake process and therefore have a small buffering-effect of Al3+ ions, e.g.,
a higher amount of the accumulated Al3+ ions remain in the soil solution.
But under these soil chemical conditions, in which the amount of cations taken
up by the root equals nearly the amount of excreted H+ ions, the competition between
H+ and Al3+ ions for the free exchanger sites defined by the pKsel values determines
the distribution of Al3+ and H+ ions in soil solution and exchanger. Documented by
the pKsel values in all soil chemical A-cases there is a high affinity of Al3+ ions for
the soil exchanger sites compared with H+ ions. In case IV-A the assumed higher
278 Trace Elements in the Rhizosphere

FIGURE 12.4A Distribution of Al3+ concentration depending on various Mb cation concen-


trations at low pHb value (soil chemical cases I-A, III-A, and IV-A; pHb 4.0, t = 4.66d, q0
= 2.5 105 Lm2s1).

affinity of Al3+ ions (in relation to H+ and compared with cases I-A and III-A) for
the soil exchanger sites leads to a higher exchanger phase concentration and a low
Al3+ solution accumulation just around the root only. This is also documented at the
clear concentration decrease at diffusion transport only compared with, e.g., case
I-A (Figure 12.4B).
Although the initial Al3+ concentrations are equal in all bulk soil concentrations
of subcases A, the temporal development of the accumulation levels on the root
surface are different (Figure 12.4B).

4. Al3+ and Al-Hydroxo Complexes Concentration Changes at


High Bulk pH and Various Mb Cation Concentrations
At high bulk pH (pHb 4.9) the greatest part of the Al bulk concentration in soil
solution consists of hydroxo-Al complexes, within which Al(OH)2+ is of highest
concentration (Table 12.3). Due to low NO3 availability pH decreases have been
calculated in all cases (Figure 12.3A). Consequently, hydroxo Al complexes trans-
ported toward the root surface via water flux are continuously dissolved into the
basic ions Al3+ and OH. Therefore, according to the partly drastic pH decreases of
up to 2.5 mm distance from the root surface, there is a pH-depending partial
transformation of Al-OH complexes into Al3+ ions (Figure 12.5B). This results in
concentration decreases of hydroxo Al ions and a simultaneous increase in Al3+
concentrations (Figure 12.5A).
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 279

FIGURE 12.4B Temporal distribution of Al3+ ions at the root surface at various high Mb
cation concentrations and convective and diffusive transport (pHb 4.0; soil chemical cases
I-A, III-A, IV-A; q0 = 2.5 105 Lm2s1).

FIGURE 12.4C Distribution of equivalent fraction of Al3+ ions on soil exchanger at various
Mb cation concentrations (pHb 4.0; soil chemical cases I-A, III-A, IV-A; q0max = 2.5
105 Lm2s1).
280 Trace Elements in the Rhizosphere

FIGURE 12.5A Distribution of Al3+ ion concentration at various Mb cation concentrations


and high initial pH values (pHb 4.9; soil chemical cases I-B (low Mb cation concentration),
II-B (medium Mb cation concentration), III-B (high Mb cation concentration), t = 4.66d; q0
= 2.5 105 Lm2s1).

The dissolution of hydroxo-Al complexes transported toward the root should


be considered as a transport-influenced, pH-dependent source of Al3+ and OH ions,
on the one hand, and a sink of hydroxo-Al ions, on the other. The change in the
chemical bonding form of aluminum is connected with a change in the transport
behavior (the self diffusion velocity of hydroxo-Al complexes are higher than the
diffusion mobility of Al3+ ions and no sorption-induced retardation of Al-OH com-
plexes is assumed) and subsequent chemical reactions, e.g., a higher amount of
Al3+-ions has a reduced diffusion mobility and they participate in exchange reactions
with the soil exchanger. This dynamic equilibrium is also influenced by the diffusion
of Al-OH ions toward the root and the back diffusion of Al3+ ions. The near identity
of the Al3+ ion accumulation levels in the various soil chemical conditions reflects
the fact that the various amounts of Al3+ ions, resulting from the dissolution of Al-
OH complexes, just fill up the Al3+ concentration level. This results from the
convective transport of Al3+ bulk ions to about the same accumulation level in all
cases considered.
The occupation of Al3+ ions of the soil exchanger sites (Figure 12.5C) is reduced
compared with the situation at low bulk pH (Figure 12.4C). This is caused by a
higher affinity of protons for the soil exchanger sites (compare pK sel H / Al values in
subcases A and B).
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 281

FIGURE 12.5B Distribution of hydroxo-Al complexes at various Mb cation concentration


and high pHb value (soil chemical cases I-B, III-B, and IV-B; pHb 4.9; t = 4.66d; q0 = 2.5
105 Lm2s1).

B. INFLUENCE OF SOIL CONDITIONS: INCREASING NO3


CONCENTRATIONS
1. H+ and Aluminum Concentration at Fluctuating
Daily-Patterned Water Fluxes

The following calculation results are based on soil chemical case I-A but progres-
sively replace Cl concentration by an increasing NO3 concentration; q0max; a daily-
patterned water flux with a q0max = 7.5 105 Lm2s1 is used.
Due to its high root uptake rate, increasing NO3 availability causes a reduced
H or an increased OH root excretion rate (Figure 12.6C), resulting in a smaller
+

pH decrease or a pH increase in the rhizosphere compared with pHb (Figure 12.6A).


Already at NO3 concentration of 0.25 mmol L1, the pH at the root surface only
slightly decreases to a value of 3.9 compared to a pH-value of 3.78 with low NO3
availability (Figure 12.2A). As already demonstrated (Figure 12.2C), also at
increased NO3 availability, the dynamics of the H+ soil exchanger fraction is char-
acterized by accumulations and depletions during the diffusion periods and water
flux periods, respectively. But the higher desorption rate at increasing NO3 concen-
tration results in a continuous H depletion of the exchanger occupation during the
simulation period (Figure 12.6B).
The pH changes and the H+ equivalent fraction on the soil exchanger are due to
the H+/OH root excretion rate shown in Figure 12.6C (positive values for proton
282 Trace Elements in the Rhizosphere

FIGURE 12.5C Distribution of the equivalent fraction of Al3+ ions on the soil exchanger at
various Mb cation concentrations and high initial pH (pHb 4.9; soil chemical cases I-B, III-
B, IV-B; q0 = 2.5 105 Lm2s1).

excretion and negative for OH root excretion). Increasing NO3 availability shows
reduced or increased OH root excretion rates. It is seen that during the water flux
periods increased OH excretion and higher H+ excretion rates are calculated.
At increasing NO3 availability, a decreasing Al3+ ion accumulation level at maximum
water fluxes is calculated; Figure 12.7A shows a reduction in the Al3+ ion root surface
concentration from 0.95 mmol L1 (NO3 = 0.25 mmol L1) to 0.55 mmol L1 (NO3
= 0.75 mmol L1) on the root surface. This is due to the increased buffering of Al3+
ions by the soil exchanger; an increased amount of soil solution Al3+ occupies the
soil exchanger sites, which becomes free through the desorption of nutritional cations
and in this situation, additionally due to desorption of H+ ions from the soil
exchanger.
But at the highest NO3 concentration (1.0 mmol L1) the concentration of
previously accumulated Al3+ on the soil exchanger decreases again (Figure 12.7B).
At the given NO3 concentration, the initial high H+ desorption rate from the soil
exchanger at the beginning of the calculation process continuously decreases during
the course of the process. Therefore, the continuous pH increases as a response of
the root-released OH ions causing a desorption of protons from the soil exchanger.
Consequently, at the highest NO3 concentration, the resulting high pH increase
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 283

FIGURE 12.6A Distribution of pH depending on the NO3 concentration at daily-patterned


water flux (soil chemical case I-A, t = 3.54d; q0max = 7.5 105 Lm2s1; t = 2.33d at NO3 =
1.0 mmol L1 and t = 4.66d in other cases).

(Figure 12.6A) causes a change in the chemical bonding form of the Al3+ ions. An
increasing amount of solution Al3+ ions is transformed into hydroxo-Al complexes
by their convective transport toward the root. Thus the accumulated Al ions at the
root surface are not only present as Al3+ ions, but rather to a larger extent, as Al-
hydroxo complexes.
Furthermore, this continuous reduction of Al3+ ion soil solution concentration
causes a desorption of previously adsorbed Al3+ ions just around the root as a result
of the cation competition for the soil exchanger sites. Also, this input of desorbed
Al3+ ions is transformed into hydroxo-Al complexes and is transported via retrodif-
fusion.
But as the pH in the rhizosphere has a clear spatial gradient, so are the Al
hydroxocomplexes partially transformed back to dissociated Al3+ ions; consequently,
the Al3+ ions are partially adsorbed by the soil exchanger sites. The spatial concen-
tration distribution of Al3+ on the soil exchanger in Figure 12.7B shows the described
Al3+ depletion just around the root as well as a slight increase of Al soil exchanger
fraction behind this small depletion zone.
The following calculations have been introduced with a constant water flux rate
(q0 = 2.5 105 L m2 s1). The simulation results represent the distribution of H+
ion and Al components at various soil chemical bulk conditions at low pHb (soil
chemical cases I-A, II, III-A and IV-A).
284 Trace Elements in the Rhizosphere

FIGURE 12.6B Distribution of equivalent fraction of H+ ions on the soil exchanger depending
on NO3 concentration (soil chemical case I-A, t = 3.54d; q0max = 7.5 105 Lm2s1; compare
Figure 12.6A).

FIGURE 12.6C Temporal development of the H+ (positive values) and OH (negative values)
root excretion rates depending on the NO3 concentration (soil chemical case I-A; q0max = 7.5
105 Lm2s1).
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 285

FIGURE 12.7A Distribution of Al3+ concentration depending on NO3 concentration (soil


chemical case I-A; q0max = 7.5 105 Lm2s1; t = 2.33d at NO3 = 1.0 mmol L1 and t = 4.66d
in other cases).

FIGURE 12.7B Temporal dynamics of Al3+, hydroxo-Al complexes, and AlTotal on the root
surface at an NO3 concentration of 1.0 mmol L1 (soil chemical case I-A, q0max = 7.5 105L2s1).
286 Trace Elements in the Rhizosphere

FIGURE 12.8A Distribution of pH at various concentrations of Mb and Ma cations and high


NO3 concentrations (soil chemical cases I-A (NO3 = 1.125 mmol L1), II (NO3 = 1.4 mmol
L1), III-A (NO3 = 1.15 mmol L1), and IV-A (NO3 = 1.25 mmol L1); t = 4.66d; q0 = 2.5
105 Lm2s1).

2. pH Changes at Various Mb and Ma Cation Concentrations


and Bulk pH Values

Figure 12.8A shows that high nitrate concentrations at various soil chemical bulk
conditions cause various pH increases (Figure 12.8A and 12.8B). First of all the
different NO3 concentrations produce various rates of OH excretions. At the nitrate
concentration values of 1.125 mmol L1 and 1.4 mmol L1 in the soil chemical case
I (low Mb cation concentration) and II (high Ma cation concentration), respectively,
the highest OH rates are calculated (Figure 12.8C). Increased Mb cation availability
reduces the OH excretion rates also at high nitrate initial concentrations (cases III-A
and IV-A). At the beginning of calculation, even an H+ ion excretion at high Mb
cation availability is produced (case IV-A). Hence it can be stated that high NO3
concentrations alone do not necessarily produce an OH excretion (e.g., in case IV-A
an NO3 concentration of 1.0 mmol L1 causes an H+ excretion during the whole
calculation period; not documented) but the changing uptake ratio of anions, in
particular of NO3, and cations determine the amount of excreted H+/OH ions
(Figure 12.8C).
Second, the calculated root surface pH resulting from OH root excretion has a
clear temporal pattern (Figure 12.8B). The first partly drastic pH increase in all soil
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 287

FIGURE 12.8B Temporal development of the pH at various concentrations of Mb and Ma


cations and NO3 concentration (soil chemical cases I-A (NO3 = 1.125 mmol L1), II (NO3
= 1.4 mmol L1), III-A (NO3 - 1.15 mmol L1), and IV-A (NO3 = 1.25 mmol L1); pHb
4.0; q0 = 2.5 105 Lm2s1).

chemical cases is caused by the initial adsorption on the soil exchanger, e.g., also
at the initial H+ root excretion in case IV-A there is a pH increase. The subsequent
period is characterized by the transport-influenced formation of gradients of all ions
involved. During this period a continuous increase in pH is observed. Depletion of
H+ ions in soil solution due to the neutralization by root excreted OH ions leads to
a desorption of protons from the soil exchanger (Figure 12.8D). In case II the
depletion of H+ ions on the soil exchanger includes a large spatial extent; this H+
ion input into the soil solution prevents a higher pH increase also at the highest OH
root excretion rate. Also in soil chemical case I-A the calculated highest pH increase
corresponds to a clear H depletion on the soil exchanger (Figures 12.8A and 12.8D).

3. Aluminum Concentration at Various Mb and Ma Cation


Concentrations and Different Bulk pH Values

The dynamics of Al3+ concentration at higher NO3 availability is determined by the


buffering of the soil exchanger and transformation of Al3+ ions into hydroxo-Al
complexes. In contrast to the situation at low NO3 concentration (Figure 12.4A),
288 Trace Elements in the Rhizosphere

FIGURE 12.8C Temporal development of pH on the root surface at various concentrations


of Mb and Ma cations and NO3 concentrations (soil chemical cases I-A (NO3 = 1.125 mmol
L1), II (NO3 = 1.4 mmol L1), III-A (NO3 1.15 mmol L1), and IV-A (NO3 = 1.25 mmol
L1); q0 = 2.5 105 Lm2s1).

there is nearly no significant accumulation of Al3+ on the root surface even at high
Al3+ bulk concentrations. The amount of Al ions transported via water flux toward
the root are lower as the adsorption rates on the soil exchanger and the transformation
rates of Al3+ into complexed Al bonding forms.
Figure 12.9A shows the gradients of Al3+ ions concentration on the root surface
at various soil chemical conditions (cases I-A, II, III-A, IV-A). As already observed
from the proton concentration distribution, the Al3+ concentration in soil solution in
all soil chemical cases drops immediately at the beginning of the uptake process.
This is due to the buffering effect of the soil exchanger. Also at high Ma cation
concentrations the Al3+ solution concentration decreases continuously within the
whole rhizosphere. These patterns are correlated with the development of the pH
values in the rhizosphere and particularly on the root surface (Figure 12.8A). At a
high H+ concentration on the soil exchanger (and a low occupation with Mb cations,
soil chemical case II) the occupation of exchanger sites, which became free due to
H+ desorption (Figure 12.8D), constitutes the only buffering effect other than the
release of Mb cations from the soil exchanger. In the soil chemical cases III-A
Modeling the Dynamics of the Rhizosphere Aluminum Chemistry 289

FIGURE 12.8D Distribution of equivalent fraction of H+ ions on the soil exchanger at various
concentrations of Mb and Ma cations and NO3 (t = 4.66d; soil chemical cases I-A (NO3 =
1.125 mmol L1), II (NO3 = 1.4 mmol L1), III-A (NO3 = 1.15 mmol L1), and IV-A (NO3
= 1.25 mmol L1); q0 = 2.5 105 Lm2s1).

(medium Mb cation concentration) and IV-A (high Mb cation concentration), the


concentration of protons on the soil exchanger is much lower, but the main buffering
effect is due to the higher amounts of desorbed Mb cations. Also in these cases the
amounts of competing H+ ions are partly considerably reduced due to the OH root
excretion; hence the Al3+ buffering extent is clearly increased compared with low
NO3 bulk concentrations. In the soil case I-A the release of free exchanger sites is
small because of less concentrations of H+ (compared with case II) and Mb cations
(compared with cases III and IV); hence the Al3+ buffering extent is small (compare
Figure 12.4A). Consequently, at low NO3 and high NO3 concentrations, high
solution concentrations of Al3+ on the root surface are calculated (Figure 12.1A).
But a high pH increase here effectuates a transformation of Al3+ ions delivered toward