(Books in Soils, Plants, and The Environment) M.N.V. Prasad-Metals in The Environment - Analysis by Biodiversity (Books in Soils, Plants, and The Environment) - CRC Press (2001) PDF

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Preface
Metal contamination and pollution in the environment, the significance of biodiversity conservation, and the root causes of biodiversity loss are emphasized in a
wide array of works. However, information about the utility of phytodiversity
and microbial diversity for the management of metal-contaminated and -polluted
ecosystems is rather scanty. Therefore, the collective work presented in this volume spotlights the potentiality of biodiversity for monitoring and abatement of
metal pollution in the environment, and also explores the emerging issues and
initiatives concerning metals in the environment.
Heavy metals are being enriched in all aspects of the environment, viz.,
air, water, and soil, by anthropogenic as well as natural processes. For example,
effluents from industrial and mining wastes have increased several thousand-fold
the metal concentrations in river water and sediments. Aquatic systems (freshwater, marine, and estuarine) act as receptacles for several metals. Serpentine soils
contain heavy metals for nickel (averaging 10 mg per g of soil), as well as cobalt
and chromiumboth of which are present at lower levels than nickel and are
characterized by high concentrations of iron and magnesium and low nutrient
levels. Some of the best-studied nickeliferous biotopes are the New Caledonian
soils, which are inhabited by nickel-resistant bacteria as well as metal-tolerant
plants, many of which act as nickel hyperaccumulators. An interesting ecosystem
is established in these biotopes driven by a nickel cycle, in which hyperaccumulating trees extract nickel from deep soil and rock layers and subsequently store
it in their leaves (up to 1% Ni in leaf dry matter). When the leaves are shed from
the trees, the nickel is leached out into the surrounding topsoil. The solubilized
metal exerts a localized selective pressure on the topsoil microflora, which aciii
iv
Preface
quire resistance to high levels of nickel (20 mM). In fact, the predominant
cultivable microflora found in the topsoil (bacteria, fungi, and protozoa) adapt
to the high levels of nickel. Interestingly, the microflora that were not found
directly beneath the canopy but in the same soil showed tolerance to lower levels
of nickel (3 mM) compared to the resistant population. Thus, the nickel selection
pressure existing as a gradient around the hyperaccumulator plants has a dramatic
effect on the composition of the local microbial population. The most successful
monitoring methods for metals in the environment are based on gene- and proteinbased bacterial heavy metal biosensors.
Likewise, several associations of mosses, liverworts, and ferns are capable
of growing on metal-enriched substrates. Each of these groups of plants possesses
certain anatomical and physiological properties that enable representatives to occupy unique ecological niches in natural and man-made metalliferous environments. The best documented of these are the groups of specialized bryophytes
found on substrates enriched with copper; so-called copper mosses are found
worldwide and come from widely separated taxonomic groups. Other bryophytes
are associated with lead- and zinc-enriched substrates. No distinctive bryophyte
flora were observed on serpentine soils. Pteridophytes are associated with serpentine substrates in various parts of the world, and several African species grow
on copper-enriched soils or on soils polluted by metal-smelter emissions. The
existence of these diverse groups is a clear indication of the ability of bryophytes
and pteridophytes to adapt to extremes of metal content of their growth substrate,
either by avoidance of the toxic constituents or by expressing resistance/tolerance
to metals at an organismal or cellular level.
Among angiosperms, the hyperaccumulators are reported in Brassicaceae,
Cyperaceae, Cunoniaceae, Caryophyllaceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae, and Violaceae. About 400 metal-hyperaccumulating species
have been identified, and this number might increase in the near future. The
consequences and ecological role of metal hyperaccumulation are expanding rapidly. Different schools of thought have their own convictions. The salient views
are:
Hyperaccumulation is possible when plants have hypertolerance properties
that may be the result of chelation and vacuolar compartmentalization.
In general, the root of a plant contains more metal than shoot. In plants
that hyperaccumulate heavy metals, the roots must transfer the bioaccumulated metals to the shoots. It has been reported that in normal plant
root, Zn, Cd, or Ni concentrations are 10 or more times higher than shoot
concentrations, but in hyperaccumulators, shoot metal concentrations
may exceed those in roots. Mn and Zn are reported to concentrate up to
1% and more in leaf dry matter in the case of hyperaccumulators.
Preface
Hyperaccumulators can resist drought and can interfere with neighboring

plants.
It is also suggested that hyperaccumulators provide protection against fungal and insect attack. Recent studies suggests that Ni hyperaccumulation
has a protective function against fungal and bacterial pathogens in Streptanthus polygaloides and folivores in S. polygaloides and Thlaspi montanum. An antiherbivory effect of Zn has been found in the Zn hyperaccumulator T. caerulescens.
The fundamental aspects of microbe/plant stress responses to different
doses of metals coupled with breakthrough research innovations are amenable
for advancing environmental biotechnology. Accordingly, the chapters in this
work are organized into two parts:
Chapters 19 are based on taxonomic/habitat criteria: bacteria, mycorrhizal
fungi, freshwater algae, saltmarsh metallophytes, lichens, bryophytes and
pteridophytes, angiosperms (Brassicaceae, Caryophyllaceae, Fabaceae,
and Poaceae [the tolerance of monocots to heavy metals is generally
higher than that of dicots and requires a rather long exposure time to
heavy metals]), and aquatic macrophytes.
Chapters 1015 are based on functionality and utility of biodiversity: angiospermous and gymnospermous tree rings and dendroanalysis, tree
crops, tree bark, heavy-metal interactions with soil microbes, consequent
implications, subsoil acidification and plant responses to aluminum, and
heavy-metal behavior in soils emphasizing certain remediation strategies.
Tree rings are the result of a rhythmic activity of the cambium. In spring
and early summer the cambium is active and new wood is formed, while in autumn and winter the cambium is dormant. The mineral composition of the tree
rings depends partly on the mineral uptake of the tree. The extent of mineral
uptake may reflect the availability of the elements in the trees environment. In
fact, several of the gymnosperms (Cedrus, Chamaecyparis, Cryptomeria, Gingko,
Juniperus, Larix, Pinus, Pseudotsuga, Picea, Taxodium, Thuja, and Tsuga) are
being used as retrospective biomonitors of trace-metal pollution.
Bark enclosed in tree trunks, known generally as tree bark pockets, provides invaluable information for monitoring metal pollution in the atmosphere.
Because bark pockets were not previously considered useful parts of a tree, tree
trunks with bark pockets were discarded as waste. Many forest trees with bark
pockets have therefore been left standing and not harvested.
Several plants and microbes are being employed for remediation of the
metal-polluted ecosystems. For example, constructed wetlands, reed beds, and
vi
Preface
floating-plant systems are quite common for the treatment of wastewaters and
industrial effluents containing toxic heavy metals. In situ and on-site remediation
of metallic residues is gaining considerable global significance. As civilization
progresses we need to conserve biodiversity for biomonitoring and decontaminating the environment.
I fervently believe that this work will stimulate future research in the enchanting field of metals in the environment, leading to progress in conserving
the unknown for the prosperity of humankind.
I am extremely grateful to Padma Bhushan Professor P. Rama Rao, ViceChancellor, University of Hyderabad, for inspiring me to focus my research on
biodiversity and for his constant encouragement. I thank Professor T. Suryanarayana, Dean, School of Life Sciences, for supporting my academic endeavors.
I am grateful to all the contributors for comprehensive reviews which culminated in the present text. I am pleased with the excellent cooperation of my wife,
Savithri. The superb and skillful job of Ms. Annie Cok and timely action from
Mr. Jeff Stockton, Ms. Marilyn Ludzki, and Mr Russell Dekker at Marcel Dekker,
Inc., produced this work in a timely manner.
M. N. V. Prasad
Contents
Preface
iii
Contributors
ix
Bacteria
Daniel van der Lelie and C. Tibazarwa
Mycorrhizal Fungi
Jan V. Colpaert and P. Vandenkoornhuyse
37
Freshwater Algae
Barbara Pawlik-Skowronska and Tadeusz Skowronski
59
Salt Marshes
Isabel Cacador and Carlos Vale
95
Lichens
Cristina Branquinho
117
Bryophytes and Pteridophtes

Nicholas W. Lepp
159
vii
viii
Contents
Angiosperms (Asteraceae, Convolvulaceae, Fabaceae and

Poaceae; other than Brassicaceae)
Anna Siedlecka, Anna Tukendorf, Ewa Skorzynska-Polit,
Waldemar Maksymiec, Magorzata Wojcik, Tadeusz Baszynski,
and Zbigniew Krupa
171
Brassicaceae
Luigi Sanita` di Toppi, Maria Augusta Favali,
Roberto Gabbrielli, and Patrizia Gremigni
219
Aquatic Macrophytes
M. N. V. Prasad, Maria Greger, and Bruce N. Smith
259
10
Aluminum Toxicity in Acid Soils: Plant Responses to

Aluminum
Hideaki Matsumoto, Yoko Yamamoto, and S. Rama Devi
11
Tree Crops
Tracy Punshon
12
Tree Bark: Tree Bark Pockets as Pollution Time Capsules

for Historical Monitoring
Kenichi Satake
13
Tree Rings and Dendroanalysis

C. Nabais, J. Hagemeyer, and H. Freitas
14
Heavy Metal Interactions in Soils and Implications for Soil

Microbial Biodiversity
R. Naidu, G. S. R. Krishnamurti, N. S. Bolan, W. Wenzel, and
M. Megharaj
15
Behavior of Heavy Metals and Their Remediation in

Metalliferous Soils
Arun B. Mukherjee
289
321
353
367
401
433
Biodiversity Index
473
Subject Index
483
Contributors
Tadeusz Baszynski Department of Plant Physiology, Maria Curie-Skodowska

University, Lublin, Poland
N. S. Bolan Institute of Natural Resources, Massey University, Palmerston
North, New Zealand
Cristina Branquinho
Oeiras, Portugal
Environmental Department, Universidade Atlantica,
Isabel Cacador Institute of Oceanography, University of Lisbon, Lisbon, Portugal

Jan V. Colpaert Center for Environmental Sciences, Limburgs Universitair
Centrum, Diepenbeek, Belgium
S. Rama Devi Research Institute for Bioresources, Okayama University, Kurashiki, Japan
Maria Augusta Favali Department of Evolutionary and Functional Biology,
Section of Plant Biology, University of Parma, Parma, Italy
H. Freitas Departamento de Botanica, Universidade de Coimbra, Coimbra,
Portugal
ix
Contributors
Roberto Gabbrielli Department of Plant Biology, University of Florence, Florence, Italy

Maria Greger Department of Botany, Stockholm University, Stockholm,
Sweden
Patrizia Gremigni Center for Legumes in Mediterranean Agriculture
(CLIMA), University of Western Australia, Perth, Western Australia, Australia
J. Hagemeyer Department of Ecology, University of Bielefeld, Bielefeld, Germany
G. S. R. Krishnamurti Remediation of Contaminated Environments Program,
CSIRO Land and Water, Urrbrae, South Australia, Australia
Zbigniew Krupa Department of Plant Physiology, Maria Curie-Skodowska
Nicholas W. Lepp School of Biological and Earth Sciences, Liverpool John
Moores University, Liverpool, United Kingdom
Waldemar Maksymiec Department of Plant Physiology, Maria Curie-Skodowska University, Lublin, Poland
Hideaki Matsumoto
Kurashiki, Japan
Research Institute for Bioresources, Okayama University,
M. Megharaj Remediation of Contaminated Environments Program, CSIRO

Land and Water, Urrbrae, South Australia, Australia
Arun B. Mukherjee Department of Environmental Protection, University of
Helsinki, Helsinki, Finland
C. Nabais Departamento de Botanica, Universidade de Coimbra, Coimbra,
Portugal
R. Naidu Remediation of Contaminated Environments Program, CSIRO Land
and Water, Urrbrae, South Australia, Australia
Barbara Pawlik-Skowronska
Sciences, Lublin, Poland
Institute of Ecology of the Polish Academy of
Contributors
xi
M. N. V. Prasad Department of Plant Sciences, University of Hyderabad,

Hyderabad, India
Tracy Punshon Advanced Analytical Center for Environmental Services, Savannah River Ecology Laboratory, Aiken, South Carolina
Luigi Sanita` di Toppi Department of Evolutionary and Functional Biology
Section of Plant Biology, University of Parma, Parma, Italy
Kenichi Satake Department of Global Environment, National Institute for Environmental Studies, Tsukuba, Japan
Anna Siedlecka Department of Plant Physiology, Maria Curie-Skodowska
Ewa Skorzynska-Polit Department of Plant Physiology, Maria Curie-Skodowska University, Lublin, Poland
Tadeusz Skowronski Institute of Ecology of the Polish Academy of Sciences,
Lublin, Poland
Bruce N. Smith Department of Botany and Range Science, Brigham Young
University, Provo, Utah
C. Tibazarwa Department of Environmental Technology, Flemish Institute for
Technological Research (Vito), Mol, Belgium
Anna Tukendorf Department of Plant Physiology, Maria Curie-Skodowska
Carlos Vale Research Institute for Fisheries and Sea Research, Lisbon, Portugal
P. Vandenkoornhuyse Laboratorie de Genetique et Evolution des Populations
vegetales, Universite de Lille, Paris, France
Daniel van der Lelie Department of Environmental Technology, Flemish Institute for Technological Research (Vito) Mol, Belgium
W. Wenzel Remediation of Contaminated Environments Program, CSIRO
Land and Water, Urrbrae, South Australia, Australia and University of Agricultural Sciences Vienna (BOKU), Vienna, Austria
xii
Contributors
Magorzata Wojcik Department of Plant Physiology, Maria Curie-Skodowska University, Lublin, Poland
Yoko Yamamoto Research Institute for Bioresources, Okayama University,
Kurashiki, Japan
1
Bacteria
Daniel van der Lelie and C. Tibazarwa
Flemish Institute for Technological Research (Vito), Mol, Belgium
INTRODUCTION
This chapter focuses on the way in which bacteria respond to heavy metals in
the environment, either from natural sources or due to anthropogenic activities.
Metal resistance in bacteria is an area whose potential exploitation in the areas
of bioremediation, environmental monitoring, and environmental cleanup technologies is progressively being realized. The inclusion of microorganisms and
their products in metal recovery and environmental biotechnologies has been the
subject of ever-increasing attention during the past few decades. Biologically,
bacterial heavy metal resistance mechanisms and their regulation are interesting
phenomena in themselves, since many metals cations are necessary requirements
in cellular metabolism; thus, in the presence of abnormally high levels of metal
cations, the bacteria must strike a fine balance between uptake of the required
amounts of metal cations and expulsion of the excess. This is referred to as heavy
metal homeostasis.
van der Lelie and Tibazarwa
ECOLOGICAL RELEVANCE OF BACTERIAL

INTERACTIONS WITH HEAVY METALS
Bacteria have been interacting with heavy metals since their early evolutionary
history. Bacterial interactions with heavy metals have been best studies in extreme environments where the emphasis has been to assess bacterial adaptation,
metabolism, tolerance, and resistance to heavy metals. In general, the types of
interactions are directly dependent on the biological role of the heavy metal species in the bacteria: some heavy metals are required as essential cofactors for
protein activity or to stabilize protein conformations, while most heavy metals
tend to be toxic at high concentrations (reviewed in 1). Under these extreme
environments, a selective advantage is conferred to those organisms that have
adopted resistance mechanisms to withstand the toxic effects of high concentrations of heavy metals.
For a given biotope or ecological niche, these types of interactions are also
indirectly affected by the utilization of heavy metals by other organisms within
that biotope. Within the dynamics of ecological evolution, the biological availability (bioavailability) and speciation of heavy metals can act as the main selective pressure determining the composition of the microbial population. It has been
observed for a number of biotopes that if one or more commensals of the biotope
are able to immobilize the heavy metal species in that environment, then the
biological inactivation of the metal species would reduce its bioavailability,
thereby relieving the metal selection pressure. As a result, newer or alternative
populations that would not necessarily require resistance traits to survive could
then flourish on this biotope. Thus, the speciation of heavy metals in a biotope
determines the nature of their interaction with bacteria, which, in turn, may be
wholly influenced by their interaction with other organisms. This phenomenon
has been described for biotopes of both natural and anthropogenic (man-made)
origin.
2.1
Natural Metal-Rich Biotopes
Examples of natural metal-rich biotopes include serpentine (or ultramafic) soils,

which commonly result from the weathering of serpentinite rocks (2). These soils
tend to be enriched in the heavy metals nickel (averaging 10 mg per gram soil),
cobalt, and chromiumthe last two of which are present at lower levels than
the firstand are typified by high concentrations of iron and magnesium and
low nutrient levels (2,3). Some of the best studied nickeliferous biotopes are the
New Caledonian soils (4), which are inhabited by nickel-resistant bacteria as well
as metal-tolerant plants, many of which act as nickel hyperaccumulators (3). An
interesting ecosystem is established in these biotopes driven by a nickel cycle,
in which hyperaccumulating trees extract nickel from deep soil and rock layers
and subsequently store it in their leaves (up to 1% Ni in leaf dry matter). When
Bacteria
the leaves fall off the trees, the nickel is leached out of the plant tissues into the
surrounding top soil; the solubilized metal exerts a localized selective pressure
for a top soil microflora resistant to high levels of nickel (20mM). In fact, the
predominant cultivable microflora found in the top soilfrom bacteria to fungi
and protozoaare resistant to high levels of nickel (4). Interestingly, the microflora, which was not found directly beneath the leaf canopy but in the same
soil, showed tolerance to lower levels of nickel (3 mM) in comparison with the
resistant population. Thus, the nickel selection pressure that exists as a gradient
around the hyperaccumulator plants has a dramatic effect on the composition of
the local microbial population.
2.2
Biotopes Rich in Heavy Metals Resulting

from Anthropogenic Activities
A different situation exists in metal-rich anthropogenic biotopes, resulting from

mans activities, since the metal selective pressure is not necessarily constant.
Such biotopes are found to occur in soils close to leaching activities for mining
of metal ores (5) and also in the vicinities of metal-processing factories (68).
The anthropogenic sites tend to contain particularly high metal concentrations in
the range of 103 105 mg/kg soil (see also 7,9). Some of the best studied are the
pyrite-like biotopes, in which the interaction of Thiobacillus bacteria with iron
and other heavy metals is directly related to a sulfur cycle, necessary for the
maintenance of a balanced ecosystem within this biotope. The predominant and
best characterized species in this genus is Thiobacillus ferrooxidans, which is
widely used in biological mining operations (reviewed in 5,10,11). This autotrophic obligate chemolithotroph derives its energy and reducing power from the
oxidation of ferrous iron and reduced sulfur compounds under strict acidic (low
pH) conditions (11). All thiobacilli are capable of oxidizing reduced sulfur compounds to meet their energy requirements: T. ferrooxidans utilizes ferrous iron
as an electron donor, and the key enzymes require sulfate ions as cofactor components. This organism is a commensal of pyrite-like (iron-rich) biotopes where it
plays an important role in the cycling of Fe and S (11). This cycling involves
the oxidation of reduced sulfur compoundsincluding elemental sulfur, sulfides,
and sulfitesto sulfuric acid. Pyrite-like biotopes, which contain high levels of
insoluble iron sulfides, can be oxidized by the bacteria to yield soluble iron sulfates, thereby leaching the metal into solution (12). This process also occurs
in the presence of other minerals, including nickel, copper, lead, gallium, zinc,
cobalt, and copper, and can result in the release of high concentrations of soluble
heavy metal fractions into the surrounding environment. In the case of iron, the
organism deals with the change in the biological availability of this cation by
generating and regenerating the oxidant Fe3 from Fe2, which can be utilized
for its own energy production, and as a micronutrient by other commensals of
this biotope. The leaching metabolism of this organism can be detrimental

to the organism if the solubilized metal cations reach toxic concentrations or if
extremely toxic cations, such as arsenic present in arsenopyrite minerals, are
released in a more bioavailable form. To overcome the environmental constraints
caused by these heavy metals, Thiobacillus spp. have evolved resistance mechanisms to a variety of toxic metals, including arsenic and mercury. For example,
the resistance to mercury volatizes the metal thereby removing it from solution.
Nevertheless, it is clear that although the organism has a significant ecological
advantage in being able to solubilize and consequently make trace elements available for its metabolism, this same property can have detrimental effects on its
own proliferation and those of other commensals in the ecosystem if a foreign
selective pressure was to be introduced into the ecosystem, such as that posed
by increased contamination of a toxic heavy metal.
The sulfate-reducing bacteria are found in different biospheres of both natural origin, such as thermal sulfur springs, ocean and sea beds), and of anthropogenic origin, such as mining waters from metal sulfide deposits (reviewed in 13).
This group includes Desulfovibrio spp., Desulfobulbus spp. (14), Desulfobacteriaceae and other strains within the -proteobacteria, gram-positive species as well
as archaebacteria, and are also implicated in the sulfur cycle (their phylogeny is
presented in 15 and 16). The Desulfovibrio bacteria are chemoorganotrophic,
strictly anaerobic, gram-negative organisms that are able to utilize sulfates and
other partially oxidized forms of sulfur for anaerobic respiration. The resulting
metabolite is hydrogen sulfide, which reacts easily with heavy metal cations forming metal sulfides of low solubilitythe inverse metabolism to that of the thiobacilli. In addition, various sulfate-reducing bacteria can directly reduce metal
oxianions, such as arsenate and chromate, that are subsequently precipitated as
metal-sulfate complexes. Thus, these two groups of organisms have opposite effects on heavy metal solubility and therefore biological availability: the thiobacilli
and other sulfur-oxidizing bacteria increase the bioavailability by leaching out
heavy metal sulfates whereas the sulfate-reducing bacteria reduce the bioavailability by forming poorly soluble metal sulfides, which can mineralize out of
solution with organic matter. In acid mine drain waters, the occurrence of both
types of bacteria would affect the speciation of heavy metals, which in turn is
dictated largely by the Fe and S element cycles, and would therefore influence
the biological makeup of the ecosystem.
2.3
Sites of Metallurgical Industries
Bacterial interactions with heavy metals in contaminated sites resulting from metallurgical processing and related industries has been a major area of interest during the past decades, particularly in industrialized nations where the unclean practices of the metallurgy industry during the 19th and 20th centuries resulted in a
Bacteria
legacy of large areas of soil contamination that are unsuitable for life. The
Maatheide soil in Lommel, Belgium is one such example, characterized by heavy
contamination with zinc (700040,000 ppm), aluminum (39,000 ppm), lead
(2000 ppm), cadmium (20 ppm), and copper (1000 ppm) (6,8). In these soils,
Zn was found to be highly abundant. It was also the most biologically available
heavy metal and the main selection pressure for bacteria inhabiting this biotope
(17). The microbial diversity of these soils has been monitored periodically during the past 12 years. Early studies revealed that the majority of bacteria on this
biotope were metal-resistant Ralstonia eutropha-like strains (40% of the population), showing high levels of zinc resistance. Samples taken 10 years later revealed that this subgroup made up only 14% of the population and had been
replaced by other gram-negative and even gram-positive bacteria that showed
reduced resistance to heavy metals, including zinc (8). The progressive decrease
in zinc toxicity of these soils as a result of natural weathering processes, mans
attempts at remediation, as well as the microbial interaction with the heavy metals
has been attributed to this succession in microbial populations. The Ralstonia
eutropha-like strains, of which the type strain CH34 is the best characterized,
are gram-negative (-protobacteria) soil bacteria displaying resistance characteristics to a variety of heavy metals, and are thus well suited for these types of
biotopes (1820, reviewed in 2123). As described later in this chapter, the interactions of these bacteria with heavy metals can result in alteration of the metal
speciation as well as its bioavailability to other organisms. Similar types of heavy
metal resistance, such as the czc cobalt, zinc, and cadmiumresistant determinant,
are found in Ralstonia eutropha DS185, the dominant heavy metalresistant Ralstonia eutropha strain isolated from the Maatheide (8,20). The reduced bioavailability of Zn, Cd, Ni, Co, and Pb has relieved the selection pressure on the microbial populations so that, with time, what were the pioneering and dominant
metalloresistant bacteria have been succeeded with metallotolerant species (8,20).
Eventually, these metallotolerant populations may be succeeded by metallosensitive organisms, which are already present in the soil, albeit in low numbers. This
was demonstrated during in situ heavy metal inactivation experiments using the
soil additives beringite and steelshots to immobilize the bioavailable fraction of
heavy metals present in Maatheide (17). For untreated soils, a strong increase in
the metal-resistant subpopulations was observed with increasing metal contamination. However, 12 months after the application of the soil additives, up to a
100-fold decrease in the heavy metalresistant subpopulations was observed for
beringite- and beringite-plus steelshots-treated soils. However, treatment with
other soil additives resulted in hardly any decrease of the heavy metalresistant
subpopulations. It was hypothesized that the observed decrease in heavy metal
resistant bacterial subpopulations was directly reflecting the efficacy of in situ
metal immobilization. Further physicochemical and biological evaluation confirmed the sustainability of zinc and cadmium immobilization by beringite and
beringite plus steelshot treatment, while the inefficient heavy metal immobilization by the other soil additives was also confirmed. This shows that the ecology
of heavy metalresistant soil bacteria, which is directly affected by the bioavailability of the heavy metals, can be used as a tool to predict the efficacy of in
situ heavy metal immobilization.
3
HEAVY METALRESISTANT BACTERIA
The speciation of a heavy metal determines the resultant interaction with bacteria.
Being able to withstand high concentrations of bioavailable heavy metals clearly
offers an advantage for surviving harsh environmental conditions caused by increased heavy metal concentrations, and it is not surprising that metal resistance
determinants and metal tolerance capabilities are widespread in different bacterial
genera. Strains isolated from natural and anthropogenic biotopes have been assigned to the gram-negative -proteobacteria, which include the Ralstonia eutropha strains (now termed Ralstonia metallidurans, and formerly Alcaligenes eutrophussee 20), and -proteobacteria including many Pseudomonas strains.
Both the - and -proteobacteria exhibit heavy metal resistance phenotypes varying widely in the micromolar to millimolar range, which can be of plasmid or
chromosomal origin. However, the highest resistance levels are found in members
of the subgroup (reviewed in 7, 21). The other classes isolated from anthropogenic biotopes include low-GC gram-positive species, which show heavy metal
resistance in the micromolar concentration range, and some high-GC content
gram-positive species (e.g., Arthrobacter spp.; 24), which can exhibit resistance
phenotypes in the low millimolar range. The heterogeneous group termed Burkholderia (which are still awaiting proper classification using a molecular taxonomy approach) predominates in some natural nickeliferous biotopes, although
other strains have also been isolated, belonging to the genus Acinetobacter, Agrobacterium, Comamonas, and Arthrobacter (24). However, the highest heavy
metal resistance phenotypes are found in the archaebacteria and in the Thiobacillus genus, which are mostly acidophilic oligate chemolithotrophs and which have
been isolated from highly polluted sites where metal concentrations can reach
molar levels and are highly bioavailable due to the low pH (2) conditions.
Heavy metal resistance has now been characterized in many gram-negative
and gram-positive bacteria, particularly those with a high-GC content, and, to a
lesser extent, in the archaebacteria. Microbiological data on the types and numbers of bacterial populations in an environmental sample has been used with some
degree of accuracy to assess the heavy metal pollution of the sample (reviewed in
7,17). However, due to the inaccuracies of sampling from the environment as
well as the complex nature of the endogenous microbial populations, strategies
are being pursued that rely principally on understanding the bacteria-heavy metal
interactions at the molecular level.
The interactions between bacteria and heavy metals have been widely ex-
Bacteria
ploited: the most successful applications include the utilization of the acidophilic,
sulfur-oxidizing T. ferrooxidans in leaching activities for the extraction of various
heavy metals from low-grade ores (reviewed in 5,10). The resistance mechanisms
of some other bacteria, particularly the - and -proteobacteriaceae, have also
been used as the underlying principles for the development of various environmental technologies: therefore, these will be discussed in greater detail.
3.1
Bacterial Heavy Metal Resistance Mechanisms
The physiological role of the metal is a key determining factor in its fate when
it is encountered by a bacterium. Some metals are tolerated at high levels because
they are (bio)chemically unreactive or simply do not pose a toxicity threat even
at high concentrations, whereas the majority exhibit some sort of toxicity above
threshold concentrations (reviewed 1,2527). Metal toxicity may result from the
fact that (1) high concentrations of some cations can competitively inhibit the
normal functioning of analogous metals that are essential for cellular metabolism,
e.g., as enzyme cofactors; (2) some metals are not tolerated even at low concentrations because they can interact with cellular components and damage them; (3)
they can form strong interactions with key proteins in the cell, thereby inhibiting
or hyperstimulating their activity; (4) DNA-metal complexes can damage the
structural integrity of DNA, which can induce mutations. Bacteria have adopted
the following mechanisms to deal with potentially toxic concentrations and types
of metal species (also reviewed in 28):
1. Pumping out the metal before it can accumulate in the bacterial cytoplasm, typically involving an elaborate protein pump system in the
membrane.
2. Actively taking up the metal by specific uptake proteins that immediately channel the toxic metal to a specific detoxification pathway.
3. Exclusion of the metal by a permeability barrier; exclusion can be total
or partial.
4. Enzymatic conversion of the metal species to a less toxic form (detoxification) or into an alternative form that can activate rapid removal
from the cell.
5. Sequestration or immobilization of the metal in a compartment of the
cell or on the outer cell.
Examples of how these mechanisms can interact to confer resistance to specific
heavy metal cations in bacteria are summarized in Table 1 and will be described
below. It is important to note that many of these mechanisms are encoded by
extrachromosomal elements, mainly plasmids or on mobile genetic elements, and
these are generally the best characterized. Nevertheless, chromosomally encoded
resistance mechanisms have also been described in the literature. Some organisms
have evolved both chromosomal and plasmid-encoded resistance mechanisms
TABLE 1 Examples of Bacterial Resistance Mechanisms to Heavy Metalsa
Ni
Zn
Cd
Co
Hg
Pb
Cr
As
(Sb)
a
ATPases
CDF
Enzymatic
reduction
Sequestration
Undefined
efflux
Others
cnrYXHCBA
nccYXHCBAN
nreAB
czcNICBADRSE
czrRSCBA
zntRA
ziaRA
cadCA
smtBA
czcNICBADRSE
czrRSCBA
cadCA
smtBA
nccYXHCBA
cnrYXHCBA
czcNICBADRSE
czrRSCBA
coaRA
silPCBARSE
copSRABCDGFH
copABCDRS
pcoABCDRS
copYZAB
merRTPCAD
merRTPAD
merRTPABD
pbrTRABCD
cadCA
chrDCAB
chrAB
chrAB
arsRDABC
arsRBC
CnrCBA
NccCBA
X
CzcCBA
CzrCBA
X
X
X
X
CzcCBA
CzrCBA
X
X
NccCBA
CnrCBA
CzcCBA
CzrCBA
X
SilCBA
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
ZntA
ZiaA
CadA
X
X
X
CadA
X
X
X
X
X
CoaA
SilP
CopF
X
X
CopA, CopB
X
X
X
PbrA
CadA
ChrD
X
X
ArsA
X
X
X
X
CzcD
X
X
X
X
X
CzcD
X
X
X
X
X
CzcD
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
MerA
MerA
MerA
X
X
X
?
?
ArsC
ArsC
?
?
X
CzcE
X
X
X
X
SmtA
CzcE
X
X
SmtA
X
?
CzcE
X
X
SilE
CopH
X
PcoE
X
X
X
X
PbrD
X
X
X
X
X
X
X
X
NreAB
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
ChrA
ChrA
ChrA
ArsB
ArsB
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
CzcN, I
X
X
X
CopABCD, G
CopABCD
PcoABCD
X
MerT, P, C
MerT, P
MerT, P, B
PbrT, B, C
X
ChrB, C, D
ChrB
ChrB
X
X
Genes present in the operon but not specified in the table are involved in regulation.
Genomic
localization
Plasmid
Plasmid
Plas./chrom.
Plasmid
Chromosome
Chromosome
Chromosome
Plasmid
Chromosome
Plasmid
Chromosome
Plasmid
Plasmid
Plasmid
Plasmid
Chromosome
Chromosome
Chromosome
Plasmid
Plasmid
Plas./chrom.
Chromosome
Plas./chrom.
Plas./chrom.
Plas./chrom.
Plasmid
Plasmid
Plasmid
Plasmid
Chromosome
Plasmid
Plas./chrom.
Ref.
21,35
21,36
36,67,68
32,62
37
56,104
58
25,27
94
32,62
37
25,27
94
21,36
21,35
32,62
37
105
38
22
60,61
60,61
59,60
70
70
70
55
25,27
62
84
83,85
27,75
27,109
Ag
Cu
Proton/
cation
antiporter
Cation
Operon
structure
Bacteria
even for the same metal; however, the physiological interplay between both types
of systems is rarely well understood, and in this chapter chromosomally and
plasmid-encoded systems will be considered as separate, unless identical genes
are involved. The evolutionary significance of chromosomal, plasmid, mobile
element localization of resistance operons is assessed in 28.
Ralstonia eutropha CH34 is perhaps a model organism in terms of bacterial
resistance mechanisms and interaction with heavy metals in soils. CH34 is the
type strain of a group formerly known as Alcaligenes eutrophus but whose taxonomic classification was recently revised to Ralstonia eutropha (20) and is currently being renamed to Ralstonia metallidurans (Mergeay, personal communication). This strain and its derivatives were originally isolated from the metal-rich
sediments of a zinc factory in Belgium (18). One of the most remarkable properties of CH34 is its ability to thrive in metal-rich environments: this subject has
been extensively reviewed, most recently in (9,21,22). In order to survive in
metal-rich biotopes, CH34 has evolved an astounding number of genetic determinants that encode for resistance to many heavy metals and metal compounds
(18,21,22). These determinants, which are mainly localized on two megaplasmids, pMOL28 (18,29) and pMOL30 (18), include resistance determinants to
combinations of cobalt and nickel (cnr), cadmium, zinc, and cobalt (czc), chromate (chr), mercury (mer), thallium (tll), copper (cop), lead (pbr), and others
awaiting definition.
The resistance mechanisms of CH34 and other metal-resistant bacteria, as
well as their genetic organization and regulatory control, are detailed below. The
interactions with essential metals, including Co, Cu, Ni, and Zn, are differentiated
in comparison with interactions with the highly toxic metals and metalloids such
as Ag, Cd, Cr, Hg, Pb, Sb, and Bi.
3.1.1
Three-Component Cation/Proton Antiporter Systems

for Metal Efflux
These systems are members of the major facilitator superfamily of transporters

(reviewed in 69). The model system for three-component cation/proton antiporters is the Czc system of R. eutropha CH34, which confers resistance to cadmium,
zinc, and cobalt by pumping out these metals when their intracellular concentrations exceed a certain threshold (22,3033). This metal pump is encoded by
the structural gene czcCBA. Other examples of three-component cation/proton
antiporters involved in heavy metal resistance include CnrCBA, mediating efflux
of cobalt and nickel in CH34 (34,35), NccCBA, with substrates nickel, cobalt,
and cadmium in R. eutropha strain 31A (36), CzrCBA, with substrates zinc and
cadmium in P. aeruginosa (37), and SilCBA mediating efflux of silver in Solmonella typhimurium (38).
The mechanism of efflux has been described for the Czc system (22,30
32,39,40), and it has been demonstrated to be essentially a zinc efflux system.
10
Three membrane proteins are involved in transporting the divalent cations

zinc, cadmium, cobaltacross the inner membrane. The cytoplasmic membranespanning A protein, which constitutes the actual cation/proton antiporter, is
a member of the RND (resistance/nodulation/division) superfamily of transporter
proteins (3943). Typical of the RND proteins, the A protein translocates the
metal cation from the cytoplasm in one channel with the concomitant uptake of
protons from the cytoplasm into the periplasm through a separate, adjacent tunnel (27,31,40,41). This system of transport of a substrate differs from the ATPdriven mechanisms of ATP-binding cassettes (ABC transporters; 44) where the
driving force for metal cation transport comes from the translocation coupling
of uptake of protons and expulsion of cations into the periplasm. The CzcA/
CzrA/NccA/CnrA proteins are highly homologous, showing conservation in the
regions corresponding to the proton tunnels; however, the metal translocating
region has evolved specifically for the cation substrate. The proton uptake results
in an increased pH in the extracellular environment, which is related to the metal
concentration, and for R. eutropha CH34 alkanization of the extracellular medium
contributes to the extracellular immobilization of the metal cations.
The second protein in the cation translocation pathway is the periplasmic
B protein (which is anchored at its N terminus to the outer membrane), which
has the specific function of ensuring the outward passage of the metal cations
from the cytoplasm across both membranes, without leakage into the periplasm
(31,41). The B protein is a member of a family known as membrane fusion proteins, or MFPs (31,41,45) and is functional as a dimer in the CzcCB2A complex
(31). Therefore it is hypothesized that all B subunits of related metal resistance
exporters would also be functional as dimers.
The final passage of the metal cations through the bacterial membrane is
mediated by the C protein. The latter is a member of the outer membrane
factors or OMF family (41,43,45), which is anchored to the outer membrane and
protrudes into the periplasm (31). The function of this protein is to translocate
the metal cation from the MFP B protein toward the cell exterior. In Czc-mediated
efflux, an outer membrane protein (Omp) protein is believed to carry out the
terminal step in removing the cation from the outer membrane surface (31), and
this is also likely to be the case for the other related metal resistance systems.
This translocation function across the outer membrane is specific for the cation
substrate, which explains the lower homology of the C protein, even for operons
sharing a common substrate. The membrane orientation of the C protein is believed to be important in the ultimate translocation of the cation from CzcC to
an Omp, which would transport the cations away from the cell surface (31).
Recently, cation/proton antiporter systems for metal cation efflux have also
been described in gram-positives species. Interestingly, these units contain only
two components, which are homologues of the A and B proteins of the gramnegatives species and seem to lack the OMF, the C protein. Examples include
Bacteria
11
the newly described NrsAB proteins that are involved in nickel, cobalt, and zinc
homeostasis of Synechocystis sp. (46).
3.1.2
Efflux Based on Cation Diffusion Facilitation
This resistance mechanism is based on metal cation efflux by an inner membrane

protein termed the cation diffusion facilitator, or CDF (39,41,43). CDFs typically
act in concert with other cation efflux mechanisms (Table 1), but alone they can
confer a low-level resistance phenotype by way of efflux of metal cations across
the cytoplasmic membrane resulting in reduced accumulation in the cytoplasm
(39,43). The best characterized bacterial CDF is CzcD, which spans the cytoplasmic membrane of R. eutropha CH34 (39) and is implicated in resistance to
zinc, cadmium, and cobalt as well as in the regulation of this resistance mechanismalthough this has yet to be fully understood (39). CzcD is thought to be
functional at very low metal concentrations, where it may act in the uptake of
metal cations into the cytoplasm, while at high concentrations of metals, the protein would sponge up metal cations through high-affinity binding by its numerous
His residues and exporting the metal substrates across the cytoplasmic membrane
(39). It has yet to be determined whether CzcD is itself a regulatory protein or
simply an accessory protein whose export activity affects one or more regulators
(33,39,47). Eukaryotic homologues of CzcD include COT1 (48) and ZRC1 (49)
of Saccharomyces cerevisiae, which efflux cobalt and zinc, respectively, from
the cytoplasm to the mitochondria.
3.1.3
Heavy Metal P-Type Efflux ATPases
ATPases are protein pumps that utilize the energy derived from ATP hydrolysis
to actively transport substrates from one cellular compartment to another; their
enzyme activity is limited to ATP hydrolysis, which means that the substrate is
left unchanged. All known ATPases involved in transport of heavy metals for
resistance purposes are membrane-spanning, typically integral proteins of the
cytoplasmic membrane, and function in the outward translocation of the metals,
i.e., expulsion from the cell interior to the exterior. The requirement for ATP
hydrolysis taxes the organism for energy, which could explain why many of
the metal resistance ATPases function in the elimination of highly toxic metal
cations.
Some of the best characterized ATPases involved in heavy metal resistance
are those belonging to the P-type CadA family (50), which occur ubiquitously
in both gram-positive and gram-negative genera. CadA of Staphylococcus aureus,
the model system, functions in cadmium, lead, and bismuth efflux (reviewed in
2527); it achieves this by turning over an aspartyl phosphate intermediate during
the catalytic cycle (51,52; reviewed in 53 and 54). Other members of this subfamily include PbrA, functioning specifically for lead efflux in R. eutropha CH34
(55); SilP for silver efflux in S. typhimurium (38); ZntA for zinc, cadmium, and
12
lead efflux in Escherichia coli (56,57) and the closely related ZiaA for zinc efflux
in Synechocystis (58); and CopF for copper efflux in R. eutropha CH34 (22).
The other types of ATPases involved in metal efflux include the chromosomally encoded P-type ATPase CopB for copper export in E. hirae (which acts
in concert with the CopA uptake ATPase; 59, reviewed in 60 and 61), ChrD for
chromate efflux in R. eutropha CH34 (62), and ArsA of E. coli (reviewed in 63).
In R. eutropha CH34, the amino acid sequence of the newly described ChrD
protein, which has been implicated in the resistance mechanism to Cr6, shares
homology with ATPases of ABC transporters (62). However, the precise role of
this ATPase function in relation to the main efflux-based resistance mechanism
of the ChrAB proteins (see below) has yet to be demonstrated. The gram-positive
ATPases are generally able to pump out the metal cations directly from the cytoplasm, whereas the gram-negative ATPases require additional protein functions
to ensure that the cations are translocated across both the inner and outer membranes unidirectionally toward the outside, without leakage into the periplasmic
space. Thus, examples of ATPase accessory proteins include ArsB, which complements ArsA for arsenite efflux in E. coli (8,64), and PbrB, a putative outer
membrane lipoprotein that likely channels the translocation of Pb2 ions from
the PbrA ATPase across the outer membrane of R. eutropha CH34 to the cell
exterior (55).
3.1.4
Other Types of Efflux Mechanisms
Copper resistance in both gram-positive and gram-negative bacteria typically involves a combination of uptake, efflux, and, in some cases, storage. Copper is
an essential micronutrient for bacteria; its utilization and movement in the cell
are intricately controlled because free copper ions can be very toxic. The best
characterized resistance determinants include the pco of E. coli (reviewed in 65),
the cop operons of Pseudomonas (53,60,66) and R. eutropha CH34 (22,23), while
copper tolerance has been characterized for the chromosomally encoded cop operon of E. hirae (59; reviewed in 60,61). The common features of the E. coli,
R. eutropha, and Pseudomonas copper resistance systems include the ABCD
structural genes that encode an inner membrane uptake protein (PcoD/CopD),
an outer membrane uptake protein (PcoB/CopB), and two periplasmic copperbinding proteins (PcoA/CopA and PcoC/CopC). Both the CopA and CopB bind
copper with high affinities: for example, the CopB protein of R. eutropha CH34
can potentially bind 23 Cu2 cations per monomer (22). As described in a previous section, R. eutropha CH34 also mediates efflux by the CopF ATPase; however, the physiological interplay between this and the CopABCD system has yet
to be defined.
Broad host range expression of nickel resistance in proteobacteria is mediated by the NreAB proteins (36,67,68). The mechanism is based on efflux, which
is thought to be principally carried out by the NreB protein (68). In R. eutropha
Bacteria
13
31A, the Nre system occurs together with the Ncc cation/proton antiporter system, resulting in the expression of high-level nickel resistance (36,68). In Synechocystis sp., the NrsD protein, which shows high homology with the NreB protein
of R. eutropha 31A (46), was shown to be a membrane-spanning divalent cation
transport protein of the major facilitator superfamily (reviewed in 69) that binds
nickel with high affinity.
3.1.5
Enzymatic Reduction
Enzymatic reduction is a highly specific resistance mechanism adopted by bacteria for the transformation and removal of highly toxic metal species. Enzymatic
reduction of metal cations is highly energy-dependent, and in some examples of
resistance, chemical reduction can result in a metal species that is more toxic.
The best characterized is mercury resistance, which is also the single most
widespread of all antimicrobial resistances. The Tn501-localized mer operon of
E. coli is perhaps the best studied of all metal resistance systems (see review
in 70). Mercuric ions are extremely toxic to bacteria because they can interact
nonselectively with the thiol groups of proteins. Therefore, a complex mechanism
of resistance has evolved, which has been relatively well conserved among eubacteria (25,26). The mechanism involves uptake of Hg2 into the cell interior
to avoid its deleterious interaction with cellular components, and this is carried
out first by the MerP protein in the periplasm and then the MerT protein that
spans the inner membrane (70).
The mercuric reductase protein, MerA, which is localized in the cytoplasm,
enzymatically reduces the toxic Hg2 ions to Hg0: this nontoxic end product simply volatizes out of the cell. Mercuric reductase is a highly specific metalloenzyme reacting only with Hg2; therefore, in bacteria harboring a broad-spectrum
resistance to inorganic Hg2 as well as organomercurial compounds the reductase
functions in concert with an organomercurial lyase enzyme, MerB, which cleaves
the Hg2 moiety prior to chemical reduction by the MerA protein (32,71,72).
Arsenate reductase, or ArsC, is the main component of the plasmid-encoded
arsenate resistance mechanism of S. aureus (27,73,74), E. coli (75), and chromosomally encoded mechanisms of some Pseudomonas species (76) and E. coli
(77). This cytoplasmic metalloreductase reduces intracellular arsenate (As5) to
the more toxic arsenite (As3). The latter is extremely reactive with the thiol
groups of proteins and is therefore rapidly expunged by the ArsB protein, which
in S. aureus transports the arsenite across the cell membrane in an ATPaseindependent manner, while in E. coli and other gram-negatives bacteria the ArsA
and ArsB proteins combine to form an efflux ATPase (64,78; reviewed in 27).
Metabolically, it seems paradoxical for the organism to intentionally convert the
already toxic As5 to the more toxic As3. However, the reasoning may be related
to maintaining phosphate homeostasis; since arsenate is structurally similar to
the phosphate oxyanion, the organism alters the contaminating metal to arsenite,
14
which has no analogue that is essential to metabolism, and can therefore be rapidly removed from the cell. A Desulfomicrobium strain has been described that
utilizes the reduction of arsenate to arsenite to meet respiration requirements
rather than as a resistance mechanism (79).
Arsenate reduction is the more widespread resistance mechanism for arsenicals; however, some bacteria confer resistance by oxidizing arsenite and other
arsenocompunds to the less toxic arsenate, with the most effective arsenite oxidizer being Alcaligenes faecalis (80). Also, there are two recent, independent
reports of new types of chemolithoautotrophic gram-negative species that are
capable of oxidizing arsenite to arsenate (81,82), which in the strain NT-26 is
effected by the periplasmic-located arsenite oxidase (82). Bacteria have also been
described that can oxidize arsenite to elemental arsenic (reviewed in 27).
Chromate resistance has been shown to include a reduction of the toxic
Cr6 cation to the less toxic and less soluble Cr3. As yet, no specific chromate
reductase has been linked to chromate resistance, and it is believed that a chromosomal reductasemost likely one involved in sulfate metabolismcarries out
this function in a variety of bacteria (62,8387).
3.1.6
Other Mechanisms of Heavy Metal Resistance
Described below are mechanisms that in themselves are unique due to the specific
toxicities or cellular utilization of the metal, or simply because they have only
recently been described and await detailed characterization. For the toxic metals,
a recurring theme of the resistance mechanism is cation uptake, immediately
followed by transformation and then export from the cell. Specific examples are
given below.
In the case of chromate, which is considered to be highly toxic to biological
systems because it can interfere with sulfate transport systems, both plasmid and
chromosomal determinants have been reported. The ChrA protein is an as-yet
uncharacterized transmembrane efflux protein that functions in the expulsion of
the toxic chromate in P. aeruginosa (84), Bacillus spp. and Enterobacter cloacae
(86) and R. eutropha. Chromate is capable of entering the cell interior via the
sulfate uptake systems. Under sulfate-starved conditions, chromate is readily
taken up into the cell and rapidly reduced by the chromosomally encoded sulfate
reduction systems (87). The rapid turnover of Cr(VI) to the less soluble Cr(III)
(which is not an energetically favorable enzymatic process for the bacteria) does
not allow for the induction of the chr genes, which are activated by Chr(VI)
(87). However, under sulfate-saturated conditions, chromate uptake into the cells
occurs more slowly. Concomitantly, the sulfate-reducing systems become inactivated and this results in the intracellular accumulation of Cr(VI)the trigger for
induction of chr expression. In R. eutropha, ChrA activity has been shown to be
induced under sulfate-saturated conditions (87). Thus, ChrA activity is inducible
on accumulation of intracellular Cr(VI) which occurs under sulfate-saturated con-
Bacteria
15
ditions, and this triggers a rapid expulsion of the toxic oxy anion. The function
of the ChrB protein, which is also essential for chromate resistance, has yet to
be defined (30,62), whereas the ChrC protein shows homology with a manganesecontaining superoxide dismutase and ChrD is likely to be an ATPase homologous
to those of ABC transporters.
3.1.7
Complexation, Sequestration, and Precipitation

of Heavy Metals
Sequestration of heavy metals in different compartments of the bacterial cell can

serve two purposes in dealing with high concentrations of heavy metals: (a) to
immobilize the metal to prevent it from moving freely within the cell, which can
be reversible or not, and (b) to concentrate or precipitate the metal out of solution
irreversibly in order to completely abolish its toxic activity.
The cellular sequestration of metals that occurs in concert with resistance
mechanisms largely depends on the metal species itself, on how the metal is
exported out of the cell, and on the metal concentration. Sequestration of the
metal typically involves high-affinity proteins as well as nonspecific interactions
with polymeric substances or other types of biological adsorbents produced by
the cell.
3.1.7.1 Extracellular. In CH34, postefflux sequestration of the metal cations at the cell surface is an important mechanism that prevents reentry of the metal
into the cell, particularly when the extracellular concentrations are high (32,88,89).
In cultures of CH34, zinc and cadmium removal results in immobilization of the
metal cations as complexes with carbonates, bicarbonates, and hydroxides. These
processes were found to be induced by carbon dioxide emitted by the metabolic
activity as well as alkalinization of the extracellular environment resulting from
the cation/proton antiporter activity, while in other R. eutropha strains secretion of
extracellular polysaccharides has also been implicated. These metal complexes are
precipitated as crystals at the cell surface (21,32,88,89). This bioprecipitation process precedes the release of extracellular polysaccharides and nucleation proteins,
which also act to sequester the metal at the cell surface, as demonstrated for the R.
eutropha-like strain ER121 (42). As described later in this chapter, the physical
immobilization of the metals outside the bacterial cells has been exploited in bioreactor designs for the removal and recovery of metals from metal-contaminated
wastes (32,90). It was recently demonstrated that cnr- and ncc-mediated resistance
mechanisms also result in metal sequestration, which in accumulation experiments
with nickel could result in 48% removal of the metal from culture supernatants (S.
Taghavi, personal communication). Furthermore, when CH34 is pregrown in the
presence of zinc or cadmium and transferred to a medium with high levels of nickel,
metal precipitation at the cell surface is observed (32). Thus, a common postefflux
sequestration/immobilization mechanism seems to be operable in CH34; the pre-
16
cise nature of this process has yet to be pinpointed, although precipitation kinetics
implicate high-pH-induced complexation in the form of metal carbonates as being
a major underlying cause (32,88,89,90). It follows that because cation/proton antiporter efflux mechanisms by their nature lead to high localized concentrations of
metal at the cell surface, then all CBA-type mechanisms can potentially induce
some type of postefflux immobilization of their substrates.
Some gram-negative organisms have evolved resistance mechanisms that
allow for the sequestration of high concentrations of heavy metals in the periplasmic space. For essential cations, such as copper, a newly assigned gene is copH
whose gene product shares high homology with CzcE of R. eutropha CH34,
which may function in cadmium, zinc, and cobalt sequestration in the periplasm
(van der Lelie et al., unpublished data). The function of CopH is still under investigation; however, preliminary data indicate that CopH is essential for copper
resistance since copH mutants displayed a reduced copper resistance phenotype
(van der Lelie, unpublished data).
In P. syringae var. tomato, excess copper is stored in the periplasmic space
in its blue form (Cu2) by an unknown mechanism that may involve high-affinity
copper-binding proteins (60). In E. coli containing plasmid pRJ1004, no periplasmic copper storage has been observed (91), although an additional periplasmic
protein, PcoE, has been described which is a high-affinity copper-binding protein
that may be implicated in this function (92). PcoE is homologous to the silverbinding periplasmic SilE protein, whose crystal structure was recently solved
(38). The SilE protein structure is novel: a periplasmic protein, SilE, binds four
Ag cations per monomer. Apo-SilE has very little secondary structure that likely
maintains it in an inactive state, while metallized SilE adopts a highly ordered
structure rich in -helical content, resulting in protein activation (Silver, personal
communication). This type of structural control on protein activity prevents fortuitous activation by cations other than Ag, which renders the system highly specific for silver cations.
3.1.7.2 Intracellular Intracellular sequestration of heavy metals is a
mechanism shared between prokaryotic and eukaryotic systems. This is due to
a family of small proteins called metallothioneins that are typically short polypeptides of approximately 60 residues. These proteins are rich in cysteine residues
and bind heavy metals with high affinity, thereby immobilizing them within the
cytoplasm (reviewed in 27). Metallothioneins are widely dispersed in eukaryotes,
the best characterized being that of the Norwegian rat, Rattus norvegicus (93),
while the best characterized for bacteria is SmtA which effects zinc resistance in
the cyanobacterium Synechococcus (94). Most eukaryotic metallothioneins bind
nonspecifically to certain families of metal species, whereas SmtA binds with
high affinity to its substrates Zn2 and Cd2 (with a preference for the former)
Bacteria
17
in metal-binding clusters, which are organized at the N and C termini of the

protein (94).
Lead resistance in S. aureus and Citrobacter freundii has also been shown
to involve the detoxification of Pb2 cations to a less reactive species and the
subsequent intracellular immobilization of the metal as lead-phosphate complexes (95). This prevents the detoxified lead from participating in any cellular
activity that could be potentially harmful to the organism and also permits the
organism from wasting energy in pumping out the cation. The recently described
lead resistance operon of R. eutropha CD34 may also mediate sequestration of
the metal, and PbrD, a presumed Pb-binding protein, is believed to be involved
in this function since deletion mutants abrogated lead sequestration in CH34 derivative strains (55).
3.2
Physiological Role of the Metal Determines the

Resistance Mechanism
Resistance to heavy metals that are required as trace elements for bacterial metabolism poses an interesting paradox for bacteria. On the one hand, resistance
against toxic levels of the metal confers a selective advantage of the bacteria as
compared with sensitive commensals within a given biotope; however, the bacteria must ensure that resistance does not jeopardize the normal homeostatic utilization of the metal required for normal growth. The evolution of bacterial resistance
and tolerance to metals has been widely debated during the past decade for several
reasons: (a) there seems to be common features in the driving forces by which
bacteria recruit heavy metal and drug resistance genes; (b) the evolutionary
choice of the mechanism of resistance that the bacteria adopts seems to be closely
related to whether the metal is essential or toxic (reviewed in 28). The essential
metals include zinc, nickel, cobalt, and copper, while the highly toxic metals
include cadmium, mercury, silver, arsenic, chromate, thallium, lead, strontium,
and bismuth. Most resistance mechanisms are inducible by metal, which enables
the organism to expend energy only when absolutely necessary. Thus, in some
regulation mutant strains, such as the R. eutropha CH34 derivative AE963, which
constitutively expresses the cnr operon and as a result becomes nickel-deficient
(96), the organism can become dependent on the metal presence for normal
growth.
3.2.1
Regulation of Heavy Metal Resistance
As a general principle, bacterial resistance mechanisms must necessarily take

into account the metabolic requirements of the organism and the physiological
role of the metal. A finely tuned regulation of the resistance mechanisms is therefore a prerequisite so as not to jeopardize homeostasis.
18
As suggested by Table 1, most metal resistance mechanisms can be generally divided into those based on efflux, enzymatic conversion, or sequestration.
The majority of the coding regions (structural genes) seem to exist as modules
(Fig. 1). However, the genes of regulation seem to have been acquired by a mix
and matching of different genetic systems, which may have taken place at different evolutionary stages (Table 2) (reviewed in 1,9799).
3.2.1.1 Regulation of Cation/Proton Antiporters As shown in Table 2,
the main types of regulatory modules controlling cation/proton antiporter-based
resistance systems are the two-component responder/sensor units. Examples include PcoRS and CopRS, controlling copper resistance of E. coli and Pseudomonas, respectively (reviewed in 60); CzrRS, controlling cadmium and zinc resistance in P. aeruginosa (37); and SilRS, controlling silver resistance in S.
typhimurium (38). The sensor or S protein is a histidine kinase that autophosphorylates itself at a His residue on interaction with its metal substrate and is
typically membrane spanning. Autophosphorylation of the S protein causes an
allosteric conformational change in the C terminus of the protein that is transduced across the inner membrane to the cytoplasmic responder protein. The latter
is activated by phosphorylation of an Asp residue by the kinase activity of the
S protein. In the inactive state, the responder will typically act as DNA-binding
repressor, preventing transcription of the structural genes. The responder protein
is activated by phosphorylation, which relieves DNA repression by abrogating the
DNA binding activity. In general, all two-component sensor-responder systems
constitute a highly efficient on-and-off switch for expression of resistance,
allowing a fine-tuning of the level of induction in proportion to the metal concentration. This is certainly true for the czc operon of R. eutropha CH34, where
regulation by the two-component CzcRS allows the bacteria to respond optimally
in a micromolar to millimolar concentration range of Zn2 (87). The lower micromolar threshold allows CH34 to take up zinc freely for growth requirements, and
the effective working range of the regulatory unit allows the organism to modulate efflux of excess metal in proportion to the extracellular levels. CH34 also
FIG. 1 Genetic relationships between bacterial metal resistance mechanisms. Boxes and lines indicate the relationships based on amino acid sequence homology. Vertical lines indicate homology between blocks of genes.
The resistance operons are as follows: cnr (cobalt and nickel), czc (cadmium,
zinc, cobalt), ncc (nickel, cobalt, and cadmium), czr (cadmium and zinc), sil
(silver), cop, pco (copper), chr (chromate), cad (cadmium), zia, znt (zinc), ars
(arsenate), pbr (lead), mer (mercury). mgt encodes a presumed Mg2 transport protein. C and P indicate chromosomal and plasmid-encoded, respectively.
Bacteria
19
20
TABLE 2 Best-Studied Regulatory Modules Involved in Regulation of Heavy Metal Resistance Mechanisms
in Bacteria
Structural Resistance
Cation/proton
antiporter
MerR transcriptional regulators
Two-component responder-sensors
ATPase
Enzymatic
reduction
ZntR
CoaR
PbrR
Sequestration
PbrR
CzcRS
CzrRS
SilRS
CopRS
CopRS
PcoRS
ECF-Based
ArsR transcriptional regulators
CadC
ZiaR
ArsR
SmtB
Others
CzcNI
ArsD
MerD
138,104
105
55
33,47
37
38
22,60
60
60
35,100
36
112
58
110
114
22,47
119
108
CnrYXH
NccYXH
Ref.
Bacteria
21
makes use of the additional functions of CzcD (and perhaps CzcN and Czcl),
which are thought to control the upper limits of transcription of the resistance
genes to prevent excessive metal efflux (47,87). For czc, which effluxes zinc,
cadmium, and cobalt, regulation is chiefly controlled by the zinc concentration,
which is the most potent inducer of CzcRS activity, and this allows the organism
to modulate expression of resistance without affecting the homeostasis.
Interestingly, the cation proton antiporter systems encoded by the cnr and
ncc operons of R. eutropha strains CH34 and 31A, respectively, are controlled
by a newly described three-component regulatory unit, based on alternative
factors. The Ncc and Cnr systems are closely related; both are essentially nickel
efflux systems and are therefore mainly regulated by nickel (35,96,100). The
regulation of cnr was recently described and seems to involve an intricate interplay between CnrH, an alternative factor of the ECF family; CnrY, a putative
anti- factor that antagonizes CnrH activity; and CnrX, a periplasmic protein
that may function as a metal sensor (100,101). The CnrH protein is a DNAbinding activator protein that specifically directs transcription of the cnr genes
in the presence of RNA polymerase (100,102). The activity is controlled at the
protein level by the CnrY and CnrX proteins, which constitute the metal-sensing
unit. CnrH is capable of activating cnr transcription up to 1000-fold between 100
to 300 M of nickel, and can modulate transcription activation as a function
of both nickel and cobalt concentrations in the micromolar to millimolar range
(100,101). This ECF-based regulation has yet to be described for other metal
resistance operons, although CnrH has homologues, including RpoE (or SigE),
which also controls operons involved in extracellular sensing and stress responses
(reviewed in 102,103).
3.2.1.2 Regulation of ATPases As shown in Table 2, three main families
of regulators have been described and include the MerR family and ArsR subfamilies of transcriptional regulators as well as the two-component responder/sensor
units. Both the MerR and ArsR classes are metal-binding, DNA-binding proteins,
which repress gene transcription in the absence of inducer metal and whose function is abrogated by the specific binding of their cognate metal cation ligand.
The MerR family of proteins includes MerR, which controls the merencoded mercury resistance found ubiquitously in eubateria; ZntR, which controls the zinc resistance, znt operon of E. coli (104); CoaR, controlling cobalt
resistance in Synechocystis (105); ZiaR, controlling the ZiaA zinc ATPase of
Synechocystis sp. (58); and PbrR, controlling the pbr operon encoding lead resistance in R. eutropha CH34 (55). Only MerR, PbrR, ZntR, and CoaR have been
characterized at the protein level.
MerR is a small, dimeric protein with an N-terminal DNA binding domain
and a mercury binding site at its C terminus. In the absence of inducer Hg2,
MerR binds to an operator site (a region of dyad symmetry), which lies between
22
the 35 and 10 regions of the merT promoter (PT) and actively represses RNA
polymerase holoenzyme from transcribing merR or the structural genes, thereby
actively repressing all mer genes, including its own. Activation of the mer operon
is effected by the binding of a single Hg2 to the MerR dimer (106); structural
changes that result from the binding of the Hg-MerR complex to its cognate PT
promoter cause a localized distortion of the DNA in the promoter region thereby
allowing the 35 and 10 regions to align adequately as to be recognized by
core RNA polymerase. The distortion is detrimental to the structural integrity of
the merR promoter, PR, so that it becomes inactive (107). Downregulation of mer
expression at low mercuric ion concentrations is effected by the MerD protein,
which can bind the operator region in PT, resulting in reduced transcription from
this promoter (108). This mechanism of transcriptional control is one of the best
examples of tight regulation known in eubacteria. ZntR has only recently been
identified and characterized (104). Like MerR, ZntR activates transcription from
PzntA, the promoter of zntA; both Cd2 and Hg2 have a low induction effect (in
the micromolar range) on transcription from PzntA. Nevertheless, induction of transcription from this promoter was observed to reach a maximum of about 100-fold
induction in the presence of 1 mM Zn2, compared with 100,000-fold induction
resulting from MerR activation from the Pmer promoter (70). One reason for this
lies in the observation that ZntR does not actively repress transcription from its
cognate promoter as does MerR (104), which in the absence of inducer Hg2
represses transcription up to 10-fold (65). This difference in promoter strengths
between the mer and znt operons reflects the importance of maintaining the homeostatic balance of Zn2 compared with the need to expulse toxic levels of
mercury.
The ArsR subfamily of repressors includes ArsR, which controls expression
of the ars operon encoding arsenate resistance in a variety of eubacteria (109
111); CadC, which regulates the cad operon encoding resistance to cadmium,
zinc, and lead in a large number of gram-positive and gram-negative bacteria
(57,112,113); and SmtB, whose crystal structure was recently resolved and which
regulates the expression of the SmtA metallothionein of Synechococcus sp.
(94,114). This group of proteins are homodimeric DNA- and metal-binding proteins, that have a unique pair of cysteines near the C-terminal region, which is
implicated in both DNA binding and cation sensing (25,26,112,114117). In the
absence of inducer metal, the repressors bind to an operator region downstream
of their own promoter, thereby inhibiting transcription of the structural genes as
well as their own transcription. In the case of CadC, the repressor has a high
affinity for DNA; however, its interaction with metal cations, including Mn2,
Zn2, Cd2, Bi2, and Pb2, cause the repressor to dissociate from the DNA molecule, thereby allowing transcription of the resistance genes (112,113). This allows
CadA ATPase to respond to a variety of potentially toxic metal cations. The
Bacteria
23
crystal structure of SmtB was recently solved and found to be a homodimeric

protein (61,114).
Two-component systems are also involved in the regulation of copper resistance ATPases and include CopRS, which might regulate the CopF ATPase of
R. eutropha CH34 (22). The CopYZ unit that regulates the cop operon of E.
hirae is also two-component, although it is not a classical responder/sensor system. CopY is a DNA-binding repressor protein and CopZ an antirepressor protein
(118). This regulatory mechanism, which is unique for a metal resistance operon,
is effected by the CopY aporepressor, which cannot bind to its cognate operator/
promoter DNA in the absence of Cu (the preferred substrate in vitro). However,
at physiologically tolerable levels of the inducer metal cation, the complex
(CopY-Cu)2 is converted to a DNA-binding repressor. At higher metal concentrations, the antirepressor CopZ binds Cu and the CopZ-Cu complex interacts
with (CopY-Cu)2 to form an inhibitory complex that disrupts the DNA binding
activity of the latter. This regulatory mechanism allows a fine-tuned response
to extracellular copper, since it allows the expression of uptake proteins at low
concentrations of the metal and the expression of the efflux proteins when physiological tolerable thresholds are surpassed. Thus, metal homeostasis and resistance
mechanisms are simultaneously controlled (118).
3.2.1.3 Regulation of Enzymatic Reduction As mentioned above, enzymatic reduction is an effective resistance mechanism for the detoxification of
highly toxic metal cations. Therefore, most regulators of these mechanisms tend
to be highly cation-specific transcriptional regulators. Examples include ArsR,
controlling expression of the ArsC arsenate reductase (109111), and MerR, controlling expression of the MerA mercuric reductase (70).
Regulation of expression of the enzymatic reduction processes seems to
involve an additional regulator that limits the maximum expression levels of resistance above the control levels of the main transcriptional regulators. Examples
include the DNA-binding repressors ArsD (119,120) and MerD (108), which set
the upper limits of expression of the ars and mer operons, respectively.
3.2.1.4 Regulation of Metal Sequestration Processes In most cases, sequestration is a complex process involving proteins, polysaccharides, and so
forth; therefore, the underlying mechanisms of control are not always well defined. In the case of copper sequestration, the Pco and Cop systems of gramnegative species are likely to be under the control of the two-component
responder/sensor regulatory units that respond specificically to Cu cations and
that control expression of the resistance gene (see above). Sequestration of the
SmtA metallothionein is controlled by the SmtB protein (see above), which responds to both Zn2 and Cd2.
24
APPLICATIONS BASED ON BACTERIAL INTERACTIONS

WITH HEAVY METALS
4.1
Metal Leaching
Metal leaching is based on metabolic activity of the bacteria in the presence of

heavy metals. Metal leaching activities for the purposes of mining from metal
ores has been recently reviewed (5). The key organisms implicated include members of the Thiobacillus and Leptospirillum genera. Thiobacillus ferrooxidans is
the most frequently used and, as described earlier, this is due to its interesting
metabolism that allows it to oxidize ferrous iron and other heavy metals as part
of a sulfur cycle required by the organism for its energy production. Under strict
acidic conditions, this results in the production of soluble metal sulfates (reviewed
in 5, 10, 11), a process also known as leaching (12). This process occurs
naturally in iron-rich sulfide deposits. However, the presence of heavy metal
impurities, including nickel, copper, lead, gallium, zinc, cobalt, and copper, has
been shown to cause the release of high concentrations of soluble heavy metals.
This process is used at an industrial scale for leaching of metals from low-grade
ores. This is typically carried out in heaps (heap or dump leaching), where
the ore is stacked as a heap on a slope or inclination, and acidified water is poured
over this heap to lower the pH and to stimulate the activity of the acidophilic
bacteria. The aqueous metal fraction that leaches out of the ore is collected at
the bottom of the slope. Similar principles have been applied to construct continuous-flow bioreactors where inoculants of T. ferroxidans were used to leach out
contaminating metals from sewage sludge resulting in the solubilization of more
than 90% of the otherwise insoluble metal fraction, thereby decontaminating the
sludge for safe disposal (121,122).
Leaching of heavy metals from solid contaminated wastes has also been
described based on citric acid excretion by P. putida, which could effectively
solubilize zinc from industrial filter dust (123).
4.2
Decontamination of Soils and Water
Biological treatment methods for decontamination of metal-polluted soils have

been given increasing attention since traditional remediation techniques are often
ineffective at desorbing the metal contaminants from soil particles.
4.2.1
Applications Based on Ralstonia eutropha CH34

and Related Strains
The interaction of CH34 with metals is a feature that has been exploited in the
development of various technologies for environmental applications; these have
been widely documented and were recently reviewed in 9,22, and 23). Some
applications are based on the function of the metal resistance structural proteins
Bacteria
25
and metal precipitation; others exploit the capability of heavy metals to control
gene activity in CH34. In addition, CH34 is capable of degrading certain aromatic
xenobiotic compounds, including biphenyl (124), which has extended the range
of applications of this organism for the treatment of a wide range of industrial
wastes containing mixed pollution (125).
4.2.1.1 Heavy Metal Removal and Recovery from Contaminated Effluents
One of the more successful technologies developed is the BICMER, or Bacteria
Immobilized Composite MEmbrane Reactor (42,88,89,125). The design of the
BICMER is founded on the underlying mechanism of postefflux metal precipitation by CH34. This novel type of bioreactor consists of a specially designed
Zirfon membrane on whose surface the active bacteria are immobilised, thus
forming a biofilm. The waste stream contaminated with heavy metals and/or
organic xenobiotics contacts the bacteria within a tubular structure or a flat sheet
support while a separated feeding stream keeps the bacteria viable. CH34 activity
results in metal precipitation as insoluble crystals on the microbial biofilm that
are subsequently recovered on glass bead columns. This bioreactor concept has
been successfully applied for the removal and recovery of various heavy metals,
including Zn, Cd, Co, Ni, Cu Pb, Y, and Ge, from wastewaters or process streams.
The BICMER has many advantages over other related technologies, including
the following: (a) the concentration effect of the pollutant on the membrane reduces the sludge volume; (b) the insoluble metal carbonate crystals allow direct
recovery of the metal; and (c) the ability of CH34 to degrade certain organic
compounds means that the biofilm can tolerate wastewater loads containing recalcitrant organic compounds, including polyaromatic hydrocarbons (PAHs) (125).
A similar concept based on the biosorption and bioprecipitation properties
of the Ralstonia biofilms has been applied in the development of a moving-bed
sand filter where the biofilm is allowed to form on sand grains (90). The resulting
metal-loaded biomass, i.e., bacteria containing immobilized metals even up to
10% of dry matter, is recovered and collected. These sand filters are being developed as a polishing technology for treatment of heavily contaminated effluents
(90).
4.2.1.2 Soil Bioremediation Two key features of CH34 physiology have
been exploited in the development of the BMSR or Bio Metal Sludge Reactor
for contaminated soils, namely, that the organism can sequester heavy metals
and adsorb them onto its outer surface and that it can also stimulate soil settling,
including the fraction of fine organic matter, while the heavy metalloaded bacteria remain in suspension. The BMSR concept involves a direct inoculation of
the soil with the metal-resistant bacteria into a stirred reactor. Interestingly, CH34
activity alters the colloidal properties of the treated soil by improving its settling
capacity, thus allowing the treated soil to be separated and recovered from the
heavy metalloaded biomass consisting of bacteria charged with heavy metals
26
which remain in suspension. This biomass can subsequently be removed from

the process water. The heavy metal content of the resulting biomass can attain
0.5% on a weight-for-weight basis (90) while the overall performance of the
BMSR can result in up to 85% of the bioavailable metals from the soils to be
extracted within 48 h.
4.2.1.3 Metal Immobilization Using Biomolecules This is based on the
immobilization of the metals in the soil using biopolymers or biosurfactants. Biopolymers bind metals with high affinity and can be physically separated from
waste effluents or cleansed soils, thus allowing metal recovery. Examples include
exopolysaccharides, cyclodextrins, and biosurfactants. These biopolymers complex divalent metal cations and render them more soluble in aqueous solution,
which can be further exploited in soil washing techniques for maximal removal
of the metal contaminants in the soil (reviewed in 126).
4.2.2
Environmental Monitoring and Metal Sensing:

Heavy Metal Biosensors
The application of bacterial resistance mechanisms to develop heavy metal

sensing biological tools has been widely explored and is now reaching full potential. With the recent advances made in our understanding of the bacterial heavy
metal resistance mechanisms, it has been possible to engineer bacteria that produce a measurable and quantitative signal when brought in contact with specific
metal species (127131).
4.2.2.1 Gene-Based Biosensors To date, the most successful metal biosensors are the gene-based biosensors constructed by the fusion of metal resistance regulators with the bioluminescent luciferase genes of Vibrio spp.
(127,129,132,133) and of Photobacterium phosphoreum (reviewed in 134). The
most important criterion for such transcriptional fusions is a metal-responsive
promoter, which ideally should be significantly activated in the presence of metal.
Transcriptional fusions incorporating both a metal-inducible promoter and a gene
encoding a regulator can allow for a fine-tuned transcriptional response by the
biosensor construct on activation by metals and these (reviewed in 129). Promoterless vector plasmids have been constructed based on the luciferase reporter
genes, luxCDABE, and these have facilitated the cloning of metal-responsive
genes so that in biosensor constructs light emission occurs as a function of metalinduced transcription (128,129,131,133).
Using different regulatory regions of CH34 resistance determinants, it has
been possible to develop an amalgam of BIOMET (biological metal) sensors for
different metals and families of metals (23,131). These are summarized in Table
3. The gene-based biosensors have a unique niche in environmental monitoring
applications since they are one of the few techniques that can give information
on the bioavailability of a metal species in a particular sample (129,130). Quanti-
Bacteria
TABLE 3 Gene-Based BIOMET Biosensor Strains Developed in R. eutropha CH34
Strain
AE1239
AE1433
AE1433
AE2440
AE2450
AE2515
Cation
specificity
Metalresponsive
genes
Detection limit
(mg/kg dw)
Industrial soil/
sediment
(mg/kg dw)
Background soil
(mg/kg dw)
Ref.
Cu2
Zn2
Cd2
Cr6
Pb2
Ni2
cop
czc
czc
chr
pbr
cnr
18.7
5.9
6.37
1.21
0.83
2.2
6731365
24067159
18109
6831319
21925788
5381731
1429
50148
0.52.9
3261
3593
722
128
133
133
131
131
135
All constructions are based on the bioluminescent lux reporter genes of Vibrio fischeri. The biosensor strains have been standardized
for their response to specific metal cations in soil and other environmental samples. The detection limit indicates the lowest metal
concentration for a quantifiable biosensor response. Also included in the table are data on the typical contamination levels in industrial
soils/sediments as well as the norms in noncontaminated soils.
27
28
tation of the bioavailability of a metal species is perhaps the most important

parameter for assessing the relative toxicity of a sample to biological systems
(130). The BIOMET tests were found to be a determining criterion in the evaluation of in situ inactivation of metal-contaminated soils following treatment with
chemical additives (17); the usefulness of these tests was maximally realized
when used in conjunction with chemical and biological tests based on phytoxicity,
zootoxicity, and bacterial ecology (135). Other applications of the BIOMET sensors include the routine monitoring of bioavailable metal levels in soils subjected
to remediation strategies (23,90) and measurement of metal content in industrial
wastes/byproducts, such as incinerator fly ashes (131,133).
4.2.2.2 Protein-Based Biosensors The next generation of biosensors
will likely be the protein-based biosensors that are currently being developed
alongside the gene-based variety (131). These sensors are based on the immobilization of metal-responsive proteins (typically metal-binding metalloregulatory
proteins) or other metal-binding proteins onto electrodes; the sensing quality is
measured as a change in capacitance when the protein interacts with the metal
(131). Biosensors based on MerR (70,136) have been successfully applied for
sensing of low Hg2 concentrations, whereas a recombinant form of SmtA could
sense Cu2 levels between 105and 101 M (131).
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2
Mycorrhizal Fungi
Jan V. Colpaert
Limburgs Universitair Centrum, Diepenbeek, Belgium
P. Vandenkoornhuyse
Universite de Lille, Paris, France
INTRODUCTION
Mycorrhizal symbiosis is the most widespread symbiosis between plants and microorganisms. Between 80% and 90% of all seed plant species have fungi in their
roots, forming structures known as mycorrhizas (1). Mycorrhizas are a functional
part of the plant roots where the fungus provides an interface between the roots
and the soil. Mycorrhizas are very effective in assimilating nutrients, including
essential metals and their analogues, many of which are present in toxic concentrations on contaminated soils. Mycorrhizas range widely in form and in type of
fungus involved, demonstrating that they represent not a single class of symbiosis
but rather a type of plant-fungus association that has evolved repeatedly, in response to distinct selection pressures (2). Mycorrhizas have been classified in
various ways, such as into ectomycorrhizas, typically formed between some longlived woody plant species and long-lived fungi (generally Basidiomycota and
Ascomycota) (Fig. 1), and endomycorrhizas. The latter contain the most ubiquitous mycorrhizal association, the arbuscular mycorrhizas (Fig. 1), involving a
37
38
Colpaert and Vandenkoornhuyse
FIG. 1 Longitudinal section of an ectomycorrhiza and an arbuscular mycorrhiza. (Ref. 105.)
very wide range of plants and a small group of fungi in the Glomales (Zygomycota) (3). A much smaller group of plants form the ericoid endomycorrhizas. In
the following paragraphs, we describe the three types of mycorrhizas that are
commonly found in metal-polluted habitats.
Arbuscular mycorrhizal (AM) fungi are strict biotrophs. In exchange for
carbon compounds, these symbiotic fungi improve the uptake of phosphorus in
plants (1,4,5), a highly immobile element in soil. In some circumstances, AM
fungi can also benefit plants by increasing the uptake of nitrogen (4,6) and micronutrients (5,7). In general, AM fungi have a positive effect on plant health and
resistance to stress factors (8).
The ectomycorrhizal (ECM) fungi are typically associated with woody
plant species in boreal and temperate forest ecosystems. Most members of the
important tree families (Pinaceae, Fagaceae, Betulaceae, Salicaceae, Tiliaceae)
in these climatic regions are predominantly ECM, although species of the (sub)tropical families Myrtaceae and Dipterocarpaceae also appear to be mainly ECM.
Mycorrhizal Fungi
39
In contrast to the situation found in the AM symbiosis, a large number of fungi

have been reported as forming ectomycorrhizas (9). The majority of these species
belong to the Basidiomycota, but a significant proportion of Ascomycota are also
involved, as are a few zygomycetous fungi. Most ECM fungi are biotrophs in
their natural habitats, although some species can be cultured in vitro. They increase the fitness of their host trees in very similar ways as the AM fungi of
herbaceous plants. However, ECM fungi seem to be better adapted to organic
soil layers in forests, and it is generally accepted that they have better access to
organic P and N compounds than plant roots or AM fungi (1).
The third distinct form of mycorrhizas can be recognized in members of
the Ericales: the ericoid mycorrhizal (ERM) fungi. The very fine hair roots of
these plant species are usually intracellularly colonized by a limited (?) number
of fungi, mainly ascomycetes belonging to the genera Hymenoscyphus and Oidiodendron. Ericaceous plants are most strongly associated with nutrient-poor soils
in which the major growth-limiting nutrient is nitrogen. In the Arctic regions
these plants become dominant in soils where a recalcitrant litter accumulates as
raw humus. The ERM mycobionts play a key role in the nitrogen nutrition of
the plants, and it is thought that the fungi protect the plants against the high metal
concentrations (Al, Fe, Mn) present in many habitats of the Ericaceae (1).
DO HEAVY METALS IMPOSE A SELECTION PRESSURE

ON MYCORRHIZAL COMMUNITIES?
Heavy metals in soils are known to be toxic to most organisms, including plants
(this volume, next chapters, 10), when present in excessive concentrations. However, metal tolerances in mycorrhizal fungi have been investigated in much less
detail than in their hosts. This is despite the fact that they may be key to plant
survival on contaminated soils, not only for potential conferred or enhanced metal
resistances but for their role in nutrient and water acquisition as well (1113).
The impact of metal toxicity on vegetations can easily be demonstrated and there
is ample evidence for the evolution of adaptive metal tolerance in some higher
plants, e.g., in grass species. Other plants, typical metallophytes, are only found
on metal-contaminated soil (14). Similar evidence is far less convincing when
mycorrhizal communities or even soil-borne microorganisms in general are considered. An important reason for this lack of knowledge is the obvious practical
difficulties in assessing the active below-ground microbial communities and populations (15). Recent developments in molecular ecology now provide us with
some tools that allow for the detection and accurate identification of belowground fungal partners (16). The accuracy and feasibility of mycorrhizal community studies have been greatly enhanced.
40
Although there is evidence that some microorganisms are more sensitive

to heavy metal stress than plants growing on the same soil (17,18), heavy metal
pollution-mediated changes in plant and fungal communities have seldom been
studied in detail on the same site. In many fungal groups, there is little evidence
for intraspecific adaptive changes between populations from polluted or nonpolluted habitats (19). Reviewing the effect of metals on soil microbial communities,
Duxbury (20) concluded that there is rarely a correlation between the metal tolerance of microorganisms under laboratory conditions and the metal concentrations
at the site of origin, unless extremely high metal concentrations are present in
the soil. Many of the soil-borne fungi that occur on polluted soils are supposed
to have a constitutive high level of metal tolerance, so that selection pressure
for more adaptive tolerance is seldom exhibited (19,21). Evidence for the
evolutionary adaptation to heavy metals among mycorrhizal fungi is scarce. In
many cases, heavy metal pollution did not result in an increase in metal tolerance
of mycorrhizal fungi, although it is likely that there is selection for those species
that possess a high constitutive metal tolerance (21,22).
Nevertheless, adverse effects on soil microbial parameters (e.g., specific
microbial respiration rate, N2 fixation) and on mycorrhizal communities can be
observed in metal-polluted environments (18,23). Such changes might have significant implications for ecosystem functioning and sustainability.
3
3.1
MYCORRHIZAL FUNGI IN HEAVY METAL

POLLUTED SOILS
Arbuscular Mycorrhizas
For 20 years the natural occurrence of AM fungi in soils highly polluted by heavy
metals has been repeatedly reported (2434). Considerable AM fungal species
diversity can exist in metal-contaminated soils (30,32,35,36). In mine spoils containing elevated levels of Zn and Cd, Gildon and Tinker (24) found that the
indigenous clover had high AM colonization levels. In a calamine spoil covered
with a natural vegetation of metal-adapted plant species, Pawlowska et al. (32)
found extremely high colonization levels in some plant species as well as spores
of six different AM fungi. Different species of Glomus were also found in the
rhizosphere of the zinc violet, Viola calaminaria, a metallophyte plant that colonizes metalliferous soils and ancient mining sites (33,34). Zak et al. (25) found
that the number of spores of AM fungi in mine soils in Canada was very heterogeneous and probably leads to differences in the genetic pool of AM fungi. By
using Medicago sativa as host plant, a mycorrhizal infection potential in mine
spoils was demonstrated (37). The beneficial effect of mycorrhizal symbiosis on
Zn tolerance of the grass Andropogon gerardii was proposed by Shetty et al.
(36). The authors also showed that the AM fungi isolated from Zn-contaminated
Mycorrhizal Fungi
41
soil surrounding mine tailing were more effective in increasing the plant biomass
at high levels of Zn, whereas AM fungi from a noncontaminated site promoted
plant growth only in the soil containing lower Zn concentrations. In soils severely
polluted by atmospheric deposition of metals from smelters, an abundant mycorrhizal colonization has often been found in metal-adapted plants (27,29,38,39).
Griffioen et al. (38) highlighted the fact that the yield of AM fungal infection of
Agrostis capillaris was comparable for the plants grown on uncontaminated soil
and on a Zn/Cd-polluted soil. However, a negative correlation between infection
rate and Cu concentration in soil was observed.
In agricultural soils polluted by sewage sludge amendments or by emissions
from smelters, high levels of mycorrhizal colonization are also observed (40,41).
Some studies revealed that in these polluted fields the mycorrhizal colonization
of crops could be reduced or delayed (28,42,43), whereas in other studies the
addition of metal containing sludge did not affect in situ AM development (44).
Therefore, it has been hypothesized that AM fungal ecotypes can exhibit varying
degrees of tolerance to metals (43). The effects of different inputs of metal-contaminated sewage sludge to an agricultural field experiment (Braunschweig, Germany) have been studied. For plots that received the highest sludge amendment,
the total number of spores in soil decreased significantly. However, metal concentrations in these soils remained below the European Communitys maximum allowable concentration limits for metals in soils (45,46). As pointed out by Del
Val et al. (43), the inhibition of mycorrhizal colonization in the contaminated soils
could be due in part to the inhibition of fungal spread. In addition, modification of
the soil pH, P concentration, and CEC can be correlated with the mycorrhizal
colonization (41,42,47). Leyval et al. (11) emphasized that the mycorrhizal infectivity of different heavy metalpolluted soils is linked to the amounts of
bioavailable/extractable metals from soils rather than total metal concentration.
Although this idea is now accepted, the availability of metals has been rarely
measured, so that comparisons between studies are difficult to make (11).
3.2
Ectomycorrhizas
Ectomycorrhizal fungi can be found everywhere potential host trees can colonize
metal-contaminated soils. However, relatively few tree species can survive on
metalliferous soils with high metal loads. Metallophyte vegetations on the naturally metalliferous soils in western and northern Europe are slowly invaded by
ectomycorrhizal Betula sp., Salix sp., Pinus sp. (32,48,49), and by arbuscular
mycorrhizal Acer sp. (50). Related taxa can be found on mining sites in America
and in soils severely polluted by atmospheric deposition of metals from smelters.
In most cases, tree growth is seriously affected by metal stress, and metals in
leaves or needles can reach toxic concentrations. In 1975, a field site was designed
as a test plot for the reforestation of the zinc desert in Lommel (Belgium).
42
The site is polluted with nonferrous metals emitted by a zinc smelter, which was
dismantled in 1973 (39). Container plants of Pinus sylvestris, P. nigra, Betula
pendula, Robinia pseudoacacia, Prunus serotina, and several Acer spp. were
planted in homogeneous blocks of 20 20 m on a 1-ha plot, devoid of vegetation
due to Zn toxicity. Saplings were planted in large planting holes (10 L) filled
with the original sandy soil amended with a metal immobilizing substance (loess,
marl, or compost). Although there are a few miserable survivors of most tree
species after 25 years, only the ectomycorrhizal genera Pinus and Betula were
able to form a real forest. In 1985, we found basidiocarps of 7 ECM fungal
species (6 genera) in these plots. In 1999, species richness had increased to 17
ECM species from 11 genera. The most abundant sporocarps in both years were
from Suillus luteus and Amanita muscaria. A considerable number of reports
mention the presence of basidiocarps of several ECM species on severely metalpolluted soils, but systematic field studies are scarce. In addition, there is clearly
a need for more below-ground ECM community studies in these highly contaminated environments. Vralstad et al. (51) found that a dominant ECM morphotype
on seedlings of Pinus sylvestris, Betula pubescens, and Salix phylicifolia growing
at the edge of a copper mine spoil in Norway was formed by an ascomycete
from the Hymenoscyphus ericae aggregate. This mycobiont probably never forms
sporocarps, and this finding suggests that we only have a very incomplete view
on the biodiversity of mycorrhizal symbionts in metal-polluted environments.
Screening experiments in vitro and in symbiosis have confirmed considerable
variation in heavy metal sensitivity among ECM fungi (22,5256).
3.3
Ericoid Mycorrhizas
The dwarf shrub Calluna vulgaris is a characteristic ericoid host plant that is
a successful colonist of some acidic Cu-, Zn-, and/or Pb-polluted soils in Europe
(12,48,51,57). On these soils, it appears that the plants are consistently colonized
by a mycorrhizal symbiont, Hymenoscyphus ericae (Pezizella ericae), which
clearly is essential for the survival of the plants in these contaminated habitats
(57). Another ericoid mycorrhizal fungus from metal contaminated soils is Oidiodendron maius, which was present in roots of Vaccinium myrtillus (58).
4
HEAVY METAL TOLERANCE IN MYCORRHIZAL FUNGI
The in situ occurrence of AM fungal spores and/or mycorrhizal colonization even

in highly polluted soils reported in numerous papers strongly suggests the possible metal tolerance of AM fungi. Gildon and Tinker (59) reported the first evidence of a possible tolerant state of an AM fungal isolate. These authors, along
with others, have tested the growth performance of AM fungi by measuring different mycorrhizal parameters such as AM root colonization, and the ratio of
Mycorrhizal Fungi
43
coils, arbuscules, and vesicles within the root cortical cells for different AM fungal isolates and different host plants (24,43,59,60). Since AM fungi cannot complete their life cycle in axenic cultures (strict biotroph), metal tolerance has also
been estimated from the ability of AM spores to germinate in a polluted substrate
(6163). Because the preinfective stages of the AM fungi are very sensitive to
the presence of heavy metals (64), such an in vitro biotest could be very efficient
to detect metal toxicity. By using one of these strategies, a higher tolerance to
Zn, Cd, Cu, and/or Pb of indigenous fungi isolated from polluted soils in comparison to isolates from unpolluted soils has been clearly demonstrated in several
FIG. 2 Zn and Cd tolerance of isolates of the ectomycorrhizal fungus Suillus

luteus. Tolerance indices (%) calculated for in vitro biomass production (dry
weight). Open symbols, isolates from a metal polluted site (Lommel, B);
closed symbols, isolates from a nonpolluted site (Paal, B).
44
studies (5961). Elsewhere different susceptibility to heavy metals was highlighted among isolates from the same metal-polluted soil (46). By using a Cdtolerant Glomus mosseae, Joner and Leyval (65) have shown that the growth of
extraradical hyphae was not affected by high concentration of Cd added to the
soil of the hyphal compartment. Nevertheless, to date there is no published evidence of the stability of metal tolerance in AM fungi.
Metal tolerance in ECM fungi has been studied repeatedly (21,22). From
these studies it appears that ECM fungi from metal-contaminated soils often display constitutive tolerance, although it cannot be excluded that some species only
colonize less polluted microsites in the polluted soil. In most cases, there is little
evidence for intraspecific adaptive changes between ECM populations from polluted or nonpolluted habitats (22). Nevertheless, adaptive tolerance to Zn and Cd
was observed in some ECM fungi at the Zn- and Cd-polluted Lommel site in
Belgium (53). These results were recently confirmed on the population level of
Suillus luteus, and it also appears that the trait is genetically stable (66). In Fig.
2, the in vitro Zn and Cd tolerance is shown for S. luteus isolates from a polluted
and nonpolluted site. Adaptive Al tolerance was also found in the same mycobiont (67) and in another pioneer ECM fungus: Pisolithus tinctorius (68).
Adaptive Zn tolerance has also been observed in mycorrhizal endophytes
of the Ericaceae from polluted soils (58).
5
PLANT PROTECTION TO METALS: FUNCTIONS OF

MYCORRHIZAL FUNGI AND MECHANISMS INVOLVED
There has been increasing interest in the functions of AM fungi in heavy metal
polluted soils. There is circumstantial evidence that plants can benefit from AM
colonization on highly contaminated soils. Nevertheless, it remains a matter of
debate whether AM fungi protect their host from exposure to metal contaminants
by reduced assimilation or transfer of metals to the host (12). AM colonization
is maintained on mine plant vegetations for the same reasons that fungi are maintained in uncontaminated environments (e.g., phosphorus acquisition). Coevolution of hosts and AM fungi in metal-contaminated environments can nevertheless
be expected and this process might lead to a further increase of the metal tolerance
of a host.
Using a compartmentalized system separating roots and extraradical mycelium, it has been demonstrated that the extraradical mycelium of AM fungi can
transport 65Zn (69) and 109Cd (65) from soil to roots. Nevertheless, the metal
transfer from roots to shoots was limited (65). The authors hypothesized that Cd
was immobilized within the roots. To estimate the capacity of hyphae to bind
metals, Joner et al. (70) performed an experiment with excised mycelium of different Glomus sp. isolates with different history of exposure to Zn and Cd. They
showed that AM hyphae have a high metal sorption capacity (up to 0.5 mg of
Mycorrhizal Fungi
45
Cd per mg of dry biomass) and that the metal sorption capacity was related to
the adaptation of the fungus to high levels of heavy metals. Other authors have
studied the sorption and subcellular localization of Cu and As in the extraradical
mycelium of different isolates of Glomus sp. by transmission electron microscopy
combined with X-ray microanalysis (EDAX) (71). The results showed that both
Cu and As were sequestered within the hyphal walls. In agreement with Joner
et al. (70), Gonzalez-Chavez et al. (71) showed differences in the levels of sorbed
Cu highlighting functional differences among AM fungi that can be related to
the history of the exposure of the isolates. Tolerant AM fungi isolated from polluted soils sorbed more metals than nontolerant fungi, resulting in a limitation
of metal transfer to plants (70,71).
In 1994, Medeiros et al. (72) reported differences in efficiency of mycorrhizal fungi in the uptake of metals by plants. In addition, Shetty et al. (30) showed
that mycorrhizal grasses were more successful than nonmycorrhizal grasses in
the colonization of mine soil. The main ecological implication of these investigations was that the obvious plant metal tolerance might be conferred by the fungal
symbiont. Recent studies have enforced this hypothesis (33,34,73). Metal-tolerant
fungi allow the establishment of plantsof even nonadapted speciesin highly
metal-polluted soil. According to Wilkinson and Dickinson (49), there are more
opportunities for genetic changes that match environmental constraints in fungi
then in short- or long-lived higher plants. Therefore, one of the major functions
of mycorrhiza would be to allow the adjustment to local soil conditions and acclimation of plants.
Coevolution of ectomycorrhizal fungi and their long-lived tree hosts for
more metal tolerance in polluted environments is probably a very slow process.
There is little evidence that on metalliferous soils the tree species have adapted
genetically to a similar extent as some herbaceous plant species did in the same
habitats (49,74,75). The occurrence of Zn and Pb tolerant ecotypes of birch on
metalliferous soils has been reported by Brown and Wilkins (76) and by Eltrop
et al. (48). ECM fungi may adapt more rapidly than trees due to their shorter
life cycles. Selection of more resistant ECM fungi is likely to be involved in the
survival of highly mycotrophic trees such as pines and birches in metal-polluted
environments. As with the AM symbiosis, there is evidence that ECM fungi have
beneficial effects in the alleviation of metal toxicity in forest tree seedlings (13).
Some authors argue that ECM fungi do not confer increased metal resistance to
their host but that the symbiosis fulfills its normal ecological function (12). We
believe that this conclusion cannot be generalized since particular ECM fungi
are able to restrict transfer of metals to the host while maintaining normal nutrient
transfer to their host tree (54,77,78). Metal transfer to shoots is, however, a complex process that is partly affected by the transpiration stream (13). However, as
long as the mycorrhizal root system remains functional, a reduced transfer of
metals to the host might promote survival of trees in polluted environments (79).
46
More convincing evidence that ECM fungi can endow their hosts with metal
resistance was recently demonstrated in a dose-response experiment in which
mycorrhizal and nonmycorrhizal pine seedlings were exposed to four Cu concentrations. Plants were grown in semihydroponics with P as the growth-limiting
element. Biomass production, P content, and P uptake rate were severely inhibited
with increasing Cu concentrations in nonmycorrhizal (NM) seedlings, whereas
plants colonized with Thelephora terrestris or Suillus bovinus were respectively
not at all or much less affected by the Cu treatments. Cu transfer to shoots remained lower in mycorrhizal plants than in NM seedlings. Results of this experiment are illustrated in Fig. 3 showing the Cu response on the ratio of ECM/NM
plants for biomass, P content, and Cu burden in the needles. In the absence of
FIG. 3 Response of Pinus sylvestris seedlings to toxic Cu concentrations. Ratio of ectomycorrhizal (ECM)/nonmycorrhizal (NM) plants for total biomass
(), total P () and total Cu in shoots (). Seedlings were grown under P
limitation and were exposed to elevated Cu for 36 days. (A) Plants mycorrhizal with Thelephora terrestris. (B) Plants mycorrhizal with Suillus bovinus.
Mycorrhizal Fungi
47
elevated Cu, the ECM/NM ratio for biomass and P content is close to 1, indicating
no difference between NM and ECM plants. With increasing Cu exposure, the
ECM/NM ratio for biomass and P content consistently increased, whereas total
Cu in the shoots was relatively lower in ECM plants. These results showed that
both mycobionts could protect root development and functioning of their host.
At the highest Cu concentration (47 M), transfer of Cu (and other nutrients)
was impaired in NM plants because of a complete inhibition of root growth.
Mycorrhizal colonization of the short roots was hardly affected by the Cu
treatment. The mechanisms involved in the amelioration of metal stress remain
unclear and are probably diverse. For a good overview of possible mechanisms,
the reader is referred to the reviews of Leyval et al. (11) and Jentschke and
Godbold (13). From our own experience, we suggest that ECM fungi that are
able to survive at elevated metal exposure and that can avoid excessive uptake
of metal in the symplast theoretically can reduce metal exposure of the roots of
their host. In this respect it should be stressed that sporocarps of ECM fungi from
metal-polluted sites generally do not accumulate the toxic metal, certainly when
it is an essential element (66,80,81). Low metal concentrations or a low mobility
of metals in mycelia might be important to reduce metal exposure to host roots.
Prevention of excess metal uptake might be realized by excretion of metalimmobilizing substances, extracellular sequestration, or well-regulated uptake
systems coupled to stable plasmalemmas.
6
DIVERSITY OF MYCORRHIZAL FUNGI IN HEAVY METAL

POLLUTED SOIL
Because mycorrhizal fungi constitute a living bridge for the plant nutrition and
resistance to different environmental stresses, there is an increasing interest in
the diversity of mycorrhizal fungi in soils.
The species richness of AM fungi has been analyzed for both nonpolluted
soil (8287) and heavy metalpolluted soil (30,32,34,36,45,46,88,89,90) (Table
1). Species richness is probably dependent on the type of soil, but it nevertheless
seems to decrease when the soils are polluted (Table 1). In the few studies that
focused on old, nondisturbed mine vegetations, spores of several AM fungi were
found (32,34). At present, it is not clear whether some of these fungi have a
preference for such habitats. In agricultural soils, the AM fungal species richness
is usually low (45,46,86) compared with the number of species found in natural
grasslands (82,83). Soil tillage and agricultural inputs of pesticides and fertilizers
are suspected to deplete AM fungal diversity (91,92). In most cases, the species
richness measured is the result of cumulated local diversity(90).
In two different field experiments, a long-term sewage sludge application
with increasing concentrations of metals lead to a decrease in both size and diversity of AM fungi populations (45,46). For soils receiving intermediate rates of
TABLE 1 Analyses of the AM Fungal Species Richness in Different Ecosystems
48
Location, surface (if known), sampling strategy, reference
Total no.
of species
AM genera (%)
G
Gi
Sc
12
21
43
14
26
25
58
67
63
57
38
44
25
5
9
14
8
24
0
5
0
14
4
4
0
14
7
0
11
16
17
9
21
14
38
12
Acidic mine soil, West Virginia (60 m2), S 1 sample, TC (88)

Dunes, Joaquina, Brazil (600 m2), S 1 sample (89)
Industrial soil, North Carolina (75 m2), S 1 sample, TC (90)
Mine spoil, Kansas (1 ha), S 1 sample (30)
Calamine spoil, metal tolerant vegetation, Poland (32)
Disturbed site, S 3 samples
Undisturbed site, S 3 samples
Mine spoil, Kansas (1 ha), S 1 sample (36)
Control soil, S 1 sample (36)
Sewage sludge amended field experiment (old arable field trial),
Braunschweig, Germany (85.5 m2) (45)
Control plot, S1 sample, TC
Moderately polluted soil, S1 sample, TC
Highly polluted soil, S1 sample, TC
Sewage sludge amended field experiment (ex-woodland field trial),
Braunschweig, Germany (85.5 m2) (46)
Control plot, S1 sample, TC
Moderately polluted soil, S1 sample, TC
Highly polluted soil, S1 sample, TC
Mine spoil, rhizosphere zinc violet (Viola calaminaria) S 1 sample, TC (34)
5
12
24
3
20
33
42
100
60
17
29
0
0
0
0
0
0
8
12
0
20
42
17
0
2
6
3
5
50
83
67
60
0
0
0
0
50
17
33
40
0
0
0
0
0
0
0
0
6
6
5
100
100
100
0
0
0
0
0
0
0
0
0
0
0
0
3
4
2
4
100
100
100
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
a
Studies performed in nonpolluted soils are reported on the top of the table. Taxonomic analysis performed directly on the sampled
soil (S) or on trap cultures (TC). Ratio of the number of species that belong to Glomus (G), Acaulospora (A), Entrophospora (E), Gigaspora (Gi), and Scutellospora (Sc).
Prairie, Central Iowa (14336 m ), S 32 samples (82)

Konza Prairie, Kansas (35 km2), S 6 samples (83)
Apple plantings, 18 states of the USA, S 18 samples and TC (84)
Dunes, Great Lake, Wisconsin (600 km), S 10 samples (85)
Dunes, East coasts of the USA (355 km), S 19 samples (86)
Cedar Creek Natural History Area, Minnesota (18 km2), S 15 samples (87)
Mycorrhizal Fungi
49
sludge contamination, Del Val et al. (46) found a decrease of the total spore
number whereas the diversity index increased. On the other hand, Vandenkoornhuyse (45) highlighted an increase of the AM fungal population size and constant
species richness in moderately polluted soil. A similar response to stress was
revealed on the same field experiment for Rhizobium leguminosarum bv. trifolii
(93).
Additional analyses on the diversity of spores isolated from the soil of
the different plots of the field experiment were performed. The analysis of the
intraspecific diversity of two indigenous Glomus species by ISSR fingerprints on
single spores revealed a high diversity whatever the AM fungal population (94).
The number of haplotypes was correlated with the size of the population. For
both species, the population genetic analyses showed a high likelihood of occurrence of recombination events for the populations of spores isolated from the
most polluted soil. This mechanism might allow the fungi to generate more genetic diversity so that they can adapt more rapidly to the environmental stress
(94).
A hypothetical model of the effects of stress on an AM fungal population
can be proposed. In this model, we assume that in stable and uniform environments the competition might allow the neutral emergence of competitive species or genotypes with high fitness. A moderate stress may modify the previous
equilibrium by a possible decrease of the fitness of the individuals leading to a
proliferation of more types (15,18). For a higher level of stress, a progressive
extinction of nonadapted organisms ends in the loss of diversity.
The number of ECM fungi that can associate with ectomycorrhizal trees
is considerably larger than in the AM symbiosis. However, as pointed out above,
we have a very incomplete view on the biodiversity of mycorrhizal symbionts in
metal-polluted environments. Nevertheless, in expectation of more below-ground
community studies, we would like to refer to two community studies based on
above-ground sporocarp observations, along metal gradients in northern and
southern Sweden (81,95). Although molecular studies have demonstrated a considerable lack of correspondence between the above- and below-ground communities of ECM colonizers (16), the field studies of Ruhling suggest that particular
mycorrhizal guilds disappear with increasing metal stress. Sporocarp production
in Picea abies forests along a Cu-Zn gradient was investigated (95). The average
number of species of macrofungi per plot (1000 m2) decreased significantly along
the gradient: 35 species at a Cu concentration of about 100 g/g organic matter,
25 species at 1000 g/g, and only 15 species at 10,000 g Cu/g organic matter.
Some ECM taxa decreasing along the gradient were Chantarellus cibarius, Cortinarius sp., Dermocybe sp., Gomphidius sp., Hydnum sp., Lactarius sp., Paxillus
involutus, and Russula sp. Taxa that were not affected or that increased in frequency include Albatrellus ovinus, Amanita sp. Cantharellus tubaeformis, Laccaria laccata, and Leccinum sp. In northern Sweden, a similar study was performed
50
along a more complex metal (As, Cu, Pb, Cd, Zn) gradient and very similar
results were obtained as well (81). In the least polluted plots, 4.4 species of macrofungi were found per 100 m2, whereas only 1.3 species were observed in the
most heavily polluted plots. The number of observations of the genus Amanita
increased in the most heavily polluted area, whereas Cortinarius, Lactarius, and
Russula showed decreasing numbers of observations. The reduced sporocarp production and the decreasing above-ground diversity do not necessarily mean that
the percentage of root colonization decreases over the same gradient. It is certainly possible that the frequency of other nonfruiting fungi (Hymenoscyphus
ericae) increases on the most polluted sites, as was observed in the study of
Vralstad et al. (51). Other ectomycorrhizal taxa that have been frequently found
on heavily polluted soils include Hebeloma sp., Pisolithus tinctorius (96), Rhizopogon sp. (97), Scleroderma sp. (98), Suillus sp. (22,66) and the Cd-accumulating
Amanita muscaria (53,80).
AM FUNGI AND SOIL QUALITY ASSESSMENT
The different ways by which AM fungi influence their host plants and their terrestrial ecosystems illustrates fairly well the importance of these fungi for soil quality. However, little attention has been paid on this group of microorganisms in
the assessment of soil quality. In agricultural soils, different practices may harm
the potential AM colonization (and consequently functions) (92). Phosphate fertilization (99), soil pH, availability of carbon and nitrogen (87), soil tillage (91),
crop rotation (100), and pesticide application (101) all can modify or reduce the
AM formation. Among current agricultural practices, the application of sewage
sludge and associated heavy metal pollution is becoming an important problem.
Since numerous factors can modify the AM fungi in soils, the measurement of
a toxic effect on AM fungi is difficult to assess properly. In a field experiment
with different plots of the same soil structure and the same crop history, it is
possible to study the long-term effect of metal-contaminated sewage sludge applications on the indigenous AM fungi. In Table 2 we show the metal concentrations
measured in an old arable field experiment (45) and in an ex-woodland field
experiment of Braunschweig (Germany) (46). In both studies, the metal concentration in the most polluted plot was below the European Communitys maximum
allowable concentration limits for metals in sludge-treated soils (Table 2). As
described before, a toxic effect was nevertheless observed on the populations of
AM fungi in these soils (45,46,94). Metal toxicity is even suspected in moderately
polluted soils. The measurement of fluxes of free metals in soil solution (102)
demonstrated that Zn and Cd are the most available elements. However, it is
difficult to determine which factors are responsible for the toxicity. We and others
(18,103) argued that decisions on soil protection should be based on knowledge
Available heavy metals pFss

(pFss log Fss)
Total metal conc. (mg/kg)
Old arable soil:

C
MP
HP
Old woodland soil:
C
MP
HP
Regulations:
European communityb
United Statesc
Zn
Cd
Ni
Cu
Pb
Zn
Cd
Ni
Cu
Pb
56
163
329
0.4
0.8
2.7
8.5
16.3
32
11.9
42.9
93.7
22
29
95
3.3
2.9
1.2
5.0
4.9
3.4
3.8
3.6
2.1
3.6
3.4
2.5
4.2
4.1
3.6
43
88
295
0.3
0.6
2.8
8.6
8.3
29
9.8
17
92
29
30
111
2.4
1.9
1.1
3.9
3.8
3.0
3.5
3.1
1.8
3.6
3.3
2.5
3.8
3.7
3.4
150300
1400
13
20
3075
210
50140
750
50300
150
Mycorrhizal Fungi
TABLE 2 Concentrations of Metals in an Agricultural Soil Treated with Sewage Sludge (Braunschweig, Germany)a
a
Heavy metals in a control plot (C), a moderately polluted plot (MP), and a highly polluted plot (HP) in old woodland soil (43,46) and
in old arable soil (45,94). The flux of metal from solid soil to solution (Fss) in ng cm2s1 estimates the available heavy metal fraction
(102). Maximal concentrations of heavy metals allowed in agricultural soils are included for comparison.
b
Commission of the European Communities. Council directive (86/278/EEC) on the protection of the environment, and in particular
of the soil, when sludge is used in agriculture. Off J Eur Comm L181(annex 1A) pp 612, 1986.
c
Calculated from maximum cumulative pollutant loading limits without taking into account background concentrations of the elements
in soils. U.S. environmental protection agency. Standards for the use or disposal of sewage sludge. Fed Regist 58:92489415, 1993.
51
52
of the risk of damage of microbial functions and processes. It has been proposed
that legislation in many countries could be more restrictive concerning the rate
of metal accumulation in soil (104).
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3
Freshwater Algae
Barbara Pawlik-Skowronska and Tadeusz Skowronski
Institute of Ecology of the Polish Academy of Sciences, Lublin, Poland
INTRODUCTION
Algae are phototrophic organisms that are very diverse in size and morphology.
Most of them are aquatic and inhabit both fresh and saline waters. They are
classified in the following divisions: Chlorophyta, Chromophyta, Rhodophyta.
Depending on the classification, prokaryotic bluegreen forms (Cyanobacteria,
Cyanophyta) and Prochlorophyta may be also included.
Some metals are required for algal metabolism and physiology as macroelements (K, Mg) or microelements (Co, Cu, Fe, Mn, Mo, Ni, V, Zn), and must be
obtained from the external environment. However, the essential elements, such
as Cu, Mn, Ni, and Zn, can be toxic at high concentrations. Other metals, such
as Cd, Hb, Pb, have no known biological function and are always toxic. The term
heavy metals, used by ecologists, generally refers to the elements possessing a
density greater than 6 g/cm3 and connotations of toxicity, e.g., Ag, As, Cd, Cu,
Hg, Mn, Ni, Pb, and Zn (1). As reported in several reviews (28), all of these
and related metals as well as metalloids may be accumulated in algae and have
an impact on them.
59
60
Pawlik-Skowronska and Skowronski
ACCUMULATION OF METALS BY ALGAE
Algae, primary producers and essential constituents of setting materials in aquatic

ecosystems, are often the dominant vectors for heavy metals. Plankton particles
(including cyanobacteria and eukaryotic algae) play an important role as biosorbents influencing the environmental fate of metals, their chemical speciation and
bioavailability (911). For example, during a plankton abundance maximum
sorption of cadmium occurred, with phytoplankton playing a significant role.
Both nanoplankton and phytoplankton were largely responsible for transferring
cadmium from the water column to the sediments (12). On the other hand, algae
as primary producers can introduce heavy metals into the aquatic food chain and
this phenomenon may have important consequences for ecosystem structure and
function (13).
Algae possess the capacity for taking up heavy metals from the water, producing an internal concentration greater than that of their surroundings. For example, the samples of Cladophora glomerata collected from different rivers and
streams in northeastern England contained 133 g Cu/g; 6.631130 g Zn/g;
0.0426.97 g Cd/g; and 0.141330 g Pb/g; whereas the metal concentrations
in the filterable water were 3.117.9, 2167; 0.048.3, and 0.522.4 g/L, respectively. Significant correlation between the concentration of metal in Cladophora and water was stated (14); therefore, the algal ability for heavy metal accumulation may be useful for environmental monitoring, and chemical analyses of
these organisms may give valuable information about contamination of the
aquatic environment (1). Metal concentrations in algal biomass can vary widely.
For instance, Zn concentrations in different taxons of algae and cyanobacteria at
different locations ranged from 5.8 to 219,000 g/g (4); Cd concentrations from
1 to 340 g/g (5); and Pb concentrations from 1 to 14,000 g/g (6). As recently
reported (15), the cellular Cu concentration in the green alga Scenedesmus subspicatus can range from 2 to 950 g/g at the external free Cu concentrations
1014 107 M, and from 33 to 6500 g Zn/g at the external free Zn concentrations
109 105 M.
The metal bioaccumulation ability of algae in the environment can reflect
to some extent a concentration factor (CF), i.e., the ratio between metal concentration in the cells and that in the surrounding water. This factor varied for various
species and for different metals in a broad range (Table 1). CF is highly dependent
on many environmental variables affecting the metal sorption level (free metal
concentration in the solution, physiological status of the cell, algal cell density,
kind of water, interference with other ions, etc.). Therefore, it is difficult to compare the CF values directly. The heavy metal uptake capacities of different algal
species can be better described by sorption isotherms (1619) showing a relationship between the metal concentration in the biomass and the residual metal concentration in water.
Freshwater Algae
61
TABLE 1 Studies Showing the Variety of Concentration Factors of Heavy

Metals in Freshwater Algae
Alga
Chlorella pyrenoidosa
Chlorella vulgaris
Cladophora
Nostoc 586
Scenedesmus obliquus
Hydrodictyon reticulatum
Tribonema
Cladophora
Tribonema
Nostoc 586
Anacystis nidulans
Ulothrix
Coccomyxa
Nostoc 586
Tribonema
Metal
Concentration
factors
Ref.
Cd
Cd
Cd
Cd
Cd
Cr
Cu
Cu
Cu
Fe
Fe
Hg
Hg
Mn
Ni
Ni
Pb
Pb
Zn
16,70032,000
863900
500
92.5
105
132425
13,00083,000
18003500
4983300
140,000290,000
2676949
24,00047,300
318
37154945
195056,130
140
31,000
91.4
5201100
57
16
58
57
57
59
60
60
59
60
59
57
57
59
61
60
60
57
60
The mechanism of heavy metal uptake by algal cells is complex process

comprising two phases: a fast, metabolic independent surface binding of physicochemical nature and a metabolism-dependent phase, whereby the metal ions are
transported across the cell membrane to the cells (3,2027). From the quantitative
point of view, in the case of planktonic green and bluegreen algae the surface
sorption may be the largest proportion of the total metal uptake (about 8098%),
as reported for Cd, Cu, Zn, and Pb (16,23,27,28). However, in the case of very
low concentrations of essential metals (15), up to 80% of the total cellular copper
in Scenedesmus subspicatus was located intracellularly and only 20% was located
on the cell surface. With the increasing external free Zn concentrations from 109
to 105 M, the amount of superficially adsorbed Zn increased from 5% to 80%
of the total cellular Zn content in this alga.
The large surface area of microalgae may highly affect the metal equilibrium concentration in the surrounding water and, in consequence, the metal availability to other organisms. This concerns both living and dead algal biomass;
dead cells can often adsorb metal ions in larger quantities than living biomass
62
(19,23) because more binding sites are available. Algal surfaces possess various
functional groups, such as carboxyl, amino, phosphate, hydroxyl, and sulphydry,
capable of binding heavy metals (2931). In sorption process (where an ion exchange mechanism usually predominates), metals usually bind to anionic sites
by displacing protons from acidic groups or existing metals from anionic sites
(29,32). For example, this has been reported for freshwater filamentous green
algae Vaucheria, Tribonema, Spirogyra, Oedogonium (33), as well as some unicellular green and bluegreen algae (19). Some metals and metalloids (e.g., tin,
technetium, germanium, arsenic, selenium) can exist in the form of anionic complexes. They can be also taken up by algae because certain functional groups;
such as amines and imidazoles, can be positively charged when protonated and
may electrostatically bind negatively charged metal complexes (31,34).
Particular taxonomic algal groups and species exhibit considerable variations of the surface properties and hence different metal binding characteristics.
The structural and chemical variations of cell surface components as well as
various cell morphologies may be responsible for different metal sorption capacities observed. For example, Eisenia bicyclis was more effective in binding aluminum at pH 2 than were Cyanidium caldarium, Spirulina platensis, and Chlorella
pyrenoidosa, while Cyanidium caldarium bound more copper at pH 2 than did
Eisenia bicyclis, Spirulina platensis, and Chlorella pyrenoidosa (31). There are
marked variations in specific surface values (m2 cm3) between individual algal
species (35). For example, the cyanobacteria Anabaena inaequalis and Anabaena
lutea displayed especially high Cd adsorption per surface unit, whereas some
Chlorophyceae, i.e., Klebsormidium sp., Ulothrix gigas) had rather low surface
adsorption capability. The cation binding capacities of the unicellular cyanobacteria Anacystis nidulans, Aphanocapsa sp., Synechocystis aquatilis, and the green
algae Stichococcus bacillaris and Chlorella kessleri were much higher than those
of the filamentous macroalga Vaucheria sp. (Xanthophyceae), which possess
much lower ratio of the surface to volume (19,36).
The metabolism-independent surface sorption of heavy metals reported for
many eukaryotic and prokaryotic algae is rapid (3,16,17,19,22,23,28,3739). Algae bind most metals from the solution within a few minutes and sorption equilibrium is soon reached. This passive phase of the metal uptake is usually described
by sorption isotherms (the Langmuir or Freundlich model). Heavy metal sorption
by algal cells may be influenced by many environmental variables, and pH is
one of the most important factors because of its effect on the charge of functional
groups and chemical speciation of metals. Low external pH decreased algal sorption of such metals as Cu, Zn, Cd, Pb, and Co (5,17,19,31). However, sorption
of anionic metal complexes such as TcO4 increased with the decreasing external
pH (34). Temperature effect on the surface sorption of heavy metals is relatively
small (17). However, other environmental factors, such as competing metal cations and complexing ligands, may strongly affect this process.
Freshwater Algae
63
The second, metabolic-dependent (active) phase of metal uptake occurs

obviously only in the living algal cells. There have been demonstrated the active
uptake of Mn2 into Chlorella pyrenoidosa cells (40), energy-dependent Cd transport into S. bacillaris cells (24,41), Ni2 transport into Anabaena cylindrica cells
(42), Cd and Zn transport into Chlorella vulgaris cells (43), Cd transport into
Synechocystis aquatilis cells (26), Zn active uptake by Selenastrum capricornutum (44) and Chara corralina (45), as well as Cu active transport into the cells
of Nostoc calcicola (38). The intracellular transport of metal ions studied both
in green and bluegreen algae runs usually more slowly than surface sorption.
In darkness, intracellular Cd, Zn, Cu, and Pb uptake was reduced as observed in
both eukaryotic algae such as Chlorella, Stichococcus, Vaucheria (2224,37),
and prokaryotic ones (26,38). In both the bluegreen algae Anabaena cylindrica
(42), Nostoc calcicola (38), Synechocystis aquatilis (26), and the green algae
Stichococcus bacillaris (24) and Chara corralina (45), membrane potential, proton gradient across the cell membrane, and ATP may be involved in intracellular
uptake of heavy metals such as Cd, Cu, Ni, and Zn. The intracellular transport
of divalent metal cations is concentration-dependent, frequently shows saturation
with the increasing metal concentration, and follows the Michaelis-Menten kinetics. It was reported for Cu transport into Nostoc calcicola cells (38), Cd uptake
into Anacystis nidulans (46) and Synechocystis aquatilis (26), Ni transport into
Anabaena cylindrica (42), and Zn uptake into Selenastrum capricornutum (44).
On the contrary to the surface sorption, metal intracellular transport into
the algal cells is strongly temperature-dependent (26,41). Cd transport into the
green alga Stichococcus bacillaris cells was completely inhibited at 4C and increased with the temperature increase by 35C (41).
There is the assumption based on the competitive uptake studies between
metal cations that nonessential metals (Cd, Pb) share in algal cells transport systems for essential elements like Ca, Mn, Zn (26,40,41,47,48). However, the process of essential metal uptake in algae is not fully recognized. It has been suggested (49) that the Ca influx into the cells of Chara corralina occurs via voltagedependent calcium channels; however, it is not clear if there are specific channels
for Ca. Essential elements like Zn may be accumulated intracellularly into algal
cells using high- or low-affinity transport systems (44,45).
The pH of the surrounding environment can strongly influence not only
the metal surface sorption (as mentioned earlier) but the metabolic-dependent
intracellular transport as well because of its effect on metal speciation and cellular
membrane potential (2). There are scarce data on this subject. As reported for the
green alga Stichococcus bacillaris (41) and the bluegreen alga (cyanobacterium)
Synechocystis aquatilis (26), the intracellular Cd transport occurred at pH 7 but
was strongly inhibited at the acidic pH values (pH 45) (Fig. 1). However, there
was no effect of changing external pH on the Ca influx into the cells of Chara
corralina (49).
64
FIG. 1 Cd2 transport (1) and 14C fixation (2) in the presence of cadmium in
S. aquatilis at different external pHs. Total cadmium concentration (8.9 M)
and 14C fixation in the control without Cd at each pH values were taken as
100%. Transport measurements were made after 40 min. (From Ref. 91.)
Some metals and metalloids, such as Pb, Hg, Sn, and As, can occur as
organometallic compounds, which can be also accumulated by algae and may
exert even stronger toxic effects than inorganic forms of these elements
(25,50,51,52). The accumulation of organotin compounds was reported for a few
freshwater green algae like Ankistrodesmus falcatus (53), Scenedesmus obliquus,
Chlorella vulgaris (54), and some cyanobacteria (55). The accumulation of organocomplexes of Pb, Cu, Cr, and Tl in neutral lipids and plastoglobuli of the
green alga Cladophora has also been reported (50). However little is known about
the uptake mechanisms of organometallic compounds. There are suggestions that
lipophilic organometallic compounds (like methylmercury) can rapidly pass algal
cell membranes via passive diffusion (56).
3
EFFECTS OF METALS ON ALGAL GROWTH

AND METABOLISM
The primary impact of heavy metals on the algal cells is at the biochemical and
physiological levels. Heavy metals can interact with cellular macromolecules or
Freshwater Algae
65
disturb cell membrane integrity, leading to inhibition of essential cellular metabolic or physiological process and ultrastructural changes. Uptake of heavy metals can result in inhibition of several enzymes (62). Generally, as mechanisms
of metal action on enzymes, binding of the metal to the sulfhydryl group involved
in the catalytic action or structural integrity of enzymes and deficiency of essential
metals in metaloproteins or metal-protein complexes combined with substitution
of the toxic metal for the deficient element, are proposed. Strong affinity to SH
groups is exhibited particularly by such metals as Cd, Pb, and Hg (62). Besides,
heavy metal uptake can disrupt cell transport processes; also, in some cases oxidative stress is possible (63). Free radicals generated by some transition metals can
damage cell compartments leading to decrease in growth or cell death. In the
presence of Cu, Co, Pb, and Ni, the increase of peroxidase activity in Selenastrum
capricornutum to prevent cell damage was reported (64). For these reasons, heavy
metals may interrupt the normal metabolic processes and integrity of algal cells
in a number of ways.
The uptake of toxic amounts of metals by algae can resulting among other
things, inhibition of photosynthesis, as well as alteration of respiration and adenylate metabolism. In addition, several ultrastructural and morphological changes
can be observed in the algal cells. Metal stress can affect total cell volume, number and relative volume of polyphosphate bodies, lipids, vacuoles, cell wall, and
periplasmalemmal space, among other factors (65). Enlarged algal cells, unable
to divide, have been often observed (6669). The mentioned effects lead to the
inhibition of algal growth. Some toxic effects of heavy metals on the algal cells
are presented in Table 2. The toxic effects of heavy metals on algae depend on
the condition of exposition, time, and heavy metal dose. Generally, the toxicity
of most metals is a function of free metal ion concentration in the solution, rather
than total metal concentration, and can vary in the presence of complexing agents
(70,71). In the aquatic systems, a number of environmental factors affect metal
toxicity through the influence on metal chemical speciation and metal uptake
process. One of the most essential factors is pH. Generally, toxicity of such metals
as Cd, Cu, and Zn toward algal cells decreased as the pH of surrounding solution
decreased (2). For example, cadmium exerted toxic effects on the green alga
Stichococcus bacillaris at pH 67, whereas it did not affect the biomass yield
substantially at pH 3 and 9 (72). Cobalt, copper, and nickel were less toxic to
the green alga Chlamydomonas reinhardti at pH 5 than at pH 7 (71). Also the
decrease of copper and uranium toxicity at pH 5.7 compared to those at pH 6.5
was reported for the freshwater alga Chlorella sp. (73). Apart from hydrogen
ions, other cations and anions can influence the toxic effect of metal on the aquatic
organisms through various types of competitive interactions. These other ions
(Ca2, Mn2) may protect the organism from metal uptake or from its toxic expression. For example, they markedly reduced cadmium toxicity to the green alga
Stichococcus bacillaris (74). However, in some cases the presence of other ions
66
TABLE 2 Some Toxic Effects of Heavy Metals on Green and BlueGreen Algae
Organism
Metal
Observed effect
Growth
inhibition
inhibition; cell division inhibition
inhibition
inhibition
Cd
Cd, Zn
Cu
Pb and organolead complexes
Growth
Growth
Growth
Growth
Cu and Cu complexes
Zn, Cd, Hg
Cu
Ni, Hg
Zn
Cu, Cd, Zn
Cd
Scenedesmus acutus
Euglena gracilis
Cu
Zn, Cd, Hg
Chlorella sp.
Hg, Zn
Cu
Pb
Chlorella sp.
Scenedesmus acutus
Chlorella sp.
Hg, Zn
Cu
Hg, Zn
Growth inhibition
Cell division inhibition
Cell division inhibition
Growth inhibition
Growth inhibition
Growth inhibition
Photosynthesis
Inhibition of O2 evolution; inhibition of CO2
fixation
Inhibition of O2 evolution
Inhibition of O2 evolution; inhibition of CO2
fixation
Inhibition of O2 evolution
Inhibition of CO2 fixation
Inhibition of chlorophyll biosynthesis and O2
evolution
Destruction of chlorophyll
Destruction of chlorophyll
Inhibition of the rate of chlorophyll synthesis; reduction of thylakoid surface area
80
66,67,84
82
50
69
83
69
84
77
28
80
82
85
86
69
87
86
82
86
Stichococcus bacillaris
Scenedesmus acutus
Chlorella fusca
Cladophora glomerata
Euglena gracilis
Anabaena inaequalis
Microcystis aeruginosa
Chroococcus paris
Ref.
Cd
Ni, Hg
Cu, Cd, Zn
Anabaena flos-aquae
Synechocystis aquatilis
Cd
Cd
Chlorella fusca
mixture of metals: Al, Fe, Cu,

Zn, Ni, Mn
Pb and organolead complexes
Chlorella fusca
Cladophora glomerata
Al
Plectonema boryanum
Anabaena flos-aquae
Anabaena variabilis
Anabaena flos-aquae
Cd
Zn
Cd
Synechocystis aquatilis
Chlorella sp.
Euglena gracilis
Cd
Zn, Hg
Zn, Cd, Hg
Pb
Cu, Cd
Changes in subcellular structure and larger

proportion of heterocysts
Increased number of polyphosphate bodies
Increased number of polyphosphate bodies
Reduction in subcellular structures; cell wall
lysis
Other Metabolic Processes
Changes in cellular adenylate metabolism
Respiration inhibition
Respiration inhibition
Stimulation of dark respiration
88
84
89
90
75,91
92
50
93
68
94
95
90
26
86
85
87
67
Monoraphidium
dybowski
Stichococcus sp.
Anabaena 7120
Reduction of thylakoid surface area

Photosynthesis inhibition; pigment bleaching
Inhibition of Hill activity and photosynthetic
oxygen evolution
Reduction in number of carboxysomes
Reduced CO2 fixation, O2 evolution and carbonic anhydrase activity
Ultrastructural Changes
Chloroplast damage; disruption of thylakoidal membranes
Breakdown of organelles; distendent endoplasmic reticulum; shrinkage of chloroplast;
multinucleate cells; changes in cell wall
structure
Detachment of plasmalemma from the cell
wall; destruction of cell wall; vacuolization
Freshwater Algae
Anabaena flos-aquae
Anabaena inaequalis
Anacystis nidulans
68
may enhance the toxic effect. Among inorganic ligands phosphate and chloride
ions are persistent complexing agents of heavy metals. They are important complexing factors also in freshwaters, particularly in eutrophic waters or these contaminated with mining effluents and sewages of high salinity. As reported for
the bluegreen alga Synechocystis aquatilis in solutions containing cadmium
chloride complexes (CdCl, CdCl2, CdCl3), Cd toxicity was significantly limited
(75). In the aquatic environment the dissolved organic matter has also been demonstrated to affect metal toxicity (76). Various chemical forms of metal differ in
their availability to living organism (77). On the other hand, the algal sensitivity
to metals is species- or strain-specific (78,79).
The stress caused by heavy metals in algal cells induces quantitative and
qualitative changes in the structure and functioning of algal communities.
4
DIVERSITY OF ALGAL COMMUNITIES IN THE

FRESHWATER HABITATS POLLUTED WITH METALS
Although high concentrations of heavy metals are known to be toxic to biota,

reports on the freshwater algae surviving in the environments with elevated concentrations of heavy metals are common (96105). An important aspect of ecological investigations of algal communities in the metal-contaminated freshwaters
is the descriptive studies of algal flora in the polluted rivers, streams, and lakes.
Freshwater streams and rivers may carry heavy metals due to the leaching of
natural outcrops or industrial (mining, smelting) and agricultural (artificial fertilizers, pesticides) activities. Most studies of algal communities in the metalcontaminated waters were carried out in the polluted streams or rivers situated
in the Cu, Pb, and Zn mining regions (100,101,103,106108).
4.1
BlueGreen Algae (Cyanobacteria)
In the metal-contaminated freshwater habitats of high pH, bluegreen algae (cyanobacteria) are often abundant. The dominant genera reported in the zincenriched waters were filamentous nonheterocystous Oscillatoria, Phormidium,
Plectonema, and Schizothrix (109,110). Bluegreen algae were found in the habitats of very high zinc concentrations (10100 mg Zn/L) but only of pH higher
than 5 (111). Low pH (5) severely limits their survival (112). Yasuno and
Fukushima (113) showed that the bluegreen algae Chamaesiphon minutus and
Phormidium luridum usually appeared in the copper- and zinc-polluted rivers in
Japan. As reported by Takamura et al. (103), different species of Phormidium
occurred in three Japanese rivers polluted with Cu and Zn. Also in the water of the
urban river Miyata of alkaline pH, polluted with Cu and Zn (108), the following
cyanobacteria species were identified: Chamaesiphon subglobosus, Phormidium
foveolarum, and P. uncinatum.
Freshwater Algae
4.2
69
Green Algae
In the river water in Wales (U.K.) polluted with Zn, Cd, Cu, and Pb, McLean
and Jones (97) reported the occurrence of the filamentous green algae Hormidium
rivulare, Ulothrix spp., and Spirogyra spp. Generally, the green macroalgae in
the zinc-enriched streams included such species as Hormidium rivulare (98), Stigeoclonium tenue (114), Ulothrix moniliformis and Mougeotia sp. (109). Two
hundred isolates comprising 87 species of Chlorophyta were obtained from the
sites along the rivers that drain the ancient Cu and Pb mining region in Cornwall
(U.K.) (100,101). They were classified mainly into the following taxonomic
groups: Ulotrichales (Microspora stagnorum, M. pachyderma, M. willeana, M.
tumidula, Ulothrix spp., Hormidium spp., Stigeoclonium tenue, Cylindrocapsa,
Stichococcus spp.); Zygnematales (Mougeotia, Spirogyra), Volvocales and Tetrasporales (Chlamydomonas, Gleococcus, Chlamydocapsa, Asterococcus), Chlorococcaceae (Chlorella, Hypnomonas, Pleurococcus, Trochiscia); Oocystaceae
(Oocystis, Pseudococcomyxa), and Scenedesmaceae (Ankistrodesmus, Selenastrum, Scenedesmus). As reported by Whitton (1), abundant growths of the filamentous green alga Stigeoclonium combined with the complete absence of Cladophora was often associated with high concentrations of heavy metals combined
with relatively high nutrient concentrations. Conversely, massive growths of Cladophora glomerata usually indicated that heavy metals were present in flowing
waters only at very low concentrations (14). The algal survey in three Japanese
rivers polluted with Cu and Zn showed (103) that green algae were represented
by different species of Chlorella, Oocystis, Scenedesmus, Chlamydomonas,
Ulothrix, Klebsormidium, Stigeoclonium, Stichococcus, and Cosmarium. In the
Cu- and Zn-polluted river Miyata in Japan, among a very limited number of
periphytic algal flora Stigeoclonium aestivale and Oocystis lacustris (Chlorophyta) also appeared (108). The green microalgae Chlorella fusca var. vacuolata and Scenedesmus acutiformis were isolated from lake water in Canada,
contaminated with Cu and Ni (96). The natural algal assemblages from the lakes
(USA) contaminated with arsenic consisted of planktonic species of Chlorophyta:
Chlorella sp., as well as mucilage sheath forming Chlamydocapsa bacillus and
Chlamydocapsa cf. petrify (115). The isolated algae showed different sensitivity
to various chemical species of arsenic, which can play a role in the selection
of the phytoplankton communities. Recently, the periphytic green alga Oocystis
nephrocytioides was identified as dominant in the freshwater communities exposed to high Cu concentrations (105). An abundant growth of Stigeoclonium
sp. (104) accompanied by minor growths of a few species of green microalgae
Scenedesmus spp., Chlorella sp., and Dictyococcus sp. (Pawlik-Skowronska, personal communication) was observed in the fast-flowing water of a mine drainage
stream of elevated Zn, Cd, and Pb concentrations in southern Poland.
70
4.3
Diatoms
In addition to Cyanophyta and Chlorophyta, some Bacillariophyceae (Chromophyta) also were reported in heavy metalpolluted freshwater. In slower flowing, metal-rich waters, planktonic microalgae included several diatom genera,
e.g., Eunotia, Pinnularia, Navicula, and Synedra (109). In the mountain stream
in Canada (water pH about 7), subjected to Zn, Cu, and Cd contamination, Deniseger et al. (102) reported the occurrence of such periphytic species of Bacillariophyceae as Achnanthes minutissima, Achnanthes microcephala, Synedra filiphormis, and Nitschia sp. The same diatom species were present both above and
below heavy metal mining operations; however, at the downstream contaminated
site the virtual absence of Chlorophyta was observed as compared with upstream
control site. The Bacillariophyceae isolated from three Japanese rivers polluted
with Cu and Zn were represented by 20 species belonging to the following genera:
Achnanthes, Nitschia, Synedra, Gomphonema, Navicula, Pinnularia, Eunotia,
and Cymbella. In the water of the urban river Miyata running through the abandoned copper mining region in Japan (108), among the very restricted periphyton
species three diatoms were present: Achnanthes minutissima, Nitschia palea, and
Surirella angustata. The river water was alkaline (pH was approximately 8) and
polluted with Cu (0.070.22 mg/L) and Zn (0.130.55 mg/L). Achnanthes minutissima was also reported to be dominant in other copper-polluted rivers in Japan
(113). As can be seen, all of the above-mentioned diatoms have been classified
as the Naviculales order, and independently of the geographic location the same
diatom species appeared in the heavy metalpolluted waters. However, it has
been stated (116) that the differentiation in species composition can be associated
with the local environmental conditions, such as pH of the water. For example,
among diatoms, domination of Eunotia spp. in the acid streams and rivers is
striking (117,118), and for the higher pH, Achnanthes spp., Nitschia spp., Navicula spp., and Gomphonema spp. are typical (117). Species richness and total
biomass in freshwaters have been shown to diminish with the pH decrease
(116,119). As reported by Kwandrans (120), in the acidic stream (U.K.) polluted
with coal mine wastes (pH 2.63.3) the number of species was very limited and
the algal community consisted only of Klebsormidium rivulare, Microthamnion
strictissimum (Ulotrichales), Eunotia exigua, Pinnularia acoricola, Nitschia sp.
(Bacillariophyceae), mucilage sheathforming Gleochrysis turfosa (Chrysophyceae), and the green microalgae Euglena mutabilis and Chlamydomonas acidophila. However, among the diatoms inhabiting freshwaters of various pH values
only a few taxa proved resistant to heavy metals; i.e., Achnanthes microcephala,
Achnanthes minutissima, Achnanthes linearis, Eunotia exigua, Synedra filiformis,
and Nitschia sp. (102,105,108,113,121123).
Freshwater Algae
4.4
71
Metal Effects on Algal Communities
As reported by many authors (100,102,105,123,124), both algal abundance

and algal diversity as measured by the number of species were reduced in high
metal-polluted sites. Associations of species that were evident in the field samples
were correlated with water metal levels (Table 3). For Cu, Pb, and Fe, the metal
concentrations in algae were positively correlated with water metal levels (100).
As observed by Foster (100), the degree of floristic stability correlated with the
degree of heavy metal pollution rather than the polluting metal per se. This was
shown by the close similarity between the algal flora on the copper-polluted river
and the lead-polluted river. At high metal pollution (Cu, Cd, Pb, and Zn) an
association of filamentous algae of the genus Microspora (Microspora stagnorum, M. pachyderma, M. willeana) accompanied by the microflora of Pseudococcomyxa adhaerens, Microthamnion kutzingianum, and Chlamydomonas vulgaris was observed (100). In the sites of low metal pollution the typical
filamentous flora was Spirogyra, Mougeotia, Microspora floccosa, and the variety
of planktonic species belonging to Scenedesmaceae, Oocystaceae, and Desmidiales. The moderately polluted sites had flora consisting of Zygogonium ericetorum, Microspora tumidula, Hormidium spp., Microthamnion spp., Stigeoclonium tenue, and many Volvocales and Tetrasporales species.
As follows from many papers (97,98,100,102), there are no species invariably indicative of heavy metal pollution; however, it seems that there are taxonomic groups of green algae, e.g., Ulotrichales (Microspora, Hormidium, Stigeoclonium), mucilaginous Chlorophyceae (Chlamydomonas, Chlamydocapsa,
Oocystis), and some pennate diatoms (Achnanthes, Nitschia, Navicula) abundantly occurring in the freshwater sites encompassing large ranges of heavy metal
concentrations. These algae may be characterized by their abilities to adapt to
high metal concentrations but cannot be estimated as strictly indicative of them.
For instance, various Stigeoclonium species were found in the toxic metalfree
areas (125,126) as well as in the streams and rivers polluted with various heavy
metals (104,108,127,137). It is still difficult to make generalizations about the
influence of heavy metal pollution on algal species and community composition.
The individual situations in nature are very diverse, ranging from lakes to fastflowing streams, from one metal to a mixture of many, from nutrient-poor to
nutrient-rich water of different pH values and load of suspended matter. In heavy
metalpolluted, fast-flowing waters, periphyton communities well attached to the
substrate dominate and the sort of substrate can be also responsible for species
composition of the periphyton community (97). The water pH is a very important
factor influencing directly (on cell physiology) or indirectly (on water chemistry)
the algal development (116). It is also known that the algal community structure
72
TABLE 3 Algal Associations from the 64-Species Analysisa

Metal levels
Low
Moderate
Moderate-high
High or variable
Chlorella fusca
var. vacuolata
4 m
Ulothrix variabilis
Batrachosperum
vagum
Staurastrum punctulatum
Ankistrodesmus
falcatus var.
mirabilis
Pleurococcus vulgaris
Spirogyra laxa?
Chlorella fusca
var. fusca
Hypnomonas lobata
Hormidium rivulare
Chlorella vulgaris
var. vulgaris
Chlamydomonas
hebes
Mougeotia gracillima?
Zygogonium ericetorum
Chlamydomonas
debaryana
Chlamydomonas
botryopara
Microthamnion
kutzingianum
Chlorella saccharophila var. ellipsoidea
Pleurococcus
rufescens
Stigeoclonium
tenue
Oedogonium 7
m
Pseudococcomyxa adhaerens
Microspora
pachyderma
Microspora stagnorum
Mougeotia parvula?
Microthamnion
strictissimum
Hormidium scopulinum
Hormidium pseudostichococcus
Hypnomonas
chlorococcoides var. incrassata
Spirogyra nitida
Spondylosium
pygmaeum
Chlamydomonas
debaryana var.
micropapilli
Closterium striolatum
Low-moderate
Low-moderate
Cu, high Zn,
Pb, Fe
Microspora tumidula
Chlamydomonas
globosa
Euglena gracilis
Approximate metal concentrations

(mg L1)
Low
Cu
Pb
Zn
Fe
0.1
0.1
0.5
0.2
Moderate
High
0.10.2
0.10.2
0.51.0
0.22.0
0.20.4
0.20.4
1.02.5
2.0
Freshwater Algae
Ankistrodesmus
falcatus var. tumidus
Scenedesmus serratus
Chlamydomonas
terricola
Hypnomonas
chlorococcoides
Chlamydomonas
heterogama
Asterococcus limneticus
Scenedesmus
armatus
Oocystis parva
a
Species with similar loadings for two or more principal factors have been grouped according to their distributions at the sites. The
approximate metal concentrations represented by the descriptive assignment are also given (mg L1 total concentrations).
Source: Ref. 100.
73
74
of freshwaters depends on the season of the year (126), and spring or summer
communities may react differently to heavy metal contamination. Dissimilarity
index reported by Deniseger et al. (102) for the heavy metalpolluted stream
community was high in summer and low in spring. However, there is evidence
(128) that generally the dissimilarity of algal periphyton communities in the
stream mesocosm increased with the increase of Zn concentration. Despite the
complex condition in the freshwater ecosystems, the heavy metal impact is usually reflected by qualitative and quantitative changes in the algal community composition (129,130).
Taking into account the above-mentioned data, it seems possible to distinguish some taxonomic groups that occur more often in heavy metalpolluted
sites, i.e., some filamentous green algae belonging to Ulotrichales and Chaetophorales, microalgae like Chlorococcaceae, Oocystaceae, Scenedesmaceae, Volvocales, some Cyanophyta occurring in the alkaline or neutral waters, and some
pennate diatoms belonging to Naviculales, e.g., Achnanthes spp. in the neutral/
alkaline waters and Eunotia exigua in the acidic environment.
4.5
Development of Metal-Resistant/Tolerant
Communities
Although many biotic environmental factors are responsible for the algal community composition, heavy metals can act as agents of selection leading to establishment of the metal-tolerant ecotypes under characteristic local conditions. It was
observed in nature, where metal-tolerant algae were isolated from the metalenriched sites (e.g., Zn-tolerant filamentous green algae H. rivulare and Stigeoclonium tenue (98,127), Zn-resistant coccoid green algae Oocystis elliptica
and the bluegreen alga Phormidium (128), Cd-tolerant Chlamydomonas spp.
(101), Cu-tolerant Oocystis nephrocytioides (105,146), As-tolerant Chlorella sp.,
Chlamydocapsa spp. (115), and under the laboratory conditions Zn-tolerant Anacystis nidulans (131) and Selenastrum capricornutum (132). The tolerance levels
(determined as heavy metal sensitivity of algal photosynthesis) of freshwater Bacillariophyceae, Charophyceae, and Chlorophyceae showed significant positive
correlations with Cu concentrations in the field, whereas for Cd and Zn the correlations were not so clear. As shown in Fig. 2, the algae (excluding bluegreen)
isolated from the metal polluted sites were less sensitive to the polluting metals
than those from the unpolluted sites (103).
Pollutants such as heavy metals may affect communities because of the
exclusion of sensitive species, or by the replacement with tolerant or opportunistic
life forms. In aquatic environments subjected to pollution stress, increased tolerance has been found in the populations of algae. The concept of pollution-induced
community tolerance (PICT) proposed by Blanck et al. (133) states that exposure
of a community to a toxicant above its effect threshold exerts a selection pressure
Freshwater Algae
75
FIG. 2 Relative photosynthesis (%) of Achnanthes minutissima isolated from

a polluted site (left) and an unpolluted site (right) of the River Miyata. (From
Ref. 103.)
TABLE 4 Structural and Physiological Effects of Cu on Microalgal

Communities in Freshwater Enclosures
Time (days)
Culow
Cuhigh
No effects on biomass, photosynthesis activity, or

short-term tolerance.
Small change in species
composition.
214
Decrease in photosynthesis
activity.
1420
Increased tolerance for copper in short-term test. Cotolerance for zinc.
Very strong reduction in biomass and photosynthesis

activity. Increased shortterm tolerance. Strong
change in species composition.
Continuation of the effects
seen during the first 2
days.
Increased biomass and photosynthesis activity. High
tolerance for copper and
cotolerance for zinc.
Source: Ref. 135.
76
on sensitive species or individuals leading to their exclusions. As a result, the

community will consist of more tolerant life forms and the overall tolerance of
the community will increase as a consequence of this toxicant-induced succession. This has been already validated for the periphyton communities under arsenate stress (134), for the microalgal communities under copper stress (Table 4),
and for the stream epilithon exposed to Zn (136). Also, in a field study of the
heavily zinc-contaminated stretch of the river Dommel in Belgium (137), the
microbenthic algal communities showed high tolerance to Zn. These elevated
tolerance levels might be due to a long-term selection, favoring tolerant organisms and leading to a tolerant community as predicted by the PICT concept (134).
As reported recently by Paulsson et al. (123), the species composition of riverine
algal community changed slightly at low Zn concentrations but was strongly
affected at high Zn stress (Fig. 3). In the case of copper effects on the structure
FIG. 3 Periphyton species richness after long-term exposure to zinc. Species

richness represents the number of algal taxa or groups of taxa found in any
of the 50 fields counted on a glass disc (n 23) at each concentration. Zinc
concentrations are based on analyzed total zinc in aquaria water. Bars represent standard deviations. The horizontal solid line indicates the mean baseline of no effect. The solid regression line is based on values between 0.28
and 25 M of zinc (n 17; P 0.001). The dotted lines indicate 95% confidence limits for the regression. (From Ref. 123.)
Freshwater Algae
77
of freshwater algal periphyton communities, it has been also stated (105) that
the Cu-induced structural impacts on the periphyton communities can be evidenced as an increased tolerance to copper. As shown in Table 5, the long-term
exposure of the periphyton communities of the river Glatt to Cu caused a dominance shift from Cyanophyceae to Chlorophyta, while the relative abundance of
Bacillariophyceae was stable independent of the Cu treatments. Each class of the
studied algae showed broad interspecific differences in sensitivity to Cu. Among
Cyanophyceae the most tolerant was Pseudoanabaena catenata (dominant at 1
M Cu) while among Chlorophyta the most tolerant was Oocystis nephrocytioides, whose relative abundance increased from less than 1% (in control) to 56%
in 5-M Cu treatments (105). Of the diatoms again Achnanthes was the dominant
genus at 5 M Cu. As shown in Table 5, upon long-term Cu exposure the most
sensitive algal species were eliminated and the tolerant-species dominated. The
TABLE 5 Relative Abundance (%) of Algal Taxa in Periphyton

Communities After 12 Weeks of Exposure to Copper a
Copper conc. (M)
Chlorophyta
Cosmarium sp.
Geminella interrupta
Mougeotia sp.
Oedogonium sp.
Oocystis nephrocytioides
Phacotus lenticularis
Scenedesmus spp.
Others
Bacillariophyceae
Achnanthes sp.
Fragilaria sp.
Tabellaria fenestrata
Others
Cyanophyceae
Gomphospheria sp.
Microcystis sp.
Oscillatoria sp.
Pseudoanabaena catenata
Control
0.05
0.1
0.5
1
7
3
1
1
1
4
9
1
3
6
14
14
1
5
3
1
18
7
2
8
1
9
7
1
11
11
4
22
1
2
13
4
3
1
0
20
1
0
13
0
0
0
0
56
5
0
13
6
2
3
3
6
1
2
8
2
2
4
5
4
1
3
11
0
3
3
15
14
2
1
3
10
27
25
1
7
6
21
3
24
3
0
8
3
6
0
8
2
0
0
34
0
0
5
0
a
Values represent means of three measurements done with composite samples taken
from each of three treatment replicates.
Source: Ref. 105.
78
observed species selection and structural shifts of the community due to the presence of pollutant also support the concept of the pollution-induced community
tolerance. However, the relationship between heavy metal concentration and
community response, such as shifts in the tolerance to metals, is confounded
by the influence of chemical speciation on metal bioavailability. Metal species
formation is influenced by various local physicochemical conditions such as acidity, salinity, inorganic and organic ligands, and particulate matter (138).
VARIETY OF MECHANISMS OF METAL RESISTANCE/

TOLERANCE
The ability of algae to survive and reproduce in the heavy metalpolluted waters
has not been explained so far and may rely on intrinsic properties of the organisms
as well as the physical and chemical nature of the habitat. It can also depend on
genetic adaptation over the extended time periods by mutation, genetic exchange,
selection, etc., or on changes in the organism physiology resulting from metal
exposure (139). Metal resistance can be metal-specific, e.g., the green alga Spirogyra sp. isolated from the Cu- and Zn-polluted river showed specific resistance
only to Zn, while some pennate diatoms and the green alga Klebsormidium klebsii
showed specific resistance only to Cd (103). Since metal pollution is rarely confined to one metal, combined resistance to several metals (multiple resistance,
cotolerance) can be expected to occur among algae from such environments.
Cotolerance to zinc and cadmium was evident in some freshwater algal populations, and this resistance can result from the presence of both metals in the
polluted sites (98,101). The Cu-resistant algae studied by Foster (101) were significantly Pb-resistant; however, the Pb-resistant algae were Cu-sensitive.
Cu-tolerant Bacillariophyceae and Cd-tolerant Chlorophyceae isolated from the
polluted river tended also to be Zn-tolerant (103). Freshwater phytoplankton communities exposed to the increased copper concentrations also showed increased
tolerance for zinc (135). Algal periphyton communities exposed to Cu (105) displayed an increased cotolerance to Zn, Ag, and Ni. This phenomenon has been
explained (101) as resistance achieved by a mechanism to cope with excessive
levels of another metal. However, there is no firm evidence that resistance to
different metals can depend on the same genes.
Within algal populations surviving under the chronic metal exposition, different evolutionary strategies, conveying different types and degrees of metal
resistance, appear to coexist. Regardless of whether it is constitutive or induced
upon exposure, metal resistance is usually a heritable phenomenon. According
to Stokes (140), metal resistance is used to describe the algal species in which
all populations are normally able to tolerate metals, while algal tolerance occurs
in the species of widespread distribution, with populations in the unpolluted envi-
Freshwater Algae
79
ronments being less metal tolerant. It is assumed that tolerant populations are
genetically distinct from those that are not. However, tolerance and resistance
are arbitrary terms often used interchangeably (141). Following the Levitt definition (142), heavy metal resistance can be achieved in two ways: avoidance or
tolerance. Avoidance is defined as the ability to prevent excessive metal uptake.
The tolerance is an organisms ability to cope with metals that are excessively
accumulated within its body.
5.1
Metal Avoidance
There are some assumptions that extracellular organic material can be involved
in the enhanced heavy metal resistance of some strains of algae. For instance,
the extracellular material produced by Gleocystis gigas ( Chlamydomonas ampla) (143) and Chlorella vulgaris (144) reduced cupric ion activity and thus metal
toxicity. Similar observation has been made recently for the Cu-resistant green
alga Oocystis pusilla (145). However, Cu complexation was not essential for Cu
tolerance of another species Oocystis nephrocytioides (146). High levels of cell
wallassociated copper have been reported for other Chlorophyta (147); in the
freshwater green algae such as Cladophora, Chlorella, Scenedesmus, Spirogyra,
and Chara, cell walls played important role in heavy metal binding (45,148,149).
The surface metal binding may protect cells to some extent from the toxic effects
by reducing the concentration of free metal cations in surrounding water. As
mentioned before, the surface of algae contains a number of functional groups
with high affinity for metal ions. Some of these groups are inert adsorption sites,
not involved in metal transport across the cell membrane.
Decreased internal accumulation of heavy metals has been also proposed
as a mechanism of resistance in certain algae. In the green alga Scenedesmus
acutiformis, Cu transport across plasmalemma was extremely slow compared
with a sensitive strain (150). Also, the Cd resistance of Euglena gracilis (151)
can rely on the modification of membrane permeability. The decreased affinity
of membrane permeases to Cd ions reduced the metal uptake. A similar explanation has been proposed by Rai et al. (152) for the Cu tolerance of the bluegreen
alga Anabaena doliolum. The heavy metal resistance of the green alga Dunaliella
acidophila resulted from the reduced uptake of metal cations due to the positive
potential of the plasma membrane (153). More recently, a low Ni uptake in
acid-tolerant Chlorella vulgaris, brought about by a change in the membrane
potential and permeability, was offered as a resistance mechanism to metals
(194). It is in agreement with the observation (155) that the Cd, Zn, Cu, and Ni
resistance in the mutant strains of Chlamydomonas reinhardtii was associated
with reduced active metal uptake. It has also been shown that the development
of Cu resistance in Euglena can be related to the changes in structural carbohydrates in the cell surface (156).
80
Metal exclusion as another mechanism of Cu and Zn resistance both in

green and bluegreen algae has been proposed (157,158,159). In prokaryotic
bluegreen algae, however, the metal resistance mechanism would rely on the
plasmid-encoded metal efflux (160,161), as in the Cd-resistant bacteria Pseudomonas putida (162).
5.2
Metal Tolerance
Metal tolerance relies on detoxification mechanisms operating within algal cells.

The possibility of internal detoxification in freshwater algae has received more
attention recently.
5.2.1
Production of Intracellular Metal-Binding Compounds
A common response of algae upon exposure to heavy metals is the synthesis of

intracellular metal-binding compounds, which may function in detoxification of
these metals (150,163165). Eukaryotic algae synthesize sulfur-rich oligopeptide
phytochelatins similar to those found in higher plants (166168). Phytochelatins
are produced in algal cells intracellularly in response to several heavy metals and
metalloids, though Cd appears to be the most effective inducer. They are small
peptides typically having the structure ( Glu-Cys)n-Gly, n 211 (163), synthesized enzymatically. Phytochelatins can complex metals intracellularly via sulfhydryl groups of cysteine. As shown in Table 6, a number of metals, such as
Cd, Cu, Zn, Pb, Hg, and Ag, induce phytochelatins in different taxons of freshwater algae (27,63,104,166174). However, there have been reported some interspecies differences (166,174) concerning both total levels of these peptides and particular oligomer concentrations (Fig. 4) as in marine phytoplankton (175,176).
Most studies concerning the phytochelatin synthesis in freshwater algae exposed to heavy metals were carried out under laboratory conditions
(27,166,168,170,171,174). However, recently phytochelatins were identified and
determined in the field population of freshwater algae in the metal-contaminated
lakes in Switzerland (173) and in the field populations of Stigeoclonium sp. growing abundantly in a mine drainage stream of elevated Zn, Pb, and Cd levels in
Poland (104). Intracellular binding of Cd by phytochelatins in some algal cells
was stated in vivo (166,167), and a considerable proportion of incorporated Cd
in the unicellular green alga Chlamydomonas reinhardtii was sequestered by
complex formation with phytochelatin oligomers in both cytosol and chloroplasts
(172). Intracellular metals were found to be of magnitude order higher than phytochelatin levels in the algae inhabiting the heavy metalpolluted waters (104,173).
It suggests that phytochelatins cannot be the only tolerance mechanism; however,
they still may be a very important part of a detoxification process as the first line
of defense together with glutathione (GSH). GSH plays a role as a precursor of
phytochelatin synthesis; however, there are some data showing that GSH may
Freshwater Algae
81
TABLE 6 Freshwater Algae that Produce Phytochelatins in Response to

Heavy Metals
Organism
Metals
Ref.
Navicula pelliculosa (Bacillariophyceae)

Cd
166
Fragillaria crotonensis (Bacillariophyceae)
Cd
166
Thalassiosira pseudonana (Bacillariophyceae)
Cd, Zn, Cu
63
Euglena gracilis (Euglenophyceae)
Cd
166
Chlorella fusca (Chlorophyceae)
Cd, Pb, Zn, Ag, Cu, Hg
166
Chlorella sp. (Chlorophyceae)
Cd
170
Scenedesmus acutiformis (Chlorophyceae)
Cd, Pb, Zn, Ag, Cu, Hg
166
Scenedesmus quadricauda (Chlorophyceae)
Cd
168
Scenedesmus subspicatus (Chlorophyceae)
Cu
173
Chlamydomonas reinhardtii (Chlorophyceae)
Cd
166, 167, 172
Chlamydomonas reinhardtii (Chlorophyceae)
Hg
167
Stichococcus bacillaris (Chlorophyceae)
Cd
166, 171
Stichococcus bacillaris (Chlorophyceae)
Pb
27
Monoraphidium minutum (Chlorophyceae)
Cd
166
Stigeoclonium sp. (Chlorophyceae)
Metal mixture
104
(Zn, Pb, Cd)
Stigeoclonium tenue (Chlorophyceae)
Metal mixture
104
(Zn, Pb, Cd)
Bumilleriopsis filiformis (Xanthophyceae)
Cd
166
Vaucheria compacta (Xanthophyceae)
Cd
174
Vaucheria debaryana (Xanthophyceae)
Cd
174
Mixed lake phytoplankton
Metal mixture
173
(Cu, Cd, Zn)
sometimes play an essential role in heavy metal detoxification. In the case of

metals such as Hg, Ag, and Pb, which create mercaptide complexes of high stability (177,178), an excessive production of GSH besides phytochelatins was observed in some freshwater Chlorophyta (27,167). It is also likely that phytochelatins and probably GSH can serve in algae as a shuttle of metals to the vacuole,
as has been demonstrated in the yeast and plant tissue (179,180), or to the cytoplasmic membrane where they may be exported from the cells with metal ions.
The latter possibility was suggested for Cd and marine diatom (181) as well as
for Pb and freshwater green alga Stichococcus bacillaris (27). It has recently
been evidenced in higher plants and yeasts (182) that genes encoding the enzyme
phytochelatin synthase mediate Cd detoxification in eukaryotes. In the case of
prokaryotic blue-green algae like Synechococcus Cd and Zn tolerance involves
intracellular binding of metals to ligands similar to some extent to eukaryotic
metallothioneins (183185). Class II cyanobacterial metallothionein, however,
does not appear to play a role in Cu detoxification.
82
FIG. 4 Effect of external Cd concentration on phytochelatin (PC) production

in V. compacta (A) and V. debaryana (B) after 3-day exposure. Temperature
of Cd exposure: (A) 16C; (B), 18C. In control cultures (Cd-free) PCs were not
detected. (From Ref. 174.)
Freshwater Algae
5.2.2
83
Metal Compartmentalization
It has been reported for several algae (both prokaryotic and eukaryotic) that intracellular metal sequestration may involve precipitation within the specific intraprotoplast sites. The most frequently reported sites are polyphosphate granules in
algae, which store inorganic phosphate and metabolically essential metals
(92,148,186188). The ability of polyphosphate bodies to accumulate a number
of heavy metals like Fe, Zn, Cd, Cu, and Pb may serve as protection of algal
cells from metal toxicity. The other cell compartment that plays a role in heavy
metal sequestration in plant organisms is the vacuole (189). There are scarce
data, however, concerning the accumulation of metals in algal vacuoles. Silverberg (190) reported Pb in the peripheral vacuoles of the filamentous green
alga Stigeoclonium tenue in Pb exposed cells. X-ray microanalysis performed on
cells of flagellate green alga Dunaliella bioculata revealed that Cd was associated
with sulfur in vacuoles (191). Recently, the rapid transfer of zinc to vacuole of
the giant freshwater alga Chara corralina was also reported (45).
5.2.3
Metal Transformations
Microorganisms can carry out chemical transformations of heavy metals such as

oxidation, reduction, alkylation, and dealkylation, and some of these have been
proposed as mechanisms of metal resistance (192,193). However, there are only
a few data showing the involvement of algae in metal transformation. For instance, in the Hg-resistant Chlorella, enzymatic reduction and volatilization of
Hg were reported (158). Debutylation of toxic tributyltin (53) and demethylation
of trimethyllead were reported in the freshwater chlorophyte Ankistrodesmus falcatus. Biomethylation and excretion of arsenic have been reported in the Asresistant Chlorella vulgaris (194). These scarce data do not give any idea about
the role of metal biotransformations in the development of heavy metal resistance
in algae. There are reports showing that organic-metal complexes of Sn, Pb, or As
can be more toxic for algae than inorganic forms of heavy metals (50,115,195).
The phenomenon of heavy metal tolerance in freshwater algae is still not
well recognized and can rely on several mechanisms operating simultaneously
in the exposed organism.
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4
Salt Marshes
Isabel Cacador
University of Lisbon, Lisbon, Portugal
Carlos Vale
Research Institute for Fisheries and Sea Research, Lisbon, Portugal
SALT MARSHES
Salt marshes are present in moderate- to low-energy intertidal zones of macroand mesotidal estuaries and bays. These areas have a great ecological value for
the ecosystem, namely in terms of nutrient regeneration, primary production, habitat for fish and birds as well as other wildlife, and as shoreline stabilizers. The
tidal flooding causes a transport of dissolved and particulate material from the
estuarine waters to the salt marshes, and consequently seawater constituents, like
sodium chloride, are progressively incorporated in the sediments (13). These
conditions favor the colonization of salt marshes by vascular plants (Fig. 1) belonging to a few cosmopolitan genera (4). The dominant factor determining the
composition of salt marsh flora (5) is the ability to withstand a sediment environment characterized by high salinity. Halophytes that colonize these areas possess
a well-developed aerenchyma system through which atmospheric oxygen is transported from the leaves to the roots. The oxygen that is not consumed in the roots
95
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Cacador and Vale
FIG. 1 Pancas salt marsh: a pure stand of Spartina maritima.
during respiration diffuses into the surrounding sediment and forms an oxidized
rhizosphere (68). These oxidative microenvironments are important to the plant
because they may increase nutrient availability and act as a protective barrier to
heavy metal uptake (9).
Cities and industrialized areas were often installed in the proximity of salt
marshes in estuaries and coastal lagoons. In these cases, flooding transports large
quantities of contaminants in both dissolved and suspended particulate forms to
the salt marsh areas. Anthropogenic metals are incorporated in the sediments,
decreasing their availability in the water column. Several studies have proposed
that salt marshes act as natural sinks for metals (1012). However, a number of
factors may cause postdepositional mobilization of metals from industrial and
urban origin. Diagenetic reactions related to oxidation of organic matter that usually occurs in high levels in salt marshes and oxidation of metal sulfides in the
rooting zone (13) lead to the mobilization of metals. Complex interactions between salt marsh plants and sediments result in the redistribution of metals in
the sediment-root system (14). Metals are taken up by the roots and translocated
to the above-ground parts of the plants; when the plants die the biosynthesized
organic matter is oxidized and the metals returned to the sediments. Since roots
accumulate larger quantities of metals (15,16), the interactions between belowground biomass and sediments are extremely effective and may have a strong
influence on the form and concentrations of metals in the rhizosphere (7). Removal or substitution of degradation products by the flooding water that perco-
Retention of Heavy Metals in Salt Marshes
97
lates the upper sediment layers during the flood tide (17) forces the export of
material to the water column.
THE TAGUS ESTUARY SALT MARSHES: A CASE STUDY
The Tagus estuary is one of the largest estuaries on the Atlantic coast of Europe,
covering an area of 300 km2 at low tide and 340 km2 at extreme high tide (Fig.
2). The southern and eastern parts of the estuary contain extensive intertidal mudflat areas with the presence of Spartina maritima (Poales: Poaceae), Halimione
portulacoides (Caryophyllales: Chenopodiaceae), and Arthrocnemum fruticosum
(Caryophyllales: Chenopodiaceae). Contrary to many cases in Europe where pollutants from industrial regions are discharged into rivers and brought to the estuaries via the rivers, in the Tagus most pollutants are discharged directly into the
estuary. The estuary receives effluents from about 2.5 million inhabitants living in
the greater Lisbon area, together with the discharges from industries (chemicals,
steelmaking, and shipbuilding). Previous studies showed that Tagus salt marshes
incorporate large quantities of anthropogenic metals into the sediments. Concen-
FIG. 2 The Tagus estuary and the salt marsh area: sampling sites.
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Cacador and Vale
trations of copper (Cu), zinc (Zn), cadmiun (Cd), and lead (Pb) in the upper
sediment increase 312 times relative to preindustrial levels, which indicates that
a substantial quantity of anthropogenic metals is incorporated into the sediment
(18).
METAL CONTAMINATION OF THE TAGUS SALT

MARSHES SEDIMENTS
Vascular plants in salt marshes are determinant to the dynamics of the estuarine
ecosystem. Plants act as sediment traps, facilitating an important settling of suspended estuarine material and their associated metals, and influence the retention
and the accumulation processes of metals in salt marsh sediments (18). Several
studies point to metal contamination of surface sediments from the salt marsh
areas in the Tagus estuary (12,18). Higher contaminations were found close to
the anthropogenic sources, highlighting the role of salt marshes in the retention
of metals in this system (19). As in many other coastal systems located in the
proximity of urban and industrial zones, metal contamination decreases with the
sediment depth. The metal concentrations in surface sediments are often related
to contamination of the coastal environment. An enrichment factor (EF) may be
calculated by the ratio between metal concentration in the first sediment layer
(05 cm) and in deeper sediment layers (4555 cm). This depth is below the
plant roots, avoiding the influence of the roots on metal redistribution, and sediments were deposited before the industrial period in Portugal (18). Consequently,
the EF represents the increment of metal concentrations in the sediments due to
environmental contamination (20). The calculated EFs for Zn, Pb, Cu, and Cd
in the Tagus salt marshes are present in Fig. 3. The higher values were registered
in Corroios (C), Talaminho (T), and Rosario (R), which are the salt marshes
nearby metal anthropogenic sources. Lower values were found in areas P and B
located in the Natural Reserve of the Tagus estuary. The calculation of EF allows
assessing the retention of anthropogenic metals in the salt marshes along the
estuary. In spite of the clear picture obtained, it should be stressed this calculation
is a simplistic approach when changes of metal concentrations due to early diagenetic processes (19) are not taken into account.
The sedimentary organic matter is considered to have an important role in
the retention of metals in sediments. The relationships between metal concentrations and organic matter content, here estimated from the loss on ignition (LOI)
at 550C (20) are shown in Fig. 4. These figures were plotted with values from
three sediment layers: surface (05 cm), subsurface (515 cm), and deeper sediment layers (4555 cm). In general, the deeper sediment layer has lower organic
matter content and lower metal concentrations than the upper two layers. Metal
99
FIG. 3 Enrichment factors: heavy metal concentration (05 cm)/(4555) cm

(g g1), sediment layer for Zn, Pb, Cu, and Cd in Pancas (P), Barco (B), Sarilhos Pequenos (S), Rosario (R), Paio Pires (p), Talaminho (T), and Corroios
(C) salt marshes.
concentrations are not linearly correlated to LOI, which indicates that organic
matter content is not the dominant factor for metal concentration in upper sediment layers of salt marshes. Though vegetated litter is the main source of organic
matter to salt marshes (2), as well as an important vehicle of metals that have
been uptaken by plants during their growing process to the sediment (11), anthropogenic inputs appear to be superimposed. Furthermore, metal concentrations
and LOI in sediment sites colonized by Spartina maritima, Halimione portulacoides, and Arthrocnemum fruticosum did not show consistent differences, rein-
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Cacador and Vale
FIG. 4 Relationships between the concentrations of Zn, Pb, Cu (mol g1),

and LOI (%) in sediments from three layers: (05) (), sub-superficial 515
), and 4555 cm () of the Tagus salt marshes.
cm (
forcing the hypothesis of external sources in the geographic distribution of metal

concentration in the upper sediment layers.
The amount of metal extracted by the DTPA is assumed to be an estimation
of the metal quantity potentially available to the plants (21). These amounts are
relatively small in comparison to the total metal concentration, being lower than
10% for Cd and Cu and not exceeding 30% for Zn (7). Moreover, the extracted
metal concentrations changed with the salt marsh. When metal fractions extracted
by the DTPA solution are plotted against LOI (20), inverse relationships are
obtained (Fig. 5). The best fitting curves have similar shapes and the general
equation is [Me]extracted A[LOI]b. This indicates that metals are more available
in sediments impoverished in organic matter than in organically rich sediments.
For example, available Zn was 10 times higher in sediments with LOI lower than
10% than in sediments containing more than 20%. These sediments showed low
proportion of Pb, Cu, and Cd, while in sediments with low organic matter content
the proportions were generally higher and varied with the metal. Otherwise, rooting sediments containing higher organic content have lower metal extracted by
the DTPA solution.
101
FIG. 5 Relationships of extractable Zn, Pb, Cu, and Cd (% to the total mass)
and LOI (%) in sediments between roots (515 cm) of the Tagus salt marshes.
EFFECT OF PLANT ROOTS ON SEDIMENT CHEMISTRY
Roots may affect substantially the chemistry of sediment layers containing high
below-ground biomass. The interaction between roots and sediments is exceedingly complicated, covering a wide range of chemical, physical, and biological
processes (22). Plants take up nutrients and other minor and trace elements from
the sediments, causing a flux of dissolved chemical species from the sediments
toward the root surfaces. Furthermore, oxidants, exudates, and gases liberated by
the roots change the surrounding sediment environment. Rooting sediments in
the Tagus salt marshes are richer in organic matter and have a lower pH and
higher redox potential than unvegetated bulk sediment (7) from nearby areas (Fig.
6). Vertical profiles of Zn, Pb, and Cu concentrations in vegetated areas differ
in shape and concentrations of those found in nonvegetated sediments (Fig. 7).
The major characteristic is the appearance of a subsurface maximum of several
metals. Concentration peaks are formed at depths of higher rooting density, and
levels in the upper 30 cm were higher than those in the corresponding layers of
nonvegetated sediments. These differences are clearly related to the presence
and activity of roots. Metal incorporation in salt marsh sediments is therefore
substantially different from that observed in nonvegetated intertidal and subtidal
areas (18). Apparently, the larger amounts of metals retained in the rooting sedi-
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Cacador and Vale
FIG. 6 Redox potential (Eh) vs. pH, and Organic matter content (LOI) vs. pH
in sediment between roots, corresponding mainly to the 5- to 15-cm layer
(), and in nonvegetated sediment (including sediment below roots) (*) at
Corroios and Rosario Tagus salt marsh.
103
FIG. 7 Vertical profiles (mean SD, n 3) of total Zn, Pb, and Cu concentrations (g g1 dry weight) in sediments with () and without () vegetation
from less contaminated site 1 and more contaminated site 2.
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Cacador and Vale
FIG. 8 Distribution of Zn, Pb, and Cu among operationally defined fractions

in three layers in vegetated and nonvegetated sediment from sites 1 and 2.
Sediment fractions are exchangeable (), carbonates ( ), oxides ( ), organic
matter ( ), and residual ().
105
ment layers are not easily available to the plants. When compared with the sediments not colonized by vascular plants (12), sediments around roots have lower
Pb, Zn, and Cu concentrations in mobile chemical forms (Fig. 8). In fact, the
amount of metals taken up by the plants depends on their availability, which is
a function of several factorsnamely, organic matter, pH, redox potential, metal
speciation, and root-sediment interactions (22)which change drastically in the
vicinity of the roots (rhizosphere). These results indicate higher percentages of
metals unavailable to plants (12).
5
FORMATION OF METAL-RICH CONCRETIONS AROUND

THE ROOTS
By releasing gaseous oxygen to the rhizosphere, vascular plants modify dramatically the sediment characteristics in the areas colonized by salt marsh vegetation.
FIG. 9 Concentrations of Zn, Pb, Cu, and Cd in rhizoconcretions (0) obtained

with sieves of 2, 1, 0.5, and 0.25 mesh size and in sediment between roots
(-).
106
Cacador and Vale
(A)
(B)
FIG. 10 (A) Rhizoconcretions on the roots of Aster tripolium in the Tagus
estuary. (B) Enlarged view of a concretion showing that root remains in its
interior.
107
The presence of oxygen at the root-sediment interface creates local oxidizing

conditions in otherwise reducing sediments. This affects in particular the chemistry of iron and manganes, whose soluble reduced forms diffuse toward these
interfaces, where they are precipitated as insoluble iron and manganese oxides.
Thin reddish brown deposits on plant roots have been observed on several floodtolerant plants and attributed to the oxidative capacity of roots (23). Iron oxyhydroxides have been identified in these deposits (24). Other metals are also associated with these iron plaques (25), reaching higher concentrations than those found
in the bulk sediments (Fig. 9). In regions where salt marsh plants remain active
most of the year, like the Tagus estuary salt marshes (26), much thicker iron
oxide coatings around the roots have been observed (25,27). Iron precipitates in
tubular structures (Fig. 10), forming tubular concretions with a few millimeter
size, so-called rhizoconcretions. These concretions are formed over a period of
months (8) and contain more than 10% iron, doubling the concentrations of the
bulk sediments (25). Rhizoconcretion formation thus contributes to salt marshes
acting as natural sinks for trace elements from anthropogenic origin.
6
METAL ACCUMULATION IN ROOTS

AND TRANSLOCATION
Besides sediments, salt marsh plants are also important temporary sinks for metals. Transition elements are accumulated in vegetation during the growth season
(28). Roots, stems, and leaves of the salt marsh plants contain distinct levels of
Zn, Pb, Cu, and Cd (Fig. 11). Zinc was the most abundant metal in all analyzed
parts of the plants. The highest metal concentrations were recorded in the root
system, and only small fractions were found in the above-ground parts of the
plant. The accumulation partition was more pronounced for Pb and Cd, since
these metals were more efficiently stored in the roots, than for Zn and Cu, which
are translocated to the upper parts. The metal concentrations varied with the plant
species and with the salt marsh. The results obtained in an annual study of the
Tagus salt marsh indicate that Halimione portulacoides accumulates more Zn,
Pb, Cu, and Cd than Spartina maritima (Fig. 11), and metal concentrations in
their roots and sediments varied seasonally. The interaction between plant roots
and sediments in salt marshes increases during the growing season and drops in
winter, when plants decrease their activities (9,26). The temperature and salinity
of the upper layer of Tagus salt marsh sediments rise in summer, to 30C and
50C, respectively. However, temperature is always positive in winter and salinity does not decrease drastically (27). Consequently, the salt marsh plants in the
Tagus estuary remain active during the almost entire year. Sediments around
Spartina maritima root were more acid and reductive (Fig. 12) than those around
Halimione portulacoides, which indicates a stronger influence of that plant on
sediments (9). However, pH and redox potential values did not show a pro-
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Cacador and Vale
FIG. 11 Mean concentrations of Zn, Pb, Cu (mol g1 dry weight) and Cd

(nmol g1 dry weight) in roots ( ), stems (), and leaves () of Spartina
maritima and Halimione portulacoides (n 8) between July 1991 and July
1992.
nounced seasonal variation, as would occur if plants stopped interacting with the
sediment chemistry in winter. Daily inundation of the salt marshes by tide may
also alter the sediment pore water chemistry, but its effect should be considered
minor because conditions in the different plant areas vary. However, root biomass
increased substantially between March and August/September, and the metal
concentrations in both plants showed the same pattern: lower values in winter
109
FIG. 12 Mean and standard deviation of the organic matter content (LOI), pH,
and Eh (July 1991 to July 1992) of the sediments colonized by Spartina maritima () and Halimione portulacoides (), n 8.
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Cacador and Vale
and an increase with the biomass. This means that growing process induces an
increase of metal uptake by the roots. The root system is clearly the major pool
of Zn, Pb, Cu, and Cd for Spartina maritima and Halimione portulacoides, since
metal concentrations were higher in roots than in stems and leaves. These results
are in agreement with the efficient storage of metals in roots that was observed
for several salt marsh plants (28). The progressive increase of biomass from 3
to 8 kg m2 (Fig. 13) implies that a considerable amount of metals was taken up
by the roots: In autumn/winter part of the root system dies and metals returned
to the sediments. These exchanges can only be registered in the bulk sediments
when root biomass is abundant, as in the Tagus where the ratio of root to sediment
reached 1:4. The increase of Zn, Pb, and Cu concentrations in the sediment that
was registered in January, when levels in the roots and biomass were lower,
corresponded to the transfer of metals from the plant to the sediments. In the
spring, metal levels decreased in the sediment between roots (Fig. 14), as result
of an inverse transportation of metals. This pattern was observed for the two
studied plants (28). Cadmium showed a different seasonal distribution of the
other three analyzed metals, suggesting that root-sediment exchange is more complicated.
In spite of the high root density in sediments it is still a simplistic approach
to consider that exchanges of metals involve roots and sediments exclusively. A
mass balance of metals exchanged between sediment and roots was calculated
for each 2 months for 1 year. Changes of metal concentrations in sediments were,
FIG. 13 Root biomass (kg m2) of Spartina maritima () and Halimione portulacoides ( ), n 3.
111
FIG. 14 Mean and standard deviation of concentrations of Zn, Pb, Cu (mol

g1 dry weight), and Cd (nmol g1 dry weight) in sediments between the roots
of Spartina maritima ( ) and Halimione portulacoides ( ) (n 8) between
July 1991 to July 1992.
in general, more accentuated than in roots. Processes other than uptake by the
plants should occur in the sediments, namely, oxidation of insoluble sulfides (13)
and formation of insoluble oxides (6) in microenvironments. These processes
may interfere with the trace metal mobility (8,30). Only in winter were the Pb
variations in sediments and roots comparable, suggesting a simpler transfer be-
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Cacador and Vale
tween the two compartments. The uptake process by the roots involves the soluble
metals in pore water, and their concentrations are influenced by several factors,
namely, tidally induced advection, diffusion, and reactivity in the rhizosphere.
In addition, the variation of mobile metals in sediments and roots may be poorly
expressed by the total metal concentration. These geochemical processes and
physical mechanisms all contribute to complicate a simple metal exchange between roots and sediments.
Apart from the complexity of those exchanges, it is clear that Halimione
portulacoides acumulates more metals than in Spartina maritima, and that plant
appears to be a better temporary sink for metals than Spartina maritima. Our
results are in agreement with a general rule that states that dicotyledons (Halimione portulacoides) have the capacity for higher accumulation of metals that monocotyledons (Spartina maritima) (31).
7
ROLE OF HALOPHYTES ON METAL CYCLING IN SALT

MARSHES: A PROPOSED MODEL
The results obtained in these investigations allow the proposal of a simplified

model for the retention of contaminants in the sediments and their interaction
with the plants. A schematic representation is shown in Fig. 15. The steps of
metal dynamics in salt marshes include the following:
1. Import of metals from the estuary in dissolved forms and associated
with suspended sediments and biogenic particles transported by the
tide. Higher enrichment in surface sediments occurs in the proximity
of anthropogenic sources, highlighting the contribution of the salt
marshes to remove anthropogenic metals from the contaminated estuarine waters.
2. Metal remobilization and subsequent alterations on metal fractionation in the sediments due to diagenetic reactions. The high quantity
of sedimentary organic matter favors the internal metal redistribution.
3. Take-up of metals from contaminated sediments during the growth season and accumulation in the plant tissues. Basically through their subterranean components vascular plants in salt marshes act as temporary
sinks for heavy metals.
4. Releasing oxygen to the rhizosphere, vascular plants alter the biogeochemistry of the sediments and modify dramatically the rooting sediments. Results from this interaction include modification of metal speciation around roots and buildup of iron-rich rhizoconcretions in which
trace elements are sorbed. Accumulation of heavy metals in these structures may greatly exceed concentrations found in nearby sediments.
113
FIG. 15 Schematic representation of factors affecting uptake of heavy metals.

The model means illustrate that bioavailability of the elements is one of many
factors determining uptake by plants and that the influence of root biochemistry is a key factor.
SUMMARY
Salt marsh vegetation influences the dynamics of the estuarine ecosystem and
retains efficiently anthropogenic metals discharged to the system. Basically metal
cycling in salt marshes includes retention of suspended particles and associated
anthropogenic metals transported by the tides; taking up of metals from contaminated sediments during the growth season; accumulation in the plant tissues,
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Cacador and Vale
mostly within the root system. In this way, vascular plants act as temporary
sinks for heavy metals. Roots also act as an important vector to the incorporation of metals in salt marsh sediments. By releasing oxygen to the rhizosphere,
vascular plants influence the biogeochemistry of the sediments and greatly modify the sediment characteristics of salt marshes. These interactions cause changes
of metal speciation in the rhizosphere and buildup of tubular concretions around
the roots rich in iron hydroxides. These concretions accumulate metals in concentrations higher than those found in nearby sediments, contributing to salt marshes
that act as natural sinks for these elements. In unstable sediment environments
such as the Tagus salt marshes, several processes change the availability of the
metals to the plants. However, changes induced by the presence of roots are most
prominent. Sediments around the roots are richer in organic matter and have a
lower pH and higher redox potential than unvegetated bulk sediments. When
compared with sediments not colonized by vascular plants, the metals in rooting
sediments have lower concentrations of mobile chemical forms and, therefore,
lower percentages of metals available to plants. The accumulation of metals in
sediments and vegetation, together with the root-sediment interactions, all contribute to an overall reduction of metal bioavailability to plants. This suggests
that salt marshes in the Tagus estuary may be important areas to help reduce the
environmental contamination caused by the industrial and urban zones. Further
investigation of the biogeochemistry of sediments and root-sediment interactions
is needed to yield a better understanding of the reduction of metallic pollution
in estuarine environments.
REFERENCES
1. P Leendertse. Impact of nutrients and heavy metals on the salt marsh vegetation in
the Wadden Sea. PhD thesis, Vrije Universiteit, Amsterdam, 1995.
2. ML Otte. Heavy metals and arsenic in vegetation of salt marshes and foodplains.
PhD thesis, Vrije Universiteit, Amsterdam, 1991.
3. C Vale. Temporal variations of particulate metals in the Tagus river estuary. Sci
Tot Environ 97/98:137154, 1990.
4. P Adam. Salt Marsh Ecology. New York: Cambridge University Press, 1990,
p. 445.
5. W Stephen, DE Seneca, W Woodhouse. Tidal salt marsh restoration. Aquatic Bot
32:122, 1988.
6. BL Haines, EL Dun. Coastal marshes. In: BF Chabot, HA Mooney, eds. Physiological Ecology of North American Plant Communities. London: Chapman and Hall,
1985, pp. 323347.
7. I Cacador, C Vale, FM Catarino. Accumulation of Zn, Pb, Cu, Cr and Ni in sediments
between roots of the Tagus estuary salt marshes, Portugal. Est Coast Shelf Sci 42:
393403, 1996.
8. B Sundby, C Vale, I Cacador, F Catarino, MJ Madureira, M Caetano. Metal-rich
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concritions on the roots of salt-marsh plants. Mechanism and rate of formation. Limnol Oceanogr 43(2):245252, 1998.
ML Otte, J Rozema, L Koster, MS Haarssma, RA Broekman. Iron plaque on roots
of Aster tripolium L.: interaction with zinc uptake. New Phytol 111:309317,
1989.
O Oenema, R Steneker, J Reynders. The soil environment of the intertidal area in
the Westerschelde. Hydrobiolo Bull 22:2130, 1988.
RA Orson, RL Simpson, RE Good. A mechanism for the accumulation and retention
of heavy metals in tidal freshwater marshes of the upper Delaware River estuary.
Est Coast Shelf Sci 34:171186, 1992.
I Cacador, C Vale, FM Catarino. The influence of plants on concentration and fractionation of Zn, Pb, and Cu in salt marsh sediments (Tagus Estuary, Portugal). J
Ecosyst Health 5:193198, 1996.
MJ Madureira, C Vale, ML Simoes. Effect of plants on sulphur geochemistry in the
Tagus salt-marsh sediments. Mar Chemistry 58:2737, 1997.
B Tinker, P Barraclough. Root-soil interactions. In: O Hutzinger, ed. Reactions and
Processes, 2: Part D. Berlin: Springer-Verlag, 1988, pp. 154171.
JJ Albertsl, MT Price, M Kania. Metal concentrations in tissues of Spartina alterniflora (Loisel) and sediments of Georgia salt marshes. Est Coast Shelf Sci 30:
458, 1990.
J Rozema, ML Otte, R Broekman, G Kamber, H Punte. The response of Spartina
angelica to heavy metal pollution. In: AJ Gray, ed. Spartina angelica. London:
HMSO, 1990, pp. 3947.
M Caetano, M Falcao, C Vale, MJ Bebbiano. Tidal flushing of ammonium, iron and
manganese from inter-tidal sediment pore waters. Mar Chem 58:203211, 1997.
I Cacador, C Vale, FM Catarino. Effects of plants on the accumulation of Zn, Pb,
Cu and Cd in sediments of the Tagus estuary salt marshes, Portugal. In: J-P Vernet,
ed. Environmental Contamination. Amsterdam: Elsevier, 1993, pp. 355365.
PM Chapman. The sediment quality triad approach to determining pollution-induced
degradation. Sci Total Environ 97/98:815825, 1990.
I Cacador, C Vale, F Catarino. Relationships between metal concentrations and organic matter content in the Tagus estuary salt marsh sediments halophyte uses in
different climates, I. In: A Hamdy, et al. eds. Leiden: Backuys Publishers, 1999,
pp. 103110.
A Piccolo. Reactivity of added humic substances towards plant available heavy metals in soils. Sci Tot Environ 81/82:607614, 1989.
BJ Alloway. Soil processes and the behavior of metals. In: BJ Alloway, ed. Heavy
Metals in the Soils. Glasgow: John Wiley & Sons, 1990.
IA Mendelssohn, M Postek. Elemental analysis of deposits on the roots of Spartina
alterniflora Loisel. Am J Bot 69(6):904912, 1982.
AA Crowder, SM Macfie. Seasonal deposition of ferric plaque on roots of wetland
plants. Can J Bot 64:21202124, 1986.
C Vale, FM Catarino, C Cortesao, MI Cacador. Presence of metal-rich rhizoconcretions on the roots of Spartina maritima from the salt marshes of the Tagus estuary,
Portugal. Sci Total Environ 97/98:617626, 1990.
FM Catarino, MI Cacador. Producao de biomassa e estrategia de desenvolvimento
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em Spartina maritima e outros elementos da vegetacao dos sapais do estuario do
Tejo. Bol Soc Broteriana 2a ser 54:384403, 1981.
I Cacador. Acumulacao e retencao de metais pesados nos sapais do Estuario do Tejo.
Tese de Doutoramento, Universidade de Lisboa, 1994.
I Cacador, C Vale, FM Catarino. Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritima and Halimione portulacoides
from Tagus estuary salt marshes. Marine Environmental Research 49:279290,
2000.
WHO Ernst. Element allocation and (re)translocation in plants and its impact on
representative sampling. In: H Lieth and B Markert, eds. Element concentration cadasters in ecosystems. Weinheim: VCH Verlagsgesellschaft, 1990, pp. 1740.
WG Beeftink, J Rozema. The nature and functioning of salt marshes. In: W Salomons, BI Bayne, EK Dursma, and U Forstner, eds. Pollution of the Northsea: An
Assessment. Berlin: Springer-Verlag, 1988, pp. 5987.
WHO Ernst, W Mathys, J Salaske, P Janiesch. Aspekte von Schwermetallbelastungen in Westfalen. Abhandl. Landesmus. Naturk Westf 36(2):130, 1974.
5
Lichens
Cristina Branquinho
Universidade Atlantica, Oeiras, Portugal
1
1.1
INTRODUCTION
Understanding the Singularity of Lichens
Lichens are by definition symbiotic organisms composed of a fungal partner, the

mycobiont, and one or more photosynthetic partners, the photobiont, which may
be either a green alga or a cyanobacterium (1). The nature of the lichen symbiosis
is still not totally clear: although most authors consider lichens to be a classic
example of mutualism, in which all partners gain benefits from association, others
suggest that lichens are an example of controlled parasitism (1). The degree of
lichenization varies significantly from photobiont cells loosely attached to a fungus to a more complex organization that in no way resembles the bionts that
form it (1). Lichens are a very successful symbiosis; they are found in almost
all terrestrial habitats, including tropical, desert and polar, and in fresh and salt
water habitats. Lichens are dominant life forms in 8% of the earths terrestrial
surface (2).
The mycobiont, a heterotrophic organism, benefits from the carbohydrates
produced by the photobiont. In the case of nitrogen-fixing cyanolichens, the mycobiont also gains a nitrogen source. The fungus probably functions as a reservoir
117
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Branquinho
of inorganic nutrients for the photobiont and also creates a humid environment,
enhancing water uptake, offers a physical support, and substantially reduces the
light intensity for the photobiont.
On the basis of their overall habitat, lichens are traditionally divided into
three main morphological groups: crustose, foliose, and fruticose. Crustose lichens are tightly attached to the substrate with their lower surface and may not
be removed from it without destruction. Foliose lichens are leaf-like, flat, and
only partially attached to the substrate. Fruticose lichens always stand out from
the surface of the substrate (Fig. 1). Their thallus lobes are hair-like, strap-shaped,
or shrubby and the lobes may be flat or cylindrical. Lichens may also be classified
according to the substrate they occupy: as endolithic if they grow inside rock;
epilithic if they grow on rock surfaces; terricolous if they grow in soil; and epiphytic if they grow on trees.
Lichens are extremely slow-growing and long-lived organisms whose
growth rates mostly depend on climate conditions. Lichens of extreme climates,
such as desert, polar, or alpine ecosystems, have only short periods in which full
metabolic activity and growth can occur. Consequently, only very low cell turnover rates and minimal annual size increases are recorded (1 mm/year). However, the majority of lichens in temperate or subtropical to tropical climates have
annual radial growth in the range of millimeters to a few centimeters. Although
FIG. 1 The fruticose lichen Evernia prunastri.
Lichens
119
extreme ages of lichens are estimated in the range of millennia (3), the majority
develop over years or decades. Estimates of lichen longevity should be interpreted
critically because the metabolically active cells may be not as old as the thalli.
1.2
Chemical and Physical Properties of Metals
The position and classification of the chemical elements in the periodic system
of the elements (PSE) do not indicate their acute or chronic toxicity to living
organisms (46), since the PSE is based on purely physicochemical characteristics (6,7). Proposals have been made for a classification of metals more relevant
to biological systems, based on the chemical behavior of the metals cations and
especially on their ability to become part of a chemical complex (4,5,79). Nieboer and Richardson (4) divide elements into three groups: (a) class A elements
showing preference for ligands containing oxygen, including all of the macronutrient cations of terrestrial plants (e.g., K, Mg, Ca), as well as some other alkaline
and earth metals (e.g., Cr and Fe); (b) class B elements showing preference for
ligands with nitrogen or sulfur (e.g., Ag, Au, and Hg); and (c) borderline elements, with intermediate characteristics between class A and class B (e.g., Cu,
Co, Ni, and Pb).
1.3
Rationale of the Chapter
From the above the complexity and singular nature of these organisms are obvious. Moreover, there is a great lack of information relating to both the nature of
the lichen symbiosis and the interactions between the bionts. Thus, lichens should
always be regarded as complex, mostly unknown biological systems. The role
of lichens as sensitive indicators and monitors of atmospheric pollution is of
undeniable importance. Despite the great number of publications dealing with
the interaction of lichens with heavy metals (10), a detailed knowledge of the
physiology of metal absorption in these organisms, i.e., uptake, accumulation,
retention, localization, release, tolerance, and toxicity of metals, is needed before
lichens can be extensively and reliably used for monitoring of metal deposition
patterns (1114). In this chapter a critical review of the more pertinent aspects
of the interaction between lichens and metals will be given with emphasis on
effects of metals on lichen communities and on physiological performance; their
tolerance mechanisms; sources, retention, and release of metals; uptake and location of metals; sources of variability during lichen sampling; and calibration of
lichens against dust gauges.
2
LICHENS AS GOOD BIOINDICATORS

AND BIOMONITORS OF METAL POLLUTION
Lichens are the most studied bioindicators and biomonitors of air pollution. Some
lichens are extremely sensitive to atmospheric pollution and have been used as
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Branquinho
good bioindicators of such pollution for more than a century (1517). The importance of this role is evidenced by the large number of publications on this topic
(10). Lichens have also been used extensively to biomonitor heavy metal contamination (11,15,16,18). Bioindicators can provide information on qualitative variations in the environment, while biomonitors supply quantitative information.
Both uses are based on organisms, communities, morphology, cellular structure,
metabolism, and biochemical processes, etc. (19,20). The concentrations of given
pollutants, measured within the organism, are used to reconstruct the spatial and
temporal deposition patterns of the pollutants deposited at a location (18,19).
2.1
Lichens as Bioindicators
Epiphytic lichens have traditionally been used as bioindicators of atmospheric

pollution because they show differential sensibilities to air pollution, i.e., the most
sensitive lichens tend to disappear from polluted areas whereas the most tolerant
species can be seen in areas with moderate pollution emissions (9,15,17,21,22).
The first documented reports of the disappearance of lichens from city centers were made in the 1800s (23). Nylander (24) suggested that the absence of
the lichens was due to air pollution. In 1970, Hawksworth and Rose (25) correlated the declines of lichens in urban areas with mean winter SO2 measurements
and developed a semiquantitative scale for English forests relating the occurrence
of approximately 50 lichen species. Since then almost every city in Europe and
some in Canada and the United States have been mapped using lichen biodiversity
data (19). Today the list of air pollutants related to the decline of lichens in urban
and industrial areas is much longer, including hydrogen fluoride (HF), heavy
metals, acid rain, radionuclides, oxidants and organic compounds, and it is not
complete (23).
2.2
Lichens as Biomonitors
Lichens have been used extensively to biomonitor heavy metal contamination

from anthropogenic sources: roads, mines, and industrial facilities in urban and
rural locations (11,15,16,18). However, they have also been used to assess natural
sources of metals, such as volcanoes (26). Many authors have pointed out that
lichens have several of the characteristics required of the ideal biological monitor
(11,15,16,18,19,27). They have a wide geographic distribution, occurring in rural,
urban, and industrial areas, thus allowing comparison of pollutant concentrations
from diverse regions. The morphology of lichens does not vary seasonally, so
that pollutant accumulation can occur throughout the year. They are photosynthetically active throughout the year. Their ability to accumulate heavy metals has
been widely demonstrated (11,28,29). Lichens are poikilohydric organisms, lacking a developed cuticle, which have very limited control of the uptake and loss
of water and solutes from atmospheric deposition. Furthermore, they depend
Lichens
121
mostly on atmospheric deposition for nutrition because their root structures

do not function as in higher plants. They lack mechanisms for active absorption
from the substrate. On the other hand, in contrast to higher plants, they have no
excretion mechanisms. In situ lichens are slow-growing, long-lived organisms,
which facilitates their use as long-term integrators of atmospheric deposition.
Lichens are easily handled and transplanted, thus permitting biomonitoring for
defined periods of time and the assessment of short-term atmospheric deposition.
Both bioindicators and biomonitors may be considered as organisms or
communities of organisms that react to variations in environmental conditions
with a change in their signs of activity (20). These changes may manifest
themselves:
1. In the structure or dynamics of the population or community of organisms;
2. In changes in the organisms functional response that may lead to
changes in vitality;
3. In concentrations of elements that influence the organism.
3
MAPPING LICHEN BIODIVERSITY TO ASSESS

METAL POLLUTION
Lichens may be used as bioindicators whenever air pollution causes compositional changes in their communities. The principal outputs in this area are biodiversity maps based on all lichen flora and on the use of several indices of atmospheric purity (IAPs). Over the last 30 years, numerous studies have assessed
the effect of air pollution on lichens, which supposedly reflect the local air
quality. This is usually accomplished by mapping of lichen communities. Lichens have been used as bioindicators to evaluate pollution in many urban and
industrial areas; more than 50 cities around the world have already been mapped
(19). Several regional and national studies have been performed in England, the
Netherlands, Finland, Denmark, Sweden, Slovenia, Estonia, southern California,
northern Italy, etc. (19). The importance of lichen biodiversity data was recently
demonstrated by Cislaghi and Nimis (30), who compared such data with mortality
maps of a large part of northeastern Italy. The results strongly supported a relationship between air pollution measured by the lichen biodiversity data and lung cancer.
One of the most common approaches to mapping lichen biodiversity is the
use of IAPs. The IAP originally developed by LeBlanc and De Sloover (31) is
based on the presence of lichens, on their frequency, and on an ecological index
for the species. This toxitolerance factor for each species is determined on a
somewhat subjective basis (17,32). Furthermore, there is evidence that the tolerance of a given species to air pollution may differ according to the general climatic conditions (17). Thus, even in the rare cases where a toxitolerance factor
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Branquinho
for calculating the IAP is based on experimental data, its extrapolation to another
area, with different climatic conditions, may not be accurate and cannot be recommended (17). Both the lichen frequency and the number of lichen species have
been shown to be less subjective parameters that are correlated with changes in
atmospheric pollution (14,19,32).
The declines in lichen communities found in the majority of biodiversity
studies were mainly due to gaseous pollution (mainly HF and SO2) (19). Whenever there is a mixture of both gaseous and metal pollution, most authors have
difficulty in distinguishing the relative contribution of metal pollution to lichen
disappearance or to the injury symptoms observed under field conditions. The
distribution of epiphytic lichen species has been studied in the area of a copper
smelter in Canada and around a zinc smelter in the United States (33,34). These
studies suggested that the lichen distribution was influenced more by SO2 emissions than by metal pollution.
Few studies have reported the effects on lichen flora of metal pollution
without gaseous emissions. Folkeson and Andersson-Bringmark (35) studied the
lichens in the coniferous forest surrounding the brass mills at Gusum, southeastern Sweden, which is heavily polluted with Cu and Zn but not with SO2. The
author concluded that the deterioration of the lichen vegetation was due to Cu
and Zn. The effects on the disappearance of some species of the genus Cladonia
due to metal pollution were clearly observed (35). Loppi et al. (36) showed the
results of a retrospective study performed in the Chianti region in central Italy
using lichen biodiversity data and concentrations of elements accumulated in the
lichen Parmelia caperata. The results of sampling in 1980 and 1996 showed that
the general decrease in concentrations of metals with time was associated with
an increase in the number and frequency of lichen species (36).
Herzig et al (32) combined lichen biodiversity data with multielemental
analysis in lichens and found that many elements correlated well with the IAP18 (based on the frequency of more than 40 lichen species). They suggested that
biodiversity and multielemental analysis are complementary, enabling detailed
statements on biological effect and total air pollution in general and on single
pollutants in particular.
Another study evaluated the impact on lichen flora of an underground copper mine located in southern Portugal (14,37). The mine processes sulfide ore to
produce Cu concentrates that are transported by rail and shipped to smelters overseas. The main source of atmospheric pollution at the mine site is fugitive dust
emissions from the stockpiles and waste heaps. Since the mine is located in a
rural area distant from any other industry or major city, there are no other significant local sources of air pollution (14,37). This study used lichen biodiversity,
measured by the parameter number of lichen species, to identify the zones of the
mine subject to long-term emissions (Fig. 2). In this case study, the fruticose
Lichens
123
FIG. 2 Estimated spatial distribution of the total number of epiphytic lichens

in the surroundings of a copper mine obtained by kriging of data based on
19 sampling points () during 1993. The center of the mine is represented
by , the Cu-concentrate stockpile by , and the waste heaps by . The studied area was divided into classes with different numbers of lichen species:
10, 1018, and 18 species. (From Ref. 37.)
lichens appeared to be the most sensitive to Cu pollution, followed first by the

foliose and then by the crustose forms (14).
One of the difficulties with mapping lichen biodiversity is that it requires
specific training in lichenology. Recorders must be competent at identifying and
distinguishing a wide range of lichens in different stages of development. The
use of lichen data on biodiversity maps to evaluate the impact of atmospheric
pollution should be accompanied by the concentration of elements accumulated
in the lichen thallus to avoid incorrect interpretations (14,32). Lichens are also
bioindicators of several other environmental factors such as climate, so that
changes in the composition of a lichen community may not be solely due to
changes in pollution levels. Once the primary pollutants that cause damage to
lichen communities have been identified, the levels of the pollutants that cause
those changes should be evaluated (38).
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The main advantage of lichen biodiversity mapping is that it combines a

high number of sampling points with relatively low costs, enhancing the data
quality. As a first approach, such studies allow the easy identification of the highrisk areas to be further examined in future studies. An example is the optimization
of the location of chemical and physical samplers whose numbers are limited
due to their high costs. Biodiversity mapping studies are also important as effective early warning systems to detect environmental changes.
4
PHYSIOLOGICAL RESPONSES OF LICHENS TO METALS
The main topics considered in physiological studies are morphological changes,

variations in vitality, and changes in functional response. Under field conditions
it is difficult to determine the effect of heavy metals on the physiology of lichens
because their deposition is often linked with acid rain (SO2) or the presence of O3,
nitric oxides (NOx), peroxyacetyle nitrate (PAN), HF, or other anthropogenic gases
(39). Most studies on the effects of metals on lichens were performed under controlled conditions, and only a few have been confirmed under field conditions (37).
4.1
Effect on Cell Membranes
The cell membrane is an obvious initial site for toxic metal action, and the loss
of ions, particularly K, from the cell interior in lichens has been widely used as
an indication of membrane integrity (1214,37,4044). This is based on the fact
that most of the K is located intracellularly. A proportion of Mg is also readily
available within the cell, and this cation may also be used as an indicator of
severe membrane damage (1214,37,45,46). The recovery of soluble elements
from incubation media or washing solutions, measured by conductivity, has been
used to indicate cellular damage: by metals in the laboratory or by air pollution
in the field (39,4750). Estimation of K loss into such washing solutions might
overestimate leaching from the cell if there is a source of this element in the
environment. At sites with very high levels of dry deposition, K deposited on
the surface or bound to the wall of the lichen may reach 50% of the total K
concentration (Fig. 3). Thus, the conductivity may not be always related to K
loss from inside the cell (51). The sequential elution procedure was shown to be
valuable for distinguishing between elements acquired from the environment and
those physiologically released (see Sec. 6). Some authors wash the lichen a few
seconds before the conductivity measurements as described by Garty et al. (39),
but care must be taken because a small proportion of K might still be bound to
the wall and be displaced by other cations with a stronger affinity for those binding sites.
The ion loss is a measure of the physiological state of the mycobiont, which
composes approximately 90% of the lichen dry weight (12,52). Tarhanen et al.
Lichens
125
FIG. 3 Concentration of Ca (A and B), Mg (C and D), and K (E and F) in extracellular soluble, intracellular soluble, and particulate fractions in () marginal
zones and () central zones of the lichen Xanthoria parietina collected near
a cement mill (A, C, and E) and at Lisbon (B, D, and F). Columns are means
and bars are the standard deviations of five replicates. For the same element,
site and location values with different letters are significantly different for
P 0.05 using a one-way ANOVA. (From Ref. 51.)
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(50) supported the hypothesis that ergosterol is a fungal-specific component and

there is a significant correlation between ergosterol content and the K leakage.
It is the dominant sterol in lichenized fungi and a principal constituent of the
fungal plasma membrane; its concentration is indicative of the metabolically active fungal biomass. Changes in sterol composition can alter the physical properties of membranes and fatty acid component of lipids, and can collectively influence enzyme activity and ion permeability.
The effect of metals on lichens depends primarily on the species and on
the concentration applied. Under controlled conditions, the cations Hg, Tl, and
Ag have been shown to cause severe membrane damage in lichens (29,40). Levels
of Ni, Mn, Co, and Cd only cause membrane damage under unrealistic conditions,
such as low pH and very high concentrations (29,40).
However, one of the elements that undoubtedly causes severe membrane
damage in lichens is Cu (13,14,29,37,40,42,44,50,53). This element also causes
membrane damage in higher plants (8,54), fungi (5), and hepatics (55). The effects of supplied Cu on the release of physiological elements from the lichens
Usnea spp. and Ramalina fastigiata were investigated to establish the extent of
Cu-induced membrane damage (13). The study hypothesized that Usnea spp.
would be more sensitive than R. fastigiata to Cu because a lichen biodiversity
study around a Cu mine in southern Portugal had found the latter to have a wider
distribution than the former (14,37,56). To establish the relative sensitivity of
the lichens to Cu uptake, intracellular Cu concentrations were correlated with
alterations in membrane integrity (Fig. 4). It was shown that Usnea spp. was
more sensitive to Cu uptake than R. fastigiata, with greater K loss occurring at
lower supplied Cu concentrations (Fig. 4). In both lichens losses of intracellular
K (Fig. 4 and Table 1) appeared to occur as a result of intracellular Cu uptake.
The authors found no losses of intracellular Mg (Fig. 4) and suggested that Cu
induces specific K loss in lichens rather than nonselective membrane damage
(46), arguing that Mg is also lost when generalized membrane damage occurs,
as with desiccated samples (13,41). In an experiment performed under field conditions, Tarhanen et al. (50) also reported membrane damage, measured through
K leakage and ergosterol concentration, when the metal content of Bryoria fuscescens thalli exceeded 6.3 mol g1 Cu and 1.7 mol g1 Ni.
The effect of Pb on membrane integrity is controversial: some authors have
reported slight membrane damage at very high concentrations (40,42), whereas
others have not (12,14). Nieboer et al. (42) reported a slight K efflux due to Pb
uptake on Umbilicaria muhlenbergii. However, these authors did not discriminate
between extra- and intracellular K, and some of the K efflux may have derived
from extracellular sites. Branquinho et al. (12) investigated the effect of extraand intracellular Pb concentration on membrane integrity of several epiphytic
lichen species with different photobiont associations (cyanobiont, phycobiont,
and phycobiont with cyanobacterial cephalodia) and thallus growth forms (foli-
FIG. 4 Response on incubation for 2 h in different Cu concentrations of intracellular mean concentrations of Cu (), K (), Mg () and Ca () in Ramalina
fastigiata (open symbols) and Usnea spp. (closed symbols). The control sample (c) was incubated in DDW for 2 h. Symbols are means and bars are the
standard deviations of five replicates. (From Ref. 13.)
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TABLE 1 Linear Correlation Coefficients (Pearson r)

of Intracellular Cu Concentrations with Changes
in the Distribution of Intracellular K, Mg, and Ca
Concentrations in Ramalina fastigiata and Usnea sppa
Lichen species
R. fastigiata
Usnea spp.
Mg
Ca
0.82**
0.57*
0.47
0.24
0.13
0.09
a
Cation measurements were performed after incubation for 2
h in solutions of different Cu concentrations. Significant linear
correlations are marked * for 0.1 P 0.05 and ** P 0.05,
n 9.
Source: Ref. 14.
ose, fruticose) (Fig. 5). Increasing Pb concentration was not observed to alter
lichen membrane permeability, as measured by the loss of intracellular K and Mg,
in the species studied (Fig. 5 and Table 2). Also, no differences were observed in
lichen membrane permeability with time after Pb incubation (120 h) (Fig. 6),
and so no effect of Pb uptake was observed on the mycobiont (12).
Under field conditions the concentrations of several elementsB, Al, Cr,
Fe, Si, Ti, Zn (49,57) and Mg (58)have been correlated with membrane damage
as measured by electrical conductivity. However, this was not subsequently confirmed in the laboratory. This is important since the most dominant pollutant in
several of these studies is SO2 (39,49,53,57), which also causes severe membrane
damage, and it is difficult to isolate the effect of metals with certainty.
4.2
Effect on Photosynthetic Apparatus
Chlorophyll in lichens is usually determined by the method proposed by Ronen

and Galun (59), who showed that the ratio of optical density between 435 nm
and 415 nm is a reliable parameter for estimation of chlorophyll degradation in
DMSO pigment extracts (48,53,60). Several authors indicate that metals increase
the chlorophyll degradation (53,60). The metals responsible for significant increases in chlorophyll degradation in both the field and the laboratory are Pb,
Fe, Zn, Mn and Cu (49,53,60). It is also suggested that the presence of K ions
protects the photobiont chlorophyll against degradation and possibly stimulates
chlorophyll synthesis (53). No effect was observed in chlorophyll degradation
with increasing concentrations of Ca, Cl, Zn, St, and P. A displacement and a
decrease in the absorption spectrum of chlorophyll with Cu, Hg, and Ag under
controlled conditions was observed (40). Bartok et al. (61) found that when transplanted for 150 days to an industrial area of Romania (cellulose manufactures and
Lichens
129
FIG. 5 Intracellular (A, B, C) K (A), Mg (B), and Ca (C) concentrations (mol

g1) in lichens, immediately after 2 h incubation in different supplied Pb concentrations (presented in a logarithmic scale). For each species and cation,
the control sample (c) was incubated in DDW for 2 h. The symbols show the
average and the bars the standard deviation of three replicates for each lichen
species: Peltigera canina (), Lobaria pulmonaria (), Parmelia caperata ()
and Ramalina farinacea (). (From Ref. 12.)
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TABLE 2 Correlation Coefficients (Pearson r) of

Intracellular Pb Concentrations with the Concentration
of Intracellular K, Mg, and Ca in Four Lichen Species
Species
Peltigera canina
Lobaria pulmonaria
Parmelia caperata
Ramalina farinacea
Mg
Ca
0.44
0.58
0.25
0.86
0.92*
0.76
0.88*
0.84
0.03
0.46
0.42
0.65
Significant linear correlations are marked for * P 0.1, n 5.

Source: Ref. 14.
refractory material plants), the lichen Xanthoria parietina underwent a significant

decrease in the content of pigments, particularly chlorophyll a, which significantly correlated with accumulation of Mg, Cr, Fe, and Cd.
Photosynthesis is more sensitive to Cu, Cd, and Zn in cyanobiont than in
phycobiont lichens (62). This sensitivity appears to be related to the prokaryotic
nature of the cyanobacteria; the algal component is thus more sensitive to metals.
For Puckett (40), Ag and Hg followed by Cu were the metals which cause the
greater reductions in 14C fixation, while for Richardson et al. (63), only Cu caused
substantial reduced 14C fixation in Umbilicaria muhlenbergii, which was not affected by uptake of Sr, Mg, Ca, Ni, Zn, and Pb.
Chlorophyll fluorescence has been used to study the effect of contaminants
on the electron transport chain associated with photosystem II (PSII) in lichens
(1214,37,6467). The ratio of variable chlorophyll fluorescence to maximal
fluorescence (Fv /Fm) has been widely used as a parameter to assess the state of
the photosynthetic apparatus and to reflect the efficiency of the primary photochemical reactions in PSII in higher plants (6870) and in lichens (71,72). The
effects of pollutants on lichens showed significant reductions in Fv /Fm with no
changes in other photosynthetic parameters, showing that chlorophyll fluorescence was a more sensitive indicator of damage (64,66).
The effect of extra- and intracellular Pb concentration on chlorophyll fluorescence in several epiphytic lichen species with different photobiont associations
(cyanobiont, phycobiont, and phycobiont with cyanobacterial cephalodia) and
thallus growth forms (foliose, fruticose) has been investigated (12). It was shown
that Pb uptake resulted in a decrease in PSII photochemical reactions, particularly
in cyanobiont lichens (Fig. 7) (12). A significant reduction of Fv /Fm in L. pulmonaria and P. caperata only occurred 48 h after Pb incubation, whereas in P.
canina the decrease was observed immediately (Fig. 7). Although Pb is currently
considered to be a metal with very low mobility (73), transfer of some wallbound Pb to the cell interior, as shown for other elements (Zn, Cu, and Cd) (43),
Lichens
131
FIG. 6 Effect of storage after Pb incubation in intracellular K (A) and Mg (B)

concentrations, in different lichen species: Peltigera canina, Lobaria pulmonaria, Parmelia caperata, and Ramalina farinacea, submitted to three different
treatments: () control samples assessed immediately after incubation in
H2O; ( ) control samples assessed 120 h after incubation in H2O; and ( )
samples assessed 120 h after incubation in 4.8 mM PbNO3. Columns are averages and bars are standard deviations of three replicates. For each species
and element, treatments with the same letter are not significantly different
using a Tukey test for P 0.05. (From Ref. 12.)
may have occurred in L. pulmonaria and P. caperata. The substantial decrease

in Fv /Fm observed for L. pulmonaria might be related to a higher sensitivity of
Dictyochloropsis algae to Pb uptake than Trebouxia (P. caperata and R. farinacea). Thus, the studied effects of Pb in lichens were shown to be dependent
only on the nature of the photobiont. Our results showed that lichen PSIIphotochemical reactions were more sensitive to Pb than indicators of cell membrane damage (Figs. 47) (62,74).
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FIG. 7 Changes in average chlorophyll a fluorescence parameter (Fv /Fm) with

time after Pb incubation (h) in different lichen species: Peltigera canina, Lobaria pulmonaria, Parmelia caperata, and Ramalina farinacea. Prior to the
measurements the samples were incubated in different Pb concentrations for
2 h (mM): control (), 0.005 (), 0.05 (), 0.48 (), and 4.83 (). (From Ref.
12.)
The effect of the Cu on lichen PSII photochemical reactions has been investigated under both field and laboratory conditions (37). There was a significant
correlation between the decrease in Fv /Fm and the increase in intracellular Cu
concentrations under field conditions (Fig. 8). In fact, at intracellular Cu concentrations above about 2.0 mol g1, the photochemical reactions of the lichen R.
fastigiata were completely inhibited at the PSII level (Fig. 8). These results are
in agreement with controlled Cu uptake experiments performed on R. fastigiata
Lichens
133
FIG. 8 Variation of chlorophyll fluorescence parameter (Fv /Fm) with intracellular Cu concentration in the lichen Ramalina fastigiata collected at different
distances from the mine (r 0.88; P 0.05; n 14). Symbols represent
the means and bars the standard deviations of five replicates. (From Ref. 37.)
(Fig. 9). The Fv /Fm values of R. fastigiata samples subjected to Cu addition under
controlled conditions were zero for supplied Cu concentrations above 1.6 mM
(with intracellular Cu concentrations between 3.7 and 4.3 mol g1) (Fig. 9). A
highly significant (P 0.01) linear correlation (correlation coefficient r 0.90,
n 10) was found between the Fv /Fm values and the intracellular Cu concentration. It was concluded that total inhibition of PSII photochemical reactions occurred in R. fastigiata under field and controlled conditions when intracellular
Cu concentrations exceeded a threshold of about 2.0 mol g1. No samples of
this species have been found under field conditions beyond the Cu threshold (37).
These results are in agreement with the Cu threshold concentrations obtained for
other lichen species by Folkeson and Andersson-Bringmark (35), which found
the first signs of reduction in epiphytic lichen cover on pine trunks and twigs
when concentrations of Cu in Hypogyminia physodes exceeded 2.0 mol g1.
Thus, the fluorescence parameter Fv /Fm proved to be a good indicator of the
survival capacity of R. fastigiata under field conditions and a useful parameter for
determination of the sensitivity of lichens (photobiont) to Cu pollution. It would
be useful to determine whether other lichen species have the same Cu threshold.
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FIG. 9 () Response of chlorophyll fluorescence (ratio Fv /Fm) in the lichens

Ramalina fastigiata after incubation for 2 h in different Cu concentrations.
Symbols are the means and bars the standard deviations of three replicates.
() Response on incubation for 2 h in different Cu concentrations of intracellular mean concentrations of Cu in Ramalina fastigiata. Symbols are means
and bars are the standard deviations of five replicates. The control sample
(C) was incubated in deionized water for 2 h. (From Ref. 37.)
4.3
Other Effects
Current information on ultrastructural effects due to metals alone is insufficient

(75). Copper and nickel (Ni) treatments applied under field conditions induced
ultrastructural changes in algal and fungal cells of the lichen Bryoria fuscescens
(75). The response depended on the nature of the metal, its concentration, and
climate. The photobiont was the most sensitive component, and the main ultrastructural changes in alga cells were located in the chloroplasts and mitochondria
(75). The photobiont developed ultrastructural injuries very quickly when thallus
metal concentrations exceeded 0.91.2 mol g1 Cu and 0.1234 mol g1 Ni
in combination with acidity, or more than 20 mol g1 Ni in the absence of
acidity.
Recently, Garty and co-workers (39,58) used the spectral reflectance response of lichens as an indicator of the physiological status of the alga cells,
stating that spectral reflectance correlates empirically with photosynthetic activity, fractional vegetation cover, green leaf biomass or leaf area, primary produc-
Lichens
135
tivity, and carbon in standing biomass. The authors demonstrated that remote
sensing methods effectively detect lichens under stress induced by air pollutants
such as heavy metals and SO2 (39,58). The normalized difference vegetation
index (NDVI) was positively correlated with K concentration in the lichen thallus
and negatively correlated with the concentration of Mn, Ni, Pb, and S.
Other authors have used the production of ethylene as an indicator of metallic stress in lichens (58,76). Lichens display an increase in the ethylene production
when exposed to air polluted with heavy metals (11,58,76,77); this increase reduces the lichens growth rate. Endogenous auxins in lichens 1 year after transplantation to an air-polluted urban site were lower than at the control site (77).
Bartok et al. (61) found that when transplanted for 150 days to an industrial
area of Romania (cellulose manufactures and refractory material plants), the lichen Xanthoria parietina showed a decrease in the respiration rate (measured
through dehydrogenasic activity) with signs of total exhaustion. The respiration
rate was correlated with the distance from the pollution source. The lichens also
significantly accumulated Mg, Cr, Fe, and Cd.
Glenn et al. (78) studied the influence of vehicular traffic on lichens growing in the natural reserve of Montseny, Catalonia, Spain, and found signs of
damage to the lichens. These were mainly due to arthropod feeding and fungal
parasites, which were substantially elevated on roadsides (1433%), in comparison with a control site (10%). They also found elevated levels of Pb, Zn, and
Cu in large thalli near the roadsides.
4.4
Comment
In general, the primary effects of heavy metals are dependent on the nature of the
photobiont, the symbiotic partner most sensitive to heavy metals. The cyanobiont
lichens have been shown to be more sensitive than the phycobiont ones. Of the
most studied elements, Cu and Hg have been shown to be most toxic to lichens.
The applicability of the chlorophyll fluorescence Fv /Fm ratio as a monitor
of metal pollution effects on lichens has been demonstrated. The method has the
advantage of being nondestructive and noninvasive, facilitating repeated measurements.
One of the main problems of using lichens as bioindicators of atmospheric
metal pollution is the difficulty of demonstrating the extent to which morphological and physiological changes observed under field conditions are due to metal
pollution alone. Lichens are also quite sensitive to variations in other abiotic
factors, such as moisture, light, temperature, and pH. When evaluating lichen
biodiversity or the physiological effects of pollutants under field conditions, care
must be taken to determine whether other abiotic factors have also changed and
to confirm the effect of the pollutants under controlled conditions. On the other
hand, ecophysiologists should also verify pollution conditions before attributing
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the decrease in lichen performance to changes in climate, microclimate, pH, forest

degradation, and so forth.
The various authors are not always in agreement as to the physiological
effects of metals on lichens. It is suggested that future studies consider the metal
concentrations that actually enter the cells to justify stated physiological impacts;
otherwise it will be difficult to define threshold values for the organisms.
5
SOURCES, RETENTION, AND RELEASE

OF METALS IN LICHENS
The concentration of elements in lichens is the result of the equilibrium between

uptake and release from and into the surrounding environment. One of the drawbacks of using biological systems for monitoring is that the mechanisms of element uptake and retention by biomonitors are still not sufficiently known (27).
5.1
Wet Deposition
Elements in the atmosphere are scavenged from the air and intercepted by lichens
through precipitation and occult precipitation (fog and dew). The latter is very
important to lichens, and concentrations of nutrients and contaminants may be
substantially higher in fog and dew than in rainfall because more dilution occurs
in the formation of rain (79). Few studies have considered the effect of occult
precipitation on the concentrations of elements in lichens. Concerning the rainfall
process, there is no consensus regarding the contribution to element concentration
in lichens.
The effect of rainfall on Cu and Fe concentrations in lichens transplanted
for 3 months to a Cu mine site in the south of Portugal was studied over 3 years
(Table 3). Total Cu and Fe concentrations increased in R. fastigiata and Usnea
spp. with increasing rainfall volume, particularly if the rainfall had occurred during the previous 30 days (Table 3). It was then suggested that the chemical nature
of lichen surfaces may change with rainfall events, giving rise to a higher particle
retention efficiency (14). Other authors have reported losses of elements caused
by rainfall in places with long dry deposition periods (8082). However, these
authors did not discriminate between soluble and particulate elements. In general,
after a long dry period the first moments of rain produce very concentrated metal
solutions, whereas later rainfall produces more dilute solutions. Elements with
low affinities for wall binding sites may become bound in the first moments of
a rainfall event before being washed. Elements in the precipitation will compete
with those already bound to the wall exchange sites. Concentrations of the elements in the rainfall and affinity for the binding sites will be the major factors
determining the new chemical composition of the lichen wall after rainfall. In
general, it may be concluded that in rainfall events the retention of elements by
Lichens
137
TABLE 3 Correlation Coefficients Between the Concentration of Cu and Fe

in Ramalina fastigiata and Usnea spp. During Each Transplantation Period,
with Variables Related to Daily Rainfall During the Same Transplantation
Periodsa
Ramalina
fastigiata
Rainfall variables (mm)
Total rainfallb
Average daily rainfall
Cumulative rainfall 10 days before
transplant retrieval
Usnea spp.
Cu
Fe
Cu
Fe
0.83**
0.83**
0.85**
0.82**
0.82**
0.82**
0.63*
0.64*
ns
0.71*
0.72**
0.67*
0.91**
0.88**
0.69*
0.73**
0.95**
0.92**
0.80**
0.83**
** P 0.05; * 0.1 P 0.05; ns P 0.1; for Usnea spp. n 8; for R. fastigiata n 7;

a
The lichen samples were transplanted to 760 m from the center of the mine.
b
Cumulative rainfall calculated for each transplantation period.
the lichen surface depends on element solubility (depending also on the emission
source); particle size; nature of the elements (particularly their binding affinity
for the lichen exchange sites); form of the lichen surface (structure and chemistry); and climate (duration of the dry period and the type of rainfall).
5.2
Dry Deposition
Atmospheric dry deposition of metals may occur through sedimentation and

impaction (79). In a study of a Cu mine in the south of Portugal with a very
dry climate, transplanted lichens intercepted more particles through impaction
than by sedimentation (14). Nevertheless, sedimentation probably makes a greater
contribution in countries with more humid climates. Copper particles trapped in
the deposit dust gauges showed no correlation with Cu intercepted by the transplanted lichens (14,83). The particles measured by deposit dust gauges normally
fall into the devices as a result of their own settling speed or are carried in with
precipitation. Furthermore, these deposit dust gauges are only satisfactory for the
collection of particles greater than 100 m diameter; smaller particles usually
only settle by impacting (15). This kind of gauge tends to underestimate deposition during high wind speed periods (15). However, concentrations of the same
element intercepted by directional dust gauges significantly correlated with the
total Cu intercepted by the lichens (14,83). The directional gauges were capable
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of collecting not only direct settling dust but also wind-driven particles. Thus,
in a dry climate lichens intercept most of the elements by impaction. Several
authors state that dry deposition is substantially greater at the end of the summer,
having accumulated during the dry period (14,80,84).
5.3
Substrate Sources
Although lichens have long been used as biomonitors, studies of element sources
other than air pollution have been limited. Recent research on uptake by lichens
suggested that lichens may take up elements from the substrate (8587). Lichens
may cause weathering of rock surfaces by both mechanical and chemical processes. After nutrients have been solubilized, elements may be taken up. Lichens growing on certain metal-rich rocks produce secondary substanceslichen
acidsto complex metal ions. Lichens produce a wide range of secondary products, many of which occur as extracellular crystals within the lichens. Most are
exclusive products of lichen symbiosis; some are useful in medicine and cosmetics.
Purvis et al. (88) showed that lichens growing in Cu-rich substrates (5%
Cu dry weight) produced norstictic acid and psoromic acid within the lichen thalli
to avoid the toxic effects of Cu. Lichens were found to grow directly on uranium
minerals and concentrate uranium within their tissues by McLean et al. (89), who
suggested that melanin-like pigments were involved. Other authors have found
significant differences in the concentrations of some elements between Flavoparmelia baltimorensis growing on granite and tree bark (86). For a review on lichens
growing in metal-enriched environments, see Purvis and Halls (90).
Contamination from the soil underneath terricolous lichens can occur by
capillary diffusion from more basal tissues, soil or litter (91), or from local windblown particles. In terricolous lichens the bases are more influenced by the soil
whereas the state of the tips mainly reflects atmospheric deposition. Epiphytic
lichens are subjected only to wind-blown dust; the relative influence of these
particles depends on the height at which they grow. On a local scale, particles
derived from factories, mines, and traffic are important for monitoring heavy
metal deposition. At background areas the particles measured in biomonitors are
simply soil or rock dust carried out by wind, rain splash, or animal activity (92).
Goyal and Seaward (85) showed that rhizinae of Peltigera canina are capable
of absorbing, accumulating, translocating, and regulating soluble metals, which
then are able to move freely to and from the upper thalli surface. The poikilohydrous water relations of lichens, with cyclic wetting and drying, presumably accelerate the uptake of soluble mineral material from the substrate by the diffusive
mechanism (91).
Iron, aluminum, and titanium are often used as indicators of soil contamination (16,87,93,94). Under natural conditions, elevated levels of these elements
Lichens
139
appear mainly to indicate local wind-blown dust, particularly in areas with surface
soil disturbance (93,94). In very dry and unvegetated regions, a stronger dispersal
of these elements occurs, and direct comparison of metal concentrations and biomonitor results between different climates is not possible. In fact, Loppi et al.
(87) showed that in remote areas of the Mediterranean the elemental composition
of unwashed epiphytic lichen samples was affected by soil contamination; lithogenic elements (Al, Fe, and Ti) in soils were correlated with the same elements
in the lichens. They also concluded that dust contamination is highly variable
and probably depends on local site characteristics.
Elements dissolved in precipitation may be taken up by epiphytic lichens
through scavenging from the atmosphere or leaching of the canopy or tree trunk
where the lichen stands. Moreover, the growth form or the position in the tree
of a lichen may influence its interception of elements. A fruticose lichen is in
less contact with the substrate than, for example, a foliose or a crustose. On the
other hand, lichens located in the runoff area of a tree trunk and lichens located
at the ends of small branches are exposed to different levels of elements. Farmer
et al. (95) showed that epiphyte tissue chemistry responded to seasonal changes
in stem flow chemistry. Different tree canopies have different abilities to intercept
metals, so that lichens growing in different tree species are submitted to different
chemical environments. Some elements (N, K, Ca, Cu, Fe, and Zn) in lichens
growing in several Quercus species were shown to be influenced by the substrate
element content (86). Oliveira et al. (51) found that epiphytic lichens collected
from different phorophytes (tree bark) showed significant differences in their
contents of Ca, Mg, K, Zn, and Fe, though no significant differences between K
concentrations were observed. Potassium is mainly located within the cell and
thus less related to environmental conditions. The differences observed for the
other elements could be related to differences between canopy architectures. Variations in bark pH might also be responsible for some of the observed differences
between the element contents of lichen samples (95,96).
5.4
Influence of Lichen Interception Surfaces
Branquinho (14) reported that the capacity of Ramalina fastigiata to intercept

Cu was approximately twice that of Usnea spp., due to a greater efficiency in
intercepting particles. The proportion of soluble to particulate Cu depended on
the lichen species and was a reflection of the capacity of each species to differentially take up soluble or particulate Cu. There are few studies on the efficiency
of capture of various particles by different plant surfaces (15). It is frequently
claimed that differences between collecting surface characteristics, such as
roughness, explain differences in the capture and retention of metal-rich particles;
hence deposition onto pubescent leaves is expected to be greater than deposition
onto smooth leaves (15). Fine hairs on the surfaces of leaves project through the
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Branquinho
viscous sublayer and trap particles that would otherwise have insufficient inertia
to reach the vegetation surface (15,96,97). The results of the study of Branquinho
(14) were unexpected, since Usnea spp. shows greater hairiness than R. fastigiata.
Higher efficiency of collecting particles is also normally explained by higher
surface volume ratios of thalli (15,91), which appeared to be greater in Usnea
spp. than in R. fastigiata. Thus, in this case, it was suggested that the ability of
the different lichen species to intercept particles might be related to chemical,
adhesive, and water retention characteristics of the thalli rather than structural
features (15,97,98). Future studies of thallus surface chemistry should elucidate
differences in lichens efficiencies of interception and retention of particles from
dry deposition.
5.5
Metal Release Processes
Lichens may release metals by losing biomass through fragmentation by wind,

consumption by arthropods, or release of reproduction structures (isidia and soredia). Unbound elements on the lichen surface may be lost during rainfall periods
(80,81). Redistribution of elements bound to the wall occurs through input of
elements from rainfall dry deposition and particle dissolution. Particles may give
rise to soluble material, though to date no studies have determined rates of release
of metals from lichens. Deruelle (99) showed that Pb acquired by lichen transplants at sites contaminated with automobile exhaust was lost within months once
they had been returned to their original uncontaminated site.
6
UPTAKE AND CELLULAR LOCATION

OF ELEMENTS IN LICHENS
The analysis of air pollution uptake by biomonitors is important because only

those pollutants that are absorbed in living cells will directly affect metabolism
(100). In addition, analysis of air pollution uptake can provide insight into the
air pollution sink strengths of ecosystem components.
6.1
Metal Uptake
The mechanisms of metal uptake in lichens have been reviewed by several authors (9,14,38,91,101103) and can be summarized as follows:
1. Extracellular ion exchange. Early studies on metal uptake by lichens
emphasize the fact that it mainly occurs at extracellular sites through
cation exchange processes. These extracellular sites are presumed
to be in the cell walls and on the outer surface of the cell membrane
(91,102), and it has been suggested that the lichen thallus is similar to
an ion exchange resin (29,104,105). The lichen wall binding sites are
Lichens
141
probably carboxylic acid in nature, and part of the protein component

of the fungal cell walls and membranes (106). This process is physicochemically regulated, rapid, and reversible. Dissolved metal ions are
rapidly bound to extracellular sites (38).
2. Intracellular uptake. This requires the passage of an element across
the plasma membrane, using an appropriate carrier system. Uptake is
slower and more selective than by ion exchange, and the rate of uptake
remains approximately linear for a longer period than for extracellular
uptake.
3. Particle entrapment. Trapping of particles contributes significantly to
the elemental levels found in lichens (91). Particles are accumulated
onto and within the lichen, and may later be solubilized to some extent
by secondary lichen products. The occurrence of metals in particles in
lichen thalli has been demonstrated by Garty et al. (28). Partial dissolution, binding to exchange sites, and incorporation into the cell are all
possible (43,45).
It has been suggested that the accumulation and retention of high concentrations of metal pollutants by lichens can be explained by their high cation exchange capacity and morphological features (surface, structure, and roughness),
which allow interception and retention of particles (11,16,103). Most studies dealing with lichens as biomonitors have up to now provided evidence about the total
amount of metals accumulated in the lichen thallus. The elements in the particulate form will have a short-term impact only if some particulate dissolution occurs, giving rise to soluble material. On the other hand, soluble elements, if located extracellularly, are assumed to be less important for immediate metabolic
effect than those located intracellularly (5,102). Therefore, the potential of any
element to affect metabolic processes directly cannot be assessed by an analysis
of whole-lichen element concentrations (44,74,107). Hence, the fact that a thallus
has a high content of a toxic element does not necessarily indicate that the organism possesses some cellular tolerance mechanism. Understanding the availability
to the lichen (estimated through quantification of element solubility) and the cellular location of potentially toxic compounds is therefore an important step for
establishing whether or not such elements affect cellular mechanisms (102). It
is also essential that lichen responses to a particular pollutant or pollutant mixture
are clearly defined and understood before setting out a field experiment (18).
To this end, considerable controlled plant screening to establish dose-response
relationships is required.
6.2
Sequential Elution Techniques
Quantification of metals associated with extracellular exchange sites on the cell

wall of plants, fungi, bryophytes, and lichens has been achieved through cation
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Branquinho
exchange techniques (5,12,54,108). In lichens, the absence of a cuticle allows the

location and quantification of extracellular and intracellular metals by a sequential
elution technique (12,13,44,46,109). The cations bound to the wall can be displaced efficiently by other cations with a strong affinity for those binding sites
(110,111) or by chelating agents (44). The quantities of metals subsequently released correspond to the amounts of intracellular elements retained by the cell
membrane (110). A suitable elution method requires that the displacing agent
should not cause membrane damage in order to avoid ambiguity as to the location
of the added metal (110). Other workers have employed electron microscope
techniques, which may involve some redistribution of elements during sample
preparation to visualize, but not exactly quantify, elements at the same location.
Nickel has been found to be a suitable displacing agent for most class A
physiologically important elements (e.g., Na, K, Mg, and Ca) and many more
borderline heavy metals (e.g. Cd, Zn, and Co) (4). Strontium has also been
used for the displacement of most of the class A elements. Elements with
considerably greater class B characteristics (e.g., Pb, Cu) can be displaced
with Na2-EDTA (dissodium ethylenediaminetetraacetic) (13,14,44).
6.3
Cellular Location of Elements Under Field Conditions
Under field conditions, metals are delivered to the lichens dissolved in precipitation and in particulate form (16). Until now, only a few in practical biomonitoring
studies using lichens have discriminated between soluble and insoluble metals,
as well as intra- and extracellular location of the soluble metals (14,37).
Sequential elution of samples of field-polluted lichens growing near a busy
road revealed that most of the metals intercepted by the lichens were located
extracellularly in a soluble form (14). Moreover, the total analysis did not provide
information on species differences for metal uptake. Determination of the location (extra-/intracellular) and availability (soluble/insoluble) of the elements Pb,
Zn, and Cu in the studied lichens allowed detection of differences between species, which were not revealed by analysis of whole-lichen element concentrations.
In temporal studies the effects of rainfall events on extracellular element concentrations were shown to be important, since K and Mg appeared to be leached
from the lichen thallus, whereas other elements (i.e., Pb, Zn, Cu, and Ca) appeared
to remain bound to the lichen (14). Boonpragob and co-workers (80,81) also
reported that lichen total metal content shows no seasonal pattern, whereas data
on leachable elements showed seasonal variations with pollution.
Knowledge of the proportion of physiological elements in the different fractions is necessary to quantify and evaluate possible physiological alterations that
might change the response of lichens to their environment and thus affect their
biomonitoring capacity. The concentrations of K, Mg, and Ca in either extra- or
intracellular locations near a busy road and a Cu mine were measured (14). How-
Lichens
143
ever, only extracellular soluble and particulate K, Mg, and Ca were found to
decrease with increasing distance from the pollution sources (14). The soluble
intracellular elements remained relatively constant (unless alterations in membrane permeability occurred due to toxic effects) and did not reflect the variations
with distance from the source. Thus, even in the case of macronutrients (i.e., K,
Mg, and Ca), which have high baseline concentrations, it is possible to evaluate
the influence of the environment whenever their location (intra- and extracellular)
and form (soluble/particulate) is taken into account (14). The authors of a study
of the impact of deposition of saline elements on the southwestern coast of Portugal (84) found that only the concentrations of extracellular ions decreased with
increasing distance from the coast. The intracellular fractions of these elements
are relatively independent of the ions on the surface and bound to the cell wall
due to physiological control by the organisms.
The intracellular location of Cu, K, and Mg can explain the physiological
changes and species survival in the surroundings of a copper mine (14). The
proportions of particulate and extracellular soluble Cu in lichens allowed a comparison with the availability of Cu in the suspected source; discrimination between soluble and particulate Cu and the location of soluble elements at extraor intracellular sites in the lichens provided more relevant information on the
nature of the intercepted pollutants and their effects on lichen survival than total
analysis. Extracellular and particulate metals are dependent on environmental
sources, whereas those located within the cell are more related to physiological
processes (107).
7
MECHANISMS OF TOLERANCE TO METAL POLLUTION
Though several hundred publications have dealt with lichens and metals, only a
few describe lichen species as pollutant-resistant or pollutant-tolerant, illustrating
our lack of knowledge of these organisms mechanisms of tolerance to high metal
content (112,113).
Sarret et al. (113) described the mechanisms of lichen resistance to metal
pollution by comparing Diploschistes muscorum, a hyperaccumulator of Pb and
Zn, and Xanthoria parietina, a Pb-tolerant species. Under normal conditions D.
muscorum produces oxalate, but synthesis is enhanced by exposure to metal pollution, accounting for the hyperaccumulation of Zn and Pb (1.2- to 2-fold) from
the substrate (113). It is also suggested that other cations, such as Fe, Cu, Cd,
and Ca, may also be complexed to oxalate. Moreover, the authors propose that
lichens excrete the oxalates and so this is an extracellular immobilization mechanism. In contrast to D. muscorum, X. parietina does not increase oxalate production in polluted conditions. Sarret et al. (113) concluded that Pb in X. parietina
was predominantly complexed to the fungal cell walls by a passive ion exchange
process.
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Branquinho
Purvis et al. (88,114) also showed that lichens growing in Cu-rich substrates
(with 5% Cu dry weight) produced norstictic and psoromic acids within the lichen
thalli for the formation of metal-lichen acid complexes. The presence of lichen
acids explains how some lichens may tolerate metals by means of a detoxification
mechanism, arising from the formation of an insoluble metal-lichen substance
complex.
It is generally accepted that X. parietina is able to tolerate high levels of
pollutants, including heavy metals (48,67,112,113), and that Usnea spp. is more
sensitive to general air pollution (Figs. 4, 8, and 9) (14,37). In order to evaluate
the cation exchange capacity of different lichen species, samples of several species were submitted to saturated concentrations of Pb and Cu (Fig. 10), elements
with the greatest affinity for the lichens wall binding sites (4). The samples were
then submitted to a sequential elution to displace Pb and Cu from the extracellular
sites, which is a measure of their cation exchange capacity (Fig. 10). The results
show that the cation exchange capacity values obtained for the different species
were in agreement with the previously determined susceptibility to pollution.
Thus, X. parietina had the lower cation exchange capacity, meaning that its toler-
FIG. 10 Lichen cation exchange capacity measured by the Pb and Cu displaced from the extracellular sites after lichen samples of several species
have been submitted to Pb and Cu saturation concentrations for 2 h. The
extracellular fraction was obtained by elution of the lichen samples with 20
mM dissodium ethylenediaminetetraacetic (Na2-EDTA). Columns are means
of 510 replicates.
Lichens
145
ance is probably based on a metal avoidance mechanism. On the other hand,

Usnea spp. showed the highest cation exchange capacity (Fig. 10), which is also
in agreement with its relative susceptibility to metal pollution (13,14,37). The
wall-bound metals may enter the cell more easily, causing negative physiological
effects.
In another experiment, Silberstein et al. (112) compared X. parietina, a
resistant lichen, with the more sensitive Ramalina duriaei in relation to possible
air pollution protection mechanisms. They found that the former has a multitude
of possible protective systems that can be classified in two categories: (a) induced
or stimulated by exposure to pollutants; and (b) constitutive defense. The first
may occur through (a) oxidation of SO2 to nontoxic sulfate; (b) an increased
glutathione content; (c) increase in amino acid synthesis, mainly proline and arginine; or (d) an increase in enzymatic detoxification of active oxygen forms. The
second are enabled by (a) efficient buffering capacity, (b) high K content, and
(c) antioxidation by parietin. Since X. parietina is a foliose species and R. duriaei
is fruticose, the former has a greater ratio of surface exposed to the atmospheric
pollution to volume.
Lichens subjected to air and soil pollution may have three mechanisms of
metal immobilization: (a) entrapment of metal-rich particles, which depends on
their surface/volume ratio; (b) intracellular uptake with likely complexation to
S-containing peptides and metallothioneins; and (c) extracellular complexation
within fungal cell walls, the metal being complexed to hydroxyl, carboxyl, phosphate, amine, or sulfhydryl groups. In the case, the low cation exchange capacity
might be a strategy to avoid metals entering the cells. The authors also suggest
that metals may be immobilized in lichens by organic acids and lichen substances,
particularly the oxalates due to their strong metal binding ability, as is the case
of D. muscorum and lichens growing in Cu-rich substrates. For instance, parietinic acid, which is an anthraquinone produced exclusively by X. parietina, possess several functional groups that can bind metals (113). As in higher plants,
these ligands may be involved in excretion and immobilization of metals within
the vacuole.
Apparently, lichens have differing strategies to avoid metals. The mycobiont is the most important partner in terms of metal accumulation, and it seems
that more comparisons with other biological models such as mycorrhiza could
benefit lichen biomonitoring techniques.
8
SOURCES OF VARIABILITY WHEN SAMPLING LICHENS
Another drawback to the use of biological systems as monitors is that their reaction depends on their specific characteristics (genetic, age, state of health, type
of reproduction, etc.) and on environmental parameters (elemental availability,
substrate, topography, micro-/macroclimatic conditions, etc.) (7,18). For this rea-
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son the standardization of biological monitors is difficult (7). Lichens are one of
the most frequently used organisms for biomonitoring of atmospheric pollution.
However, there is a lack of knowledge of the causes of sample variability, which
is an important source of error in biomonitoring analysis (6,20).
Before starting a biomonitoring survey, it is necessary to test the effect of
different phorophytes on levels of elements in the epiphytic lichens growing below (51,86). This should cover canopy effects and the relative position of the
lichen in the phorophyte (see Sec. 5.3). If differences are observed between phorophytes, care should be taken to choose only one or to intercalibrate lichens
concentrations between phorophytes.
Atmospheric heavy metal pollution gives rise to anomalous high concentrations of metals in the environment, originating from a variety of anthropogenic
sources as well as natural geochemical processes. Heavy metals occur naturally
in rock-forming and ore minerals, and so a range of normal background concentrations of these elements can be found in soils, sediments, water, and living
organisms. Lichens may also be contaminated by wind-blown dust from the surrounding soils (see Sec. 5.3). However, these background values may very substantially at local, regional, or national scales. Soil in areas rich in minerals (e.g.,
serpentine and calcareous areas) may contain high levels of metals. Lichens may
also become naturally polluted in volcanic areas or on coastal regions. Grasso
et al. (115) found that levels of As, Sb, Br, and Pb in lichens collected in the
volcanic areas of Mt. Etna and Vulcano Island in Italy reflected the local gaseous
emissions (plume and fumaroles). Thus, for all studies, the background levels of
pollutants must be evaluated at the study site; comparison with other sites in the
region may not be appropriate.
It is important to consider that sampling of lichens for biomonitoring studies may take a few days, if not weeks, due to the high number of sampling points
normally involved. It is advisable to perform the sampling during the shortest
time possible under stable climatic conditions. Rainfall or a long dry deposition
period (see Secs. 5.1 and 5.2) might substantially change the concentrations of
elements in lichens (14,51,80,81,84,116), such that spatial comparisons are not
possible.
When the pollutants under consideration also have an important physiological role, it may be important to discriminate between insoluble, soluble extracellular, and soluble intracellular compounds in order to distinguish between environmental sources and physiological requirements (see Sec. 6).
Several authors have shown that metal deposition is usually a local phenomenon (37,67,84), whereas gaseous deposition occurs over longer ranges. Several sampling networks have distances between sampling points of 1030 km
(117,118). This type of sampling network does not allow identification of all the
possible sources of metallic pollution. Sites where the local deposition of metals
may occur include roads (99), mines (14,37), and cement mills (67). The possibility of unknown sources of pollution being identified by such a network is very
Lichens
147
small, considering that most of the metal pollution sources previously described
have an impact radius not greater than 2 km. Thus, a sampling network to measure
metallic atmospheric deposition should be based on local factors, such as location
of known sources, topography, altitude, predominant winds, etc., and should be
designed so as to allow subsequent identification of the known and unknown
pollution sources.
Some of the problems described above can be ameliorated by the use of
transplanted lichens, which are homogeneous because they are all collected from
the same location. They are especially useful for biomonitoring studies where
lichens are not normally present, or when the study of the dynamics of deposition
is intended (16). Sampling of lichen transplants is useful in pinpointing sources
of pollution and can reveal dispersion patterns of particulate material (15,57,99).
When sampling lichens for biomonitoring purposes it is important to consider the age of the thalli to be analyzed. Since there is no way of identifying
the exact age of a certain part of a lichen, we suggest testing whether there are
differences between young and old parts of the lichen thalli before implementing
biomonitoring programs. Oliveira et al. (51) established the relative importance
of several sources of variability in sampling lichens for biomonitoring: variations
in the chemical composition of the lichen thallus with ages; and the influence of
the substratum in chemical composition of epiphytic lichens (growing on tree
bark). Levels of Ca, Mg, and K in marginal and central zones of the lichen thallus
exposed to different pollution conditions were compared, taking into account the
solubility and the location of the elements in lichen thalli (Fig. 3). The central
zones of the thallus had significantly higher concentrations of particulate Ca, Mg,
and K than the marginal ones (Fig. 3). Central parts, which are older, had been
exposed to dust particles for a longer period. This could be related to the continual
entrapment of particles by fungal hyphae during lichen growth. Lichens collected
near a cement mill contained higher concentrations of extracellular soluble elements than those from an urban environment (Lisbon) (Fig. 3); dust production
may have caused a higher deposition of elements on the lichen surface at the
cement mill. It has been reported that some elements may be released from inside
the lichen cell, particularly K due to desiccation damage (41). However, intracellular K concentrations in lichens from the cement mill were similar to those
from Lisbon, suggesting that membrane damage had not occurred (Fig. 3E, F).
Variations in intracellular concentrations are mainly related to physiological requirements and are more difficult to interpret in terms of environmental sources
(14) (see Sec. 6.2).
9
CALIBRATION LICHENS WITH DUST GAUGES
Another difficulty in the use of lichens as biomonitors is that it has very rarely
been possible to find a linear relationship between concentrations of pollutants
in lichen thalli and in the atmosphere (119). This is important because regulations
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Branquinho
always define threshold levels in terms of atmospheric concentrations. Moreover,

the application of biomonitors has several advantages over the use of physical
measurements: common occurrence in the field; easy sampling and transplanting;
greater aptitude for pollutant accumulation; and no requirement for expensive
technical equipment (7,15). However, in order to determine absolute atmospheric
concentrations, the relation between the concentration of elements intercepted by
the lichens and atmospheric deposition rates must be calibrated. Some qualitative
(93,116,120,121) and quantitative (14,82,83,122126) comparisons of biomonitoring methods with physical and chemical measurements of atmospheric deposition have been published.
FIG. 11 Plots of observed vs. predicted values obtained by linear multiple

regression models between Cu concentration in R R. fastigiata (A and C)
and U Usnea spp. (B and D) and total volume of rainfall, TR (C and D)
with the dependent variable, Cu concentration intercepted by the directional
gauges (Dir): A (Dir 0.83 17.40*R); B (Dir 7.52 28.25*U); C (Dir
3.14 14.43*R 0.13*TR); and D (Dir 3.81 24.67*U 0.11*TR). (From
Ref. 83.)
Lichens
149
In order to transform the concentrations of Cu and Fe in lichens (expressed

in mol g1 dry weight) into atmospheric deposition values (mg m2), a calibration between the Cu concentrations in lichens and dust gauges was attempted for
comparable periods (14,83). Levels of Cu in the directional gauges were significantly correlated with the total Cu intercepted by the lichens (14,83), a significant
fraction of which was of particulate origin. The introduction of rainfall variables
improved the regression model, demonstrating that element levels in lichens were
dependent on this environmental factor. Among the rainfall-related variables, total rainfall and average rainfall were the ones with the greatest influence in Cu
deposition (see Sec. 5.1) (14,83). A calibration model was obtained that permits
the transformation of element concentrations in lichens (mol g1) into atmospheric deposition rates (mol m2 month1). The model performance measured
by observed vs. predicted values seems quite good, considering that biological
material was involved and that the variability of data from biological systems is
normally very high (Fig. 11).
10 FINAL REMARKS
As seen before, lichens are extremely complex organisms and their basic biology
is still not totally clear. However, when using them as biomonitors there is a
tendency to simplify such complex systems. I would like to point out that reliable
data based on biomonitors must be supported by more research, particularly in
the following areas:
1. It is suggested that researchers do more work on the location of metals
in lichens to allow determination of the threshold intracellular concentrations above which metals cause physiological changes in lichens.
On the other hand, both the insoluble (particulate material) and the
extracellular (surface and wall bound) metal concentrations are more
dependent on environmental exposure than on total concentrations in
the lichens.
2. Careful planning is required to avoid the high variability associated
with sampling lichens for biomonitoring purposes. However, the degree of error associated with biological variability can be offset by the
use of a large number of sampling points, permitting the production
of reliable pollution models that could not be obtained by instrumental
recording owing to high unit costs and the scarcity of the sampling
points.
3. Use of lichen biodiversity data to map the impact of atmospheric pollution should be accompanied by the concentrations of elements accumulated in the lichen thallus to allow distinction between climatic and
pollution factors. Finally, models of the interaction of metals with lichens should be confirmed under field conditions; laboratory-based
data may be unrealistic.
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Branquinho
4. Lichens are able to integrate their physical, chemical, and biological

exposure to atmospheric metals. The fact that biomonitors integrate
this information should be considered an advantage because it takes
into account the interaction between different kinds of pollution. The
use of biological monitors is more representative of the field conditions
and of the ecosystem than physical monitoring devices such as deposit
gauges (15). Dust gauges or high-volume samplers emphasize the
physical aspects of the pollution and do not consider the chemical and
biological aspects, which in some cases are more significant (19). Nevertheless, whenever possible the concentrations of elements in lichens
should be translated into atmospheric deposition rates to allow comparison with legislation based on dust gauges or high-volume samplers.
ACKNOWLEDGMENTS
I would like to acknowledge Dr. D. H. Brown and Prof. F. Catarino for discussing some of the data presented here. I would like to acknowledge the helpful
comments made on a draft of this manuscript by Drs. Cristina Maguas, Gisela
Oliveira, and Steve Houghton.
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6
Bryophytes and Pteridophytes
Nicholas W. Lepp
Liverpool John Moores University, Liverpool, United Kingdom
INTRODUCTION
There are several well-known associations that occur between mosses, liverworts,
and ferns that grow on metal-enriched substrates. These groups of plants each
possess certain anatomical and physiological properties that enable some representatives to occupy unique ecological niches in natural and man-made metaliferous environments. The best documented of these are the groups of specialized
bryophytes that are found on substrates enriched with copper; so-called copper
mosses are found worldwide (1) and come from widely separated taxonomic
groups. Other bryophytes are associated with lead- and zinc-enriched substrates.
Serpentine soils do not appear to possess a distinctive bryophyte flora. Some
species of pteridophytes are associated with serpentine substrates in various parts
of the world (24), while several African species grow on copper-enriched soils
or on soils polluted by metal smelter emissions (5). The existence of these diverse
groups is a clear indication of the ability of Bryophytes and Pteridophytes to
adapt to extremes of metal content of their growth substrate, either by avoiding
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Lepp
the toxic constituents or by expressing resistance/tolerance to metals at an organismal or cellular level.

2
BIOLOGY OF BRYOPHYTES AND PTERIDOPHYTES

IN RELATION TO POTENTIAL FOR METAL TOXICITY
AND TOLERANCE
Because both groups of plants possess motile gametes, they require water for
sexual reproduction. In addition, both possess two distinct growth forms (protonema and gametophyte/sporophyte in bryophytes, prothallus/gametophyte in pteridophytes) and have less developed water relations than vascular plants. Each
of these properties makes bryophytes and pteridophytes potentially more vulnerable to metals in their growth substrates than vascular plants.
Bryophytes do not acquire inorganic ions and complexes by means of root
uptake. No species of foliose bryophyte possess a cuticle; leaves are generally
a single cell in thickness and desiccate readily. Plants are attached to the substrate
by means of nonabsorptive rhizoids, which serve no other function than attachment. Uptake of water and dissolved solutes occurs over the whole plant surface,
with the bulk of inorganic solutes arriving via precipitation enriched with foliar
wash-off or leachates from plant canopies above the bryophytes. Bryophytes
show two different patterns of water uptake, depending on their growth form.
The branched, mat-forming pleurocarpous species are ectohydric, taking up water
over the whole body surface. In contrast, the upright, nonbranching, tuft-forming
acrocarpous species take up water on the outer surface of the stem, with water
moving up or down by capillary action. There are no selective barriers to metal
uptake in bryophytes, except for the cell walls. Substrate metal content only plays
a role in determining bryophyte distribution where soluble metals have direct
access to biological processes in the cell cytoplasm or where there is the direct
potential for interference with either the sexual reproductive process or the establishment of asexual propagules (gemmae, tubers) (6).
Pteridophytes have different water relations to bryophytes. In addition to
possessing well-developed cuticles, they possess root systems that absorb water
and dissolved ions from the soil and efficient conducting systems that connect
roots and shoots. In these respects they are similar to higher plants. However,
pteridophytes still depend on water for sexual reproduction, with the sex organs
developing on prothalli which, in turn, have developed from spores shed by the
mature plant. A film of water is essential for reproduction to take place, so that
soluble metals have the potential to impact pteridophytes at a critical stage in
their life cycle, despite the possession of similar barriers to unlimited metal access
to those found in higher plants. Asexual reproduction is less common in pteridophytes than in other lower plant phyla (7).
161
MOSSES AND LIVERWORTS ASSOCIATED

WITH METAL-ENRICHED SUBSTRATES
Several species of bryophytes can be classed as obligate metallophytes, their

distribution being inextricably linked to soils or substrates rich in various heavy
metals. The best known of these are the so-called copper mosses, a group of
acrocarpous mosses and liverworts that appear to be exclusively associated with
substrates enriched with copper and other heavy metals. These species are either
localized endemics or possess worldwide highly disjunct distribution, both reflecting the uncommon nature of their preferred substrates (8).
The most widely investigated copper mosses are those in the genera Mielichhoferia and Scopelophila (Merceya). These are associated almost exclusively
with copper-rich or other metal-rich substrates, either natural outcrops or substrates enriched as a result of human activity. Of the two genera, Scopelophila
is most frequently and incontrovertably associated with metal-enriched substrates. The six known species are almost certainly all copper mosses and S.
ligulata is always reported as associated with metal deposits; these include copper, iron, and silver, as well as soils associated with sulfur-enriched waters from
hot springs (9). In Europe, S. cataractae is associated with soils contaminated
by metal processing industries, especially those that processed sulfide-based ores.
All of the habitats at which this species occurs in Europe are artificial, e.g., spoil
heaps, soils contaminated with metal particulates or slags or railway embankments, or ballast associated with metal-processing industries. All European populations also appear to be sterile or exclusively male. The substrates with which
S. cataractae is associated are also less acidic (pH 5.66.8) than those reported
for other copper mosses (10). Other populations of S. cataractae have been studied in Japan. Here the species is associated with copper mines in Honshu and
Shikoko as well as with some surprising artificial substrates. Populations are recorded growing on rocks receiving drainage from copper roofs as well as occurring on the base of a bronze statue of Buddha (11). Elsewhere in Japan, a
single natural population has been recorded on an outcrop rich in limonite (iron
oxide), but other occurrences not directly connected with copper-enriched substrates are in the vicinity of hot springs (11). Regrettably, there are no detailed
chemical analyses of the substrates that support S. cataractae in those sites not
directly contaminated by human activity. The other European species, S. ligulata,
has a more restricted distribution in the Alps and Pyrenees (10).
There is debate as to the copper requirements of the other genus of copper
moss Mielichhoferia. Both M. elongata and the closely related M. nitida have
been widely reported to be associated with copper deposits, especially pyrites and
its weathering products, in several parts of Europe. Some analysis of substrates
associated with these species indicates the presence of elevated copper concentra-
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tions, but in other cases, concentrations are not remarkable. North American species (M. mieilichhoferi, M. macrocarpa) may occur on substrates with some degree of mineralization, but the 14 species of Mielichhoferia found in South
America (mostly in the Andes) do not appear to be associated with copper enrichment. Japanese species have not been reported to be associated with copper-rich
substrates, but for both South America and Japan, there is a lack of recent chemical analytical data of both plants and soils. In common with Scopelophila, Mielichhoferia species are also associated with sulfur-enriched waters and substrates.
Whether this is a primary determinant of their distribution or a secondary aspect,
as many of the heavy metals have been derived from sulfide ores, is not clear
(1).
Two species of the genus Ditrichum, both endemic to the United Kingdom,
are totally confined to metal-enriched substrates. D. cornubicum, found in only
two localities in Cornwall (southwestern England) occurs on copper-enriched
substrates at disused mine sites, whilst D. plumbicola occurs on lead mine spoil in
Northern Wales, Northumberland, and the Isle of Man (12,13). As with European
populations of S. cataractae, both of these species have never been found in a
fertile state, but D. cornubicum is known to aestivate by means of tubers. The
moss Grimmia atrata is also closely associated with acidic, copper-bearing rocks
in the United Kingdom and elsewhere in Europe and Japan (12).
Several species of the leafy liverwort Cephaloziella are also closely associated with metal-enriched soils and substrates. C. nicholsonii is a UK endemic
species, confined to old copper mine workings in southwestern England and
Wales, while the closely related C. masalongi is confined to similar substrates
but with a wider distribution in Europe and North America (14). UK populations
of C. integerrima are confined to copper soils in Cornwall, but the species has
a wider European distribution, whereas the more common C. rubella, C. hampeana, and C. stellulifera are all found on old mine spoils in the United Kingdom.
Neither C. nicholsonii nor C. masalongi are reported as fertile in their UK sites
(14). Both species produce gemmae, so asexual reproduction may have perpetuated apparent copper dependency in both species. The leafy liverwort Gymnocolea acutiloba has also been reported to occur predominantly on copper-enriched
substrates in several parts of the world, but its single UK location is nonmetalliferous (13). There is a possibility that this may not be a distinct species.
Studies on bryophytes associated with areas of copper mineralization and
processing have been reported from Shaba province, Zaire (15). Three species
(Brachymenium acuminatum, B. philonotula, and Campylopus bequartii) occurred most frequently, while other taxa (Bryum arachnoideum and unidentified
species of Bryum and Pottia) occurred much less frequently. There is no indication if any species in the first group are exclusively cuprophilic.
Shacklette (16) described in some detail the bryophyte communities associated with various types of mineralization in Alaska. Two species, the leafy liver-
163
wort Nardia scalaris and the acrocarpous moss Oligotrichum hercynicum, were
found to occur in pure stands on several areas of copper-enriched soil (0.20.6%
Cu), while the leafy liverwort Gymnocolea acutiloba was only found to occur
on such substrates. An exposure of galena/sphaelerite ore (PbS/ZnS) was found
to be exclusively colonized by the leafy liverwort Cephalozia bicuspidata. No
significant bryophyte associations were reported from the vicinity of old Mercury
(cinnabar) mining activity.
Despite the wealth of information on the unique assemblages of vascular
plants that occur on serpentine substrates, there appear to be no serpentinespecific bryophytes reported in the literature. Indeed, a depauperate bryoflora has
been reported for at least one UK serpentine site at Keen of Hamar, Shetland
(17).
In many instances, bryophytes associated with metal-enriched substrates
are also abundant on other substrates. Several ubiquitous urban taxa (Bryum argenteum, Funaria hygrometrica, and Pohlia nutans) are frequently found on
metal-polluted soils. P. nutans is particularly associated with natural and polluted
copper-rich soils. It has been reported to form a pure turf in a copper swamp
forest in New Brunswick, Canada (18) in situations where total soil copperconcentrations were 310%; trees and other vascular plants found these conditions phytotoxic. This species was also a dominant constituent of the bryoflora
in copper-contaminated turf at the BICC copper rod plant at Prescot, Merseyside,
United Kingdom (19), occurring where tolerant grasses (Agrostis sp.) failed to
flourish. Bryum argenteum has also been reported to flourish on copper-rich substrates (20).
There have been few systematic field investigations of the direct effects of
heavy metals on the ecology of bryophytes. In a field study at a well-characterized
site (the BICC copper rod plant, Prescot), Lepp and Salmon (19) found clear
differences in the bryoflora that could be attributed to differences in soil copper
concentrations. Where these exceeded a threshold (total HNO3-extractable
Cu 350 mg kg1 dry wt), pleurocarpous moss species disappeared, to be replaced by acrocarpous species (Pohlia nutans, Barbula recurvirostra, and Bryum
rubensthe latter where the substrate was alkaline due to the presence of limestone chippings). This was attributed to either a reduction in grass cover, leaving
gaps in a closed sward for invasion by more metal- and drought-resistant mosses,
or reduced potential for metal access due to the ectohydric water relations of the
acrocarpous species. In addition, routine mowing of the grassland would have
prevented colonization of bare patches by annual or perennial phanaerogams. It
would appear that where soil metal concentrations have a directly adverse effect
on higher plants, some bryophyte species are clearly able to colonize and thrive
on contaminated substrates. These do not tend to be species confined to such
substrates, but a number of common mosses and leafy liverworts appear to be
competitive in such situations.
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Lepp
(a)
(b)
FIG. 1 (a) Bioconcentration of different elements in Scapania undulata (mg/
kg dry wt) before and after bioassay with when cultivated in solutions containing 70100% sewage accumulated lead 85 times in 100% sewage and 58
times in 70% sewage. Mercury content increased 40 times in 100% sewage
and 20 times in 70% sewage. (b) Folds of bioconcentration of different metals.
(From Ref. 21.)
165
Experiments with the liverwort Scapania undulata collected from a clean,

montane forest stream when cultivated in solutions containing 70100% sewage
accumulated lead 85 times in 100% sewage and 58 times in 70% sewage. Likewise, mercury content increased 40 times in 100% sewage and 20 times in 70%
sewage. Increased contents of cadmium, chromium, copper, and nickel were observed (Fig. 1) (21). It was observed that this liverwort when exposed to 70%
sewage did not show significant toxic symptoms and hence suggested its possible
use in removal of toxic metals from water.
4
PTERIDOPHYTES ASSOCIATED WITH METAL-RICH

SUBSTRATES
There is a dearth of information on this topic. Few systematic investigations have

considered the interactions between members of the various groups of pteridophytes and heavy metals, and there are no groups that are comparable with the
copper mosses referred to above. Brooks and Malaisse (5) list several species of
pteridophyte associated with different types of metal-enriched substrates in southcentral Africa. In Zimbabwe, ferns are very rare on nonserpentine Ni-rich soils;
only Pellea calomelanos and Cheilanthes hirta were reported. No ferns were
reported from chromium- or arsenic-rich soils, but Pteris vittata may often occur
on arsenical mine dumps (2225). This latter species is also found on arsenical
mine dumps in West Africa (26), while As accumulation has been demonstrated
in Ceratopteris cornuta from the same area (27).
Ferns are more frequently encountered on copper-enriched substrates. Wild
(28) reports 10 taxa that occur on such substrates in Zimbabwe, and Jacobsen
(29) lists an additional 7 species. These totals make the pteridophytes the fourth
most abundant group (in terms of species richness) of plants associated with
copper soils. This may be influenced by the rocky nature of many copper-rich
sites, allowing ferns to flourish in the shaded and moist crevices that predominate
in this type of terrain. A similar argument has been advanced to account for the
presence of P. vittata at Ghanaian mine sites, as this species is absent from the
remainder of the West African tropical region (26).
Studies on the flora of metal-enriched soils in Shaba province, Zaire have
demonstrated the significant proportion of the native pteridophytes that grow in
such conditions. Of the 90 pteridophyte taxa known to occur in the region, at
least 22 occur over mineralized ground, representing 8.4% of the metallicolous
flora of the province (5). There is some debate as to the presence of any obligate
metallophytes among this group; Cheilanthes inaequalis var. lanopetiolata reportedly confined to copper/cobalt deposits is now considered synonymous with
the more widespread and substrate-indifferent var. inaequalis. Mohria lepigera
appears to be a locally good indicator of copper deposits in this region, but occurs
off copper elsewhere in Zaire (5). Pteris vittata is also abundant on copper-rich
166
Lepp
damp sandy clay soils, frequently forming a monoculture (5). The majority of
the pteridophytes associated with metalliferous soils in Shaba province do not
appear to accumulate metals in shoot tissues [see table in Brooks and Malaisse
(5) for details], with the exception of Ophioglossum lancifolium which appears
to accumulate both Co and Cu from its substrate.
Ferns are also recorded from serpentine and other ultramafic soils. Wild
(28) records seven taxa from the Great Dyke region of Zimbabwe, including
Pellea calomelanos and Cheilanthes hirta. However, it would appear that ferns
of the genus Asplenium are the most typical group with close associations with
such soils in more temperate regions.
Two species, the very rare and disjunctly distributed A. presolanense (found
in isolated populations in Switzerland and Canada) and the more widespread A.
cuneifolium, are considered to be serpentine endemics (4,30). While A. cuneifolium is absent from Britain, there is a very characteristic assemblage of other
Asplenium spp. that are found at serpentine and other metal-enriched sites. The
most common pteridophyte at UK serpentine sites is A. adiantum-nigrum, which
is present as a distinct morphological form. The characteristics of serpentine populations are carried over in cultivation, and while this species will grow in ordinary soil, it is less vigorous than when cultivated in a serpentine substrate (4).
A. viride, which is considered an indicator of ultrabasic soils in Scandinavia, is
only found on highly calcareous substrates in the United Kingdom (4). Equally
noteworthy are the occurrences of several very rare interspecific Asplenium hybrids at UK serpentine sites [A. x alternifolia (A. septiontrionale x A. trichomanes), A. x contrei (A. septiontrionale x A. adiantum-nigrum), A. x murbeckii
(A. septentrionale x A. ruta-muraria)]. It has been hypothesized that these may
persist due to the lack of competition from other ferns and vascular plants (4).
A. cuneifolium and the heliophile fern Nothalaena marantae are reported as abundant in shaded situations on serpentine rocks in southeastern France. The latter
species is predominantly Mediterranean in its distribution and only occurs in
isolated populations on serpentine substrates in central Europe, at the northern
limits of its distribution (2).
Other pteridophytes reported as typical for old mining areas in the UK,
include A. ruta-muraria, A. trichomanes, and A. viride at alkaline sites, these
being replaced by A. adiantum-nigrum and A. septiontrionale where conditions
are more acidic (4). There are also characteristic pteridophyte communities associated with the metal-rich and acidic former tin mine settling ponds in southwestern England. Here, Anthyrium filix-femina is locally abundant and the horsetail
Equisetum palustre dominates the pond margins (4).
5
GENETIC DIVERSITY
Studies on genetic variation in Mielichhoferia elongata, a species of copper moss

with a widely disjunct distribution, have revealed some interesting facts. Most
167
populations seem to possess a low degree of internal variability, but there is great
variation between populations. Thirteen of the investigated populations seemed
to consist of a single clone, but populations in Colorado possessed a tremendous
resevoir of genetic variation. The genetic structure of M. elongata suggests repeated dispersal and founding of populations (31). Similar studies on a second
copper moss Scopelophila cataractae, which has recently extended its range into
Europe, show similar trends (32). European populations seem to be homogenous,
as are populations from copper-enriched soils in the vicinity of Japanese temples,
but other Asian and North American populations are very diverse. Long distance
dispersal may account for the current world distribution of S. cataractae, it is
probably native to both Asia and North America, but appears to be a recent immigrant to its western European sites. This species produces no sporophytes in its
North American and European populations (33) but is fertile at its Asian sites.
Studies on North American populations showed that 50% contained no gametangia and that male and female plants were never found in the same population.
Plants from different populations showed extensive morphological variability
when cultivated on differentially metal-contaminated substrates, indicative of genetic polymorphism. All populations grew best on the most contaminated substrate, with significant variations in growth being evident as substrate metal concentrations decreased (33).
6
CONSERVATION
Metal-enriched soils are under threat internationally. Areas of natural mineralization may be mined, while sites where soils have been polluted by metal extraction
or processing are being increasingly rehabilitated to comply with national guidelines on acceptable concentrations of hazardous substances. Many isolated metalliferous outcrops in Zaire are being exploited, with no action taken to safeguard
the unique plants that have evolved at such sites (A.J.M. Baker, pers. commun.).
In the United Kingdom, old mine sites in former mining areas are being reclaimed, usually to some form of amenity grassland, while urban contaminated
sites are frequently redeveloped in a manner that precludes the survival of any
metallophytes.
In the United Kingdom, several endemic and nationally endangered bryophytes are confined to a handful of former mine sites in Wales and southwestern
England. Detailed conservation action plans have been drawn up to manage such
locations to ensure the continued survival of the leafy liverworts Cephaloziella
calyculata, C. integerrima, and C. nicholsonii and the mosses Ditrichum cornubiensis and Pohlia andalusica. The liverworts are threatened by landscaping or
tidying of former mine sites as well as capping of old shafts and the associated
building work. Ditrichum cornubiensis has more specialized requirements. This
species is a colonist of bare, unshaded, copper-rich soils, such as track edges,
eroding banks, and soil dug from ditches. The plant spreads by means of under-
168
Lepp
ground tubers and requires a succession of bare habitats to colonize. Any changes
to the management of tracks or ditches that significantly reduce these types of
habitat will seriously threaten the two extant populations of this global rarity
(34).
In North Wales, Ditrichum plumbicola is confined to lead mine spoil at
approximately 20 locations in the Conwy Valley. It occurs on silty or peaty soils
on stable waste tips, confined to sparsely vegetated areas and forming pure tufts
on rough surfaces that may have been created by frost heave. It only occurs on
acidic spoil tips and is absent where the tip has a pH 6.5 (13). This species is
clearly highly vulnerable to any management strategy that reduces bare ground or
neutralizes soil acidity. In the same area, the cuprophile liverwort Cephaloziella
masalongi, once frequently associated with copper mining sites, is now considered to be very rare, due to the cessation of mining and the gradual loss of suitable
habitat (13).
7
CONCLUSIONS
Several species of bryophytes and pteridophytes are able to flourish on metalenriched soils and substrates. In some cases, species are solely confined to such
sites, but other widespread species become dominant where substrate metal concentrations exert powerful selective pressures. Both plant groups have received
significantly less attention than metallophytic vascular plants, leaving some important unanswered questions. There is a clear need for more detailed and controlled investigation of the copper mosses to establish the exact nature of the
interaction between copper, sulfur, and pH in determining the ecological preferences of these species. In addition, there is also a pressing need for conservation
of vulnerable metallophytic species, in a similar fashion to the detailed schemes
proposed for endemic and endangered bryophytes of copper soils in the United
Kingdom.
REFERENCES
1. HT Shacklette. Copper mosses as indicators of metal concentrations. US Geol Surv
Bull 1198G, 18pp, 1967.
2. P Duvigneaud. (1966) Note sur la biogeochimie des serpentines du Sud-Ouest de
la France. Bull Soc Roy Bot Belg 93:271329, 1966.
3. W Ernst. Schwermetallvegetation der Erde. Stuttgart: Fischer, 1974.
4. C Page. Ferns. London: Collins, 1988, pp. 270278.
5. RR Brooks, F Malaisse. The Heavy Metal-Tolerant Flora of Southcentral Africa.
Rotterdam: Balkema, 1995.
6. DHS Richardson. The Biology of Mosses. Oxford: Blackwell, 1981.
7. HA Hyde, AE Wade, SG Harrison. Welsh Ferns, ed. 4th Cardiff: National Museum
of Wales, 1969.
169
8. H Persson. Studies in copper mosses. Hattori Bot Lab 17:118, 1956.

9. A Schatz. Speculations on the ecology and photosynthesis of the copper mosses.
Bryologist 58:113120, 1955.
10. A Sotiaux, Ph De Zuttere. Le genre Scopelophila (Mitt.) Lindb. en Europe. Cryptogamie Bryologie Lichenologie 8:95108, 1987.
11. A Noguchi. On some mosses of Merceya with special reference to the variation and
ecology. Kumamoto J Sci 2:239257, 1956.
12. AJE Smith. The Moss Flora of Britain and Ireland. Cambridge, UK: Cambridge
University Press, 1978.
13. MO Hill. A bryophyte flora of North Wales. J Bryol 15:377491, 1988.
14. AJE Smith. The liverworts of Britain and Ireland. Cambridge, UK: Cambridge University Press, 1990.
15. A Empain. Heavy metals in bryophytes from Shaba province. In: RR Brooks, F
Malaisse, eds. The Heavy Metal-Tolerant Flora of Southcentral Africa. Rotterdam:
Balkema, 1985, pp 103117.
16. HT Shacklette. Bryophytes associated with mineral deposits and solutions in Alaska
US Geol Surv Bull 1198C 18pp, 1965.
17. DR Slingsby. The Keen of Hamar, ShetlandA long-term site-specific study of a
classic serpentine site. In: AJM Baker, J Proctor, RD Reeves, eds. The Vegetation
of Ultramafic (Serpentine) Soils. Andover, UK: Intercept, 1992, pp. 235241.
18. DC Fraser. A syngenetic copper deposit of recent age. Econ Geol 56:961962, 1961.
19. NW Lepp, D Salmon. A field study of the ecotoxicology of copper to bryophytes.
Environ Pollut 106:153156, 1999.
20. P Wilkins. Observations on the ecology of Mielichhoferia elongata and other copper mosses in the British Isles. Bryologist 80:175181, 1977.
21. A Samecka-Cymerman, AJ Kempers. Bioaccumulation of heavy metals by aquatic
macrophytes around Wroclaw, Poland. Ecotox Environ Safe 35:242247, 1996.
22. H Wild. Geobotanical anomalies in Rhodesia. 3The vegetation of nickel-bearing
soils. Kirkia 7 Suppl. 162, 1970.
23. H Wild. Indigenous plants and chromium in Rhodesia. Kirkia 9:233241, 1974.
24. H Wild. Geobotanical anomalies in Rhodesia. 4The vegetation of arsenical soils.
Kirkia 9:243264, 1974.
25. H Wild. Arsenic-tolerant plant species established on arsenical mine dumps in Rhodesia. Kirkia 9:265278, 1974.
26. JB Hall. Pteris vittata Linn. A gold mine fern in Ghana. Nigerian Field 35:19,
1970.
27. EH AmonooNeizer, D Nyamah, SB Bakiamoh. Mercury and arsenic pollution in
soil and biological samples around the mining town of Obuasi, Ghana. Water, Air
Soil Pollut 91:363373, 1996.
28. H Wild. The flora of the Great Dyke of Southern Rhodesia with special reference
to the serpentine soils. Kirkia 5:4986, 1965.
29. WGB Jacobsen. A checklist and discussion of the flora of a portion of the Lomagundi
district, Rhodesia. Kirkia 9:147207, 1973.
30. JC Vogel, FJ Rumsey, JJ Schneller, SJ Russell, JS Holmes, JA Barrett, M Gibby. The
origin, status and distribution of Asplenium presolanense spec. nov. (Aspleniaceae,
Pteridophyta). Botanica Helv 108:269288, 1998.
170
Lepp
31. AJ Shaw, RE Schnieder. Genetic biogeography of the rare copper moss Mielichhoferia elongata (Bryaceae). Am J Bot 82:817, 1995.
32. AJ Shaw. Genetic biogeography of the rare copper moss Scopelophila cataractae
(Pottiaceae). Plant System Evol 197:4358, 1995.
33. AJ Shaw. Population biology of the rare copper moss Scopelophila cataractae. Am
J Bot 80:10341041, 1993.
34. Cornwall Biodiversity Initiative (2000). Metalliferous mines action plan. http:/ /www.
wildelifetrust.org.uk/cornwall/wow/audit2/act aal.htm (accessed 31.01.2000).
7
Angiosperms (Asteraceae, Convolvulaceae,
Fabaceae and Poaceae; other than
Brassicaceae)
Anna Siedlecka, Anna Tukendorf, Ewa Skorzynska-Polit,
Waldemar Maksymiec, Magorzata Wojcik, Tadeusz Baszynski,
and Zbigniew Krupa
Maria Curie-Skodowska University, Lublin, Poland
Heavy metals are defined as metallic elements with atomic number higher
than 20, but 12 of them are widely known as environmental pollutants due to
their release by industry: Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sn, and Zn.
Within this group Cd, Cu, Fe, Hg, Ni, Pb, and Zn are usually considered as
potentially hazardous for plants and animals, despite the fact that most of them
(Cu, Fe, Ni, and Zn) are known as essential elements necessary for plant and
animal metabolism (13). In general, biodiversity in plant response to heavy
metals will be discussed in this chapter. Nonetheless, some particularly important
responses to Alnonheavy, but well known as a very toxic metalare also
described. Some plants, like Arabidopsis sp. (a model organism in plant molecular biology), Thlaspi sp. (hyperaccumulator), or such popular experimental material as Brassica sp., Lactuca sp., or Raphanus sp. belong to the Brassicaceae
171
172
Siedlecka et al.
family, and their responses to metals are described in detail by Di Toppi et al.
in Chapter 8 of this book.
1
1.1
METALS UPTAKE
Soil Factors
Biodiversity in plant response to heavy metal stress results from a number of

factors: the metal itself, the plant species, and even the plant growth stage. The
content of heavy metals in natural, uncontaminated soils is very different and
depends on a number of factors, such as lithosphere (base rock), soil age, soil
type, and covering flora. In general, heavy metals can be accumulated in higher
amounts in the sorption complex of soils rich in organic matter, but their release
to soil solution is much slower than in mineral soils due to high affinity of soil
organic compounds to heavy metals (1). Heavy metals solubility in soil and thus
their availability to plants is controlled also by soil pH, type of mineral colloids,
and many other important factors, such as microbial activity, redox potential, and
aeration (1,2) (Fig. 1). Some of these factors act in several ways; an increase
FIG. 1 Soil and root factors modifying heavy metals uptake from soil.
Angiosperms
173
of Ca2 content in soil (liming) decreases heavy metals uptake by roots due to
physiological antagonism of Ca2 /heavy metal ions, but heavy metals uptake is
decreased even more by increased heavy metals retention by soil colloids (1)
(Fig. 1). Availability of free ions has been widely considered as a primary factor
of metallic nutrients uptake by plants. There is evidence now that this relationship
is not so obvious (4). Free ion concentration dependent uptake of divalent cations
is easier than when metallic nutrients are present in chelated form (5) (Fig. 1).
Nonetheless, if the quantity of the metal-complexing ligand is limited (the case
of soil solution ligands, which often are of low affinity and low selectivity in
respect to individual elements) or the ligand is very diluted, chelation may have
no influence or may even enhance a divalent cation uptake (4). However, chelation has a very stimulating effect on root uptake and translocation to shoots of
trivalent cations, like Cr, Ga, or In (5) (Fig. 1). It is also not possible to select
the most important soil factors influencing metals availability to plants because
their importance can vary among elements; for Zn it seems to be a Zn-buffering
mechanism in the soil, whereas for Fe and Mn it is soil pH and soil redox state
(2,6,7).
1.2
Plant Factors
Almost all heavy metals can be taken up by plants in two ways, which are mostly
concentration-dependent (2,3,712):
Nonmetabolic uptake by energy-independent mechanisms. Intact membranes are effective barriers for ions and uncharged molecules, but when
solutes are more concentrated at the one side of the membrane they can
diffuse down the concentration gradient with the aid of membrane carriers or even aqueous pores. This transport is known as passive and takes
place when heavy metal is present in the root environment at a high
amount. It may be also stimulated by lowering the free ion level in cytoplasm due to its incorporation into organic structures, deposition in some
cell compartments, transport to other cells, or bounding to charged
groups. This process often takes place in meristematic tissue of root
tips.
Metabolic uptake by energy-dependent mechanisms. This active uptake is
involved in taking up ions against their concentration gradient. In this
mechanism, a proton motive force (ATP-driven H pumps) creates pH
and electropotential gradient which stimulate ion passing to the cell
through selective ion channels or carriers.
Higher plants have sophisticated and specific mechanisms for Fe uptake. Iron,
despite being widely spread in the lithosphere, usually predominates in soils in
174
Siedlecka et al.
nonsoluble and oxidized forms, and plants have developed two strategies of its
uptake involving two main problems: availability and necessity of its presence
in plants in reduced form (1,2,1318).
Strategy I, typical for most higher plants (all dicotyledonous and monocotyledonous except Graminae), is a three-step, complicated process. The
first stepmobilizationmeans release of protons, organic acids, and
phenolic compounds to the soil resulting in evolving free Fe3 from the
soil complex and its chelation by phenolics to keep it in solubilized form.
The next step is reduction by root membrane reductasesconstitutive
proteins whose activity increases even by several orders of magnitude
in Fe deficiency conditions. Reduction of iron from its ferric to ferrous
form takes place in the rhizosphere, outside roots. The last step is ferrous
ion uptake by roots, mainly through plasmalemma of special transfer
cells. The number of these cells increases significantly in Fe deficiency
conditions.
Strategy II, present only among Graminae, is much simpler than strategy
I. Roots of grasses release to soil specific chelating substances called
phytosiderophores, which are mainly derivatives of mugineic acid synthesized from nicotianaminea nonprotein amino acid. They extract and
chelate ferric ions from soil sorption complex; the whole complex with
oxidized Fe ions is taken up by plant. Ferric ions are released and their
immediate reduction takes place inside root cells.
Strategy II is more effective and more resistant to unfavorable environmental
factors than strategy I because two important steps (Fe release from chelate and
Fe reduction) are shifted from the rhizosphere into homeostatic conditions inside
living root cells.
1.3
Interactions in Uptake
For plants of both strategies heavy metals toxicity results in decreased Fe uptake
and Fe deficiency despite a good availability of this essential element. This effect
is so strong that external symptoms of heavy metal toxicity and Fe deficiency
are very similar for a number of elements (2,1922). Inhibition or disturbances
in functioning of root plasmalemma ATPase and oxidoreductase as well as young
root tip damage and increased root Fe immobilization belong to the main mechanisms of Fe deficiency induction in strategy I plants (2,3,9,2226). Strategy II
plants are less sensitive to heavy metalsinduced Fe deficiency due to better
efficiency of this strategy, as described above. Nonetheless, inhibition of mugineic acidferric complex uptake in barley roots by Cu, Zn, and Co was also
reported (27).
Angiosperms
175
Toxic influence of heavy metals is connected not only with inducing Fe

deficiency but with numerous disturbances in plant mineral composition, resulting in complicated effects both of indirect and direct nature, leading to
changes in plant metabolism (2,3,2226,2830). In general, relationships between toxic metals and other nutrients, including heavy metals essential for plant
growth and development, can be divided into three categories (3,31) (Fig. 2):
Additionwhen there are no effects for plant growth and metabolism of
introducing yet another element to the environment
Antagonismwhen the introduced element is of beneficial influence for
plants
Synergismwhen the new element results in multiplication of plant stress
symptoms
FIG. 2 Plant responses to combination of metals in the growth medium. Different arrows show direction of relationship documented: , same relationship occurs for both metals; , one metal influences another one. (Data taken
from Refs. 10, 3135.)
176
Siedlecka et al.
PHOTOSYNTHESIS
The sensitivity of higher plants to heavy metals stress is revealed largely by its
interference in functioning of the photosynthetic apparatus (36, 37 and references
therein). Alterations of the chloroplast function and structure in plants exposed
to heavy metals depend on the individual heavy metal, its concentration in the
plant growth medium, uptake to plant organs, and on many mechanisms involved
in detoxification processes after the metal has entered the plant as well as its final
concentration found in chloroplasts. Plant species, their genetic and individual
features, duration of heavy metals action, the age and physiological condition of
plants during the treatment are also greatly significant. The photosynthetic apparatus seems to be affected by a number of direct and indirect actions of the metal
ions resulting in functional and structural disorders.
2.1
Photosynthetic Pigments
One of the toxic effects resulting from excess supply of heavy metals is a change
in chloroplast pigments. Reduced accumulation of chlorophyll as an effect of the
action of heavy metals such as Cd, Cu, Hg, Pb, Mn, Ni, and Zn in many plant
species has been well documented in both in vitro and in vivo experiments (37
45).
Chlorosis, the most apparent symptom of heavy metals effect in leaves, may
result from inhibition of chlorophyll synthesis caused by reaction with constituent
biosynthetic enzymes as well as chlorophyll degradation. Another reason of chlorosis may be a strong interaction between heavy metals and Fe (34, 37 and references therein, 40, 46, 47). Heavy metalsinduced Fe deficiency obviously affects,
among others things, photochemical activity of thylakoid membranes, including
all Fe-containing complexes of the photosynthetic electron transport chain (3, 18
and references therein). The opinion that heavy metals, such as Cd, Hg, or Pb,
interfere with chlorophyll biosynthesis by inhibition of -aminolevulinic acid
(ALA) formation, ALA dehydratase activity as well as protochlorophyllide photoreduction is based on experiments with short-term action of the metal in detached leaves or their segments as well as in whole plants (4855).
It was recently postulated that Cd does not affect chlorophyll biosynthesis
itself but interferes with chlorophyll integration into stable chlorophyll-protein
complexes of thylakoid membranes required for normal protosystem II (PSII)
activity (43). The metal, reducing chlorophyll content in leaves, also influenced
the expression and assembly of chlorophyll-binding proteins. A strong reduction
of all chlorophyll-containing complexes in cucumber under Cd treatment was
found in the order of PSI LHCII PSII core and to a lesser extent under Pb
stress, where LHCII appeared somewhat more sensitive than PSI (45,47). Cadmium effects on disturbances of LHCII oligomerization process correlated with
Angiosperms
177
the level of trans-16:1 fatty acid in phosphatidylglycerol were hypothesized (56

58). Most recently, Cd affected LHCII accumulation by drastically reducing the
steady-state level of Lhcb transcripts was demonstrated on the basis of hybridization analysis (59).
Catabolism of chlorophyll in heavy metaltreated plants was also sporadically considered. The expected mechanism of chlorophyll degradation related to
stimulation of chlorophyllase activity in heavy metalstressed plants has not been
univocally documented. An increase in hydrolytic activity of chlorophyllase in
rice leaves was induced by Hg, Zn, and Cu (60), in contrast to its decrease with
increasing Cd concentration found in primary leaves of barley (61). Involvement
of Cd-induced lipid peroxidation in chlorophyll degradation based on a positive
relationship between increase of lipoxygenase activity and decrease in chlorophyll content was recently proposed (62). The participation of galactolipase,
whose activity is enhanced in Cd-treated plants, was also taken into consideration
in diminished chlorophyll accumulation (63).
Chlorophyll accumulation in plants stressed by heavy metals depends on
their sensitivity or tolerance to metal action. A differentiated step of chlorophyll
synthesis inhibition was observed in sensitive (significant chlorophyll decrease)
and tolerant (almost unchanged chlorophyll level) population of spinach as a
response to Cu toxicity (64,65). Also, in a Cu-tolerant population of Silene cucubalus chlorophyll content was not affected by Cu, whereas sensitive plants became chlorotic (66). These toxic effects of the metal were confirmed later when
chlorosis in a Cu-sensitive population of Minuartia hirsuta and permanent green
leaves in a Cu-tolerant plant were demonstrated (67). In wheat cultivars, which
differ in their tolerance to Mn, chlorophyll concentration declined under Mn treatment. However, a higher pigment concentration was maintained in tolerant than
sensitive cultivars (68). Also, three genotypes of Phaseolus vulgaris with contrasting tolerance to Mn toxicity showed increased chlorosis in the more sensitive
genotype (69).
Differentiated resistance of plants to heavy metals, resulting in changes of
the plastid pigment content, depends on the developmental stage of the plants in
the time of treatment. In runner bean plants treated with Cu and Cd at the early
growth stage the chlorophyll content increased on the leaf area basis, while chlorosis was observed in plants treated at the final stage of primary leaf growth (70
74). A reduced leaf area, an increased leaf density, smaller palisade parenchyma
cells, and reduced mesophyll cell volume in young leaves may suggest a lower
sensitivity of the pigment to Cd and excess Cu than processes of leaf growth.
Chlorophyll content depending on the developmental stage was observed in leaf
sections of different ages of monocotyledonous plants, such as maize (75) and
rye (76). The relationship between the developmental step of pigeon pea exposed
to Cd and Ni and chlorophyll content was indicated by almost unchanged pigment
178
Siedlecka et al.
content in plants when treated with heavy metals at the vegetative and flowering
step. However, drastic reduction in chlorophyll content, greater in Cd- than in
Ni-treated plants, was observed at the pod-filling stage (77,78).
2.2
Chloroplast Ultrastructure
Changes in chlorophyll accumulation are usually accompanied by heavy metal

induced drastic ultrastructural damages of the chloroplast fine structure, mainly
at the thylakoid level (46,64,65,75,7996). During a long-term action of heavy
metals, the decomposition of the chloroplast architecture, such as degradation of
grana stacks and stroma lamellae, increase in the number and size of plastoglobuli, and occurrence of intrathylakoidal inclusions, is the most frequently observed symptom in heavy metalstressed plants.
In damages of chloroplasts as a response of plant to heavy metals, the
developmental stage of plants during the treatment as well as plant sensitivity to
their action seem to be of great importance. Small or no changes in chloroplast
ultrastructure of runner bean plants treated with Cu and Cd at their early stage
but great alterations at the end of growth stage have been observed (87,90). The
tissue sections at different stages of maturity along secondary maize leaf revealed
that at high Cd concentrations ultrastructural alterations of chloroplasts were
more distinct in mature tissue of both the mesophyll and the bundle sheet (75).
These observations can suggest dicots and monocots to be more resistant to heavy
metals at the early growth stage. Nevertheless, the tolerance of monocots to heavy
metals is generally higher than that of dicots and requires rather a long exposure
time to heavy metals.
Heavy metals affect chloroplast ultrastructure to a greater extent in sensitive
than tolerant wheat cultivars (82) and spinach populations (64,65). Delayed etioplast transformation into chloroplast caused by Pb (94) and Cd (85) was also
reported.
2.3
Thylakoid Membrane Polypeptides and Acyl Lipids
Disruption of the chloroplast ultrastructure in heavy metalstressed plants seems

to be related to disorganization of protein and acyl lipid composition of thylakoid
membranes. Heavy metalaffected damage to the structure and composition of
the thylakoid membranes induces changes in lipid matrix, while concomitant release of fatty acids probably leads to dissociation of thylakoid polypeptide components. Decomposition in chlorophyll-protein constituents of PSII was found in
older spinach and bean plants exposed to Cu (65,97). A lower accumulation of
some extrinsic polypeptides of PSII particles is age-dependent as shown in runner
bean plants treated with Cd and excess Cu (70,74,89). In older chlorotic leaves
it concerns a high decrease in the polypeptide content both of the oxygen evolving
complex (OEC) and PSII core antenna. In leaves treated with the metal at a
Angiosperms
179
younger age only 17- or 17- and 23-kDa polypeptides of OEC changed noticeably. Polypeptide release from thylakoid membranes in rice exposed to excess
Cu was also noted (98). These data correspond well to changes in PSII activity
confirming the donor side of PSII as a most sensitive site of heavy metal action
along the photosynthetic electron transport chain proposed earlier (37 and references therein).
Experiments in vitro are in agreement with this statement. Disorganization
of the donor side of PSII is indicated by the release of OEC polypeptides from
thylakoid membranes of spinach (99) and Vigna unguiculata (100) incubated
with Cd. Similar conclusions resulted from significant depletion of 17- and 23kDa polypeptide and partially of the 33-kDa polypeptide in both Pb- and Znincubated spinach chloroplasts (101).
Heavy metalinduced damages in thylakoid membranes were visualized
also by alteration in the lipid fraction of the membrane. The changes in the content
and composition of acyl lipids forming the lipid environment of photosystem
complexes were confirmed by several authors. A decrease of acyl lipids and
changes in their fatty acid composition in the chloroplast membranes were observed after exposure of Phaseolus vulgaris and Zea mays to Pb (102104), Lycopersicon esculentum, Raphanus sativus, and Phaseolus coccineus to Cd
(56,58,63,74), Spinacia oleracea, Phaseolus coccineus, and Triticum durum to
excess Cu (70,95,97), as well as Pinus banksiana to V and Ni (105). An alteration
in the lipid environment around PSII was taken into account as the cause of the
loss of PSII activity following Cd treatment (37,57,58). Some of these changes
that implicate photosynthetic activity in chloroplasts depended on the time of
metal action, its concentration and application to the nutrient solution, and the
age/growth stage of plants. The destruction of the thylakoid membrane shown
in spinach chloroplasts was interpreted as a result of lipid peroxidation mediated
by excess Cu (106). Significant changes in acyl lipid correlated with a distinct
loss of core antenna PSII polypeptides and OEC subunits mentioned above characterize spinach and bean plants exposed to Cu and Cd by the end of the intensive
growth stage of primary leaves (74,97).
2.4
Photosynthetic Electron Transport
Photochemical reactions of PSII have been shown to be more susceptible to heavy

metal stress than those of PSI. On the basis of in vitro and in vivo studies, the
PSII reaction center and PSII electron transport were recognized as strongly affected by Cd (37 and references therein, 107) (Fig. 3). For a long time PSI was
considered as relatively resistant to Cd. In earlier in vivo studies, only a small
decrease in PSI activity in Cd-treated plants was found (81,85). Later on, at higher
concentrations of the metal, a significant inhibition of both PSII and PSI in chloroplasts of Cd- and Pb-treated clover and lucerne seedlings was reported (108) and
180
Siedlecka et al.
FIG. 3 Toxic influence of metals on the photosynthetic apparatus of C3 higher

plants. Abbreviations: CA, carbonic anhydrase; CF0 , chloroplast coupling factor 0; CF1, chloroplast coupling factor 1; cyt, cytochrome; Fd, ferredoxin;
FDPase, fructose-1,6-bisphosphatase; FNR, ferredoxin-NADP oxidoreductase; GAPDH, NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase; LHC I, light-harvesting chlorophyll a/b protein complex I; LHC II, lightharvesting chlorophyll a/b protein complex II; OEC, oxygen evolving
complex; PC, plastocyanin; PCR, photosynthetic carbon reduction cycle
(Calvin cycle); PGA, 3-phosphoglyceric acid; PQ, plastoquinone; PR kinase,
ribulose-5-phosphate kinase (phosphoribulokinase); PS I, photosystem I; PS
II, photosystem II; RA, Rubisco activase; Rubisco, ribulose-1,5-bisphosphate
carboxylase/oxygenase; SdBPase, sedoheptulose-1,7-bisphosphatase; TM,
thylakoid membrane; TPI, triosephosphate isomerase. (Data taken from Refs.
18,35,37, 39,52,77,79,115,130138). Cu influence on Rubisco and CA in
Phaseolus vulgaris plantsSiedlecka and Krupa (unpublished data).
Angiosperms
181
also confirmed in chloroplasts of Cd-exposed runner bean plants (111). At the

same time, new data showing that application of a low, non toxic level of Cd to
wheat plants modifies the primary photochemistry of PSI as that of PSII were
obtained, although its sensitivity to Cd was lower than that of PSII (112). A
similar conclusion was drawn from PSI activity inhibition in respect of Cd-Fe
interaction in bean plants (18 and references therein, 35) (Fig. 3). Recently, it
was demonstrated that in maize seedlings Cd strongly interacts with Fe leading
to Fe deficiency, thus decreasing the level of ferredoxin and markedly affecting
PSI activity on its reducing side (24). The sensitivity of the PSI reducing side
was suggested in earlier in vitro studies on spinach chloroplasts incubated with
Cu, showing not only PSII disorder on its donor side but also PSI inhibition due
to interaction with ferredoxin (111). However, the PSI oxidizing side affected
by heavy metals was recognized in vivo when a decrease in plastocyanin content
in chloroplast of rice exposed to excess Cu (112) as well as in Ocimum basilicum
plants treated with Ni and, to a lower extent, Zn was shown (113) (Fig. 3). Decrease in ferredoxin content in chloroplasts of O. basilicum indicates that both
sides of PSI are Ni-sensitive. The sensitivity of both PSII and PSI to Cu was
also found in Cu-treated intolerant spinach (65) and ecotype of Thlaspi ochroleucum (42), but changes in PSI after exposition to Cu were smaller than those
occurring in PSII.
PSII, essential for photosynthetic regulation, is strongly affected by heavy
metals at different target sites (37, 39, 48, 114 and references therein) (Fig. 3).
As shown in in vitro studies, the water-oxidizing system of PSII was the most
affected, but OEC was postulated as the primary target of heavy metals toxicity.
This was supported in in vivo examinations of photosynthetic activities inhibition
in plants under Cd, Pb, and excess Cu stress (81,85,108). The replacement of
Mn ions in the OEC by Cd (81) and Cd- or Cu-induced alteration in the lipid
composition of the thylakoid membrane resulting in release of fatty acids causing
OEC subunits disturbance were taken into consideration as the reason for the
loss of PSII activity. Inhibition of photosynthetic electron transport by Cu, mainly
at the PSII before the DPC (1,5-diphenylcarbohydrazide) donor site, was shown
in rice chloroplasts (115) (Fig. 3). The PSII electron transport on both the donor
and acceptor side influenced by heavy metals, like Cu, was concluded elsewhere
(67,116). The antenna chlorophyll a molecule of PSII was also proposed as a
primary site of Cu inhibition (86).
The inhibition site for Co, Ni, and Zn in chloroplasts isolated from pea
leaves was located on the PSII acceptor side at the level of the secondary quinone
acceptor, QB (117) (Fig. 3). This was also indicated by increase in QA reduction
state and thermal energy dissipation due to excess Mn limiting energy utilization
in the Calvin cycle found in white birch leaves (118). The acceptor side as a site
of Cu action in chloroplasts of runner bean plants was also confirmed in in vivo
studies (70,71).
182
Siedlecka et al.
The toxic effect of heavy metals on the photosynthetic processes in leaves

of developing and mature plants seems to be different. In hydroponically cultivated runner bean plants supplied with excess Cu various responses depending
on the growth stage at which the metal was added to the nutrient medium were
noticed (70,71). In plants treated with Cu at the young development stage, the
inhibitory effect on the acceptor side of PSII, (due to induced inhibition of the
Calvin cycle) and down-regulation of the electron transport was suggested in
the absence of changes in the primary PSII photochemistry. In plants treated at
a more developed stage a low PSII activity may result from alteration in the
donor side and PSII reaction center as well as in the acceptor side of PSII.
In addition, the effect of Cd on PSII activity was shown to be considerably
modified by the developmental stage of plants during treatment (73,74). In runner
bean plants treated with Cd at an early growth stage of primary leaves no disturbances of PSII efficiency and a small increase in heat dissipation of excitation
energy were observed. Cadmium treatment at the end of the growth stage affected
PSII photochemistry, electron transport, and dark reactions correlated with degradation of thylakoid membranes.
The mechanism of heavy metals action on the photosynthetic apparatus can
be influenced by the level of Ca2 accumulation in plants. Enhancement of Cd
toxic effect, including primary photochemistry of PSII, in runner bean plants
grown in Ca2-deficient medium was documented as well as reduction of Cd
toxicity in Ca2 excess (119). Similarly, in young runner bean plants Ca2 deficiency increased the toxic effect of excess Cu on the photosynthetic apparatus
(120122). However, in older plants such an effect was observed at increased
Ca2 accumulation. It is hypothesized that in the first case Cu can substitute
weakly bound Ca2 present in OEC and/or coupling factor (123125); in the
other case, Cu through Ca2 accumulation develops senescence processes (126
and references therein). A mechanism of partial prevention of Cu-induced PSII
inhibition by Ca2 was postulated for PSII particles depleted of OEC subunits
(127).
In heavy metalinduced degradation of the photosynthetic apparatus, disturbances of the turnover of D1 reaction center polypeptide of PSII has been
signalized (128 and references therein). In bean plants, after exposure to excess
Cu, the equilibrium between photoinhibition and repair resulted from increase in
the quantum yield of photoinhibition (116). Inhibition of the turnover of D1 protein in plants under Cd stress depending on the plant species and the time of Cd
exposure was also postulated (129).
2.5
2.5.1
Photosynthetic Carbon Assimilation

C3 Plants
According to Weigels observations (139,140), inhibition of the dark reactions

of photosynthesis by Cd precedes that of the photosynthetic electron transport.
Angiosperms
183
Numerous data obtained from chlorophyll a fluorescence induction kinetics measurements were recently published, leading to the conclusion that the Calvin cycle
is probably the primary target of heavy metals toxicity (37 and references
therein). Changes in the primary photochemical reactions and electron transport
seem to be secondary effects, at least for young plants. The proposed mechanism
of heavy metals toxicity concerns limitation of ATP and NADPH consumption
in the Calvin cycle causing an increase in proton gradient across thylakoid membrane, finally leading to down-regulation of both PSII photochemistry and linear
electron transport (see 37 and references therein, 141) (Fig. 3). Similar mechanism of heavy metals toxicity on the photosystems in stressed plants was also
proposed in regard to the action of Ni, Cu, Pb, Fe, and Cd (35,44,47,70,74,82,
119,141,143).
Weigel concluded that further steps of the Calvin cyclereduction and
regenerationare the most sensitive targets of Cd toxicity (139,140). Inhibition
of some important enzymes of these phases of the Calvin cycle was confirmed
in Cajanus cajan, Phaseolus vulgaris, and Triticum aestivum in the presence
of some heavy metals (52,77,132,134), (Fig. 3). Nonetheless, based on current
information, Rubisco and carboxylation process carried out by this enzyme seem
to be not only the most important but also the most sensitive step in plant response
to heavy metals (Fig. 3). Rubisco is the most abundant protein in chloroplasts
(more than 60% of total leaf protein) and its activity depends on next two enzymes
present in excessive amounts in stroma: Rubisco activase (RA), 5% of total leaf
protein, and carbonic anhydrase (CA), 2% of total leaf protein (137,144,145) (Fig.
3). Rubisco activase regulates correct conformation of Rubisco-active center. The
pool of Rubisco reaction centers that are activated and have proper conformation
to act as enzyme is called the Rubisco activation state (RAS) and is expressed
as a percentage of all Rubisco-active centers. Usually RAS is at the level of 60
70% (137,144). Depending on the Cd concentration, two different mechanisms
of Rubisco activity regulation are proposed (18, 137 and references therein):
At low Cd level, CA activation is enough to maintain full Rubisco activity,
even with accelerated Rubisco light activation at the beginning of light
day period (136,138).
At higher Cd concentrations, inhibition of CA occurs and ATP-dependent
(probably RA-dependent) mechanism results in increase of RAS up to
100%. However, this mechanism is not as efficient as the CA-dependent
one, so despite this effort Rubisco activity decreases (136,138).
An interesting phenomenon is that the above-described mechanism of CAdependent maintenance of Rubisco activity seems to operate only at moderate
Cd stress. Other investigated metals, i.e., Fe (136) and Cu (Siedlecka and Krupa,
unpublished data), even at low toxic concentrations caused substantial decrease
in CA and Rubisco activities (Fig. 3). It is also confirmed that monocotyledonous
plants showing C3 metabolism, like rye or wheat, are less sensitive to heavy
184
Siedlecka et al.
metals stress than dicotyledonous plants, also with respect to the dark phase of
photosynthesis (30,39).
2.5.1
C4 Plants
Among C4 plants the primary carboxylation process, catalyzed by phosphoenolopyruvate carboxylase (PEP-carboxylase), is the most sensitive target of heavy
metals toxicity (37, 39, 146 and references therein). In Zea mays PEP-carboxylase
appeared to be very sensitive to Cd, Cu, Pn, and Zn (130). On the other hand,
the C4 plants are well known as less sensitive to heavy metals stress than C3
plants, including C3 monocots. The reason is probably two-step carboxylation:
the first step is PEP-carboxylase dependent and sensitive to stress conditions, but
as long as this carboxylation occurs, the Rubisco-dependent secondary carboxylation is maintained by the usual CO2-concentrating procedure, resulting in efficient
metabolism despite stress conditions.
3
3.1
RESPIRATORY PROCESSES
Photorespiration
It is well known that heavy metals stress may cause a shift in Rubisco activity
from carboxylation to oxygenation (39; for review, see 137). On the other side,
the protective role of photorespiration to the photosynthetic apparatus in environmental stress conditions was also recently reviewed (147). Peroxysomal enzymes
of Phaseolus vulgaris were inhibited by toxic Zn concentrations (133). Interesting
changes in peroxysomal NADP-hydroxypyruvate reductase (HPR1) were recently observed in Phaseolus vulgaris, Secale cereale, and Arabidopsis thaliana
in the presence of Cd. At low Cd concentration an increase in the amount and
activity of this enzyme was observed, while a high Cd concentration resulted in
decreased HPR1 activity (148). For rye plants changes in HPR1 activity were
the smallest, confirming a higher resistance of monocots to heavy metals stress
conditions.
3.2
Respiration
So far much less attention has been paid to effects of heavy metals stress on
respiration than on photosynthesis. Mitochondria are known to be much more
resistant to heavy metals than chloroplasts, and they remain undisturbed even at
Cd high concentrations (149). Nonetheless, it is obvious that respiration may be
affected by heavy metals in both direct and indirect ways. Direct inhibition results
from inhibition of enzyme activities (for review, see 48). Indirect inhibition
comes from decreased gas exchange due to limited stomata conductance (146).
Insufficient production of ATP and NADPH by heavy metalsdamaged chloroplasts causes energy imbalance in plant cells. If the stress is not too strong the
Angiosperms
185
plant tends to adapt by increase in respiration, which was confirmed by detected

increase in some Krebs cycle enzymes content and/or activity in Silene italica
and Glycine max under Cd, Pn, and Ni toxicity (150; for review, see 48).
4
ROLE OF BIOCHEMICAL MEDIATORS IN

DIFFERENTIATION OF METABOLIC RESPONSE
OF PLANTS TO HEAVY METALS STRESS
Several groups of substances, such as Ca2 ions, systemin, salicylic acid (SA),
ethylene, and jasmonians (JAs), act as different stress signals within the plant
organs. These signal intermediates have various effects on metabolic processes
depending on plant species, growth properties, and individual character and
strength of the signal factor (151156).
Copper ions, involved in many oxidoreductive processes, are known as
inducing ethylene synthesis in spinach and Scenedesmus plants (105). A high
level of ethylene, resulting from inhibition of photosystems, can increase senescence processes observed in dicots (spinach and bean) and monocots (wheat)
plants treated with Cu2 at the final growth stages (7072,120) or after a longer
exposure to the metal (83,97). However, in bean plants increased lipoxygenase
activity caused by Cu2 (and also Zn2) ions, accompanied by increased ethylene
production, was observed (157). It indicates that Cu2 can also induce free radical
processes in cell membrane components directly (158162) and in consequence
triggers the JAs signal pathway (126). In many cases JAs stimulate ethylene
biosynthesis (163165); however, its inhibition has also been found in cocklebur
seeds (166). More likely JAs stimulate ethylene synthesis (or increase in ethylene
sensitivity) and simultaneously accelerate senescence processes, particularly after
a wounding stress or in older plants. At present, data from one study indicate
that in rice and Arabidopsis Cd and Cu ions can induce octadecenoic pathway
(Table 1). However, in Rauvolfia serpentinea culture cells this effect was not
observed and the ethylene content was decreased (182). Phaseolus coccineus is
also sensitive to excess Cu or Cd, but this sensitivity depends on the plant growth
stage (119,126). It indicates that Cd and Cu can influence this plant species
through JAs-independent pathway. The induction of ethylene synthesis by Fe2
ions varied in Oryza sativa plants (Table 1). In this case, plants previously exposed to wounding stress showed increased sensitivity of ethylene production to
excess metal (179). Among the investigated metals only Co ions showed inhibited
ethylene synthesis (180,181), whereas Ag affected ethylene action (183). These
results indicate that such factors as growth conditions, stress intensity (186), and
probably plant species may change the ways of heavy metals action.
Many heavy metals may elicit proteins similar to PR (pathogenesis-related)
proteins, which potentially may also be induced by signal mediators such as JAs
or SA. They are Cu, Cd, Zn, Al, Fe, La, Ag, and Ga ions investigated in Triticum
186
Siedlecka et al.
TABLE 1 Heavy Metals Stress and Expression of Signaling Pathways in

Higher Plants.
Physiological effect
[Ca2]c concentration
(Ca-dependent
signalling):
Increased
Decreased
Expression of
stress-responsive gene:
Induced
Not induced
Ethylene content:
Increased
Decreased
Metal
Al
Al
Cu, Al, Cd, Co

Cd
Cu, Cd, Zn, Al,
Fe, Zn, Ga, La
Ag, Pb, Cu, Cd
Cu, Cd, Zn
Cu
Cu
Cu, Zn
Fe2
Co
Cd, Cu
Ag
Octadecenoic pathway:
Induced
Not induced
Cd, Cu
Cu
Cd, Cu
Plant
Refs.
Hordeum vulgare
Triticum aestivum
Amaranthus tricolor
Triticum aestivum
Tobacco cells
167
168
169
170
171
Hordeum vulgare
Datura inoxa
Triticum aestivum
172
173
174
175
176
177
178
Lupinus luteus
Mimulus guttatus
Glycine max
Spinacia oleracea
Phaseolus vulgaris
Oryza sativa
Cucumis sativus
Nicotiana tabacum
Rauvolfia serpentina
Cucumis sativus, Lycopersicon aesculentum
105
157
179
180
181
182
183
Arabidopsis thaliana
Oryza sativa
Rauvolfia serpentinea
184
185
182
Angiosperms
187
TABLE 2 Relationships Between Some Plant Hormones and Heavy

Metals Toxicity
Plant hormone
GA3
ABA
Auxin
Effect
Plant
Ref.
Partially reverses the effect of

Cd or Ni
Enhances plant growth inhibition by Cd or Ni; does not affect the influence of Al on
growth
Similarly to Cd, Cu, and Ag, induces accumulation of the
same heat shocklike mRNA
class
Oryza sativa
192
Oryza sativa
192,193
Glycine max
194
aestivum (174,175,187), Datura innoxia (173), and Lupinus luteus (176). However, in Mimulus guttatus and Glycine max, Cu, Cd, and Zn did not induce such
a phenomenon (177,178). Such differences may be connected with plant species
or with methodological incoherence, and require further studies.
The cytoplasmic free Ca2 ([Ca2]c) is probably related to Ca-dependent
signal transduction pathway. Aluminum ions, a very potent plant growth inhibitor, affected this pathway when compared with heavy metals (171) (Table 1).
Depending on the plant species (Table 1), external Ca content in the medium,
as well as plant organs (167), Al ions affected cell metabolism, cytoskeleton
structure, cytoplasmic pH through an increase or decrease in [Ca2]c, and in consequence inhibited growth processes (188,189). Some heavy metals also strongly
affected Ca content in the plant tissues (67,119122,190,191).
Minor data about heavy metalsplant hormone interactions, except those
of ethylene, are summarized in Table 2. They indicate that so far only in the case
of ABA (in many cases similar in its action to JAs) and auxin has the response
of the plant hormones to heavy metals stress been shown.
5
5.1
BIODIVERSITY AND OXIDATIVE STRESS

Reactive Oxygen Species
Oxygen is evolved during photosynthesis and its consumption, more than 85%
used by the cell, occurs in mitochondrial respiration. Molecular oxygen in its
ground state is unreactive, but it can be reduced by four electrons to H2O. During
O2 reduction, intermediates such as superoxide radical (O2 ), hydrogen peroxide
(H2O2), and hydroxyl radical (OH), known as reactive oxygen species (ROS),
188
Siedlecka et al.
can be generated. Singlet oxygen (1O2) is another kind of ROS, which is formed
due to excitation energy transfer from chlorophyll to O2. ROS are capable of
reacting at the site of their generation (or some of them can diffuse to another
part of the cell) with all cellular compounds, causing lipid peroxidation, protein
damages, and DNA mutation, forming chain reactions of free radicals. Generation
of free radicals leads to peroxidative damage of membranes and often to irrepairable metabolic dysfunction and cell death. To prevent such situations, an active
antioxidant system is present in plant cells, that keeps ROS formation under control. Antioxidant system includes such enzymes as catalase (CAT), peroxidases
(POXs), superoxide dismutases (SODs), glutathione reductase (RG), and low molecular nonenzymatic compounds. They act as scavengers, first of all O2 , H2O2,
and 1O2 to prevent generation of the most toxic ROS, i.e., the hydroxyl radical
(OH). ROS are also formed in higher plants treated with heavy metals, but unfortunately this has been shown only in a few papers, so that it is difficult to discuss
the biodiversity in ROS formation under this kind of stress. Some experiments
were carried out on isolated chloroplasts or detached plant organs using the infiltration technique, but only a few were done on heavy metals treatment plants
growing in nutrient solutions. Yruela et al. (195) showed in vitro OH formation in
chloroplasts membranes of Beta vulgaris treated with Cu. Lead (only at sublethal
concentrations) caused an increase in the total pool of free radicals in lupine roots
(199). An increase of H2O2 was found in leaves Pisum sativum infiltrated by Cd
(197) and in leaves of Phaseolus vulgaris treated with Zn (198). A short-term
treatment of potato tuber discs with CdCl2 increased the concentrations of H2O2
and O2 . Moreover, in the susceptible potato species the increased H2O2 level
lasted longer than in the tolerant one (199). Age-dependent level of ROS was
observed in primary leaves of Phaseolus coccineus grown in the nutrient solution
containing Cd. The increase in O2 level was observed in Cd-treated young plants
(Skorzynska-Polit et al., unpublished).
5.2
5.2.1
Enzymes of Oxidative Stress

Superoxide Dismutase
Superoxide radical can spontaneously dismute to H2O2 and O2 in reaction accelerated by SOD. Superoxide dismutases are the family of enzymes containing different metals in their active sites. SODs containing Cu and Zn (Cu/Zn-SOD) are
generally found in cytosol of eukaryotic cells and chloroplasts, while those containing Mn (Mn-SOD) are found in the matrix of mitochondria and in prokaryotes
(200). Iron-containing SODs (Fe-SOD) are mainly present in prokaryotic organisms and some eukaryotic algae. They have been also found in some families of
higher plants: Gingkoaceae, Nymphaceae, Cruciferae, Aceraceae, in Phaseolus
vulgaris and Lycopersicon esculentum (201203). In roots, increase in total activity of SODs upon different heavy metals stress, such as Cu, Al, Pb, and Cu, was
Angiosperms
189
generally independent of plant species (soybean, lupin, Pisum sativum, Silene

cucubalus) (196,204207). Cadmium and copper caused an increase in total SOD
activity in leaves of Pisum sativum and Phaseolus coccineus (197, 208, Skorzynska-Polit et al., unpublished). Both metals and Zn decreased or had no influence
on SOD activity in seedlings and leaves of Phaseolus vulgaris (62,198,209).
Lower activity under Zn stress resulted from inhibition of Mn-SOD and/or FeSOD rather than from Cu/Zn-SOD (209). In Mn-tolerant genotype of Phaseolus
vulgaris excess Mn caused increased leaf SOD activity, lower than in the susceptible one (210). Its excess also induced Mn-SOD activity in soybean and Pisum
sativum (211213). Palma et al. (208) showed an increase in peroxisomal MnSOD activity in Cu-tolerant Pisum sativum, suggesting that this dismutase may
function in the molecular mechanisms of plant tolerance to Cu. Copper excess
caused increase in Cu/Zn-SOD activity in seedlings of Triticum vulgaris, especially in the thylakoid-bound type of the enzyme (214).
5.2.2
Catalases
Despite the heavy metals applied (Cd, Cu, Zn, Pb, or Alat higher concentrations), catalase (CAT) activity always decreased in roots of Phaseolus vulgaris,
Lycopersicon esculentum, soybean, and lupin, although in roots of Allium cepa
the enzyme activity was enhanced under Hg treatment (196,215218). Somashekaraiah et al. (62) observed a decrease in CAT activity after 4 or 6 days of
Cd action during germination of seedlings of Phaseolus vulgaris; however, in the
seedlings of Phaseolus aureus (219) or in young plants of Phaseolus coccineus
(Skorzynska-Polit et al., unpublished) treated with Cd, an increase in CAT activity was reported. An increase in CAT activity was also measured in peroxisomes
of Cu-treated Pisum sativum (208) and in leaves of Nicotiana plumbaginfolim
under Fe excess (222). Short-term stimulation of CAT activity was also measured
in leaves of Cu-treated Phaseolus vulgaris (217). Toxic concentrations of Mn
and Zn increased CAT activity in celluler extracts from rice plants and in leaves
of Pisum sativum (98,213).
5.2.3
Peroxidases
Peroxidases (POXs) have a much higher affinity to H2O2 than CATs (223). In
higher plants, different isoenzymes of POX 3 can be distinguished. They participate in scavenging of H2O2, cell wall biosynthesis, regulation of IAA (indole3-acetic acid) degradation, synthesis of ethylene, and plant defense against
pathogens. According to van Assche and Clijsters (48 and references therein),
peroxidase induction is a general response of higher plants to uptake of heavy
metals. The appearance of new isoperoxidases was observed when Oryza sativa
was treated with Zn, Cu, and Hg. Zinc-dependent induction of two anionic POXs
was observed in leaves but not in roots of Phaseolus vulgaris. However, Cu
treatment of plants led to the reverse situation when Cd induced these isozymes
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in roots and leaves (48). An increase in nonspecific activity of POX was measured
in lupin roots treated with Pb (196). Induction of guaiacol-dependent peroxidase
was also observed in roots of Cu-treated Lycopersicon esculentum (217) and in
soybean treated with Al (216). The appearance of two new anionic isozymes was
observed in stems of Cd- or Zn-treated Phaseolus vulgaris where the induction
of one of these iso-guaiacol POXs was Zn-specific (215). In seedlings of Phaseolus aureus (219), in leaves of Pisum sativum (197), and in both young and older
plants of Phaseolus coccineus (Skorzynska-Polit et al. unpublished), an increase
in guaiacol-dependent POX was measured upon Cd stress. Chaoui et al. (215),
in contrast to the results reported by van Assche and Clijsters (48), did not observe
changes in POX activity in leaves of Cd-treated Phaseolus vulgaris. This discrepancy seems to be a result of the duration of Cd action and/or its various doses
used in experiments.
5.2.3.1 Ascorbate Peroxidase Plants also have ascorbate-specific POX,
which acts in cytosol and chloroplasts. This enzyme participates mainly in the
ascorbate-glutathione cycle and plays a key role in scavenging H2O2 in chloroplasts. The activity of ascorbate peroxidase (POA) did not change in roots of
Cd- and Zn-treated Phaseolus vulgaris (215), Cu-treated Lycopersicon esculentum (217), or decreased in lupin at higher concentrations of Pb (196). However,
application of a lower Cu dose caused an increase in POA activity in roots of
Phaseolus vulgaris (224). Dependence on metal concentration seems to be the
case in Zn-treated Phaseolus vulgaris, where a lower concentration did not affect
POA activity (215); however, at a higher metal content the activity increased
(198). There have been several other reports of diverse responses of POA to
heavy metals stress depending on plant species, specific organ, metal (Cd, Cu,
Mn, Fe) and its concentration, plant sensitivity to metal, and experimental conditions (98,214,215,217,219222).
5.3
Ascorbate/Glutathione Cycle
Ascorbic acid (AA) can be the electron donor to reduce H2O2 to water in reactions
catalyzed by POA and, as mentioned earlier, participates in the ascorbateglutathione cycle, in addition to such enzymes as glutathione reductase (GR),
dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase
(MDHAR). The influence of heavy metals on this cycle has been very poorly
explored. In roots of Phaseolus vulgaris treated with excess Cu total ascorbic
dehydroascorbic acid (AA DHA) and reduced oxidized glutathione (GSH
GSSG) increased along with increase in all enzymes activity (MDHAR, DHAR,
GR) (232). In leaves of sunflower treated with Cu, Cd, or Fe, decreased DHAR
and GR activities and GSH content were observed (221). In Fe-treated Nicotiana
plumbaginifolia a decrease in AA and GSH and an increase in DHA and GSSG
was measured with no total increase in AA DHA and GSH GSSG (222).
Angiosperms
191
Ascorbate and GSH are themselves very good nonenzymatic scavengers.

They react easily with free radicals (ROS, free radicals of proteins, lipids, and
other compounds), and as a result of such reactions, the damage that occurred
in different cell compounds of the cell may be repaired. Total pool of AA in
tolerant type of Phaseolus vulgaris was maintained on a higher level under excess
Mn stress than in the sensitive one (210). Yamaguchi et al. (225) showed a protective effect of endogenous GSH on Al toxicity in suspension-cultured tobacco
cells. They suggested that GSH protects cells from oxidative membrane damage
both by direct consumption of GSH and its oxidation.
In all cases described in this chapter, symptoms of oxidative stress were
observed in plants exposed to heavy metals stress. Their responses depended on
plant species, age, tolerance or sensitivity to metals, time of exposure to stress,
and concentration of heavy metal. The general condition of the photosynthetic
apparatus seems to be related to ROS formation and, in consequence, competence
and efficiency of the antioxidant system.
6
RESPONSES OF PLANTS TO HEAVY METALS:

AVOIDANCE AND TOLERANCE MECHANISMS
Plants developed two types of strategies in response to heavy metals in the environment, i.e., stress avoidance and stress tolerance. Stress avoidance relies on
reducing or preventing metal uptake by plants or its quick exclusion from protoplast by the excretion or sequestration mechanisms. Stress tolerance includes the
mechanisms that deal with toxic metal ions present inside cells.
6.1
Avoidance
One of the strategies of stress avoidance is metal excretion from protoplast. An

active excretion of metals (Fe, Cu, Zn) from leaf cells can occur via multicellular
salt glands (Armeria maritima), hydathodes (Minuartia verna), or ectodesmata
(Silene vulgaris) (226). Metabolism-dependent exclusion of Al from the root meristem of Al-tolerant Triticum aestivum was also shown (227). Restricted ion uptake can also result from plasma membrane lipids and sterols alterations, which
is often described in bacteria, cyanobacteria, and algae. In higher plants this phenomenon does not play an important role, although it was shown in Cu-tolerant
Silene cucubalus (66). A mechanical barrier against Al ions influx to protoplast
is callose (1,3--glucan), deposited in the outer cortical cell layers of root tips.
Its presence was noted among other factors in Glycine max, Zea mays, and Triticum vulgare (228). The cell wall is a mechanical and a chemical barrier against
ion uptake as well as a compartment for deposition of ions that penetrated into
the protoplast. Depending on the plant species and the kind of metal, it can retain
from a small amount to more than 90% of the total metal taken up. Metals can be
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electrostatically bound to carboxylic functions of pectins and to charged groups

of wall proteins, or even more complex chemical bonds can occur. Moreover,
amorphous metal precipitates as phosphate, carbonate, and silicate deposited between cellulose micelles have been reported. The affinity of metal ions to polygalacturonic acid decreases in the order Pb Cr Cu Ca Zn (238). A similar
reduction of uptake of Al and elements chemically similar to Al is caused out
by mucilage, consisting mainly of polysaccharides and polyuronic acids (pectins)
deposited on the root surface of, for example, Vigna unguiculata (230). Diminution of toxic ions uptake by plants is associated with rhizosphere modification
by pH changes induced by plants or redox barrier in the plasma membrane of
root cells. The redox barrier is formed as a result of oxidizing activity of plant
roots and associated microorganisms. A number of wetland plants (Oryza sativa
L., Spartina alterniflora, Phragmites communis, Typha latifolia) form so-called
iron plaque on their roots by oxidizing Fe(II) to less soluble and less toxic Fe(III).
A similar mechanism is present for Mn(II) immobilization in the rhizosphere
(231). Mycorrhizae fungi can also be a mechanical and chemical barrier for heavy
metal penetrating into plant roots (232). Plants also excrete metal-chelating ligands to the environment. Excretion of malic and citric acids to the rhizosphere
by Helianthus annuus L., Triticum aestivum L., or polypeptides by Triticum aestivum L. in the presence of Al was observed (233235). In Helianthus annuus L.,
excretion of malate and citrate was found in response to Zn (233). Heavy metals
can be also deposited in idioblasts (226), hairs (236), and similar structures by
which the heavy metal load can be withdrawn from plant metabolism. The phenomenon of seasonal intensive metal translocation to leaves and other plant parts
and subsequent exclusion of metal from the organism by shedding these organs
is also known. It occurs, for example, in Zn excluders Anthyllis vulneraria L.
and Biscutella laevigate L., in Cu excluder Becium homblei (229,237), and in
Ni-tolerant Indigofera setiflora (238).
6.2
Tolerance
The essence of intracellular mechanisms of tolerance is free heavy metal ions

detoxification by their chelation in cytosol or storage in the vacuole. In most
plant cells the vacuole comprises more than 8090% of the cell volume and acts
as a central storage compartment for ions. Some amino acids may also chelate
metals in cytosol. The major ligands for Cu are asparagine (Asn) and histidine
(His), and for Ni histidine, glutamine (Gln), and proline (Pro). Apart from their
function in detoxification attention is paid to their role in metal transport in the
xylem and phloem. Stability of these complexes can prevent precipitation of
transported metals or their adsorption to cell walls (230,240). Organic acids are
very effective chelators for toxic metal ions in cytosol and vacuole. The most
important role in Zn tolerance is attributed to malic acid. According to the zincmalate shuttle hypothesis of Mathys (241), this organic acid is a cytosolic ligand
Angiosperms
193
for Zn, participating in the metal transport to the vacuole. In the vacuole, Znmalate complex is dissociated and malate is retransported into cytosol, whereas
vacuolar Zn is bound to ligands more potent at low pH, such as citrate or oxalate,
and also to phytate, anthocyanidines, or mustard oil glucosides, or is stored as
Zn crystals. Zn-tolerance is also associated with enhanced production of citrate
in the grass Deschampsia caespitosa (242). Both malate and citrate as well as
malonate play a role in Ni detoxification (229,239). Organic acids are not a common means for Cu, Zn, and Pb detoxification and storage. An intensive synthesis
and accumulation of high amounts of organic acids is not specific for the kind
of metal or the plant species, and does not always correspond to tolerance to a
given metal. For example, Zn-tolerant Agrostis capillaries and Silene vulgaris
exhibit a high level of malic acid but are only merely Ni-tolerant. On the other
hand, Ni-tolerant Alyssum bertolonii, rich in malate, is not Zn-tolerant (229).
Another form of metal storage in the vacuole, especially of Zn but also of Cd,
is chelation by phytin or phytate, a mixed salt of myoinositol hexaphosphoric
acid or phytic acid. Metal complexes with phytin are found in seeds, cotyledons,
leaves, and roots as dispersed in the protein matrix or aggregates, called globoids,
and in immature cells as sheet-like deposits composed mainly of Cd and S
(229,243).
Studies on the role of proteins in heavy metal binding in plants were initiated per analogiam to such complexes, called metallothioneins (MTs) in animals.
The first report about the presence of MTs in plants concerned Cu-MT in roots
of Agrostis gigantea (244). Application of procedures appropriate for isolation
and purification of such anionic complexes revealed that their structure and properties differ considerably from those of animal metallothioneins. That is why
metal-protein complexes isolated from different plant species were no longer
called MT but rather MT-like (245). In 1985, Grill et al. characterized Cd-binding
peptides in Rauwolfia serpentina and named them phytochelatins (PCs) (246).
PCs were designed to MTs class III and are defined as atypical nontranslationally
synthesized metal thiolate polypeptides (247). They occur commonly in the whole
plant kingdom (248) with the structure based on -Glu-Cys units repeated 211
times. The presence and kind of carboxy terminal amino acid were the criterion
of distinguishing five families of -Glu-Cys peptides (240):
Phytochelatins-(-Glu-Cys)n-Gly, found in all plant species studied
Iso-PC (Ala)-(-Glu-Cys)n-Ala, found in some species of Fabales (250)
Iso-PC (Ser)-(-Glu-Cys)n-Ser, found in Poaceae (251)
Iso-PC (Glu)-(-Glu-Cys)n-Glu, found in Cd-treated maize roots (252)
desGly-PC-(-Glu-Cys)n, found in species of Poaceae (251,252), Silene vulgaris and Lycopersicon peruvianum (253)
Phytochelatins [(-Glu-Cys)n-Gly], which are glutathione derivatives, are
the most widespread family responding at the earliest to increased intracellular
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Siedlecka et al.
metal concentration. That is why they are considered to play the main role in
metal detoxification. Other families of -Glu-Cys peptides, although sometimes
accumulated in greater abundance than PCs, play only an auxiliary though undoubtedly important role in the process of metal detoxification (251,254,255).
The -Glu-Cys peptides are synthesized enzymatically by the action of a specific
-Glu-Cys dipeptidyltranspeptidase trivially named phytochelatin synthase. This
enzyme is activated by the presence of free metal ions, especially Cd(II), Ag(I),
Bi(III), Pb(II), Zn(II), Cu(II), Hg(II), and Au(I). No enzyme activation was detected with Al(III), Ca(II), Fe(II), Mg(II), Mn(II), Na(I), or K(I) (256). A model
for metal-dependent phytochelatin synthase function was recently proposed
(257). Conversion of GSH into cadystins, structural analogues of plant phytochelatins, by the action of carboxypeptidase Y in vitro was also reported (258), but
the capacity of metal ions binding by these peptides was not studied.
Different functions for PCs in plant cells were proposed. PCs appear to be
part of the homeostatic system that regulates the availability of Zn and Cu
ions for apo forms of metal-requiring enzymes (259). They can also participate
in assimilatory sulfate reduction as sulfo group acceptors from adenosine-5phosphosulfate sulfotransferase (260). The most important function of PCs, however, seems to be their role in heavy metals detoxification and tolerance. Toxic
metal ions present in cytosol induce rapid synthesis of PCs, binding metals in
nontoxic complexes. It protects metal-sensitive groups such as -SH or histidyl
groups of catalytic or structural proteins. Complexing by PCs is a mechanism
especially important in Cd and Cu detoxification (261), but Pb and Zn, although
they induce the formation of PCs, are not capable of forming stable complexes
with them (253). First of all, efficient transport of metal-PC complexes to the
vacuole plays an important role in plant tolerance to heavy metals. A model for
PCs function in Cd transport to the vacuole was proposed by Vogeli-Lange and
Wagner (262) and further developed by others (236,263). According to this, phytochelatins synthesized in cytosol bind Cd in so-called low molecular weight
(LMW) complexes that are moved across the tonoplast by an ATP-binding cassette-type transporter. Inside the vacuole, more Cd, apo-PCs, and acid-labile sulfide are added to LMW complexes to form more stable sulfide-rich chelates called
high molecular weight (HMW) complexes.
Recently, more attention has been focused on searching and investigating
MTs in plants. The intensity of those studies diminished after PCs characterization in plants (246). Although 64 genes predicting MT-like proteins from a variety
of plants were already found and transcripts of some of them were detected in
all plant organs, only one protein has been isolated and characterized so far (243
and references therein). It is Ec protein isolated from mature wheat embryos and
belonging to MTs class II (i.e., peptides in which the position of the Cys residues
is different from the archetypal mammalian MT and among each other) (247).
Its presumable function is Zn(II) ions homeostasis. Whether this protein and other
Angiosperms
195
FIG. 4 Mechanisms of heavy metal tolerance and avoidance in plants.
MT-II gene products participate in heavy metals detoxification in plants remains

to be examined. However, studies on the expression of plant MT genes in
transgenic bacteria, cyanobacteria, yeast, and some plant species suggest that MT
gene products can affect an increase in metal accumulation (especially Cd and
Cu) in these organisms and enhance their metal tolerance (252 and references
therein). The mechanisms of heavy metal tolerance and avoidance in plants are
summarized in the Figure 4.
6.3
Biodiversity in Adaptation Mechanisms
Plant reaction to a given metal always involves many tolerance mechanisms and
reveals that the predominant mechanism depends on plant species, growth phase
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Siedlecka et al.
or even plant organ or tissue, and the kind of metal, time of its action, concentration, and many other exo- and endogenous factors. However, some general tendencies for metals may be observed.
Zinc is considered to be detoxified mainly by organic acids effectively
transporting it from cytosol into the vacuole where it is stored. For Cd detoxification phytochelatins appear to be the most important, although some data suggest
that the highest accumulation of this metal occurs in cell wall (264). The cell
wall is the main compartment of Pb and sometimes Cu immobilization. An important role in Cu detoxification is also attributed to amino acids and metal-binding
proteins. Such function for Ni is attributed to organic and amino acids, mainly
histidine.
The Poaceae family appears to be one of the most metal tolerant families
in the plant kingdom. Apart from this, some grass species (e.g., Holcus lanatus,
Agrostis capillaries, Festuca rubra, Deschampsia caespitosa) can very quickly
adapt to metalliferous soils (265). Many tolerant ecotypes are also found in
Caryophyllaceae, Brassicaceae (see Chapter 8), and Asteraceae. These families
are especially widespread on metal-enriched soils of temperate zone, whereas in
the tropics a much greater range of families is represented, including Fabaceae
and Lamiaceae. However, it is interesting that some common and large plant
families, such as Ranunculaceae, Leguminosae, Rosaceae, and Apiaceae, have
not apparently evolved metal tolerance and only rarely occur naturally in metalenriched soils (265,266).
The pattern of metal uptake, transport, and accumulation differs in each
kind of metal or in various plant species and could be related to different mechanisms of metal tolerance at the whole-plant level. Three types of plant-soil relationships have been identified (267):
Excluder strategy. Metal concentration in shoots is maintained at a constant
low level until a certain critical soil concentration above which unrestricted metal transport to the shoots is observed. The shoot/root concentration ratio is much less than 1.
Indicator strategy. Metal uptake and transport to shoots are regulated or
passive uptake occurs so that the internal concentration reflects the external level. The shoot/soil concentration ratio is near 1.
Accumulator strategy. Metal is actively concentrated within plant over the
full range of soil concentrations. Analysis of plant organs suggests a
general tendency for accumulators to translocate most metal taken up
from roots to shoots, and the shoot/root concentration ratio is greater
than 1.
The term hyperaccumulator was first used by Brooks et al. (268) for
plants accumulating more than 1000 g/g (0.1%) Ni in their shoot dry mass when
growing in the natural habitats. However, the threshold of hyperaccumulation
Angiosperms
197
varies considerably for different metals and amounts over 10,000 mg Zn and Mn
(1%); 1000 mg Ni, Cu, Co, Cr, and Pb (0.1%); 100 mg Cd (0.01%); and 1 mg
Au (0.0001%) per 1 kg of plant shoot dry mass (238). Metal concentrations in
some hyperaccumulators can be as high as 0.02% for Cd to 5% for Ni, Mn, and
Zn (269). Unquestionably, the most impressive accumulator is an endemic tree
Sebertia acuminata from New Caledonia in which 25.74% Ni was found in dried
latex (239). It causes blue coloration of the tree, which is why its local name is
seve bleue. Nowadays, more than 400 hyperaccumulators are known, including about 300 accumulators for Ni, 26 for Co, 24 for Cu, 19 for Se, 16 for Zn,
11 for Mn, 45 for Pb, 1 for Tl, and 1 for Cd (270). The extensive lists of
hyperaccumulators were given by Baker and Brooks (238), Brooks (271), and
Baker and Walker (237). In the temperate zone most hyperaccumulators belong
to Brassicaceae (see Chapter 8); in the tropics to Euphorbiaceae.
Hyperaccumulators can thrive in extremely hostile edaphic environments
that would kill many other species. That is why they have been the subject of
several investigations involving, among other things, their utility in detection of
environmental pollution (see below), mineral prospecting (272), phytoarchaeology (using plants to search and investigate sites of ancient human activity) (273),
phytoremediation (using plants to environment decontamination) (269), and phytomining (using plants to explore low-grade metal ores or highly mineralized
soils) (270).
7
BIOMONITORING
Plants have been more and more widely used in the detection of heavy metals
impact on the air, soil, and water caused by both natural processes and human
activity. Assessing the environmental quality using plants as well as other living
organisms is less expensive and often more efficient than using physical and
chemical analyses.
7.1
Metallophytes
Due to developing different tolerance mechanisms (see above), some plants have
adapted to the growth on metalliferous sites like mineralized soils: serpentine
(rich in Ni, Cr, Mn, Mg, Co) or calamine (rich in Zn and Cd) soils, mineral
deposits and outcrops, ancient and contemporary mining and smelting areas. Such
plants are called metallophytes (274276). Facultative metallophytes can grow
both on metalliferous and nonmetalliferous soils. Examples of such plants are
Anthoxanthum odoratum, Agrostis canina, A. capillaries, A. stolonifera,
Deschampsia caespitosa, D. flexuosa, Festuca rubra, F. ovina, Holcus lanatus,
and Silene vulgaris (274). Obligatory (or strict) metallophytes are strictly confined to metal-enriched soils, e.g., Haumaniastrum robertii, H. katangense, Viola
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Siedlecka et al.
calaminaria. That is probably due to their sensitivity to fungal attacks and inability to withstand interspecific competition on normal soils (276).
7.2
Indicators
Plants growing on heavy metalsenriched soils differ from those from surrounding sites. The vegetation structure and diversity is reduced. Usually there
are no trees or the trees are stunted. Many xeromorphic features, dwarfed growth
forms, decoloration of leaves (chlorosis, anthocyanous purple coloration), as well
as other morphological changes can be observed in these plants. Endemic or
disjunct species and edaphic ecotypes are of frequent occurrence (274,277,278).
Many species are hyperaccumulators. That is why such plants can be used in
biological monitoring of heavy metals pollution and also in mineral prospecting
and phytoarchaelogy.
In general, indicators can be divided into two groupsgeobotanical and
biogeochemical indicators. Biogeochemical indicators are used to detect mineralization by means of chemical analysis of the metal content in their tissues (277).
Bioaccumulation studies seem most powerful in large-scale surveys when the
expected date and localization of the pollution are unknown. They are the only
possibility for detecting a chemical if its concentration in the environment is
below the sensitivity level of the equipment (279) or is too low to cause characteristic symptoms of acute metal toxicity in plants (280). Geobotanical indicators
are species that achieve their greatest abundance, occur exclusively or preferentially, or have a distinctive appearance (see above) or phenological pattern (delayed leaf flush, earlier flowering, premature senescence) on heavy metals
contaminated soils (277). A number of geobotanical and biogeochemical indicators have been reported so far (272, 276, 281, and references therein). Classic
examples of geobotanical indicators are copper flowers of Shaba province,
IndiaBecium homblei and Haumaniastrum katangense. Both grow only on
soils rich in Cu ( 0.01%) and used to be successfully used for prospecting for Cu
deposits (275,276,282). Other copper flowers, or soil Cu indicators, include
Haumaniastrum robertii, Bulbostylis pseudoperennis, Arthraxon quartinianus,
Aeollanthus subacaulis var. linearis from South Central Africa (276,282), Polycorpaea spirostylis from Australia (283), Elsholtzia haichowensis from China
(273). Silene cucubalus and Minuartia verna have colonized soils enriched by a
variety of heavy metals, including Zn, Pb, Cd, and Ni in Europe (281). Plants
used as geobotanical indicators represent all three strategies of plant-soil relationships, i.e., excluder, indicator, and accumulator, whereas biogeochemical indicators belong mainly to accumulators and indicators.
The usefulness of vegetation in predicting the concentration of the elements
in the soil decreases in the order herbs shrubs trees (272). It can probably
be due to different radius of the root systems and distance for metal translocation
between roots and leaves of herbs and trees. Plant organs most easily collected
Angiosperms
199
and analyzed for heavy metals content are leaves and also twigs or shoots,
whereas roots, bark, and wood are difficult to collect. The pattern of metal accumulation in leaves and twigs varies depending on the metal. Aery and Tiagi (272)
reported that leaves in the case of Zn and stems in the case of Pb, Cd, and Cu
accumulated more of these metals than the others.
If a plant occurring on metalliferous soils can be shown to be a hyperaccumulator of some element, although it is not confined to such substrates, the biogeochemical method of prospecting soil metals may be applicable. On the other
hand, if a hyperaccumulator plant is also endemic to metalliferous soil, either
absolutely or regionally, the geobotanical method of prospecting can be used.
Biomonitoring of environmental pollution with heavy metalssensitive plants can
be also used. Morphological, physiological, and biochemical changes in these
plants can give some information about poor environmental condition. Such a
phenomenon can be easily observed in water ecosystems using, says, Lemna
minor (284).
7.3
Biomonitoring in Experiments
Metal biomonitoring in the environment can be carried out not only studying
individuals or communities in situ or samples taken from a natural population.
Biological material may be also used in experiments (279). Some test plants of
known physiology and reaction to heavy metals can be grown on polluted soil,
both in the natural environment and under control conditions, to assess its toxicity. For instance, Phaseolus vulgaris L. cv. Limburgse vroege was successfully
used to estimate the phytotoxicity of such soils. On the basis of its morphological
(shoot length, leaf area, root weight) and physiological changes (the capacity of
enzymes: peroxidase, POX; glutamate dehydrogenase, GDH; isocitrate dehydrogenase, ICDH; malic enzyme, ME), four classes of soil phytotoxicity were distinguished (280). Another test plant, commonly used to indicate Pb and other metals,
e.g., Zn, Cd, Cu, Cr, Co, Hg is Lolium multiflorum var. italicum. Plants grown
under standard conditions in unpolluted environment were exposed for 14 days
to the air of the studied area and then are analyzed for concentrations of the
individual elements (279). Solidago canadensis is used as an indicator of lead.
Further terrestrial plants proposed for use are Anthriscus cerefolium, Medicago
sativa, Raphanus sativus, Solanum lycopersicum, Volerinella locusta and Zea
mays (279). In laboratory studies, the most widely used parameter to measure
heavy metals toxicity/tolerance is root growth. This is because of the simplicity
of this test and because this organ usually responds most rapidly to the metal
and the response is the consequence of a direct effect of the metal on the root
itself. Different variants of rooting techniques have been proposed, like IT
(index of tolerancethe percentage of root elongation in metal solution compared with control solution), EC [the lowest effect concentrationthe metal concentration at which root elongation is inhibited in 50% (EC50) or completely
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Siedlecka et al.
(EC100)] (229,274). Other parameters often used in metal tolerance tests include
shoot elongation, water content, chlorophyll content, enzymes level and capacity,
and many others (241,254,280,283).
Adaptation to growth on heavy metal-enriched sites has some costs. It is
accounted for by the fact that tolerant individuals, colonizing metal mine waste
soils, have reduced fitness when grown on uncontaminated soils. It indicates the
selection against metal tolerance on unpolluted soils. In general, tolerant mine
plants are smaller and have lower biomass production and lower flowering in
comparison with their nontolerant counterparts. Two hypotheses can explain how
a cost of metal tolerance could be manifested. The trade-off hypothesis suggests that the energy or resources used in the tolerance mechanism are diverted
from other processes, e.g., growth and biomass production (285). In the metal
requirement hypothesis tolerant plants are suggested to have an enhanced metal
requirement, since tolerant individuals display a stimulatory response in growth
and enzyme activity to essential metals (Cu, Zn, Ni), to which they are tolerant.
On uncontaminated soil they are less efficient in the uptake, distribution, or utilization of these metals and their deficiency may occur. This argument forms the
basis in the metal requirement hypothesis for explanation of the lower fitness of
tolerant individuals when grown in normal soil (286). However, slow growth and
other features of plants growing on metalliferous soils must not necessarily be
a symptom of costs of tolerance to heavy metals. They may also result from
adaptation to other stressful conditions in such environments, such as nutrient
deficiency or drought (237,274).
The survey presented here does not deal with all possible mechanisms of
different heavy metals detoxification or tolerance in plants but rather with those
reported most often. Among the tolerance mechanisms discussed in this chapter,
changes in the plant metabolism to avoid metal toxicity inside the cell were not
involved. These changes include activation of alternative metabolic pathways,
increased enzyme synthesis, and changes in enzyme structures (274). Many
taxon-specific responses to each heavy metal discussed have not been mentioned,
but a great diversity in plant response to heavy metals has been demonstrated.
ACKNOWLEDGMENT
The financial assistance of the Polish Committee for Scientific Research (Grant
6P04C.064.15) is gratefully acknowledged.
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8
Brassicaceae
Luigi Sanita` di Toppi and Maria Augusta Favali
University of Parma, Parma, Italy
Roberto Gabbrielli
University of Florence, Florence, Italy
Patrizia Gremigni
University of Western Australia, Perth, Western Australia, Australia
THE BRASSICACEAE FAMILY
The Brassicaceae ( Cruciferae) are a typical example of a particularly homogeneous plant family, which comprises about 170 species already recognized by
Linnaeus and termed Tetradynamia. The species belonging to this family are
linked by a number of distinctive features, including the fairly uniform floral
structure: the calyx and the corolla consist of four sepals (K2 2) and four petals
(C4), respectively, which are arranged as a cross (hence the name Cruciferae).
The stamens, four long and two short ones, form the androecium (A2 4), while
the gynoecium is formed by two carpels (G2) and a superior ovary. The fruit,
which also has an uniform structure, is a capsule (siliqua or silicle, depending
on the length/breadth ratio) divided into two locula by a spurious septum which
is not present in all species. Although the basic fruit structure is fairly uniform,
219
Sanita` di Toppi et al.
220
it is characterized by a wide range of modifications that are relevant for classification at any level.
The modern classification of the Brassicaceae is based on the revision by
Schulz (1936) (1), who greatly contributed to the knowledge of this family in
his work Cruciferae, published one year after his death. Janchen (1942) (2)
published a rather thorough revision of Schulzs system, in particular he reduced
the number of the familys tribes from 19 to 15. Nowadays the Brassicaceae
family comprises 13 tribes.
The Brassicaceae have a wide distribution and are present on almost every
continent, mainly in the extratropical regions of the boreal hemisphere. In particular, the Alysseae and the Brassiceae tribes occur in several habitats, such as sunny
and open places, walls, rock clefts, barren hills, fields, meadows, ruderal environments, river banks, and so forth. A number of Brassicaceae species occur in
habitats with high anthropogenic pressure, and some species are of ornamental
and horticultural importance.
1.1
Alyssum L.
The genus Alyssum L. is widespread in many regions of Europe and Asia. In

Turkey, particularly in Anatolia, it achieves a high degree of diversity expressed
by the greatest number of species. For the genus Alyssum, Dudley (1964) (3)
reported six sections: Meniocus (Desv.), Psilomena (Meyer), Alyssum, Gamosepalum (Hausskn.), Tetradenia (Spach.), and Odontarrhena (Meyer). Dudley also
reported a putative A. americanum (probably corresponding to A. obovatum that
occurs in Siberia and in Turkey), which is indicated to be the only indigenous
American representative of this genus.
The genus Alyssum represents an interesting field of study for many botanists (4,5). However, at present the fame of Alyssum is not derived only from its
taxonomic background but also from the ability of some species to survive on
ultramafic substrates and to accumulate very high Ni concentrations. Already in
the 16th century, Andrea Cesalpino (1583) (6), a doctor and botanist from Arezzo
(Italy), described a Lunaria quarta alias Alysson as growing only over the
black stones of Montauto, an ofiolitic outcrop of the Upper Tiber Valley in
Tuscany. Later, Pier Antonio Micheli mentioned this plant as A. perenne in a
manuscript on the flora near Florence. It was finally identified and classified as
A. bertolonii by Desveaux (1814) (7). Then Amidei in the third meeting of the
Italian Scientists (Florence 1841) (8) stressed the close relationship between some
plant species and serpentine soils in Tuscany. However, in that time and up to
the first half of the 20th century, the interest in some Alyssum species was more
general. Many botanists were attracted by the peculiarity of the serpentine vegetation and its morphological alterations (i.e., dwarfism, glaucousness, etc.), mainly
Brassicaceae
221
attributed to the extreme edaphic conditions to which these plants were adapted
(9,10).
1.2
Brassica juncea
Brassica species have been cultivated for a long time and nowadays a number
of varieties are widely used in various continents (11). Thus, as a result of this
extensive spreading of mustard species throughout the world, there is a considerable variability for both morphological and biochemical traits (12).
In particular, Brassica juncea (L.) Coss. and Czern. (Indian mustard)
(Fig. 1) is an important crop species mainly in eastern Europe, India, Pakistan,
China, and Japan. B. juncea has a great variability in the leaf morphology, and,
consequently, it includes several cultivars. Some varieties are important as vegetables and for production of dressing oils. Most authors consider B. juncea to be
of Asiatic origin, with centers of main biodiversity in China and perhaps in India.
Morinaga (1934) (13) postulated different interrelation schemes among Brassica
species and suggested that B. juncea has originated as a natural amphiploid hybrid
(aploid chromosome number equal to 18) between B. nigra (L.) Kock (aploid
chromosome number equal to 8) and other species with aploid chromosome number equal to 10, such as B. campestris L., B. rapa L., B. chinensis L., B. pekinensis
Rupr., and B. japonica Sieb. Artificial synthesis of pseudo-Juncea forms, very
close to the natural species, has been obtained by Frandsen (1943) (14) and by
Ramanujam and Srinivaschar (1943) (15).
1.3
Thlaspi L.
The name Thlaspi was firstly used by Dioscorides Anarzabeus in his De medica
materia (16,17) to describe a plant with specially shaped fruits (from the Greek
thlao, to flatten, and aspis, shield). The genus Thlaspi includes about 75 herbaceous species with 30 annuals and 45 perennials (1,18). Thlaspi is primarily defined by fruit characters: fruit an angustiseptate silicule, the valves keeled and
usually winged; locules containing 26, rarely 110, seeds (19), but the taxonomic classification of the genus is difficult and controversial (20). In his radical
revision of the taxonomy of Thlaspi sensu lato (1,21), Meyer (17) analyzed the
anatomy of the seed testa in taxa from Europe, Africa, and the Middle East. He
split the genus into 12 segregate genera because the differences among them
were too large to include all of them in one single, broad genus. More recently,
Mummenhoff and colleagues used molecular techniques to study the controversial phylogenetic aspects of Thlaspi (19,20). The isoelectrofocusing (IEF) analysis of the enzyme Rubisco was applied to 14 species of Thlaspi (20). Preliminary
results confirmed the existence of three major groups in the genus Thlaspi
Thlaspi sensu strictu (s. str.), Microthlaspi, and Noccaea (20)consistent with
222
FIG. 1 (a) Thlaspi caerulescens; (b) Brassica juncea
Brassicaceae
223
three of the genera previously defined by Meyer (17). Thlaspi s. str. includes
only annual species, whereas Microthlaspi comprises both annual and perennial
species and supports Meyers results. Noccaea shows great heterogeneity and
does not coincide with any previous systematic classification based on morphological and anatomical characters (20).
Zunk et al. (19) investigated the genetic variation among 28 populations
from metalliferous (subsp. caerulescens) and nonmetalliferous soils (subsp. calaminare) of the metallophyte T. caerulescens (Fig. 1). The isozyme analysis
of aspartate aminotransferase, phosphoglucomutase, leucine aminopeptidase, and
phosphoglucoisomerase indicated a very low degree of genetic variability in T.
caerulescens and no correlation of genetic data with either the environmental
factors such as Zn contamination or the geographic distribution of certain genotypes (19).
The analysis of chloroplast DNA restriction site variation in 45 populations
representing 20 Thlaspi species (22) and of sequences of the internal transcribed
spacer regions of nuclear ribosomal DNA in 16 Thlaspi accessions (23) confirmed
the presence of three major groups, thus supporting Meyers segregates: Thlaspi
s. str., Microthlaspi, and Noccaea (including Raparia), with the greatest differences detected between Thlaspi s. str. and all other species (Table 1).
Interestingly, the metal accumulating characteristics may be of taxonomic
significance, since all the species belonging to Thlaspi s. str. are incapable of
hyperaccumulating Ni or Zn, whereas several species of the lineages Noccaea
and Raparia, which were originally part of Thlaspi [T. caerulescens J. et C. Presl.,
T. goesingense Halacsy, T rotundifolium. (Wulfen) Kock subsp. cepaefolium (L)
Greuter & Burdet, T. praecox, T. alpestre], are metal hyperaccumulators
(19,24,25). However, the present chapter will refer to these Noccaea taxa as
Thlaspi sensu lato (Table 1).
The genus Thlaspi is distributed worldwide, primarily in the subartic and
the north temperate zone of Eurasia, but also in eastern Africa, western North
America and South America, northern Asia and Japan, whereas no species have
been recorded in Australia (1,17,25,26).
2
HEAVY METAL ACCUMULATION AND DETOXIFICATION
There are about 300 Ni hyperaccumulator plant species, 26 Co hyperaccumulators, and 24 for Cu, 19 for Se, 16 for Zn, 11 for Mn, 2 for Tl, and 1 that hyperaccumulates Cd (27). Many Brassicaceae are known for their heavy metal accumulation and detoxification capability, in particular by species of the genera Alyssum,
Brassica, and Thlaspi.
TABLE 1 Intrageneric Systems for Thlaspi s.l. Following Schultz (1), Clapham (26), and Meyer (17)
Claphamb,c
T. sect. Nomisma DC.

T. arvense
T. sect. Chaunothlaspi O.E. Schultz
T. alliaceum
T. sect. Carpoceras DC.
T. ceratocarpum
T. sect. Nomisma
T. arvense
T. sect. Nomisma
T. sect. Apterygium
T. cepaefolium subsp. rotundifolium
T. sect. Pterotropis DC. (incl. Neurotropis, DC.)
T. montanum
T. sect. Apterygium
T. cepaefolium subsp. rotundifolium
T. sect. Thlaspi (sect. Pterotropis,
Neurotropis)
T. montanum
T. caerulescens subsp. caerulescens subsp. calaminare
T. macranthum
T. alliaceum
T. bulbosum
T. perfoliatum
T. perfoliatum
Thlaspi s. str.
T. sect. Thlaspi
T. arvense
T. sect. Chaunothlaspi
T. alliaceum
T. sect. Carpoceras
T. ceratocarpum
Noccaea Moench
N. sect. Noccaea
N. rotundifolia L. Moench subsp.
rotundifolia
N. sect. Pterotropis
N. montana (L.) F. K. Meyer
N. caerulescens (J. & C. Presl)
F. K. Meyer
N. macrantha (Lipsky) F. K. Meyer
Raparia F. K. Meyer
R. bulbosa (Spruner) F. K. Meyer
Microthlaspi F. K. Meyer
M. perfoliatum (L.) F. K. Meyer
M. natolicum (Boiss.) F. K. Meyer
M. granatense (Boiss. & Reuter)
F. K. Meyer
T. caerulescens and T. macranthum were not recognized by Schultz (1936).

Thlaspi (Microthlaspi) granatense and T. (Microthlaspi) natolicum were not recognised by Schultz (1936) and Clapham (1964).
c
T. ceratocarpum does not occur in the area treated by Clapham (1964).
d
Meyer did not recognize subspecies with Noccaea caerulescens.
Source: Modified from Ref. (23), courtesy of NRC Research Press, April 2000.
b
T. bulbosum
Meyerb,d
224
Schultz a,b
Brassicaceae
2.1
225
Alyssum
In 1948, while studying the mineral nutrition of the Impruneta ophiolitic outcrop
vegetation (near Florence), as part of a geobotanical survey, two Florentine researchers, Minguzzi and Vergnano, discovered the unusual accumulation of Ni
in A. bertolonii Desv. (28). Despite the discovery in Russia of a second Ni
plant, A. murale (29), these results received little acknowledgment by the scientific community for a long time.
Much later, after the discovery of other plants that are capable of accumulating high Ni concentrations (3033) and especially thanks to the work of Brooks
and co-workers and their strategy regarding the analysis of small herbarium samples (34), metal-accumulating plant species received further attention. At present,
the term hyperaccumulator, coined to characterize the plants with Ni concentrations higher than 1000 g g1 in dry matter (34), is widely accepted.
A wide investigation recognized that the genus Alyssum contains the greatest number about 48 of Ni hyperaccumulator species (35), 14 of which belong
to the European flora (36). It is interesting to note that 13 of these European
Alyssum and 22 Anatolian Ni hyperaccumulators (Table 2) were included in section Odontarrhena (35), demonstrating the combined high degree of endemism
and resistance to high Ni concentrations of this group. On the other hand, it has
been observed that the environmental pressure exerted on the adaptation capability of this genus may be due not only to high Ni concentrations but also to other
factors causing the infertility of the serpentine soils. These include high levels
of Cr, Co, and Mg, and a deficiency of nutrients like Ca, N, P, and K (42).
Nevertheless, considering the similar effects observed in the same plants grown
on ophiolitic substrates and in Ni culture solutions (43), and the several factors
affecting the features of the serpentine soils, it is generally accepted that the
presence of high Ni concentrations plays an important role in the evolution of a
serpentine flora (28,44). A particular case is represented by Alyssum hyperaccumulators, in which a very high Ni tolerance has evolved.
A. bertolonii showed a Ni tolerance higher than the one required by other
nonaccumulator species that occur at the same ophiolitic outcrop (45). This suggests that metal hyperaccumulation must have a function besides the ability to
survive on serpentine soils. Some hypotheses regarding the meaning of Ni accumulation have been proposed (46), but one of the most persuasive ecological
explanations seems to be the Ni defensive role against herbivores or pathogens
(47). This function, which might be similar in other hyperaccumulators, can be
improved if the metal is localized in the outer layers of leaves and roots. As in
other Ni accumulators, such as Hybantus floribundus (48), Senecio coronatus
(49), and Thlaspi montanum var. siskiyouense (50), in A. bertolonii Ni has been
evidenced in leaf epidermal cells as a red-stained Ni-dimethylglyoxime complex
(51). Furthermore, microprobe analysis has shown that leaf hairs have the highest
226
TABLE 2 Alyssum Hyperaccumulators
Species
akamasicum Burtt
alpestre L.
anatolicum Hausskn
argenteum All.
bertolonii Desv
subsp. Scutarium Nyar.
callichroum Boiss. & Buhse
caricum T.R. Dudley & Huber-Morath
cassium Boiss.
chondrogynum Burtt
cicilicum Bois & Balansa
condensatum Boiss. &, Hausskn.
constellatum Boiss
crenulatum Boiss.
cypricum Nyar.
davisianum T.R. Dudley
discolor T.R. Dudley & Huber-Morath
dubertretii Gombault
eriophyllum Boiss. & Hausskn
euboeum Halacsy
fallacinum Hausskn.
floribundum Boiss. & Balansa
Ref.
Cyprus
S. Europe (Alps)
Anatolia
Italy
Italy (Tuscany)
Italy
Anatolia
Anatolia
Anatolia
Cyprus
Anatolia
Iraq, Syria
Anatolia, Iraq
Anatolia, Syria
Cyprus, Anatolia
Anatolia
Anatolia
Anatolia
Anatolia
Euboea
Crete
Anatolia
9090
4480
8170
29400
13400
10200
10900
16500
20000
6300
13500
4090
18100
10400
23600
19600
11700
16500
11500
4550
3960
7700
35
36,37
35
35,37,38,39
28,36,37
39
35
35,39
35
35
35
35,39
35
35
35
35
35
35
35
36,39
36
35
1
2
3
4
5
6
7
8
9
10
11
12
13
14
16
17
18
19
20
21
22
23
Location
Max. Ni in
leaves (g
g1 DW)
giosnanum Nyar
heldreichii Hausskn.
hubermorathi T.R. Dudley
janchenii Nyar.
lesbiacum (Candargy) Rech. f.
malacitanum T.R. Dudley
markgrafii O.E. Schulz.
masmenaeum Boiss.
murale Waldst. & Kit.
obovatum (C.A. Mayer)
oxicarpum Boiss. & Balansa
peltarioides Boiss subsp. virgatiforme
penjwinensis T.R. Dudley
pinifolium Nyar. T.R. Dudley
pintodasilvae T.R. Dudley
pterocarpum T.R. Dudley Rech. f.
robertianum Bernard ex Gren. & Godr.
samariferum Boiss. & Hausskn
singarense Boiss. & Hausskn.
smolikanum Nyar.
syriacum Nyar.
tenium Halacsy (Tinos)
trapeziforme Waldst.
troodii Boiss.
virgatum Nyar.
Anatolia
Greece
Anatolia
Albania
Lesvos
Spain
Albania
Anatolia
Balkans
Russia
Anatolia
Anatolia
Iraq
Anatolia
Portugal
Anatolia
Corsica
Samar
Iraq
Grece
Syria
Tinos
Anatolia
Cyprus
Anatolia
7390
12500
13500
9610
22400
10000
13700
24300
7080
4590
7290
7600
7860
21100
9000
22200
12500
18900
1280
6600
10200
3420
11900
17100
6230
35
36
35
35
35
34
35,39
35
29,35,36
35
35
35,39
35
35,39
36,40,41
35,39
35
35,39
35
36
35
36
35
35,39
35
Brassicaceae
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Adapted courtesy of AB Academic Publishers, April 2000.
227
228
Ni concentrations (52). Similarly, in A. lesbiacum, Ni was found in the epidermis

and in leaf trichomes by a micro-PIXE technique (53).
Probably the Ni accumulation capability is also closely connected to the
degree of adaptation and restriction to ultramafic soils in order to avoid direct
competition with the plants outside these sites (54). An increase in this very
strong feature may have reduced the geographic spreading of Alyssum hyperaccumulators. This can be observed in the ultramafic soils of Turkey, where the distribution of the species of section Odontarrhena seems inversely related to its Ni
concentration (35).
The ability to concentrate metals in plant above-ground parts, reaching levels higher than the substrate metal concentrations, is a feature of metal accumulators and, to a greater extent, of the so-termed hyperaccumulators (55). Two important processes contribute to metal accumulation efficiency: a high root metal
uptake and a high rate of long-distance transport. Among hyperaccumulators,
Alyssum species show a general tendency to take up very high amounts of Ni.
This metal is rapidly taken up from the serpentine soil and translocated to the
leaves, where it is progressively stored. When Alyssum plants are grown in solutions with increasing Ni concentrations, the maximum accumulation level in the
leaves is reached following a saturation trend (56,45). In A. bertolonii (28,57)
and A. heldreichii (56), the Ni concentration is lower in the roots than in the
leaves. However, the rate of Ni uptake in the roots is still higher in hyperaccumulator than in non-hyperaccumulator species. Ni concentration is also higher in
the extractable fractions of ophiolitic soils than in normal soils (58). In the hyperaccumulators a great amount of Ni is immobilized in the cell walls of the root
cortex due to its high cationic change capability. A decrease of pH can stimulate
the release of this element into the apoplast. However, at present there is no
evidence that the Ni fraction of the root cell wall contributes to the uptake into
the cells.
The relationship between plasma membrane transport and root uptake of
metals has not been elucidated yet. Recently, evidence was found that Zn hyperaccumulation in T. caerulescens is associated with a plasma membrane transport
mediated by specific types of selective channels (59). In Alyssum spp., besides
the physical-chemical conditions favoring the release of Ni from the soil, a similar
mechanism might support the preferential accumulation of this element rather
than of other metals also present at high concentrations in serpentine soils. On
the basis of a comparative study in A. troodii on the ability to accumulate Ni
and Co, the same mechanism was suggested to be essential for the control of the
competitive uptake of both the elements (60). Ni and Co have very similar chemical properties and both can be accumulated in leaves; however, Co accumulation
in Alyssum plants is usually much lower than Ni accumulation. A highly selective
uptake across the plasma membrane through an hypothetical ternary complex,
like a protein-organic acid with tightly bound Ni, was proposed by Still and
Brassicaceae
229
Williams (1980) (61). Great attention has been addressed to the chemical forms
that ensure the nontoxicity of Ni ions. The mechanisms of Ni tolerance and accumulation can be interpreted as two aspects of the same process because the formation of stable nontoxic complexes is essential to Ni long-distance transport and
storage in plant cells.
The ability to bind Ni ions in organic acid complexes has been suggested
to be a tolerance strategy for a number of Ni hyperaccumulator plants (62). In
water extracts from A. bertolonii leaves, Ni was found to be present in the same
gel filtration fraction containing mainly malic and malonic acids (63). Further
evidence on the relationship between Ni and these organic acids was obtained
comparing A. bertolonii grown in serpentine soils and in ordinary garden soils
(64). Higher levels of both malic and malonic acids were present in the leaves
of plants grown on serpentine soils, compared with plants from garden soil. In
the Iberian hyperaccumulators A. pintodasilvae and A. malacitanum, Ni was separated as a polar complex associated with the same gel filtration fraction containing
citric, malic, and malonic acid. By contrast, very small amounts of organic acids
were present in A. serpillifolium, a nonaccumulator species closely related to the
two Iberian hyperaccumulators (65).
In all of these cases, the presence of both metal and organic acids in the
same extract fractions has been asserted as accounting for the existence of a Ni
complex, but this has not been supported by clear direct evidence. Nevertheless,
since metals are mainly accumulated in the vacuoles (66), it is very likely that
Ni in Alyssum is bound to organic acids. However, the formation of Ni complexes
with other compounds, such as amino acids, could be favored by their higher
stability constants, in sites where the organic acid concentration is much lower
than in vacuoles (67).
Kramer et al. (1996) (68) demonstrated that concentrations of Ni and histidine are highly correlated in the xilem sap of A. lesbiacum, A. murale, and A.
bertolonii. The involvement of a histidine complex in the Ni accumulation process was directly supported by the X-ray absorption fine structure (EXAFS) analysis of xylem sap in A. lesbiacum. Further evidence of the involvement of this
amino acid in Ni tolerance was given by the increase in biomass production
and root elongation induced by exogenous histidine supplied to nontolerant A.
montanum species cultured in solutions with toxic amounts of Ni. These findings
suggest a mechanism of Ni accumulation in the genus Alyssum (Fig. 2), based
on a nontoxic Ni-histidine complex for the transport of apoplastic Ni through the
xylem sap to the leaves, where the metal is compartmentalized in the vacuoles
by means of high concentrations of organic acids (62).
It is also important to evaluate other factors and conditions that regulate
the plants ability to accumulate Ni. In the hyperaccumulator species of Alyssum,
different Ni contents have been observed (54). There is no easy way to evaluate
if this characteristic is related to the natural genetic variability among species or
230
FIG. 2 Nickel hyperaccumulation mechanisms in the Ni pump plant

A. bertolonii.
if it is attributable to soil mineral composition. Nevertheless, different values of

the maximum accumulation capacities were found in several Alyssum species
grown in solutions with increasing Ni concentrations (56). In samples of A. bertolonii collected from an ophiolitic outcrop, the degree of Ni accumulation seemed
to be related to the length of the growth period rather than to the Ni content of
the soil (57). This relationship has been recently studied through mathematical
modeling in A. bertolonii seedlings grown for 30 days in nutrient solutions (69).
The suggested model of plant growth and Ni hyperaccumulation indicates that
in A. bertolonii Ni uptake and tolerance are directly correlated only during the
plant active growth phase. A. bertolonii accumulated up to 12,000 g g1 dry
weight Ni in the leaves without showing the symptoms of growth inhibition.
Interestingly, A. bertolonii plants grown in their natural habitat showed a similar
upper limit of Ni accumulation.
Another interesting aspect of Ni detoxification is represented by the regular
shedding of the oldest leaves with the highest Ni content (57). This event represents a detoxifying process per se (70) and contributes to the establishment of
the maximum metal accumulation level typical of each Alyssum species.
2.2
Brassica juncea
Dushenkov et al (1995) (71) reported the high capability of hydroponically cultivated roots of B. juncea to take up heavy metals up from nutrient solutions. For
Brassicaceae
231
instance, roots of this species accumulated Cd and Pb, respectively, 134 and 563
times more than the initial concentrations in the solution.
It was recently demonstrated that root cells of B. juncea mutants with increased Pb uptake have a higher amount of cell wall per gram of fresh weight
than the normal root cells (72). According to the authors, this is an essential
mechanism that allows the roots of this mutant to hyperaccumulate Pb. Cell wall
desorption by EDTA further supported this hypothesis.
B. juncea translocates Pb and other heavy metals from solutions (73) or
soils (74) to shoots. It has been demonstrated that roots and shoots of B. juncea
can accumulate various amounts of Pb according to the metal concentration supplied to the culture medium (75). Perhaps B. juncea shows a special capability
to accumulate the highest levels of Pb in the shoot, as well as the ability to
accumulate and detoxify significant amounts of Cd, Cr(VI), Cu, Ni, and Zn, compared with other heavy metal hyperaccumulators (73). In a comparative study
between B. juncea and T. caerulescens, the former produced at least 20 times
more biomass than the latter and showed a rapid growth rate under field conditions (76). However, T. caerulescens had a superior capability to accumulate all
metals except Cd in the shoots (Table 3). The relative difference in metal root
uptake between these two species was less dramatic than differences in shoot
uptake. It is clear now that B. juncea first accumulates Pb in its roots and then
translocates and concentrates the metal in the shoots (and hypocotyls) (77).
In the presence of Cd ions, plants of B. juncea take up this metal by means
of the root system and translocate part of it to the shoot, in particular to the
leaves. Cd loading into the xylem sap of B. juncea displays saturation kinetics
TABLE 3 Shoot and Root Bioaccumulation Coefficientsa of Brassica juncea

and Thlaspi caerulescens b
Shoot
Metal
Cd (5)c
Cu (1)
Cr (0.4)
Ni (1)
Pb (5)
Zn (3)
Brassica
175
159
80
587
3
49
16
32
8
115
1
31
Root
Thlaspi
59
623
89
2739
29
770
12
265
15
383
23
320
Brassica
20574
55809
5486
11475
1432
1816
4295
9221
393
125
1409
1739
Thlaspi
4258
60716
8545
8425
7011
2990
168
21510
2677
4220
3616
1424
Bioaccumulation coefficient is the ratio of metal concentration in plant tissue (g g1

DW) to initial metal concentration in solution (mg l1).
b
Hydroponically grown plants were exposed to metal solution for 8 days.
c
Initial concentration of metal in the solution (mg L1).
Source: Ref. 76. Courtesy of Nature Biotechnology, March 2000.
a
232
(78), suggesting that it is facilitated by membrane transporter systems (79). The

movement of Cd in the xylem seems to be driven by the transpiration process
(78). The accumulation of Cd in roots and leaves of B. juncea induces the synthesis of phytochelatins (78,80). The formation of Cd-phytochelatins complexes is
the main system of Cd detoxification in leaves and roots of B. juncea. General
mechanisms of Cd detoxification in higher plants have been reported by Sanita`
di Toppi and Gabbrielli (81). EXAFS analysis in extracts from roots of B. juncea
has demonstrated the Cd-S4 coordination. Differently, in the xylem sap Cd is
complexed by oxygen and/or nitrogen: thus, Cd is more likely to be transported
by organic acids than phytochelatins in the xylem sap (78). In the presence of
Cd, glutathione synthetase (GS) is the rate-limiting enzyme for the synthesis of
glutathione and phytochelatins in B. juncea. Therefore, overexpression of this
enzyme might represent a powerful tool for the production of plants with the
highest heavy metal accumulation capacity (82). Furthermore, the analysis of the
expression of O-acetylserine(thiol)lyase (OAS-TL), -glutamylcysteine synthetase (-ECS), and GS in B. juncea has shown that the Cd-induced phytochelatin
synthesis might be at least partially explained by the overexpression of glutathione biosynthesis genes (83). The Cd induction of a mitochondrial -ECS isoform
in roots of B. juncea further supports this hypothesis (84). The response to Cd
in roots and leaves of B. juncea is probably based on coordinated gene expression
events tightly correlated to sulfur assimilation and glutathione synthesis (85).
B. juncea is also an accumulator of Se (86). As briefly mentioned above
with regard to Cd detoxification, high levels of S uptake and assimilation are
also involved in Se metabolism in plants. As a matter of fact, Se-tolerant plants
form acid-labile sulfide (S2), and this has been demonstrated to be the mechanism for heavy metal detoxification also in B. juncea (80). Se toxicity might be
counteracted by the incorporation of S-containing amino acids into proteins rather
than their Se isologues (87,88). The incorporation of Se into proteins is probably
the most important cause of Se toxicity in plants (89,90). In Se-nontolerant plant
species, both selenocysteine and selenomethionine are formed and included in
the elongating protein chain, where they replace the two S-containing amino acids
cysteine and methionine. The presence of these abnormal amino acids in the
proteins is responsible for Se toxicity in nontolerant plants (91).
An increased Zn supply augmented the activities of guaiacol peroxidase,
superoxide dismutase, catalase, and various components of the ascorbate-glutathione cycle in shoots of B. juncea seedlings. However, despite the prompt intervention of these detoxifying molecules, plants showed a significant increase in
lipid peroxidation and tended not to survive (92). Interestingly, in Zn-stressed B.
juncea plants, overexpression of glyoxalase I transcripts was also noticed. As
reported by the authors, overexpression of glyoxalase I would diminish the level
of methylglyoxal under heavy metal stress and allow the reformation of glutathione from its hemithioacetal (93).
Brassicaceae
2.3
233
Thlaspi
Thlaspi heavy metal hyperaccumulating taxa have colonized specialized habitats,

such as base metal deposits, undisturbed outcropping mineralizations, and heavy
metalpolluted soils. Together with the typically heterogeneous vegetation of
these metalliferous substrates, which reflects the wide variation of heavy metal
concentrations over short distances, Thlaspi has probably developed to the present status within the last 150 years (94). However, T. goesingense is well represented on both mineralized and calcareous soils, whereas T. caerulescens and T.
calaminare are represented only on metal-rich substrates and can be used as effective geobotanical indicator species in mineral exploration (95). The genus Thlaspi
comprises Zn, Ni, and Pb hyperaccumulators (96) (Table 4).
About 10 species have been identified as Zn hyperaccumulators (Table 4).
The two closely related species T. calaminare (97,62) and T. caerulescens, which
belong to the Zn-tolerant Galmei flora of western Germany and eastern Belgium
(98100), have been extensively studied (62). T. calaminare is one of the most
important metallophytes of the Thlaspion calaminaris association confined to the
calamine (Zn carbonate) deposits of western Europe. T. caerulescens has been
the elective taxon for phytoremediation trials because of its efficiency in accumulating more than 4% dry weight Zn in the aerial parts (101,102). T. caerulescens,
which now includes the variants originally grouped into T. alpestre (103), is also
the most represented member of the so-called Minuartio-Thlaspietum alpestris
association of the abandoned Pb mines of England, within the Pennines, Mendip
Hills, and North Wales. Here the vegetation is affected not only by the high levels
of soil Pb, Cd, and Zn, but also by soil instability, low organic matter status, and
severe nitrogen and potassium deficiencies. Within the southern Pennines, the
distribution of T. caerulescens strongly correlates with the location of Pb mines.
Also in the mining areas of Mendip Hills T. caerulescens is the ubiquitous metal
hyperaccumulator. T. caerulescens is also a well-known Cd hyperaccumulator
species (more than 0.01% Cd in dried leaves) (62).
Other Zn accumulators are T. rotundifolium subsp. cepaeifolium, widespread in a Pb/Zn mine of the Cave of Predil region (northern Italy) (104) and
T. goesingense, which is also a Ni hyperaccumulator and is endemically restricted
to a few serpentine outcrops in East Austria (105,106). Other five species of
Thlaspi contain comparable amounts of Zn (more than 1% Zn in dried leaves),
both in plants from Zn-rich substrates and from rocks with much lower levels
of Zn (104).
With a Pb content of 700 g g1 in dry shoots, T. rotundifolium subsp.
cepaeifolium is the only relevant Pb accumulator species within the genus Thlaspi
(Table 4) (104). This taxon has colonized the Raibl Mine (Pb/Zn mine) of the
Cave of Predil region in northern Italy, with intermediate occurrence in other
locations scattered around the confluence of the Freddo and Silizza rivers. The
234
TABLE 4 Thlaspi Hyperaccumulators: Geographic Distribution and

Maximum Metal Concentrations (% shoot dry weight)
Species
Zn
Thlaspi alpestre L.
brachypetalum Jordan
bulbosum Spruner ex Boiss.
caerulescens J. et C. Presl
calaminare (Lej.) Lej. Et Court
limosellifolium Reuter
praecox Wulfen
rotundifolium subsp. capaeifolium
stenopterum Boiss. Et Reuter
tatraense Zapal
Pb
T. rotundifolium subsp. cepaeifolium
Ni
Thlaspi alpestre L. subsp.virens (Jord.)
Hook f.
alpinum Crantz subsp. sylvium
(Gaud.) Clapham
bulbosum Spruner ex Boiss.
cyprium Bornm.
elegans Boiss.
epirotum Halacsy
goesingense Halacsy
graecum Jordan
japonicum Boiss.
jaubertii Hedge
montanum L.
var. californicum
var. montanum
var. siskiyouense
ochroleucum Boiss. Ex Heldr.
oxyceras (Boiss.) Hedge
rosulare Boiss. & Bal.
rotundifolium (L.) Gaudin
var. corymbosum (Gay) Gaudin
sylvium (Gaud.) Clapham
tymphaeum Haussknecht
Location
% Metal
Ref.
UK
France
Greece
Germany
Germany
France
Bulgaria
Italy
Spain
Slovakia
2.50
2.00
1.05
2.73
3.96
1.10
2.10
2.10
1.60
2.70
98
98
98
98
98
98
98
105
98
98
Central Europe
0.16
105
Central Europe
0.40
105
Central Europe
0.41
105
Greece
Cyprus
Syria, Turkey
Greece
Austria
Greece
Japan
Turkey
0.20
0.51
2.08
0.30
1.20
1.20
2.44
2.69
105
26
26
105
105
105
26
26
Western USA
Western USA
Western USA
Greece
Syria, Turkey
Turkey
Central Europe
Central Europe
Central Europe
Greece, Albania
0.79
0.55
1.12
0.40
3.56
0.52
1.83
0.20
3.10
0.40
26
26
26
105
26
26
105
105
105
110
Brassicaceae
235
mine tailings have been transported some distance from the river and accumulated
at various sites downstream. The metallophytes are often found in these accumulations (62,104). T. rotundifolium subsp. cepaeifolium is a dominant member of
the metal-lophyte plant community Thlaspietum cepaefolii associated with the
Raibl Mine and described by Ernst (107,108). According to the author, the soil
supporting this Thlaspi subspecies contains Pb (0.060.41%) and Zn (0.7
14.6%), with small amounts of Cu (00.12%).
Several Ni hyperaccumulator taxa of Thlaspi occur worldwide, particularly
concentrated in the Mediterranean temperate regions and Turkey. T. goesingense
Halacsy is restricted to the Austrian Alps, where it is widespread on both metalliferous and nonmetalliferous soils (105,106). T. caerulescens is occasionally found
also on serpentine soils in Scotland (109). At least three varieties of T. montanum
that hyperaccumulate Ni (varieties californicum, montanum, and siskiyouense)
were identified on the serpentine outcrops of North America (95). T. montanum
californicum and siskiyouense are probably neoendemic forms, with variety montanum as a precursor (62,95).
The genus Thlaspi includes both metal hyperaccumulators and nonaccumulators. The exceptional ability of the hyperaccumulator species to concentrate in
their tissues a wide range of metals from both metalliferous soils and nutrient
solutions has been extensively investigated (110,112,113). The physiological and
genetic aspects of metal tolerance in Thlaspi are not fully understood, although
they have been investigated in several Thlaspi species.
2.3.1
T. caerulescens
T. caerulescens is a very polymorphic species, with marked morphological differences among populations, sometimes even within the same geographic area (110).
Wild populations of T. caerulescens from different locations of the British Isles
were found to contain up to 21,000 g g1 Zn in dried shoots (110). When grown
in nutrient solutions, plants accumulated up to 48,000 g g1 Zn and 10003000
g g1 Cd, without showing visible symptoms of metal toxicity (102). In a field
trial with soil plots polluted with both Zn and Cd, T. caerulescens concentrated
these two metals in the shoot tissue 10 times compared with the soil levels (111).
With its extraordinary ability to hyperaccumulate Zn, Cd, and Ni, T. caerulescens
(formerly T. alpestre L.) has become an ideal model for study of the mechanisms
of heavy metal tolerance and hyperaccumulation in plants (112,113) and has a
great potential in phytoremediation of polluted soils (104,114,115).
Zn tolerance and hyperaccumulation are constitutive properties of T. caerulescens, although they are two physiologically and genetically independent traits
(111,116118). The mechanism used by T. caerulescens to mobilize soil Zn is
not completely understood, and recent studies (119,120) show that rhizosphere
acidification is not the main mechanism of Zn hyperaccumulation in this species.
There is also little information on the mechanism of Zn transport and accumula-
236
tion in the shoot, although the preferential accumulation of Zn in the epidermal

cells of young and mature leaves has been conclusively demonstrated (121). Recently, Lasat and Kochian (112,113) used 65Zn2 to quantify root fluxes, compartmentalization, and Zn translocation to the shoot in seedlings of the hyperaccumulator T. caerulescens and in the nonaccumulator T. arvense grown in nutrient
solution. T. caerulescens accumulated significantly higher levels of Zn than T.
arvense in the shoots, whereas the latter accumulated more Zn than T. caerulescens in the roots. However, both Zn accumulation in the roots and translocation
to the shoot were faster in T. caerulescens than in T. arvense (113). The compartmentalization study at the root level resulted in similar amounts of Zn accumulated in the cell wall and cytoplasm of both species, whereas two- to fourfold
more Zn was sequestered in the vacuole of T. arvense and made unavailable for
translocation to the shoot. Furthermore, a higher Zn concentration was present
in xylem sap of T. caerulescens and leaf sections of this species accumulated
more Zn than those of T. arvense (113). These findings, together with the large
deposits of Zn observed in the leaf vacuole of T. caerulescens (121,122), support
the hypothesis that in the hyperaccumulator species a combination of stimulated
transport systems, operating both at the leaf plasma membrane and the tonoplast
level have different kinetic properties than in the nonaccumulator T. arvense
(123). Molecular studies of Zn transport genes in T. caerulescens isolated and
identified ZNT1 as one of the micronutrient transport genes, with high sequence
homology with other Zn transport genes isolated from yeast (124) and from Arabidopsis (125). Zn hyperaccumulation in T. caerulescens relies on the ZNT1 gene,
which encodes a high-affinity Zn transporter. This gene is constitutively expressed at a much higher level in T. caerulescens than in T. arvense, where its
expression is stimulated by Zn deficiency. In fact, plant Zn status is shown to
alter the normal regulation of Zn transporter genes in T. caerulescens. An important aspect of Zn hyperaccumulation and tolerance in T. caerulescens is also the
production of low molecular weight compounds involved in Zn detoxification in
the cell (cytoplasm and vacuole) and in the long-distance transport of Zn in the
xylem vessels. Citrate was not shown to play an important role in Zn chelation,
and malate reached constitutively high concentrations in the shoots of both the
accumulator T. caerulescens and the nonaccumulator T. ochroleucum (118,126).
More recently, direct measurements of the in vivo speciation of Zn in T. caerulescens using the noninvasive technique of X-ray absorption revealed that histidine
is responsible for the transport of Zn within the cell, whereas organic acids (citrate
and oxalate) chelate Zn during long-distance transport and storage (127). Another
constitutive aspect of T. caerulescens is the high Zn requirement for maximum
growth, compared with other species (118,120,128). This probably depends on
the strong expression of the metal sequestration mechanism, which would subtract a large amount of intracellular Zn to normal physiological processes, even
when the Zn supply is low (118).
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237
T. caerulescens, together with T. goesingense (129) was shown to have a

great ability of accumulating the nonessential metal Cd (102,119,129131). In
a French population of T. caerulescens, Cd shoot concentration was exceptionally
high (up to 10,000 g g1 dry weight) in hydroponically grown plants, and neither
symptoms of Cd toxicity nor reduced plant biomass were observed (129). The
current opinion is that nonessential metals can enter the plant cells only because
of their chemical or physical similarities to plant nutrients, probably using Ca
channels and broad-range metal transporters (132). The ability of T. caerulescens
to accumulate a range of heavy metals, including Cd, cannot be explained any
longer with the presence of a nonspecific uptake mechanism, side effect of an
efficient mechanism of Zn hyperaccumulation, which can be adapted to different
metals (110). Cd accumulation is a selective process, which does not share either
common binding sites or similar compartmentalization mechanisms with Zn
(119,130) and does not occur at the expense of a reduced Zn accumulation
(102,129). The physiological and genetic basis of Cd tolerance in this species
must be further clarified (102). Preliminary results suggest that in T. caerulescens
Cd is stored mainly in the root apoplast and, to a lesser extent, in vacuoles, where
it co-occurs with Ca and Fe (131). The highest efficiency in Cd hyperaccumulation may occur in populations characterized by more transporter sites at the root
cell membrane (129). Differences in Cd tolerance observed among various populations correspond to differences in Cd hyperaccumulation ability, showing that
Cd tolerance and accumulation are probably directly linked (129). Finally, the
observed differences between Zn and Cd uptake in different varieties of T. caerulescens may be advantageous for improving phytoextraction of Cd through selection of plant populations, without losing efficiency in Zn phytoextraction
(102,129).
Some populations of T. caerulescens have an exceptional ability to take
up other metals, such as Pb, Mo, Cr, Ag, Mn, Al, Co, Fe, and Cu, listed in
increasing order of the biaccumulation coefficient (metal concentration in wholeplant dry matter/metal concentration in nutrient solutions) (111). Similarly to Zn,
Cd, and Ni, Co and Mn absorbed by the root are easily translocated to the shoots,
while the greater fraction of Cr, Cu, Al, Fe, and Pb is immobilized at the root
level (111).
2.3.2
T. rotundifolium subsp. cepaeifolium
Previously termed T. cepaeifolium (133), T. rotundifolum is an effective hyperaccumulator of various metals (Cd, Ni, Pb, Zn) (104,106108). Zn concentrations
were found to be higher in leaves than in stems or roots (133). Interestingly,
Wenzel and Jockwer (106) found that the amount of Cd, Pb, and Zn extracted
from the soil by the best performing individuals of selected populations matched
almost exactly the levels of phytoavailable metals in the soil (which is accessible
also to nonaccumulating species). Based on these data, they suggested to change
238
the lower limit of Cd concentration that qualifies plants as Cd hyperaccumulators

from 100 (134) to 50 g g1 dry weight (106).
2.3.3
T. goesingense
Two different varieties of T. goesingense are known. One occurs on Austrian

ultramafic (serpentine) outcrops and is capable of hyperaccumulating up to
12,500 g g1 Ni dry matter in the shoot (105,106). The other variety is well
represented on calcareous soils and is also a Ni hyperaccumulator when grown
on ultramafic soils (24). The ability to hyperaccumulate Ni in the shoots is thought
to be a constitutive property of this species (111). Recently, Kramer et al. (135)
compared the rate of Ni transport and tolerance in the Ni hyperaccumulator T.
goesingense and the related nonaccumulator T. arvense. They found that the hyperaccumulating phenotype observed in hydroponic cultures did not depend on
different rates of Ni transport from root to shoot in the two varieties, but probably
on a specific cellular mechanism of Ni tolerance in the leaves of the hyperaccumulator (135). On the other hand, the exudation of histidine and citrate in the rhizosphere, which plays an important role in Ni detoxification in Alyssum hyperaccumulators (135), is not essential for Ni hyperaccumulation in T. goesingense,
whereas the exudation may be responsible for decreasing Ni uptake and toxicity
in T. arvense and other nonaccumulator species (136). Furthermore, Pearsans et
al. (137) demonstrated that, although genes involved in histidine biosynthesis are
expressed at the root and shoot level, they are not regulated by Ni concentrations
and time of Ni application. The concentration of histidine in root, shoot, and
xylem sap after Ni exposure is similar in T. goesingense and T. arvense, and so
it is when histidine is measured directly in plant tissues of both Ni accumulator
and nonaccumulator species (137). The role of histidine in the detoxification and
transport of Ni, as suggested in the Ni hyperaccumulator A. lesbiacum (138), was
not demonstrated in T. goesingense and T. arvense. T. goesingense is very efficient in accumulating also Cd concentrations up to 830 g g1 in dried shoots
(129), well above the concentration usually considered to qualify a Cd hyperaccumulator (100 g g1) (134).
2.3.4
T. montanum
The species T. montanum includes the Ni hyperaccumulating varieties montanum,

californicum, and siskiyouense, which occur on serpentine soils of North
America. Also T. montanum var. fendleri is reported to accumulate both Ni and
Mn (139). T. montanum var. montanum is peculiar because it occurs both on
serpentine and normal soils (140). Similarly to T. goesingense, in T. montanum
var. montanum metal hyperaccumulation is a constitutive trait, which confers to
the plant adaptive advantages on metalliferous soils (140). The nonserpentine
populations are latent hyperaccumulators: with their relatively high Ni concentrations in the leaves (3345 g g1 Ni in dried shoots) on nonmetalliferous
Brassicaceae
239
soils, they show a predisposition for metal hyperaccumulation and can easily
hyperaccumulate metals on metalliferous soils (140). Since most Ni hyperaccumulators show also high levels of Zn in their shoots, Ni hyperaccumulation may
be the consequence of an effective nutrient uptake mechanism on fertile soils,
which can also be used with Ni when this species occurs on Ni-rich soils (140).
One of the advantages of accumulating Ni in the leaves is an efficient defense
against predators and pathogens, which can be killed by the toxic effect of Ni
(141143). There is clear evidence that the Ni hyperaccumulator T. montanum
var. siskiyouense accumulates the highest Ni concentration at the leaf surface and
particularly in the subsidiary cells surrounding guard cells and in other elongate
epidermal cells, and this also supports the defense hypothesis (144).
2.3.5
T. ochroleucum
T. ochroleucum is a pioneer plant on some metalliferous soils (24) and is highly

tolerant to such heavy metals as Ni and Zn; however, it is not a hyperaccumulator
(111,120,145). In T. ochroleucum Zn is accumulated in the root and Zn tolerance
is lower than in T. caerulescens due to a less efficient root-to-shoot transport of
the metal (118). This ecotype very rarely occurs on Cu-rich soils, and tolerance
to Ni and Zn does not confer to T. ochroleucum tolerance to Cu. Cu has adverse
effects on germination, seedling survival, and seedling growth (146) and causes
severe damage primarily at the root level (147).
2.3.6
T. sylvestre Jord. subsp. calaminare
T. sylvestre Jord. subsp. calaminare occurs on the Belgian calamine soils, accumulates Zn and requires high levels of Zn for germination and normal growth
(20 and 50 mg L1 in nutrient solution, respectively) (148).
2.3.7
T. arvense
T. arvense is a nonaccumulating species that has been widely used to compare the
performance of metal hyperaccumulators and non-hyperaccumulators (113,149,
150). T. arvense is a widespread weed in Eurasia and North America and is a
pioneer plant on abandoned lands. It is an aggressive competitor that causes severe losses in agricultural crops as well as a popular food plant, especially in
Europe (16). With its succulent leaves, it is capable of surviving in extreme
drought conditions, probably due to a typical CAM metabolism (151).
3
PHYTOREMEDIATION
For many years, the species that accumulate unusual amounts of heavy metals
have been considered by several scientists as potential indicators of metalliferous
soils and interesting models in which to study the capacity of plants to adapt to
extreme environmental conditions. In the last decade, the increasing interest in
240
phytoremediation, whereby certain plants are used to remediate contaminated

sites, has also emphasized the great potential of metal hyperaccumulators (152).
These plants could represent an appropriate and cost-effective tool for removing
heavy metals from polluted sites. Furthermore, in the case of the termed phytomining practice, they can give an economic return through the recovery of
the metals from low-grade ores (62). The efficiency of phytoremediation of heavy
metalcontaminated soils requires that plants have (a) great ability to take up and
translocate metals; (b) high efficiency in detoxifying the metal in plant tissues; (c)
high production of above-ground biomass. Many authors used the total metal
translocated (metal concentration in shoot biomass/dry weight) as a measure of
the effectiveness of metal hyperaccumulating plants in extracting heavy metals
from contaminated soils. Among Brassicaceae there are several species of potential high interest for phytoremediation.
3.1
Alyssum
The potential use of A. bertolonii in soil phytoremediation has been evaluated

in terms of the conditions capable of improving its efficiency in taking up and
accumulating Ni ions (153). In fact, reduced growth rate and biomass production,
which are usually associated to Ni tolerance and accumulation (154), may limit
the use of many metal-hyperaccumulating species to clean up contaminated soils.
A. bertolonii plants grown on an ultramafic soil and treated with appropriate
fertilizers (N, P, K), had an increased biomass productivity without changes in
Ni concentration. By contrast, the addition of calcium carbonate, which reduced
Ni and Mg availability, decreased the Ni content in A. bertolonii plants. Generally
speaking, Alyssum hyperaccumulators have been considered useful tools, capable
of competing economically with traditional practices of heavy metal decontamination (153,155). Alyssum species appear to be interesting tools also in remediating sites contaminated with other heavy metals, in particular Co, that can be
usually found in ultramafic soils together with Ni (155). To optimize the use of
Alyssum in metal recovery, appropriate conditions have been individuated: (a)
lowering of the soil pH; (b) maintenance of low calcium levels or lowering of
calcium levels in the soil by leaching; (c) use of ammonium-containing or ammonium-generating nitrogen fertilizers; (d) distribution of chelating agents to the
soil (155).
3.2
Brassica juncea
About 1 g dry weight from roots of B. juncea, placed in 400 ml of water supplied
with 300 g ml1 of Pb, reduced Pb levels in the solution to less than 1 g ml1
in a few hours. Simultaneously, the concentration of Pb in the roots exceeded
10% of the tissue dry weight (156). In other experiments, B. juncea plants were
shown to accumulate Pb in the root system (75) or in stems and leaves (157).
Brassicaceae
241
Kumar et al. (158) suggested that B. juncea could respond even further to increasing Pb levels. In particular, the Vardan genotype concentrated Pb in different
plant parts, showing a better ability in Pb uptake than other genotypes. Consequently, the Vardan genotype seems to be the most suitable for Pb phytoextraction. However, for phytoremediation of Pb-contaminated soils, the chelateassisted phytoextraction strategy (79) appears to be in general more effective
than the natural heavy metal phytoextractive ability of Pb hyperaccumulators.
EDTA promptly chelates Pb ions in the soil, then the Pb-EDTA complex is taken
up by B. juncea roots and translocated, following the transpiration stream, to the
leaves, where it is accumulated at very high levels (159). The use of chelating
agents (EDTA, EGTA, etc.) might contribute to a more effective phytoremediation of Pb-contaminated soils. Interestingly, Epstein et al. (160) observed significantly higher levels of Pb in B. juncea plants grown on a soil containing 4.8
mmol kg1 Pb and treated with 1 mmol kg1 EDTA than in plants from a soil
containing 1.5 mmol kg1 Pb and treated with 5 mmol kg1 EDTA. The authors
conclude that a higher ratio of Pb to EDTA in the soil solution will result in
more Pb taken up by the plant. However, the addition of EDTA to metal-polluted
soils has the undesirable effect of reducing the plant growth significantly. For
instance, B. juncea plants grown in the presence of 5 mmol kg1 EDTA in the
soil produced a twofold greater biomass than plants treated with 10 mmol kg1
EDTA. Furthermore, at the higher EDTA concentration, the leaves started wilting, had a transpiration rate diminished by 80%, and had a relevant abscission
(160).
Interestingly, several strains of Pseudomonas and Bacillus stimulated Cd
accumulation in B. juncea seedlings from hydroponic solutions (76). Pilon-Smits
et al. (161,162) overexpressed the E. coli -glutamylcysteine synthetase (-ECS)
and glutathione synthetase (GS) enzymes in B. juncea. These transgenic plants
concentrated more Cd than normal plants in their shoots. They might represent
a strong tool for developing effective Cd phytoremediation strategies (82,83),
possibly also toward Pb and Hg ions. Differently, scarce improvement of Cd
accumulation in B. juncea with manipulated sulfate metabolism has been reported in experiments from Lee and Leustek (163). These authors conclude that
changes in gene expression may not always be indicative of a physiologically
relevant adaptive response to an environmental condition, in full agreement
with the concepts discussed by Sanita` di Toppi and Gabbrielli (81) and Meharg
(164).
Brassica spp., which tend to accumulate S, have also high ability to take
up Se, due to the chemical similarity between SO42 and SeO42. Se is usually
present in the environment under the following forms: elemental Se, selenide
(Se2), selenite (Se4), selenate (Se6), and organic Se (selenocysteine and selenomethionine; see Sec. 2.2) (165). Furthermore, dimethylselenide and dimethyldiselenide are forms of volatile Se (166). The oxidized forms, SeO32 and SeO42,
242
are soluble in water and consequently fairly available to plants. Possible variations in the metabolism of Se in different varieties of B. juncea should be taken
into account when screening for a specific variety able to clean up Se-contaminated soils (167). In general, B. juncea leaves accumulate Se to the highest concentration when Se is supplied as SeO42, followed by selenomethionine and
SeO32, whereas selenomethionine accumulation is favored in roots, followed
by SeO32 and SeO42. In addition, Se volatilization is closely correlated to Se
accumulation in roots (168).
In general, Brassica spp. may improve their ability of accumulating Se if
high levels of SO42 are present in the soil solution. B. juncea seems to be one of
the best plant species for Se phytoremediation because it grows rapidly, produce a
large biomass and efficiently volatilizes and accumulates Se (169,170). Phytoremediation of Se-contaminated sites involves accumulation of Se in plant tissues
(171), together with volatilization of Se both by plants (172) and by soil microrganisms. On average, Se can be accumulated in B. juncea tissues up to 1000 mg
kg1 dry weight (166). Interestingly, B. juncea cleaned up a SeO42-contaminated
soil in 3 years, removing up to 50% of Se within a depth of 75 cm (173). Recent
studies have shown that SeO42 accumulation in B. juncea root system may be
rate limiting for Se assimilation and volatilization, which at the same time can
be efficiently stimulated by rhizosphere bacteria (174,175). Since bacteria appear
to increase Se accumulation in B. juncea only when this heavy metal is supplied
in the chemical form of SeO42 (not in the form of SeO32 or selenomethionine),
it is likely that different Se uptake mechanisms are expressed in B. juncea plants
(176179). Thus, B. juncea plants in association with some rhizosphere bacteria
seem to be good joined forces in phytoremediation of SeO42-contaminated
sites.
In phytoremediation of Cr(VI)-contaminated soils, the reduction in planta
of the toxic Cr(VI) to the less toxic Cr(III) may be useful. Preliminary results
obtained by means of X-ray absorbance spectroscopy (XAS) revealed that roots
of B. juncea, as well as other mustard species (196), are capable of reducing
Cr(VI) to Cr(III). Thus, B. juncea may be of relevant interest also in remediating
Cr-polluted soils.
Marked potential in U hyperaccumulation in the presence of citric acid has
been shown in B. juncea, B. narinosa, and B. chinensis. U accumulation is a fast
process: U can be found in the shoots within 24 h after the treatment with citric
acid, and it reaches a maximum in about 3 days. B. juncea plants, when used for
U phytoextraction, could be harvested just a few days after being supplied with
citric acid. As reported by the authors, with this technique U accumulation in
shoots of B. juncea raised by more than 1000-fold within a few days. They conclude that applying this technique in the field will speed up the removal of U
from contaminated soils and will provide a cost-effective soil decontamination
strategy (180).
Brassicaceae
243
B. juncea may also be effective in phytoremediation of Zn-contaminated

soils. Interestingly, Ebbs et al. (181,182) used barley and B. juncea simultaneously, since the former had the capability of hyperaccumulating very high levels of Zn and the latter, when Zn was supplied in association with EDTA, was
able in turn to accumulate Zn not previously taken up by barley.
Finally, Anderson et al. (27,183) reported hyperaccumulation of Au in B.
juncea plants cultivated in Au-treated pots in the presence of ammonium thiocyanate. In these experimental trials, interesting preliminary results of possible Au
phytoextraction were obtained.
3.3
Thlaspi
Thlaspi has attracted the attention of many scientists because several hyperaccumulating species within this genus are suitable for phytoremediation of soils polluted with high levels of Zn, Cd, Ni, and Pb. A preliminary hydroponic or pot
screening needs to be developed to identify the best accessions within a species,
in terms of metal accumulation and best potential performances in phytoremediation, to overcome the great variability seldom observed among populations of
metal hyperaccumulators of the same species (102,181).
The superiority of T. caerulescens toward other plants was demonstrated
and explained with its extraordinary efficiency in sequestering metals in the shoot
(101,114). Furthermore, this species offers the possibility of multiple metal removal due to its proven ability to accumulate high levels of Zn, Cd, and Ni.
Initially, the time span required for complete or limited removal of metal contaminants from the soil was calculated assuming that subsequent crops would remove
metals at the same rates as the first crop (111,114). Baker et al. (111) calculated
that about 13 annual croppings were needed to reduce soil Zn content of a polluted
soil from 444 to 300 g g1 (limit set by the Commission of the European Community, 1986). However, if the metal dissolution rate in the soil decreases below
the potential extraction rate by the plant, due to possible changes in metal partitioning in the soil, the effectiveness of phytoremediation may decrease with time
(106). In a different approach, Robinson et al. (184) calculated the total number
of annual croppings of T. caerulescens required for removing half of the soil
metal, based on the assumption that this was the maximum amount that could
be extracted by the plant. Soils contaminated with less than 500 g g1 Zn or 20
g g1 Cd could be remediated to half these values in about 10 and 2 years,
respectively (184). Certainly the duration of metal phytoextraction is still an
overly long process in this case to be commercially attractive (111,185).
Although T. caerulescens is very efficient in taking up high levels of Zn,
Cd, and Ni from the soil as well as accumulating these metals in the shoots, the
use of this species for commercial scale phytoremediation (115,152) is limited
by its small size (about 15 cm high), slow growth rate, and rosette growth habit
244
(185,186). The mechanical harvest of these plants is very difficult, the dry weight
yield is too low (6-month-old plants of T. caerulescens yielded about 34 t ha1
dry weight) (111,185). One possible solution to this shortcoming might be the
transfer of Zn-hyperaccumulating properties from T. caerulescens into a high
biomass-producing plant (101) as a realistic possibility of making phytoremediation a commercial technology. Other species of the genus have been shown to
be very efficient in accumulating Zn from Zn polluted soils in a greater plant
biomass (185) but perhaps not particularly resistant to the high levels of metal
accumulated (187). On the other hand, the use of molecular techniques to transfer
the desired genes for metal hyperaccumulation from T. caerulescens into larger
plants is premature because, despite the fact that research has been done in this
direction, genes responsible for plant metal tolerance and hyperaccumulation
have not been fully identified in metal hyperaccumulators. Studies of the genetics
of metal hyperaccumulation in T. caerulescens have shown that Zn tolerance and
hyperaccumulation are separate traits (117) and generally metal tolerance is a
polygenic trait in other species of plant accumulators (188). Another possibility,
which has been successfully explored in two laboratories (186,187), is the production of somatic hybrids through fusion of protoplasts from parents bringing the
desired traits. Sexual incompatibilities between parents with widely divergent
backgrounds can be overcome, even though the somatic hybrids obtained might
be sterile or not viable. But the few fertile hybrids that survive can be backcrossed to one parent, and genes can be transferred between different genomes
via nonhomologous recombination (189). Most of the intertribal somatic hybrids
that were produced between T. caerulescens and Brassica napus (186,187) had
intermediate size. One of the two fertile hybrids obtained (hybrid 60/31) showed
both the desired characters of vigorous growth and highly increased resistance
to Pb, and the amount of Pb that this hybrid was able to extract from the soil
was similar to that of both parents (187). Also, some Zn-tolerant hybrids with
fast growth and erect growth habit were obtained and will be back-crossed with
B. napus to get viable seed (186).
Attempts to increase plant biomass production by growing T. caerulescens
in a more fertile environment (190,191) were not always successful. Different
varieties of T. caerulescens grown in pot and field experiments had different
responses to soil fertilizer application: one population produced significantly
higher plant biomass and extracted more Zn and Cd from polluted soils amended
with N, whereas the other produced more biomass but had reduced Zn and Cd
uptake when grown either on P or on N fertilized soils (191). In other field experiments with T. caerulescens (190), the plant proved to be adapted to growing in
an ultramafic environment. Only the addition of N fertilizer increased plant biomass, but it slightly reduced Ni and Zn concentrations in the shoot. Furthermore,
Ni and Zn levels in plant tissues were much lower than those found in wild plants,
probably due to a too short growing period and to the high growth rates achieved
Brassicaceae
245
under the experimental conditions. However, T. caerulescens showed a potential

in phytoremediation of soil Ni (190). Furthermore, the application of metal complexing agents such as EDTA to contaminated soils to enhance metal phytoextraction (181,192), which has been successful in increasing metal uptake in B. juncea
(74), might be used to increase metal uptake by T. caerulescens as well (193).
T. rotundifolium subsp. cepaefolium is a well-known Pb hyperaccumulator,
which accumulates up to 8200 g g1 Pb in the shoots (24). However, it is not
suitable for Pb phytoextraction because of its very low growth rate. Recently, B.
juncea has been found to have a more efficient translocation system that concentrates up to 34.5 mg g1 Pb in the shoot (73).
Progress in the area of soil phytoremediation is closely dependent on a
better understanding of the physiological mechanisms involved in Zn transport
and accumulation in root and shoot of hyperaccumulating plants. While several
studies on metal long-distance transport and storage in the leaf of T. caerulescens
have been published in the past few decades, only recently did Schwartz et al.
(194) and Whiting et al. (195) highlight the importance of root development and
function in T. caerulescens. In this species, roots showed a high affinity for Zn
and exhibited two major types of morphologies, depending on the presence of
Zn in the soil, with cluster roots in the presence of high Zn levels and finer
and longer roots in noncontaminated soils (194). T. caerulescens root architecture
seems to depend specifically on Zn form, concentration, and localization in the
soil. Plant roots tend to explore a rather large soil volume and colonize preferentially soil spots with high Zn concentration to ensure that the plants high
requirements for Zn are satisfied (194). Similar root proliferation toward localized
soil Zn enrichments was observed in T. caerulescens but not in T. arvense by
Whiting et al. (195). In the nonaccumulator T. arvense roots were shorter and
smaller than in the hyperaccumulator T. caerulescens and tended to proliferate
toward the noncontaminated soil. The discovery of these sophisticated Zn-scavenging mechanisms in T. caerulescens roots is essential for a better understanding
of metal acquisition in plants.
Future screenings for the selection of hyperaccumulating plants suitable
for phytoremediation will have to take into account not only plants with high
shoot biomass but also those with high root/shoot mass ratio and high specific
root length (195). The trait metal hyperaccumulator may result from efficient
root foraging for metals and preferential production of root biomass toward soil
metal sources.
ACKNOWLEDGMENTS
Thanks to Drs. H. Lambers, M. Tomaselli and L. Pignotti for their helpful revision
and advice. We are also grateful to Drs. C. Gonnelli and E. Torricelli and to Miss
C. Padovani for their thorough assistance with the artwork.
246
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9
Aquatic Macrophytes
M. N. V. Prasad
University of Hyderabad, Hyderabad, India
Maria Greger
Stockholm University, Stockholm, Sweden
Bruce N. Smith
Brigham Young University, Provo, Utah
INTRODUCTION
Angiospermous families, such as Cyperaceae, Potamogetonaceae, Ranunculaceae, Typhaceae, Haloragaceae, Hydrocharitaceae, Najadaceae, Juncaceae,
Pontederiaceae, Zosterophyllaceae, Lemnaceae, Typhaceae, etc., have aquatic
and semiaquatic habitats. While a great diversity of freshwater aquatic plants
carry out their complete life cycle floating on the surface (e.g., Eichhornia and
Lemna), others live on the surface but retain an attachment to the sediments
(Nymphaea). Many others are emergent with only the roots submerged (Ranunculus, Typha, Carex, etc.). A few species live completely submerged (Potamogeton,
Najas, Ruppia, etc.) while retaining a connection to sediments from which minerals are absorbed (Fig. 1a,b). All aquatic angiosperms have terrestrial ancesters
259
260
(a)
(b)
Prasad et al.
Aquatic Macrophytes
261
but, like dolphins and whales, have adapted to life in the water (1). While minerals
can be absorbed from water by leaves, for many plants rooted in sediments, root
uptake is paramount.
Macrophytes take up metals from water, producing an internal concentration several fold greater than their surroundings. They play a crucial role in biogeochemical cycling of trace metals. The submerged plant thickets in polluted
lakes are reported to accumulate trace metals to the tune of 103 104 or reduce
the water velocity there by accelerating sedimentation of the suspended fine particulate trace metals, which otherwise are toxic to the biota when present in the
interstitial waters in available form (2,3).
METAL UPTAKE BY DIFFERENT SPECIES OF

AQUATIC PLANTS
Heavy metals are enriched in all compartments of the environment (air, water,
and soil) by anthropogenic activities (4). Finally, the aquatic systems act as receptacle for several of these metals. For example, the metal concentration in river
waters increased several thousand fold by effluents from mining wastes. Background levels of metals in freshwater and seawater, as well as in sediments of
marine and lacustrine origin, are depicted in Figs. 2 and 3 (5).
FIG. 1 Vertical zonation in a tropical wetland plant assemblage. (a) A generalized representation of zonation consisting of seven zones that occur at the
land-water interface and the various life forms of plant species that occupy
each zone, including the position of above-ground structures and roots in
relation to the sediment surface and water table. (b) A typical zonation in
tropical wetland macrophytes. Floating islands consisting of Eichhornia crassipes, Lemma, Wolfia, etc. species, euhydrophytic zone comprising landemergent plants species encompassing Typha latifolia, Nymphea, rooted
submerged Vallisneria spiralis; and submerged free-floating, e.g., Ceratophyllum demersum. Aquatic macrophytes can be broadly divided into three categories: (i) Free-floating plants: Except roots, the whole body of the plant is
above the water, e.g., Eichhornia crassipes (water hyacinth), Ludwigia sp.
(water primrose), Pistia stratiotes (water lettuce), Lemma sp., Wolffia, and
Spirodela, etc. (duckweeds), Ceratophyllum demersum (coontail), Salvinia
sp. and Azolla (water ferns). (ii) Submerged (rooted) plants: These remain
submerged in water, e.g., Egeria densa (brazilian elodea), Elodea canadensis
(elodea), and Hydrilla, Vallisneria (tape grass). (iii) Emergent (rooted) plants:
These are rooted to sediments but emergent above the water, e.g., Alternanthera philoxeroides (alligator weed), Typha latifolia (cattail), Phragmites communis (reed).
262
Prasad et al.
FIG. 2 Background concentration of metals in seawater and freshwater (5).
Many environmental factors are known to modify the availability of metals

in water to aquatic plants. Such factors are chemical speciation of the metal, pH,
organic chelators, humic substances, particles and complexing agents, presence
of other metals and anions, ionic strength, temperature, salinity, light intensity,
and oxygen level, and thereby the redox potential (Eh) (Fig. 4) (4).
In lakes, pH is important for speciation and thus also for the availability
of metals to macrophytes. Most metal concentrations in water increase with decreasing pH, with the highest pH value of about 4. Especially in aquatic systems,
the redox potential (Eh) is important. At low redox potential, metals become
bound to sulfides in sediments and are thus immobilized. In water, salinity affects
the availability of metals, since high salinity causes formation of metal-chloride
complexes, which are hard for plants and other organisms to take up (56).
An equilibrium is maintained in the interface between the metals in water
and metals in the sediment (Figs. 5 and 6). Plants are able to take up metals
from these media. Sediment are considered as sinks and metals are therefore
accumulated in sediments in high concentrations. Table 1 details the terminology
used for transfer of metals from sediments to interstitial waters and plants. Water
and air function more or less as transport media for elements. It is known that
Aquatic Macrophytes
263
FIG. 3 Background concentration of metals in marine and fluvial sediments

(5).
metals have a shorter retention time in air and water compared with that of sediments. However, this depends on the element. For example, Pb has a very short
retention time in water, whereas for Zn it is longer. Therefore, plant uptake of
metal as well as the distribution within the plant tissues is influenced by the
retention time. Furthermore, there is an equilibrium in the interface between the
metals in the water and the metals in the sediment. This means that if some
external factor influences the equilibrium, the uptake of metals by plants may
also be affected (79). Such factors include resuspension, bioturbation, addition
of organic matter, and changed salinity. It has been shown that salinity decreased
the uptake of Cd, Cu, and Zn in submerged plants in hydroponic conditions (9).
However, if the system also included sediment, salinity gave an opposite effect,
increasing the Cd uptake in the submerged plant. The explanation was that in
the first case, CdClx complexes were formed in the water, and these complexes
are hard for the plant to take up. In the second case, addition of salt released Cd
bound in the sediment, probably by exchange with Cd and Na on the colloid,
thereby changing the equilibrium toward a higher Cd concentration in the water.
Thus, uptake of Cd by the shoot increased (9).
264
Prasad et al.
FIG. 4 Model of metal (Me) translocation in freshwater macrophyte-watersediment (9).
FIG. 5 Model of metal (Me) translocation in saline environment (9).
Aquatic Macrophytes
265
FIG. 6 Various useful methods for monitoring heavy metals using aquatic
macrophytes.
Different plant species take up different amounts of metals (919) (Tables

26). The concentration found in various plant materials varies from site to site
due to environmental pollution situation at that site. The variation within the
same species can therefore be large (Tables 2 and 5). There are also differences
in uptake between various metals. Not only the uptake but also the toxicity of
TABLE 1 Transfer of Metal from Sediments to Interstitial

Waters and Biota (33)
Term
Bioconcentration
Bioconcentration factor
Bioaccumulation
Biomagnification
Biominification
Explanation
Concentration in organisms greater than concentration in medium, i.e., concentration factor 1
(Shoot concentration shoot biomass root
concentration root biomass)/[(shoot biomass root biomass) duration of growth in
days] (28)
Increase in concentration with age or size of organisms
Increase in concentration in organisms at successively higher trophic levels
Decrease in concentration in organisms at successively higher trophic level
266
Prasad et al.
TABLE 2 Concentration Range of Lead and Copper in Different

Macrophytes Collected from 11 Sites of Two English Lakes, Ullswater and
Coniston Water (20)
Cu (g g1 dry wt.)
Pb (g g1 dry wt.)
Macrophyte
Shoot
Root
Shoot
Root
Potamogeton crispus
Potamogeton perfoliatus
Potamogeton, other spp.
Myriophyllum alterniflorum
Elodea canadensis
Littorella uniflora
Isoetes lacustris
1937
1327
14510
8490
9460
1480
1990
1860
1016
13550
11165
250490
18140
45330
95500
25180
37950
26590
18930
7310
151200
1406600
71700
312400
291000
34110
351500
405700
metals varies. Toxicity of different metals to Elodea canadensis and Myriophyllum spicatum is highest with Cu Hg As Cd Zn Pb (8).
Submerged plant leaves have a very thin cuticle. The leaves of submerged
plants are therefore very good at metal uptake directly from the water. The foliar
uptake of Cd by Potamogeton pectinatus was nearly 10 times higher than that
of Pisum sativum (9). Welsh and Denny (20) showed that lead was accumulated
in the shoots via absorption from the water.
Macrophytes take up heavy metals via roots from the sediment and via
shoots directly from the water. Therefore, the integrated amounts of bioavailable
metals in water and sediment can be indicated by using macrophytes (Fig. 7).
Plants can also evolve ecotypes pretty soon and thereby encounter unfavorable
conditions. Plants are also stationary, long-lived, and accumulate metals; therefore, they are suitable in monitoring of polluted sites. Metal concentration in
TABLE 3 Concentration Factors for Some Elements in Macrophytes

Element
Macrophyte
Elodea
Myriophyllum
Potamogeton
Najas
Nuphar
Phragmites
Zn
Fe
Co
Cu
6190
2670
1760
1080
530
1770
1950
1600
740
560
570
350
8770
6280
4064
3513
1268
3388
18.0
19.9
8.0
3.8
1.7
2.5
189
181
98
45
64
52
Aquatic Macrophytes
267
TABLE 4 Leaf Tissue Mn and Pb Concentrations (g g1 dry wt.) of

Representative Roadside Plant Species from Moderately High Traffic
Volume Roadsidesa
Plant species
Aquatic
Ranunculus aquatilis
Potamogeton pectinatus
Ruppia maritima
Lemna minor
Scirpus acutes
Chara spp.
Herbaceous
Asclepias speciosa
Kochia scoparia
Cardaria draba
Convolvulus arvensis
Lactuca serriola
Medicago sativa
Graminaceous
Distichlis spicata
Agropyron cristatum
Agropyron repens
Manganese
Lead
3
5
3
3
3
3
13,680
3,414
1,755
569
393
489
1,153
240
339
81
6
23
8.0
15.6
56.3
14.5
8.0
37.0
0.1
3.3
8.2
1.1
2.1
2.8
3
13
3
7
6
5
117.5
106.2
85.0
97.7
43.8
44.2
9.6
14.3
26.7
21.7
7.7
3.1
5.0
5.2
14.7
1.6
5.3
10.2
1.4
1.8
2.8
0.8
3.1
4.3
3
26
7
104.0 11.3
42.7 3.4
46.1 7.4
3.0 2.1
7.1 1.4
12.6 2.2
Each value represents the mean SE of n 3 plant samples.

Source: Data from Ref. 91.
plants must be related to the time of the year since the metal concentrations vary
in season (21). Aquatic plants also release metals through their leaves. Plants
that are used as bioindicators must retain the metals in their plant body. Ceratophyllum demersum, Myriophyllum spicatum, Potamogeton pectinatus, P. perfoliatus, and Zannichellia palustris have been proposed as bioindicators (2228).
3
REDISTRIBUTION DUE TO UPTAKE BY SEDIMENT
Bottom sediments have been polluted with heavy metals by industrial outlets
over decades. Due to the pollutants, but probably mainly due to reduced light
intensity caused by increased density of particles in the water column, the ability
of plants to grow on these bottoms will be low. The metals have been bound to
organic particles and sedimentated to the bottom. In the sediment, the redox potential is low and therefore most metals are firmly bound to sulfides.
When the industrial outlet has been shut down, new unpolluted sediment
has been deposited on top of the contaminated one and plants are able to colonize
268
TABLE 5 Concentration Range of Heavy Metals in Different Macrophytes Collected from 47 Sites of Shallow Costal
Areas, Stockholm, Sweden
Cd
Species
Potamogeton perfoliatus
Elodea canadensis
Ranunculus baudotii
Myriophyllum spicatum
Potamogeton pectinatus
Cu
Pb
Zn
Ni
Cr
0.72.5
1.31.7
2.02.8
0.73.3
0.52.9
0.94.0
1.82.6
2.64.7
0.47.3
0.62.7
7.358
2122
9.937
4.532
3.826
3.310
1826
1475
6.734
2.833
74520
8493
88377
65386
43171
20719
66106
112124
35185
17106
0.351
623.6
9.817
0.357
0.354
0.373
7.5109
2532
0.7148
0.3115
3.728
3446
6.512
4.925
5.517
2.330
1477
8.014
5.850
3.934
0.055.7
1.63.2
0.050.9
0.054.7
0.33.1
0.054.6
0.86.3
1.01.7
0.16.4
0.054.4
S, shoot; R, root.
Prasad et al.
(mean SE, n 3) in Utah, USAa

Category
Areas
Provo River
Utah Lake
Bear River
Sevier River
Clear Lake
Fish Springs
Plants
Ranunculus aquatilis
Potamogeton crispus
P. filiformis
P. pectinatus
Ruppia maritima
Najas marina
Carex spp.
Scripus maritimus
Distichlis spicata
Chara spp.
Cadmium
Iron
Lead
Manganese
2
3
0.7
0.5
0
0.8
0.2b
0.2b
0.2a
0.5a
0a
0.2a
1649
1748
489
948
93
127
277b
125b
79ab
392ab
13a
17a
17
65
7
1
10
5
4b
11c
1ab
0a
1ab
1a
3660
445
177
70
51
18
682b
143a
71a
13a
11a
2a
3
3
2
2
0.6
2
0.3
1.3
0.3
1
0ab
0.2ab
0a
0.2a
0.2a
0.7a
0.3a
0.2a
0.3a
0.4a
756
1035
4686
818
437
89
72
43
80
157
321ab
226b
367b
110ab
173a
25a
9a
19a
12a
36a
8
21
12
12
10
8
1
9
1
8
3a
11a
2a
3a
3a
3a
0.2a
1a
0.4a
2a
13,680
3741
3552
1458
243
23
40
305
27
10
1632b
2014a
271a
330a
137c
23c
17c
66c
11c
3c
Aquatic Macrophytes
TABLE 6 Aquatic Plant Tissue Metal Concentrations (ppm dry wt.) by Species and Wetland
a
Listed in approximate descending order of human impact. Means within columns not sharing a letter are significantly different,
P 0.05.
269
270
Prasad et al.
FIG. 7 Factors regulating metal bioavailability in sediments.
the bottom area. Colonization of macrophytes on shallow bottoms brings about

increased redox potential in the rhizosphere caused by the photosynthetic oxygen
which has been translocated down to the roots by the lacunar system (2931).
The increased redox potential increases the bioavailability of heavy metals to the
plant roots, thus facilitating the uptake of heavy metals by the roots. Part of the
metals up taken will then be translocated to the leaves and thereafter transferred
to grazing animals. Thus, macrophytes can increase the metal circulation in the
aquatic environment. Several macrophytes not only serve as indicators of metal
pollution but also redistribute the metals in the aquatic ecosystem (3239).
FACTORS INFLUENCING METAL UPTAKE
The uptake of metals, both by roots and by leaves, increases with increasing
metal concentration in the external medium. However, the uptake is not linear
in correlation to the concentration increase. This is due to the fact that the metals
are bound in the tissue, causing saturation that is governed by the rate at which
the metal is conducted away. The uptake efficiency (or accumulation factor) is
highest at low external concentrations. This is shown for Cd in solution culture
(22). Greger also demonstrated increased uptake from one and the same metal
concentration with increasing root absorption area (root mass). [External factors,
such as temperature and light, not only influence growth but also affect metal
uptake (Fig. 4).]
In aquatic macrophytes, the metal removal is accomplished through uptake
by roots, chemical precipitation, and ion exchange with or absorption of settled
clay and organic compounds (4042). Metal uptake is enhanced due to the pres-
Aquatic Macrophytes
271
ence of metal-binding ligands such as thiols or synthesis of metal-chelating

peptides/proteins, i.e., phytochelatins (4351) or surfactants such as linear alkylbenzenesulfonate (52), or combinations of metals in wastewaters may exert synergistic or antagonistic influence on metal uptake (53).
Aquatic sediments have a low oxygen content, and since metals are more
soluble and readily taken up in anoxic and low pH environments, aquatic macrophytes typically have much higher metal contents than nearby terrestrial plants,
even when the total metal content of the soils is equivalent. Risk assessment of
toxic trace metals in aquatic biota and use of inbuilt water-renovating strategies
on ecologically acceptable principles has gained considerable significance in the
field of environmental biotechnology and biotechnological methods of pollution
control (5457).
Aquatic macrophytes such as water hyacinth (Eichhornia crassipes) and
several duckweeds have attracted the attention of scientists for their ability to
accumulate trace metals. Using E. crassipes, heavy metal uptake from metalpolluted water, metal specificities on introduction and binding affinities of heavy
metalbinding complexes in root, purification and characterization of the metalbinding complex, and sorption of heavy metals from metal-containing solutions
have been extensively investigated (58).
Metals found in nature in more than one valance state are more readily
taken up by plants in the reduced form. Often the metal is reoxidized within the
plant tissues. People studying mineral nutrition in plants have usually distinguished mobile and immobile elements. Heavy metals, or even lighter metals in
excess, are often toxic to plants. Even at sublethal concentrations, physiological
tests, such as changes in pigment composition, photosynthesis, and respiration,
can reflect stress and predict future plant damage (5961).
5
WATER-SEDIMENT INTERACTIONS, METAL

AVAILABILITY, AND PARTITIONING
Sediments (particularly lacustrine) act as an important reservoir for metals of

anthropogenic source. The fluxes of metals to sediments may be gravitational,
i.e., settling of particulate material with its associated metals, or diffusive, i.e.,
in cases where dissolved metal concentrations in the water column exceed those
in the sediments interstitial waters, notably in acid lakes. Thus, in the upper strata
of lake sediments, higher levels of metals would be available than those in the
overlying water column (3239).
In aquatic ecosystems, the sediments and water interface serve as the habitat
for a diverse community of whatever aquatic macrophytes are prominent. Metals
present in surficial sediments in particulate form may exist either as constituent
elements present in the essentially insoluble products of physical weathering
form, i.e., lattice-bound (metal in detrital or residual minerals), or in a variety
272
Prasad et al.
of secondary forms [adsorbed on surfaces (iron/manganese oxides, clays, humic

flocs)], associated with organic form matter, sulfides, etc. The secondary forms
are more reactive and are more likely to be bioavailable. The cardinal factors
affecting the metal bioavailability to macrophytes in aquatic ecosystems are temperature, salinity, pH, Eh, and metal-metal interactions (Fig. 7).
Aquatic macrophytes have paramount significance in the monitoring of
metals in aquatic ecosystems (6265). In wetland ecosystems, a wide variety of
processes ranging from physicochemical to biological operate that can provide
a suitable situation for removal of metals (Fig. 7). For example, in the case of
acidic metal-rich mine drainage, the principal processes include oxidation of dissolved metal ions and subsequent precipitation of metal hydroxides, bacterial
reduction of sulfate and precipitation of metal sulfides, coprecipitation of metals
with iron hydroxides, adsorption of metals onto precipitated hydroxides, adsorption of metals onto organic or clay substrates, and, finally, metal uptake by growing macrophytes.
6
CONSTRUCTED WETLANDS FOR REMOVAL OF

METALS WITH MACROPHYTES
Constructed wetlands with reed beds and floating-plant systems have been common for the treatment of various types of wastewaters for many years. This strategy is currently gaining importance globally and expanding to address
contaminated/polluted soils and water bodies (Table 5). Bioconcentration of trace
metals by aquatic macrophytes is of special concern to human welfare and for
environmental protection and conservation (6668).
Natural wetland ecosystems are inherently complex. Hence, for the purpose
of treatment of metal-contaminated waters it is advantageous to construct separate
tanks within the treatment system, with each tank designated to perform a particular function maximally (occasionally more than one would be beneficial). The
design of wetlands constructed for the treatment of metal-contaminated waters
represents an attempt to identify and optimize the key processes that promote
the removal of a specific targeted metal. Alternatively, this includes suppression
of potentially interfering and competing processes.
Treatment of wastewaters/natural waters containing a single metal, such
as iron, can be achieved using a constructed wetland designated to optimize only
one of the possible factors. For example, removal of iron involves precipitation
of iron hydroxide in an aerobic environment. In contrast, if the water contains a
mixture of metals, e.g., iron and zinc, in high concentrations, the constructed
wetland may have to adapt different strategies, such as application of aerobic
and anaerobic processes. An aerobic environment promotes the precipitation of
aluminum and iron hydroxides and coprecipitation of arsenic. An anaerobic situa-
Aquatic Macrophytes
273
tion promotes the reduction of sulfates and the consequent precipitation of sulfides primarily for copper, cadmium, and zinc.
The precipitation of hydroxides is regulated by pH and the availability of
oxygen, which can be ensured as follows:
1. Construct shallow wetlands with a maximal water depth of about 3 m.
2. Minimize use of organic detritus because it demands oxygen for decomposition. It is preferable to use large inorganic substrate.
3. Design the landscaping into ridges and gullies to ensure continual mixing of the water within the system so as to prevent stratification of
water into oxygen-rich and oxygen-depleted zones.
4. Incorporate cascades (Fig. 8) at the point of influent to promote oxygenation of air.
5. Utilizing reed beds comprising Phragmites australis (common reed),
Typha latifolia (cattail), etc., which have the ability to transfer oxygen
to the root zone (Fig. 9).
Revegetation of mine tailings is a challenging task in view of the fact that
the metal mine tailings are usually very poor in nutrients, rich in toxic metal
content, and have little capacity to retain water. Furthermore, wind erosion of
mine tailings poses a serious environmental problem. All of these problems would
be averted if tailings were revegetated with wetlands using metal-tolerant wetland
plants. Wetlands have been used and constructed for the treatment of metal-con-
FIG. 8 Diagrammatic sketch of a cascade model of constructed wetland for

removal of contaminants using aquatic macrophytes, i.e., Typha spp. and
Scirpus lacustris. (Redrawn based on 79.)
274
Prasad et al.
FIG. 9 Generalized plan of the cross section of large-scale constructed wetland.
taminated water in recent years, indicating that wetland plants that hyperaccumulate metals can tolerate elevated metal concentrations (Fig. 10).
Glyceria fluitans (floating sweetgrass) is an amphibious plant found growing in the tailings pond of an abandoned lead-zinc mine in Glendalough, County
Wicklow (38). Greenhouse experiments demonstrated that G. fluitans could grow
in sand culture treated with high zinc sulfate solution. Further research confirmed
that two populations of G. fluitans, one from a metal-contaminated and the other
from a noncontaminated site, could be grown successfully on mine tailings with
a high zinc content. G. fluitans and two other wetland plants, Phragmites australis
and Typha latifolia, have since been grown on both alkaline and acidic zinc mine
tailings in field conditions under fertilized and nonfertilized conditions. Research
findings obtained so far indicate that G. fluitans can be easily established on zinc
mine tailings. It appears also to have a very low nutrient requirement, thus keeping fertilizer costs to a minimum during rehabilitation of mine tailings.
Wetlands have been constructed in Ireland for the passive treatment of
tailings water originating from a lead-zinc mine. Water originating from mine
tailings (slags) is often characterized by high metal and sulfate concentrations in
comparison with background levels. Conventional methodology of tailing water
treatment involves chemical treatment, which is a costly procedure requiring intensive chemical and labor inputs.
Therefore, more recently both constructed and natural wetlands have been
utilized for metal removal and wastewater quality control. Wetlands with their
Aquatic Macrophytes
275
FIG. 10 Plants exhibiting four strategies when exposed to elevated concentration of metals. Indicators: Plants in which uptake and translocation reflect
metal concentration in interstetial water and show toxic symptoms. Excluders: Plants that restrict the uptake of toxic metals into shoot over a wide
range of background concentrations. Accumulators: Plants in which uptake
and translocation reflect metal concentration in interstitial water without
showing toxic symptoms. Hyperaccumulators: Plants in which metal concentration is more than 0.1% in dry matter. (Redrawn based on 9.)
diversified macrophytes are known to retain substances such as metals from water
passing through them. Aquatic macrophytes encompass many common weeds
enable cost-effective treatment and remediation technologies for wastewaters
contaminated with inorganics and organics (7882).
7
ACID MINE DRAINAGE
Acid mine drainage (AMD) significantly impairs the quality of aquatic ecosystems. The exposure and oxidation of iron sulfide from coal mining results in
AMD. AMD is a significant source of water pollution for the Appalachian region
of the United States. There, estimates by the Bureau of Mines indicate that about
20,000 km of US. streams and rivers are impaired by AMD. Therefore, in recent
years several low-cost preventive and passive technologies have been developed
that utilize biological and natural chemical processes to remediate the contamined
276
Prasad et al.
mine waters without chemical treatment. In this regard, constructed wetlands

and its assemblage, anoxic limestone drains (ALDs), and successive alkalinityproducing systems (SAPS) have potential, and promising results have been obtained in several instances (83,84).
8
METAL TOXICITY AND TOLERANCE
Induction of phytochelatins is one of the principal mechanisms of metal detoxification in aquatic macrophytes, e.g., Hydrilla verticillata, Certophyllum demersum, and Vallisneria americana. The induction of phytochelatins concomitantly
reduced the cellular glutathione (4351). The well-established mechanisms of
metal toxicity and tolerance are depicted in Fig. 11 (8590).
FIG. 11 Sources of metals and metal speciation in aquatic ecosystem with

concomitant manifestations of toxicity and tolerance. Plant sources of heavy
metals are mining (smelting, river dredging, mine spoils and tailings, metal
industries, etc.), industry (plastics, textiles, microelectronics, wood preservatives, refineries, etc.), atmospheric deposition (urban refuse disposal, pyrometallurgical industries, automobile exhausts, fossil fuel combustion, etc.),
agriculture (fertilizers, pesticides, etc.), and waste disposal (sewage sludge,
leachate from land fill, etc.). Once metal ions are taken up by the cells, they
are complexed in the cytosol with glutathione and the derived phytochelatins
are transported into vacuole. Another notable cellular response is that some
metals interact with genes having metal-regulating elements at the promotor
region as found for metallothionein genes of animals (8590).
Aquatic Macrophytes
277
Methylcyclopentadienyl manganese tricarbonyl (MMT) has largely replaced tetraethyl lead as the antiknock compound in gasoline sold in North
America. Manganese and lead uptake by plants growing near highways was much
greater in aquatic plants than in terrestrial plants (Table 4) (91). Proximity to the
roadway and volume of traffic greatly contributed to plant concentrations of metals. The Mn concentration increased over a growing season in Potamogeton pectinatus. By October the Mn levels exceeded levels known to be dangerous to a
number of animals, including man (92). In June, the form in the leaves was Mn2.
By October it had shifted to predominately the Mn3 form (93). In iron and manganese the Mn2 form is mobile whereas the Mn3 form is immobile.
The concentration of Mn in aquatic plants is directly related to the density
and proximity of human activity (Table 6) (94). Time of exposure to the metal
is also an important factor (46). Plant tissues can accumulate very high levels of
Mn and other metals before showing visible symptoms (95). Long before visible
symptoms of toxicity appear, metabolic response to metals can be measured.
Lemna minor L. treated with methyl mercury for just 12 h showed altered metabolism (Fig. 12) (94). Both respiration and photosynthesis were inhibited by concentrations of methyl mercury greater than 0.1 g ml1. At lower concentrations
(0.0001, 0.001, and 0.01 g ml1) of methyl mercury respiration was stimulated
to levels higher than control plants, probably due to mobilization of defenses
against free radicals. Frond exposure to KCN and SHAM (salicylhydroxamic
acid) under methyl mercury treatments revealed variation in the cytochrome oxidase pathway (Fig. 12). Low levels of methyl mercury stimulated the cytochrome
pathway. Little change was noted in SHAM-sensitive respiration.
9
IRON PLAQUE AND ITS SIGNIFICANCE IN

AQUATIC MACROPHYTES
Several macrophytes have shown the deposits of ion oxides or hydroxides on

their root surface; these are called iron iron plaque. It is believed that wetland
plants exhibiting iron plaque are expected to tolerate the metal-contaminated environment. Iron plaque acts as a barrier for the uptake of toxic metals that get
adsorbed or immobilized by the plaque. The role of plaque in different species
and geochemical settings of the sediments warrants further attention (96).
In macrophytes of Bull Island, Dublin, Ireland, the anaerobic conditions
caused iron plaque formation on roots of plants due to oxidation of iron in the
rhizosphere. Using six different species of grasses and flowering plants, a comparison has been made on the degree of iron plaque formation. The techniques employed include light microscopy using JB-4 resin and staining with potassium
ferrocyanide. It was observed that metals accumulated in the rhizosphere of wetlands and in the burrow walls of sediment infauna. Preliminary studies have suggested that salt marsh plants and sediment infauna could significantly affect the
278
Prasad et al.
FIG. 12 Respiratory carbon dioxide from Lemna minor L. In the dark at different concentrations of methylmercury (MeHg) after 30-min exposures to the
metabolic inhibitors potassium cyanide (KCN) and salicyl hydroxamic acid
(SHAM), compared with control (94).
retention capacity of wetlands for metals through oxidation of the rhizosphere/

burrow wall. It was hypothesized that the thin oxidized soil zones around salt
marsh-plant roots and invertebrate burrows provide a source of iron oxyhydroxides an otherwise anoxic, chemically reduced soil, which would result in the
accumulation of various heavy metals and metalloids in the direction of the root/
burrow. At Bull Island salt marsh (Dublin, Ireland), Fe and As concentrations
were significantly higher in the rhizosphere of the two plant species (Spartina
townsendii and Atriplex portulacoides) and in the burrow wall of the lungworm
Arenicola marina, compared with the adjacent bulk soil. Zn showed the same
trend. A similar study, conducted at Rogerstown Estuary (Dublin, Ireland), found
that both Zn and As accumulated in the rhizosphere of the same plants, but Fe
did not. There are suggestions that the high organic matter content of this marsh
may somehow interfere with oxidation processes involving Fe and/or with the
binding of Zn and As to iron oxyhydroxides. More recent studies carried out at
two marshes in South Carolina, USA, have shown no significant accumulation
of Fe, Zn, or As in the rhizosphere of Spartina alterniflora (short and tall form)
Aquatic Macrophytes
279
or in the burrow walls of Uca pugnax and Arenicola christata. It seems that the
hypothesized accumulation processes that were observed at Bull Island cannot
be assumed to occur universally. The study highlights the heterogeneous chemical nature of salt marsh soils, even to within a few millimetersa factor that
must be taken into consideration when sampling in such systems and when managing wetland ecosystems (9799).
10 FOOD
Some aquatic plants are used as food and feed. Water spinach (Ipomea aquatica)
is commonly used as a vegetable and pig food in Thailand. It grows fast, easily,
and is cultivated wherever there is water, as well as in industrial areas and big
cities. It takes up and accumulates heavy metals to such an extent that its possible
threat to human health has been discussed (17,70). Resent studies have shown
FIG. 13 Productivity (t ha1 yr1) of selected aquatic macrophytes under specific conditions that enable them to act as potential agents of phytoremediation.
280
TABLE 7 Examples of Phytoremediation of Aquatic Ecosystems Using Macrophytes

Location
Application
Metal(s)
Medium
Chevron Corporation
Western USA
Phytoremediation Arsenic
Photovolatilization Selenium
Wetlands
Water
California, USA
Stockholm, Sweden
Photovolatilization Selenium
Phytoremediation Cadmium, copper
lead, and zinc
Drainage, water
Storm water ditch,
sewage wetland
Ukraine uranium tailings Rhizofiltration
137
Cesium
Strontium
226
Radium
Water
90
Elliot Lake, Canada
Phytoremediation
Watershed
Plant(s) used
Typha, Nymphea (water lily)
Variety of wetland plants and
rhizosphere bacteria
Salicornia
Phlaris arundinaceae
Typha latifolia
Scirpus sylvaticus
Hydroponic culture of crop
plants
Typha latifolia
Prasad et al.
Aquatic Macrophytes
281
that plants collected in Thailand, close to Bangkok, contained up to 530, 350,

and 123 (g g1 DW) of Pb, Hg, and Cd, respectively (Gothberg, Bengtsson,
Greger, and Karlsson, unpublished). The biggest problem seems to be Hg. According to FAO, the intake of Hg shall not overcome 43 g/week, which means
that not more than 250 g of plant per day can be eaten. This plant has also been
tested for removal of metals from wastewaters (100).
11 CONCLUSIONS
Aquatic macrophytes listed above have several negative characteristics, i.e., hardiness, ability to survive under adverse environmental conditions, and high productivity (Fig. 13). This enables them to act as potential agents of phytoremediation (Tables 7 and 8). Furthermore, an anaerobic environment in the water and
sediments renders elements in less oxidized forms. This increases the uptake
by aquatic plants and may make it possible to effectively use aquatic plants in
phytoremediation of wetlands. The partial pressure of oxygen in water is only a
small fraction of the 21% oxygen in air; the aquatic environment itself is a strong
defense against free radical formation. Unfortunately, low levels of oxygen also
greatly reduce the availability of energy liberated by catabolism. This can hinder
growth, particularly in stagnant ponds or other situations lacking natural aeration.
The aquatic environment, especially bottom waters and sediments, often
have a low oxygen content. Metals are more easily taken up by plants in reduced
form. Once in the plant they may be oxidized and become immobile. Thus,
aquatic macrophytes generally have a much higher metal content than terrestrial
plants and can be usefully employed in phytoremediation. Aquatic macrophytes
both living and nonliving serve not only as accumulators and indicators of pollution but also mitigate metal pollution to a considerable extent (101112).
TABLE 8 Wetlands and Their Service for the Clean-up of

Metal-Contanimated Aquatic Ecosystems
Wheal Jane tin mine in Cornwall, UK
Tennesse Valley Corporation, USA
West Glamorgon, Wales, UK
Ranger uranium mine in the northern territory, Australia
Weir International Mining Consultants, West Virginia, USA
Note: The above list is not exhaustive.
282
Prasad et al.
ACKNOWLEDGMENTS
One of the authors, MNVP, is grateful to the Department of Science and Technology, New Delhi (No. SP/SO/A21/97) dt 28-3-97, the Ministry of Environment
and Forests, New Delhi, Govt. of India (No.19/33/95-RE) dt 28-3-97, Council
of Scientific and Industrial Research, New Delhi (No. 38(901) 95/EMR-II dt 3010-1995.); the Third World Academy of Sciences, Italy (RGA No: 98-167 RG/
BIO/AS dt 21.12.98) and the Swedish International Development Agency, Sweden for providing assistance to deal with research in the area of trace metals.
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10
Aluminum Toxicity in Acid Soils
Plant Responses to Aluminum
Hideaki Matsumoto, Yoko Yamamoto,

and S. Rama Devi
Okayama University, Kurashiki, Japan
1
1.1
GENERAL ASPECTS OF ALUMINUM STRESS IN ACID

SOILS
Introduction to Acid Soils
Acidity is a major degradative factor of soils and covers an extensive area of

both tropical and temperate regions (Fig. 1). Acid soils occupy nearly 30% (3.95
bha) of the arable land area (1) in both tropical and temperate belts. Depending
on the degree of weathering and soil acidity, the world acid soils are classified
into eight groups (entisols, inceptisols, andisols, spodosols, alfisols, ultisols, oxisols, and histosols). Among them, oxisols, ultisols, and alfisols represent the majority of the acid soils in the tropical region (1). The cold and temperate acid
soils are mainly dominated by spodosols, alfisols, inceptisols, and histosols, and
the tropical belt is dominated by ultisols and oxisols.
Oxisols are characterized by having mainly the oxides of Fe and Al; they
consist of clay and highly insoluble minerals, such as quartz. This soil condition
289
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FIG. 1 Distribution of acid soils in the world. (Courtesy of Kluwer Academic

Publishers, London.)
represents the advanced stage of weathering and has a very low water-holding
capacity.
Ultisols are the most widespread type of soil in the warm humid regions.
Kaolinite is the major predominant clay mineral with minor amounts of silicates.
Alfisols are also the major predominant soils in the humid regions, with
kaolinite as the predominant clay mineral with admixtures of small amounts of
silicates.
The factors involved in producing acid soils are the prolonged leaching by
rainwater, soil-forming processes, and climatic conditions (2). In addition to these
natural processes, even the agricultural farming processes and management practices, such as high use of nitrogen fertilizers, removal of cations by harvested
crops, leaching and runoff of cations, resulted in lowering of soil pH (3). In many
industrialized areas, the atmospheric deposition of sulfur and nitrogen compounds
is a major source of proton influx to soils (2). More than the low pH of the soils,
the major problem associated with acid soils is the toxicity of aluminum (Al)
and manganese and the deficiency of phosphorus, calcium, magnesium, and potassium (4). In addition to these nutritional factors, the acid soils are also characterized by low water-holding capacity due to compaction of soils. Apart from
mineral toxicities, soil acidification is also known to change the species spectrum
of the forest soils by changing the microbial activity of the soils (5). Acidification
is also known to reduce the degradation of soil organic matter and alters cation
and nutrient flow in the ecosystem. In general, most of the acid soils have low
291
exchangeable bases (e.g., Ca, Mg, and K) mainly because of the low cation exchange capacity (CEC) of the soils (6). The oxides of Al and Fe in wet acidic
soils fix a large fraction of the phosphate, making it unavailable to plants leading
to lower crop yields. Therefore, Al toxicity is the major agronomic problem in
acid soils.
Since Al is the major component of acid soils, this chapter mainly focuses
on the understanding of Al toxicity and tolerance mechanisms in plants, which
would be highly useful for selecting and breeding plants for Al resistance and
to reclaim acid soils.
1.2
Al is the most abundant element in Earths crust, and globally soils with Al
problems cover up to 67% of the total acidic soils. In acidic soils, the phytotoxic
species of Al is solubilized to the levels that inhibit root growth and in turn
increase crop losses. Because of its agronomic importance, much research has
been focused on basic and applied aspects of Al toxicity and tolerance. Recently,
considerable effort has been made to understand the Al toxicity and tolerance at
whole-plant, cellular, subcellular, and molecular levels (7,8).
1.2.1
Chemistry of Aluminum
The chemistry of Al in water and soil is very complex as it exists in various

forms depending on the pH of the solution and its complex interactions with soil
and plant system (9). In the soil, Al exists in various forms, e.g., hydroxyoxides,
aluminosilicates, and aluminophosphates (10). Under acidic conditions, the toxic
Al species are released into solution. The released Al in soil solution can be
phytotoxic and poses a major environmental risk (11). There is a lot of ambiguity
about the toxic forms of Al that inhibit the plant growth. In solution, Al forms
numerous complexes with organic and inorganic ligands (12). It is assumed that
monomeric Al3 is more toxic than hydroxy mononuclear forms Al(OH)2 and
Al (OH)2 (13). Moreover, recently it has been demonstrated that under a certain
low pH, polynuclear Al, such as Al13 can also exist in solution, which is much
more toxic than the monomeric Al3 form (14). However, the possibility of the
existence of such polynuclear forms in soil solution is thought to be very low
(9).
1.2.2
Sites of Aluminum Toxicity in Plant Cell
In addition to its complex chemistry, there is controversy with regard to Al toxicity sites in plants (11,15). However, several reports reveal that Al is largely associated with cell walls and plasma membrane because of negative charges of the
cell wall pectin and phospholipids of the plasma membrane (7,16). Al toxicity
mainly depends on the Al concentration and on the duration of treatment and plant
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Matsumoto et al.
species. However, whether the toxic effect of Al is exhibited in the apoplasm or

symplasm is still not clear. In spite of many studies on the sites for Al toxicity,
no concurrent evidence for any one particular site specific to Al toxicity has been
known.
1.3
Aluminum Toxicity in Plants
The toxic effects of aluminum include stunted root growth, poor root hair development, swollen root apices, and stubby and brittle roots resulting in the decrease
in translocation of water, nutrient elements, and phosphate from roots to shoots
(7,8). However, there exists a wide variation among the species or the cultivars
of the same species with regard to Al tolerance. The primary toxic effect of Al
is the inhibition of root growth. In most situations, Al appears to be toxic to
actively dividing and elongating cells and not to mature cells (17). This has been
demonstrated with many plant species. In Al-sensitive maize cultivar, it has been
shown that the transition zone of the root apex where cells undergo rapid division
and elongation is more sensitive to Al toxicity than other parts of the roots (18).
Blancaflor et al. (19) also reported that Al toxicity is associated with elongation
zone of maize roots. In addition to roots, suspension-cultured tobacco cells are
more sensitive to Al toxicity at the actively dividing log phase than the stationary
phase, indicating that actively dividing cells are the targets for Al toxicity (20).
However, the cytotoxicity varied widely depending on the concentration and duration of Al treatment. In Al-sensitive plants, Al at concentrations as low as 10
M inhibits root growth within 10min after the Al application at pH 4.5 (21).
Al exists mostly as Al3 at this pH, but other forms of Al such as Al(OH)2
and Al (OH)2 also exist. However, the short-term results of Al toxicity mostly
correlated with monomeric forms of Al.
As the effect of Al varies with the concentration and duration of the treatment, Al is assumed to have multiple sites of action. Moreover, whether these
effects are primary or secondary responses is difficult to analyze. Since root
growth inhibition is the immediate response of the plants to Al, Al toxicity seems
to be external or apoplastic in nature (mainly the cell wall and outer portion of
plasma membrane). One of the proposed mechanisms for the toxicity of Al is
Al-mediated rapid and irreversible displacement of Ca from cell walls, especially
Ca bound to pectin. Because of the greater affinity of Al to pectin compared with
Ca, it is likely that the binding of Al makes the cell wall more rigid and prevents
its loosening, which is essential for cell expansion (21,22). It is also known that
Al alters the cellular Ca homeostasis and the blockage of Ca channels of the
plasma membranes (23). In tobacco BY-2 cells, the growth inhibition by Al is
linked to Al-induced decrease in cytosolic Ca levels (23). In addition to cell walls,
Al is known to displace Ca from the bridges between phospholipid head groups,
causing increased phospholipid packing due to its small size and trivalent charge.
293
In addition to displacement, Al is also known to lower the uptake of calcium by

cells (2427). Since Ca is a key element involved in the signaling process, the
change in Ca levels would affect various metabolic processes, which are linked
to Ca-dependent signal transduction (25,28).
Another important phenomenon associated with Al toxicity is the accumulation of callose. This has been considered as one of the sensitive markers of Al
toxicity. Several studies have shown that callose (-1,3-glucane) accumulation
starts immediately after the exposure to Al, and a strong correlation between Al
toxicity and callose accumulation has been observed (29). However, in Al-tolerant Arabidopsis mutants, no obligatory relationship has been observed between
Al uptake and callose accumulation (30).
Although several studies support the idea that a major portion of Al is
associated with cell walls or apoplast, recent studies with gaint cells of Chara
indicated that Al is able to pass through the plasma membrane and enter into
symplast (31). In addition to cell wall pectin, another potential site of Al binding
is the phospholipids of the plasma membrane (32). In spite of a few attempts to
understand the Al effects on membrane functions, no conclusive evidence has
been obtained. Al-mediated changes in the lipid composition have been further
demonstrated by Zhang et al. (33) who observed genotypic-dependent changes
in lipid composition using Al-sensitive and tolerant plants. However, it is still
uncertain whether there is any relationship between Al toxicity and the changes
in lipid composition. In addition to the changes in lipid composition, Al is also
known to induce some membrane proteins (34). However, the implication of their
role in Al tolerance is not clear.
1.4
Aluminum Tolerance in Plants
There has been considerable variation among the plant species in Al tolerance.
This is due to the variations in the accumulation of Al and the tolerance to the
toxic effects of Al. Al tolerance in plants is achieved by either avoidance or
internal tolerance (7,8). The plants acquire Al tolerance simply by avoiding the
metal accumulation. This has been achieved by exuding organic acids such as
malate, citrate, and oxalate from the roots, which chelate the toxic Al rendering
it nontoxic to plant (3537). On the other hand, plants developed some internal
resistance mechanism(s) against Al. The factors that favor Al exclusion include
(a) release of organic ligands such as malate, citrate, oxalate; (b) increase of
rhizospheric pH by roots; (c) increased binding to cell walls; (d) decreased permeability of plasma membranes to Al influx; and, (e) binding of Al in the mucilage.
Among them, there is increasing evidence for the chelation of Al by organic
acids. This is evident from the studies showing that the external application of
organic acids protects the plants from Al toxicity. The exclusion of organic acids
by Al has been demonstrated in several plants. However, the type of organic acid
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Matsumoto et al.
secreted varies with the plants. The high resistance of buckwheat plants to Al
has been ascribed to internal detoxification as well as rapid exclusion of oxalic
acid from the roots (38). In wheat, malate exudation is important for the chelation
of Al (39). Using near-isogenic lines, Delhaize et al. (35) convincingly demonstrated the role of organic acid excretion in Al tolerance. In maize, Pellet et al.
(40) demonstrated the release of citrate from the Al-tolerant cultivar. These results suggest that organic acid exudation play a major role in Al tolerance. However, studies with wheat cultivars showed that malate exudation did not always
correlate with the root growth in the presence of Al, suggesting that there could be
other resistance mechanisms operating in conferring Al tolerance. For instance, in
Al-tolerant Arabidopsis mutants, Al is known to increase the rhizospheric pH by
increased influx of H ions at the root tip region (41). Degenhardt et al. (42)
provided the first evidence in support of the role of increased rhizospheric pH
in Al tolerance using Arabidopsis mutants. In addition to rhizospheric pH change,
some of the mutants of the same plant released organic acids in response to the
exposure to Al, indicating that more than one tolerance mechanism exist in plants
(41).
In addition to these metal exclusion processes, it has been postulated that
Al binding to mucilage could play an important role in Al resistance (43).
Although more information is being added to the literature, there is no
convincing evidence to support any of these hypotheses. However, with recent
technical advances in dealing with Al, satisfactory insight into Al toxicity has
been gained. The mechanism of Al toxicity and tolerance are discussed in detail
in subsequent sections of this chapter.
2
2.1
PLANT RESPONSES TO ALUMINUM STRESS: THE

WHOLE PLANT
General Aspects of Plant Growth in Acid Soil
Aluminum (Al) is the most abundant metal in soil, comprising approximately

7%. A genotypical difference in Al toxicity or tolerance exists among plant species, and the growth of plants in acid soil is better in the tolerant genotypes.
Therefore, the effect of acid soil on plant growth is strongly related to the toxicity
of Al. Al has been known to inhibit the root growth of barley and rye in acid
soil since 1918 (44). Soil acidity seems to be gradually increasing due to environmental problems, such as acid rain. Furthermore, the anticipated burst of the
global population in the future has drawn attention to Al toxicity and tolerance
in acid soil in the past few decades (8,45,46).
2.2
Inhibition of Root Elongation by Aluminum
Inhibition of root elongation is the first visible symptom under Al stress. Inhibition of root elongation in Al-sensitive maize occurred within 30 min (47). In
295
most plant species, root elongation is markedly inhibited by Al at a micromolar

level at low pH such as 4.5 in a simple solution containing Ca2 alone. However,
the rate of inhibition is decreased by coexistence of other ions. A change of
the negativity of the root surface upon the association with cations lowers the
accessibility of Al to the root.
The inhibition of root elongation by AlCl3 in Al-tolerant (Atlas 66) and
Al-sensitive (Scout 66) wheat is shown in Fig. 2. Root elongation in Scout 66
was apparently inhibited by a 3-h treatment with 5 M Al but that of Atlas 66
was not inhibited up to almost 10 times higher concentration (48). The inhibition
of root elongation was accompanied by a decrease of cell viability, which was
detected by staining with propidium iodide, due to the damage of plasma membrane under Al stress. The decrease of cell viability coincided with the suppression of root elongation (49).
2.2.1
Localized Accumulation of Aluminum in Roots
The region of Al accumulation and inhibition of root elongation has been suggested to be restricted to the root apex including the root cap, meristem, and
elongation zone (7,50). When Al-tolerant (Atlas 66) and Al-sensitive (Scout 66)
wheat were treated with 50 M Al and stained with hematoxylin, only the apices
of root were stained in both cultivars. In Atlas 66, the root apex exposed to 20
M Al for 48 h was strongly stained but that exposed to 5 M Al was only
faintly stained. On the contrary, the root apex of Scout 66 exposed to 5 M
was stained strongly (49) (Fig. 3). These results indicate that the amount of Al
accumulated in the growing root tissue is related to the sensitivity of the plant
to Al and that tolerant wheat has a specific mechanism to excrete Al from the
root apex.
2.2.2
Morphological Change of Cells Induced

by Aluminum Toxicity
Since the shortening of roots caused by Al is accompanied by an increase in the

diameter of the root tip, the diameter of the second layer cells of the cortex in
Atlas 66 was measured. In control roots, the cells in the portion 0.63.2 mm
from the root cap junction are markedly long and small in diameter (Fig. 4). This
indicates that this portion is the elongation zone. Al treatment inhibited the increase in the cell length but increased the diameter of root cells (50). The ratio
of length to diameter of cells in the control root was three to four times larger
than that in the Al-treated roots. In particular, the cells in the second and third
layers of the cortex were highly swollen laterally. The swollen cells were characterized by the drastic accumulation of lignin on their cell wall. These results
indicate that the root zone where the elongation was inhibited by Al is the zone
where Al is accumulated. Recently, it was found that the distal part of the transition zone of the root apex, where the cells are undergoing a preparatory phase
for rapid elongation, is the primary target of Al in corn (18).
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FIG. 2 Seedlings of Al-tolerant (Atlas 66) and Al-sensitive (Scout 66) wheat.
Seedlings were grown in the presence or absence of Al (pH 4.5) in 100 M
CaCl2 solution for 5 days. Top (Scout 66): 0, 1, 2, 5, and 10 M Al from left;
bottom (Atlas 66): 0, 5, 10, 20, and 50 M Al. (From Ref. 48.)
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FIG. 3 Hematoxylin-stained roots of Atlas 66 and Scout 66. Seedlings were

grown in the presence or absence of Al for 48 h. (A) Atlas 66 treated with 0,
5, and 20 M Al from the left. (B) Scout 66 treated with 0, 1, and 5 M Al from
the left. Bar indicates 1.0 mm. (From Ref. 49.)
2.2.3
Inhibition of Cell Division by Aluminum
Cell division in the meristematic zone of the root measured by counting the metaphase cells is inhibited by Al (51,52). Compared with the time required for the
inhibition of root elongation, a longer time is needed for the inhibition of cell
division under Al stress. This means that the primary action of Al is the inhibition
of root elongation. The contribution of cell division is only 12% of the root
elongation. However, inhibition of root elongation is substantially caused by the
inhibition of the elongation of the cells in the elongation zone, which is derived
from the adjacent meristematic zone; Al toxicity kills the plant root by inhibiting
cell division. Thus, the binding of Al to DNA or chromatin that induces the
condensation of their structure concomitant with decreased template activity may
cause the inhibition of cell division (5355). Additional evidence for the inhibition of cell division is the disruption of phragmoplast and spindle microtubules
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FIG. 4 Effects of Al on the length and diameter and the ratio of length to
diameter of root cells in the second layer from surface in Atlas 66. Roots were
treated with or without 20 M Al for 24 h. Data are means of five or six samples ( SE). (From Ref. 50.)
299
in the logarithmic growth cells under Al stress (56). Also, the observation of the
extrusion of nucleolor materials from the nucleus to cytoplasm supports the above
speculation (57).
2.3
2.3.1
Physiology of Aluminum Toxicity

Aluminum Toxicity and Cell Wall
It is important to elucidate the localization of the absorbed Al in the cell in order

to understand the toxicity of Al. The major portion ranging from 30% to 90%
of the absorbed Al is found in the apoplast (58). Externally applied Al binds to
the binding site in the apoplast immediately. A significant correlation between
the amounts of Al and phosphate was found in the cell wall. Thus, Al phosphate
in the cell wall may act as a barrier for the transport of Al into the cytosol. Pectin
has been proposed to be a candidate of the cell wall component to which Al
binds (58,59). However, research on the binding of Al to pectin in vivo is scanty
(60). Blamey et al. showed that the addition of Al to an artificial Ca pectate
membrane reduced water permeability (59). This may be related to the change
of topology of apoplast in vivo, which may regulate the apoplastic transport.
2.3.2
Callose Formation
Callose formation on plasma membrane is a specific phenomenon under Al stress.

Synthesized callose is deposited in the apoplast. Supply of 50 M Al for only
30 min induced the callose formation in the root apex in soybean seedlings (61).
When roots were exposed to Al at a concentration greater than 10 M, the callose
concentration in the apical 1030 mm of the root tip was inversely correlated
with the root elongation in cow pea. Therefore, callose formation is related to a
potent parameter for Al toxicity. Recently, callose was found to accumulate in
plasmodesmata in wheat root, causing the inhibition of cell-to-cell trafficking of
molecules through plasmodesmata (M. Sivaguru et al., personal communication,
2000).
2.3.3
Plasma Membrane
The plasma membrane is the first candidate for the target of Al because of its
outermost location in the cell and high content of phosphorus as phospholipid.
Al has a 560-fold higher affinity for the phosphatidylcholine surface than Ca2
(62). Al3 at 5 106 M can neutralize the negative surface charge of the plasma
membrane and cause a shift of potential of the membrane surface from 30
mV to 11 mV. Interestingly, the depolarization of the surface charge ( potential) occurred mostly at the root apex where Al was also localized the most in Altreated squash (Ahn et al., personal communication, 2001). In Al-tolerant wheat
cultivar Dade, the membrane potential was rapidly and significantly depolarized
by exposure to Al at high concentrations. The maximum depolarization was 150
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Matsumoto et al.
mV at 150 M AlCl3. In contrast, the membrane potential of the Al-sensitive

cultivar Romano was depolarized only slightly by Al (63). Binding of Al to
plasma membrane caused the decrease of membrane fluidity (64). Lipid peroxidation in the root tips of soybean was enhanced only after a long-term treatment
with Al (65). This lipid peroxidation was intensified by Fe2. The enhanced lipid
peroxidation by oxygen free radicals is a consequence of the action of Al on
membrane structure. The physiological function of plasma membrane is also affected by Al. Plasma membrane H-ATPase activity was inhibited concomitant
with depolarization of the membrane surface potential in root apex in Al-treated
squash (Ahn et al., personal communication, 2001). In cortex cells in the intact
roots of Northern red val (Quercus rubra), Al significantly altered the activation
energy required to transport water (72%), urea (9%), and monoethyl urea
(7%) across the cell membrane. Ion transport process was also altered by Al.
At 100 M Al, the influx of Ca2, NH4 and K was inhibited by 69%, 40%,
and 13%, respectively, but the flux of NO3 and PO43 was enhanced by 44%
and 17%, respectively (66). This is caused by the Al-induced positive charge of
the membrane. Furthermore, Al blocked the inward-rectifying K channel. Contrary to plasma membrane, the effect of Al on tonoplast is scarcely investigated.
In barley root, the ATP- and PPi-dependent H transport activity of the tonoplast
vesicle is increased by Al stress. The defense mechanism of barley may be related
to the maintenance of H homeostasis in the cytoplasm through the sequestration
of Al into the vacuole by the activation of H transport activity (67).
2.3.4
Calcium
Much work has been done on Al toxicity in terms of Ca metabolism (68). Ca2
influx was rapidly inhibited at the root apex in correlation with the inhibition of
root growth in wheat (21). It is interesting that the inhibitory effect of Al on Ca2
transport is stronger in Al-sensitive wheat (Scout 66) than in Al-tolerant wheat.
The Al-induced inhibition of Ca2 uptake was rapidly recovered by the
removal of Al from the solution. Not only influx but also translocation of Ca2
from root apex to the basal part was similarly repressed by Al (69). The antagonistic effect of the large amount of Ca2 on Al toxicity is known (70) (Fig. 5). Al
increased membrane permeability to nonelectrolytes such as urea but decreased
the membrane permeability to lipids and water. The effects of Ca2 on the permeability were opposite those of Al3 (71). One of the plausible inhibitory mechanisms of Al on Ca2 metabolism is the displacement of apoplasmic and membrane
Ca2 by Al. In the isolated cell wall equilibrated in 50 M Ca2 at pH 4.4, 100
M Al displaced more than 80% of the Ca2 with a half-time of 25 min (72).
Recently, several researchers cast doubt on the disorder of Ca2 metabolism by
Al. Treatment of wheat with Al severely inhibited root growth but not Ca2 uptake. Some ameliorating cations that severely inhibit root growth depress Ca2
uptake in the presence of Al but not in its absence. The conclusion of Kinraide
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FIG. 5 Ameliorating effect of Ca2 on the inhibition of pea root elongation

caused by Al. Three-day-old pea seedlings were treated with 5 M AlCl3 and
CaCl2 at various concentrations for 24 h. Each point is the mean of results
from five samples in each case. (): control, (): Al treatment. (From Ref. 70.)
et al. was that the Ca displacement hypothesis fails to explain Al toxicity, and
that amelioration by cations occurs because of decreased membrane surface negativity and the consequent decrease in Al3 activity on the membrane surface (73).
Furthermore, an increase in cytoplasmic free Ca2 or [Ca2]cyt, was greater in Alsensitive wheat than Al-tolerant wheat, in spite of the general understanding that
Ca2 influx is inhibited by Al. The Al-related increase of [Ca2]cyt was correlated
with the inhibition of root growth and Al-induced increase in [Ca2]cyt was recovered by removing Al. This disturbance of [Ca2]cyt homeostasis can be one of
the mechanisms of Al toxicity (74). However, there is still a discrepancy in the
involvement of Ca2 metabolism in Al toxicity, and more work is needed to
clarify the feature of Al-Ca interaction.
2.4
2.4.1
Aluminum Tolerance
Genes Responding to Aluminum
Unlike animals, higher plants are usually fixed in the soil by their roots and thus
cannot escape from the stress. Therefore, plants have been forced to develop a
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specific way to avoid stress in the soil. Al-tolerant genes have been searched at
different levels. At the chromosomal level, genes for Al tolerance in the mediumtolerant wheat variety Chinese Spring were found to be localized on chromosome arms 6AL, 7AS, 2DL, 3DL, 4DL, and 4BL, and on chromosome 7D (75).
Major genes for the tolerance in rye seems to be located on 3R and 6RS. In Altolerant Atlas 66 wheat, D genome determined the tolerance to acid soil and
consequently contributed to the increased adaptation of the hexaploid wheats
during their evolution. However, not all genes for Al tolerance in Atlas 66 are
located on the D-genome chromosome (76). From the molecular research, genes
encoding glutathione S-transferase, peroxidase, blue copper-binding protein, phenylalanine ammonia lyase, 1,3--glucanase, cysteine proteinase, etc., were found
to be induced by Al stress (7779). However, various materials and experimental
conditions were used in these studies, and further research is needed to evaluate
whether these Al-induced genes are relevant to the mechanism of tolerance from
an agricultural point of view. So far many mechanisms are proposed for the
tolerance of plant against Al toxicity. Studies on the mechanism of exclusion of
Al from the rhizosphere which represses Al toxicity may be interesting.
2.4.2
pH Regulation in the Rhizosphere
The solubility of Al depends on the solution pH. The most toxic Al3 is solubilized at a pH lower than 4.5. Therefore, plants can reduce Al toxicity by increasing the pH in the rhizosphere, resulting in the formation of less toxic Al. The Altolerant Arabidopsis mutant alr-104 alkalinized the rhizosphere in the presence of
Al but the wild type did not. Alr-104 induced a twofold increase in net H influx
only at the root tip, causing a rise in the pH at the root surface by 0.15 unit.
Thus, the Al resistance in alr-104 is caused by the Al-induced pH increase in the
rhizosphere. In the absence of Al, no difference in root H fluxes was observed
between wild type and alr-104 (42).
2.4.3
Mucilage
Al forms a chelate complex with ligands and becomes less toxic. Mucilage and
organic acids are considered to be natural Al ligands that are excreted from the
roots. Root apices of most plant species are covered with a mucilaginous substance that is exuded from root cap cells. Mucilage consists mainly of polysaccharides containing uronic acid and has a high Al binding capacity. Fifty percent of
the total Al of root apices was associated with mucilage in cowpea (80). Al bound
to mucilage accounted for approximately 2535% of the Al remaining after desorption in citric acid solution in the wheat root. It has been postulated that the
binding of Al to mucilage is one of the Al-tolerant mechanisms. This was supported by the demonstration that removal of mucilage prior to treatment with Al
facilitated accumulation of Al in root apices and enhanced Al rhizotoxicity in
cowpea (80). On the contrary, mucilage of Zea mays was not effective in pre-
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venting Al-induced root inhibition in hydroponic culture. The total binding capacity of the mucilage was too small, although mucilage strongly binds Al and the
bound Al was not phytotoxic (81).
2.4.4
Organic Acids
The most powerful candidate for the natural Al-chelating substance is an organic
acid such as citrate, malate, and oxalic acid. The first related evidence is the
excretion of citrate from snapbean root under Al stress. The root of the Al-resistant cultivar excreted 70 times more citrate in the presence of Al than in the
absence of Al and 10 times more citrate than the Al-sensitive cultivars (82). Altolerant wheat cultivars excreted 5- to 10-fold more malic acid instantly after
exposure to Al stress. The root apex is the primary region of malic acid excretion,
and the excretion is specifically induced by Al not by other trivalent cations.
Organic acids are supposed to be excreted through the anion channel of the
plasma membrane. Buckwheat, which is tolerant to acid soil, excretes oxalic acid
promptly after exposure to Al (83). Taro also excretes oxalic acid. The mixture
of citric acid and Al at a molar ratio higher than 1 : 1 did not inhibit the root
elongation in wheat (Fig. 6). The excretion of citrate is a rather slow process in
comparison with that of malate and oxalic acid and needs the biosynthesis by
increased activity of citrate synthase. In this respect, it was recently reported that
the transgenic plants of tobacco and papaya to which the citrate synthase (CS)
gene from Pseudomonas aeruginosa was introduced together with the 35S promotor of calliflower mosaic virus was tolerant to Al. They accumulated a large
amount of citrate and excreted it from the root (84).
However, the regulation of the excretion of organic acids by the Al signal
is important because continuous excretion of organic acids due to the overexpression of the genes may consume an extraordinarily large amount of energy and
carbon. This regulative mechanism should be solved from a practical point of
view. It is interesting to know why excretion of organic acid under Al stress is
different among plant species. The effect of organic acid on Al detoxification
depends on the stability constant of the complex of organic acid and Al.
2.5
Beneficial Effect of Aluminum
Some plants are known to grow better in the presence of Al. The beautiful blue
color of hydrangea (Hydrangea macrophylla) is caused by the specific pigment,
delphinidin diglycoside, which chelates with Al. Hydrangea can grow under high
Al stress and contains a large amount of Al in the cell sap of leaves. Al was
found to be chelated with citrate at a molar ratio of 1: 1 (Al/citrate) forming less
toxic Al (85). Tea contains as high as 30,000 ppm Al in epidermal cells of old
leaves but only 600 ppm in young leaves (86). Al is localized in epidermal cells
of old leaves. The shape of epidermal cells varies considerably between old and
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Matsumoto et al.
FIG. 6 Effect of molar ratio of Al to citric acid on the root elongation of wheat,
Scout 66. The Al concentration was 50 M with 0.1 mM CaCl2 (pH 4.5). Seedlings were cultured in the solution containing Al and citric acid at various
ratios for 9 h. Error bars represent SD (n 3). (From Ref. 91.)
young leaves. The epidermal cells expand horizontally and the transversal length
is greater than the longitudinal length in old leaves. The well-thickened cell wall
is a feature of epidermal cells of old leaves. Al may be accumulated in the cell
wall during thickening (Fig. 7). Also, the strategy of tea plants against Al toxicity
is that most of Al is bound to catechine, although some Al binds to phenolics
and organic acids (87).
Al promotes the growth of tea, which is characterized by the stimulation
of new root formation (Fig. 8). The mechanism of the promotive effect of Al is
unknown, but one explanation is the regulation of phosphorus utilization by the
formation of an Al-phosphate complex because the tea plant is sensitive to an
excess amount of phosphorus, which is harmful for the growth of tea (88). Growth
of Melastoma malabathricum, Melaleuca cajuputi, Acacia mangium, Hydrangea
macrophylla, Vaccinium macrocarpon, Polygonum sachalinense, and Oryza sat-
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FIG. 7 Upper epidermal and palisade cells of old (left) and young (right) tea
leaves, 2800. Transverse sections were embedded in epoxy resin and
stained with iodine. (From Ref. 86.)
iva are stimulated more or less by Al. Plants whose growth is stimulated by Al
are classified according to the criteria of Al accumulation: (a) Al excluders such
as M. cajuputi, A. mangium, O. sativa; (b) Al root accumulators such as V. macrocarpon and P. sachalinense; and (c) Al accumulators such as M. malabathricum and H. macrophylla (89). The mechanism of tolerance in H. macrophylla
has been investigated intensively and the internal chelation of Al with citrate
(mol ratio 1 :1) was detected. However, the mechanism of growth stimulation
by Al is not known.
M. melabathricum, M. macrocarpon, and A. mangium are well adapted to
low-pH soils, such as peat soils and acid sulfate soils in the lowland tropics of
Thailand and Malaysia. V. macrocarpon and P. sachalinense are also well
adapted to peat soil and low-pH soils in the temperate region. Growth of tropical
plants is enhanced by Al and NH4 application. In all plant species, the pH of the
culture solution is decreased and the concentration of soluble Al and P is increased by the application of NH4 , which promotes growth (90). Roots of M.
cajuputi excrete a large amount of citrate, and the excretion is slightly increased
by Al treatment. The mechanism by which Al benefits the growth of plants is
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Matsumoto et al.
FIG. 8 Tea seedlings grown in the cultural solution for 100 days with (right)
or without (left) 103 M AlCl3. (From Ref. 86.)
not clear and may vary depending on the plant genotype and growth condition,
especially mineral composition in the medium.
3
3.1
PLANT RESPONSES TO ALUMINUM STRESS: PLANT

CELLS IN SUSPENSION CULTURE
Cultured Plant Cells as a System to Analyze Cellular
Metal Toxicity and Tolerance
Plant cells in suspension culture are useful to analyze responses to toxic metal
ions (eg., Al, Na, Cd, Cu) at cellular and molecular levels. The physiological
307
conditions of cells in suspension culture can be more easily adjusted than those
of whole plants in water culture. Thus, it is possible to prepare a physiologically
homogeneous cell population in large quantities. Furthermore, we can easily isolate cell lines exhibiting a metal-tolerant phenotype. However, it is not always
possible to examine whether the tolerant phenotype selected is heritable or not,
since cells in suspension culture gradually lose the capacity to regenerate intact
plants. In addition, in suspension culture the cells are always shaken to supply
oxygen, which is also a kind of physiological stress. In spite of these disadvantages, cultured plant cell systems are useful for supporting studies of whole-plant
systems. Several research groups have successfully used cultured plant cells for
physiological and genetic studies of Al toxicity and tolerance. Here we describe
the general precautions for handling a suspension cell system for analyses of
metal toxicity and tolerance. Then we describe the mechanisms of Al cytotoxicity
and tolerance elucidated by use of suspension-cultured cells.
3.2
3.2.1
General Precautions for Handling Suspension-Cultured

Cells for Stress Studies
Cell Lines
Plant cells in suspension culture are often derived from the callus generated from
plant tissues (92). At the beginning of the suspension culture, there may be a
mixture of cells showing heterogeneous characters, but the cells may maintain
the capacity to regenerate intact plants. Thus, the cells during the primary cultures
should be used for the isolation of stress-tolerant cell lines, if the regeneration
of intact plants from selected cells are planned (see Sec. 3.4.1). Instead, established cell lines, which were purified and have cultured for a long time without
apparent changes of phenotype, seem to be composed of homogeneous cells.
Thus, the reproducible responses to metal ions can be expected in the established
cell lines, indicating that the established cell lines are useful for analyses of stress
physiology (see Sec. 3.3) as well as for the isolation of stress-tolerant cell lines
or genes (see Secs. 3.4.2 and 3.5).
Several established cell lines from several species are available. We have
used a tobacco cell line SL originally generated from pith callus of Nicotiana
tobacum L. cv. Samsun (93,94). Another tobacco cell line, BY-2, is a kind of
standard cell line, which has been widely used for physiological studies
(95).
3.2.2
Cellular Growth Conditions
Plant cells in suspension culture are maintained by the transfer of portion of a

fully grown cell suspension into new medium. In the case of tobacco cell line
SL, cells do not grow just after the transfer (lag phase), begin to grow logarithmi-
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cally for a few days (log phase), and gradually stop growth (stationary phase)
(20).
Physiological conditions of cultured cells seem to change depending on the
growth phase. Actually, Al sensitivity of tobacco cells was higher in a log phase
than that in a stationary phase (20). Therefore, it is necessary to check the growth
condition of cells before the start of experiments. The growth rate of cells can
be roughly estimated by the population doubling time.
3.2.3
Treatment Medium
The metal ion of interest may interact with medium components and may affect plant
cells differently depending on the treatment medium. Al ions have strong affinity
with oxygen donor ligands such as phosphate (Pi) and carboxylate groups. Therefore,
Al forms insoluble precipitates with Pi in medium and exhibits lower toxic effect.
When the treatment time with Al is within 24 h, tobacco cells are not
starved of Pi (96). Therefore, Pi can be withheld from the treatment medium
(97). EDTA should be removed from the treatment medium for Al because it
chelates Al (97). Other cationic ions may interfere with Al3, since the ameliorative effect of cationic ions (eg., H, Ca2, Mg2, K, Na) on the rhizotoxicity
of Al3 in wheat (Triticum aestivum L.) roots seems to be due to the reduction of
the negativity of the cell surface electrical potential by the cations (73). Therefore,
concentrations of cationic ions of treatment medium should be taken into consideration. Furthermore, it should be remembered that some metal ions interactively
exhibit cytotoxicity. A combination of Al and Fe ions synergistically enhances
lipid peroxidation in tobacco cells (see Sec. 3.3.2).
Several metal ions change ionic species depending on medium pH. Below
pH 5.0, Al ions are monomeric ions that can interact with cells and are toxic.
Therefore, medium pH should be adjusted below 5.
3.2.4
Determinations of Viability
Cellular viability can be estimated quantitatively by (a) the uptake of Evans blue,
(b) the formation of formazan from 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl
tetrazolium bromide (MTT), or (c) the degree of posttreatment growth (98). Uptake of nonpermeable dyes, such as Evans blue, is caused by the loss of plasma
membrane integrity, which is thought to be a marker of cell death (99,100). The
formation of blue formazan from MTT is a marker of active cells, since MTT
is a permeable dye and is reduced to form blue formazan by active mitochondria
(101,102). The alteration of growth capability by metal treatment can be directly
estimated by the relative growth of the treated cells to control cells during posttreatment culture (20,98).
The general information for handling plant cells in suspension culture (92)
and the experimental conditions for the study of Al stress using plant cells in
suspension culture were also previously discussed (103,104).
3.3
3.3.1
309
Mechanisms of Aluminum Cytotoxicity

Responses of Cultured Tobacco Cells to Aluminum
3.3.1.1 Aluminum Accumulation The degree of the accumulation of Al

in tobacco cells depends on medium components. In a simple medium containing
3 mM CaCl2 and 3% sucrose (pH 4.5), the accumulation of Al in cells initiated
immediately after a start of Al exposure and increased until the most Al in medium accumulated in cells (105). This kinetic pattern was similarly observed in
plant roots (106). In nutrient medium without Fe ions, the accumulation of Al
was very limited. However, in nutrient medium with Fe ions, a burst of the accumulation of Al started after prolonged Al exposure (8 h), which is due to the
loss of integrity of the plasma membrane caused by the Al-enhanced Fe-mediated
peroxidation of lipids (107) (see Sec. 3.3.2). It was also reported that the higher
the CaCl2 concentration in medium, the lower the Al uptake in a tobacco cell
line BY-2 in a simple medium containing 10 mM MES, 5 mM sucrose, 5 mM
KCl, and CaCl2 (0.2, 2.0, 20 mM) (pH 4.5) (108).
3.3.1.2 Callose Production Callose synthesis is the most sensitive indicator of Al effect in plant roots (29,109). The Al-induced callose synthesis was
identically detected in a simple calcium medium in both cultured soybean (Glycine max) cells (110) and tobacco cells (105). In soybean cells, the production
of callose was detected as early as 15 min after the start of Al exposure.
3.3.1.3 Other Responses In Soybean cells, Al reduced net K efflux in
a short-term exposure (110). The possibility that Al increases [Ca]cyt has not been
fully examined because of the technical difficulty of detecting a transient change
of [Ca]cyt (see Sec. 2.3.4). In a case of tobacco cell line BY-2, Al induced a
decrease in [Ca]cyt , suggesting that Al may act to block Ca2 channels at the
plasma membrane (23).
3.3.2
Aluminum-Enhanced Peroxidation of Lipids
Al ions have a strong affinity for the surface of biomembranes (62), which causes
the rigidification of the membrane (111). The Al-induced rigidification of the
plasma membrane seems to affect the metabolism in the plasma membrane. Al
stimulates the Fe(II)-mediated peroxidation of membrane lipids, and this nonenzymatic reaction was proposed to be due to the Al-induced rigidification of the
membrane (112). The Al-enhanced peroxidation of lipids has been reported in
various systems including phospholipid liposomes (112), soybean root tips
(65,113), and cultured tobacco cells (114). In the cultured tobacco cells treated
with Al in a simple calcium solution (see Sec. 3.3.1.1), the accumulation of Al
in cells itself did not cause the peroxidation of lipids; but, the addition of Fe(II)
to the Al-accumulated cells immediately resulted in lipid peroxidation, followed
by loss of integrity of the plasma membrane several hours later (105). Thus, it
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Matsumoto et al.
seems that the accumulation of Al sensitizes the membrane to the Fe(II)-mediated

peroxidation of lipids and that the Al-enhanced peroxidation of lipids is a direct
cause of cell death (Fig. 9). The Al-enhanced peroxidation of lipids was also
observed in pea (Pisum sativum L.) roots as an early response to Al without
external supply of Fe ions (our unpublished results).
The Al-enhanced peroxidation of lipids leads to the apoptosis-like cell
death in cultured tobacco cells (115). In animal cells, cell death is a scheduled
event (so-called programmed cell death or apoptosis) during a range of biological
processes involving a relatively limited number of execution pathways. Internucleosomal fragmentation of DNA is the hallmark of apoptosis. In addition, an
increase of intracellular Ca2 and the activation of endogenous proteinases are
known to participate in signaling pathways to promote apoptosis. Tobacco cells
that had been treated with Al in the presence of Fe(II) in nutrient medium exhibited cell shrinkage and internucleosomal DNA fragmentation, which was detected
as DNA ladders by agarose gel electrophoresis in the form of bands corresponding to increasing multiples of approximately 150-bp fragments (Fig. 10). Furthermore, the cell death process required extracellular Ca2 and endogenous protein-
FIG. 9 Responses to Al of tobacco cells in Ca medium. The exposure of cells

to Al in Ca medium leads to the accumulation of Al in cells and the deposition
of callose, whereas the plasma membrane is apparently normal. The addition
of Fe2 to the Al-accumulated cells induces the Fe2-mediated peroxidation of
lipids, which results in leakage of K ions, enhancement of callose deposition,
and loss of integrity of the plasma membrane (or cell death). (From Ref. 105.)
311
FIG. 10 DNA fragmentation in tobacco cells treated with Al in the presence

of Fe2. Cells were treated with various concentrations of AlCl3 (0, 50, 100,
150 M) in nutrient medium containing 100 M FeSO4 (pH 4.0) for 24 h at a
cell density of 10 mg fresh weight mL1. After the treatment, protoplasts were
prepared and nuclear DNA was isolated. DNA was analyzed by agarose gel
electrophoresis followed by ethidium bromide staining. -Phage DNA digested with HindIII (designated as M) and 100-bp molecular ruler (indicated
on the right side) were used for DNA markers. (From Ref. 115.)
ase. Thus, the cell process initiated by the Al-enhanced Fe(II)-mediated

peroxidation of lipids is involved in the apoptosis-like cell death program.
3.4
Isolation and Characterization of Aluminum-Tolerant

Cell Lines
Since responses to Al in cultured plant cells are similar to those in whole plant
roots in water culture (see above), it is likely that some of the mechanisms of
tolerance to Al are mediated at the cellular level. Based on this idea, several
attempts have been carried out for the isolation of Al-tolerant cell lines under
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various screening conditions from various species (see below). The Al-tolerant
phenotype expressed in some selected cell lines was elucidated to be due to genetic alterations (mutation) by the regeneration of the plants that maintained Al
tolerance. The mechanism of Al tolerance was also investigated by use of Altolerant cell lines.
3.4.1
Isolation of Aluminum-Tolerant Cell Lines

and Regeneration of Aluminum-Tolerant
Plants
A large number of Al-tolerant variants were selected from nonmutagenized cell

suspension cultures of Nicotiana plumbaginifolia Viv. (116). Furthermore, fertile
plants were regenerated from the variants that retained stable Al resistance in
callus culture, and all of them transmitted Al tolerance to their seedling progeny
in segregation ratios expected for a single dominant mutation.
Two types of Al-tolerant cell lines were selected from carrot (Daucus
carota L.) cells in suspension culture (117). One type was tolerant to ionic Al,
and the other was not tolerant to ionic Al but grew with insoluble Al-phosphate
as a sole source of Pi. Both tolerant phenotypes were passed on from cells to the
regenerated plants or plantlets (117).
The reverse approach to getting Al-tolerant cells in suspension culture from
Al-tolerant plant cultivars has not been successful. Cell suspensions were generated from the explants derived from an Al-sensitive (Romano) and an Al-tolerant
cultivar (Dade) of Phaseolus vulgaris L. Although Al sensitivity was evaluated
in these cultures using Al-induced callose synthesis, differences in callose production were not expressed between cell cultures (104).
3.4.2
Mechanisms of Aluminum Tolerance Expressed in

Aluminum-Tolerant Cell Lines
In acid soils, the availability of Pi is reduced because of the precipitation of Alphosphate (45). The cell line selected from carrot cells (see Sec. 3.4.1) grew
normally in Al-phosphate medium by secretion of citrate into medium. Citric
acid chelates the Al in Al-phosphate, which results in the formation of nonphytotoxic Al-citrate and the solubilization of Pi. Furthermore, the cell line showed
higher activity of mitochondrial citrate synthase (118), and the overexpression
of mitochondrial citrate synthase gene of Arabidopsis thaliana in carrot cells
improved the growth in Al-phosphate medium (119). Thus, the enhanced activity
of mitochondrial citrate synthase seems to increase the productivity of citrate,
which may increase cell growth in the presence of insoluble Pi source such as
Al-phosphate.
A tobacco cell line tolerant to the Al-enhanced peroxidation of lipids was
isolated (120). Tobacco cells (a cell line SL) were mutagenized with ethyl methanesulfonate (EMS) to increase mutation frequency and then exposed to a lethal
313
dose of Al in nutrient medium containing Fe ions. The survivors were grown in

Al-free medium, and the Al-tolerant cell line was isolated. Compared to the parental cell line (SL), this cell line exhibited a 65-fold higher specific activity of
phenylalanine ammonia lyase and contained 10-fold more phenylpropanoids. The
phenylporpanoids (mainly caffeoylputresine) exhibited antioxidant activity
against Al-enhanced peroxidation of lipids in the parental cell line (unpublished
results). Thus, the phenylpropanoids that accumulated in the Al-tolerant cell line
seem to protect cells from the Al-enhanced peroxidation of lipids. This cell line
was also tolerant to the peroxidation of lipids caused by hydrogen peroxide, tertbutylated hydroperoxide, and A23187 (unpublished results).
3.5
Aluminum-Inducible Genes Isolated from Cultured

Cells
Treatment with Al in the presence of Fe(II), which enhances the peroxidation of

lipids in tobacco cells (see Sec. 3.3.2), induced genes for glutathione S-transferase, peroxidase, and GDP dissociation inhibitor (77,78,121). The possible
involvement of these genes in Al tolerance was investigated by transferring these
genes into Arabidopsis thaliana. All three genes conferred a degree of Al tolerance in A. thaliana, suggesting that these genes are involved in Al tolerance
mechanisms (122).
4
SUMMARY
Suspension-cultured tobacco cells are useful for (a) investigation of the details
of Al cytotoxicity at cellular and molecular levels, (b) isolation of a large variety
of Al-tolerance cell lines, and (c) elucidation of possible mechanisms of Al tolerance and of possible Al tolerance genes. This system also seems to be useful for
the study of toxicity and tolerance of other metals in plant cells.
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11
Tree Crops
Tracy Punshon
Savannah River Ecology Laboratory, Aiken, South Carolina
INTRODUCTION
The preface to a recent text on biodiversity states that the biota of the earth is
being altered at an unprecedented rate. We are seeing massive changes in landscape use that are creating even more abundant successional patches, reduction
in population sizes, and in the worst cases, losses of species (1). A plethora of
anthropogenic activities have forced changes in biodiversity, most notably
changes in land use, unsustainable exploitation, and global pollution. Any organism unable to adapt to the new anthropogenic environments being created will
either move or perish. Biodiversity is a measure of the number and abundance
of species within an ecosystem and is therefore considered an indication of environmental quality (2). Natural forest ecosystems are believed to have the highest
biodiversity, as they representin successional termsa climax community; the
last of many ecological seres. Environmental stresses that negatively affect
growth and reproduction will reduce biodiversity, forcing out unadapted species
by a process of either migration or extinction, while being balanced by an increase
in biodiversity caused by the adaptation of other species to unique, localized
contaminated niches (3). Much attention has been focused on the various environ321
322
Punshon
mental problems caused by widespread industrialization and urbanization, and

the impact can be seen in every component of the biosphere. Global warming,
caused by overproduction of CO 2 , is responsible for significant increases in temperature that impact species distribution. Combustion of coal for energy production releases large quantities of sulfur and nitrogen dioxides into the air, which
combine with water to form acid rain, acidifying lakes and causing forest decline.
Energy production from coal combustion also produces hundreds of millions of
tons of ash waste per year [100 million tons in the United States alone (4)], the
majority of which is stockpiled on land. A huge range of chemical waste products
from various industrial and domestic sources is emitted in to the air, soil, and
water. In an attempt to clarify how these various sources of contamination affect
the biosphere they have generally been studied separately, although the effects
observed on the wider environment are a result of highly complex interactions
between contaminants and conditions, which is almost impossible to predict.
However, the overall effect of all of these environmental stresses operating simultaneously is negative, possibly because the rate at which the environment is being
altered is faster than the rate at which the majority of species can adapt.
Continued selection pressure from chronic environmental contamination
can prompt adaptational changes as well as toxicity. The survival of long-lived
woody species depends on the ability to adapt in the short term to environmental
change; and there is mounting evidence to suggest that some species may be able
to take advantage of polluted environments and in some cases ameliorate them
(5). Trees are now the focus of a great deal of research dealing with low-cost
environmental clean-up, specifically for controlling hydrology, degrading organic
contaminants in groundwater plumes, stabilizing eroded sediments, removing
heavy metals from contaminated soil, and as biological filters for sewage sludge
disposal.
The research reviewed in this chapter focuses on adaptive changes of trees
to heavy metal stress, with respect to their potentially beneficial uses for the
remediation of heavy metals in the soil. The effects of metals on the physiological
processes of woody plants, how these effects act together to produce changes
on the whole ecosystem, and the mechanisms of adaptation of woody plants to
anthropogenic selection pressures are also introduced. Several other highly successful remedial applications using trees have also been mentioned with the main
aim of introducing the reader to the full potential of the use of trees for remediation, including degradation of organic chemicals, N and P removal, and wastewater purification, although they are left for others to review in detail. Their
inclusion is intended to emphasize the potential of trees to remediate complex
heterogeneous contamination rather than single-contaminant profiles, due to the
predominance of mixed wastes. It is not the intention of this chapter to restrict
the focus of the reader but rather to enlarge it.
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323
SIGNIFICANCE OF FOREST ECOSYSTEMS
Forest and related areas (e.g., woodland, shrubland, degraded forests, etc.) cover
approximately 52 10 6 km 2 of the earths surface; an area three times that of
cropland, and 75% more than grassland; estimated to be 2045% of land cover
(6,7). Forests are a vital part of the global ecosystem, producing 60% of the net
primary productivity of all terrestrial environments and regulating water regimes
by intercepting rainfall and regulating flow. They also play a vital role in enhancing soil quality, preventing soil erosion, adding organic matter from seasonal leaf
fall, and enriching the diversity and abundance of soil microbes. It is not an
exaggeration to say that the presence of woody vegetation has a profound ameliatory effect on the physical, chemical, and biological health of the soil.
Tree crops form the basis of a wide variety of important industries, including timber, processed wood and paper, rubber, fruit, and coffee. Immense quantities of wood are produced annually; for example, in 1988 the total world timber
production was conservatively estimated at 3.4 10 12 m 3; with 1.7 10 11 tonnes
of paper produced that same year. In terms of international trade, more than 2.5
10 11 m 3 of wood and 5.1 10 10 m 3 of paper was traded in 1988 alone, amounting
to $9 10 11, or 3% of world trade income (7).
SIGNIFICANCE OF HEAVY METAL CONTAMINATION
In the past, the term heavy metal referred to metals in the periodic table with
an atomic number greater than 20, excluding the alkali and alkali earth metals
(8), or metals that have specific gravities greater than 5 g cm 3. The term is
somewhat inaccurate, however, and alternative nomenclature has been suggested,
with some workers preferring the term potentially toxic elements (9). The
elements Cd, Cr, Cu, Hg, Ni, Pb, and Zn are generally considered the most important elements associated with soil pollution.
Heavy metals occur naturally in rock formations and ore minerals (Table
1); therefore, they have a natural background concentration in soil, sediments,
water, plants, and animals (10). Soils develop as a result of the gradual weathering
of rock formations and typically inherit the characteristic metal assemblage of
the parent rock. Igneous parent rock potentially contributes higher concentrations
of Cr, Mn, Co, and Ni to the soils, whereas sedimentary parent materials (especially shales) contribute Cr, Co, Ni, Zn, and Pb (11). One of the most interesting
examples of this phenomenon can be found in New Caledonia where serpentine
rock formations, rich in Ni, Co, and Cr, give rise to soils with such elevated
concentrations of metals that they are populated only with specifically adapted
flora. These plants, the majority of which belong to the genera Alyssum and
Thlaspi, have evolved a constitutional tolerance to the metals, and accumulate
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Punshon
TABLE 1 Sources (Mineral and Anthropogenic) and Characteristic

Information on the Heavy Metals
Source
Element
Cadmium (Cd)
Chromium (Cr)
Copper (Cu)
Mercury (Hg)
Nickel (Ni)
Lead (Pb)
Zinc (Zn)
Mineral
Anthropogenic
Black shales: greenockite

(CdS: 77% Cd); otavite
(CdCO 3 65.2% Cd)
Carlsbergite (CrN 78%
Cr)
Phosphatic fertilizers, spoil

heaps, sewage sludge
disposal on land
Metal finishing wastes,
wood preservatives, sewage sludge
Mining and smelting,
metal processing (wire
manufacturing), sewage
Smelting processes, spoil
heaps, sewage sludge
disposal
Iron, steel and pyrometallurgical industry wastes
Chalcocite (Cu 2 S: 79%

Cu) Bornite; atacamite,
azurite
Kenhsuite (Hg 3 S 2 Cl 2: 82%
Hg); montroydite; metacinnabar
Ultramafic (serpentine
anomalies) millerite
(NiS)
Galena (PbS: 73% Pb)
Sphalerite [(Zn, Fe)S:

67.1% Zn]; wurtzite
Automobile exhausts, spoil

heaps and tailings, fossil
fuel combustion
Spoil heaps and mining,
pesticides, textiles and
electronic waste
Sources: a Swaine (129); b Allaway (130); c Bohn et al. (131).
such high concentrations that they can constitute 1% of their leaf dry weight
(12) and are described as hyperaccumulators. The majority of hyperaccumulators
consist of small herbaceous plants, although the tree Serbertia acuminata (se`ve
bleue) is notable for its bright blue sap, containing approximately 11% nickel as
the citrate complex.
Elements such as Cu and Zn are required by biological systems for normal
function; in plants, these concentrations are lowapproximately 6 and 20 mg
kg 1 DW, respectivelyand are rarely deficient. Copper is required for several
enzymes involved in oxidation and reduction reactions, e.g., cytochrome oxidase
(13), whereas zinc is required in greater quantities, with more than 80 plant enzymes containing this metal (14). The anthropogenic sources of heavy metals
(Table 1) can be broadly grouped into metalliferous mining and smelting, industry, atmospheric deposition, agriculture, and waste disposal on land (11).
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325
Degradation of soil quality affects forestry efforts as well as natural woodland ecosystems. Contamination of the soil with heavy metals, radionuclides, and
organic compounds is widespread. The United States alone has a total of 1283
soil contamination sites on the National Priority List of the Superfund program
(15). These sites include chemical plants, contamination from urbanization,
groundwater contamination, industrial waste treatment facilities, landfill, sites
contaminated as a result of military operations, radioactive operations, and mine
tailings. The extent, diversity, and complexity of the contamination is enormous.
When comparing the quantity of heavy metals released to the environment as a
result of anthropogenic activities to the natural background, Campbell et al. (16)
showed that anthropogenic input was 15 times higher than natural for Cd, 100
times higher for Pb, 13 times higher for Cu, and 21 times higher for Zn.
One factor that highlights heavy metals as particularly important contaminants is their immutable nature, i.e., they cannot be degraded and in some cases
their complexes are just as toxic as the free metal ion. In the case of Hg, for
example, organic complexes are considered to be more toxic than inorganic
forms, as demonstrated by Picea abies exposed to HgCl 2 and methyl-HgCl 2 in
nutrient solution (17). In this case, heightened toxicity was shown to be a result
of the chemical form of Hg rather than an increase in uptake.
Heavy metals also have a considerably longer residence time within various
components of the biosphere than organic contaminants. In situations where
heavy metal deposition has decreased through stricter pollution controls, their
presence in forested ecosystems remains a problem. The environmental toxicity
of heavy metals is governed by their bioavailability. Put simply, the bioavailable
heavy metal concentration is that which is readily accessible for uptake by any
biological organism. In the soil, plant roots tend to take up heavy metals present
in soluble forms. If the metals are insoluble they can remain bound to soil colloids
without entering the soil solution and can be considered in the meantime to be
relatively harmless, e.g., the complexed forms CdCl 2 and Zn-EDTA are more
bioavailable due to their increased solubility. Heavy metals remaining in a nonavailable form within the soil profile may be solubilized by a variety of processes,
the most prominent being changes in soil pH. Generally, the more acidic the
soil matrix becomes the more bioavailable the heavy metals become. Hence, the
widespread incidence of acid precipitation in many European and North American forest systems becomes a major factor in exacerbating heavy metal contamination. Studies of catchment areas in Sudbury, Ontario have shown that the effects of mining and resource recovery activities, now ceased, have left a legacy
of elevated Cu and Ni concentrations in the soil expected to persist for the next
1000 years (18). There is a great deal of experimental evidence confirming that
acidic precipitation increases the concentration of bioavailable metals; Roemkens
and Salomons (19) demonstrated that in forest soils, Cd and Zn concentrations
increased at a pH below 5.5. Multiple linear regression of their data showed that
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Punshon
the change in cation exchange capacity (CEC) and pH explained 79% and 83%
of the measured distribution coefficients (K d ) for Cd and Zn, respectively. Soils
with an inherently low acid buffering capacity, such as the highly weathered
ultisols of the southeastern Atlantic coastal plain, are far more susceptible to
increases in metal bioavailability under conditions of acidic precipitation. Cu in
particular binds very strongly to organic material, and can remain in the soil and
continue to affect ecosystems many years after the contamination episode (20).
4
4.1
EFFECT OF HEAVY METALS ON WOODY PLANTS

Physiological Effects
A great deal of research has been dedicated to the study of how metals affect
woody plants, ranging from their effect on specific cellular processes to their
effect on nutrient cycling within forest ecosystems. Heavy metals impact woody
plants via two main pathways; through the root system from metals entering the
soil, and via aerial deposition. By far the most direct effect is produced through
aerial contamination (21), whereby metals are deposited onto the surface of the
leaves. Aerial deposition of heavy metals also impacts the soil because deposited
metals are washed down the trunk and accumulate around the base of trees (22).
Heavy metals entering the soil can go through a wide variety of species
changes as a result of the prevailing edaphic conditions controlling bioavailability
(such as pH, CEC, and organic matter content) (23). Interaction with soil microbes and reactions with root exudates also change the species and bioavailability of heavy metals (24). Once metals reach root tissues, their uptake is dependent
on certain physiological factors, including characteristics of the heavy metal in
particular. Metals that also function as essential micronutrients, such as Cu and
Zn, have established mechanisms of uptake, and increasing external concentrations frequently result in increased uptake; for example, the roots of pine seedlings growing on soils spiked with 200 M ionic Zn accumulated up to 73 M
g 1 of Zn (0.48% dry weight) (25). Typical metal concentrations of metals within
soil solution and plants are shown in Table 2.
Nonessential metals, such as Cd, can enter plant cells via nonmetabolic
pathways (passive diffusion as well as active uptake); work on passive Cd uptake
in excised barley roots described a saturation effect where Cd floods into the
root cells unhindered above specific concentrations (26). This may result from
cell membrane damage caused by the high ambient metal concentrations, which
disrupts the selective uptake mechanisms, allowing an uncontrolled efflux (27).
It seems likely that mixtures of nonmetabolic and metabolic mechanisms are
responsible for Cd uptake. Solution culture experiments comparing Cd uptake
by Fagus sylvatica L. (common beech) showed high uptake at 20C, although
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327
TABLE 2 Typical Concentration of Heavy Metals in Uncontaminated Soil

Solutions and Plants
Normal conc.
(g kg 1 DW)
Element
Soil (total) a
Plants b
Toxic conc.
(soil solution; mg L 1) c
Cadmium (Cd)
Chromium (Cr)
Copper (Cu)
Mercury (Hg)
Nickel (Ni)
Lead (Pb)
Zinc (Zn)
0.017
51000
2100
0.020.2
101000
2200
10300
0.20.8
0.0215
415
0.0050.5
0.035
0.110
8400
0.001
0.001
0.040.3
0.001
0.05
0.001
0.005
Sources: a Swaine (129); b Lindsay (132); c Allaway (130).
Cd efflux was still detected after the temperature was reduced to 0Cwhen
most metabolic processes had ceased (27). Punshon and Dickinson (28) found
that Cd uptake in Salix trees occurred during exposure to an incremental supply
of Cd (0.15 mg L 1 increasing to 1.5 mg L 1 over 128 days), although in all
tissues the Cd concentration remained consistent at approximately 100 g g 1.
This indicated thatfor all Salix species investigated in the studyCd influx
may be restricted by a mechanism that did not place demands on metabolism
(i.e., passive) because both leaf and shoot biomass were unaffected by elevated
concentrations of Cd when compared with control-grown plants. In the same
experiment, Cu accumulated in the root tissues (300 g g 1) and Zn accumulated in leaves, stems, and roots. In Salix burjatica Nazarov. cv. Aquatica leaf
Zn concentrations greater than 600 g g 1 were accompanied by severe signs of
toxicity.
Once inside the symplasm, heavy metals inhibit growth and reproduction
via a variety of mechanisms. Root elongation is frequently used as a first indication of a plant response to soil-borne metals because this is the organ that first
comes into contact with soil contamination. Metal resistance characteristics of
plants were initially based on differential root elongation indices in metal-treated
and untreated solutions (29). The exact mechanisms of growth inhibition are not
as yet fully described (30), but it is likely that mechanisms are specific to the
metal(s) and the plant species involved, and also as a function of the external
metal concentration. In general, heavy metals seriously disrupt a variety of physiological processes because they bind very strongly to enzymes. Van Assche and
Clijsters (31) summarized the effects of metals on enzyme systems, reporting
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Punshon
that they bind to functional ligands (mainly sulfhydryl groups) that regulate structure and catalytic action, also inducing deficiencies of other metal ions by substitution into metalloproteins.
Cross-linkage enzymes responsible for cellular elongation (32) may be also
affected in this way by metals, subsequently inhibiting the elasticity of developing
root cells. The loss of elasticity as a result of metal toxicity has been demonstrated
in the cells of herbaceous plant roots, specifically Phaseolus vulgaris (33) and
Festuca rubra (34). Metal contamination of the soil may also inhibit the overall
development of root biomass, as reported in Picea abies in response to Cd and
Zn (35) and in a variety of other forest species (e.g., Pinus strobus, Pinus taeda,
Liriodendron tulipifera, Prunus virginiana and Betula alleghaniensis) in response
to Cd (36). Often the reductions in root biomass are severe and occur at relatively
low bioavailable concentrations. Wickliff and Evans (37) reported that in nitrogen-free solutions, as little as 0.03 ppm Cd resulted in a 44% reduction in root
biomass and accumulation of up to 61 mg kg 1 Cd within dried root tissues.
Other effects of heavy metals include a change in root architecture, inhibition of
root hair formation, and reduced root initiation (30). These symptoms are not
universal to all species; adventitious roots of Salix spp. cuttings respond to exposure to Cu, Cd, and Zn in solution by significantly increasing the number of roots
(28,38,39). Heavy metals can also reduce the percentage of mitotic cells in the
root apical meristems (40), a reduction in root hair formation, root membrane
damage (41), and decreased transpiration rate (42). Therefore, the reduction in
root biomass is the overall effect of several individual physiological effects
caused by heavy metals. Populus spp. trees exposed to elevated concentrations
of Ni (5 mg L 1) in nutrient solution have a drastically reduced transpiration
rate in comparison with control plantswith control tree cuttings transpiring
approximately 0.51 L plant 1 day 1, and Ni-treated plants only used several
milliliters per day (Punshon and Adriano, unpubl.). Metals also affect photosynthesis by inhibiting enzymes, producing visible symptoms of toxicity such as
stunting, chlorosis, and necrosis (31).
4.2
Effects on Forest Ecosystems
In the late 1970s, evidence of widespread environmental damage began to appear

in the coniferous forests of northern and northwestern Europe. This took the form
of visible toxicity symptoms such as yellowing of the needles and advancing tree
mortality, causing a large-scale decline in the health of conifer plantations. First
observed in spruce forests of Bavaria, West Germany, forest decline has spread
across Europe and into the United States (43). By the early 1980s, 2025% of
European forests were classified as moderately or severely damaged from seemingly unknown causes. Research indicated that forest decline may be caused primarily by air pollutionspecifically SO 2 and NO x , although correlations between
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329
tree mortality and any one specific stress factor could not be found (44). Schulze
(43) believes that forest decline is a response to a complex set of physiological
effects triggered by ozone, SO 2 , NO x , ammonia, and organic pesticides. Until
recently, heavy metals were not implicated as a contributory factor in forest decline, although workers have now found elevated concentrations of phytochelatins (produced during metal stress to chelate metals within the cell) in the needles
of red spruce collected in the United States, suggesting that heavy metals may
be partly responsible (45).
There is a wealth of information in the scientific literature about the dynamics of heavy metal movement and accumulation in forest ecosystems. Studies
tend to concentrate around northern Europe and the former USSR, where acid
rain has amplified the effects of metal deposition (19), although studies have
spread to the United States where the effects of decline are beginning to emerge.
Many studies focus on the effect of aerial deposition from nearby smelting plants
on existing forested ecosystems (18,20,4648). One effect common to many studies is the effect of heavy metals on decomposition processes. Nutrients cycle
through the forest ecosystems via breakdown of leaf litter, representing a major
process for returning nutrients to the soil. The recovery of nutrients from senescing leaves can contribute 6080% of nutrients required by the following season
(49,50). In metal-contaminated situations the rate of litter decomposition drops
dramatically and nutrient cycling is diminished. Often this is the result of direct
toxicity of heavy metals to the detritivores that feed on leaf litter and convert it
into humic material (51), and decomposition is reduced to such an extent that
large quantities of litter accumulate on the surface. Nikliska et al. (52) found that
treatment of forest litter with Cd, Cu, Pb, or Zn significantly reduced the respiration rates (used as a measure of degradation metabolism). Respiration rates were
even lower after the contaminated litter was stored for progressively longer periods of time. There is evidence to suggest that a reduction in decomposition may
be a common response of many different forest ecosystems and that metals tend
to be trapped within the system because nutrient cycling slows down; this characteristic trapping of metals in the decomposer system has been shown in the
mangrove swamps of southeastern Brazil. Silva et al. (53) studied the cycling of
Zn, Mn, and Fe through the production, decomposition, and export of litter in
the Itacurussa Experimental Mangrove Forest and found that less than 0.01% of
metals were exported from the system via the leaf litter degradation pathways.
They concluded that this represented a biogeochemical barrier to metal transfer.
However, it is likely that in aerobic soils metal availability and hence metal export
may be much higher, and the retention of bioavailable metals within the system
will have a deleterious effect.
Heavy metals also affect the microbial communities in forest soils. Transection studies along pollution gradients have shown that the community structure
and the tolerances of bacterial species change on a distance gradient from a metal
330
Punshon
smelter (54). These studies showed that bacterial species were less sensitive to
heavy metals than fungal species; that with increasing proximity to the metal
smelter, fungal phospholipid fatty acid content of the soil (used as an indication
of the magnitude of fungal biomass) was significantly reduced; and that bacteria
in this region of the gradient exhibited increased metal resistance in comparison
with populations farther away. Since fungal species in the soil are important for
mycorrhizal colonization of tree roots and in decomposition of leaf litter, there
are severe repercussions to increased heavy metal loading of forest systems.
5
ADAPTATIONS OF WOODY PLANTS TO HEAVY

METALS
Heavy metal tolerance of plants as a result of anthropogenic pollution has been

studied since the 1930s when stable ecotypic adaptation was demonstrated in the
herbaceous plant Silene dioica growing on a metalliferous mine site (55). It is
thought that through a process of natural selection certain plants can take advantage of metal-contaminated niches and retain tolerance traits within their population, forming an ecotype. Laboratory studies have shown stability of tolerance
traits in herbaceous plants, by exposing tolerant and normal ecotypes of the same
species to metals in nutrient culture and observing their differential growth (3,56).
Normal populations of plant species contain individuals that express specific genetic mutations dependent on the extent of genetic variability. Certain mutations
allow individuals to survive elevated concentrations of heavy metals in the soil,
although they may only number one or two for every thousand individuals within
a population (57). Although seemingly insignificant, these individuals can initiate
colonization of contaminated niches. For trees, however, there is little evidence
that they can develop tolerance in the classic sense, i.e., tolerance that is passed
on to progeny (30). An important example of this was reported by Watmough
and Dickinson (58), where chronic aerial Cu contamination from a nearby smelter
impacted a mature sycamore (Acer pseduoplatanus L.) woodland, resulting in
the induction of Cu resistance traits in established trees but effectively halting
any seedling regeneration. In this case, certain adaptive mechanisms were employed by the mature trees, which were not passed on to their seed, indicating
that true (heritable) tolerance was not responsible for their continued survival.
Instances of metal tolerance via compartmentalization of Zn as an organic
complex within the cell vacuole have been reported for barley (Hordeum vulgare
L.) (59) and other grasses (60), whereas reported instances of woody plant resistance to metals were mediated through symbiotic mycorrhizal association, shown
primarily in Betula (6163). Jones and Hutchinson (64) also worked with birch
(B. papyrifera Marsh.) inoculated with the mycorrhizal fungus Scleroderma flavidium E. & E., known to increase plant resistance to Ni. They found that inoculated plants were more metal-resistant than controls, not because the fungus al-
Tree Crops
331
tered the uptake and distribution of Ni within the plant as was previously thought
but because it enhanced uptake of P and Mg within root tissues. The importance
of mycorrhizal association cannot be overstated when discussing the relationship
between woody plants and heavy metal contamination. Remediation schemes
involving trees are dependent on the use of mycorrhizal incoluation for their
success (65). Some species of trees naturally invade industrially contaminated
or disturbed land, and several workers have found Betula and Salix species growing of mine spoils enriched with heavy metals such as Pb and Zn (66) as well
as Cd (67). There are examples of tree species with much higher metal resistance
capabilities than others, many of which involve Betula or Salix species. In nonmycorrhizal studies involving a variety of willow species, Punshon and Dickinson
(68) found wide variation in responses, consistently higher metal resistance in a
mine ecotype of Salix caprea, taken from a Pb-Zn mining site (Trelogan, Clywd,
UK). This ecotype was able to withstand plant-available concentrations of Cu,
Cd, and Zn in solution culture in which nonadapted species could not survive;
root elongationbased tolerance indices in excess of 100% for the ecotype indicated that growth in metal-enriched solutions was greater than in controls. The
mine spoil substrate in which the ecotype had been growing contained total concentrations (g g 1 DW) of 72.2 (40) Cu, 116.6 (7.1) Cd, 1468 (88) Zn,
in addition to 13,488 (527) Pb, and only contained 4% organic matter (39).
Further analysis of the roots and the presence of fruiting bodies indicated that
they were associated with a mycorrhizal fungus, thought to be Hebeloma spp.,
indicating that even given elevated metal resistance, trees still require mycorrhizal
association, especially when growing in contaminated soil.
Physiological studies of trees have revealed that they respond developmentally to changes in their environment. For example, they respond to variation in
light incumbent on the canopy by changing the shape of their leaves and even
the concentration of chloroplasts within the leaves. These sun-and-shade leaves
have alterations in the surface area to either maximize photosynthetic area (required in the shade) or reduce it (required during long hours of sun exposure)
(69). A similar response in leaf morphology is seen in eastern cottonwood when
exposed to different soil moisture regimes. In this case, leaf size increased from
xeric provenances in the west to the mesic provenances in the east (70). These
morphological adaptations can be seen within an individual; branches represent
independent meristems arising from the central trunk and respond independently
to stresses presented by their immediate microclimate. This is known as the theory of genetic mosaicism (71). Such predisposition of woody plants to adaptation
within the lifetime of the individual may be a product of their longevity and/or
a way of avoiding the metabolic costs of heritable tolerance (72).
Considering this, it seems feasible to assume that if a tree species possesses
the appropriate genetic variability it can potentially adapt to environmental stress,
such as heavy metal contamination, and that this adaptation can be manipulated.
332
Punshon
Several studies have shown that trees from the genus Salix (including Populus)
are highly variable, have a predisposition for poor and contaminated soils, and
can be acclimated to withstand increasing metal concentrations over relatively
short periods of time (28). The occurrence of Salix spp. on industrially contaminated sites (73) is evidence that they possess the genetic variability to invade
these niches. Presuming that long-lived woody plants need to adapt to stress
within their lifetime, other mechanisms of tolerance, such as phenotypic plasticity, have been considered as an alternative mechanism. Phenotypic plasticity is
the variable expression of the phenotype in response to stress and allows rapid
adaptation without being passed on to progeny. In a review, Thompson (74) implicates phenotypic plasticity as a possible component in the development of
metal resistance in woody plants (75).
6
BENEFICIAL USES OF TREES IN SOIL REMEDIATION
The various environmental benefits of the establishment of woody vegetation has

already been mentioned, with emphasis on the fact that trees perform a variety
of vital functions in the maintenance of ecosystem health. It is the application
of these functions to contaminated soils that has given rise to the use of trees as
agents of environmental clean-up. There are two main innovations that use plants
to remediate metal contaminated soils, specifically involving fast-growing trees
such as Salix (willows and osiers) and Populus (poplar), but which also use Alnus
(Alder) and in some cases Eucalyptus (Eucalypts). They are short-rotation forestry (SRF) as a means of disposing of sewage sludge and phytoremediation.
SRF was initially used as a means of producing CO 2-conservative energy but
later developed into a means of sewage sludge disposal when the high nutrient
and water requirement became costly. The heavy metal resistance of the trees
used in SRF then became significant due to the extremely high heavy metal concentration of domestic and industrial sludges.
SRF was pioneered by the International Energy Agency, an initiation involving Austria, Canada, Denmark, Finland, Italy, the Netherlands, Sweden,
United Kingdom, United States, and Brazil (76). This emerged from the rapid
development of SRF in Sweden. This resulted from the introduction in 1991 of
a new agricultural policy that introduced lower grain prices, compensation for
willow plantations on set-aside land, and higher taxes on fossil fuel. Sweden now
has 10,000 ha of commercialized energy (or biofuel) plantations, with the
further development of 200,000 ha in progress to replace imported oil traditionally used for heating purposes (77). During 1994 in Sweden, about 80 TWh of
energy was generated using biomass fuel, i.e., approximately 20% of the total
energy supply (78). However, the energy forestry program was originally established in 1978 with the aim of acting as an advisory body for the dissemination
of information on high and sustainable production of woody biomass that is
Tree Crops
333
ecologically acceptable. The large quantities of fertilizer and water required to

sustain the intensive management of bioforestry prompted the use of sewage
sludge, having the additional benefit of providing a valuable avenue for waste
disposal. Information on the use SRF as a means of disposing of wastewaters
and sludges was brought together in 1994, prompting wider research on the dynamics of heavy metal uptake and tolerances of various species within the genus
Salix (79).
At the same time the science of phytoremediation was being developed.
The study of heavy metal tolerance indicated that plants could have beneficial
practical applications in metal removal from the soil and was coupled with the
search for specific hyperaccumulator plants for a variety of toxic metals (80).
Phytoremediation initially used herbaceous plants, most notably Brassica juncea
(Indian mustard), with a great deal of success (8185).
Trees belonging to the genus Salix have characteristics that recommend
them for remediation of inorganic contaminants, especially of Cd, to which they
are particularly tolerant (28,8688). To date, there is still a lack of research targeted at systematically identifying exactly which Salix and Poplar species (and
hybrids) are most suitable for use in heavy metal processing and which species
could remediate mixed wastes, i.e., organic and inorganic contaminants. Even
more important for phytoremediation, information on the use of fast-growing
trees in various aspects of environmental remediation, agronomic practices, and
their effects on the environment have not been previously collated in a form
that custodians of contaminated sites can easily use. Without this knowledge the
commercial use of trees to remediate various types of contaminants in a realistic
heterogeneous soil environment is still far off.
6.1
Short Rotation Forestry and Waste Disposal
The use of willows, poplars, and other fast-growing trees in the disposal of wastewaters and sludges is based on the practice of SRF, which has been used successfully in Sweden for the production of biofuel for the past 20 years (89). SRF takes
its name from the short rotation time typical of poplar and willows compared with
conifers (60120 years) or broadleaved trees (3550 years); typically between
3 and 6 years (90). Under optimum conditions (i.e., intensive management strategies), SRF can yield between 12,000 and 20,000 kg dry matter (DM) ha 1 yr 1
(91), although in general, lower yields of between 60009000 kg DM ha 1 yr 1
are more likely (90) (Table 3). If SRF is used for the generation of energy, approximately 4.5 MWh of energy can be produced from burning 1 ton DM (50% moisture) (92).
There is now a substantial body of data that indicates that the irrigation of
biomass plantations with sewage effluent may not only increase and accelerate
the growth of the trees but also provides a valuable avenue for recycling an
334
TABLE 3 Tree Species Used in Short-Rotation Forestry (SRF) Around the World
Country
Austria
Salix spp., Populus spp., Alnus spp., Betula spp.
Canada
Populus euramericana (DN17; DN74, DN177, DN181);

Populus nigra maximowiczii (NM6)
S. viminalis
S. dasyclados Wimm., S. viminalis, S. myrsinifolia
P. deltoides Bartr. P. nigra P. nigra, P. deltoides, P.
alba
Usually P. euramericana, also P. deltoides nigra and
P. trichocarpa deltoides clones
Salix, Populus, Alnus, Betula, S. viminalis, S. dasyclados
Salix, Populus, Alnus, Eucalyptus, P. deltoides nigra,
P. trichocarpa deltoides, P. trichocarpa, P. tachamahaca trichocarpa, P. alba tremuloides
P. deltoides, P. balsamifera, P. trichocarpa, P. tremuloides, Robinia pseudoacacia, Geditsia trianthus, Leucacena refusa, Prosopis glandulosa, Prosopis alba, Betula pendula, Alnus glutinosa, Alnus rubra,
Eucalyptus grandis, E. saligna
Denmark
Finland
Italy
Netherlands
Sweden
United Kingdom
United States
Yield (t DM ha1 yr1)
Tree species (and clone) used
5 without intensive management;

2324 with; average 1012
No figures available
7.6 (range: 6.78.1)
4.7 to 910
3.59
810
5.610
3.610
4.516.7
Punshon
Note: All yields are based on approximate ranges irrespective of the species of clone used. Source: Handbook on How to Grow Short
Rotation Forests, International Energy Agency, 1997 (76).
Tree Crops
335
abundant waste stream. Production of sewage sludge is approximately 6 million

t yr 1 in the United States, 2.5 million t yr 1 in Germany, and 1.07 million t yr 1
in the United Kingdom, and as such its disposal has often been problematic because it requires extensive treatment before it is considered safe. This includes
the removal of excess N, heavy metals, organic chemicals such as pesticides,
and pathogenic bacteria prior to application onto agricultural land (93). Current
research on the use of sludge as a fertilizer for SRF indicates strongly that willow
plantations may be capable of performing the majority of the required clean-up
steps. Perttu (94) suggested that SRF could be used as vegetation filters to
utilize the nutrients in municipal sewage sludge, wastewater, leakage water, and
bioash (wood ash), although dredged sediments containing organic chemicals and
pathogenic bacteria have also been effectively purified following application
on to biomass plantations.
The tree species used in biomass forestry vary between areas in which they
are grown. A summary of the species used (Table 3) indicates that in warmer
countries, such as the United States, species such as Eucalyptus, which are native
to Australia, can be successfully planted. As previously mentioned, the amount
of woody biomass produced in SRF varies with species, management practices,
and location, although maximum yield values of 2324 t DM ha 1 yr 1 have
been noted in Austria (76) (Table 3). Many of the SRF species commonly used,
especially Populus spp., are those that have also shown considerable potential
in the remediation of groundwater contaminated with organic chemicals, specifically the P. deltoides nigra and P. trichocarpa deltoides clones (95). SRF
fulfills several important environmental and ecological factors; there is no net
CO 2 contribution to the atmosphere compared to energy production using fossil
fuel, and there is a small net uptake. Pesticide use is lower than on conventional
agricultural crops, and the growth of willows for SRF improves the condition of
the soil as well as biodiversity (96,97).
Primary requirements of SRF are a rich supply of nitrogen and water. The
ability of fast-growing trees to remove nutrients and water can also be applied
to wastewater treatment; excessive concentrations of nutrient chemicals pose environmental problems when present in excess, such as eutrophication of streams
and lakes, causing algal blooms and a progressive reduction in potability. Standards usually monitor the biological oxygen demand (BOD), total N, ammonia
N, and total P in water (98), and often these strict regulatory limits cannot be met
by conventional treatment methods alone. The elimination of P from wastewater
generally involves the use of large quantities of chemical agents, which are difficult to clean up once the process has been completed. Obarska-Pempkowiak (99)
found that Salix viminalis and S. arenaria could significantly reduce the nitrogen
concentration of municipal wastewater; in particular, S. viminalis reduced the
total N concentration of wastewater from more than 35 gm 2 to less than 5 g
m 2 over a period of 270 days. Typical N and P contents of applied sludges are
336
Punshon
approximately 4.5 kg N t 1 and 2.8 kg P t 1 in the raw cake, of which 1.5 and
1.4 kg t 1 N and P, respectively, are available in the first cropping season (100).
Kutera and Soroko (98) found that willow and poplars irrigated with sewage
sludge showed an increased yield and that the trees took up almost 95% of the
N and P load of the effluent, simultaneously lowering the BOD of the effluent
by an average of 96%.
Removal of water by fast-growing trees remains an important issue, especially for those wishing to use the trees to obtain some degree of hydrological
control, e.g., in contaminated groundwater seepline interception. This involves
the use of fast-growing phreatophytes to utilize contaminated groundwater, degrade the organic chemicals, and dry out the seepline area, where there is often
standing water. Exact figures of water use vary, and depend very much on the
soil conditions, especially the presence of other heavy metals contaminants. SRF
studies use water use efficiency (WUE) as a means of measuring transpiration
rates, measuring the amount of dry biomass produced per unit of water. Studies
on the average long-term WUE of a stand of Salix viminalis showed a production
of 6.3 g dry biomass per kg of transpired water, which is high compared with
other tree species (101). However, for hydrological control and seepline interception, species choice criteria obviously include a tree with a lower WUE, i.e., the
highest throughput of water per gram of dry biomass. Information about nitrogen
status is important when considering water use because foliar nitrogen concentration affects stomatal conductance (101). Work by Blake et al. (102) showed that
WUE varies widely between clones within a particular species and demonstrated
this for Populus. They found that the cottonwood P. nigra N-80 (section Aegeiros) had a low WUE, in that biomass production was lower per kg transpired
water, whereas the balsam poplar P. maximowiczii M-4 (section Tacamahaca)
and the white poplar P. alba A-499 (section Leuce) had twice the dry matter
production for the same amount of transpired water.
6.2
Removal of Heavy Metals
Ecological studies have identified fast-growing trees invading metalliferous mine

spoils (67); therefore, the scope of their use in waste disposal and soil amendment
extends also to their removal of metals. One of the most notable features of Salix
is their seemingly innate ability to take up concentration of Cd within their tissues
far in excess of the range considered normal. Heavy metals taken up during biological purification of sewage sludge were investigated by Nielsen (103) who
found that S. viminalis accumulated significant concentrations of Cd within root
and shoot tissues. Studies have emphasized that SRF can be used for collecting
metals from the soil, in effect using the trees as biological filters. Goransson and
Philippot (104) found that almost all of the Cd applied during sludge application
was taken up by Betula pendula and that theoretically the trees could remove
Tree Crops
337
1.5 kg Cd ha 1 yr 1 when watered with sludge. Landberg and Greger (105) found
that net transport of heavy metals to the shoots varied widely between 1% and
72%, therefore, certain clones were capable of accumulating more metals than
others, indicating the need for clone selection for resistant trees (106). Systematic
screening of fast-growing trees to heavy metals is lacking; discovery of species
able to tolerate and remove metals remain serendipitous, and responses of Salix
to metals suggest a species-specific response. In hydroponic experiments, Salix
cuttings were exposed to varying levels of Ni for 28 days with measurements
taken of the length of the longest root and shoot. Responses of these important
SRF clones to Ni supplied at external concentrations of between 05 mg L1
show that above 1 mg L1 growth inhibition is severe (Figure 1). Maintenance
of sub-lethal external metal concentration is a pre-requisite in the use of nonhyperaccumulator species, especially where harvesting the woody biomass is
used as a means of metal removal. External metals concentrations affect the rate
FIG. 1. Effect of varying concentrations of nickel on leaf and root growth in

Salix, grown hydroponically and harvested after 28 days. Data are means of
longest root and shoot SE where n 5.
338
Punshon
of uptake, with lower concentrations allowing a greater total accumulation. Resistance induction studies using Cu, Cd and Zn have shown that if low initial external concentrations of metals are used, and doses are gradually increased over
time, growth responses can be considerably enhanced in comparison with sudden
exposure. By increasing concentrations ten-fold over a 128 day period, the SRF
clone Salix viminalis showed no signs of root growth inhibition (Figure 2) in
response to heavy metals (39). There is very little information on metal uptake
characteristics of Alnus (37,107) or Eucalyptus (108,109). To date, these species
have not been screened for their ability to remove heavy metals from the soil.
The efficiency with which willows can remove metals from sewage sludge
fertilized soils has been demonstrated in several key studies. Table 4 summarizes
two studies on willow vegetation filters to give an indication of the metal concentration contributed to the soil by application of sewage sludge and how much
willows can remove. In a field experiment using S. viminalis, S. viminalis
triandra, and S. dasyclados, Riddel-Black (106) found that the trees were able
to remove 2.73.3% of the total Ni added via sludge application; 3.24.5% of
the Cu, up to 45% of the Zn and most notably a maximum of 426% of the Cd
added via sludge was removed by S. viminalis triandra Q83. Table 4 also
confirms this affinity for Cd, which indicates that of the 5.2 g Cd ha 1 added
FIG. 2. Effect of gradually increasing nutrient solution concentrations of

heavy metals on root length in the biomass willow Salix viminalis. Data are
mean longest root length SE where n 54. Initial and final metal doses
are indicated.
Tree Crops
339
TABLE 4 Comparison of Concentrations of Heavy Metals Added by

Sewage Sludge Fertilization and Removed by SRF a
Heavy metals
Source
1. Nielsen [103]
Using S. viminalis (78112)
Added via sludge (6,833 kg ha1
DW) (g ha1)
Removed with biomass (g ha1)
1. Riddel-Black (106)
Using S. viminalis and S. triandra hybrids (Bowles, SQ
683, Triandra Q83)
Added with sludge (200 m3
ha1) (g ha1)
Removed with biomass (g ha1):
viminalis Bowles Hybrid
viminalis SQ 683
viminalis triandra Q83
dasyclados
a
Cu
Cd
Pb
535
5.2
447
4.613.1
2.06.0
16.247.7
140
58.6
63.1
81.5
73.6
53
34.3
76.7
6.01
Ni
93
13.4
9.3
11.1
13.8
Zn
419
1.56
0.954
1.36
1.21
Values are expressed as g ha1 except where stated otherwise.
(103), up to 13.1 g Cd ha 1 was removed. Therefore, it was calculated that the

number of years of willow SRF practices required to reduce heavy metals loads
of the soil to the target concentration is far shorter for Cd than for the other
metals (106). There is evidence to suggest that fast-growing trees may be capable
of playing a major role in reducing the concentration of organic chemicals in
contaminated sludges, soils, and groundwater (see next section). Using landfarming techniques, Harmsen et al. (110) used willows to remove polyaromatic
hydrocarbons from dredged harbor sediments, concluding that the high yield of
biomass was instrumental to reducing the costs. Willows and poplar trees were
also used in a multispecies wetland system to remove bacteria and other pathogens from municipal wastewater.
6.3
Fast-Growing Trees in Phytoremediation
Phytoremediation is a relatively new technology that involves the use of green

plants to ameliorate the condition of the soil. Usually the amelioration takes the
form of degradation or removal of contaminants, although it can also be extended
to include improvement of the physical condition of the soil or immobilization
of contaminants. The study of phytoremediation is generally divided in three
340
Punshon
distinct areas: phytoextraction, phytodegradation, and phytostabilization. Phytoextraction involves the removal of contaminants from the soil solution and in
to the tissues of the plant and is generally associated with inorganic contaminants,
such as heavy metals. Phytodegradation techniques are usually applied to organic
contaminants, where aspects of the plant metabolism are used to convert contaminants to benign byproducts. Phytostabilization is the use of the physical presence
of plants to improve soil quality and can entail erosion prevention in situations
where contaminated material is being spread via the action of wind or water.
Heavy metalcontaminated sediments are usually accessible to either phytoextraction (to remove metals) or phytostabilization (to immobilize the contaminated
soil or the contaminants within it). Studies of phytoremediation also often include
the term rhizofiltration to describe the use of plants to remove heavy metals from
polluted waters.
One of the most attractive features of phytoremediation is the incredibly
low cost in comparison with conventional clean-up technologies. Hazardouswaste clean-up is projected to cost at least $400 billion in the United States alone;
$7.1 billion on sites contaminated solely with heavy metals and $35.4 billion on
sites contaminated by metals and various types of organic contaminants (111).
In addition, the environmental impact of some of the conventional technologies
means that the soil is eroded, degraded, or biologically inert once clean-up has
been completed, and therefore it still requires revegetation and stabilization in
addition.
In a review of phytoremediation of heavy metals, Salt et al. (111) state that
the optimum plant for the phytoextraction process should not only be able to
tolerate and accumulate high levels of heavy metals in its harvestable parts but
also have a rapid growth rate and the potential to produce high biomass in the
field. Initially, however, hyperaccumulator plants and plants from the family
Brassicaceae were the main species to be used in phytoextraction field trials. This
choice was prompted by solution culture studies in which the hyperaccumulator
Thlaspi caerulescans accumulated 2739 ( 323) g g 1 DW Ni after 8 days of
growth in a solution containing 1 mg Ni L 1 (111), although the biomass production of the plants was very low. Fast growing trees, such as willows (e.g. Salix
caprea) grown in Ni-enriched hydroponic solution, took up less Ni over a longer
period of time than Thlaspi; 120 g g1 DW in the leaves of plants grown in
solution containing 4 mg Ni L1 for a period of 21 days (88) (Fig. 3). However,
despite this, willows produce several orders of magnitude more biomass than the
hyperaccumulators, and therefore metal removal using these plants may still be
feasible. Unadapted nonmycorrhizal willows accumulated Ni above the normal
range of 0.025 g g1 in hydroponics (11). From these studies it would appear
that S. caprea could accumulate reasonably high concentrations of Ni provided
the solution concentrations was not toxic; accumulation was still approximately
20 times higher than background at this treatment level. Heavy metal uptake
Tree Crops
341
using nonadapted plants is achievable as long as excess toxicity is avoided. Nickel

availability in the soil varies with the prevailing edaphic conditions but is generally several orders of magnitude below the total concentrations (112), and given
a plentiful supply of water and available N, fast-growing trees may theoretically
be useful for phytoextraction in situations of low to moderate metal contamination, while producing enough woody biomass to make the process economically
feasible. Other phytoremediation studies have indicated that hybrid poplars may
be useful for heavy metal clean-up; Banuelos et al. (113) tested a range of hybrids
in culture using saline-, boron-, and selenium-enriched drainage water and found
that P. trichocarpa deltoides performed best in terms of the lack of toxicity
symptoms.
Hybrid poplars (members of the genus Salix) have been widely studied
for their ability to degrade organic contaminants, most notably trichloroethylene
(TCE) (95,114) but also tetrachloroethylene (PCE) (115), trinitrotoluene (TNT)
(116,117) atrazine (118), and 1,4-dioxane (119). The phytodegradation of volatile
organic compounds (VOCs) is not likely to be due to the possession of a unique
metabolic pathway ablesuch as in the case of TCEto perform reductive dechlorination, but rather a function of rapid growth, a deep root system that can
rely entirely on groundwater aquifers (i.e., they are phreatophytes), and a high
transpiration rate in comparison with that of many other tree species. Other species, such as Salix and Eucalyptus, are also phreatophytes and have a characteristic dimorphic root morphology. This refers to the two types of root systems they
typically possess: shallow lateral roots that primarily obtain water from precipitation (during the wet season), and the deeply penetrating tap (sinker) roots, which
obtain water almost exclusively from the underlying water table (to a maximum
of about 30 feet) during drier periods. This innate ability to degrade organic
chemicals may provide useful in the remediation of mixtures of organic and inor-
FIG. 3. Metal concentrations (g g 1 ) in tissues of Salix caprea after 21 days

growth in solution culture. Data are means where n 3 (88).
342
Punshon
ganic contaminants. However, there is a shortfall of research information on how

this may be achieved and which clones could be used; organic degradation relies
on high transpiration rates, whereas many heavy metal contaminants severely
reduce water uptake. It also indicates that if phytodegradation schemes are to be
successful, they can only be carried out on sites without toxic levels of heavy
metal contamination.
Eucalyptus has been traditionally used to dry out water-logged soils because of its extremely high transpiration rates, although only a relatively small
number of studies have been carried out using Eucalypts for soil remediation.
One specific study utilized their high transpiration rates as conduits for mercury
transpiration (120), and another studied Eucalypts for reclamation of mine spoils
(121124) with good rates of survival and biomass production. Mishra et al.
(125) used hybrid Eucalypts in reclaiming coal fly ash and achieved successful
long-term site revegetation. However, there is little additional evidence to suggest
that Eucalypts have an ability to tolerate elevated concentrations of inorganic or
organic contamination and still maintain their high transpiration rates. Abouelkhair (126) found that Cd concentrations up to 16 mg kg 1 decreased seedling
height of Eucalyptus camaldulensis inoculated with the ectomycorrhizal fungus
Pisolithus tinctorus, although it was also noted that stem diameter, weight, leaf
area, and water uptake were not affected. Furthermore, Mitchell et al. (127) used
Eucalyptus eximia as a sensitive species in assessment of phytotoxic copper, triallate, and anthracene concentrations. It is therefore unlikely that Eucalypts
although ideal for biomass and hydrological controlcan be used in remediation
programs where heavy metals are a concern.
7
CONCLUSION
It is clear that the extent to which fast-growing tree crops can be used for phytoremediation of heavy metals has not been fully explored. Our ecological knowledge of these plants indicates that certain species possess useful metal resistance
characteristics and can rapidly establish on degraded contaminated land, ameliorating the contamination over successive cropping periods and stabilizing the
soil. Their innate ability to metabolize organic contaminants and tolerate elevated
concentrations of heavy metals, such as Cd, should stimulate research into mixed
waste remediation, as it is the complex heterogeneous substrates that provide the
greatest challenge for phytoremediation. By far the largest portion of the projected cost in conventional environmental clean-up ($35.4 billion) is taken up by
these sites. The potential for the production of biomass during phytoremediation
schemes may become an important selling point for a technology for which enduser acceptance remains a problem. The information reviewed in this chapter is
offered as an attempt to introduce further considerations in the use of trees for
Tree Crops
343
environmental clean-up, such as water use, nutrient requirements, and other potential applications, because it is this information that will ensure the success of
clean-up schemes using fast-growing trees. The presence of these two very similar fields of research developing concurrently but independently suggests that
more rapid developments may be possible if the economical, ecological, and agricultural lessons learned from SRF are used as a starting point for phytoremediation. There is some evidence to suggest that the initial signs of crossover are
occurring; work using land-farming techniques (biomass production for energy using willow crops) have been suggested for the removal of polyaromatic
hydrocarbons and mineral oils from dredged sediments (110). This represents one
of the first applications of acknowledged SRF practices to organic contaminant
remediation.
There is every indication that biomass production and disposal of both organic and metalliferous wastes is theoretically possible, opening the way for plantations that are specifically established to receive waste material and produce
energy from it. The final step in the use of trees to manage our waste products
is the safe combustion of the wood for energy and the possibility that inorganic
contaminants can be rescued from the ashes and recycled. Researchers in Sweden
are already making progress toward the development of safer biomass combustion technology; using a heavy metal separation technique involving high-temperature cyclone separation of metals while retaining Ca, Mg, P, and 75% of K
within the ash for reuse (128), recycling valuable nutrients into the intensively
managed soil.
The ability of fast-growing trees to take up heavy metals from the soil in
the presence of organic contaminants remains to be tested in either the laboratory
or the field, and it is this interdisciplinary research that will be instrumental in
making phytoremediation a more acceptable clean-up technology. The immense
variation, hybridization, and adaptability that exist within fast-growing trees
could provide economically viable solutions for a wide variety of contamination
issues (113).
ACKNOWLEDGMENTS
The author gratefully acknowledges the assistance of Dr. John Seaman, Dr. Ken
McLeod, Dr. Nick Dickinson, and Prof. N. W. Lepp for their helpful comments
on the manuscript.
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12
Tree Bark
Tree Bark Pockets as Pollution Time Capsules
for Historical Monitoring
Kenichi Satake
National Institute for Environmental Studies, Tsukuba, Japan
INTRODUCTION
Historical monitoring of environmental pollution combined with present day

monitoring is essential for assessing pollution levels in a world that has different
levels and types of human activity and a diverse environmental history.
Recently in Japan, bark enclosed in tree trunks, known as tree bark pockets, has been shown to provide some of the most readily available historical
specimens for monitoring air pollution (1). Bark pockets are common in tree
trunks. The phenomenon is well recognized by forestry technicians, saw millers,
and dendrologists because trunks with bark pockets often have problems with
lack of strength, discoloration, and microbial decomposition of the xylem layer.
Bark pockets have not previously been identified as being useful parts of a tree.
Their potential value for scientific research has previously been overlooked. Harvested trunks with bark pockets have mostly been discarded as waste. Therefore,
many forest trees identified as containing bark pockets have been left standing
and not harvested.
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Satake
This chapter describes the scientific use of bark pockets for environmental
research as pollution time capsules. This approach opens a new frontier in
methods of historical monitoring in the world.
TREE BARK POCKETS FOR HISTORICAL MONITORING

OF AIR POLLUTION
Tree bark provides ideal specimens for directly measuring levels of local pollution. This is because a tree bark accumulates air pollutants directly onto its surface
(Fig. 1). However, despite this suitability for providing direct evidence of air
pollution, bark has not been used to monitor historical trends in air pollutants
because it is also exposed to polluted air emitted by modern sources, thereby
confounding historical interpretation. The presence of bark pockets within the
trunk removes this disadvantage. During tree growth bark can be incorporated
into the trunk to form a bark pocket by many mechanisms that are described in
the next section. One of the important aspects of using bark pockets as pollution
time capsules is that the bark pockets are located between annual rings, thus
allowing extremely accurate dating.
MECHANISMS OF BARK POCKET FORMATION
There are many physical and biological causes of the formation of bark pockets
as pollution time capsules. The mechanisms are classified as follows:
3.1
Mechanism (1): Bark PocketType 1, Encapsulation

of a Wound
Figure 2 shows a wound in a tree trunk caused by physical damage and the
subsequent development of covering the wound with newly formed bark and
xylem. Figure 3 shows bark pockets formed by this process.
Encapsulation of a wound is one of the main mechanisms of bark pocket
formation. A wound in the process of encapsulation by surrounding bark is called
a bark pocket to be. The process of encapsulation of the polluted outer bark
leads to the enclosure of pollutants into the trunk by the trees annual rings, thus
allowing accurate dating. In some cases, biological materials, such as shoots of
epiphytic bryophytes, are found between the two bark layers in the bark pocket.
Epiphytic bryophytes are potentially indicators of atmospheric pollution (2). Information about the past environment can be obtained from species found in
samples through analysis of the pollutants contained in the biological materials.
In addition to this, the outermost xylem in the bark pocket (Fig. 3) is available
for samples that retain pollutants, provided microbial decomposition is limited.
Tree Bark
FIG. 1 Accumulation of air pollutants on the surface of tree.
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Satake
FIG. 2 A wound in a tree trunk caused by physical damage and the subsequent development of covering the wound with newly formed bark and
xylem.
3.2
Mechanism (2): Bark PocketType 2, Joining of Two

Tree Trunks (Branches)
Figure 4 shows the initial contact between two tree trunks or branches and the
radial section of bark pockets formed by this mechanism (X m X n). Figure 5
shows radial section of bark pocket. Figure 6 shows a vertical section of the bark
pocket (Y 0 Y n).
This type of bark pocket provides at least three replicates of continuous
time series data on the outer bark samples because the barks are formed symmetrically on both sides of the horizontal and vertical axes. The bark pockets cover
long spans of successive years, from old to new, permitting monitoring of continuous, year-by-year, historical changes in pollution.
3.3
Mechanism (3): Bark PocketType 3, Encapsulation

of a Branch
Figure 7 show the steps in the encapsulation of a branch. Shaping and/or pruning
of branches are the main cause of this type of bark pocket, which is usually
observed in trees planted on roadsides and in parks, gardens, and artificial forests.
Tree Bark
FIG. 3 Bark pocket type 1 in a tree trunk.
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Satake
FIG. 4 Initial contact between two tree trunks or branches (a) and radial section of bark pockets formed by this mechanism (Xm Xn) (b).
FIG. 5 Radial section of bark pocket formed by the joining of two branches
(x m x m).
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359
FIG. 6 Vertical section of the bark pocket formed by joining two branches
(y 0 y n).
The appearance of the outer bark formed in this encapsulation process differs
from that of normal outer barks. This type looks like a cats eye but it is a
trees eye for watching the environment (Fig. 8). Trees eye is an indicator
of the location of bark pockets in a tree. Some trees eyes in the process of bark
pocket formation can be called bark pockets-to-be because they change to bark
pockets after encapsulation.
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Satake
FIG. 7 Steps in the encapsulation of a branch after pruning.
FIG. 8 Cats eye ( trees eye bark pocket-to-be) formed on the outer bark
after pruning the branch.
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361
The physical and chemical characteristics of outer bark differ markedly

with different tree species. For example, the outer bark of black pine (Pinus
thunbergii), red pine (P. densiflora) and white birch (Betula tauschii) peel off
from the trunk. The barks of some species exfoliate with characteristic periodicity. In this case, it is difficult to estimate trends in air pollution using outer bark
and bark pockets because the exposure period of the outer bark is shorter than
the time estimated from the annual rings enclosing the bark pocket. However,
the section of the cut end of the branch or wax rosin layer in the bark pocket
remains available as an accumulator of air pollutants provided that the xylem of
the section is not microbially decomposed. In this case, the annual rings of the
cut end of the branch and the record of pruning help with correct dating of the
section.
3.4
Mechanism (4): Bark PocketType 4, The Joining

of Uneven Trunk
Figure 9 shows the bark pocket formed in the process of growing on an uneven
trunk. This type of bark pocket is often observed in beech (Fagus japonica, Fagus
sylvatica), which has an uneven trunk. This type of bark pocket in the trunk
forms in the same compass direction as the continuously jointed outer bark. Bark
pockets arranged in the same compass direction in the trunk offer an advantage
for understanding historical changes in pollution because there is a directional
relationship between the source of pollutants and the tree that accumulates the
pollutants.
FIG. 9 Bark pocket formed in the process of growing on an uneven trunk.
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3.5
Satake
Other Mechanisms (5): Bark PocketType 5
There are other occasional causes and characteristic mechanisms of bark pocket
formation. Artificial materials such as wire, nails, and fences can damage a tree
and become encapsuled to form bark pockets as the tree grows. Wood fire, thunder attack, and cracks formed in the trunks by freezing during cold winters are
other causes of characteristic bark pocket formation.
4
CHARACTERISTICS OF BARK POCKETS AS POLLUTION

TIME CAPSULES
Natural and artificial materials have been used for historical monitoring of pollutants transported into terrestrial and aquatic ecosystems (Fig. 10). Typical of these
materials are lake sediments, polar ice, herbarium specimens, peat, tree rings,
bones, freshwater shells and corals. However, these materials are not always fit
for historical monitoring of air pollution. Difficulties often exist in the use of
such specimens for monitoring.
Major problems include (a) dispersal and translocation of the pollutants;
(b) contamination during natural and/or artificial preservation periods; (c) difficulty in sourcing materials with a suitable time scale from inhabited monitoring
sites such as urban areas, and also from remote and background areas; (d) incor-
FIG. 10 Typical natural and artificial materials for historical monitoring of

pollutants transported to terrestrial and aquatic ecosystems. (Examples are
lake sediments, polar ice, herbarium specimens, peat, corals and tree rings.)
Tree Bark
363
rect dating. Corresponding examples of these problems are as follows: (a) Human
activities, such as fishing by net and bioturbation by bottom fauna, often disturb
the laminar structure of lake sediments formed by the deposition of particulates.
(b) Herbarium specimens are in contact with the current atmosphere during their
storage. Exchange of pollutants between bones and soil or contamination by soil
may occur. (c) Polar ice from the Antarctic may provide information on background areas or global pollution, but it is difficult to get information on pollution
in populated urban or rural areas. (d) Evaluation and application of dating methods suitable for the selected materials are required.
One of the practical dating methods usually used is Pb-210 dating. The
half-life of Pb-210 is about 22.2 years. Therefore, dating is limited to between
several and 150 years, and the resolution depends on the accuracy of analysis.
Another method is the C-14 method. The half-life of C-14 is about 5600 years.
Thus, the dating is from several thousand years to 20,00030,000 years. There
are some other dating methods, such as chronological methods using annual rings
in tree trunks, shells, or corals. This dating, especially the use of a trees annual
rings, is possible to more than several thousand years (3). Consequently, if we
want to know about changes in pollution that occurred during and after the industrial revolution, dendrochronological dating is best fitted for this purpose.
5
DIFFERENCE BETWEEN BARK POCKETS AND ANNUAL

RINGS AS POLLUTION TIME CAPSULES
There are many reports of heavy metals that accumulate in tree rings corresponding to historical changes in pollution (4). Trees form a growth ring every year.
Many publications have analyzed the pollutants in annual rings and calculated
the age of the rings, but this presents problems because most atmospheric pollutants that accumulate in tree rings are limited to water-soluble compounds transported by way of soil and roots. Also, there are time lags, sometimes more than
10 years, between soil pollution and the count of annual rings.
Most air pollutants in the form of wet and dry depositions on trees accumulate directly on the outer bark. The concentration of pollutants in the annual rings
is very low compared with that of the outer bark because of the dispersal that
occurs in the process of pollutant transportation through soils and trees. Furthermore, lateral movement of pollutants may occur between adjacent rings (5).
Therefore, it is difficult to obtain direct information on air pollution from
annual rings, although some historical trends in pollution are reflected in the
concentration of pollutants in the annual rings.
In the case of bark pockets, there are no time lags, and dispersal of pollutants and translocation of pollutants from bark pockets after encapsulation is considered to be limited because the outermost parts of the two barks in contact with
each other consist of the dead tissue in the bark pockets and the bark pocket is
not a passageway for water from the roots to the leaves.
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Satake
EXAMPLES OF HISTORICAL MONITORING USING BARK

POCKETS
For our research using bark pockets for historical monitoring, a 350-year-old
Japanese cedar (Cryptomeria japonica) that had suffered typhoon damage and a
226-year-old Japanese cedar were collected, respectively, from Nikko in 1990
and Yakushima Island in 1992. Nikko, located about 100 km north of Tokyo, is
famous for its Toshogu shrine (a World Heritage site) which was built about 360
years ago. Also at this time, avenues of C. japonica were planted along the roadside. Yakushima Island, famous for many C. japonica trees older than a thousand
years, includes a national park and is also a World Heritage site.
The bark pocket collected at Nikko was formed around 17601780 (240
220 years ago based on 2000) and the bark pocket collected at Yakushima was
formed around 17861809 (214191 years ago). The concentrations of lead as
a pollutant in the bark pockets and outer bark were determined, since the use of
leaded gasoline as a fuel for automobiles has increased drastically since World
War II. Many countries still use it and lead pollution is still spreading, although
manufacturing and sales of leaded gasoline are already banned in Japan. The
results of the analysis showed a marked difference between the amount of lead
contained in the bark pockets from the Edo era and that in the outer bark, which
reflected modern lead pollution. In the case of the cedar trees from the roadside
in Nikko, the concentration of lead in modern samples was about 1000 times
FIG. 11 Relative concentration of lead in the bark pockets collected from

Nikko and Yakushima.
Tree Bark
365
higher than that of the Edo era (Fig. 11). In the case of Yakushima Island, a
remote island located in southern Japan, the concentration of lead in modern
samples was about 1020 times higher than that of the Edo era (1).
In addition to these samples, trees containing bark pockets were collected
in the precincts of Muro Temple in Nara prefecture in Japan, about 60 km south
of Kyoto and about 37 km south of Nara. The analysis of bark pockets of C.
japonica from Muro Temple using laser ablation ICP-MS revealed clearly the
historical changes in mercury and lead pollution over a period from 140 to 70
years ago and up to the present time (6). The respective concentrations of lead
and mercury in the modern outer bark were about 40 and 4 times higher than
those of 140 years ago.
7
NEW PROJECT CONCERNING ATMOSPHERIC CHANGES

IN THE WORLD
While environmental pollution and destruction of nature are expanding on a

global scale, it is becoming increasingly important to grasp the influence of human activities on a global environment, especially in the world. In the historical
monitoring using bark pockets, heavy metals were analyzed as pollutants. However, similar monitoring are possible for other inorganic and organic pollutants.
Therefore, we are planning to propose a new project for historical monitoring of
atmospheric changes. In addition, we are planning to select the forests for historical monitoring (Time Capsule Forests) throughout the world. The Nakagawa experimental forest, Hokkaido University, located at Latitude 444444, is the first
Time Capsule Forest for this purpose.
REFERENCES
1. K Satake, A Tanaka, K Kimura. Accumulation of lead in tree trunk bark pockets as
pollution time capsules. Sci Tot Environ 181:2530, 1996.
2. K Satake, K Kimura, A Tanaka, V Virtanen. Two-hundred-years old shoots of the
epiphytic moss Brotherella henonii preserved in a bark pocket of the conifer Cryptomeria japonica. J Bryol 18:815832, 1995.
3. FH Schweingruber. Tree Rings. Dordrecht: Kluwer Academic, 1988.
4. CF Baes III, HL Ragsdale. Age-specific lead distribution in xylem rings of tree genera
in Atlanta, Georgia. Environ Pollut Ser B 2:2135, 1981.
5. JR Donnelly, JB Shance, PG Schaberg. Lead mobility within the xylem of red spruce
seedlings: implications for the development of pollution histories. J Environ Qual 19:
268271, 1990.
6. K Satake, R Idegawa, M Obata, N Furuta, N. Historical environmental monitoring
using bark pockets as pollution time capsules. Proceedings of the Fifth International
Conference on the Biogeochemistry of Trace Elements; Vienna 99, Vol. 2. 1074
1075, 1999.
13
C. Nabais and H. Freitas
Universidade de Coimbra, Coimbra, Portugal
J. Hagemeyer
University of Bielefeld, Bielefeld, Germany
DENDROANALYSIS: DEFINITION AND FUNDAMENTAL

PRINCIPLES
Trees in temperate regions produce annual growth rings (1). Such rings are the
result of a rhythmic activity of the cambium. During spring and early summer
the cambium is active and new wood is formed, whereas in autumn and winter
the cambium is dormant (2).
The mineral composition of the wood rings depends partly on mineral uptake of the tree. The extent of mineral uptake may reflect the availability of the
elements in the environment surrounding the tree (3,4). This has lead to the idea
of using tree rings as retrospective biomonitors of trace metal pollution; the
method is known as dendroanalysis (5).
To use dendroanalysis as a tool for biomonitoring environmental pollution,
some conditions must be meet: (a) a constant relation must exist between the
element concentration in the environment and the amount of the element present
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Nabais et al.
in the wood tissue; (b) incorporation of the element into a certain annual ring
must occur within a limited period of time; (c) it must be known to which rings
the element is transported, either only to the outermost and youngest ring (e.g.,
in ring-porous trees water conduction takes place primarily in the youngest
growth ring) or to a number of older rings; (d) after incorporation in the annual
ring no subsequent remobilization or radial transport should occur; (e) the radial
distribution pattern of an element should not change its profile over a long period;
(f) the radial distribution of a certain element should be similar in different parts
of the same tree and in different trees growing in the same area (3,4).
LITERATURE REVIEW OF DENDROANALYSIS
A summary of the available publications reporting radial distribution patterns of

elements in tree rings is given in Table 1. Some dendroanalytical studies were
interpreted as supporting the feasibility of dendroanalysis, while others questioned the method (Table 1). From the published articles cited, 41% state that
the method of dendroanalysis to monitor environmental pollution is possible,
26% state that monitoring is not possible, 21% find the method questionable, 8%
do not give an opinion on the validation of the method, and 4% conclude that
some trace metals can be used to monitor environmental pollution (Table 1).
2.1
Research Supporting Dendroanalysis
In some cases correlations were found between patterns of tree ring heavy-metal
contents and temporal records of pollution from other sources, e.g., data of industrial or traffic activities. Several studies have found that Pb concentrations in soils
and tree rings declined sharply with increasing distance from the road (6,7). The
wood rings of Acer saccharum (8), Quercus nigra (9), and Q. velutina (9) appear
to accurately record annual changes in atmospheric metal deposition.
Pinus ponderosa growing along rivers with contaminated sediments from
the discharge of heavy metalladen waste showed higher concentrations of Zn
and Cd than trees growing at uncontaminated sites (10). The decline of Zn and
Cd concentrations in the younger growth rings was explained by the reduction
of mining activity and implementation of pollution control technologies that reduced the Zn and Cd pollution in the rivers (10). However, the authors do not
exclude the radial translocation of Zn and Cd as a possible influence on the radial
distribution patterns.
In Quercus robur the radial distributions of Cd and Pb in wood rings growing in the surroundings of a factory showed a good correlation with the calculated
emission history (11). However, Cd content in the oak wood continued to increase, even after close-down of the factory. This suggests that Cd was accumu-
Author
Year
Wright & Will (74)

Orman & Will (75)
Furukawa (76)
1957
1960
1961
Schroeder & Balassa (77)

Furukawa (78)
1961
1963
Furukawa (79)
Galligan et al. (80)
Ault et al. (81)
Holtzman (82)
Ishizaki et al. (83)
McMillin (84)
Fossum et al. (85)
Janin & Clement (86)
Szopa et al. (87)
Wardell & Hart (48)
Hampp & Holl (88)
Rolfe (89)
Ward et al. (90)
1964
1965
1970
1970
1970
1970
1972
1972
1973
1973
1974
1974
1974
Tree genus
Monitoring
Ca, K, Mg, N, Na, P

Ca, K, N, P
Ca, K, N, P
n.m.
n.m.
n.m.
Cd, Pb
Ca, K, N, P
n.m.
Ca, K, N, P
Mn
Pb
Pb
Cd, Zn
Ca, Fe, K, Mg, Mn, Na, P
Ca, Mg, Mn
Ca, K, Mg, P
Pb
Ca, Cl, K, Mg, Mn, P, S
Pb
Pb
Pb
n.m.
n.m.
?
?
n.m.
n.m.
n.m.
369
Pinus
Pinus
Cedrus, Chamaecyparis,
Cryptomeria, Gingko,
Larix, Pinus, Taxodium
Ulmus
Ailanthus, Alnus, Cercidiphyllum, Cinnamomum,
Fagus, Fraxinus, Idesia,
Lithocarpus, Paulownia,
Phellodendron, Prunus,
Quercus, Robinia
Cryptomeria, Pinus
Pseudotsuga
Quercus
Carya, Quercus, Ulmus
Cryptomeria
Pinus
Pinus
Populus
Pinus, Quercus
Quercus
Acer, Robinia, Tilia
Acer, Pinus, Quercus
Acer, Aesculus, Fraxinus,
Platanus, Quercus, Ulmus
Element
TABLE 1 List of Publications Presenting Original Data of Radial Distribution Patterns of Elements in Tree Stems a
370
TABLE 1 Continued
Year
Tree genus
Basham & Cowling (91)
1975
Hall et al. (92)

Ricci (93)
1975
1975
Picea, Pinus, Populus, Quercus

Fraxinus, Quercus
Pinus
Sheppard & Funk (94)
1975
Pinus
Suzuki (95)
Barnes et al. (96)
Clement & Janin (97)
Dollard et al. (98)
Faucherre & Dutot (99)
Gilboy et al. (5)
Lepp (100)
1975
1976
1976
1976
1976
1976
1976
Pillay (101)
1976
Cryptomeria
Carpinus, Picea
Populus
Acer
Pinus
Ulmus
Acer, Fagus, Populus, Tilia,
Ulmus
Gleditsia, Prunus, Quercus,
Syringa, Thuja, Tsuga
Tian & Lepp (102)

Tout et al. (103)
Ward et al. (104)
Herrmann et al. (105)
Hincman et al. (106)
1977
1977
1977
1978
1978
Acer, Larix
Cedrus, Ulmus
Beilschmiedia
Quercus
Pinus
Kardell & Larsson (107)
1978
Quercus
Element
Monitoring
Mg, P
Pb
Ag, Al, Ba, Br, Ca, Cl, Co,
Cr, Fe, Hg, K, Mg, Mn,
Na, Rb, V, W, Zn
Ag, Au, Co, Cr, Fe, Hg, K,
La, Mn, Na, Rb, Sb, Zn
Cd, Pb, Zn
Pb
Ca, K, Mg, Mn, P, Zn
Pb
Zn
K
Cu, Pb
?
?
Ag, Al, As, Au, Ca, Cl, Co,

Cr, Cu, Fe, Hf, Hg, K, La,
Mn, Na, Rb, Zn
Pb, Zn
Br, Cl, J, K, Mg, Na, Sr
Ag
Cu, Fe, Mn, Pb, Zn
Br, Fe, K, Mn, Na, Pb, W,
Zn
Cd, Pb
n.m.
?
Nabais et al.
Author
1978
1979
1979
1979
Ulmus
Pinus
Quercus
Pinus
Cl
Zn
Cd
Cd, Cu, Pb, Zn
Valkovic et al. (112)
1979
Not specified
Fergusson et al. (113)

Schrimpff (114)
Baes & Ragsdale (6)
1980
1980
1981
Robitaille (115)
Suzuki (116)
Suzuki (117)
Isermann (118)
Saka & Goring (119)
Wickern & Breckle (120)
Baes & McLaughlin (121)
Baes et al. (122)
1981
1981
1982
1983
1983
1983
1984
1984
Tilia
Abies
Carya, Liriodendron, Quercus
Abies
Cryptomeria
Pinus
Picea, Prunus, Quercus
Picea
Quercus
Pinus
Pinus
Ni, Pb, Ti, various element

ratios
Pb
Cd, Cu, Mn, Pb, Zn
Pb
Brownridge (123)
1984
Ciepal & Niemtur (124)

Greune & Fengel (125)
1984
1984
Acer, Carya, Fagus, Quercus, Robinia

Pinus
Picea
Kazmierczakowa et al. (126)

Legge et al. (127)
Ogihara & Katsumo (128)
1984
1984
1984
Fagus
Pinus
Pinus
(Cu, Pb)
(Zn, Pb)
Cu, Pb, Zn
Cd
Hg
Al, Cd, Cu, Mn, Pb, Zn
Ca, Na, S
Pb
Al, Fe, Mo, Ti, Zn
Al, Ca, Cd, Cu, Fe, Mn, Mo,
Ti, Zn
137
Cs, 40 K
n.m.
Pb, Zn
Al, Ba, Ca, Cl, Cu, Fe, K,
Mg, Mn, Na, P, S, Si, Zn
Cd, Cr, Fe, Pb, Zn
Al, As, Cl, Cu, Mn, P, Si, Zn
Al, Cd, Cr, Cu, F, Hg, Ni, Zn
?
n.m.
Tout & Gilboy (108)

Dutot & Faucherre (109)
Gilboy et al. (110)
Symeonides (111)
371
372
TABLE 1 Continued
Year
Tree genus
Taneda et al. (129)
1984
Bauch et al. (130)
1985
Castanea, Cryptomeria, Kalopanax, Prunus, Thujopsis

Picea
Braker et al. (131)
1985
Pinus
Berish & Ragsdale (132)
1985
Carya
Breckle & Schure (133)

Kosmus & Grill (134)
Matusiewicz & Barnes (135)
1985
1985
1985
Fraxinus, Quercus
Aesculus
Acer, Picea
Maurer & Peters (136)

McLaughlin & Braker (137)
Hagemeyer & Breckle (60)
Katayama et al. (138)
1985
1985
1986
1986
Picea
Picea
Quercus
Cryptomeria

Lim & Cousens (140)
Maurer et al. (141)
Meisch et al. (142)
1986
1986
1986
1986
Cryptomeria
Pinus
Picea
Fagus
Meisch et al. (143)
1986
Fagus
Element
Ca, Cu, Fe, Mg, Mn, Zn
Al, Ca, Cd, Fe, K, Mg, Mn,

Pb, Zn
Al, Ba, Ca, Cd, Cu, Fe, Mg,
Mn, Mo, P, Ti, Zn
Al, Cd, Cu, K, Mg, Mn, Ni,
Pb, Zn
Na
Cd, Cu, Pb, Zn
Al, As, Ba, Ca, Cu, Fe, Ge,
K, Mg, Na, Si, Sr, V, Zn
Ca, Pb
Fe
Cd
Al, Br, Ca, Ce, Cl, Co, Cs,
Fe, K, La, Mg, Mn, Na,
Rb, Sc, Sm, Ti, V
Cs, 137 Cs, K, 40 K, Na, Rb
Ca, K, Mg, N, P
Cl, N, F, P, S
Al, Ca, Co, Cr, Cu, K, Mg,
Mn, Na, Ni, Pb, Zn
Al, Ca, Fe, Mg, Mn, Zn
Monitoring
n.m.
?
?
?
?
Nabais et al.
Author
1986
Baes & McLaughlin (66)

Guyette & McGinnes (145)
Hofmann & Born (146)
Maclauchlan et al. (147)
McLenahen et al. (148)
Nagj et al. (149)
1987
1987
1987
1987
1987
1987
Castanea, Cryptomeria, Kalopunax, Prunus, Thujopsis

Pinus
Juniperus
Fraxinus
Alnus
Liriodendron
Pinus
Okada et al. (150)
1987
Cryptomeria
Queirolo & Valenta (151)

Truby & Zottl (152)
Arp & Manasc (153)
1987
1987
1988
Quercus
Betula, Pseudotsuga
Picea
Chigira et al. (154)
1988
Cryptomeria
Kohno et al. (155)
1988
Kyncl et al. (156)

Lukaszewski et al. (157)
Okada et al. (158)
Ragsdale & Berish (159)
Scherbatskoy & Matusiewicz (160)
1988
1988
1988
1988
1988
Chamaecyparis, Cryptomeria
Picea
Pinus
Cryptomeria
Carya
Acer, Picea
Tendel & Wolf (161)
1988
Pinus
Ca, Cu, Fe, K, Mg, Mn, Na,

Zn
Fe, Zn
Al, Cu, Fe, Mn, Zn
Pb
Ca, Cu, Fe, Pb, Sr, Zn
Ca, P
Ca, Cr, Cu, Fe, K, Mn, Pb,
Rb, S, Ti, V, Zn
Al, Br, Ca, Cl, Cr, K, Mg,
Mn, Na, Rb
Cd, Pb
Cd, Pb, Zn
Al, Ca, Cr, Cu, Fe, K, Mg,
Mn, Ni, P, Zn
Ca, Cs, 137 Cs, K, Na, Rb, 90 Sr
137
Cs
?
(Sr)
(Cs)
373
Ca, Cu, Fe, Mg, Mn, Pb

Cd, Cu, Zn
Cl, K, Mg, Mn
Pb
Al, As, Ba, Ca, Cu, Fe, Ge,
K, Mg, Mn, Na, Si, Sr, V,
Zn
Al, Ca, Cd, Cu, Fe, K, Mg,
Mn, Ni, P, Pb, S, Zn
Taneda et al. (144)
TABLE 1 Continued
Truby (162)
1988
Truby & Zottl (163)

Bondietti et al. (164)
Frelich et al. (165)
1988
1989
1989
Abies, Pseudotsuga, Quercus

Abies, Pseudotsuga
Picea, Tsuga
Acer, Pinus
Guyette et al. (166)

Hagemeyer et al. (167)
1989
1989
Juniperus
Fagus
Hasanen & Huttunen (168)
1989
Pinus
Helmisaari & Siltala (169)
1989
Pinus
Ilgen & Nebe (170)

Long & Davis (171)
1989
1989
Picea
Quercus
McClenahen et al. (55)

Queirolo (172)
Queirolo et al. (173)
Aberg et al. (174)
Bondietti et al. (13)
Hall et al. (175)
Jordan et al. (176)
Kairiukstis & Kocharov (177)
Momoshima & Bondietti (30)
Okada et al. (40)
1989
1989
1989
1990
1990
1990
1990
1990
1990
1990
Liriodendron
Quercus
Quercus
Picea, Pinus
Abies, Betula, Picea, Tsuga
Pseudotsuga
Pinus
Pinus
Picea
Cryptomeria
Tree genus
Element
Monitoring
Pb
Cd, Pb, Zn
Al, Ca, Mg, 90 Sr
Ca, Fe, K, Mg, Mn, P, Pb, S,
Zn
Mo, S
Ca, Cd, K, Mg, Na, Ni, Pb,
Zn
Al, B, Ca, K, Mg, Mn, Mo,
N, P, Rb, S, Zn
Al, B, Ca, K, Mg, Mn, N, P,
Zn
As, Cd, Cu, N, Pb, S, Zn
Al, B, Ba, Ca, Cd, Co, Cu,
Cr, Fe, K, Mg, Mn, Na, Ni,
P, Pb, Si, Sr
Ca, K, Mn, P, Sr
Cd, Cu, Pb, Zn
Cd, Pb
Ca
Ca, Mg, Mn, Sr
Ce, La, Nd, Sm
Al, Cd, Cu, Mn, Pb, Zn
K
Ca, P, cation ratios
Ca, Cl, K, Mg, Mn, Na, Rb
?
n.m.
?
?
?
Nabais et al.
Year
374
Author
Quercus
Pernestal et al. (178)
1990
Picea
Queirolo et al. (179)

Truby & Lindner (180)
Truby & Zottl (181)
Vroblesky & Yanosky (182)
Zech et al. (183)
Gilfrich et al. (184)
1990
1990
1990
1990
1990
1991
Quercus
Picea
Picea
Liriodendron
Fagus
Quercus, Sassafras
Guyette et al. (185)

Kennedy & Bergeron (186)
1991
1991
Ohmann & Grigal (187)
1991
Juniperus
Acer, Fraxinus, Populus,
Thuja, Tsuga
Abies, Acer, Pinus, Populus
Pernestal et al. (188)
1991
Picea
Ritters et al. (189)
1991
Stewart et al. (7)

Zayed et al. (190)
Chun & Hui-Yi (41)
1991
1991
1992
Abies, Acer, Picea, Pinus,

Populus
Dacrycarpus
Picea
Pinus, Quercus
Ferretti et al. (191)

Hantemirov (73)
Vroblesky et al. (193)
Wickman (194)
1992
1992
1992
1992
1992
Pinus
Fagus
Pinus
Liriodendron
Picea, Pinus
Al, Ba, Br, Ca, Cl, Cs, Cu, K,

Mg, Mn, Na, Rb
Ca, Cl, Cu, Fe, K, Mn, S, Sr,
Zn
Cd, Cu, Pb, Zn
Mn
Cd, Pb, Zn
Cl, Fe
Mg, S
Ca, Cl, Co, Cu, Fe, K, Mn,
Ni, Ti, Zn
Cd, Pb
Mn
Al, B, Ca, Cd, Cr, Cu, Fe, K,
Mg, Mn, N, Na, Ni, P, Pb,
S, Zn
Ca, Cr, Cu, Fe, K, Mg, Mn,
Zn
Al, B, Ca, Cu, Fe, K, Mg,
Mn, N, P, S, Zn
Cd, Cu, Mn, Pb, Zn
Al
Al, As, Ca, Cd, Cr, Cu, Fe, K,
Mg, Mn, Ni, P, Pb, Ti, V,
Zn
Cl, P, S
Cd, Pb, Zn
Ca, Cu, K, Mg
K
Ca, Mg, Mn, Sr, 87 Sr/ 86 Sr
?
?
n.m.
?
/
375
1990
Okada et al. (44)
376
TABLE 1 Continued
Year
Tree genus
Yanosky & Vroblesky (195)
1992
Zayed et al. (196)

Ferretti et al. (197)
1992
1993
Liquidambar, Liriodendron,
Prunus, Quercus, Sassafras
Picea
Pinus
Hemmann (198)
1993
Pinus

Kudo et al. (200)
1993
1993
Cryptomeria
Cryptomeria
Lukaszewski et al. (201)

Okada et al. (45)
1993
1993
Okada et al. (46)
1993
Wenk (202)
Balk & Hagemeyer (69)
Dittmar & Zech (203)
Forget et al. (204)
Hoffmann et al. (205)
Hofmann (206)
1993
1994
1994
1994
1994
1994
1994
Populus
Populus, Abies, Cedrus,
Chamaecyparis, Larix,
Metasequoia, Pinus
Castanea, Hovenia, Lindera,
Phellodendron, Quercus,
Sorbus, Stewartia, Zelkova
Picea
Quercus
Fagus
Picea
Fagus
Pinus, Populus
Fraxinus
Element
Monitoring
Ni
Al, Ca, Cu, Fe, Mg, Mn, Zn

Al, Ca, Cd, Cu, Fe, K, Mg,
Mn, Na, Pb, Rb, Zn
Al, B, Ca, Cd, Cu, Fe, K, Mg,
Mn, Na, Zn
As, Cu, K
137
Cs, 40 K, 239 Pu239, 240 Pu
Cd, Cu, Pb, Zn

Mn, Na, Rb, Sm, Zn
(Pu)
(Cs, K)
Al, Ba, Br, Ca, Ce, Cl, Cs, K,

La, Mg, Mn, Na, Rb, Sm,
Sr, Zn
N, S
Cd, Pb
Mg, Mn, S
Mn
Ni
Mg, Al/Mg, Pb/Mg
Pb
Nabais et al.
Author
1994
Momoshima et al. (208)

Myre & Camire (209)
Truby (210)
1994
1994
1994
Aoki et al. (211)

Cote & Camire (212)
Eklund (11)
Garrec et al. (213)
Hagemeyer (61)
Hagemeyer & Schafer (19)
Hagemeyer & Shin (68)
Kardell (214)
Momoshima et al. (12)
1995
1995
1995
1995
1995
1995
1995
1995
1995
Acer, Carya, Fagus, Liriodendron, Tsuga, Ulmus,

Picea, Pinus
Abies, Cryptomeria
Larix
Abies, Fagus, Picea, Pinus,
Pseudotsuga, Quercus
Cryptomeria
Acer
Quercus
Abies, Cryptomeria, Picea
Quercus
Fagus
Pinus
Pinus, Quercus
Cryptomeria
Shortle et al. (215)

Truby (216)
Li et al. (217)
Poulson et al. (218)
Suzuki (219)
Yanosky et al. (220)
Eklund et al. (221)
Latimer et al. (222)
Watmough & Hutchinson (223)
1995
1995
1995
1995
1995
1995
1996
1996
1996
Abies, Picea
Picea, Pinus, Quercus
Picea
Tsuga
Cryptomeria
Taxodium
Quercus
Taxodium
Acer
137
Cs,
90
Sr
Ca, K, Mg, P
Ca, K, Mg, Mn, P, Zn
Ca, Cd, Cu, K, Mg, Mn, P,
Pb, Zn
137
Cs, 90 Sr
Al, Ca, K, Mg
Cd, Pb
Cs, K, Pu
Cd
Cd, Pb, Zn
Cd, Pb
Cd, Cu, Pb, Zn
Ba, Ca, Cs, K, Mg, Mn, Na,
P, Rb, Sr,
Ca
Cd, Cu, Pb, Zn
Hg
N
Cd, Cu, Hg, V
Cl
Cd, Pb
Pb, Zn
Ca, Cd, Cu, K, Fe, Mg, Mn,
Ni, Pb, Sr, Zn
(Sr)
(Cs)
Momoshima & Bondietti (207)
377
378
TABLE 1 Continued
Year
Garbe-Schnberg et al. (14)

Jonsson et al. (224)
Oliveira et al. (225)
1997
1997
1997
Betula, Pinus
Quercus
Pinus
Watmough et al. (226)

Hofmann et al. (227)
Horn et al. (20)
Larsson & Helmisaari (228)
1997
1998
1998
1998
Acer
Picea
Pinus
Prohaska et al. (22)
1998
Picea
Schaumloffel et al. (10)

Watmough et al. (8)
Dewalle et al. (229)
1998
1998
1999
Penninckx et al. (230)

Watmough (231)
Watmough & Hutchinson (67)
1999
1999
1999
Pinus
Acer
Acer, Betula, Fagus, Larix,
Liriodendron, Picea, Prunus
Fagus
Abies, Acer, Picea
Abies
Anderson et al. (9)
2000
Quercus
Tree genus
Element
Al, Cu, Pb
Cd, Pb
Al, B, Br, Ca, Co, Cs, Fe, K,
Mg, Mn, Na, P, Rb, Zn
Cd, Mg, Mn, Pb
Al, Ba, Ca, La, Mn, P
Pb
Ca, Cu, Fe, K, Mn, Ni, Rb,
Sr, Zn
Al, Ba, Ca, Cd, Co, Cr, Fe,
Mg, Mn, Pb, Sr, Zn
Cd, K, Zn
Ca, Cd, Cr, Cu, Mn, Pb, Zn
Al, Ca, Mg, Mn
Ca, K, Mg, Mn, N, P

Ca, Cd, Cu, Pb
Al, Ca, Cd, Cu, Mg, Mn, Ni,
Pb, Zn
Pb
Monitoring
?
/
a
The column Monitoring summarizes the authors conclusions about the value of dendrochemistry in biomonitoring: () monitoring
is possible; () monitoring is not possible; (?) method questionable; (n.m.) not mentioned or monitoring was not intended.
Source: Updated from Refs. 3 and 4.
Nabais et al.
Author
379
lated in the soil profile and subsequently mobilized, absorbed by the roots, and
incorporated in the wood (Fig. 1) (11).
Japanese cedar or sugi (Cryptomeria japonica D. Don) shows a decline in
Japan probably due to acidic deposition (12). Acidic deposition in the soil can
change the availability of elements for uptake by plants. To trace back the acidic
deposition history in a forest it is necessary to understand the distribution and
mobility of cations in the xylem. Distributions of cations that are immobile in
annual rings can be used as a temporal record of nutrient availability (13). In the
annual rings of sugi the radial distribution of 90 Sr was related to the cumulative
deposition of fallout from nuclear weapon tests, while the radial distribution of
137
Cs was unrelated (12). This suggests that Sr 2 is immobile while Cs is mobile
in the horizontal direction in wood. The radial distribution of Sr 2 in sugi could
be used as a chronological index of nutrient availability (12).
FIG. 1 Radial distribution of Cd in stem wood of Quercus robur adjacent to

a factory and from an unpolluted reference site. Estimated emissions of Cd
from the factory are also presented (From Ref. 11.)
380
2.2
Nabais et al.
Research Questioning Dendroanalysis
The feasibility of dendroanalysis is not beyond doubt and in several studies the
basic assumptions of the method have been questioned.
The Cu, Al, and Pb levels in rings of Pinus silvestris and Betula sp. growing
near nonpolluted areas were similar to those from trees in the immediate surroundings of industrial pollution sources, although metal levels in the soil may
be up to 1000 times higher in the contaminated site (14). The temporal profiles
in tree rings could not be linked to the pollution history of the area.
Seasonal variations of Ni concentrations in stem wood of Fagus sylvatica
were detected (15). Highest levels of Ni were determined during the spring mobilization period in April (15). Trace element cations in the xylem tissue can be
mobile in the xylem sap or bound by electrostatic force to fixed negative charges
in the xylem vessel walls (16). Binding to fixed negative charges is a reversible
process. During the annual cycle of growth in deciduous plants, the ionic composition or the pH of the xylem sap can change and ions can be remobilized and
transported to other parts of the tree (17,18). These processes could explain the
variations of Ni concentrations in the wood of Fagus sylvatica (15). Therefore,
FIG. 2 Radial distribution of 206 Pb/ 207 Pb in growth rings and bark of Picea abies in Germany. The isotopic ratio of industrial lead used in Germany since
1980 are below 1.16, whereas before it was about 1.17. Geogenic lead in Germany has 206 Pb/ 207 Pb above 1.18. (From Ref. 20.)
381
seasonal variations of Ni concentrations in wood preclude the use of radial Ni

distributions for the reconstruction of pollution levels in the past (19).
The industrial and traffic lead emissions show very different isotopic signatures (206 Pb/ 207 Pb) from those of local soils. Hence, the isotopic rations allow
differentiation between these sources (20). It would be expected to find different
isotopic signatures of Pb in wood rings from the year 1950 onward with an increase in environmental pollution. The ratios of 206 Pb to 207 Pb in growth rings and
bark from Norway spruce (Picea abies) showed a large change from geogenic, or
pedogenic, lead isotope values (19301937) to more anthropogenic industrial
ratios only at the end of the trees lifetime in 1990 (Fig. 2) (20). However, from
1943 to 1976 the isotopic ratio remains constant, although during this period an
increase in industrial and traffic emissions was observed, with a change in 206 Pb/
207
Pb (20). Therefore there was no conservation of past trace metal pollution in
growth rings of Picea abies.
3
TRANSPORT AND DEPOSITION OF TRACE METALS IN

THE XYLEM
Trees are not passive recorders of the external environment, and the application
of dendroanalysis to environmental monitoring requires an understanding of the
factors that influence the mobility of trace metals in the xylem (21). The accumulation of elements in annually formed tree rings is controlled by such factors as
the cation binding capacity of wood, transformation of sapwood into heartwood,
and processes of radial translocation of elements in the tree stem (22).
3.1
Cation Binding Capacity of Wood
Trace metals that enter roots are transported in the xylem with the transpiration
stream. The xylem sap in a tree is the liquid that flows in the apoplast, mainly
in the conducting elements of the wood (23).
Wood is mainly composed of cellulose, pectins, and lignin (24). The cellulose microfibrils are embedded in a gel of pectic polysaccharides, the major component of which is polygalacturonic acid. These acids bear negatively charged
carboxyl groups, which are mainly responsible for the overall negative charge
of the cell wall (25). The amount of nondiffusible negative charges in the apoplast
is normally quantified by the cation exchange capacity (CEC) (26).
Most elements do not follow simple diffusion or mass flow kinetics from
the soil to the stem (27). Interactions of cations with nondiffusible anions of the
cell wall of the xylem vessels lead to a separation of cation transport from water
flow (28). The xylem operates as an ion exchange column that has the potential
to impede movement up the bole for months or even years (29,30). Changes in
382
Nabais et al.
wood chemistry sampled at breast height may therefore correspond to changes

in soil chemistry that occurred several years earlier.
The amount and proportion of ions retained in the xylem depends on the
ionic charge density of the ions (Ca 2 K ), the activity of competing cations,
the charge density of the nondiffusible anions, and the pH of the xylem sap,
which may vary from 5 to 7 (26,31). Binding to mobile organic molecules (e.g.,
carboxylic acids, amino acids, peptides) will also alter the charged state of cations
and reduce their affinity for fixed negative charges in cell walls (32). Consequently, the transport rate of di- or trivalent cations is enhanced significantly by
complexation of the cation (33).
Most of the cations retained in the cell walls are easily exchangeable (28).
However, cell walls may also contain multivalent cations precipitated or irreversibly bound to polyuronic acids and other ligands (34). Cations that are relatively
mobile in the xylem would obscure the time course changes in the trees environment (13).
The radial distributions of elements in stem wood depend partly on the
cation exchange capacity of the tissue. In stem wood of Picea rubens, the density
of fixed negative charges in cell walls is higher in the stem center (30). Most
trees show a decline in cation concentration with increasing tree age, which may
be linked more to the binding exchange properties in the woody tissue than to
the uptake of trace elements from the soil (22). The variation of the CEC with
the distance from the stem center suggests that functional groups responsible for
cation binding change with time (12). As the stem radius increases, the tracheids
are longer with a consequent wider distribution of the negative charges (30).
Element concentrations within an individual annual ring may strongly depend on the stem side. In Picea abies element concentrations within a single tree
ring varied with the direction by more than 60% (22). This variation was explained by the changed proportion of compression wood at the various sides of
the tree stem (22). Compression wood, formed only in gymnosperms, develops
in response to unbalanced mechanical pressure (35). The growth rings are wider
with higher density and appear darker due to the enhanced lignin and reduced
cellulose content. As 6070% of the cations bind to hydroxyl groups, the ligninrich compression wood provides more binding sites than the normal wood (22).
An increased concentration of elements is expected in compression wood due to
these differences in wood chemistry.
3.2
Effects of the Sapwood/Heartwood Boundary on

Trace Element Distributions
In many tree species heartwood formation is obvious from the color change of
the wood. This aging process, leading to cell death and the decomposition of
383
storage material, is also associated with an increase in content of extractives (36).

Some of these heartwood extractives, like phenolics and terpenoids, function as
decay inhibitors (37). Heartwood polyphenols are synthesized in situ at the sapwood-heartwood boundary from the breakdown of starch or from soluble sugars
(36). During the process of heartwood formation the moisture content of wood
decreases, the ray parenchyma cells die, stored starch in these cells disappears,
lignification begins much earlier, and the wood is impregnated with extractives
(38,39).
Many authors observed that the radial distribution patterns of trace elements
in tree stems are influenced by the location of the border between sapwood and
heartwood (40,41). Toxic elements like Cd and Pb showed low concentrations
near the cambium and increased levels toward the stem center. A translocation
of toxic organic substances through the rays into the heartwood was suggested
as a possible mechanism of sapwood detoxification (42).
Nutrient concentrations generally decrease with increasing distance from
the pith in heartwood (43). The pattern is disrupted at the heartwood-sapwood
boundary, where the nutrient concentration in the sapwood is much higher than
the concentration in the youngest heartwood (40,4346). This discontinuity
across the heartwood-sapwood boundary indicates the retranslocation of nutrients
from newly forming heartwood to fully functional sapwood (43,47).
Wardell and Hart (48) showed that P, K, Mn, Mg, and Ca were retranslocated out of the senescing sapwood via ray cells. Because of the high volume
of stem wood in a forest, nutrient retranslocation from forming heartwood to
functional sapwood can have a significant effect on intrasystem nutrient cycling.
Andrews and Siccama (49) reported nutrient retranslocation in Chamaecyparis
thyoides and estimated that 80% of the Ca and 78% of the Mg required for new
wood growth was recovered from dying sapwood. The degree of retranslocation
of nutrients from senescing sapwood has been shown to be inversely related to
soil nutrient availability (43).
Once metals enter trees, they are not necessarily restricted to tree rings
formed during the current year. In coniferous wood (e.g., Picea) and diffuse porous wood of angiosperms (e.g., Fagus, Acer, Populus), the water movement
passes through several annual rings in the outer part of the stemthe sapwood.
In an 18-year-old spruce tree (Picea abies) 1012 tree rings were involved
in water transportation (50). Therefore, the element accumulation of an individual
ring is not the result of the element uptake in the year when this ring was formed.
In ring porous wood (e.g., Quercus, Ulmus, Fraxinus), water movement is
mainly confined to the outermost and youngest annual ring (51). In such trees
the transport of minerals from the roots reaches mainly the current years ring.
These conditions are more favorable for an element chronology in stem wood
(3).
384
3.3
Nabais et al.
Radial Translocation
The ray parenchyma of wood is specialized for the temporary storage and remobilization of organic and inorganic reserve material (52). The rays have contact
cells with large pits to the vessel elements and central rows of isolation cells
without conspicuous pits to the vessel elements. The isolation cells play an essential role in radial transport (53). The minimum radial flux of sugars through the
tangential walls in the isolation cells of Populus canadensis was 80.7 pmol
cm 2 s 1 (52).
Donnelly et al. (54) reported that lateral movement of Pb in Picea rubens
seedlings occurred both inward and outward. Trace metals may differ in their
mobility between tree rings. In tulip trees (Lirodendron tulipifera), lateral movement is significant for such elements as Mn, K, P, Ca, Sr, and Zn but less so for
Al, Si, Fe, and Cu (55). However, to our knowledge, no investigations were
performed on the storage capacity, mobilization, and translocation rates of trace
metals in ray parenchyma cells.
In addition to the radial transport of carbohydrates along rays from the
phloem into the xylem and vice versa, exchange seems to occur between the rays
and the vessels through the contact cells (56). The contact cells differ in their
FIG. 3 Radial distributions of Cd in stem wood of Quercus robur sampled in

1983 and in 1994. The arrows indicate the transition between sapwood and
heartwood at the time of sampling. (From Ref. 61.)
385
anatomy from the other ray cells. They are connected with vessels by enlarged
pits, which are probably involved in the exchange of solutes between vessels and
rays (56). The pits of the contact cells in rays of Betula populifolia and Populus
tremuloides showed a high phosphatase activity (57). The enzymatic activity in
the contact cells began in spring, when the starch started to break down into
sugars in the ray cells and ended when the leaves were fully developed. During
this period, sugars could be found in the xylem sap, suggesting that the contact
cells of the rays were involved in an active secretion of sugars into vessels (57).
The ray cells can play an important role in the process of heartwood formation, through the production of tannins (58). Some authors suggested that the ray
cells at the heartwood-sapwood boundary have an increased metabolic activity
(59). Peaks of trace metals were observed at the heartwood-sapwood boundary
(40,4446,60). These peaks were suggested to result from centripetal movements
of substances in the sapwood (15). This centripetal transport might serve to detoxify the live part of the stem and dispose of toxic substances in the heartwood
(61). Probably the ray cells have an important role in the transport of trace metals
to the heartwood.
The heartwood-sapwood boundary moves with time and the peaks of trace
metals follow the location of that boundary (Fig. 3) (61). Therefore, the current
locations of these peaks are not related to pollution events in the trees environment.
4
BIOMONITO-RING?
Although the idea of dendroanalysis is fascinating, many problems still remain

to be solved before the potential of the method can be realistically assessed (62).
Some important factors merit further investigation, e.g., the possible pathways
of metal uptake (via roots, foliar uptake, absorption through the stem surface)
and their relative importance in the accumulation of trace metals in tree rings; the
chemical binding forms of trace metals in wood; and the mobility and transport of
trace metals in wood rings (63,64).
Some authors state that in trees growing in more acidic soils, with high
availability of trace metals, uptake by roots is the major pathway by which metals
enter into tree rings (21,65). However, in less acidic soils direct uptake through
bark and leaves can also be an important pathway to absorb trace metals (21,65).
These metals are then translocated to the outermost xylem ring (66). According
to Watmough and Hutchinson (67), metals entering via this pathway are more
likely to accurately record changes in atmospheric deposition with minimal radial
translocation. However, the actual mechanism and extent of metal uptake through
the bark and leaves into tree rings have not been determined. Even if trace metals
are incorporated in the recent tree rings through bark or leaves, radial translocation is possible.
386
Nabais et al.
One way to assess the mobility of elements in wood is to determine the

strength of their chemical binding forms by using several extractants with different binding strength to metal cations (68). The efficiencies of water, CaCl 2, acetic
acid, malic acid, citric acid, and Na 2 EDTA in extracting Cd and Pb from wood
of Pinus sylvestris and Quercus petraea were investigated (68,69). Three fractions of Cd and Pb in wood were distinguished: water-soluble, exchangeable,
and nonexchangeable.
The mobile fraction of the metals is dissolved in the xylem sap and is
extractable with water. In wood of beech trees (Fagus sylvatica), less than 1%
of the total Cd content was dissolved in the xylem sap (18).
The less mobile fraction of the metal cations is bound to fixed negative
charges in the xylem vessel walls (28). The fraction of Cd and Pb extracted with
solutions of organic acids, Na 2 EDTA, and CaCl 2 includes cations bound to fixed
negative charges in the xylem vessel walls. Extracted amounts of Cd increased
in the order acetic acid malic acid citric acid EDTA. The number of
carboxylic groups of such ligands increases from 1 (acetic acid) to 4 (EDTA)
(68). The stability constants of complexes of these ligands formed with Cd and
Pb increase in the same order (70). Such ligands compete for cations with fixed
negative charges in the wood. They extract different fractions of the bound Cd
and Pb. In the case of extraction with CaCl 2 solution, only ion exchange without
metal complexation is involved. The Ca-exchangeable fraction contained substantial amounts of Cd and Pb.
The nonextractable fraction comprised 20% of total Cd and 50% of total
Pb (68). Elements of this fraction can be precipitated as insoluble phosphates,
or immobilized in crystal structures, like oxalates.
A high percentage of Cd and Pb in wood tissue is mobile or can be remobilized. The seasonal changes of the organic acid composition of the xylem sap
may affect mineral concentrations in wood by varying cation transport and remobilization (71,72). Therefore, radial distribution patterns of trace elements in
wood can probably change their profile with time.
The total content of the chemical elements in rings cannot be used for
retrospective biomonitoring (73). Only the chemical forms of an element that are
strongly connected with the xylem can eventually be used in pollution reconstruction.
A few conditions under which dendroanalytical biomonitoring may have
a better chance to produce useful results can be noted:
1. When using an element of low mobility in plants, e.g., Pb.
2. When the selected tree species have a ring-porous wood, in which most
of the xylem sap goes through one or two rings;
3. The time passing between emission of an element into the environment
387
and its uptake by the tree can differ and this possible time shift should
be taken into account when interpreting the results.
4. Only long-term trends in the history of environmental pollution should
be considered in radial distribution patterns.
5. Places with high deposition of pollutants and abrupt changes in pollution are the most suitable for dendroanalysis.
5
CONCLUSIONS
A fundamental assumption of the dendroanalytical method is the stability of the

mineral distribution patterns, i.e., once the elements are stored in wood rings no
significant mobility should occur.
Although some studies presented good correlations between radial distributions of heavy metals in tree rings and temporal records of pollution from industry
or traffic, others failed in using dendroanalysis as a chronological record of pollution.
Radial element distributions can change with time. Mobile portions of elements in wood indicate the possibility of remobilization and retranslocation.
Therefore, tree ring analysis does not provide a reliable record of the pollution
history. Nevertheless, the method can provide information about transport and
storage of elements in trees.
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Ecol Manage 113:2333, 1999.
231. SA Watmough. Monitoring historical changes in soil and atmospheric trace metal
levels by dendrochemical analysis. Environ Pollut 106:391403, 1999.
14
Heavy Metal Interactions in Soils
and Implications for Soil Microbial
Biodiversity
R. Naidu, G. S. R. Krishnamurti, W. Wenzel,
and M. Megharaj
CSIRO Land and Water, Urrbrae, South Australia, Australia
N. S. Bolan
Massey University, Palmerston North, New Zealand
INTRODUCTION
Heavy metals are defined as those elements (some metalloids) that have atomic
density 6 g cm 3. Some of these elements include cadmium (8.65), cobalt
(8.90), mercury (13.50), nickel (8.90), zinc (7.10), lead (11.30), and copper
(11.30). Often arsenic and selenium with a specific gravity of 5.73 and 4.79,
respectively, are also included among the heavy metals. Heavy metals includes
both biologically essential (Co, Cu, Mn, and Zn) and nonessential (Cd,
Pb, Hg, As) elements. The essential elements are required in low concentrations
and hence are known as trace elements or micro elements. The nonessential
heavy metals are phytotoxic and/or toxic to animals and are known as toxic
elements. Consequently, heavy metals are extensively researched in the life,
401
402
Naidu et al.
agricultural, and environmental sciences. Among the heavy metals As, Se, Cr,
Cu, Cd, Zn, and Pb have been most commonly studied. Other heavy metals, such
as mercury (Hg) and tin (Sn), are important, especially in the mining environment
and around industries involving processing of ores containing these metals.
The interaction of heavy metals with soil mineral components has been
recently reviewed (see, for example, reviews by Naidu et al., 1995, 1996, 1997).
These studies reveal that such interactions vary with both the nature and properties of soils, with the retention of metal cations generally increasing with increasing soil pH. The implications of such interactions to soil and crop quality have
also been studied, although the role of soil chemical factors on remediation of
metal contaminated sites is challenging and still a subject of much interest. In
contrast to metal-soil mineral interactions, only limited studies have been conducted on the impact of metals on soil biodiversity, especially in relation to both
bioavailability and metal-organic interactions. Where such studies have been performed, the focus has been heavy metal toxicity and bioavailability to soil microbial populations at the population level (Brookes et al., 1986; Chander and
Brookes, 1991a,b; Martensson and Witter, 1990) or at the functional level (e.g.,
Giller et al., 1997). While metal contaminants may change microbial community
structure and species diversity, the functional ability of the community may remain unchanged. The abundance and species composition of microbial groups
reflect the total ecotoxicological, effects of heavy metals and not just the results
of bioavailability at any particular time. In this chapter, by following an overview
of sources of metals and their interactions in soils we aim to relate soil-metal
interactions to microbial biodiversity and its potential implications to soil productivity.
2
SOURCES OF METALS IN SOIL ENVIRONMENT
Heavy metals in terrestrial and aquatic environments originate from human activities and weathering of soil parent materials. Due to their low bioavailability, the
metals present in ores are often not available for plant uptake and cause minimal
impact to soil organisms. The natural process of pedogenesis leads to mineral
breakdown and translocation of the products, as well as accessions from dust
storms, volcanic eruptions, and forest fires. Often the concentrations of heavy
metals released into the soil system by pedogenic processes are largely related
to the origin and nature of the parent material. Typically soils rich in heavy metals
include those derived from serpentine. These soils are disproportionately rich in
magnesium (Mg) and heavy metals such as Ni, Cr, and cobalt (Co) and poor in
calcium (Ca). Copper-, zinc- (Zn-), and Pb-rich soils have also been reported in
a number of countries (Adriano, 1986). Along with these toxic metals, many
incidences of poisoning have been reported in northern India, West Bengal, Bangladesh, and parts of Asia where local people have been adversely impacted by
403
ingestion of arsenic (Chakroborty and Saha, 1987; Naidu and Skinner, 1999) and
selenium ingested either through water or crops produced on contaminated soils.
While Bangladesh and West Bengal poisoning have been linked to the presence
of elevated concentrations of As in groundwater, Se toxicity in northern India
has been associated with elevated levels of Se in food produced on Se-contaminated soils. The origin of Se has been linked to naturally occurring minerals
(Dhillon and Dhillon, 1990). Apart from Se and As, other metals (e.g., Cr. Ni,
Pb) derived via pedogenic processes have limited impact on soil biota and crop
quality. Unlike pedogenic inputs, heavy metals added through anthropogenic activities have high bioavailability (Naidu et al., 1996). Anthropogenic activities,
primarily associated with industrial processes, manufacturing, and the disposal
of domestic and industrial refuse and waste materials (Adriano, 1986), are the
major source of metal enrichment in soils. Atmospheric pollution from lead based
petrol is a major issue in many developing countries where there is no provision
for the introduction of unleaded fuel.
Disposal of industrial waste materials has been estimated to contaminate
thousands of hectares of productive agricultural land in countries throughout the
world. This is a particular problem in the Asian Pacific region where land based
disposal of untreated wastes is still being practiced due to the lack of regulatory
guidelines (Naidu et al., 1996). Uncontrolled mining, manufacturing, and disposal
of metals and metal-containing materials inevitably cause environmental pollution. While there are vast differences in the amounts of different metals produced,
soil concentrations of most metals, especially Ni, Cr, and Cd, have increased
over the last 60 years (Alloway, 1990).
3
FATE OF METAL IONS IN THE SOIL ENVIRONMENT
Metal ions can be retained in the soil by both sorption and precipitation reactions.
The lower the metal solution concentration and the more sites available for sorption, the more likely that sorption/desorption processes will determine the soil
solution concentration (Brummer et al., 1983; Tiller et al., 1984a,b). However,
the fate of metals in the soil environment is dependent on both soil properties and
environmental factors. Following input into the soil environment, heavy metals
interact with the soil mineral and organic phases (Fig. 1).
Both soil properties and soil solution composition determine the dynamic
equilibrium between metals in solution and the soil solid phase. The concentration
of metals in soil solution is influenced by the nature of both organic (citrate,
oxalate, fulvic, dissolved organic carbon) and inorganic (H 2 PO 4, NO 3, Cl ,
and SO 42) ligand ions through their influence on metal sorption processes (Shuman, 1986; Homann and Zasoski, 1987; Naidu et al., 1994a; Harter and Naidu,
1995; Naidu and Harter, 1997; Bolan et al., 1999a). Two reasons have been given
for the effect of inorganic anions on the sorption of heavy metal cations (Naidu
404
Naidu et al.
FIG. 1 Fate of pollutant and nutrient ions in soil ecosystem.
et al., 1994). First, inorganic anions form ion pair complexes with heavy metals
and thereby reduce the sorption of heavy metals. Second, the specific sorption
of ligand anions is likely to increase the negative charge on soil particles (Fig.
2) and thereby increases the sorption of heavy metals.
A number of studies have examined the effect of inorganic anionic complex
formation on the sorption of Cd 2 by soils (Boekhold et al., 1993; Naidu et al.,
1994b). Most of these studies have indicated that among the four common inorganic anions H 2 PO 4, NO 3, Cl , and SO 42, Cl has often been found to form
a complex with Cd 2 as CdCl , thereby decreasing the sorption of Cd 2 onto soil
particles (Naidu et al., 1994a). However, when the activity of Cd 2 was corrected
for complex formation the sorption curves for Cd 2 in NO 3 and Cl media coincided. OConnor et al. (1984) showed that while the presence of Cl ions (as
CaCl 2) decreased adsorption of Cd, SO 42 (as CaSO 4) ions increased Cd sorption
relative to comparable concentrations of ClO 4 in Typic Torrifluvents, Ustollic
Calciorthids, and Petrocalcic Paleustolls. All three soils were calcareous in nature, with pH values exceeding 7.5. Cd-chloro complexation was identified as
the active process reducing Cd retention. They attributed the increased retention
in the presence of SO 42 to the low Ca ion activity available for competition with
Cd due to the formation of the soluble CaSO 40 complex. In contrast to inorganic
ligand ions, Haas and Horowitz (1986) found that in a few cases Cd adsorption
by kaolinite, a variable-charge mineral, was enhanced by the presence of organic
matter (OM) which was attributed to the formation of an adsorbed organic layer
on the clay surface.
405
FIG. 2 Effect of solution pH and organic ligand ions on potential of an oxisol

(Naidu and Harter, 1995).
Anion-induced cation sorption has been reported for a number of cations

(Ryden and Syers, 1976; Bolland et al., 1977; Wann and Uehara, 1978; Shuman,
1986). Specific sorption of anions and cations onto variable charge components
has often been shown to increase the surface negative charge (Table 1). Shuman
(1986) observed that the specific sorption of anions, such as H 2 PO 4 and SO 42,
increased the sorption of Zn 2 by variable-charge components. Bolland et al.
(1977) and Kuo and McNeal (1984) reported that H 2 PO 4 sorption increases the
sorption of Zn 2 and Cd 2 by hydrous ferric oxide. Anion-induced cation sorption
depends on the variable-charge components of the soils. Naidu et al. (1994a) and
Bolan et al. (1999a) have demonstrated phosphate and sulfate induced Cd sorption in strongly weathered oxisols from Australia and less weathered but net
positively charged andept from New Zealand. The above studies suggest that two
different mechanisms may operate depending on the nature of the soil surface
(Harter and Naidu, 1995).
Soils that have a high affinity for ligand anions may enhance metal ion
adsorption through modifications in the soil surface charge density. In strongly
weathered soils, ligand adsorption on clay surfaces enhance metal adsorption.
This is illustrated by the recent studies of Naidu et al. (1994a) and Bolan et al.
(1999a), who reported that the adsorption of specifically sorbing ligands such as
PO 43 and SO 42 in strongly weathered and variable-charge soils can induce Cd
406
Naidu et al.
TABLE 1 Increase in Surface Charges Due to Specific Adsorption of

Anions and Cations (Bolan et al., 1999b)
Soil
constituents
Iron hydrous
oxide
Allophane
Soil
Soil
Soil
Soils
Aluminum
oxide
Soils
Soil
Soil
Solute
pH
Charge added
(mol mol 1 anion)
Phosphate
6.5
1.25
Phosphate
Phosphate
Sulfate
Phosphate
Phosphate
Phosphate
Sulfate
5.1
6.5
6.5
5.0
6.5
7.5
7.0
5.8
5.0
0.5
0.65
0.26
0.38
0.47
0.77
0.350.7
0.52
1.06
Sulfate
Calcium
Calcium
5.6
5.8
5.8
0.25
0.350.58
0.52
Ref.
Bolan et al. (1985)
Rajan et al. (1974)
Bolan et al. (1986b)
Sawhney (1974)
Schalscha et al. (1974)

Naidu et al. (1990b)
Rajan (1978)
Curtin and Syers (1990)
Bolan et al. (1993)
Ryden and Syers
(1975)
adsorption through increased negative surface charge. A similar interaction involving organic ligands was also reported by Davis and Leckie (1978) for variable-charge oxide surfaces where certain organic ligands were found to enhance
the adsorption of Cu(II) or Ag(I). An alternate mechanism, which appears to be
important in temperate soils, involves metal-ligand complexation in solution and
subsequent reduction in cation charge, which probably reduces adsorption (Naidu
and Harter, 1998). The decrease in Cd sorption onto soils in the presence of
phosphate (Krishnamurti et al., 1999) was attributed to the formation of soluble
Cd-phosphate complexes such as CdH 2 PO 4 (log K CdH2PO4 2.91, Martell et
al., 1997). This is consistent with the concept that free Cd 2 activity, rather than
total dissolved Cd concentration, is a controlling factor in Cd sorption (OConnor
et al., 1984). The relative value of distribution constant, as measured by the
Freundlich parameter a derived from the adsorption isotherm of the soils, had
shown that phosphate-retarded Cd sorption by at least one order of magnitude.
The Freundlich parameter a for the mildly alkaline (pH 7.8) soil (organic C 3.9%)
was 68,071 compared to the value of 2334 for mildly acidic (pH 6.1) soil (organic
C 3.0%). This was consistent with the behavior of a cationic heavy metal (Welp
and Brummer, 1997). This mechanism may also be operative in high-pH (limed)
tropical soils in which anion adsorption will be low and any effect on adsorption
will be through ligand-metal complexation process.
407
However, the nature of interaction between metals and soil colloidal particles varies with the nature of soil type and the sorbing surface. Depending on
the nature of metal-soil interactions, sorption reactions may be defined as nonspecific or specific. Nonspecific adsorption from outer-sphere surface complexes is
also referred to as physical adsorption (Sposito, 1984). If the physical adsorption
process was the only factor controlling metal sorption, then the maximum adsorption capacity of the soils should be dictated by their cation exchange capacity
(CEC). However, in many soils the amount of heavy metal sorbed exceeds the
CEC of the soils. This suggests that in addition to physical adsorption other processes, such as precipitation and specific sorption, also contribute to metal retention in soils.
Specific sorption refers to exchange of heavy metals with surface ligands
to form covalent bonds with lattice ion. Specific adsorption results in heavy metal
adsorption far greater than that expected from cation exchange. In specific adsorption, the ions penetrate the coordination shell of the structural atom and are
bonded by covalent bonds via O and OH groups to the structural cations. Thus,
specific adsorption of metal ion may influence the surface charge characteristics,
potential, and electrophoretic mobility of soil particles, demonstrated by lowering of the point of zero charge (Bolan et al., 1993). Specific adsorption is
strongly pH-dependent, increasing with pH for cationic species and decreasing
with pH for anionic species. However, the extent of hydrolysis is pH-dependent
and therefore influenced by the soil/solution ratio. The metal ions which hydrolyze range from hard metal ions, i.e., those which include the alkali metal
and alkaline earth metal ions, to moderately soft metal ions, such as Cd 2, Pb 2,
and Hg 2.
Precipitation appears to be the predominant process in high pH soils and
in the presence of anions such as S 2, CO 32, OH , and PO 43 and when the
concentration of the heavy metal ion is high (Naidu et al., 1997). Coprecipitation
of metals, especially in the presence of iron oxyhydroxide, has also been reported
and often such interactions lead to significant changes in the surface chemical
properties of the substrate (Table 2).
Since the product of the hydrolysis reaction is H , metal ions are generally
classed as Lewis acids (after G. N. Lewis). Pearson (1963) further classified metal
ions into two groups, depending on whether they formed their most stable complexes with C, N, or O, the first ligand atom from groups V, VI, and VII (hard
acids, class a), or whether they formed their most stable complexes with the
second or a subsequent member of each group (soft acids, class b). This classification, known as the hard and soft acid base theory (HSAB), states that hard
Lewis acids prefer to react with hard Lewis bases and soft acids prefer to react
with soft bases. Hard acids are usually small have a high positive charge, and
low polarizability, and do not contain unshared pairs of electrons in their valence
408
Naidu et al.
TABLE 2 Coprecipitation of
Minerals in the Presence of Iron
and Manganese Oxyhydroxides,
Calcite, and Clay Minerals
Mineral
Coprecipitation
Fe oxides
Mn oxides
Calcite
Clay minerals
Ni, Cu, Zn, Mo

Co, Ni, Zn, Pb
Mn, Co, Cd
Ni, Co, Zn, Cu, Pb
shells (Pearson, 1963). Trivalent metal ions such as Fe 3 and Al 3 are generally
hard because of their high ionic charge. Hard bases have high electronegativity,
low polarizability, and are hard to oxidize. Soft acid and soft bases have properties opposite that of the hard acid and bases. Table 3 lists the metal ions and the
inorganic ligand anions according to hard and soft acidbase concept. Generally,
the hard metal ions include the plant macronutrient and secondary nutrient metal
cations (Ca, Mg), whereas the borderline acids include the micronutrient elements
such as Mn(II), Fe(II), Co, and Zn.
The hydroxy species of metal ions (MOH) is more strongly retained than
the corresponding metal ions. Hence, specific adsorption increases with a decrease in the pK value for hydrolysis reaction:
Cd(pK 10.1) Ni(9.9) Co(9.7) Zn(9.0)
Cu(7.7) Pb(7.7) Hg(3.4)
Other chemical interactions that contribute to metal retention by colloid particles
include complexation reaction between inorganic and organic ligand ions. As
might be expected, the organic component of soil constituents has a high affinity
for heavy metal cations because of the presence of ligands or groups that can
form chelates with metals (Harter and Naidu, 1995; Naidu and Harter, 1998).
TABLE 3 Some Hard and Soft Acids and Bases

Hard
Acids
Bases
Soft
3
Sn Cr , Co , Fe ,
As 3
H 2 O, OH, O, ROH,
COO , CO 32, NO 3,
PO 43, SO 42, ClO 4, F
Borderline
2
Cu , Hg , Pb , Cd ,
Hg 2
H , S 2, SH, I
Fe , Co 2, Ni 2,
Cu 2, Zn 2, Pb 2
Cl , NO 2, SO 32,
Br
409
The functional groups include carboxyl, phenolic, alcoholic, and carbonyl groups
(Stevenson, 1977). With increasing pH, the functional groups dissociate, thereby
increasing the affinity of ligand ions for metal cations. The order of metal cations
complexed by organics is as follows:
Cu 2 Fe 2 Pb 2 Ni 2 Co 2 Mn 2 Zn 2
However, the extent of metalorganic complex formation varies with a number
of factors, including temperature, steric factors (e.g., geometry and flexibility),
concentration, etc. All of these interactions are controlled by solution pH and
ionic strength (IS), nature of the metal species, dominant cation, and inorganic
and organic ligands present in the soil solution. Thus, metal sorption by soils
depends on the chemical nature of the metal species present in the soil solution
and the nature of constituent soil minerals.
4
METAL INTERACTIONS VS. SOIL SOLID PHASE

COMPOSITION
Irrespective of the nature of interaction between the metals and soil colloidal
particles, following sorption, metal ions redistribute among organic and mineral
soil constituents. Speciation studies suggest that the majority of metals (Cd, Cu,
Zn, Pb, A) are associated with organic matter and inorganic Fe and Al oxides
in soils. Factors affecting the distribution of a metal among different forms include pH, ionic strength of the soil solution, the solid and solution components
and their relative concentration and affinities for the metal, and time (Jones and
Jarvis, 1981; McBride, 1991; Foerstner, 1991; Ritchie and Sposito, 1995). A very
large number of sequential extraction schemes have been used for soils, generally
attempting to identify metals held in any of the fractions: soluble, exchangeable,
adsorbed/carbonate, organically bound, Mn oxideoccluded, amorphous mineral
colloidoccluded, crystalline Fe oxideoccluded, and residual or lattice mineral
bound. The most commonly used schemes are based on that designed by Tessier
et al. (1979). Metal fractionation using the sequential extraction techniques have
primarily been used to identify the fate of the metals applied in waste sludges
and in soils contaminated with smelters and mine drainage wastes (Petruzelli et
al., 1981; Sposito et al., 1982; Lake et al., 1984; Chang et al., 1984; Hickey and
Kittrick, 1984; Dudka and Chlopecka, 1990). These studies suggest that treating
the soils with sludges or wastes shifts the solid phases of the metals away from
immobile fractions to forms that are potentially more mobile, labile, and bioavailable. For example, Dudka and Chlopecka (1990) found that residual forms of
Cd, Cu, and Zn decrease from 3443% to 634%, while the more easily available
forms increase by treating soils with sludge (Table 4).
Elaborate sequential extraction schemes and geochem modelling have frequently been used to identify the distribution of different species of the metal
410
Naidu et al.
TABLE 4 Solid-Phase Metal Fractions in Soils Determined Using

Sequential Extraction Scheme
Cd
Fraction
Exchangeable
Carbonates
Fe oxides
Organic
Residual
Control
0.15
0.08
0.2
0.15
0.5
(13)
(7)
(17)
(13)
(43)
Cu
Sludge
0.12
0.37
0.2
0.15
0.5
(15)
(25)
(14)
(10)
(34)
Control
0.11
0.2
0.16
1.8
16.5
(1)
(1)
(1)
(10)
(34)
Zn
Sludge
0.16
2.7
77.2
46.6
21.7
(0.1)
(2)
(56)
(34)
(16)
Control
2.1
11.2
18.0
29.0
25.2
(3)
(16)
(26)
(42)
(36)
Sludge
112.8
178.9
145.0
61.6
28.5
(26)
(41)
(33)
(14)
(6)
All values in mg kg 1, with the % values in parentheses, calculated on the basis of total
metal in soils.
Sources: Data from Dudka and Chlopecka, 1990.
amongst various forms of solid species (see reviews by Ross, 1994; Krishnamurti,
2000). However, very few attempts have been made to identify the particular
species of the metal that contributes to bioavailability. The mechanism which
operates the phytoavailability of solution and solid-phase species can be visualized as shown in Fig. 3.
Using an innovative extraction scheme (Krishnamurti et al., 1995; Krishnamurti and Naidu, 2000), which estimates the species associated to the metalorganic complexes, the importance of metal-organic complexes in the bioavailability of Cd in native soils was established based on the multiple correlation
FIG. 3 Phytoavailability of solution and solid-phase metal species.
411
analysis between the data on the distribution of solid species and the bioavailable
Cd (Table 5). The importance of metal-fulvic complexes in the phytoavailability
of Cu and Zn was also shown using this scheme for the speciation of solid phases
of Cu and Zn (Krishnamurti, unpublished). This scheme is a refinement toward
better understanding of solid-phase metal species in soils (Table 6). Based on
the differential FTIR spectra of the metal-organic complexes extracted by the 0.1
M sodium pyrophosphate extractant used in their speciation scheme, Krishnamurti et al. (1997a) showed that Cd in soils was apparently bonded at the COO
of the carboxyl and OH of the phenolic groups.
Compared with bulk soils, solid-phase speciation of Cd (added with fertilizer P) differs substantially in phosphate fertilizertreated rhizosphere soils
(Krishnamurti et al., 1996). The amounts of Cd species associated as adsorbed
and with metal-organic complexes of the rhizosphere soils were appreciably
higher than those of the corresponding bulk soils. The increase was attributed to
the increased amount of carbonate, a product of plant respiration, and the organic
acids released as root exudates, present in soil-root interface. The formation of
aqueous complexes of Cd with low molecular weight organic acids (LMWOAs)
of root exudates in soil rhizosphere are expected to dominate the solution chemistry of Cd. Based on differential pulse anode stripping voltametric and cation
exchange resin extraction data, the dissolved Cd in soil solutions was found to
be almost completely complexed with organic matter (del Castilho et al., 1993;
Sauve et al., 2000). The functional group chemistry of the organic solutes was
not determined in these studies but even organic acids dominated by COOH
TABLE 5 Data on Regression Analysis Between Phytoavailable Metal and

Solid-Phase Species of a Few Soils of South Australia (Krishnamurti and
Naidu, 2000)
Metal-fulvic
complexes
Exchangeable
Simple correlation coefficients

Phytoavailable Cd
0.824 (0.002)
0.735 (0.01)
Cu
0.944 (0.0001)
Zn
0.669 (0.02)
0.832 (0.002)
Multiple regression analysis
Phytoavailable Cd 0.0004 3.5676 Exch. Cd 2.6500
-Fulvic Cd
Phytoavailable Cu 1.2528 6.2022 Fulvic Cu 4.6304
Humic Cu
Phytoavailable Zn 33.582 8.8092 Exch. Zn 2.3669
Fulvic Zn
Metal-humic
complexes
0.168 (ns)
0.468 (ns)
R 2 0.915 (0.0001)
R 2 0.942 (0.0001)
R 2 0.789 (0.002)
Values in parentheses are the levels of significance; ns significant at P 0.1.
412
Naidu et al.
TABLE 6 Multistep Sequential Extraction Scheme for Solid-Phase Metal

Speciation of Soils
Step
1
2
3
4
5
6
7
8
Species
Reagent
Shaking time
Exchangeable
10 ml of M NH 4 NO 3 (pH 7)
46 h
6h
Adsorbed/carbonates
25 ml of M CH 3 COONa (pH 5)
20 h
Metal-organic complexes
30 ml of 0.1 M Na 4 P 2 O 7 (pH 10)
30 ml of the soil extract was brought to pH 1.0 with 6 M HCl and the suspension left overnight for the coagulation of humic complexes. The suspension was centrifuged at 12,000g for 10 min. Metals associated with fulvic
complexes were determined in the supernatant. Metals associated with
humic complexes were determined in the residue solubilized with 0.1 M
Na 4 P 2 O 7.
30 min
Easily reducible oxides
20 ml of 0.1 M NH 2 OH.HCl
(pH2)
2 h (85C)
Organics
5 ml of 30% H 2 O 2,2 ml of 0.02
M HNO 3 3 ml of 30% H 2 O 2,
2 h (85C)
30 min
1 ml of 0.02 M HNO 3 cool;
add 10 ml of 2 M NH 4 NO 3 in
20% nitric acid
4 h (dark)
Amorphous colloids
0.2 M NH 4 C 2 O 4 (pH3)
30 min (95C)
Crystalline Fe oxides
0.2 M NH 4 C 2 O 4 (pH3) in 0.1 M
ascorbic acid
Residual
Digestion with HF-HClO 4
The volume of the reagent was for 1 g (2 mm) sample. After each treatment, carried
out at 25C unless otherwise stated, the extract was collected by centrifugation for 10
min at 12,000g; the residue was washed once with 10 ml deionized distilled water.
Data from Krishnamurti and Naidu, 2000.
groups should facilitate complexation of Cd. Indeed, Krishnamurti et al. (1997b)

observed significant solubilization of Cd from neutral to slightly acid soils with
0.11 mM concentrations of acetic, succinic, oxalic, and citric acids. The data
suggest that the Cd release was related to the stability constant of the Cd LMWOA
complex (Fig. 4). Similar increases in the solubilities of metal cations (Hue et
al., 1986) and nonmetals (Bolan et al., 1994) with increasing stability constants
for LMWOA have been reported.
A logical solution to minimize plant uptake and to protect the quality of
the food chain is to render the trace metals in the soil immobile. The phytoavailability of the different forms of the solid phase species decreases in the order:
soluble exchangeable adsorbed metal-organic complexes organics
Fe, Mn oxides residual (fixed in mineral lattices). Immobilization of heavy
metals, such as Pb, Zn, and Cd, was achieved by additives (zeolites, Czupyrna
et al., 1989, Vangronsveld et al., 1990, Gworek, 1992; Mn oxides, Fu et al., 1991;
clay-hydroxy Al polymers, Jansen et al., 1993, Mench et al., 1994) that may not
413
FIG. 4 Relationship between Cd released from a few soils of Canada (Luseland, Waitville, and Jedburgh) by selected low molecular weight organic
acids (LMWOAs) (concentration 0.01 M) during the reaction period of 0.25
h and the logarithm of the stability constant of the Cd-LMWOA complexes.
(Redrawn from Krishnamurti et al., 1997b.)
produce any detrimental byproduct or alter the physicochemical environment of

the soils to effect the plant growth, but could fix the heavy metals in their lattice
structure. Organic immobilization may also be possible. For example, CuSO 4 is
widely used in milking yard to treat lameness in dairy cattle. The residual solution
is flushed to the effluent pond. In the pond, 99% of the free Cu 2 is immobilized
as organically complexed Cu.
BIOLOGICAL INTERACTIONS AND TRANSITIONS
An overview on biological interactions of trace metals in soils is presented in

Fig. 5. The biological components of soils, plants, and microbes control OM
dynamics via release of soluble and insoluble organic compounds and subsequent
transformation (Whipps and Lynch, 1983). Plants act as sources of solid OM via
senescence of areal parts and roots, and deposition of, e.g., muscillage in the
rhizosphere. In addition, plant roots exudate soluble low molecular weight organic compounds, including sugars, amino acids, aliphatic acids, and phenolic
compounds. Both soluble and insoluble organic compounds are utilized as substrate by microbes. Microbes, in particular microbial associations in the rhizosphere, absorb and transform OM while releasing microbial exudates and debris.
Overall, biological transformation of OM may result either in increased molecular
size and decreased solubility (humification), or in decreased molecular size, typically associated with enhanced solubility (decomposition).
414
Naidu et al.
FIG. 5 Major interactions of heavy metals with plant roots, microbes, and
organic components (solid organic matter, SOM) in soils. LMW-OM and
HMW-OM denote low and high molecular weight organic matter in the solid
phase, respectively; massive arrows denote matter fluxes or transitions, dotted arrows influences or any other kind of interaction.
The microbial transformation of OM has various implications to the fate

of trace metals in the soil environment:
Changes of the solubility and reactivity (via functional groups) of OM
can alter sorption reactions of metals onto OM and change the speciation of metals in the soil solution via the formation of metal-organic
complexes (Senesi, 1992).
Metals bound to solid OM can be released during decomposition.
Redox and acidbase reactions associated with microbial transformation of OM can enhance or decrease metal solubility through alteration
of the redox status of metals and the surface of metal sorbents (eg.
hydrous oxides of Fe and Mn), metal hydrolysis, competition of protons
or hydroxyl ions for adsorption sites, or proton/redox-induced dissolution of sorbents and metal compounds. Changes in acidity and redox
may also affect the stability of soluble and surface metal-organic compounds.
415
In addition, plant roots and microbes interact with metals via the following
major pathways:
Plant roots and microbes surfaces can physically adsorb metals (Beveridge et al., 1995).
Metals are taken up by plant roots and microbes.
Plant-microbial associations are involved in the enzymatic transformation of metal-organic compounds, including volatiles, e.g., of Hg, Se,
and As (Azaizeh et al., 1997).
Root and microbial exudates can increase metal solubility via the formation of metal-organic complexes (Gerke, 1992; Mench and Martin,
1991), associated with enhanced desorption (Krishnamurti), and
changed speciation in the soil solution (Knight et al., 1997b; McBride,
1989).
Plant roots and microbes can modify metal solubility and metal sorption
(Adriano, 1986; Ross, 1994) via excretion of protons, carboxylic acid
(Ohwaki and Sugahara, 1997), or redox-active compounds (e.g., phenolics; Marschner and Roemheld, 1994).
Plant and microbial exudates can enhance weathering and transformation of soil minerals (Hinsinger et al., 1993) and thus release trace elements from these minerals and modify their characteristics (structure,
surface area, functional groups) for sorption of trace elements.
In wetland soils, some plants with an aerenchyma can support an increased redox potential in their rhizosphere that causes coprecipitation
of trace elements along with and adsorption of trace elements on precipitated iron oxides (Doyle and Otte, 1997).
The intensity and rate of metal interactions with biota is not evenly distributed throughout the soils. Hot spots with increased activity include the rhizosphere and are typically associated with zones with large OM content (topsoil,
around plant debris, organic soil amendments). Accordingly, gradients of metal
solubility, speciation, and chemical extractability are found across the rhizosphere
of metal-hyperaccumulating (Bernal et al., 1994; McGrath et al., 1997) and nonaccumulating plant species (Doyle and Otte, 1997; Neng-Chang and Huai-Man,
1992; Youssef and Chino, 1989).
6
ADVERSE IMPACTS OF METALS ON SOIL

BIODIVERSITY
Soils are an important habitat for a wide variety of microorganisms differing

in their physiology and metabolic activity. Microorganisms are of paramount
importance in decomposition of organic matter and nutrient cycling in soil. Microorganisms themselves represent one of the largest reservoirs for essential soil
416
Naidu et al.
nutrients and thus are considered to be a living pool of soil organic matter. There
has been considerable amount of literature available on the effects of heavy metals on soil microorganisms and their functions in soil. In this section we have
attempted to provide an overview of the impact of heavy metal pollution on
microbial populations and their processes in the soil.
7
7.1
HEAVY METAL TOXICITY TO MICROBIAL

COMMUNITIES
Abundance and Biomass
The individual populations of microorganisms (bacteria, fungi, algae, actinomycetes, etc.) respond to heavy metal pollution in soils by way of increasing or
decreasing their density (numbers) and biomass. The use of viable counts as
colony-forming units as a measure of microbial abundance in soil has limited
value considering that only 0.1 to 10% of the total bacteria can be counted
using various nutrient growth media. Several studies have shown a relationship
between the metal concentration and the emergence of metal-resistant microbes
in soils (Olson and Thornton, 1982; Doelman and Haanstra, 1979; Hallas and
Cooney, 1981; Doelman et al., 1994). Diaz-Ravina et al. (1994) reported an increased bacterial community tolerance to metals (Cd, Cu, Zn, Ni with the exception of Pb) in a sandy loam soil containing 4.4% OM as determined by the thymidine (3 H) incorporation assay. In another study involving high metalcontaminated soil, viable counts (population size) and microbial biomass reached
the same level as uncontaminated control soil over a period of 420 days (Kelly
et al., 1999a) even though microbial diversity was altered.
A severe decrease in the density of algal and cyanobacterial populations
was observed in a long-term tannery-contaminated soil containing Cr as the major
contaminant (unpublished work).
7.2
Diversity
Nordgren et al. (1986) has observed the physiological groups of bacteria as more
sensitive to metal pollution in a soil taken from a smelter in Sweden. Using
multivariate analysis, these authors studied the impact of metals and pH on numbers of bacteria producing acids from substrates such as maltose, arabinose, cellobiose, and xylose. Hydrolysis of chitin, starch, cellulose, and xylan was also
considerably affected by the metals. Only few studies have indicated phospholipid fatty acid composition (PFLA) and Biolog as sensitive indicators of changing microbial communities due to metal pollution. Addition of Zn (6000 mg kg 1
soil) to soil altered the microbial community structure as evidenced by shifts in
both PLFA and Biolog metabolic profiles (Kelly et al., 1999a). The Zn-induced
effects on PLFA profiles included a relative decrease in the fatty acid signatures
417
indicative of actinomycetes and arbuscular mycorrhizal fungi. Knight et al.

(1997a), using Biolog substrate utilization patterns, have demonstrated that addition of Cu (143157 mg kg 1), Cd (3.14.3 mg kg 1), and Zn (307354 mg
kg 1) to sandy loam soils of varying pH (4.57.0) reduced the metabolic potential
of the extracted soil microbial population. In a study where soil was evaluated
for the impact of sludge-borne heavy metals on microbial communities, even
18 years after termination of sludge application a decrease in several specific
populations of microorganisms as revealed by Biolog and PLFA profiles was
found (Kelly et al., 1999b). A severe reduction in the density and species composition of algae and cyanobacteria were noticed in a long-term tannery waste
contaminated soil. In highly contaminated soil (total Cr, 63,000 mg kg 1 soil),
only one type of alga (Chlorococcum type unicellular alga) occurred, in contrast
to the control soil which had a total of eight algal species including three cyanobacteria (unpublished work).
8
EFFECTS ON MICROBIAL PROCESSES
The negative impact of metals on microbial processes is important from an ecosystem perspective, and any of such effects could potentially result in a major
ecological perturbance. Hence, it is most relevant to examine the effects of metals
on microbial processes in combination with communities. Following are some of
the examples where metals have exerted negative effects on important microbial
processes. The most commonly used indicators of metal effects on microflora in
soil are (a) soil respiration, (b) soil nitrification, (c) soil microbial biomass, and
(d) soil enzymes.
8.1
Carbon Mineralization
Soil respiration is one of the most commonly and widely used techniques to
assess microbial activity and can be measured by adding an organic substrate
(substrate-induced respiration) or by using the organic substance already present
in the soil (basal respiration). A 10% decrease in basal respiration was observed
in an organic soil (pH 6.2) by 4.98 mol of Cd and 5 mol each of Cu, Zn, and
Ni (Lighthart et al., 1983). The metal toxicity was more pronounced in a sandy
loam soil (pH 4.9) where 0.09 mol Cd, 1.57 mol Cu, 0.48 mol Pb, 0.15 mol
Zn, and 1.7 mol Ni g 1 soil decreased basal respiration by 17, 25, 25, 21, and
28%, respectively. Valsecchi et al. (1995) studied the effect of metal pollution
on the soil microflora in 16 different agricultural soils containing varying amounts
of organic matter (0.97.10%) and pH (5.77.0). Ammonium acetate-EDTA
extractable heavy metal concentrations in these soils were in the following range
(mg kg 1): Zn, 144884; Pb, 9847; Cu, 8276; Ni, 1.9530.0; Cr, 0.0531;
Cd, 0.549.1. This study showed a close positive relationship between metals
418
Naidu et al.
and organic carbon in soils, while a negative relationship was observed between
total metals and soil respiration or microbial biomass. These results clearly indicate that heavy metals cause an alteration in soil C cycle resulting in a decrease
in the net mineralization of soil organic matter and further suggests the importance of soil properties in exerting metal toxicity toward microflora.
8.2
Nitrogen Transformation
Metals have been shown to be both stimulatory and inhibitory to nitrogen mineralization in field and laboratory studies, depending on the metal dose (Baath,
1989). Nitrification is the most sensitive parameter among various steps in the
nitrogen cycle. Ammonification of organic nitrogen to ammonium ions and subsequent nitrification of ammonium to nitrate were inhibited by 100 mg kg 1 Zn,
100500 mg kg 1 Pb and Cr, and 10100 mg kg 1 Cd. Brooks (1984) observed
no effect on nitrogen mineralization in sludge-amended soils. Mineralization of
sludge-derived OM did not differ significantly among the metal-contaminated
and uncontaminated soils (Johnston, 1989). However, a low turnover of organic
matter was reported in metal-contaminated soils (Chander and Brooks, 1991a,b).
Thus, the results obtained by different investigators on the effects of metals on
nitrogen mineralization are partly contradictory (Duxbury, 1985; Baath 1989;
Chander, 1991), suggesting that caution be exercised while interpreting these
results.
Cadmium inhibited nitrogenase in root nodules of red alder (Alnus rubra)
at concentrations greater than 0.03 mol Cd g 1 soil (Wickliff et al., 1980). Interestingly, even at lower concentrations of Cd (0.090.9 mol Cd g 1 soil), nitrogenase activity was still affected while nodulation was stimulated. The addition of
Mo and Zn stimulated N 2 fixation by Vigna unguiculata in a sandy loam soil
with optimal activity occurring at 153 mol Zn ha 1 and 91 mol Mn ha 1 (Rhoden
and Allen, 1983). The stimulatory effect of Mo and Zn might be due to their
requirement as essential elements for enzymatic activity. On the other hand, Cd
has no proven biological function. There have been several reports of negative
effects of Cd, Cu, Co, Ni, and Zn on both nitrogen fixation and nodulation of
plants (Porter and Seridan, 1981; Vesper and Weidensaul, 1978; McGrath et al.,
1988).
Heterotrophic nitrogen fixation was shown to be sensitive to metal pollution. For example, 50 mg kg 1 Cr and 50200 mg Cu kg 1 soil (Skujins et al.,
1986) and 24 mg Cd kg 1 (Coppola et al., 1988) were reported as sensitive to
heterotrophic N 2 fixation. However, Lorenz et al. (1992) opined that heterotrophic
N 2 fixing bacteria are not ubiquitous or active enough to be considered as indicator organisms.
Cyanobacteria are more widespread than the other free living diazotrophic
microorganisms and thus are very important for nitrogen economy of soils. These
419
organisms grow autotrophically on the soil surface and utilise sun light as an
energy source. Brooks et al. (1986) studied the growth and nitrogen fixation by
cyanobacteria on metal-polluted soils. These investigators found no or little
growth and nitrogen fixation on metal contaminated sludge-amended soils even
after 120 days. Martensson and Witter (1990) also reported the absence of cyanobacterial growth on sludge treated soils where as farmyard-manure-amended soils
developed algal growth. Using sludge-amended soils, Fliessbach et al. (1989)
reported a large decrease in cyanobacterial counts in metal-contaminated compared to uncontaminated soils.
8.3
Soil Enzymes
The measurement of the activity of various enzymes in soil has been suggested
as an index of microbial activity. The activity of dehydrogenase has been shown
to be decreased in many metal polluted soils whereas phosphatase was unaffected
(Brooks et al., 1984; Reddy et al., 1987). Dehydrogenase is intracellular, present
only in living organisms, and represents the active biomass whereas phosphatase
is extracellular. Dehydrogenase activity has been suggested as a more sensitive
FIG. 6 Flow chart of major components involved in adverse metal impacts

on soil microbes.
420
Naidu et al.
indicator of sludge metal pollution in soil (Fliessbach and Reber, 1990). In another study, dehydrogenase activity was severely affected by Zn at a concentration of 6538 mg kg 1 soil (Kelly et al., 1999a). Kuperman and Carreiro (1997)
reported considerable inhibition of a range of soil enzymes (N-acetylglucosamidase, -glucosidase, endocellulase, acid and alkaline phosphatase) by As, Cd,
Cr, Cu, Ni, Pb, and Zn concentrations ranging from 7.2 to 48.1 m mol kg 1 soil.
However, the decreased activities of dehydrogenase observed in Cu-polluted soils
require caution in interpreting those results since dehydrogenase measurements
based on triphenylformazan formation was shown to be affected by abiotic reaction with Cu (Chander and Brooks, 1991c).
There are few examples showing that metal speciation and fractionation
may be a key to improve our understanding of metal impacts on soil microbes.
Chaudri et al. (1999) established a relation between free Zn 2 activity and inhibition of microbial activity using a bisensor test. Other approaches have employed
chemical fractionation of metals in soils to establish thresholds that may be used
also for legislative purposes in soil protection policy.
Dwelling on the above discussion we derived a flow chart showing the
major components involved in adverse effects of metals on soil microbes (Fig.
6).
9
ADVERSE IMPACTS OF METALS ON FOOD QUALITY

AND IMPLICATIONS TO ANIMAL AND HUMAN
HEALTH ISSUES
Food quality, specifically the introduction of unwanted elements such as heavy

metals into food crops, is one of the major issues concerning regulators and the
public throughout the world. Besides impacting on animal and human health,
unrealistically high levels of metals also constrict free trade between countries
leading to significant implications to socioeconomic issues. As many of the elements or compounds in question are present in foods in trace quantities, their
presence has only relatively recently been cause for concern as improvements in
analytical techniques have indicated the level of contamination. Cadmium is the
element of most concern as it has a relatively high transfer coefficient from soil
to plant (compared with As, F, Hg, or Pb), and human dietary intakes of this
element are under scrutiny in many countries. Following ingestion of food containing Cd, a large proportion of the Cd is excreted by the body. However, a
small proportion is retained, mostly in the kidneys and liver, so that it may accumulate in these organs. It is thought that above a level of 200 mg Cd g 1 of
kidney (wet), pathological symptoms may appear (Friberg et al., 1985). However,
many of the studies examining the effect of Cd on body functions have used
short-term high-dose techniques, often with pure Cd salts.
421
Most of the Cd accumulated in grazing animal is derived mainly from the

pasture intake. Bramley (1990) estimated that annually approximately 55 and
275 mg Cd is ingested per sheep and cattle, respectively, through the intake of
herbage. Work in New Zealand has indicated that cattle and sheep may ingest
110% and 30%, respectively, of their dry matter intake in the form of soil. In
areas where soil contains high Cd, e.g., as a result of sewage sludge and fertilizer
application, soil ingestion is expected to play a significant role in Cd uptake by
the farm animals. Roberts et al. (1994) have shown that even though sheep ingest
3646 kg soil per year, this only contributes 58% of the total Cd intake for the
lax- and hard-grazed flocks, respectively.
Ruminants do not have a homeostatic control mechanism for regulating Cd
absorption or excretion which is affected by the level of dietary Cd content.
Although intestinal uptake of Cd has been estimated to account for more than
90% of the total Cd absorbed, most of the Cd ingested is discarded. About 80
90% of the total ingested Cd is excreted in the feces and only 0.05% is excreted
in the urine. Most dietary Cd is bound to metallothionein and is absorbed intact
into the circulation. Over 50% of the Cd ultimately accumulates in the liver and
kidney. In animals, kidney and liver Cd accounts for 5070% of the total Cd,
with kidney having a higher Cd concentration than the liver. Other organs, such
as pancreas, spleen, heart, brain, and testis, together with muscle and fat, accumulate small amount of Cd. In blood Cd is associated with albumin-like protein
which is transported to kidney where it is filtered through the glomerulus and
reabsorbed by the proximal tubules (Friberg et al., 1985).
In animals, the effects of prolonged exposure to abnormal levels of Cd
include testicular necrosis, placenta destruction, abortion, teratogenic malformations, renal damage, osteomalacia, immunosuppression, pulmonary edema, and
emphysema. In humans, severe Cd exposure can result in emphysema, bronchitis,
ulceration of nasal mucosa, renal dysfunction, liver necrosis, and anemia, hypertension, skeletal deformities, prostate and lung cancer, and teratogenesis (Chowdhary and Chandra, 1987).
Different countries have set guidelines on the maximum permissible levels
for Cd in various meat products (Table 7). In an earlier survey of cattle, pigs,
and sheep in New Zealand, the mean Cd concentration in kidney cortex, liver,
and muscle was less than 0.4, 0.1, and 0.05 mg Cd kg 1 wet weight, respectively
(Solly et al., 1981). These values are well below both the concentration measured
in other countries (Table 4) and the maximum permissible concentration stipulated by many countries (Table 3).
More recent testing of animal offal indicated that some 2228% of sheep
and 1420% of cattle between 1988 and 1991 had kidney Cd contents greater
than permissible level of 1 mg Cd kg 1 (Roberts et al., 1993). In general, older
animals had higher kidney Cd contents as these animals had longer exposure to
422
Naidu et al.
TABLE 7 Legal Maximum Permissible Limits for

Cadmium in Muscle, Liver, and Kidney of Sheep and
Cattle in Different Countries
Maximum permissible limit
(mg Cd kg 1 fresh weight)
Country
The Netherlands
Australia
Hungary
New Zealand
Denmark
Muscle
Liver
Kidney
0.05
0.2
0.5
1.0
0.1
1.0
1.25
0.5
1.0
3.0
2.5
0.5
1.0
0.5
Cd in their environment and hence greater opportunity to consume and retain

Cd. Feral deer and sheep isolated from human intervention also showed agerelated retention of Cd in kidney tissue as a result of exposure to naturally occurring Cd in the environment.
The average annual consumption of red meat per New Zealander (70-kg
adult) is estimated to be 80 kg. Based on a median Cd content of 0.01 mg kg 1
wet weight, the annual intake of Cd is expected to be 0.8 mg, which is equivalent
to a daily intake of 2.19 g Cd. This is considerably less than the maximum safe
level of 70 g Cd day 1 (Peters, 1988) from all sources in the diet. Although
offal, such as liver and kidney, contains more Cd than other meat, the small
quantity of offal consumed by the New Zealand population is unlikely to contribute significantly to the total Cd intake. However, in sections of population and
in pets where offal forms a larger portion of the diet, Cd intake from this source
could be a major concern. The population critical concentration of Cd at which
10% of the human population would exhibit signs of renal impairment is about
160 mg Cd kg 1 wet weight (Friberg, 1984). From Table 8 it can be shown that
it is unlikely that Cd input from meat exceeds the critical concentration in the
lifetime of a New Zealander.
The effect of prolonged exposure to low levels of Cd in foods (where Cd
is often complexed by organic anions) has not been evaluated. To date the only
reported human health problems directly attributed to Cd have been caused by
occupational exposure in the mining or metallurgical industries, or by the consumption by a malnourished population of food contaminated by industrial wastes
(the famous outbreak of Itai-Itai disease in Japan). Some workers argue that
risks due to dietary intake of Cd are low (Ryan et al., 1982), although the World
Health Organization/Food and Agriculture Organization Expert Committee on
Food Additives in 1972 proposed a maximum tolerable level for intake by hu-
423
TABLE 8 Cadmium Concentration in Liver and Kidney Samples of Cattle

and Sheep in Various Countries
Cd concentration
(mg Cd kg 1
fresh weight)
Country
Animal
Liver
Kidney
Ref.
Australia
Cattle
Sheep
Cattle
Sheep
Cattle
Cattle
0.18
0.30
0.10
0.10
0.11
0.21
0.30
0.96
0.25
0.25
0.36
0.55
Langlands et al. (1988)
New Zealand
Netherlands
USA
Solly et al. (1981)

Vos et al. (1987)
Mussman (1975)
mans of 6.78.3 mg Cd kg 1 wk 1 body weight. This was revised in 1980 to 7

mg Cd kg 1 wk 1 and after review recently (1993) was modified to 5 mg Cd
kg 1 wk 1. In keeping with these regulations some countries have adopted food
regulations controlling Cd concentrations.
10 CONCLUSION
Based on the available studies some generalizations can be made regarding the
impact of metals on soil biota and their processes. These studies raise concern
about long-term effects of these metals at large concentrations in the environment
since heavy metals can persist for tens of thousands of years, unlike their organic
counterparts, which can be mineralized. It would seem that soil respiration,
organic matter degradation, nitrification, and autotrophic nitrogen fixation are
among the processes most sensitive to heavy metals, while total bacterial numbers, biomass, and enzyme activities are quite insensitive. This is because soil
microbial communities can change without any measurable change in total microbial biomass, which probably represents the metal-resistant microorganisms.
Many of the studies involving metal effects on soil biota did not include soil
analysis data, which makes it difficult to accurately interpret the results. Hence,
there is a need for inclusion of quantitative analysis of soils in long-term studies
for improved understanding of the potential impact of metal contaminants on
terrestrial biota. It is equally important to identify the bioavailable and thus potentially toxic metal fractions and species in contaminated soils and establish relations between simple predictors of such fractions and metal impact on microbial
functions related to soil quality.
424
Naidu et al.
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15
Behavior of Heavy Metals and Their
Remediation in Metalliferous Soils
Arun B. Mukherjee
University of Helsinki, Helsinki, Finland
INTRODUCTION
Soil is a part of the ecosystem. It consists of mineral materials, plant roots, microbial and animal biomass, organic compounds, water, and a gaseous atmosphere
(1). All of these are unevenly distributed; in addition, heavy metals from natural
and anthropogenic sources form great sinks in the soil bed. Soil also serves as
a chamber for the decomposition of organic wastes and production of greenhouse
gases such as CO 2 (68). The concentration of heavy metals in the environment
is decreasing as well as increasing. For example, in the developed countries, low
concentration of heavy metals in soil compartments is expected to be a new problem in the 21st century. On the other hand, underdeveloped countries must deal
with high levels of toxic metals in their air, water, and soil compartments. For
these reasons, problems related to contamination of soils in recent years have
been addressed by not only the scientific community but by decision makers and
economists as well. Heavy metals in the soil bed enter from both natural and
industrial sources. Natural sources include wind-borne soil particles, sea salt
433
434
Mukherjee
spray, volcanoes, forest fires, and biogenic processes, whereas anthropogenic

sources include power generation, industrial processes, use of metals, and vehicular discharge of pollutants. On a global scale, the emissions of Pb, Cd, V, and
Zn from industrial sources exceed those from natural sources by factors of 12,
5, 3, and 3, respectively (19). There is little information on the global flux of
heavy metals stemming from the above-mentioned sources and their deposition
in the soil bed. But there are some studies done in the 1980s that can still be
considered as state-of-the-art work (Tables 1 and 2). In addition, the spreading
of Pfertilizers, sewage sludge, lime, manure, composts, high metal content of
parent rocks, trash, ashes, and slags will add further hazardous pollutants, especially Hg, Pb, Cd, Zn, and V in soils, and these are challenging problems for
society (20). Many consumer goods, including lead car batteries, nickel-cadmium
batteries, thermometers, and fluorescent tubes, contain substantial amounts of
heavy metals that will no doubt become a threat for contamination of soils; soil
covers 30% of the surface of the earth where plant species grow, and there is
strong competition between man, nature, and toxic metals (4). Each soil type,
however, has limited capacity to hold heavy metals, which is why there is growing concern that the anthropogenic input of heavy metals in soils in different
areas of this planet may exceed the limit (21,22). Environmental preferences are
specially attributed to groups of heavy metals in different compartments of the
ecosystem due to their toxicity, including Cd, Pb, Zn, Ni, Cr, Hg, Cu, and As
for agricultural soils; Cd, Pb, Zn, Cu, and Ni for forest soil; Cd, Pb, Zn, Hg, Cu,
and As for aquatic systems; and Pb, Cd, and Hg for the atmosphere (64).
A number of studies have been made on the behavior of heavy metals in
different compartments of the environment. It is observed that each metal behaves
in a different way in the ecosystem due to different thermodynamic and physicochemical properties. For example, Cd is deposited on soils in the particulate
forms, e.g., in the solid phase, whereas Hg and As exist in the atmosphere predominantly in the vapor phase. Consequently, Hg has a tendency to be re-emitted
to the air once deposited on the surface of soils (5). Deposition rate of Cd is
maximal in industrial countries, but in other regions of the planet where industrialization is limited, Cd enters into soil compartments through fertilizers, mostly
manure, sewage sludge, and biosolids (6). It has been suggested that anthropogenic deposition of Cd is maximum (7,8), but there is some uncertainly when
considering the partitioning of emissions of Cd and other heavy metals between
natural and anthropogenic sources (9). It is clear that on the global scale atmospheric deposition of heavy metals to soils is most important, whereas on a local
or regional scale, other sources may be more significant from the perspective of
soil pollution.
To improve the physicochemical properties of arid zone soils, addition of
sewage sludge precedes the release of heavy metals whose soluble fraction may
vary between 0.5% and 7.0% of the total amount of heavy metalborne sludge.
Source
Wind-borne
soil particles
Sea salt spray
Volcanoes
Wild forest
fires
Biogenic processes
Total
As
Cd
Co
Cr
Cu
Hg
Mn
Ni
Pb
Zn
0.35.0
00.4
0.67.5
3.650
0.915.0
00.1
42400
1.820
0.37.5
3.035
0.23.1
0.27.5
00.4
00.1
0.11.5
00.2
00.1
01.9
00.6
01.4
0.829
00.2
0.26.9
0.918.0
0.17.5
02.0
00.1
01.7
4.280
1.245
02.6
0.928
0.14.5
02.8
0.56.0
0.13.8
00.9
0.319
0.315
0.47.5
01.7
01.3
0.12.2
0.16.4
02.7
4.156
0.11.7
03.4
0.416
0.611.4
4.583
2.253.8
04.9
51.5582
2.956.8
0.923.5
4.086
1.123.5
0.13.9
Heavy Metals on Metalliferous Soils
TABLE 1 Estimated Global Natural Emission of Heavy Metals (1000 t/annum)
Note: Natural fluxes of Mo, Sb, Se, and V were excluded from this table.
435
436
TABLE 2 Global Industrial Emissions of Selected Heavy Metals into Soil Compartments in the 1980s (1000 t/annum)
Source
Wastes
Chemicals
Ashes
Fertilizers
Biomass (Peat)
Atmospheric
deposition
Total
As
Cd
1.315.1
3641
6.737
00.5
8.418.0
1.114.3
0.81.6
1.513
00.3
00.1
2.28.4
25.5215
305610
149446
00.4
00.2
5.138
52.4111.6
5.637.7
4851310
Cr
Cu
Hg
Mn
Ni
39.2239
395790
93335
0.10.6
0.22.0
1436
05.1
0.60.8
0.44.8
0.64.3
95415
100500
4981655
0.10.8
5.217
7.446
19.4142
6.532
56279
0.20.6
0.23.5
1137
36.2140
195390
45242
0.42.3
0.52.6
202263
5421403
1.615
7062633
93.3494
4791039
Pb
Se
Zn
0.513.2
0.10.2
4.160
00.1
00.4
1.32.6
6.546
0.62.8
1167
00.1
0.11.7
3.221
218710
310620
112484
0.31.1
0.23.5
49135
676.5
21.4138
6891954
Note: Depositions of Mo and Sb in soils were excluded from this table.

Mukherjee
437
But it is one of the major contributions of toxic metals in soils and plants (10,11).
It is true that in each country there are regulations on heavy metal contamination
of soils based on total metal concentrations of sewage sludge, manure, or lime.
But a better picture of bioavailability can be drawn when one considers the soil
solution of a metal. The relation between solution, total metal, and total metal
species is a result of environmental conditions, such as pH, ionic strength, and
potential ligand concentration in soils (12). In addition, one should consider the
geochemical forms of heavy metals in soils that may affect their solubility and
hence influence their bioavailability (13,64). Animals play an important role in
the soil compartment through different mechanisms such as inoculation, grazing,
litter combination, and so on. These animal populations will be affected if they
live longer in the environment of toxic metals, and they may acquire exposure
resistance which will help prolong their lives. The decomposition of organic matter is regulated by the predation of one animal on another. The process can also
be hampered when heavy metals, fertilizers, sewage sludge, and pesticides are
present, causing a concomitant increase in the number of bacterial decomposers,
and as such there will be retardation of decomposition of leaf litter due to less
soil-animal interaction (14). But it is surprising that microorganisms are capable
of acting directly or indirectly with heavy metals through mechanisms that include oxidation, reduction, acidification, complex formation, and bioaccumulation (Fig. 1) (17,18).
In the last two decades, a huge number of publications have been cited
regarding contamination of soils with toxic metals, chemicals, and organic compounds from industrial and natural sources. It is now also believed that global
climate change will trigger further impacts of heavy metals on soil compartments.
The flux of these toxic metals will shift from one region of the planet to another,
which will cause energetic impact on the quality of soils, plants, and soil animals.
Polluted soils may lead to tainting of foods and drinking water, which are the
basic necessities for human beings, animals, domestic livestock, and aquatic species (69).
It is impossible to give a detailed discussion of soil metal remediation
mechanisms. However, the purpose of this chapter is to present an overview of
the behavior of heavy metals in soil compartments, their uptake by plants, and
remediation of polluted soils. Remediation processes are the innovations of engineers and microbiologists. More recently, green-plant-based processes are becoming popular in many countries. In the history of civilization, it is known that
life cycles of plants have profound effects on physicochemical and biological
processes that occur in the environment (81). In addition, remediation costs of
polluted soils have been touched on in this chapter. The chapter is based on
literature searches and discussions with people from different organizations and
institutions.
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Mukherjee
FIG. 1 Microbial weather processes in the environment (redrawn) (17,18).
SOILS
Soils stem ultimately from rocks and are a vital compartment of the ecosystem.
Rocks consist of mineral and organic solids, aqueous and gaseous compounds.
Upon weathering, rocks produce the parent material and when in turn the parent
material weathers, it produces soil. A number of factors are responsible for the
formation of soils, including parent material, climate, life, topography, and time
(15).
2.1
Parent Material
Parent materials may be cited as sandstone, shale, clay, and limestone deposited
at different places of the planet. Due to the interaction of plants and rocks, new
chemicals are formed. Clay minerals are the products of weathering on rocks
439
that have significant effects on the physical and chemical properties of the soils
(16). Soils developed from different parent materials have diverse properties,
texture, and colors. For example, soils that develop from granite bedrock are
generally sandy loams of low fertility. However, the type of material that makes
up a soil affects the movement of water and air through soil and root penetration
into the soil, as well as the looseness and workability of the soil.
2.2
Climate
The climate may have a profound impact on ingredients of soils. The changes
of temperature, and snow and rainfalls, will cause expansion and contraction of
soil materials and thus have an effect on soil properties. Temperature has many
direct and indirect effects on soil biological activity, including physiological
changes, mineral weathering rates, redox potentials, and water activity. Regarding
extreme temperature, there are different opinions concerning the significance of
microbiological activity at or near the freezing point in soil. There are various
studies that support occurrence of subfreezing bioactivity in the Arctic environment (1).
2.3
Life
One can find lichens and mosses growing in soil environment. They grow, reproduce, and die. After their death, there is formation of organic compounds that
will help to produce other plants.
2.4
Topography and Time
The topography of a country is important for the growth of forest. With less
water staying on the hills after rain, plants would grow more slowly. This means
that there is a lack of organic matter for the next generation of plants. When the
whole countryside is hilly, steep slopes develop soil slowly, whereas the flatter
areas develop soils faster. In addition, soil development can be hampered due to
too much water content in soils and forests.
There is a big question mark regarding the time required to form soils on
the earth. In Alaska, a surface layer was evident in a glacial material after only
30 years of weathering. In Finland, soil was formed from the parent rock during
the glacial period, whereas in Iowa, USA, it took about 20,000 years to develop
a mature soil from wind-blown loess material (15).
All of the above-mentioned factors for the formation of soil can be expressed as follows:
Soil (Pm, C, L, T, t)
440
Mukherjee
where is a function, Pm is parent material, C is climate, L is life, T is topography, and t time.

3
HEAVY METALS IN SOIL COMPARTMENTS
The term heavy metal is generally used in connection with toxicity. In the periodic
table there are about 70 elements considered to be heavy metals based on their
specific gravity (e.g., should be 4.5 g cm 3) (23,24). However, in recent years
it has been said that toxicity is based on (a) dose response, (b) speciation of a
metal, (c) manner of exposure, e.g., inhalation, ingestion, skin, and (d) the individual exposed (26). As 3 and As 5 are toxic to human beings when the concentration in drinking water exceeds more than 0.01 mg L 1. But a minute concentration of As is needed for the survival of human beings (111). A group of authors
(25) classified the elements in the periodic table into three groupsA, B, and
borderlinebased on their relative affinity for O-, S-, and N-containing ligand.
In addition to the classification systems mentioned, heavy metals have also been
classified according to their chemical and biological properties. In the former
case, classification is based on the concepts of chemistry, e.g., acids and bases.
In the latter, it is based on the toxicity of the metal. In connection with the term
heavy metal, it is also to be noted that occasionally this term is replaced by the
term trace element or metal or toxic metal. However, heavy metals are a
subdivision of trace elements.
Table 2 indicates that soils receive large volumes of heavy metals from the
different wastes generated by industries. Two important sources of heavy metals
in worldwide soils are ash/slag residues from power generation and the general
breakdown and weathering of commercial products (such as dry-cell batteries
Ni, Cd, and Hg) on land. The metallurgical industry, mining and smelting of
ores, incineration of trash and red bag wastes, metal manufacturing industries,
and fossil fuelfired power plants have caused soil pollution since the industrial
revolution began. From all of these sources different heavy metals, such as Cu,
Pb, Zn, Hg, Cd, Ni, Cr, and V, enter directly or indirectly into the soil compartment (16,27). It has been estimated that at the beginning of the new millennium,
about 240,000 km 2 of the worlds land area will have been contaminated by
metalliferous mining activities (33). Metalliferous ores contain many important
metals, such as Pb, Zn, Ni, Cu, Cr, Cd, and Hg. Large amounts of different kinds
of ores and slag are dumped near mining and smelting areas from which leaching
of heavy metals to groundwater aquifers is to be expected. Table 3 indicates the
nature of concentration of metals in soils in the vicinity of mining and smelting
facilities.
There is an increased demand for Pfertilizers, especially in developing
countries. World consumption of Pfertilizer in 1996/97 was 31 10 6 tonnes
P 2 O 5 (28). However, the projected demand for 2025 can be about 60 10 6 tonnes
Source
Mine
Van mine
Cu-Ni smelter a
Ni smelter
Hg mine
Nuclear weapons plant
Gold mining areas
Ammunition industry
a
Country/region
Pb
Cd
Zn
Ni
Cu
Hg
Ref.
Wales & Cornwall

Wales
Finland/Harjavalta
Canada/Ontario
Slovenia/Idrija
USA/Oak Ridge
Russia/Siberia
Sweden/Zakrisdal
6150
310
4224,329
3.6
72
5
1590
20,550
2023,695
322
3000
5800
635,128
800925
7000
1.243.59
16197
34
35
36,43
37
41
42
48
51
TABLE 3 Examples of Metal Concentration (mg kg 1) in Soils Near Mining and Smelting Areas
Data are for the year 1991.
441
442
Mukherjee
of which the major part will be consumed by Latin America, Asia, and Africa
(29). Hence Cd in soils, especially in agricultural soils, may increase dramatically
in the new millennium. In addition, due to the pressure for reuse of wastes, sewage sludge, manure, lime, and organic composts, these material flows will be
important sources of heavy metals in soils, especially on a local scale. The concentration ranges of heavy metals in sewage sludge, fertilizers, manure, lime,
and composts are given in Table 4; these indicate that sewage sludge, Pfertilizers, and composts are significant sources of heavy metals in soils. Thus, the management policies and agronomy practices in a country will also affect the total
concentration of heavy metals in soils.
Varied speciations of heavy metals have been detected in soil compartments
with the use of improved analytical techniques. These metals can be present in
the soluble form or as exchangeable elements. They are often immobile, but are
associated with soil compartments, e.g., with carbonates, iron and manganese
oxides, organic matter, and parent materials. These metals or compounds generate
toxicity problems as a result of their shifting from soil compartments to the soil
solution, plants, and groundwaters (30,31). Aluminum hydroxides can also adsorb
a number of heavy metals, and in particular soil environments they can be more
important than iron oxide (7).
Often, different analytical procedures generate different data for metal concentrations in soils. At the beginning of the 1990s, Cd, Pb, Co, and Se in agricultural soils used for growing wheat and maize were analyzed by AAAc-EDTA
for 30 countries. It was reported that mean concentration (mg L 1; n 3664)
decreases in the following order: Se(16.3) Pb(4.25) Co(3.29) Cd(0.1)
(32).
Much information is available on the atmospheric deposition of heavy metals on the global as well as local scale in forest and agricultural soils, which can
be read elsewhere.
However, the mobility and bioavailability of heavy metals are generally
influenced by many factors, including pH, redox potential, temperature, soil constituents, cation exchange capacity, and organic compounds. Nevertheless, microbial activity and the physicochemical parameters of soil will govern the behavior
of heavy metals in soil environments.
3.1
Plant Uptake of Heavy Metals
Earlier it was mentioned that heavy metals are generally immobile in soil compartments (except in very sandy soils). However, often heavy metals, including
Cd, Mo, Se, As, Pb, Zn, Cu, and Hg are taken up by plants and crops. This uptake
depends on (a) the concentration and speciation of the metal in soil solution; (b)
the uptake by root surface and hence transfer of the metal from root surface into
the root; and (c) its translocation from root to the shoot. Figure 2 indicates the
Element
As
Cd
Cr
Cu
Hg
Ni
Pb
Se
V
Sewage
sludge
Pfertilizers
226
21500
2040,600
503300
0.155
165300
503000
210
20400
21200
0.1170
66245
1300
0.011.2
738
7225
0.525
21600
Nfertilizers
2.2120
0.058.5
3.219
115
0.33
738
21450
Limestones
Manure
Pesticides (%)
0.124.0
0.040.1
1015
2125
0.05
1020
201250
0.080.1
20
325
0.30.8
5.255
260
0.090.2
7.830
6.615
2.4
2260
1250
0.842
60
45
TABLE 4 Agricultural Sources of Selected Heavy Metals in Soils (mg kg 1 DW)
Note: The authors (7) cited 28 elements of which 9 are selected for this document.
443
444
Mukherjee
FIG. 2 Schematic diagram of movement of heavy metals in soil-plant systems (Reproduced with permission from Ref. 40).
relationship of heavy metals between soil and plant compartments. This would
imply that the route of administration is critical in determining the effects of
acute toxicity. Above the critical level, Mo and Se in soils and the aquatic environment in forage crops are quite toxic to animals. As, Pb, and Hg in crops in
certain amounts are harmful to consumers, but their concentrations in soils are
also potentially dangerous due to their possible direct ingestion by children, wildlife, and domestic animals. In an acidic environment, Cu, Ni, and Zn are quite
toxic to plants, whereas Cr(VI) is mobile and toxic to plants in soils in temperate
regions (38).
4
4.1
REMEDIATION OF POLLUTED SOILS

Introduction
In the last two decades, considerable attention has been paid to the problems of
soils contaminated by heavy metals and their compounds in mining and industrial
areas as well as in forests and agricultural soils. All soil contamination is not
from single-point sources, but there are also diffuse forms of soil contamination.
445
In the latter case, a large part of the land in Australia and New Zealand can be
cited as an example of Cd spreading due to application of fertilizers (46). Nowadays it is well known that contamination of soils by heavy metals is difficult to
treat because heavy metals cannot be destroyed, while on the other hand soils
polluted by acids can be neutralized. Heavy metals associated with organic compounds in soils are also difficult to treat. In the European Union, there are at least
120,000150,000 contaminated sites, containing 1.0 10 9 m 3 of wastes and
contaminated soils (44). In the United States, 250 million tonnes of hazardous
wastes per year is generated. These toxic metals and organic compounds may
leach into the groundwaters or they can be transferred to the food chain through
crops grown in waste-amended soils; Pb can be ingested by children and animals
from the contaminated soils causing Pb poisoning (45,70).
There are two remediation methods: (a) clean-up techniques based on fundamental removal principles, e.g., conventional remediation methods; and (b)
phytoremediation and biosolids techniques. Before selecting any technique, it is
necessary to know the following (47,68):
Nature of concentration of heavy metals and their compounds
Physical state of the heavy metal pollutants
Soil type, e.g., structure, percentage of clay and humic substances
Size of site, location, and the history of polluted soils
It has been reported that often it is difficult to apply remediation technology
due to environmental, geographic, legal, financial, social, time-related, and technical reasons. If this is the case, then techniques of immobilization, isolation,
solidification, and stabilization of contaminated soils should be the aim in order
to reduce risk to the ecosystem.
4.2
Remediation Principles
Remediation techniques are based on one of several principles, e.g., ex situ and
in situ. Hence, it is important to know the behavior of polluted particles and
nature of soils before starting up the cleaning program. The main technologies
that are available are listed in Table 5.
Phytoremediation, bioremediation, as well as ex situ techniques are often
used for the remediation of polluted soils. The success of remediation depends
very much on size, location, and background information of the polluted soils.
Clean-up techniques used will also vary from slightly polluted soils to highly
contaminated soils. In addition, information is necessary on how the soils have
been polluted, e.g., emissions from a facility, dumping of wastes, or dumping of
tailings from mines. Time scale and the ways in which soils are polluted will give
valuable information of soil texture, properties of soils, geochemical conversion
processes, and the distribution of heavy metals including organic compounds.
446
Mukherjee
TABLE 5 Remediation Technologies Currently Available or Under

Development for Heavy MetalsContaminated Sites
Method
Ex situ
Solidification
Thermal treatment
Washing
Leaching
Size, shape, and density
Soil excavation
In situ
Solidification
Volatilization of pollutants
Encapsulation
Attenuation
Electrokinetics
Phytostabilization
Phytoextraction
Soil amendments
Remarks
Addition of cemented agent to convert contaminated soils into hard, nonporous and
nonleachable material
This is applicable only to mercury contaminated soils
Chelate or acid extraction
Batch or pile leaching with chelates or acids
Technique for removal of fine particles that
have the highest metal concentration
Soil removal and disposal
As described above
Thermal treatment
Cover soils with impermeable layer
Dilution with uncontaminated material
Introduction of electric current so that ions
move to electrodes
Promotes vegetable growth to immobilize
metals
Removal of metals by plants
Reduce bioavailability of metals with P and
other soil amendments
Source: Data from Refs. 47 and 49.
It is reported that the newer remediation technologies are less expensive

than current remediation methods based on excavation and transportation of contaminated soils. To select any new technologies, risk assessment is necessary.
The soil excavation (e.g., ex situ) technique is generally applied to soils that are
contaminated by Pb in residential areas. The remaining ex situ technologies may
be applied to limited amounts of highly contaminated soils. Problems arise when
larger amounts of contaminated soils are treated by ex situ techniques and these
are due to the generation of hazardous wastes from the process. It is somewhat
costly to handle the generated hazardous wastes. Nowadays phytoremediation or
phytostabilization is used in the mining and smelting areas that require remediation. It is a long-term clean-up method that is especially popular in the United
States (49,50).
447
CLEAN-UP TECHNOLOGIES (EX SITU EXTRACTION)
5.1
Extraction/Wet Classification Process
Ex situ extraction is based on unit operation, which is divided into four steps:
(a) Sieving, or removal of coarse and foreign substances from the feed; (b) Mixing
whereby contaminated soils are mixed intensively with inorganic acids (HCl,
H 2 SO 4) or organic acids (acetic acid or citric acid) of intricate agents, such as
EDTA, NTA, or a combination; (c) Separation: flotation or hydrocyclone methods used to separate cleaned soil from the extracting agent; (d) treatment whereby
physical or chemical methods are applied for the removal of residue from the
used extracting agent. A schematic diagram of the process is given in Figure 3.
5.1.1
Disadvantage of the Process
In this separation method, low pH and an oxidizing atmosphere must be maintained to remove heavy metals from the feed. Therefore, to avoid corrosion problems, special stainless steel equipment is necessary, which is somewhat expensive. Complexing agents are also very expensive. However, these extracting
chemicals are necessary for regeneration; otherwise the cleaning process becomes
complex and even more expensive. This process use is limited to sandy soils and
soils containing limited amounts of clay or organic compounds (47).
5.1.2
Advantage of the Process
With this process, heavy metals as well as organic compounds can be separated
from the polluted soils.
FIG. 3 Process schematic of wet classification (Redrawn from Ref. 54).
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Mukherjee
This wet method can be applied even for the removal of Hg from contaminated soils where instead of chemicals only a high-pressure water jet is used in
the mixing drum. The technique is based on differences in particle size, shape,
and density, settling velocity, and surface properties. Hg has affinity for organic
substances and clay particles due to which wet classification is applicable for
various ionic, organic, and elemental forms of Hg. In practice, 2040 t h 1 of
Hg-contaminated soils can be handled by this method (52,53). Hg emission problems may occur prior to the main processes. These generally occur at the unloading station, which should be enclosed to avoid personal and occupational
health hazards.
5.2
Thermal Treatment of Hg-Contaminated Soils
Hg and its compounds are volatile at low temperature (356.58C). Most thermal
soil plants are designed to remove all kind of pollutants from the soil, including
petrol, oil, polyaromatic hydrocarbons, halogenated organic compounds, PCB,
furane, dioxin, and so on. To remove Hg and its compounds, the facility works
at about 600C. A simplified process diagram is shown in Fig. 4. At first the soil
is warmed at 100C in a drying drum; this is followed by a heating drum where
the temperature is maintained at about 600C. Here the Hg is released and passed
through different units before the exhaust gas is vented into the atmosphere. The
cleaned soil is cooled and moistened (54). Different thermal techniques, such as
thermal and vacuum-fired rotary dryer, and wet classification with thermal
FIG. 4 Thermal treatment for removal of mercury by engineering methods.
449
treatment, for the removal of Hg have been addressed in the literature. Similarly
the gas cleaning system may be different from that shown in Fig. 4. For example,
the spray tower may be replaced by an activated carbon filter (47).
However, there are advantages and disadvantages in the thermal treatment
(54).
5.2.1
Advantage
The process reduces maximum Hg concentration in treated soils.
5.2.2
Disadvantages
There are problems with porous fine materials in soils, e.g., bricks and
sludge.
It is difficult to obtain permission from the regulatory authority for its use.
There are social problems in the neighborhood.
It is more expensive than any other applied technique.
5.3
Electrodialytic Remediation Method
Electrodialytic remediation is a new method for removing heavy metals from

contaminated soils. Here a DC current is used as the cleaning agent together with
a combination of ion exchange membranes. This method was developed at the
Technical University of Denmark where electrokinetic remediation method is
combined with another technique known as electrodialysis. The principal of the
technique is shown in Fig. 5. Membranes 2 and 3 are in contact with the soil on
one side of the membrane and with an aqueous solution on the other. Membranes
1 and 4 are used to better control the electrode reaction. After remediation, the
heavy metals deposit in compartments II (cations) and IV (anions). The process
can be applied for the removal of Cu, Cr, Hg, Pb, and Zn from polluted soils.
When considering the electrodialytic remediation method, the following parameters should be taken into account (55).
pH is crucial for the desorption of heavy metals from the soil; for Cu, Pb,
and Zn, pH is maintained between 3 and 4, whereas for Cr it is about
2.5 for desorption.
The current density must be kept below the limiting current density to avoid
production of hydroxide ions.
The hydroxide ions may stop the migration of heavy metals.
Soil composition and speciation of metal play an important roll in this
technology. Electroreclamation is only suitable for the in situ removal
of heavy metals from the clay soils. However, few commercial applications have so far been reported for the removal of heavy metals from
polluted soils by this method (47).
450
Mukherjee
FIG. 5 Principle of electrodialytic soil remediation. (Redrawn with permission

from Ref. 55.)
CLEAN-UP TECHNOLOGIES (IN SITU EXTRACTION)
The basic principle of in situ extraction is percolation of an aqueous extracting

agent through the contaminated soils in which heavy metals are dissolved during
percolation. With the help of special pumping equipment, the percolate is pumped
up and then treated. The extracting agent can be reused after purification. The
percolation process is continued so long as removal of heavy metals from the site
is satisfactory. When considering the in situ extraction, special attention should be
given to the following parameters: geohydrological properties of the contaminated site, origin of the pollution, solubility of pollutants, purification process of
treated chemicals, and reconditioning. Only clay and sandy soils must be cleaned
up by the in situ extraction process. The success of the process depends on the
source of heavy metals. If it is originally dispersed via the water phase, then
there is maximal chance that in situ extraction will be successful. In addition,
the heavy metals should not be strongly bound to the soil particles. If necessary,
addition of such chemicals as inorganic or organic acids, complexing and oxidizing agents, and detergents may be carried out to improve extraction. Application
of this process is limited. Figure 6 indicates the simplified process scheme of the
in situ extraction method (47,56).
There are also simpler methods for soil remediation, which are summarized
in Table 6. All of these methods are readily available but expensive. A few are
mentioned below:
451
FIG. 6 In situ extraction (modified) (47,56).
Addition of limestone to raise the soil pH to 6.5 when soils are contaminated with heavy metals, especially Cd due to application of sewage
sludge (71).
Addition of clay minerals, such as vermiculite, to contaminated soils for
immobilization of heavy metals and radionuclides, e.g., 137 Cs and 90 Sr
(72).
TABLE 6 Selected Alleviation Methods That Are Adopted for MetalContaminated Soils
Target
pollutants
Soil processes
involved
Limestone
Metals,
radionuclides
Precipitation, sorption
Zeolite
Metals,
radionuclides
Metals
Fixation, ion exchange

Sorption, precipitation
Fixation, ion exchange, sorption
Technology
Apatite
Clay mineral
Metals,
radionuclides
Restrictions
Ineffective for oxyanions; certain
crops (lettuce,
spinach, tuber,
etc.)
Insufficient data
Insufficient data
Clay type; short
term
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Mukherjee
Addition of organic compounds to help immobilization of heavy metals as

well as to supply energy to microbe communities for decomposition and
transformation of organics and metalloids (73).
Addition of synthetic zeolite to contaminated soils to hinder the uptake of
Cd and Pb in plants. This has been demonstrated in laboratory experiments in Poland (74).
Besides the above-mentioned methods, it is a well-established fact that fertilizers are stable compounds that can bind Pb in soils. This process is quite
promising in the in situ immobilization of Pb (75,76). Details may be found
elsewhere (68). It is also cited that heavy metals can be stabilized by clinoptilite,
hydroxyapatite, and Fe-oxide wastes in contaminated soils (77).
7
7.1
PHYTOREMEDIATION
Introduction
In recent years, new technology based on the use of plants for remediation of
soils, known as phytoremediation, green remediation, botanical bioremediation, and phytoextraction, has come into use. This is cost-effective
in comparison with current expensive engineering methods (5760, 102). The
technology is practiced not only in the United States but in many other countries.
It is an environmentally friendly green technology that nowadays is applied to
both inorganic and organic pollutants present in the ecosystem, e.g., soil, water,
and air (65). It is also reported that metal-containing ashes from the process can
be recycled for the production of valued metals. The concentration range of a
number of heavy metals in soils and their regulatory limits in the United States
are shown in Table 7. However, the phytoremediation of heavy metals can be
divided into three sections: (a) phytoextraction; (b) rhizofiltration; and (c) phytostabilization. It has been estimated that in 2000 the phytoremedation of heavy
metalcontaminated soils in North America and Europe may be a $400 million
per year business (60). In this chapter, phytoextraction and phytostabilization
will be discussed in detail. Because rhizofiltration is a process for removal of
heavy metals from polluted wastewater streams (62), not from soil, it will not
be covered here.
7.2
Phytoextraction
Phytoextraction is generally defined as the extraction of metals by plants from

soils into their roots and above-ground shoots. The system uses for the synergistic
relationships among plants microorganisms, water, and soil that have evolved
naturally in the terrestrial and aquatic environments over millions of years. Generally plants contribute their inherent enzymatic and uptake processes to convert
453
TABLE 7 Recently Observed Concentration Range of a

Number of Important Heavy Metals in Soil and Their
Regulatory Limits
Elements
Pb
Cd
As
Cr
Hg
Cu
Zn
Concentrate range
(g kg 1) (66)
Regulatory limit
(mg kg 1) a
10006,900,000
100345,000
100102,000
5.13,950,000
0.11,800,000
30550,000
150500,000
600
100
20
100
270
600
1500
Nonresidential direct soil clean-up criteria. In Clean-up Standards for Contaminated Sites, New Jersey Department of Environmental Protection (1996).
contaminants to neutral compounds. Plants also act as hosts for aerobic and anaerobic microorganisms. Their roots and shoots increase microbial activity in the
direct environment by providing additional colonizable surface area, increasing
readily degradable carbon substrates by discharging organics and leachates.
Physically, plants decrease the movement of contaminants in soils by reducing run-off and increasing adsorption of compounds to the roots and shoots.
Once the phytoremediation system starts acting in the environment, its biological
components are naturally self-sustaining, powered by photosynthesis. The system
can be used as a clean-up technology because it is self-sustaining, cost-effective,
and aesthetically pleasing. It is now a well-established fact that by growing plants
over a number of years it is possible to clean up pollutants from contaminated
soils or to alter the chemical and physical nature of soil pollutants so that they
do not create a risk to human health and the environment (81).
Low pH of the soil plays an important factor in controlling the solubility
of metals in soils. Many studies suggest that lowering the pH of a soil will induce
desorption of heavy metals and thus increase their concentration in soil solutions.
Increased uptake of heavy metals may be possible by maintaining lower soil pH
via the addition of soil-acidifying amendments or ammonium-containing fertilizers (77).
Many different kinds of plants are used to clean contaminated sites. Wild
plants can also be used, but unfortunately their growth rate is slow. The aim
should be to select rapid growing plants so that they have high biomass in the
field. On the other hand, some authors recommend the use of hyperaccumulator
species (58). It has been reported that several high-biomass crop species, related
454
Mukherjee
to the wild mustard family, may be used for the removal of heavy metals from
soils. Brassica juncea (Indian mustard) has been used to collect Pb in its shoots,
and the spice possesses the ability to accumulate and tolerate Cd, Cr 6, Ni, Zn,
and Cu (61). Based on hydroponic cultural system studies, it is reported that B.
juncea and T. caerulescens accumulate heavy metals in their shoots. The latter
species can accumulate higher levels of heavy metals, particularly Zn and Ni, in
the shoots than the former. But B. juncea grows faster, which means it has more
biomass that will collect more metals from soil compartments (60). Ebbis et al.
(67) studied in the laboratory the ability of 30 plants to accumulate heavy metals
from soils moderately contaminated with Cd, Cu, and Zn. These laboratory studies indicated that Brassica spp. more effective in removing Zn from soil than T.
caerulescens. The reason is that the former produced 10 times more biomass.
There are diverse opinions among authors regarding the clean-up of Pb by
phytoremediation technology. Pb is a complex metal to remove from the soil bed
because it is a soft Lews acid that bonds strongly to both organic and inorganic
ligands in soils. There are three main factors that hinder the phytoextraction of
Pb, including: (a) poor plant uptake due to low solubility of Pb in soils; (b)
ineffective translocation from Pb in roots to shoots of a plant; and (c) toxicity
of Pb to the plant tissue. Many authors cited such methods as smelting, lowtemperature ashing, incineration, and microbial degradation to handle Pb in plant
tissues, but these are mostly at the theoretical stage. As of today, no Pb-contaminated site has been fully cleaned using phytoextraction technology (110).
Much attention has been paid in recent years to the behavior of Cr(III) and
Cr(VI) in Cr-contaminated soils. In Hudson County, New Jersey (USA), 2 million
tonnes of Cr slags has been used as landfill, whereas in Finland more that 600,000
t of Cr slags has been stored near the seashore in the north. On a worldwide basis,
the dumping of commercial wastes containing Cr may be the largest contributor,
accounting for 51% of the total release into soils. These disposal practices are
normal and create toxic problems due to the complex chemistry of Cr. Oxidation
and reduction reactions can convert nontoxic Cr(III) to toxic Cr(VI) and vice
versa (3,82,83). In recent years, deep-rooted plants have been used for phytostabilization of toxic Cr (58).
It is also necessary to understand the role of chemicals and metal salt additions in the phytoavailability and bioavailability of metals added to soils. It is
accepted that Fe and Mn oxides present in biosolids can increase the uptake of
specific metals from the contaminated soils, but addition of metal salts does not
have this effect (Fig. 7). A recent study suggests that ethylenediaminetetraacetic
acid (EDTA) is an effective chelating compound for mobilizing the metals in the
soil. Hyperaccumulation of Cd and Zn was noted by T. caerulescens (Prayon,
Belgium) in the presence of EDTA (Table 8), whereas the concept of chelateassisted phytoextraction may be applied to Pb, Cu, Ni, and Zn. The authors (65)
observed 28% Pb extraction from a Pb-contaminated site by using B. juncea
455
FIG. 7 The model addressed increasing Cd concentration in response to increasing total soil Cd concentration. (A) Addition of a soluble Cd salt. (B) From
addition of a soluble Cd salt with higher Zn concentration as a soluble Zn
salt. (C) Addition of biosolids. (Reproduced with permission from Ref. 79.)
(Indian mustard plant) after application of the chelating agent EDTA. It is also
reported that with the help of chelate-assisted phytoextration methods, it is possible to remove 180530 kg Pb ha 1 yr 1. The volume of contaminated biomass
can be reduced by ashing or composting; the residue should be treated as a hazardous waste or can be used as raw material for the extraction of metal (if economical) (65,84).
7.2.1
Phytovolatilization of Arsenic, Mercury,

and Selenium
Phytovolatilization can be applied to selected metals, including As, Hg, and Se.
It is not clear if terrestrial plants can volatilize As in significant quantities. As
accumulates in the roots and small amounts are transported to the shoots. However, volatilization of As may occur by rhizopheric bacteria in the terrestrial plant
(65).
Mercury toxicity, atmospheric cycling, deposition, and accumulation as
methyl-Hg in fish, birds, and mammals is well documented. More recently, it
has been reported that a modified bacterial mercuric ion reductase (MerA) has
been introduced into transgenic A. thaliana, which converts Hg 2 into elemental
456
TABLE 8 Concentration and Uptake of Zn, Cd, and Cu by T. caerulescens After Addition of EDTA
(Mean Values SD)
EDTA
addition
Plant concentration (mg L 1)
Plant uptake (mg pot 1)
DM (g)
Zn (%)
Cd
Cu
Zn
Cd
Cu
1.50 0.17
1.47 0.19
0.58 0.20
1.07 0.54
191 21
167 54
7.4 0.8
8.6 1.0
8765 3190
15196 5839
286 61
253 116
11 1.9
12 2.5
Mukherjee
457
Hg (Hg 0). It is also added that these transgenic plants (Arabidopsis) are very
effective at volatilizing Hg (85,98). This may be confirmed from recent research
in which authors (95) used a molecular genetic approach to transfer Hg resistance
genes to a variety of plant species as a potential means of phytovolatilization of
Hg 0. Figure 8 indicates how the authors (95) transferred bacterial genes to a number
of plants using standard methods for transgenic plant development. The same authors claimed that this method has potential to remove Hg from the contaminated
soils. They suggested that it is also possible to manipulate Hg 0 as Hg 2 in plant
shoot tissues, which could be an additional option for Hg phytoremediation.
Plants and soil microbes facilitate biosynthesis and emission of Se in the
form of dimethylselenide or dimethyldiselenide. It was reported that both Se nonaccumulator and accumulator species may volatilize Se. Commercial vegetable
crops (broccoli, cabbage, rice, and other plants) are quite effective in phytovolatilization, but the presence of sulfate and salinity in soils may inhibit volatilization
mechanism (86,87). However, more studies are necessary to understand plant
and soil microbial phytovolatilization mechanisms.
7.2.2
Removal of Heavy Metals by Hyperaccumulator

Plants
The ability of plants that grow in metal-polluted soils to take large amounts of
metal into their roots and shoots has been known for at least a century (88). Some
plants can take up unusually large amounts of metals (10100 times higher than
normal crops) into their shoots. These plants are known as hyperaccumulators
(89). Many authors have discussed specific species having remarkable hyperaccumulation capacities; these are summarized below (57,58,65,78,9092).
The hyperaccumulator species generally belong to the Brassicaceae and
Fabaceae families. At least 397 species have been identified, and this number
will increase based on studies of metal-enriched environments. The ecological
role of metal hyperaccumulation is still not fully understood. Although different
authors have different opinions, salient hypotheses include the following: (a)
Hyperaccumulation is possible when plants have hypertolerance properties,
which may be result of chelation and vacuolar compartmentalization. (b) Generally, the root of a plant contains more metal than the shoot. However, in hyperaccumulator plants the roots must transfer more metals to the shoots. It has been
reported that in normal plant root, Zn, Cd, or Ni concentrations are 10 or more
times higher than shoot concentrations, but in hyperaccumulators, shoot metal
FIG. 8 Enzymatic reactions encoded by bacterial merA and merB genes with
product, (Hg 0) (95).
458
Mukherjee
concentrations (e.g., 100 mg kg 1 Cd, 1000 mg kg 1 Ni, or 10,000 mg kg 1 Zn)

may exceed root levels. (c) Hyperaccumulators can resist drought and also can
interfere with neighboring plants. (d) It is also suggested that hyperaccumulators
provide protection against fungal and insect attack. Recent studies suggest that
Ni hyperaccumulation has a protective function against fungal and bacterial
pathogens in Streptanthus polygaloides, and insect herbivore in S. polygaloides
and T. montanum. An antiherbivory effect of Zn has been found in the Zn hyperaccumulator T. caerulescens (93,94).
The authors (91) analyzed 168 species of Alyssum Linnaeus to identify the
hyperaccumulators (1000 g g 1 DM) of Ni. An additional 31 hyperaccumulators (all in section Odontarrhena) were discovered in addition to the 14 European
species. Details of these species can be found elsewhere (91). There is no doubt
that hyperaccumulation plants are useful in soil clean-up as they can remove
significant amounts of heavy metals from contaminated soils. The efficiency very
much depends on the ability of root systems of the plant to quickly transfer metals
to aerial plant parts. Soils contaminated by mining and smelting are quite complex
in nature. The phytoextraction will depend on root systems in the contaminated
soil and the nature of the contaminated zones (96).
Chaney and co-workers have done pioneering work on hyperaccumulation
of metals from contaminated soils. Field results are given in Table 9. Their study
indicated that soil acidification increased the uptake of Zn and Cd and the plant
T. Caerulescens grew well when plant competition was limited by weed control.
However, in their study, low uptake of Pb was noted which is explained by the
fact that Pb hyperaccumulation is not possible when soil is treated with fertilizers
to improve biomass yield. Some scientists added the chelating agent EDTA to
dissolve soil Pb so that Pb-EDTA with water is up-taken by attenuated roots.
Transpiration carries the Pb-EDTA from the soil into plant shoots. But the growth
of plants are seized when high level of Pb is accumulated by shoots and roots
TABLE 9 Effects of Fertilizer Addition on Metal Uptake by Shoots

of T. caerulescens and Lettuce on Revival field, St Paul, MN, USA, 1993
Treatment
S
Soil pH
0
0
1
1
NH 4
NO 3
NH 4
NO 3
7.4
7.5
6.7
6.8
Metals in Thlaspi
Metals in lettuce
Cd
Zn
Pb
(mg kg 1 dry wt)
Cd
Zn
Pb
(mg kg 1 dry wt)
9.6
9.4
11.7
8.0
1360
1260
3100
2060
0.5
4.6
1.9
1.5
5.3
4.5
7.8
7.5
58
64
86
77
0.8
0.8
2.1
1.7
459
of the plant (84). It has been stated (107) that the application of EDTA is quite
expensive and not practical. However, Pb is ubiquitous in soil compartments.
Today no suitable wild or crop plants have been identified for cost-effective phytoremediation, but rhizofiltration of Pb from effluents has been identified. These
plants can bind Pb in their roots so that it does not leach to soils (97). But Prof.
Robert Tucker of Rutgers University claimed that the phytoremediation technique seems particularly attractive for the clean up of Pb in soils (109).
It has also been reported that hyperaccumulation can be achieved by genetic
engineering. Genes deciphering the Cd-binding protein metallothionein have
been examined in plants with successful results in terms of increased Cd resistance (99,100).
8
PHYTOSTABILIZATION
In the phytostabilization process, heavy metaltolerant plants are used to reduce

the mobility of metals so that there is less risk of heavy metals to the surrounding
environment (63). These specific plant species absorb heavy metals and organic
contaminants into their roots, thus reducing the mobility of the heavy metals into
the groundwater as well as their availability to the food chain. This technique
can be used to replace an idle soil cover at a site where natural vegetation is
lacking due to the presence of high concentrations of heavy metals or physical
disturbances of surface materials. In the United Kingdom, it has been reported
that by using fertilizers and planting metal-tolerant plants, excellent vegetative
cover was developed over metalliferous mine wastes (60). Based on this study
(60), three different types of grasses were made commercially available; Agrostis
tenuis cv. Goginan for acid Pb/Zn waste; Festuca rubra cv. Merlin for calcareous
Pb/Zn waste; and Agrostis tenuis cv. Parys for Cu waste. Much work is going
on regarding stabilization of Cd- and Zn-contaminated soils with metal-tolerant
grasses in the United States (60).
Recent studies also suggest that some plants may reduce metal leaching
by converting metals from the soluble oxidation state to an insoluble oxidation
state. It has been cited that concerning Cr, roots of the plant B. juncea can reduce
available toxic Cr 6 to unavailable less toxic Cr 3 (101). The point is that phytostabilizing plant species must tolerate high levels of heavy metals and that they
should be fixed into the roots not the shoots, thus avoiding mobilization of metals
in soils as well as reducing the amount of hazardous waste biomass. Chaney and
his group (79) developed special biosolids for the effective revegetation and
soil restoration of such metalliferous soils. These biosolids contain fertilizers,
composts, lime, and high-Fe biosolids. Tailor-made biosolids were applied to
barren soils containing high concentration of Zn (1%), Cd (100 mg kg 1), and
Pb (10030,000 mg kg 1). By controlling the proportion of ingredients, the group
(79) observed very satisfactory results on the bioavailability of Cd, Zn, and Pb.
460
Mukherjee
TABLE 10 Characteristics of the Soils and Materials at Banker Hill Site
Typical soil
Soil in plots
High-N biosolids
Low-N biosolids
Wood ash
Logyard waste
Zn
(mg/kg)
Cd
(mg/kg)
50
10,000
620
1700
410
86
0.5
17
2.5
22
3.4
0.5
Pb
(mg/kg)
10
3000
75
380
55
15
pH
C
(%)
N
(%)
5.5
6.4
8.0
6.5
11
7.2
3.5
0.4
32
26
18
9
0.2
0.02
5.0
2.8
0.1
0.2
They also pointed out that all biosolids do not contain high Fe levels or high
CaO equivalent. But many industrial, urban, and agricultural products can offer
high concentration of Fe and CaO equivalent for the preparation of remediation
products. Composts have a special place in the application in amending soils
because they can kill pathogens in biosolids and do not affect the safety of children (79). It has been demonstrated that using this type of biosolid it was possible
FIG. 9 A mixture of biosolids and ash is successful material for revegetation

and erosion control (106).
461
to remediate and revegetate permanently contaminated sites. Examples are Palmerton, Pennsylvania, USA (103) and Katowice, Poland (104) where mine wastes
and smelter discharges damaged the ecosystem. After the application of biosolids,
heavy metals and other contaminants are present at safe levels for domestic livestock and animals. Sometimes, noncomposted mixtures of biosolids, wood ash,
and log yard debris are quite effective in the recovery metalliferous soils; this
has been cited for soils in Bunker Hill, Idaho, USA (105). At the Bunker Hill
site, mining and smelting of Zn, Pb, Cd, and As for 65 years had contaminated
soils. Soils have been restored by application of biosolides (Table 10). In addition,
many ameliorates are known, such as zeolites, apatites, and glauconite, that decrease the mobility of metals and their uptake by plant species through metal
hydrolysis reactions and coprecipitation with carbonate (7). Before applying any
biosolids it is necessary to study the efficiency of selected ameliorates as metal
binders or stabilizers in contaminated soils. However, it has been observed that
biosolids control metal toxicity and provide organic matter that both improves
soil cultivation and acts as a substrate for soil microbes (Fig. 9).
9
REMEDIATION COSTS
Nowadays problems with contaminated soils are immense. Clean-up technologies

have been developed and are cited in this chapter; however, unfortunately, due to
financial, technological, environmental, geographic, and social reasons, cleaning
missions are not always successful. Remediation costs based on engineering
methods cited in this study are estimated to be U.S.$50200 per tonne of soil (47).
This cost was estimated at the beginning of the 1990s. Recent studies suggest that
remediation of contamination soil by conventional engineering techniques may
cost U.S.$50500 per tonne of soil. Any clean-up costs must be based on soil
type, physical state of pollutants, and size and location of the polluted site. Special
clean-up costs may be more than U.S.$1000 per tonne of soil (81), and a group
of scientists (97) estimated clean-up costs for highly contaminated soils by heavy
metals at U.S.$13 million per acre-foot of soil (soil removal, disposal, and replacement with unpolluted soil). In Finland, a secondary Pb smelter polluted soils
in its vicinity and the neighboring gardens with Pb. In the mid-1980s, smelting
activities were stopped, and remediation costs by traditional engineering methods
for 60,000 tonnes (35,000 m 3) of Pb-contaminated soils have been estimated to
be 2040 million marks (U.S.$ 5.84 mk). This means that the clean-up costs
per tonne of soil was U.S.$57115.
On the other hand, there are claims that phytoremediation or phytostabilization is less expensive than conventional engineering approaches, such as washing
or incineration of soils. Chaney and his group (97) estimated commercial phytoremediation costs for a crop with 20 tonnes biomass per hectare per harvest containing 2.5% Zn as follows:
462
Mukherjee
500 kg/ha Zn @ $1.92/kg Zn

15 kg/ha Cd @ 7.27/kg Cd
Total
$ 960/ha
$ 109/ha
$ 1,069/ha (1 ha 100 100 m 2)
This means that the cost of phytoremediation per square meter is about $1.07
(for two metals)
These costs vary from metal to metal. In the case of Co and Ni, it is possible
to make a profit above cleaning costs by burning and recovering the metal from
the biomass ash. But for Pb, U, and Se, phytoremediation costs are higher than
those of Zn and Cd. Chaney and co-workers have estimated the costs of the
use of biosolids and their conclusion is that it is costlier than phytoremediation
techniques. In their recent experience at Bunker Hill, Idaho, remediation costs
for the application of tailor-made biosolids was about U.S.$4000 per acre (4046
m 2), e.g., U.S.$0.99 m 2 of polluted soil. For phytoremediation, a general overall
cost would be about 50 cents m 2 (pers. commun., R. Chaney, 2000). Another
study estimates costs of U.S.$80 per cubic yard of soil (about 1 tonne of soils)
for the phytoremediation process (109). The same author cited disposal amounts
of polluted soils to be huge, e.g., an area of 2.5 acres (1.01 ha) and depth 18 in.
(46 cm) generates more than 5000 tonnes of soil. In contrast, plants used to take
up the metals from this same amount of soil would have only 2530 tonnes of
biomass ash after burning. The smaller ash volume from the plants is more economical to handle that the amount of polluted soils generated by conventional
methods. However, there is not much information on costs for phytoremediation
techniques; therefore, it is very difficult to reach a definite conclusion.
10 DISCUSSION AND CONCLUSIONS
In this chapter, I have preliminary discussed sources of heavy metals in the soil
compartments, their behavior in soils, uptake by plants, and remediation of polluted soils by traditional technologies with special attention to green remediation
known as phytoremediation. This information may guide the policymakers in the
selection of cost-effective methods for remediation of contaminated soils by
heavy metals and organic compounds. Moreover, the integrated load of heavy
metals in soil compartments may provide the basis for the development of policy
recommendations for the long-term sustainable distribution of heavy metals
through fertilizers and other components in soils.
Heavy metals have been recognized as toxic chemicals causing adverse
effects in the ecosystem. However, soils have been perceived as sinks for heavy
metals released from anthropogenic and natural sources. The contamination of
soils by heavy metals started from the beginning of the industrial revolution in
the 20th century. Mining and smelting, power generation, the cement industry,
chloralkali plants, secondary metal industry, and use of fertilizers and pesticides
463
all place heavy metals into the soils directly or indirectly. It has been estimated
that by the beginning of the 21st century, 240,000 km 2 of the earths land area
will be contaminated by metalliferous mining activities. The number of contaminated sites is growing in the European Union and, for example, in The Netherlands numbers around 100,000, whereas in Finland it is more than 18,100.
Soil consists of different minerals; clays; oxides of Al, Fe, and Mn; organic
and other compounds. Heavy metals binding capacity depends on the soil composition, soil pH, and redox conditions. Organic matter is important in the immobilization of Pb. However, heavy metals behavior in the soil compartments depends
on several factors, including the geochemical characteristics of the metal, plants,
soil chemical equilibria, climate, and agricultural management (64).
As the problem of contamination of soils is increasing, it is necessary to
tackle the situation. A large number of remediation techniques have been developed over the last decade and a half. These remediation methods are based on
(a) traditional clean-up technologies and (b) phytoremediation methods. Before
selection of any method it is necessary to study (a) distribution and concentration
of heavy metals in soils, (b) physical state of the pollutants, (c) soil properties,
e.g., sand and clay/loam content values, particle size distribution and percentage
of organic compounds, humic substances in soils, and (d) background information
of the site, e.g., how the site has been contaminated and for how long it has been
contaminated.
Traditional clean-up technologies are based on incineration of soils, adsorption and desorption processes, solubility, chemical treatment, and biodegradation.
For cleaning soils by traditional clean-up technologies, then, one or more methods
are generally applied. However, the experiences gained with these processes indicates that they are not capable of solving all problems. In the Western world,
many traditional clean-up methods have been developed. Each method has its
own character based on the type of soil and the physical properties of the pollutants, respectively (108). The total number of contaminated sites found in Europe
or the United States is not clear, but it is clear that the number of sites is increasing
exponentially. The costs of dealing with contaminated soils are enormous. In The
Netherlands, the estimated cost is about U.S.$150 per tonne of polluted soils
(amount of contaminated soils: 200 10 6 t and estimated costs U.S.$30 billion).
It is noted that general costs vary between U.S.$50500 per tonne of soil, but it
may reach U.S.$1000 U.S.$ if complex contaminated soil is in question.
In recent years, selected plant species that have the ability to accumulate
high concentrations of metals and organic compounds in their roots and shoots
from polluted soils have been introduced, especially in the Western world, and
this is known as phytoremediation technology. This technology is becoming popular for the clean-up of heavy metals by successive cutting of these plants. This
green technology is less expensive and friendlier to nature. However, limited
information is available regarding its costs. A few cost results are mentioned in
464
Mukherjee
Sec. 9. This technology claims that sometimes biomass ash product can be used
as a raw material for metals recovery, especially Ni and Co. It is not possible to
successfully remove all heavy metals by applying phytoremediation technology,
especially Pb. Uptake by plants for Pb is limited. The aim should be to find or
develop plants that can remove 5001000 kg of metal ha 1 yr 1. Researchers
have developed tailor-made biosolids using composts rich in hydrous Fe and
Mn oxides, phosphates, limestone, clay minerals, wood-ash, and zeolite that can
change soil-chemical properties so that contaminants are stabilized, immobile,
and not available to plants. Highly effective revegetation was reported by
applying phytoremediation technology at Palmerton, Pennsylvania, Katowice,
Poland; Bunker Hill, Idaho; and Leadville, Colorado. But phytoremediation also
has its drawbacks. It is a time-consuming process, and it may take several years
to clean up a site. There is also very limited information regarding the behavior
of pests to phytoremediation plants and the depth to which plants can sink their
roots to clean up contamination. However, sustainable soil quality and soil protection should be one of the main aims for the government in each country in the
new millennium.
ACKNOWLEDGMENT
My sincere gratitude is expressed to Professor Dr. Alina Kabata-Pendias, Institute
of Soil Science and Plant Cultivation, Pulawy, Poland; Professor W. H. Rulkens,
Wageningen Agricultural University, The Netherlands; Dr. R. L. Chaney, USDAARS, Environmental Chemistry Laboratory, Beltsville, MD, USA; Dr. Anna
Sophia Knox, University of Georgia, Aiken, South Carolina, USA; and Fredericks Scott, U.S. EPA for supplying me with valuable documents for this project.
The assistance received in preparation of this manuscript from the staff members
of the Department of Limnology and Environmental Protection, University of
Helsinki is gratefully acknowledged.
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Biodiversity Index
Abies, 371, 374, 375, 376, 377, 378

Acacia mangium, 304, 305
Acaulospora, 48
Acer sp. 41, 42, 369, 370, 371, 372,
373, 374, 375, 377, 378
Acer pseudoplatanus, 330
A. saccharum, 368
Achanthes microcephala, 70
A. minutissima, 70, 75
Achnanthes, 71, 74, 77
Achnanthes linearis, 70
Acinetobacter, 6
Aeollanthus subacaulis var linearis,
198
Aesculus, 369, 372
Agrobacterium, 6
Agrostis, 163, 459
Agrostis canina, 197
A. capillaries, 41, 193, 196, 197

A. cuneifolium, 166
A. gigantea, 193
A. stolonifera, 197
A. tenuis cv parys, 459
Ailanthus, 369
Albatrellus ovinus, 49
Alcaligenes eutrophus, 6, 9, 29, 30,
31, 33, 35
A. faecalis, 14
Allium cepa, 189
Alnus, 338, 369, 373
A. americanum, 220
A. bertlonii, 193, 220, 225, 228,
229, 229, 230, 240
A. heldreichii, 228
A. lesbiacum, 229, 229, 238, 238
A. malacitanum, 229
473
474
A. mangium, 305
A. montanum, 229
A. murale, 225, 229
A. obovatum, 220
A. perenne, 220
A. pintodasilvae, 229
A. rubra, 418
A. serpillifolium, 229
A. troodii, 228
Alyssum, 220, 223, 225, 228, 229,
230, 238, 240, 458
Amanita, 49, 50
Amanita muscaria, 42, 50
Anabaena, 67
Anabaena cylindrica, 63
A. doliolum, 79
A. flos-aquae, 67
A. inaequalis, 62, 66, 67
A. lutea, 62
A. variabilis, 67
Anacystis nidulans, 61, 62, 63, 67,
74
Andropogon gerardii, 40
Ankistrodesmus, 69
Ankistridesmus falcatus, 64, 83
A. falcatus var mirabilis, 72
Anthoxanthum odoratum, 197
Anthriscus cerefolium, 199
Anthyllis vulneraria, 192
Anthyrium filix-femina, 166
Aphanocapsa sp., 62
Arabidopsis, 171, 185, 293, 294,
302, 457
Arabidopsis thaliana, 184, 312, 313,
455
Arenicola christata, 278
A. marina, 278
Armeria maritima, 191
Arthraxon quartinianus, 198
Arthrobacter, 6
Arthrocnemum fruticosum, 97, 99
Asplenium sp., 166
Biodiversity Index
Asplenium adiantum-nigrum,
166
A. cuneifolium, 166
A. hybrida, 166
A. limneticus, 73
A. presolanense, 166
A. ruta-muraria, 166
A. septiontrionale, 166
A. trichomanes, 166
A. tripolium, 106
A. viride, 166
Asterococcus, 69
Atriplex portulacoides, 278
Bacillus sp., 14, 241
B. subtilis, 19
Barbula recurvirostra, 163
Batrachosperum vagum, 72
Becium homblei, 192, 198
Beilsch miedia, 370
Beta vulgaris, 188
Betula, 41, 42, 330, 331, 373, 374,
378, 380
Betula alleghaniensis, 328
B. papyrifera, 330
B. pendula, 336
B. pendula, 42
B. populifolia 385
B. pubescens, 42
B. tauschii, 361
Biscutella laevigate, 192
Brachymenium acuminatum, 162
B. philonotula, 162
Brassica, 171, 221, 223
B. campestris, 221
B. chinensis, 221, 242
B. juncea, 221, 222, 230232, 240
243, 245, 333, 454, 459
B. napus, 244
B. narinosa, 242
B. nigra, 221
B. pekinensis, 221
Biodiversity Index
B. rapa, 221
Bryoria fuscescens, 126, 134
Bryum, 162
B. arachnoideum, 162
Bryum argenteum, 163
B. rubens, 163
Bulbostylis pesudoperennis, 198
Bumilleriopsis filiformis, 81
Burkholderia, 6
Cajanus cajan, 183, 205
Calluna vulgaris, 42
Campylopus bequartii, 162
Cantharellus tubaeformis, 49
Carex, 259
Carpinus, 370
Carya, 369, 371, 372, 373, 377
Castanea, 372, 373, 376
Cedrus, 369, 370, 376
Cephalozia bicuspidata,163
C. hampeana, 162
C. integerrima, 162, 167
C. masalongi, 162, 168
C. nicholsonii, 162, 167
C. rubella, 162
C. stellulifera, 162
Cephaloziella, 162
Cephaloziella calyculata, 167
Ceratophyllum demersum, 267, 276
Ceratopteris cornuta, 165
Cercidiphyllum, 369
Chamaecyparis, 369, 373, 376
Chamaecyparis thyoides, 383
Chamaesiphon minutus, 68
Chamaesiphon subglobosus, 68
Chantarellus ciliarius, 49
Chara, 79
Chara corralina, 63, 83
Cheilanthes inequalis var. lanopetiolata, 165
Cheilanthus hirta, 165, 166
Chlamydocapsa, 69, 71, 74
475
Chlamydocapsa bacillus, 69
Chlamydocapsa cf. petrify, 69
Chlamydomonas acidophila, 70
C. ampla, 79
C. bacilus, 69
C. botryopara, 72
C. debaryana var. micropapilli, 72
C. hebes, 72
C. heterogama, 73
C. kessleri, 62
C. reinhardtii, 65, 79, 80, 81
C. vulgaris, 71, 79
Chlorella, 63, 65, 69, 74, 79, 81, 83
Chlorella fusca, 66, 67, 81
Chlorella fusca var fusca, 72
Chlorella fusca var vacuolata, 69, 72
C. pyrenoidosa, 61, 62, 63, 66
C. vulgaris, 61, 63, 64, 79, 83
C. vulgaris var vulgaris, 72
Chlorococcum, 417
Chroococcus paris, 66
Cinnamomum, 369
Citrobacter freundii, 17
Cladonia, 122
Cladophora, 60, 61, 64, 79
Cladophora glomerata, 60, 66, 67,
69
Comamonas, 6
Closterium striolatum, 72
Cortinarius, 49, 50
Cosmarium, 69, 77
Cryptomeria, 369, 370, 371, 372,
373, 374, 376, 377
Cryptomeria japonica, 357, 364,
379
Cucumis sativus, 186
Cyanidium caldarium, 62
Cymbella, 70
Cylindrocapsa, 69
D. muscorum, 143, 145
Dacrycarpus, 375
476
Datura innoxia, 186, 187

Daucus carota, 312
Dermocybe, 49
Deschampsia caespitosa, 193, 196,
197
D. flexuosa, 197
Desulfobulbus, 4
Desulfomicrobium, 14
Desulfovibrio, 4
Dictyococcus, 69
Diploschistes muscorum, 143
Ditrichum, 162
D. acidophila, 79
D. cornubicum, 162
D. cornubiensis, 167
D. plumbicola, 162, 168
Dunaliella bioculata, 83
D. salina, 87
Escherichia coli, 12, 13, 18, 20
E. hirae, 19
Eichhornia, 259
E. crassipes, 271
E. eximia, 342
Eiseniabicylis, 62
Elsholtizia haichowensis, 198
Enterobacter cloacae, 14
Entorphospora, 48
Eucalyptus, 332, 338, 342
Eucalyptus camaldulensis, 342
Euglena, 79
Euglena gracilis, 66, 73, 79, 81
E. mutabilis, 70
Eunotia, 70
Eunotia exigua, 70, 74
Evernia prunastri, 118
Fagus, 369372, 374378
Fagus japonica, 361
Fagus sylvatica, 326, 361, 380
Festuca ovina, 197
F. rubra cv Merlin, 459
Biodiversity Index
F. rubra, 196, 197, 328

Flavoparmelia baltimorensis, 138
Fragaria crotonensis, 81
Fragilaria, 77
Fraxinus, 369, 370, 372, 373, 375,
376, 383
Funaria hygrometrica, 163
Geminella interrupta, 77
Gigaspora, 48
Gingko, 369
Gleditsia, 370
G. turfosa, 70
Gleococcus, 69
Gleocystis gigas, 79
Glomus, 44, 45, 49, 40, 48
Glomus mosseae, 44
Glyceria fluitans, 274, 275
Glycine max, 185, 187, 191, 213,
317
Gomphenema, 70
Gomphidius sp., 49
Gomphospheria sp., 77
Grimmia atrata, 162
Gymnocolea acutiloba, 163
H. macrophylla, 305
H. rivulare, 74
Halimione portulacoides, 97, 99,
107112
Haumaniastrum katagense, 197, 198
H. robertii, 197, 198
Hebeloma, 50, 331
Helianthus annuus, 192
Holcus lanatus, 196, 197
Hordeum vulgare, 186, 330
Hormidium sp., 71
Hormidium rivulare, 69, 72, 74
H. pseudostichococcus, 72
H. scopulinum, 72
Hovenia, 376
Hybanthus floribundus, 225
Biodiversity Index
Hydnum, 49
H. macrophylla, 303
H. macrophylla, 304, 305
Hydrilla verticillata, 276
Hydrodictyon reticulatum, 61
Hymenoscyphus, 42, 39
Hymenoscyphus ericae, 42, 50
Hypnomonas, 69
H. chlorococcoides, 73
H. chlorococcoides var incrassata,
72
Hypogyminiaphysodes, 133
Idesia, 369
Indigofera setiflora, 192
Ipomea aquatica, 279
Juniperus, 373, 374, 375
Kalopunax, 372, 373
Klebsormidium, 62, 69
K. klebsii, 78
K. rivulare, 70
Laccaria laccata, 49
Lactarius, 49, 50
Lactuca, 171
Larix, 369, 370, 376, 377, 378
Leccinum sp., 49
Lemna, 259
Lemna minor, 199, 277
Leptospirillum, 24
Lindera, 376
Liquidambar, 376
Liriodendron, 376
Liriodendron tulipifera, 328
Liriodendron, 371, 373, 374, 375,
377, 378
Lithocarpus, 369
Lobaria pulmonaria, 129, 130, 131,
132
Lolium multiflorum, 199
477
Lupinus luteus, 186, 187

Lycopersicon peruvianum, 193
L. esculentum, 179, 188, 190
M. cajuputi, 305
M. malabathricum, 304, 305
M. pachyderma, 69
Medicago sativa, 40, 199
Melaleuca cajuputi, 304, 305
Metasequoia, 376
Microcystis aeruginosa, 66
Microspora floccosa, 71
M. pachyderma, 69, 70, 71, 72
M. stagnorum, 69
M. stagnosum, 71, 72
M. tumidula, 69, 71, 73
M. willeana, 69, 71
Microthamnion, 71
Microthamnion kutzingianum, 71, 72
Microthamnion strictissimum, 70,
72
Microthlaspi, 221, 223
Mielichhoferia, 161, 162
M. elongata, 161, 166, 167
M. macrocarpa, 162
M. mieilichhoferi, 162
M. nitida, 161
Mimulus guttatus, 186, 187
Minuartia hirsuta, 177
M. verna, 191, 198
Mohria lepigera, 165
Monoraphidium dybowski, 67
Mougeotia, 69, 71, 77
Mougeotia gracilima, 72
M. parvula, 72
Myriophyllum spicatum, 267
Najas, 259
Nardia scalaris, 163
Navicula, 70, 71
N. pelliculosa, 81
478
Nicotiana plumbaginifolia, 189,

190, 213, 312
N. tabacum 186, 307
N. tabacum L cv Samsun, 307
Nitschia 70, 71
Nitschia closterium, 94
Nitschia palea, 70
Noccaea, 221, 223
Nostoc, 61
Nostoc calcicola, 63
Nothalaena marantae, 166
Ocimum basilicum, 181
Oedogonium, 62, 72, 77
Oidiodendron, 39
Oidiodendron maius, 42
Oligotrichum hercynicum, 163
Oocystis, 69, 71
Oocystis elliptica, 74
O. lacustris, 69
O. nephrocytioides, 69, 74, 77, 79
O. parva, 73
Ophioglossum lancifolium, 166
Oryza sativa, 185, 189, 192, 304,
305
Oscillatoria, 68, 77
P. syringaevartomato, 16
Parmelia caperata, 122, 129- 132
Paulownia, 369
Paxillus involutus, 49
Pellea calomelanos, 165, 166
Peltigera canina, 129, 130, 132, 138
Pezizella ericae, 42
Phacotus lenticularis, 77
Phaseolus aureus, 189, 190
P. banksiana, 179
P. coccineus, 179, 185, 188, 189,
190
P. vulgaris, 177, 179, 180, 183,
184, 186, 188, 189, 190, 199,
328
Biodiversity Index
Phellodendron, 369, 376

Pholianutans, 163
Phormidium, 68, 74
P. foveolarum, 68
P. luridum, 68
P. uncinatum, 68
Photobacterium phosphoreum, 26
Phragmites australis, 274
P. communis, 192
Picea, 370, 371, 372, 373, 374, 375,
376, 377, 378
Picea abies, 49, 325, 328, 381, 382
Pinnularia, 70
Pinnularia acoricola, 70
Pinus, 41, 369, 370, 371, 373, 372,
373, 374, 375, 376, 377, 378
Pinus densiflora, 361
P. nigra, 42
P. ponderosa, 368
P. strobus, 328
P. sylvestris, 42, 46, 380, 386
P. taeda, 328
P. thumbergii, 361
Pisolithus tinctorius, 44, 50, 342
Pisum sativum, 188, 189, 189, 190,
310
Platanus, 369
Plectonema, 68
P. boryanum, 67
Plerococcus, 69
P. rufescens, 72
P. vulgaris, 72
Pohlia andalusica, 167
Pohlia nutans, 163
Polycarpea spirostylis, 198
Polygonum sachalinense, 304, 305
P. deltoides x nigra, 335, 337
P. maximowiczii, 336
P. nigra x maximowiczi, 337, 338
P. nigra, 335
P. trichocarpa x deltoides, 335, 337,
341
Biodiversity Index
Populus, 328, 332, 369, 370, 375,

376
P. tremuloides, 385
Potomogeton, 259
P. pectinatus, 267, 277
P. perfoliatus, 267
Pottia, 162
Prunus serotina, 42
P. virginiana, 328
Prunus, 369, 370, 371, 372, 373,
376, 378
Pseudoanabaena catenata, 77
Pseudococcomyxa, 69
Pseudococcomyxa adhaerens, 71, 72
Pseudomonas aeruginosa, 9, 14, 18,
19, 303
Pseudomonas, 6, 12, 13, 18, 19, 241
Pseudomonas putida, 24, 80
Pseudotsuga, 369, 373, 374, 377
Pteris vittata, 165
Quercus, 139, 369378, 383
Q. nigra, 368
Q. robur, 368
Q. rubra, 300
Q. velutina, 368
Ralstonia, 25
Ralstonia eutropha, 6, 924
R. metallidurans, 6, 9
R. eutropha, 5
Ramalina duriaei, 145
Ramalina farinaceae, 129132
R. fastigata, 126, 127, 128, 132
137, 139, 140, 148
Ranunculus, 259
Raparia, 223
Raphanus, 171
Raphanus sativus, 179, 199
Rattus novervegicus, 16
Rauvolfia serpentina, 185, 186, 193
479
Rhizobium leguminosarum bv trifolii, 49

Rhizopogon sp. 50
Robinia, 369, 371
Robinia pseudoacacia, 42
Ruppia, 259
Russula, 49, 50
Sachharomyces cerevisiae, 11, 19,
31
S. bacilaris, 63
Salix, 41, 327, 328, 331, 332, 333,
342
S. arenaria, 335
S. burjatica cv aquatica, 327
S. caprea, 331
S. dasyclados, 339
S. phylicifolia, 42
S. triandra, 339
S. viminalis, 335, 336, 339
Sassafras, 375, 376
Scapania undulata, 165
Scenedesmus, 69, 77, 79, 185
Scenedesmus acutiformis, 69, 79, 81
S. acutus, 66
S. armatus, 73
S. obliquus, 61, 64
S. quadricauda, 81
S. serratus, 73
S. subspicatus, 60, 61, 81
Schizothrix, 68
Scirpus lacustris, 275
Scleroderma flavidium, 330
Scleroderma sp., 50
Scopelophila, 161
Scopelophila cataractae, 162
S. ligulata, 161
Scopelphila cataractae, 161, 167
Scutellospora, 48
Sebertia acuminata, 197, 324
Secale cereale, 184
Selenastrum, 69
480
Selenastrum capricornutum, 63, 65,

74
Senecio cornatus, 225
Serbertia acuminata, 324
Silene cucubalus, 177, 189, 191,
198
S. dioica, 330
S. italica, 185
S. vulgaris, 193, 197
Snechococcus, 81
Solanum lycopersicum, 199
Solidago canadensis, 199
S. aureus, 11, 13, 17
S. typhimurium, 9, 11, 18
Sorbus, 376
Spartina alterniflora, 192, 278
S. maritima, 97, 99, 107112
S. townsendii, 278
Spinacea oleracea, 179
Spirogyra, 62, 69, 71, 79
S. nitida, 72
Spirulina platensis, 62
Spondylosum pygmaeum, 72
Staurastrum punctulatum, 72
Stewartia, 376
Stigeoclonium, 69, 80
Stigeoclonium aestivale, 69
Stichococcus, 63, 67, 69
Stichococcus bacillaris, 62, 63, 65,
66, 81
Stigeoclonium sp., 71, 80, 81
Stigeoclonium tenue, 69, 71, 72, 74,
81
Streptanthus polygaloides, 458
Styphimurium, 11
Suillus, 50
S. bovinus, 46
S. luteus, 42, 43, 44
Surirella angustata, 70
Synechococcus, 16, 22
Synechocystis aquatilis, 62, 63, 64,
67, 68
Biodiversity Index
Synechocystis sp. 11, 12, 13, 19, 21

Synedra, 70
Synedra filiformis, 70
Syringa, 370
Tabellaria fenestrata, 77
Taxodium, 369, 377
Thalassiosira pseudonana, 81
Thelephora terrestris, 46
Thiobacillus, 3, 4, 6, 24
T. ferrooxidans, 3, 7, 24
Thlaspi, 171, 221, 223, 243, 323
T. alpestre, 223, 233, 234, 235
T. arvense, 236, 238, 239, 245
T. caerulescens, 222, 223, 228, 231,
233, 235, 236, 237, 239, 243,
244, 245, 252, 256, 257, 454,
458
T. calaminare, 233
T. cepaefolium, 233, 237
T. goesingense, 223, 233, 234, 235,
237, 238
T. montanum, 225, 238, 458
T. montanium var calofornicum,
235
T. montanum var fendleri, 238
T. montanum var montanum, 238
T. montanum var siskiyouense, 225
T. montanum californicum subsp.
siskiyouense, 235
T. ochroleucum, 181, 239
T. praecox, 223, 223
T. rotundifolium, 223, 237
T. rotundifolium subsp cepaefolium,
223, 233, 234, 235, 245
T. sylvestre subs. calaminare, 239
Thlaspietum cepaefolii, 235
Thuja, 370, 375
Thujopsis, 372, 373
Tilia, 369, 370, 371
Trebouxia, 131
Tribonema, 61, 62
Biodiversity Index
Triticum aestivum, 183, 186, 187,

192, 308
T. durum, 179
T. vulgare, 189, 191
Trochiscia, 69
Tsuga, 370, 374, 375, 377
Typha, 259, 275
Typha latifolia, 192, 274
Uca pugnax, 278
Ulmus, 369, 370, 371, 377, 383
Ulothrix, 61, 69
Ulothrix moniliformis, 69
Ulothrix gigas, 62
U. variabilis, 72
Umbilicaria muhlenbergii, 126, 130
Usnea sp., 126, 127, 128, 136, 137,
139, 140, 144, 148
481
Vaccinium macrocarpon, 304, 305

V. myrtillus, 42
Vallisneria americana, 276
Vaucheria, 62, 63
V. compacta, 81, 82
V. debaryana, 81, 82
Vibrio, 27
Vigna ungiculata, 179, 192, 418
Viola calaminaria, 40, 198
Volerinella locusta, 199
Xanthoriaparietina, 125, 130, 135,
143, 144, 145
Zannichellia paulstris, 267
Zea mays, 179, 191, 199
Zelkova, 376
Zygogonium ericetorium, 71, 72
Subject Index
ABC ( ATP-binding cassettes)

transporters, 10
Accumulation of
aluminum, 295
metals by algae, 60
Acid
deposition, 379
soils, 289
Adaptations of woody plants to
heavy metals, 330
Adverse impacts of metals on soil
biodiversity, 415
Alfisols, 289
Algal communities, 68
Aluminum:
accumulation, 305, 309
cytotoxicity, 309
[Aluminum]
enhanced peroxidation of lipids,
310
excluders, 305
inducible genes, 313
stress, 289
tolerance, 292, 293, 301
tolerant cell lines, 311, 312
toxicity, 289, 295
toxicity and cell wall, 299
toxicity in acid soils, 291
AM fungi, 50
Andisols, 289
Anion-induced cation sorption, 405
Anthropogenic activities, 3
Antiporter systems, 9
Arbuscular mycorrhiza, 38, 40
483
484
Archaebacteria, 6
Atmospheric changes in the world,
365
Bacteria, 1
Bacteria immobilize composite
membrane reactor (BICMER),
25
Bacterial heavy metal resistance
mechanisms, 7
bark pockets, 353, 354
Behaviour of heavy metals, 433
Beneficial uses of trees in soil remediation, 332
Biological
interactions and transitions, 413
oxygen demand (BOD), 335
BIOMET sensors Biological
metal sensors, 26
Biomonitoring, 385
Biosensors, 28
Biosolid, 460
Blue-green algae (Cyanophyceae),
68
BMSR Bio Metal Sludge Reactor, 25
Beneficial effects of aluminum, 303
Calamine soils, 40
Callose
formation, 299
production, 309
Carbon mineralization, 417
Cation exchange capacity (CEC),
291, 326, 407
Cation binding capacity of wood,
381
Cation diffusion facilitation, 11
Cation diffusion facilitator (CDF),
11
Cation/Proton antiporter, 9, 18
Subject Index
Cellular growth conditions, 307

Chr chromate resistant determinant, 9
Clean-up technologies (Ex-situ extraction), 447
Clean-up technologies (in-situ extraction), 450
cnr cobalt, nickel resistant determinants, 9
Coevolution, 45
Complexation, 15
Concentration factor, 60
cop copper resistant determinant,
9
czc cadmium, zinc and cobalt resistant determinants, 5
Decontamination of soil and water,
24
Dendroanalysis, 367, 368
Dendroanalytical biomonitoring,
386
Development of metal-resistant/
tolerant communities, 74
Diatoms, 70
Diversity, 416
Diversity of
algal communities, 68
mycorrhizal fungi, 47
ECM, 45
Ectomycorrhizas, 37, 38, 41
Effect of heavy metals on woody
plants, 326
Effect of the sapwood/heartwood
boundary on trace element
distribution, 382
Effect on microbial processes, 417
Effects of metals on algal growth
and metabolism, 64
Efflux, 11
Subject Index
Electrodialytic remediation method,

449
Endomycorrhizas, 38
Entisols, 289
Environmental monitoring, 26
Enzymatic reduction, 13
Ericoid mycorrhizal fungi (ERM),
39
Ericoid mycorrhizas, 42
Fast-growing trees in phytoremediation, 340
Fate of metal ions in the soil environment, 403
Forest ecosystems, 323
Freshwater algae, 59
Functions of mycorhizal fungi, 44
Gene-based biosensors, 26
Genes responding to aluminum,
301
Gram-negative and gram-positive
bacteria, 6
Green algae, 69
Halophytes, 112
Hard and soft acid base theory
(HSAB), 407
Heartwood
polyphenols, 383
extractives, 383
Heavy metal:
biosensors, 26
homeostasis, 1
interactions in soils, 401
resistance, 6, 17
tolerance in mycorrhizal fungi,
42
toxicity to microbial communities, 416
uptake, 61
485
[Heavy metal]
resistant bacteria, 6
soil compartments, 440
Historical monitoring, 364
Histosols, 289
Hyperacccumulator, 2, 457
Inceptisols, 289
Inhibition of cell division, 297
Inhibition of root elongation, 294
Kaolinite, 404
Land-farming technique, 339
Lead resistant determinant (pbr), 9
Lewis acid, 407, 454
Low molecular weight organic
acids, 411
Mechanisms of
aluminum tolerance, 312
bark pocket formation, 354
Mercury resistant determinant
(mer), 9
Metal:
accumulation in roots, 107
avoidance, 79
binding compounds, 80
compartmentalization, 83
contaminated niches, 330
cycling, 112
effects on algal communities, 71
immobilization using biomolecules, 26
leaching, 24
polluted soils, 40
resistance/tolerance, 78
rich anthropogenic biotopes, 3
rich biotopes, 2
rich concretions, 105
sensing, 26
486
[Metal]
tolerance, 80
transformations, 83
tolerant plants, 459
interactions vs solid phase composition, 409
Metal fusion proteins (MFPs), 10
Microbial:
communities 329
transformation of OM, 414
Mucilage, 302
Mycorrhizal:
endophytes, 44
fungi, 37, 39, 40, 41, 43, 45
symbiosis, 37, 40
Natural metal-rich biotopes, 2
Natural sinks for metals, 96
Near-isogenic lines, 294
Nitrogen transformation, 418
Organic acids, 303
Outer membrane:
factors, 10
protein, 10
Oxisols, 289
polyaromatic hydrocarbons (PAHs),
25
Pb 210 dating, 363
Periphyton communities, 77
Peroxidation of lipids, 309
pH regulation, 302
Physiology of aluminum toxicity,
299
Phytoextraction, 452
Phytoremediation, 452
Phytostabilization, 452, 459
Phytovolatilization, 455
Plant responses to aluminum stress,
306
Subject Index
Plant uptake of heavy metals, 442

Pollution time capsules, 353, 362
Pollution-induced community tolerance (PICT), 74
Precipitation, 15
Protein based biosensors, 28
Regulation of:
ATPases, 21
enzymatic reduction, 23
metal sequestration process, 23
Remediation:
polluted soils, 444
principles, 445
Removal of heavy metals, 336
Rhizofiltration, 452
RND resistance/nodulation/division, 10
Salt marshes, 95
Sediment chemistry, 101
Selection pressure, 39
Sequestration, 15
Sewage sludge, 51
Short rotation forestry, 333
Sites of metallurgical industries, 4
Soil:
bioremediation, 25
enzymes, 419
microbial biodiversity, 401
quality assessment, 50
Sources of metals in soil environment, 402
Spodosols, 289
Sulfate-reducing bacteria, 4
Suspension-cultured cells, 307
Tagus estuary, 97
Thallium-resistant determinant (tll),
9
Subject Index
Thermal treatment of Hg-contaminated soils, 448

Transport and deposition of trace
metals in the xylem, 381
Tree:
bark, 353
crops, 321
rings 367
487
Ultisols, 289
Vegetation filter, 335
Water use efficiency, 336
Willows and poplar trees, 339
Zinc desert, 41

(Books in Soils, Plants, and The Environment) M.N.V. Prasad-Metals in The Environment - Analysis by Biodiversity (Books in Soils, Plants, and The Environment) - CRC Press (2001) PDF

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Hyperaccumulators can resist drought and can interfere with neighboring

Bryophytes and Pteridophtes

Angiosperms (Asteraceae, Convolvulaceae, Fabaceae and

Aluminum Toxicity in Acid Soils: Plant Responses to

Tree Bark: Tree Bark Pockets as Pollution Time Capsules

Tree Rings and Dendroanalysis

Heavy Metal Interactions in Soils and Implications for Soil

Behavior of Heavy Metals and Their Remediation in

Tadeusz Baszynski Department of Plant Physiology, Maria Curie-Skodowska

Environmental Department, Universidade Atlantica,

Isabel Cacador Institute of Oceanography, University of Lisbon, Lisbon, Portugal

Roberto Gabbrielli Department of Plant Biology, University of Florence, Florence, Italy

Research Institute for Bioresources, Okayama University,

M. Megharaj Remediation of Contaminated Environments Program, CSIRO

Institute of Ecology of the Polish Academy of

M. N. V. Prasad Department of Plant Sciences, University of Hyderabad,

van der Lelie and Tibazarwa

ECOLOGICAL RELEVANCE OF BACTERIAL

Natural Metal-Rich Biotopes

Examples of natural metal-rich biotopes include serpentine (or ultramafic) soils,

Biotopes Rich in Heavy Metals Resulting

A different situation exists in metal-rich anthropogenic biotopes, resulting from

van der Lelie and Tibazarwa

this biotope. The leaching metabolism of this organism can be detrimental

Sites of Metallurgical Industries

van der Lelie and Tibazarwa

HEAVY METALRESISTANT BACTERIA

Bacterial Heavy Metal Resistance Mechanisms

TABLE 1 Examples of Bacterial Resistance Mechanisms to Heavy Metalsa

van der Lelie and Tibazarwa

Three-Component Cation/Proton Antiporter Systems

These systems are members of the major facilitator superfamily of transporters

van der Lelie and Tibazarwa

Three membrane proteins are involved in transporting the divalent cations

Efflux Based on Cation Diffusion Facilitation

This resistance mechanism is based on metal cation efflux by an inner membrane

Heavy Metal P-Type Efflux ATPases

van der Lelie and Tibazarwa

Other Types of Efflux Mechanisms

van der Lelie and Tibazarwa

Other Mechanisms of Heavy Metal Resistance

Complexation, Sequestration, and Precipitation

Sequestration of heavy metals in different compartments of the bacterial cell can

van der Lelie and Tibazarwa

in metal-binding clusters, which are organized at the N and C termini of the

Physiological Role of the Metal Determines the

Regulation of Heavy Metal Resistance

As a general principle, bacterial resistance mechanisms must necessarily take

van der Lelie and Tibazarwa

MerR transcriptional regulators

van der Lelie and Tibazarwa

van der Lelie and Tibazarwa

crystal structure of SmtB was recently solved and found to be a homodimeric

van der Lelie and Tibazarwa

APPLICATIONS BASED ON BACTERIAL INTERACTIONS

Metal leaching is based on metabolic activity of the bacteria in the presence of

Decontamination of Soils and Water

Biological treatment methods for decontamination of metal-polluted soils have

Applications Based on Ralstonia eutropha CH34

van der Lelie and Tibazarwa

which remain in suspension. This biomass can subsequently be removed from

Environmental Monitoring and Metal Sensing: