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ENVIRONMENTAL HEALTH - PHYSICAL, CHEMICAL
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METAL CONTAMINATION
SOURCES, DETECTION AND
ENVIRONMENTAL IMPACT
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ENVIRONMENTAL HEALTH - PHYSICAL, CHEMICAL
AND BIOLOGICAL FACTORS
METAL CONTAMINATION
SOURCES, DETECTION AND
ENVIRONMENTAL IMPACT
SHAO HONG-BO
EDITOR
Nova Science Publishers, Inc.

New York
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Additional color graphics may be available in the e-book version of this book.
Library of Congress Cataloging-in-Publication Data
Metal contamination : sources, detection, and environmental impact / editor, Shao Hong-Bo.
p. cm.
Includes index.
ISBN 978-1-61942-116-5 (eBook)
1. Metals--Environmental aspects. 2. Heavy metals--Environmental aspects. 3. Nonpoint source
pollution. I. Hong-Bo, Shao.
QH545.M45M44 2012
547'.05--dc23
2011043196
Published by Nova Science Publishers, Inc. † New York

CONTENTS
Preface vii
Chapter 1 The Non-Point Pollution of Nitrogen and
Phosphorus in Chinese Agriculture Ecosystem:
Current Status and Progress 1
Gang Xu, Hongbo Shao, Yingchun Lv,
Liye Chu and Junna Sun
Chapter 2 Some Advances in Bio-Removing Hazardous Heavy
Metals from Contaminated Soils 19
Liye Chu, Hongbo Shao, Junna Sun,
Gang Xu, Lihua Zhang and Kun Yan
Chapter 3 Immobilization of Heavy Metals in Soil
by Phosphate Treatment: A Review 43
Silvano Mignardi, Alessia Corami
and Vincenzo Ferrini
Chapter 4 Metal Contaminant Source, Transport and Fate in
the Environment and Phytoremediation Methods 81
S. H. Desouki and H. Feng
Chapter 5 Pollution Sources and Fluxes in
the South-Western Spanish Littoral 95
F. Ruiz, M. L. González-Regalado, J. M. Muñoz,
M. Abad, M. I. Prudêncio, M. I. Dias,
M. I. Carretero, M. Pozo and A. Toscano
vi Contents
Chapter 6 Controlled Organic Treatment System for Acid Mine

Drainage (AMD) and Municipal Waste Water 107
O. Pollmann, N. Pollmann and L. van Rensburg
Chapter 7 Assessment of Trace Element Contamination in
the River and Alluvial Sediments Using a Sequential
Extraction Technique and Statistical Analysis 119
Sanja M. Sakan and Dragana S. Đorđević
Chapter 8 Metal Concentrations in Marine Sediments
Influenced by Anthropogenic Activities in
Bahrain, Arabian Gulf 157
Humood Naser
Chapter 9 Comparative Study in the Bivalve Capacity for
Metal Bioaccumulation in the Balearic Islands
(Spain). Mytilus Galloprovincialis as an Adequate
Sentinel Species for Monitoring Coastal Waters 177
A. Sureda, S. Tejada, A. Box, J. Benedicto
and S. Deudero
Chapter 10 Risk Assessment for the Environment,
Population and Infrastructures of Some Abandoned
Tailing Ponds in the Mining District of
Cartagena-La Union (SE Spain) 195
À. Faz Cano, J. A. Acosta, S. Martínez-Martínez,
P. Martínez-Pagán, D. M. Carmona,
R. Zornoza and S. Kabas
Index 219
PREFACE
This book is written by internationally-famous soil biologists and

environmental scientists who are engaged in the related field of metal
contamination for many years. The book is an excellent book on the process
and environmental factors that influence the specification, availability,
mobility, transfer mechanism and toxicity of metals in the ecosystem. The case
studies of different ecosystems contribute to better understanding of metal and
pollutant sources cycling and will be useful to those planning for restoring
contaminated lands. The publication will also serve as a valuable reference
textbook to soil and agricultural chemists, environmental scientists and
ecologists and students who are interested in environmental sciences and
affairs.
Chapter 1 - The eutrophication of major lakes and water systems has been
getting worse and worse due to excessive inputs of nitrogen (N) and
phosphorus (P) in Chinese farmland. It is suggested that non-point source
(NPS) pollution from agricultural is the leading source of water pollution.
Based on a number of literatures, the paper reviewed the current
eutrophication status of major water bodies and the possible sources affected
water quality in China. The authors also provided the monitoring and the
control techniques of agricultural NPS pollution. In the end, several hot spots
of non-point pollution studies are introduced and the studies and managements
in China are proposed.
Chapter 2 - Mechanisms of four methods for removing hazardous heavy
metal are detailed and compared-chemical/physical remediation, animal
remediation, phytoremediation and microremediation with emphasis on bio-
removal aspects. The latter two, namely the use of plants and microbes, are
preferred because of their cost-effectiveness, environmental friendliness and
viii Shao Hong-Bo
fewer side effects. Also the obvious disadvantages of other alternatives are
listed. In the future the application of genetic engineering or cell engineering
to create an expected and ideal species would become popular and necessary.
However, a concomitant and latent danger of genetic pollution is realized by a
few persons. To cope with this potential harm, several suggestions are put
forward including choosing self-pollinated plants, creating infertile polyploid
species and carefully selecting easy-controlled microbe species. Bravely, the
authors point out that current investigation of noncrop hyperaccumulators is of
little significance in application. Pragmatic development in the future should
be crop hyperaccumulators (newly termed as “cropaccumulators”) by
transgenic or symbiotic approach. Considering no effective plan has been put
forward by others about concrete steps of applying a hyperaccumulator to
practice, the authors bring forward a set of universal procedures, which is
novel, tentative and adaptive to evaluate hyperaccumulators’ feasibility before
large-scale commercialization.
Chapter 3 - Heavy metal contamination of soil is an environmental and
public health issue of prime interest because metals are non-biodegradable and
accumulate throughout the food chain. Conventional heavy metal remediation
technologies include chemical precipitation, membrane filtration, ion
exchange, electrochemical treatment, etc. Recently adsorption has become an
alternative treatment method of contaminated media and a great effort has
been devoted to develop new adsorbents.
Phosphate treatment is widely considered an effective in situ treatment to
reduce metal mobility and bioavailability in soils through the formation of
highly insoluble phosphates which are stable in a wide range of environmental
conditions. The mechanisms of metal retention are still debated and include:
ion exchange, dissolution/precipitation and surface complexation. Various
types of phosphate amendments, such as synthetic and natural apatites,
phosphate rocks, bone meal and bone char have been tested. Phosphate
treatment has been shown to be effective in stabilizing Pb, Cd, Cu, Zn, Co, Ni,
Sb, Cr, V in contaminated soils. In soils phosphate stabilization has been
applied mainly to remediate Pb contamination although the method can be
used to reduce also the mobility and bioavailability of other metals. The main
factors determining the removal capacity of phosphate treatment in
contaminated soils include type of phosphate source due to their different
solubilities, rate of phosphate application, heavy metals speciation, soil pH and
Eh and grain size of the amendment. The adverse effects of phosphate
stabilization resulting in enhanced leaching of phosphate and availability in the
Preface ix
soil of contaminants, such as As, Se and W, have to be addressed for

evaluating the environmental impact of phosphate treatment.
Chapter 4 - Heavy metal pollution due to anthropogenic sources is on the
rise causing great environmental and human health problems. Although
remediation of metal contaminants can be via excavation or stabilization of the
soil or sediments, these methods can be quite costly in the long run and it is
not efficient for the small-scale removal of heavy metal contaminants.
Phytoremediation is a fairly new and “green” technology, and with a focus on
heavy metals as the contaminants in question, this method utilizes plants for
in-situ reduction and/or removal of contaminants from soil, sediments and
water. The process of phytoremediation is quite simple, and with the proper
selection of plants and appropriate site conditions, the risk from these
contaminants can be reduced and minimized over time. Phytoremediation is
energy efficient, and an aesthetically pleasing method of remediating sites
with low to moderate levels of contamination. The purpose of this paper is to
provide a general review of metal contaminant source, transport and fate as
well as current phytoremediation methods including their advantages and
disadvantages, and the future directions in research using these methods.
Chapter 5 - This paper analyzes the heavy metal distribution (As, Hg and
Ni) in shallow marine sediments of southwestern Spain. The end of the Huelva
bank is the most polluted area of this littoral (Igeo > 3 for As; Hg > 1 mg kg-1),
owing to historical acid mine processes and recent industrial sewages derived
from the catchment areas of the Tinto-Odiel rivers. The W-E littoral drift
currents distribute this pollution towards the east, mainly in very shallow areas
(< 10 m water depth). In addition, pathways of pollutants transported by the
Guadiana river to the inner shelf are described, with a ‘diffuse’ pollution in the
deepest areas studied (10-18 water depth) between Isla Cristina and the
Piedras river mouth.
Chapter 6 - Most landscapes in South Africa are affected by the process of
mining. The environmental results are large amounts of heavy metals and a
low pH in the soil, caused by acid mine drainage (AMD) and in most of the
irrigation water. Additionally, the quantity and quality of water is – like in
most African and developing countries – limited and mostly too scarce for
economical processing to drinking water quality. Mining processes and the
weather conditions affect the whole agricultural sector in terms of water
quantity and water and soil quality.
For this reason, an organic medium was tested to prove of its function
purifying water resulting from mining processes. All results showed an
increase in pH from pH 3.0 of the AMD to more than pH 5.5 and a reduction
x Shao Hong-Bo
in heavy metals of about 80%. Acid mine drainage normally has to be treated
with chemicals in order to get an acceptable quality to release the water into
wetlands. The organic treated AMD has the quality of irrigation water and
could be used for irrigation in agriculture without any negative site effects on
the existing soil or water over the long term. The same organic medium was
used to optimize the efficacy of municipal waste water treatment as a pre-
treatment. More than 30% of the full treatment was done by the organic
medium with the effect of decreasing heavy metals and minimizing
cytotoxicity and endocrine disruptors. In controlled samples the cytotoxicity,
caused by specific endocrine disruptors could be significantly reduced by the
treatment with the organic treatment system.
Chapter 7 - This study presents the results of determination Cu, Cr, Zn,
Pb, Cd, V, Ni, As and Hg content and assessment of their anthropogenic
origin, mobility and contamination level in the river sediment (Tisa River and
tributaries) and alluvial sediments (Danube alluvial formation, Pančevo). In
order to assess mobility and dominate substrates of examined elements, in this
paper was applied sequential extraction method and analysis of the content of
Ca, Mg, Fe, Mn, Ti, K as well as determination of N, C, H and S content using
elemental analysis. The results obtained by sequential extraction showed a
different mobility and dominant substrate of elements in the Tisa sediments
and alluvial sediments in the Pančevo. There was shown a high mobility of
Cu, Zn, Pb and Cd in the Tisa sediments, which may be an indicator of the
existence significant anthropogenic sources of these elements. The amounts of
the studied elements extracted in the residual fraction were much higher than
those in the non-residual fractions in the Pančevo sediment, except for Cd and
V, indicating that they were mainly from crystalline lattices. Chromium and
nickel were dominantly present in the residual fraction. Vanadium was
extracted in the more mobile fractions from Pančevo sediment, which may
indicate to the existence of contamination with vanadium.
Correlation analysis was performed on total extractable contents of
elements and contents of N, C, H and S in the sediments. The results showed
that manganese oxides and alumosilicates are of great importance for the
binding of Cu, Zn, Cr, Pb and Cd in the Tisa sediment. Vanadium has a
different behaviour, origin and substrate compared to Cu, Cr and Zn. Hg was
positively correlated with Zn and Cd. The correlation of Fe with Ni is a
consequence of their siderofile nature and suggests a common origin of these
elements. Positive correlation between As and Cr, Zn and Pb is due to their
similar geochemical characteristic and binding with clay minerals.
Preface xi
Results of correlation analysis of element content in the sediment Pančevo

show a similar geochemical origin of Cu, Cr , Zn, Ni and V in the studied
sediments. These elements are bounded to alumosilicates, as well as oxides of
Fe and Mn. There was shown that organic matter is important for the binding
of Zn and Cd. Positive correlation of C with Ca, As and Hg is the result of the
carbonate nature of those elements in the investigated sediments. S is
positively correlated with Hg, which indicates the presence of Hg in the form
of HgS in the studied sediments.
Based on distribution of extracted elements content by localities and
comparison obtained results with Quality Guidelines for sediments, there was
observed the increased contents of some trace elements on investigated
localities.
Chapter 8 - The Arabian Gulf is considered among the highest
anthropogenically impacted regions in the world. Heavy metals are considered
a major anthropogenic contaminant of coastal and marine environments
leading to degradation of natural habitats and posing threats to human health.
Concentrations of cadmium, copper, lead and zinc in marine sediments
influenced by industrial and sewage effluents, discharges from major power
and desalination plants, and reclamation and coastal modification in Bahrain
were analyzed using voltammetry technique. The overall mean values in
sediments for Cd, Cu, Pb and Zn were 1.37, 14.67, 17.43 and 40.99 mg kg-1,
respectively. Elevated concentrations of Zn and Cu were detected in areas
influenced by brine wastewater discharges, and navigational activities. Higher
concentrations of Cd and Pb that attributed to petroleum industries and
effluents from variety of factories and industrial facilities in that area were
observed off the eastern coastline of Bahrain. Despite a localized increase in
Cd, heavy metal concentrations in Bahrain are within the range of variations
that are reported in sediments from the Arabian Gulf.
Chapter 9 - Metals are major pollutants in the industrial world causing
long-term effects on marine ecosystems. Bivalves are widely used as sentinel
species for monitoring coastal environments because they have a large
capacity to accumulate pollutants in their tissues to a degree that is suitable for
measurement. Our aim was to compare the metal bioaccumulation capacity of
four different bivalves –Mytilus galloprovincialis, Venus verrucosa,
Lithophaga litophaga, Chamalea gallina- and the echinoderm Paracentrotus
lividus. Samples were collected from aquaculture farms and Pb, Cd, Cu and
Hg were determined in soft tissues. All bivalves presented higher levels of
metals than P. lividus indicating that they are good markers of
bioaccumulation. The mussel M. galloprovincialis was the bivalve that
xii Shao Hong-Bo
accumulated more quantity of metals. As consequence, a second study was

performed with caged mussels from four areas around Mallorca Island
(Alcudia, Porto Colom, Santa Ponsa and Sóller) to validate the use of the
caged mussels as biomarker of metal contamination. Metal concentration was
determined in the whole mussel and antioxidant enzymes (catalase, superoxide
dismutase and glutathione peroxidase) were determined in the digestive gland.
Although there were not significant differences between the selected stations
in antioxidant enzyme activities, a direct correlation between metal
concentration and antioxidant enzymes were evidenced. These results indicate
that mussels presented increased stressful states in the areas with the highest
metal levels. M. galloprovincialis was the species with the highest metal
concentration and this characteristic together with its easy collection and
manipulation indicates that mussels are useful biomarkers of metal
contamination in coastal waters.
Chapter 10 - The mining district of Cartagena-La Unión (South-East of
Spain) has been among the most important mining centers in Spain in
obtaining lead, silver and zinc. For more than two thousand years, different
civilizations exploited mineral resources in this area. More than eighty
structures of mining wastes (tailing ponds) coming from old mineral washing
facilities were built, being currently one of the main sources of pollution. The
goal of this chapter is to provide a geophysical and geochemical assessment of
eleven tailing ponds selected in the mining district of Cartagena-La Unión,
giving a description of the risks for the environment, population and close
infrastructures. To achieve this objective, surface soil samples were taken in
these tailing ponds where pH, texture, electrical conductivity, and total,
bioavailable and water soluble heavy metals were obtained. Geophysics was
based on laying out a series of profiles in which physical parameters of the
materials in depth were obtained. The equipment used provides 2D and/or 3D
sections of electrical resistivity changes (Ohm/m).
The geochemical and geophysical studies showed that only “Brunita”
tailing pond was structurally instable with a volume of 1000000 m3. The pH
values showed that the most acidic tailing ponds were “Descargador” (< 3.5),
“Regente” (< 3.5) and “Brunita” (3.5-4.4); oppositely, “Encontrada C. Ponce”
was the most alkaline. Regarding electrical conductivity, most ponds showed a
low salinity. Brunita was the tailing pond with highest content of salts (> 16
dS m-1). The predominant textures in the selected tailing ponds were coarse.
All tailing ponds had high total concentrations of Pb (maximum values: 21308
mg kg-1 in “ El Lirio” and 21272 mg kg-1 in “Encontrada- C. Ponce”), Zn
(maximum values: 23575 mg kg-1 in “El Lirio” and 20971 mg kg-1 in “Cabezo
Preface xiii
Rajao-La Unión”) and Cu (maximum values:1823 mg kg-1 in “Peña del

Águila-La Unión” and 1470 mg kg-1 in “Cabezo Rajao-La Unión”) which are
above the limit values for all legislation used in this study.
In general trends, the highest fractions of bioavailable and water soluble
metals were found in tailing ponds with ultra acidic conditions (pH<3.5), such
as “Regente” with 72%, 4%, 17% and 14% of bioavailable Cd, Cu, Pb and Zn,
respectively; and with 2.7%, 1.4%; 0.2% and 24% of water soluble Cd, Cu, Pb
and Zn, respectively. Therefore, significant risks can arise in these tailing
ponds, because the soil particles can reach natural or populated areas by
erosion or collapse by structural instability, adversely affecting soil, surface
and underground waters, plants, animals and human populations as well as
infrastructures close to these tailing ponds. It is important to highlight that in
the acidic tailing ponds the problems of mobility of heavy metals are increased
and so, higher environmental risks may exist. For the reasons above,
rehabilitation plans should be carried out in these tailing ponds.
In: Metal Contamination ISBN: 978-1-61942-111-0
Editor: Shao Hong-Bo © 2012 Nova Science Publishers, Inc.
Chapter 1
THE NON-POINT POLLUTION OF

NITROGEN AND PHOSPHORUS IN
CHINESE AGRICULTURE ECOSYSTEM:
CURRENT STATUS AND PROGRESS
Gang Xu 1, Hongbo Shao.1,2* ,Yingchun Lv1,

Liye Chu2 and Junna Sun1,3
1
The CAS/Shandong Provincial Key Laboratory of Coastal Environmental
Process, Yantai Institute of Costal Zone Research, Chinese Academy of
Sciences(CAS), Yantai 264003,China1
2
Institute for Life Sciences, Qingdao University of Science & Technology
(QUST), Qingdao 266042, China2
3
Graduate University of the Chinese Academy of Sciences (CAS), Beijing
100049, China3
ABSTRACT
The eutrophication of major lakes and water systems has been
getting worse and worse due to excessive inputs of nitrogen (N) and
phosphorus (P) in Chinese farmland. It is suggested that non-point source

Authors for Correspondence: shaohongbochu@126.com (H.B. Shao), Tel.:+86-532-84023984,
Posting address: Institute for Life Sciences, Qingdao University of Science & Technology
(QUST), Qingdao 266042, China.
2 Gang Xu, Hongbo Shao, Yingchun Lv et al.
(NPS) pollution from agricultural is the leading source of water pollution.

Based on a number of literatures, the paper reviewed the current
eutrophication status of major water bodies and the possible sources
affected water quality in China. We also provided the monitoring and the
control techniques of agricultural NPS pollution. In the end, several hot
spots of non-point pollution studies are introduced and the studies and
managements in China are proposed.
Keywords: eutrophication; non-point source pollution; farmland
1. INTRODUCTION
Non-point source (NPS) pollution, unlike pollution from industrial and
sewage treatment plants, comes from many diffuse sources. NPS pollution
occurs when rain or snowmelt moving over and through the ground. As the
runoff moves, it picks up and carries away natural and human-made pollutants,
finally depositing them into lakes, rivers, wetlands, coastal waters, and even
our underground sources of drinking water (Carpenter et al. 1998（EPA，
2004）.
In recent years, point source pollution has been significantly reduced by
implementing pollution control standards, regulatory enforcement and capital
investment and management in our industrial and municipal infrastructure.
With the decreasing of point source pollution, more and more attention has
been drawn to the NPS pollution (Tufford et al. 1998; Haygarth et al. 1999;
Arheimer et al. 2000; Huang et al. 2003; Nie et al. 2009). In many countries,
especially in the developed countries, NPS pollution becomes the leading
source affected water quality. It was estimated that NPS contributed
approximately 60-80% of our surveyed streams and lakes in China which are
not clean enough to meet basic uses such as fishing or swimming (Chen et al.
2003). In USA, 57% of the lakes were polluted by excessive inputs of
nutrients from agriculture (Carpenter et al. 1998). In Netherlands about 60% of
the total nitrogen load of surface waters originates from agricultural land
(UlÉN et al. 2007). In the north western European countries, amounts
phosphorus loss from agricultural can contribute 40–88% of total phosphorus
(TP) transfer. TP export in small agricultural streams is generally in the range
0.3–6 kg·ha-1·year-1, with the highest losses in Norway and UK (UlÉN et al.
2007). In an increasing number of regions, the potential risk of nutrient loss in
runoff has been increased by the continued application of fertilizer and manure
The Non-Point Pollution of Nitrogen and Phosphorus … 3
from intensive livestock operation because the large amount of nutrients has
created a nitrogen and phosphorus imbalance (Alexander et al. 2007). The
surplus of nitrogen and phosphorus become the potential threat to surface
waters. It is now well recognized that the eutrophication induced by excessive
input with P and N is a widespread problem in rivers, lakes, estuaries, and
coastal oceans all round the world. The non-point pollution especially
agricultural source is a major source of P and N to surface waters all over the
world (Ongley et al. 2010).
The non-point source pollution (NPS) has been very well studied,
especially in North America and in Europe. The evaluations of NPS pollution
in China began in the early 1980s with the study of urban runoff pollution in
Beijing and in some large polluted lakes such as Dianchi and Taihu lakes
(Ongley et al. 2010). Subsequently there has been a number of scattered
evidence for the importance of NPS to the water quality (Jin 2003; Xiangcan
2003; Ju et al. 2004; Jin et al. 2005). However, there is little information about
systematic appraisals of NPS impacts in China. In 2004, the Asian
Development Bank (ADB, 2004) released its final report of rural NPS
pollution in China. The report proposed many difficulties in making a
comprehensive NPS assessment in China. As a result, we provide a brief
overview of the current eutrophication status of major water bodies, the
possible sources affected water quality, and some policies and research gaps
need to be fully understood in China in this paper.
2. THE N AND P POLLUTION IN MAJOR WATER SYSTEM

IN CHINA
According to a state environmental survey (table 1), the seven major river
basins were under intermediate pollution in China. The rivers in Zhejiang
Province and Fujian Province were under slight pollution, while rivers in
northwest China enjoyed excellent water quality. The water quality of the
rivers in southwest China was good. The overall environmental quality of sea
water is good, 94% of sea area meet Grade I standard. However, there are
48,000 Km2 of coastal sea water quality worse than Class IV. The leading
pollutants include TN and TP mainly originated from urban runoff and
agriculture. But there was striking eutrophication problem in lakes
(reservoirs). Among 28 key lakes (reservoir) under national monitoring
program, 4 met Grade II quality standard, accounting for 14.3%; 2 met Grade
III standard, taking up 7.1%; 6 met Grade IV standard, accounting for 21.4%;
5 met Grade V standard, taking up 17.9%; 11 failed to meet Grade V standard,
taking up 39.3%(China 2008 ). The main pollutants were TN and TP. Among
these lakes (reservoir) under eutrophication monitoring, 1 was under heavy
eutrophication, taking up 3.8%; 5 were under intermediate eutrophication,
taking up 19.2%; 6 were under slight eutrophication(SE), accounting for
23.0%(China 2008 ). These data suggested that the eutrophication is a
widespread and main problem in river, lakes, estuaries and costal oceans,
caused by the pollution from N and P.
The trend of eutrophication of major Chinese lakes and water systems has
been getting worse and worse since 1970s. In the early 1970s, 91.8% of the
total area of the most important lakes was found to be in the mesotrophic state.
However, from 1978 to 1987 the proportion of oligotrophic lake area
decreased from 3.2 to 0.53%, while that of eutrophic lake area increased
massively from 5.0 to 55.1%.1 Then, through the 1990s, most of these urban
lakes faced serious eutrophication problems, with many medium-sized lakes
becoming mesotrophic or eutrophic in status. Some waters even approached
the hypereutrophic level (Tang et al. 2008).
The widespread and rapidly expanding eutrophication significantly
increased the growth of algae and aquatic weeds that interfere with use of the
water for fisheries, recreation, industry, agriculture and drinking (Huang et al.
2003; Jin et al. 2005). Eutrophication has become one of the most important
factors in the delay in Chinese economic growth, at the same time actually
threatening China desired sustainable development goals (Liu et al. 2007). The
overgrowth of algae and other aquatic plants in eutrophic lakes increases
turbidity, reduces transparence, exhausts dissolved oxygen, deteriorates water
quality and, finally, collapses the aqueous ecosystem (Jin 2003).
Eutrophication has been inflicting tremendous damage on water supplies,
fisheries, transportation and even tourism, causing huge economic losses. It is
difficult to estimate the total economic loss due to eutrophication in China
(Chen et al. 2008).
The eutrophication is mainly caused by excessive inputs of N and P. Table
2 show the changes of TN and TP concentration in three lakes in China during
the past 30 years (Zhang WL et al. 2004; Jin et al. 2005). From the data, we
can see that the mean concentrations of TN and TP had increased dramatically
since the previous survey at this site in 1980s. TN and TP concentrations
increased from 1.18mg/L to 1.98mg/L, and from 0.097 to 0.28 mg/L
respectively in Dianchi Lake while in Taihu Lake TN and TP approximately
Table 1. Changes of water quality in major water systems in China (Zhang WL et al. 2004)
Water system 1960s 1970s 1980s 1990s Current Main pollutants

Dianchi Lake(Caohai) Ⅱ* Ⅲ V Poor than V Poor than V TN, TP
Dianchi Lake(Waihai) Ⅱ Ⅲ Ⅳ V Poor than V TN, TP
Taihu Lake Ⅰ～Ⅱ Ⅱ Ⅲ V Poor than V TN, TP
Chaohu Lake Ⅰ Ⅲ Ⅳ V Poor than V TN, TP
Middle to high level
Hongzehu Lake Quite good Eutrophication TN, TP
eutrophication
Dongtinghu Lake Quite good Poor to middle level eutrophication Eutrophication TN, TP
Poyanghu Lake Quite good Middle level eutrophication Eutrophication TN, TP
Yangtse River Dam Area Quite good - Eutrophication TN, TP
(*I～V indicated the water quality form quite good to poor. More detailed description found in “Report on the State of the Environment
of China,2008”)
Table 2. Change of TN and TP concentration in major watersheds in China
TN and TP in watershed N and P2O5 loads from agriculture

Watershed Period -1 -1
TN(mg L ) TP(mg L ) N(kg ha-1) P2O5(kg ha-1)
1980s 1.18 0.097 183 101
Dianchi Lake
Present 1.98 0.28 523 370
1980s 1.11 0.052 367 118
Taihu Lake
Present 2.54 0.1 462 130
1980s 1.43 0.088 130 52
Chaohu Lake
Present 3.19 0.19 384 198
Gang Xu, Hongbo Shao, Yingchun Lv et al. 7
doubled from 1.11mg/L to 2.54 mg/L and from 0.052mg/L to 0.1mg/L,

respectively. The Chaohu Lake also witnessed almost double increase of TN and
TP concentration from 1.43mg/L to 3.19 mg/L and from 0.088mg/L to 0.19mg/L,
respectively. From table 2, we also can see that the N and P2O5 loads from
agriculture had significantly increased with the increase of TN and TP in the
watershed during the past 30 years. The comparison indicated that the increased
concentrations of TN and TP in three lakes likely reflect the impact of extensive
agricultural inputs of N and P (Zhang WL et al. 2004).
70
60 industrial waste
Contribution(%)
domestic sewage
50 agriculture
40
30
20
10
0
Taihu lake Dianchi lake Chaohu lake
TN
80
70 industrial waste
Contribution(%)
60 domestic sewage
50 agriculture
40
30
20
10
0
Taihu lake Dianchi lake Chaohu lake
TP
Figure.1. The sources of TN and TP in three lakes in China.
Based on the survey on the main sources of excessive N and P in three lakes
(Taihu, Dianchi, Chaohu), sources of N and P mainly come from domestic sewage
and agricultural runoff, industrial wastewater (Figure 1). The results show that the
contribution of TN, TP from industrial inputs ranged from 10% to 16%. On the
contrary, the agricultural runoff (range: 30%-73%) and domestic sewage
(range:17%-60%) were more important than industrial waste to TN and TP loads
in three lakes (Figure 1). The contribution of domestic sewage to TN followed the
order of Dianchi Lake (57%)>Taihu Lake (25%)>Chaohu Lake (23%) while to
TP they were in the order of Taihu Lake (60%)>Dianchi Lake (45%)> Chaohu
Lake (17%). In the three lakes water quality inventory, it was reported that
agricultural pollution was the leading source of water quality impacts on surveyed
lakes because they accounted for 63%, 59% and 33% to TN in Chaohu Lake,
Taihu Lake and Dianchi Lake respectively. Agricultural pollution make up on the
average 73%, 41%,30% to the TP in Chaohu Lake, Dianchi Lake and Taihu Lake,
respectively (Zhang WL et al. 2004; Nie et al. 2009).
3. THE MAIN CAUSES OF NPP FROM AGRICULTURE IN

CHINA
NPP of N and P is caused primarily by agricultural and domestic sewage in
China. In the NPP categories, it was reported that agricultural NPS pollution was
the leading source of water quality impacts on surveyed rivers and lakes, the
second largest source of impairments to wetlands, and a major contributor to
contamination of surveyed estuaries and ground water. Agricultural activities that
cause NPS pollution include improper, excessive or poorly timed application of
fertilizer, poorly located or managed animal feeding operations, overgrazing;
plowing too often or at the wrong time, and irrigation water and rainwater
overflow.
3.1 Fertilizer in Farmland
Nutrients such as phosphorus, nitrogen, and potassium in the form of

chemical fertilizers, manure, and sludge were used to grow legumes and leave
crop residues to enhance production. When these sources exceed plant needs, or
are applied just before it rains, nutrients can wash into aquatic ecosystems (EPA,
2004). China agriculture total fertilizer application has increased steeply form
728,000 tons in the 1960s to 39,605,000 tons in the second year of the century
(Figure 2). Great amount application of fertilizer in the farmland has resulted in
the accumulation of the excessive nutrient in Chinese agricultural soils.
Figure.2. Indicators of NPP from Agriculture, China (FAOSTAT).
3.1.1 N Application
Excessive input of N fertilization and decreasing N recovery rates by crops
have caused dramatic increases in NPS pollution from agriculture in China.
China’s N fertilizer consumption became the highest in the world in 1985 and
increased strongly in the 1990s. In 2000, the total amount of N applied was more
than 24 Mt of pure N (compound fertilizer calculated on the basis of 30% N), or
about 30% of the world’s total consumption. Assuming 130 Mha of cultivated
area and the average multiple cropping index of 1.20, the mean N application rate
per crop was 155 kg N ha-1, compared to around 60 kg N ha-1 on a world average
(Ju et al. 2004; Nie et al. 2009).
There is no doubt that applying a large amount of N fertilizer resulted in N
accumulation in Chinese soil profiles. Chinese N balance surplus in cropland
began in 1979 and increased annually. In 1998, the estimated N balance surpluses
in Chinese croplands reached 6.22 Mt N. Most of the surplus N is retained in the
soil in different forms. The proportion of residual fertilizer N in soil is usually
about 15–30% after harvest of the first crop in 15N-field experiments. According
to our research, ammonium-based N fertilizer and urea applied to soil is nitrified
within 1 or 2 weeks in semiarid farming systems in the North China Plain. The
fertilizer N accumulated in soil after harvest is mainly present in the form of
nitrate. As a result, nitrate leaching is the main cause of water pollution in high
rainfall/irrigation regions. The increasing eutrophication of water bodies and the
frequent occurrence of offshore red tides in the East China Sea both demonstrate
that large N fertilizer inputs are having negative effects on the environment(Ju et
al. 2004).
Table 3. Changes of N and P loads from different sources to arable land in main watersheds in China since 1960s
1960s 1980s Present

Source -1 -1 -1 -1
N(kg ha ) P2O5(kg ha ) N(kg ha ) P2O5(kg ha ) N(kg ha-1) P2O5(kg ha-1)
Fertilizer 5 1 135 22 368 154
Animal husbandry 19 11 101 56 128 74
Rural life 29 8 49 13 56 15
Total load 53 20 285 91 552 243
Fertilizer: Animal
1:4:5 1:5:4 5:4:2 2:6:2 7:2:1 6:3:1
husbandry: Rural life
Gang Xu, Hongbo Shao, Yingchun Lv et al. 11
3.1.2 P Application
Phosphorus (P) is an essential nutrient for crop production and is often in
short supply. China became the largest consumer (30% of the total of the world in
2002) and the second largest producer of P fertilizers. According FAO data, there
is 9% of world arable land belongs to China, while 35% of world P fertilizer was
applied in Chinese farmland (Zhang et al. 2008). Since 1980s, the critical level of
soil available phosphorus was significantly increased due to the large application
of P in farmland (Figure 3).
90
80
Proportion of farmland
1980
70
2006
60
50
40
30
20
10
0
<10 10～20 20～30 30～40 >40
Soil availble P(Olsen-P)(mg/kg)
Figure.3. Change of soil available P in Chinese farmland from 1980 to 2006.
As is shown in table 4, the remained phosphorus in Chinese farmland has

increased by 11% every year since 1980(Chen et al. 2008). During the past twenty
year (1980-1999), the remained phosphorus has enhanced by 7 times, and
increased from 4.8 kg P/ha-2 in 1980 to 37.7 kg P/ha-2 in 1999. Up to now, the
average phosphorus concentration in Chinese farmland exhibited one fold higher
than that in European farmland (Withers et al. 2001). The accumulation of soil P
in high yield vegetable planting regions in China resulted in the degradation of
water environment and eventually threats the human health in China. We must
pay much attention to the increased risk followed by high accumulation of soil P.
As a result, the mean soil available P (Olsen-P) in Chinese farmland has increased
significantly from 7.4mg·kg-1 in 1980 to 20.7 mg·kg-1 in 2006 (Lu 2003).
Table 4. The phosphorus added and remained in Chinese farmland
P added(P2O5)
Year P remained(P2O5)/kg ha-2
total/t average/kg ha-2
1980 1366000 11 4.8

1985 2006000 16.2 7
1990 3769000 30.4 13.2
1995 7470000 60.2 21.2
1999 10752000 86.7 37.7
3.2 Animal Feeding Operation
By confining animals in small areas or lots, farmers and ranchers can

efficiently feed and maintain livestock. But these confined areas become major
sources of animal waste. An estimated 238,000 working farms and ranches in the
United States are considered animal feeding operations, generating about 500
million tons of manure each year (EPA, 2005). When water flow through some
poorly managed facilities, they can pick up pathogens such as bacteria and
viruses, nutrients, and oxygen-demanding organics and solids that contaminate
water quality even the ground water environment. There is consensus in the
literature that the greatest agricultural impact on water quality in China has been
intensive animal (cattle, dairy cows, swine,egg and meat chickens) raising. Asian
development bank states that the number of animal units in operations of <50
animals is 76% of all animal units in China and leads to their conclusion that it is
the approximately one million intensive livestock operations that are the main
concern for water quality in China (Ongley 2004).
In its 2001 report, the World Bank made some rough calculations of COD
loadings from pig production. Their calculations suggest that pig wastes in the
three main pig producing areas of China amounted to 2.6 million tons (Mt) in
1996 and will grow to 8.2 Mt by 2010, representing a growth of 28 percent in
1996 to 90 percent in 2010 of urban-industrial COD loads. Clearly, such numbers,
even if only crude estimates, indicate the urgent need for a NPS monitoring and
control strategy. There is very little information on other types of NPSs such as
urban runoff which must be very large in China, especially given the poor state of
sewage infrastructure in many cities.
4. FUTURE WORKS ABOUT AGRICULTURAL NPP IN CHINA

The current trophic status caused by agricultural NPS pollutions in Chinese
water bodies was far more serious than that in developed countries. China is a
traditional agricultural country. 70% of the people lived in rural region and
depended on agriculture for life. The treatment of NPS in China is complicated by
the combination of income of farmers, infrastructure construction, and agricultural
policy. There are some fundamental mechanisms or policies should be fully
understood (Reginato et al. 2004).
4.1 The Runoff Mechanism of NPS from Chinese Agriculture
In order to reduce NPS from Chinese agriculture, much more detailed

research works to understand main reasons and generating mechanisms should be
carried out in watersheds. It is important to understand the process that will
produce runoff both in water scarce regions and typical field agriculture of south
China.
4.2 Rapid Assessment Techniques
In order to predict and estimated the accumulation of NPS in soil, and its
subsequent leaching and downward movement in the soil profile or water and the
possible environmental effects, it is important to develop models, for which long-
term information obtained under field conditions(Zhang 2010). The information
can be used to estimate the potential risk of NPS leaching and contamination of
adjacent water, to prepare guidelines, recommendations and policies to restrict the
over-use of N fertilizer and manure, and the judicious use of fertilizers and other
management practices (Pilati et al. 2009; Lee et al. 2010). There is also a need to
develop effective government programs to transfer these new
recommendations/technologies to farmers and industry through education,
presentations and the control of fertilizer distribution (Han et al. 2010).
4.3 Best Agriculture Management
As is shown in Figure 3, larger amount of fertilizer accumulated in Chinese

farmland since 1980s. In China, the scattered agriculture unit made it difficult to
take out such a best agriculture management. The main problem remain in the
imbalance of ratio of N:P:K among vegetations and regions, the imbalance among
farmers. Even in similar natural conditions, the chemical fertilizer application is
different from each other (Olarewaju et al. 2009). In the past 20 years, this inter-
regional fertilizer rates become more and more larger. In order to balance the
agricultural production and the environmental pollution, best agriculture
management should be provided (UlÉN et al. 2007). This kind of policy should be
based on local climate, hydrological, geological, topological, soil and agricultural
production conditions, water protection zones of different grades. Best agriculture
management should include source control policies and technologies for reducing
pollutants resulting from agricultural activities, cropping systems, fruit tree and
flower crops with highly nitrogen and phosphorus application rates (Alexander et
al. 2007). The continuing increase of fertilizer use and animal breeding in rural
area of important watersheds makes it even more difficult in future to restore
already heavily eutrophic aquatic ecosystems. To solve the problems, it was
suggested that the proper regulations to confine environment-unfriendly
agricultural activities should be established and implemented in different
watersheds as soon as possible.
ACKNOWLEDGMENTS
Our laboratory and team group are currently supported by the National
Natural Science Foundation of China (No.41001137;41171216) and One
Hundred-Talent Plan of CAS, Yantai Science and Technology Development
Project (2010245; 2011016; 31100313), the CAS/SAFEA International
Partnership Program for Creative Research Teams, the Strategic Priority Research
Program of the Chinese Academy of Sciences (CAS)(XDA01020304), the
Science and Technology Development Plan of Shandong Province
(2010GSF10208) and Yantai Double-hundred Talent Plan (XY-003-02).
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Sheet.pdf
Chapter 2
SOME ADVANCES IN BIO-REMOVING

HAZARDOUS HEAVY METALS FROM
CONTAMINATED SOILS
Liye Chu*1,2, Hongbo Shao1,2*, Junna Sun2,3,

Gang Xu2, Lihua Zhang2 and Kun Yan2
1
Institute for Life Sciences, Qingdao University of Science and
Technology (QUST), Qingdao 266042, China
2
The CAS / Shandong Provincial Key Laboratory of Coastal
Environmental Process,Yantai Institute of Coastal Zone Research,
Chinese Academy of Sciences (CAS), Yantai 264003, China
3
Graduate University of Chinese Academy of Sciences(CAS),
Beijing 100049, P. R. China
ABSTRACT
Mechanisms of four methods for removing hazardous heavy metal
are detailed and compared-chemical/physical remediation, animal
remediation, phytoremediation and microremediation with emphasis on
bio-removal aspects. The latter two, namely the use of plants and
*
Authors for Correspondence: shaohongbochu@126.com (Shao H.-B.) Tel.:+86-532-84023984,
Posting address: Dr. Professor Shao Hong-Bo, Yantai Institute of Coastal Zone Research,
Chinese Academy of Sciences (CAS), Chunhui Rd.17,Yantai 264003, China; Institute for
Life Sciences, Qingdao University of Science and Technology (QUST),Zhengzhou Rd.53,
Qingdao 266042, China.
20 Liye Chu, Hongbo Shao, Junna Sun et al.
microbes, are preferred because of their cost-effectiveness, environmental

friendliness and fewer side effects. Also the obvious disadvantages of
other alternatives are listed. In the future the application of genetic
engineering or cell engineering to create an expected and ideal species
would become popular and necessary. However, a concomitant and latent
danger of genetic pollution is realized by a few persons. To cope with this
potential harm, several suggestions are put forward including choosing
self-pollinated plants, creating infertile polyploid species and carefully
selecting easy-controlled microbe species. Bravely, the authors point out
that current investigation of noncrop hyperaccumulators is of little
significance in application. Pragmatic development in the future should
be crop hyperaccumulators (newly termed as “cropaccumulators”) by
transgenic or symbiotic approach. Considering no effective plan has been
put forward by others about concrete steps of applying a
hyperaccumulator to practice, the authors bring forward a set of universal
procedures, which is novel, tentative and adaptive to evaluate
hyperaccumulators’ feasibility before large-scale commercialization.
Keywords: Heavy metals; Soil; Phytoremediation; Microremediation;

Ecoenvironmental concerns; Perspective.
1. INTRODUCTION
Since the industrial revolution, anthropogenic impacts have caused more
and more hazardous heavy metals’ releasing to environment. Soils, being the
basic and most essential part of the ecological system, are heavily
contaminated, too. Until now over 20,000,000 acres of farmland in China have
been contaminated by heavy metals such as Sn, Cr, Pb, and Zn, which account
for almost one fifth of the total arable farmland area. Every year China suffers
a 10,000,000 tons’ loss of crop output due to deteriorating heavy metal
pollution [51]. Different from other organic pollutants, hazardous heavy
metals are indestructible, as they can not be chemically or biologically
degraded. Even worse, some heavy metals can concentrate along the food
chain and eventually accumulate in human body because we are at the top of
the food chain [1-3]. Therefore increasing attention has been paid to in recent
years to the remediation of polluted soils, among which the use of plants and
microbes to remove hazardous metal ions is particularly emphasized [4-6].
Phytoremediation is the use of plants to remove pollutants from environment,
while microremediation refers to the use of microbes. Usually if a plant can
accumulate more than 1,000mg/kg (or 1000ppm) of Cu, Co, Cr, Ni or Pb, or
Some Advances in Bio-Removing Hazardous Heavy Metals … 21
more than 10,000mg/kg (or 10,000ppm)of Mn or Zn, it is defined as a

hyperaccumulator[7,8]. Microbes have a larger specific surface area and are
more efficient to activate and remove heavy metals. Plants, however, have
harvestable stem and leaves aboveground, which is convenient for subsequent
post processing. Using a transgenic technology to combine the two methods,
namely symbiotic system, would be an optimum way to remove and collect
hazardous metals efficiently [9-11].
However, transgenic methods sometimes cause gene flow (the transfer of
alleles of genes from one individual to another), which is latent but dangerous
for a nonnative gene segment by spreading [12,13]. Several suggestions of
minimizing the resulting potential gene pollution are available in this article.
The authors think no country would risk their crop output to plant noncrop
hyperaccumulators. So the “cropaccumulators” stand for the developmental
tendency of hyperaccumulator plants. In the end, a novel and tentative plan is
given by the authors to evaluate the feasibility and demonstrate what
procedures are needed before large-scale commercialization.
2. MECHANISMS OF FOUR METAL-REMOVING-METHODS

To date, main four methods were proposed by researchers: chemical or
physical remediation, animal remediation such as earthworm,
phytoremediation and microremediation. Because of the obvious
disadvantages and deficiency in feasibility, wide application of the former two
methods is restricted. Summarized aspects of disadvantages of these two
methods are given in the section “3. Evaluation of four metal-removing-
methods”. In this part, mechanism of each four methods for removing
hazardous heavy metal is explained and compared, and we concerns more
about the latter two-phytoremediation and microremediation(Bioremediation).
2.1. Mechanism of Chemical or Physical Remediation
Chemical or physical method is early used and even endemically

commercialized in America. Physical methods (e.g. soil leaching method and
absorbent fixation) and chemical methods (e.g. bioreduction and chelate
extraction) are used in practice. In these methods the use of chelators can not
be avoided.
Figure1 EDTA-Pb complexes. Dotted bonds to Pb are coordinate.
By adding synthetic chelators such as EDTA (ethylenediamine-tetracetic

acid), both the solubility and bioavailability of heavy metals are improved. A
chelating reagent’s molecule can form several coordinative bonds to a certain
metal atom, increasing its concentration in soil aqueous phase and mobility.
(Figure 1) [1]. Considering some metal ions strongly bonds to the soil phase
and are less bioavailable, powerful chelating reagents are employed such as Na
salt of ethylenediamine-tetracetic acid (EDTA). However, such approach
needs not only expensive chemical reagent and machines but also many
technicians. Worse, excessively usage of chemical chelates has been proven to
pollute the ground water and negatively affect soil quality, for many necessary
ions are also chelated unselectively. For example, elements Fe and Ca are
usually lost after the spray of EDTA because their concentration in the soil is
much higher than those target heavy metals such as Pb and thus have more
access and possibility to chelation. Wenzel et al. [2] conducted an experiment
using canola (Brassica napus L) and reported that leaching losses of Cu, Pb
and Zn(polluting ground water) far exceeded the amounts of metal taken up by
plants after EDTA was applied, which indicated that under some certain
circumstance the disadvantage of chelating reagent far outweigh its advantage.
Therefore, taking reagent toxicity, unselectivity and inefficacy into account, a
careful consideration concerning ecology, economy and human health is
imperative before chelators are being put into practice [14].
2.2. Mechanism of Animal Remediation
Animal here mainly refers to earthworm because it is one of the most

important soil organisms and plays an indispensable role in improving soil
quality [15-18]. By their feeding, burrowing, excreting and metabolic redox

material, both soil texture and nutrition content are improved. Chemical
groups such as –COOH and –CO are generated and exuded, which acidify soil
and activate heavy metals. Several kinds of gel material are also excreted
which facilitate complexion and chelation of metal ions. However, because of
the relatively small amount and specific surface area compared with microbes,
such improvement is neither notable nor stable. According to Yang et al. [19],
after Eisenia foetida earthworm was inoculated, PH of cock manure decreased
by 0.7-0.9. However, if the inoculation occurred in an acidic red soil the PH
value drops only by 0.03-0.18; if the inoculation happens in a sandy soil, no
obvious decrease of PH is observed. Thus current studies imply that the
effectiveness and efficiency of earthworm depend too much on outer
conditions and may not be the optimum way of rapidly removing heavy
metals. Further investigation in this field is needed.
2.3. Mechanism of Phytoremediation
2.3.1. Accumulation and Transport

In the rhizosphere of hyperaccumulator plants, protons are released by
root to acidify the soil, which mobilize metal ions and increase metal
bioavailability. This mechanism is supported by Crowley et al. in 1991[4].
However, due to metal ions’ charge, lipohilic cellular membrane would be the
first barrier of ions’ entrance into cells. Fortunately, the following kinds of
secretion can facilitate the transportation process.
(1) Transporter proteins: Specific binding domain is existed in such
proteins, which binds to and transports metal ions from extracellular space into
cells. Lasat et al. [4] have found that hyperaccumulator Thlaspi caerulescens
had bigger capacity for Zn than its relative T. arvense. And such gap is
caused by different amount of Zn transporter proteins [5], which indicates that
transporter proteins play a crucial role.
(2) Nature chelators: As we know chelators such as EDTA can bind to
heavy metal ions and make them render uncharged. An uncharged ion is of
high mobility and is much easier to get through cellular membrane. In fact,
plants can excrete nature chelators, which is much less toxic and more
biodegradable as compared to EDTA (Table 1).
Table 1. Chelators that are commonly used or of importance
Nature helators Phytochelatin (PC) Metallothionein (MT) Organic acids

Synthetic EDTA (ethylene diamine tetra acetic acid) EGTA
Chelators (ethylene glycol tetra acetic acid)
DTPA(diethylenetriaminepentaacetic acid)
Among nature chelators, Phytochelatin (PC) and Metallothionein (MT)

interest many scientists and is well studied. Metallothioneins (MT) are
categorized into three classes: Class 1 MTs referred to polypeptides related to
mammals, which contain 61 amino acids but lack aromatic amino acid or
histidines; Class 2 MTs originally come from yeasts, and Candida albicans or
cyanobacteria [6]; An familiar chelator belonging to this class is S. cerevisiae
MT, contributing to plants’ high copper tolerance [7]; Class 3 MTs is
Phytochelatins (PC) exactly, which are composed of only three amino acid-
Glu, Cys and Gly, with Glu and Cys residues linked through a γ-carboxymide
bond. In addition, Kagi [8] have found that heavy metals such as Cd, Zn, Hg,
Ag and Pb can induce the synthesis of MTs especially in animal and plant
species. A recent study shows that the best activator is Cd followed by Ag, Bi,
Pb, Zn，Cu, Hg and Au [9].
(3) Organic acids: Several organic acids (e.g. malic acid and citrate) have
been identified as positive bio-reagents to accelerate the absorption of heavy
metals by root. Such mechanism is even more notable in the root-shoot
transportation.
Figure 2. MerB converting organic Hg to Hg(2).
Table 2. Metal ions and their transporters
ZNT protein(encoded by ZIP gene) 2 2

Zn , Cd
Nramp protein(encoded by AtNramp ene) 2 2
Cd , Fe
Protein encoded by NtCBP4 2
Pb
Aquaglyceroprins 3
As
Phosphate transporter 5
As
IRT1 (iron-regulated transporter) 2 2 2
Fe , Mn , Zn
However, substantial achievements are lacked in the root-shoot

transportation except two points: one is that root-shoot pathway is closely
related to plants’ transpiration efficiency; the other is one of chelator ligands
(histidine) is found in high levels in the xylem sap of Ni tolerant plant
(Alyssum lesbiacum) and the coordination of Ni with histidine is substantiated
by Kramer et al. [10], which implies that chelation mechanism also works in
the process of xylem transferring.
On the molecular level, accumulation and transport mechanism is partly
clarified. Many transporters encoded by specific genes are investigated and it
is common that one kind of metal ion can be transported by different carrier
(Table 2) [20-27].
2.3.2. Detoxification
As we know some hazardous heavy metals exercise a detrimental
influence on cells by binding to vital protein, interfering with cellular activities
and inhibiting regulation of cells. Luckily, hyperaccumulator plants have
evolved their own mechanisms to protect themselves from negative heavy
metal stress. Several important detoxification mechanisms are explained as
follows:
(1) Chelation: chelation plays a crucial role not only in the accumulation
and transportation of heavy metals but also in the detoxification phase. Usually
chelators have ligands (most commonly histidine and citrate) and can bind
metal ions. Combined metal ions appear uncharged and inert to react to other
substance, by which way heavy metals’ damage towards cell is reduced
significantly.
(2) Vacuolar compartmentalization: Since vacuole is widely considered as
the main storage place of heavy metals in plant cells, vacuolar
compartmentalization is quite effective in controlling the distribution and
concentration of metal ions. To compartmentalize vacuole is to “arrest and
imprison” hazardous metal ions, constricting them into a limited site. Thus
other parts of the cell have no access to those dangerous metal ions and safety
is of course ensured. This mechanism is proved to be true in the Cd
detoxification and tolerance by Salt, Wagner and Rauser [11]: Cd induces the
synthesis of PCs and then forms a Cd-PC molecule, which will be transferred
into the vacuole by a Cd/H antiport and an ATP-dependent PC-transporter.
Additionally Kramer et al. [12] have reported that by “imprisoning” most of
the intracellular Ni into vacuole, metal tolerance of hyperaccumulator T.
goesingense is greatly improved, which confirms the compartmentalization
theory, too.
(3) Volatilization: By converting metal ions into volatile state, some plant
species avoid the lasting damage caused by accumulation and long-time stay
of heavy metals. A representative example is the bioprocess of Hg, which is a
worldwide volatile pollutant and which is able to accumulate in human bodies.
However, not all the plants possess such ability and even among those innate
Hg-resistant species, the relatively small amount of accumulation and their
spatial distribution have greatly limited their wide cultivation. Thus scientists
have employed genetic engineering and several transgenic plants have showed
satisfactory performance to convert and volatilize metals. Transgenic species
expressing organomercurial lyase (MerB) have much higher tolerance to
organic Hg complex than wild type and can convert methylmercury to Hg(2),
which is 100 times less toxic than the former one.[13] Furthermore, transgenic
plants expressing both MerA (enzyme that reduces Hg(2) to Hg(0)) and MerB
have shown the highest tolerance to organic Hg(up to 10μM) compared with
MerB species’ 5μM and wild type’s 0.25μM [14].
2.4. Mechanism of Microremediation
2.4.1. Metal-Binding Mechanism

Three substances should be mentioned for this mechanism:
Metallothionein (MT), Phytochelatin (PC), and some novel metal binding
peptides. As we know from the former part of this article, MT and PC play a
crucial part in plant-metal interaction. In fact, in the microbial world such
interplay also exists. By binding to heavy metal ions MTs facilitate microbes’
absorption or transportation of metal ions, and so do PCs, who are composed
by only three amino acids (Gly, Cys and Gly). That over expression of PC
synthase in microbes is effective to the accumulation and tolerance of metal
ions has been reported by Sriprang R et al. [16]. By expressing the
Arabidopsis thalina gene encoding PC synthase, enhanced Cd accumulation is
observed in Mesorhizobium huakuii subsp. rengei B3 and E. coli cells. Recent
years novel metal-binding peptides containing histidines or cysteines have
been found and engineered. These peptides are usually of higher affinity,
specificity and selectivity for a certain metal ion. Related and in-depth study,
however, is scarce.
2.4.2. Valence Transformation Mechanism

Metals of different valence vary in toxicity. By excreting special redox
enzyme, plants skillfully convert hazard metals to a relatively less toxic state
and decrease possible metal stress and damage. For example, reduction of
Cr(6) to Cr(3) is widely studied, the latter one of which is both less mobile and
less toxic.
Additionally, Kashiwa [16] has found that Bacillus sp. SF-1 was good at
reducing high concentration of Se(6) into elemental Se. The most persuasive
example of this mechanism is the mercury-resistant bacteria, in which
organomercurial lyase (MerB) is produced. As we see from Figure 2,
methylmercury is converted to Hg(2), which is 100 fold less toxic than the
former one [27-33].
2.4.3. Volatilization Mechanism

By turning metal ions into volatile state, microbes escape possible
negative effect that dangerous metal ions bring them. However, such approach
is feasible for only a few metals such as Hg and metalloid Se.
For the majority of most other metals which have no volatile state at
natural conditions, this pathway is closed. To date, the way microbes deal with
element Hg is relatively clear. In the cells of mercury-resistant bacteria there is
a MerA enzyme, an enzyme that reduces Hg(2) to volatile form
Hg(0)[16,17](Figure 3).
Figure3. MerA converting Hg(2) to Hg(0) and facilitating volatilization.
2.4.4. Extracellular Chemical Precipitation Mechanism

Quite a number of binding substances were excreted by microbes, ranging
from simple organic acid, alcohols to large polysaccharides, humic and fulvic
acids. In fact, not only metals but also metal sulphides and oxides can be
entrapped and absorbed by an extracellular mixture of polysaccharides,
mucopolysaccarides and proteins [24]. Recent studies have found that
peptidoglycan carboxyl groups are the main cation binding sites for Gram-
positive bacterial cell walls while phosphate group for Gram-negative
microbes and chitins for fungi. No matter whether the precipitation happens in
the outer surface of the cell wall or away from it, this mechanism is successful
by keeping harmful metal ions out of cytoplasm [25,33-41].
2.4.5. Symbiotic Mechanism

A big disadvantage of microremediation is that absorbed heavy metals
would still stay in the soil, so symbiotic mechanism would be more effective
by combining both microremediation and phytoremediation. Due to the
symbiotic microbes’ large amount and specific surface area, binding reagent
such as MTs, PCs and organic acid will be excreted more by symbiotic
systems than by sole plants. Thus soil will be improved with better
acidification, which ultimately leads to a better solubility, mobility and
bioavailability of heavy metals. After heavy metal particles are activated, the
subsequent process can be divided into two ways. One is that metal ions are
accumulated by plants root, transported in the xylem and detoxified through
chelation, vacuolar compartmentalization and volatilization, just as normal
phytoremediation does. The other way is heavy metals will be accumulated in
rhizosphere and nodules. Rhizosphere bacteria’s essential role in achieving
optimum rates of selenium accumulation and volatilization has been proven by
De souza et al. [21] in an Indian mustard experiment. Another symbiotic
experiment was provided by Sriplang et al. [18] by inserting the MTL4 gene
2
into M. huakuii subsp. rengei B3. Data showed that the symbionts Cd
absorption increased by2.3-6.6 fold in nodules while obvious accumulation of
2
Cu was not observed.
3. EVALUATION OF FOUR METAL-REMOVING-METHODS

3.1. Summarized Disadvantages of Chemical/Physical
Remediation
(1) It is an expensive and labor-intensive way. Using large machine and

synthesizing a great amount of chelators could be costly and the application of
landfilling and leaching technology demands not only professional technicians
but also many hours. Someone has estimated that in order to reduce soil Pb
concentration from 1.4g/kg to 0.4g/kg in ten years, phytoremediation would
cost only $27,900. Compared with landfilling method’s $1,620,000 and soil
leaching method’s $790,000, phytoremediation is very economical;
(2) Natural soil’ structure, texture and fertility can be impaired by the
method itself and by the reagent added;
(3) Excessive use of chelators would poison both plants and microbes. The
most widely-used chelator EDTA is both toxic and nonbiodegradable;
(4) Chemical/physical method may lead to pollution by mobile heavy

metal ions leaching into ground water, since the use of binding reagents makes
metal ions more soluble and mobile;
(5) Chelators such as EDTA are usually lack of selectivity and easily
cause beneficial ions’ loss. (especially for Fe and Ca).
3.2. Summarized Disadvantages of Animal Remediation
(1) Animals such as earthworm are usually small in total number and
specific surface area ， which makes them inefficient in absorbing and
accumulating heavy metals.
(2) Animals’ rigorous demand for comfortable environment and excessive
dependence on organic substance limit their wide use and decrease their
practicality.
(3) Heavy metals are still in the soil if lack of a feasible method to collect
all the earthworms.
(4) Clear mechanism of accumulating and detoxifying heavy metal in
animals needs to be investigated by further studies.
4. FUTURE DEVELOPMENT AND OPPORTUNITIES OF

BIOREMEDIATION (PHYTO- AND MICRO-REMEDIATION)
4.1. Application of Genetic Engineering or Cell Engineering
In recent years, genetic engineering technology seems increasingly

necessary, because most nature hyperaccumulators are not satisfying（due to
its slow growing, low biomass-production and rigorous demand for growing
conditions）. Aided by this powerful tool, many heavy metal resistant genes
have been introduced into plant cells (Table 3) [19]. By over expression of
natural chelators (MTs, PCs and organic acid), not only ions’ entrance into cell
but also translocation in xylem and other part is facilitated; by excreting
certain transporter proteins, specific ions was bound and transferred; by
encoding special oxidoreductase such as MerA and MerB, heavy metals’
valence is changed into a less toxic one or a more volatile one; by horizontal
transfer of plasmid, resistant gene are exchanged among rhizosphere bacteria
and this benefits bacteria’s accumulating and transferring metal ions.
Table 3. Genes introduced into plants and their source and target. G+
stands for Gram-positive bacteria and G- stands for G-negative ones
Gene Product Source Target Gene Product Source Target
merA Hg(II) G+ Liriodendron CGS Cystathione- A. B.juncea
reductase tulipifera gamma- thaliana
Nicotiana synthase
tabacum
merA Hg(II) G- A. thaliana CS Cysteine Spinach N.tabacum
reductase ase synthase
merB Organo G- A. thaliana Gsh GSH E.coli B.juncea
mercurial 2 synthase
lyase
merA Hg(II) G- N. tabacum Gsh γ-Glu- E.coli B.juncea
reductase 1 Cys synthase
merB Organo G- N. tabacum Gsh γ-Glu- E.coli Populus
mercurial 1 Cys synthase tremula
lyase
AtPC PC A. A. thaliana NtCB Cation N. N. tabacum
SI thaliana P4 channel tabacum
APSI ATP A. Brassica SL Se- mouse A. thaliana
sulfurylase thaliana juncea Cys lyase
MT-1 MT mouse N. tabacum TaPC PC T. N. glauca
SI aesitivum
Cell engineering technology (such as cell fusion or somatic hybridization)

also shows great power. Because polyploid plants are usually bigger in size
and more active in transpiration, which does good to the transportation of
heavy metals in root-to-shoot process, this method is of great significance
(Figure4) [23].
Figure 4. Somatic hybrid (B) and its parents (A) (C).

Hybrid (B) have not only a bigger size than its parent Brassica juncea (A )
and Thlaspi caerulescens (C), but also better hyperextraction in absorbing Pb,
Ni and Zn. The amount of Pb absorbed by hybrid (B) is almost the sum of its
parents (A) and (C) [20]. However, a big disadvantage coming with transgenic
technique is the genetic pollution caused by, for example, the floating of
transgenic pollen and the horizontal transferring of plasmids among microbes.
In addition, because both pollen and plasmid is invisible to naked eyes, not
until the horrible mutant species grow up and cause severe aftermath can we
realize their damage.
Therefore biosafety aspects must be taken into account and application of
transgenic species need domestic legislation. Here the authors contribute three
feasible suggestions that minimize the potential damage caused by genetic
pollution:
1) Choosing self-pollinated plants. In a self-pollinated plant, pollen

moves to the female part of the same flower or to another flower on
the same individual plant. And this means fertilization is completed
within a closed environment. Therefore, self-pollination would be an
effective way to avoid the spreading of transgenic genes into other
species.
2) Using cell engineering technology to create infertile polyploid
species.
3) Infertile plants can not transfer any gene to the next generation and so
potential spread of gene segment becomes impossible. Technically
two species are feasible: one is allopolyploid (the combination of two
heterogeneous sets of chromosomes) and the other is triploid. Both
approaches are able to make plants infertile by lack of equation-
division mechanism of chromosomes.
4) Among microbes, transgenic technique should be limited within the
sphere of rhizosphere bacteria and symbiotic bacteria, because such
kind of microbe mainly congregates around the root and shows a very
slight distribution outside the rhizosphere. In detail, the population of
“endophytic bacteria” living within the plant tissues is easier
controlled than that of rhizospheric bacteria living on or round the
plant roots, because the former depend more on plants. Therefore by
restricting the transgenic and foreign genes in a limited area and a
finite amount, potentially harm of genetic pollution is minimized.
4.2. The Development of Crop Hyperaccumulators
Another new viewpoint towards phytoremediation is that adequate

attention must be paid to the development of crop hyperaccumulators, termed
by the author as “cropaccumulators”, such as wheat, maize and rice [30-33,37-
45]. In other words, investigations of other “noncropaccumulators” species
would be of little significance in application. A large area of farmland in the
world is contaminated by heavy metals and need remediation. Considering the
finite and precious farmland sources and big population, no country would
stop growing crops and turn to noncrop hyperaccumulators which do not
provide grain. Furthermore, it usually takes several years to completely
remove a certain heavy metal from soil. This makes the application of non-
crop hyperaccumulators more unrealistic. After all, no country could afford
zero harvest and even famine for several years.
To date, more than 400 plant species have been identified as metal
hyperaccumulators and the best hyperaccumulator varies according to regions
with different climate and soil types. To date, the most studied and promising
species is within Cruciferae family such as Brassica genus, Alyssuns genus
and Thlaspi genus, but 75% of them are Ni tolerant which grow on ultramafic
soils [23,24]. Unfortunately almost none of them can be used as crops in the
farmland. Thus it is critical that we paid immediate attention to the
“cropaccumulators” field. We recommend two ways to develop this
“cropaccumulators” species:
(1) By transgenic approach. Through the introduction of foreign resistant

genes into crops, capability for accumulating, transporting and
detoxifying heavy metals can be significantly improved. Even if the
improvement is not as notable as expected, it doesn’t matter because
“cropaccumulators” can be grown every year as normal field crops
and heavy metals are bound to be removed thoroughly after a long
period of time. As long as heavy metals do not accumulate in the
edible part such as grain, long-time growing of “cropaccumulators”
would not reduce the total crop outcome and impair the grain quality.
(2) By symbiotic approach. Finding an appropriate kind of symbiotic
bacteria is the key of this method. In a symbiotic system, heavy
metals are more likely to be accumulated in nodules. This is good for
us because generally crops bear grains aboveground and the fear that
heavy metals may enrich in edible part is eliminated. Another big
advantage is that some microbes produce antibiotics to enhance plants
immunity and some produce necessary nutrients and even plant

growth hormones [25-32,45-56]. Therefore the future of this method
would be bright.
4.3. Universal Procedures of Evaluation Before Large-Scale

Commercialization
As we discussed in part “3.2 Future development and opportunities of

bioremediation”, “cropaccumulators” created by genetic or cell engineering
technology will of great importance and hope.
Figure 5. Universal procedures of evaluation before large-scale commercialization.

However, related literature concerning concrete steps of applying a

hyperaccumulator to practice is seldom found. The author tentatively puts
forward a new evaluation plan which tells us clearly what should be done
before large-scale commercialization of a new hyperaccumulator species
(Figure 5)[49-56].
Explanations of each step in Figure 5.
Step 1: After a transgenic or symbiotic species is created, theoretical

biosafety assessment is indispensable. Since foreign genes would enter the
ecological system automatically and can not be easily removed once a seed is
planted. Based on experience and hermetic experiments biosafety can be
estimated. The following parameters are recommended: sexual compatibility
of pollens, blossom and spatial distribution of nearby plants, and the fertility of
the next generation. If the new species is eligible and biosafe to the ecology, it
is allowed to go to the next step. If not, simply destroy it (for the purpose of
eliminating nonnative genes).
Step 2: Experimental field selected should be representative. A relatively
closed environment is necessary to minimize gene flow phenomenon. If
possible the selected field should be also easily regulated in water, PH,
nutrient, and sampling for the subsequent steps.
Step 3: As we know soil acidity is closely related to the activation and
accumulation of heavy metal. Nutrient, however, also matters. Studies show
that a proper amount of N and K promote the uptake of Pb but a large amount
inhibits the Pb absorption. The addition of P doesn’t improve the accumulation
of Pb, which even counteracts the original absorbing [28]. Water supplying
relates with transpiration, which plays a great role in the transportation of the
heavy metals. Every plant and microbe has its optimum temperature, PH,
water content and nutrient supplying for living, and satisfying these demands
is important.
Step 4: We recommend natural outdoor field with sunlight to sow the new
test species and spring would be the best season for the most species. Although
simulated lab field can provide similar conditions, it is not suitable for this
evaluation process in many aspects especially in the assessment of genetic
pollution where interaction with other plants is unavoidable. Besides, another
important aim of step 4 is to monitor how well the plant or symbiotic system
survives while removing heavy metals. Several parameters are cited here,
which can be categorized into two classes. One is chemical method which is
stable, and the other one is biological parameters which are less stable but
more effective and sensitive. For example, chemical parameters can show the
total amount of a certain metal, which may be useless because only soluble or
biodegradable forms of metal ion can be absorbed. A government law
(National Standard of the People’s Republic of China) provides ways of heavy
metal measurement, which is accurate and legal. Micro-parameters reflect not
only heavy metal content and stress, but also compatibility between bacteria
and plants. Representative parameters include the ratio of microbial biomass C
(Cmic) / C (Corg) and metabolic quotient (qCO2) in biochemical level and CLPP
in community level. Usually when heavy metal concentration goes up,
decrease in C/C ratio and increase in metabolic quotient can be observed.
Step 5: Keeping parameters at an optimum value can be achieved by
several approach. To decrease soil PH value, either H2SO4 or organic fertilizer
can be used. To increase PH, lime or other alkaline substance is useful. By
regulating water supplying, both transpiration and Eh value (a redox index of
soil) is controlled. The use of element P, N and K can also affect those
parameters. Through step 5’s regulation we try to ensure that new
hyperaccumulators work at a normal and relatively high efficiency.
Step 6: Different from Step 1, expert assessment is practical because
plants have matured. Maturity means pollens have been produced and spread.
Therefore at this time biosafety issue is quite realistic. A detailed and rigorous
environmental risk assessment must be completed to avoid genetic pollution as
much as possible.
Other index should also be assessed such as biomass, total cost of the
growing, and growth rate. Besides these, experts need to estimate how long
bioremediation would take, for an excessively long duration is not suitable for
process feasibility. If the plant is qualified in this step, it can go to further
large-scale commercialization; if not, destroy it (for the purpose of eliminating
nonnative genes).
Step 7: The performance of the last step needs a national legislation
concerning transgenic species and its potential risk. If it is a novel symbiotic
system using no transgenic technology, such procedure is not needed.
Additionally, when commercializing, different kinds of hyperaccumulators can
be grown by turn since one kind of hyperaccumulators is generally good at
dealing with finite kinds of metal.
CONCLUSIONS
1) Bioremediation has more advantages and should be focused more,
especially on transgenic methods.
2) Future transgenic technology should focus on four promising targets:

over expression of natural chelators (MTs, PCs and organic acid),
transporter proteins, special oxidoreductase and resistant gene in
symbiotic microbe.
3) Several ways of minimizing genetic pollutions are put forward such as
choosing self-pollinated plants, creating infertile polyploid species
and carefully selecting easy-controlled microbe species.
4) Crop hyperaccumulators should be paid more attention for studies on
non-crop species are of limited use.
5) Universal procedures are put forward to evaluate hyperaccumulators’
feasibility before large-scale commercialization.
PERSPECTIVE
Compared to chemicophysical method, bioremediation shows great
advantage. Studies on this field are increasing and its mechanism is more and
more clear[33-41]. Using transgenic technology is a tendency in the future to
create an ideal species purposely. However, genetic pollution must be taken
into consideration, which can be avoided or minimized by using methods
mentioned above. In the future crop hyperaccumulators will be a better choice
due to its feasibility, in the field of which current emphasis is scarce.
Microbes, in many cases, are more efficient in accumulating and absorbing
heavy metals because of their astronomical amount and specific surface area.
Furthermore, technique of genetic engineering in microbes is easier and more
mature than in plant cells. Therefore, using transgenic technology to create an
optimum plant + soil + microbes combination would be a promising way in
the future development [42-50]. As to the estimation of a new species or
symbiotic system, Figure 5 offers a feasible risk assessment plan and may
accelerate the application process.
ACKNOWLEDGEMENTS
Our laboratory and team group are currently supported by the National
Natural Science Foundation of China (No.41001137; 41171216; 31100313)
and One Hundred-Talent Plan of CAS, Yantai Science and Technology
Development Project (2010245;2011016), the CAS/SAFEA International
Partnership Program for Creative Research Teams, the Strategic Priority

Research Program of the Chinese Academy of Sciences (CAS)
(XDA01020304), the Science and Technology Development Plan of Shandong
Province (2010GSF10208) and Yantai Double-hundred Talent Plan (XY-003-
02).
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Chapter 3
IMMOBILIZATION OF HEAVY METALS

IN SOIL BY PHOSPHATE TREATMENT:
A REVIEW
Silvano Mignardi*, Alessia Corami and Vincenzo Ferrini

Department of Earth Sciences, University of Rome “La Sapienza”,
P.le Aldo Moro 5, 00185 Rome, Italy
ABSTRACT
Heavy metal contamination of soil is an environmental and public
health issue of prime interest because metals are non-biodegradable and
accumulate throughout the food chain. Conventional heavy metal
remediation technologies include chemical precipitation, membrane
filtration, ion exchange, electrochemical treatment, etc. Recently
adsorption has become an alternative treatment method of contaminated
media and a great effort has been devoted to develop new adsorbents.
Phosphate treatment is widely considered an effective in situ
treatment to reduce metal mobility and bioavailability in soils through the
formation of highly insoluble phosphates which are stable in a wide range
of environmental conditions. The mechanisms of metal retention are still
debated and include: ion exchange, dissolution/precipitation and surface
complexation. Various types of phosphate amendments, such as synthetic
*
Authors for Correspondence: silvano.mignardi@uniroma1.it(Silvano Mignardi), Posting
address: Department of Earth Sciences, University of Rome “La Sapienza”, P.le Aldo Moro
5, 00185 Rome, Italy.
44 Silvano Mignardi, Alessia Corami and Vincenzo Ferrini
and natural apatites, phosphate rocks, bone meal and bone char have been
tested. Phosphate treatment has been shown to be effective in stabilizing
Pb, Cd, Cu, Zn, Co, Ni, Sb, Cr, V in contaminated soils. In soils
phosphate stabilization has been applied mainly to remediate Pb
contamination although the method can be used to reduce also the
mobility and bioavailability of other metals. The main factors
determining the removal capacity of phosphate treatment in contaminated
soils include type of phosphate source due to their different solubilities,
rate of phosphate application, heavy metals speciation, soil pH and Eh
and grain size of the amendment. The adverse effects of phosphate
stabilization resulting in enhanced leaching of phosphate and availability
in the soil of contaminants, such as As, Se and W, have to be addressed
for evaluating the environmental impact of phosphate treatment.
1. INTRODUCTION
Heavy metal contamination of soil has become an issue of major concern
worldwide in the last decades. The term “heavy metals” generally includes
both biologically essential (e.g., copper, cobalt, chromium, manganese and
zinc) and non-essential (e.g., lead, cadmium and mercury) elements. The
essential elements are required for nutrition of living organism in low
concentrations, whereas the non-essential are toxic even at low levels. Heavy
metals originate mainly from anthropogenic activities, such as mining
activities, smelting, metal plating, tanneries, battery, paint and pesticides
manufacture, etc. (e.g., Kadirvelu et al., 2001; Park et al., 2011). Natural
processes of microbial or chemical degradation can not destroy heavy metals
and their accumulation in living tissues causes various disorders and diseases.
Heavy metals can only be transformed to less biovailable forms to minimize
toxic effects to living organisms.
Current remediation technologies of contaminated soils are generally very
expensive and environmentally invasive involving excavation and disposal of
polluted soils. Conversely, in situ chemical immobilization of metals is more
economical and provides a long-term solution through the formation of stable
and low soluble compounds. In the last decades there has been a growing
interest in the conversion of heavy metals in less soluble compounds through
chemical treatment of the soil by phosphate application (e.g., Cao et al.,
2003a,b,c, 2008; Chen et al., 2007; Cotter-Howells and Caporn, 1996;
Hettiarachchi et al., 2001; Ma et al., 1993, 1994; Melamed et al., 2003; Ryan
et al., 2001). Phosphate-induced immobilization of heavy metals has been
Immobilization of Heavy Metals in Soil by Phosphate Treatment 45
recognized as an effective in situ remediation method to reduce metal

availability, to mitigate acid mine drainage and to minimize toxic metal release
to groundwater. In particular, phosphate application has been recommended
among the Best Management Practices for the treatment of firing ranges soils
to immobilize Pb (Chrysochoou et al., 2007). Several different natural and
synthetic phosphate amendments have been used for soil remediation
including apatite, hydroxyapatite, phosphate rock, soluble phosphate fertilizers
(diammonium phosphate, triple super phosphate, etc.), phosphate clay and
mixtures of these materials (e.g., Cao et al., 2003a,b,c, 2008; Chen et al., 2007;
Cotter-Howells and Caporn, 1996; Ma et al., 1993, 1994; Yang et al., 2001).
However, remediation of contaminated soils is a complex task as most of them
contain elevated concentrations of more than one single metal. In multi-
element contaminated soils phosphate treatment may have negative effects
since phosphate ions compete with other anions, such as As, Sb, Se and W for
the same binding sites, enhancing their mobility in the soil environment. In
this view, both undoubted advantages and undesirable drawbacks of phosphate
treatment should be carefully evaluated to define the best management
practices for multi-element contaminated soils.
This chapter reports an overview on practical working experiences about
the remediation of contaminated soils by means of the application of
phosphate amendments. The main purpose of this review is to contribute
improving our knowledge on this in situ soil remediation technique as the
optimization of this promising remediation option can be achieved only based
on the results of these experiences.
2. SOURCES OF METALS IN SOIL SYSTEM

Natural geogenic/pedogenic processes and anthropogenic activities release
heavy metals in soils (e.g., Bolan et al., 2003; Eze et al., 2010; Hernandez et
al., 2003; Mico et al., 2006). The amounts of heavy metals provided by natural
processes are generally low and mainly related to the origin, nature and the
processes of weathering of the parent source. On the contrary, metal
enrichment in soils is hugely related to anthropogenic activities. Natural
sources include parent rocks and metallic minerals whereas the anthropogenic
activities are represented by agriculture (pesticides, fertilizers, animal
manures, etc.), energy production (battery manufacture, power plants, etc.),
metallurgy (mining and processing of ore mineral, metal finishing, smelting,
etc.), microelectronics and waste disposal of domestic, agricultural and
industrial materials (Udeigwe et al., 2010). Metals added to soil through

anthropogenic activities have high bioavailability and can reach rivers, lakes
and groundwater resulting in the contamination of drinking water sources
(Lamb et al., 2009; Sridhara et al., 2008).
3. EFFECTIVENESS OF DIFFERENT PHOSPHATE SOURCES

The phosphate sources can be divided into three major groups: readily
soluble phosphates (phosphoric acid, PA), moderately soluble phosphates
(monocalcium phosphate, MCP; dicalcium phosphate, DCP, tricalcium
phosphate, TCP; diammonium phosphate, DAP; simple super phosphate, SSP;
triple super phosphate, TSP; potassium orthophosphate, MKP) and less soluble
phosphates (synthetic and natural hydroxyapatite, HAP; phosphate rock, PR),
along with biogenic phosphates (bone char, bone meal, BM) and waste-P
(phosphatic clay). PR is the main source for the production of all the above
phosphates, but it is the low reactive compared to all refined materials,
whereas it is the most available and cost-effective phosphate source for
remediation treatment. However, generally PR contents of As, Co, Cd, Cr and
Cu are relatively high. On the contrary, biogenic phosphates are more soluble
and contain lower concentrations of contaminants as compared to the mined
sources (Kaplan and Knox, 2004).
A variety of phosphate sources have been tested in laboratory and field
tests (Table 1). Brown et al. (2004) tested the capacity of PA and TSP in
reducing Pb, Zn and Cd in a contaminated soil. Results indicated that
phosphate treatment reduced both bioavailable and phytoavailable Pb.
Application of 3.2% P-TSP and 1% P-PA was the most effective for reducing
Pb, Zn and Cd plant concentrations and in vitro extractable Pb. However, the
authors suggested that practical phosphate application with the rates they used
could have negative effects on the eutrophication of surface waters and co-
contamination of the soil with As. Caution about excessive phosphate
treatment of contaminated soil because of the enhanced plant As uptake was
suggested also by Cao and Ma (2004).
A phosphate compound able to immobilize arsenic in aqueous solution is
synthetic Fe(III) phosphate. Lenoble et al. (2005) showed that FePO4, either
amorphous or crystalline, removed As combining As(III) oxidation by Fe(III)
and phosphate substitution by As(V) leading to the precipitation of
Fe3(AsO4)2∙8H2O and FeAsO4∙2H2O.
Table 1. Selected references on different phosphate sources for heavy

metals immobilization
Phosphate amendment Heavy metals Reference

Water-insoluble compounds
Synthetic HAP Pb Zhang et al. (1998)
Synthetic HAP Pb, Zn, Cu, Cd Boisson et al. (1999)
Synthetic HAP Pb, Ni, Cd, Co Seaman et al. (2001)
Synthetic HAP Pb Ryan et al. (2001)
Natural HAP Pb Laperche et al. (1996)
Natural HAP Pb Knox et al. (2003)
Natural HAP Pb Chen et al. (1997)
Natural and biogenic HAP Pb Kaplan and Knox (2004)
PR Pb Yoon et al. (2007)
PR Cd, Zn, Pb Knox et al. (2003)
PR Pb Geebelen et al. (2003)
PR Cd, Zn, Pb Basta et al. (2001)
PR Pb, Zn, Cu, Cd Chen et al. (2006)
Low grade PR Pb, Cu, Zn, Co Saxena and D'Souza (2006)
Water-soluble compounds
MCP Pb Cao et al. (2008)
MCP Pb Melamed et al. (2003)
MCP Pb Chen et al. (2003)
MCP Pb Xenidis et al. (2010)
PA Pb Melamed et al. (2003)
PA Pb Chen et al. (2003)
SSP Pb Cui et al. (2010)
TSP Cd Thawornchaisit and Polprasertb
(2009)
TSP Pb Hettiarachchi et al. (2001)
TSP Pb, Cu, Sb Spuller et al. (2007)
DAP Pb, Zn Cotter-Howells and Caporn
(1996)
DAP Cd, Cu, Zn Zhang and Pu (2011)
DAP Pb Ruby et al. (1994)
DAP Pb Bosso et al. (2008)
KH2PO4 Pb, Cu, Ni Conesa et al. (2010)
KH2PO4 Pb, Cd, Zn Zwonitzer et al. (2003)
Other phosphate sources
BM Ni, Zn Zupancic et al. (2004)
BM Pb, Cu, Zn, Ni Hodson et al. (2001)
BM Pb Deydier et al. (2007)
MBM Pb, Zn, Cd Sneddon et al. (2006)
The capability of another iron phosphate in removing contaminants from

polluted media was evaluated by Liu and Zhao (2007). Iron phosphate
vivianite [Fe3(PO4)2∙8H2O] was synthesized and tested for in situ
immobilization of Cu in three soils (calcareous, neutral and acidic). The results
showed that the amendment significantly reduced TCLP (Toxicity
Characteristics Leaching Procedure) leachability and PBET (Physiologically-
Based Extraction Test) bioaccessibility of Cu. The application of vivianite
resulted in a reduction of water soluble-exchangeable and carbonate bound
fractions and an increase of Cu residual fraction in the soils. Sugiyama et al.
(2003) studied the Pb, Cu, Co and Cd removal capacity of TCP, DCP, and
MCP in aqueous solutions and found that all the amendments were able to
immobilize Pb, whereas Cu, Co and Cd were favourably removed only by
DCP. MCP, DCP and DAP were used for remediation studies of contaminated
soils containing Cd, Cu, Ni, Pb, Zn, Sb and As (e.g., Spuller et al., 2007;
Theodoratos et al., 2002; Wang et al., 2001). Generally, the phosphate
treatments reduced leachable metal concentrations; however, some differences
in the result were observed. For example, a slight mobilization of Zn has been
reported by Theodoratos et al. (2002). Moreover, they found that phosphate
treatment had no effect on the uptake of contaminants in Phaseolus vulgaris
and had a marked negative effect on plant growth due to increased deficiency
of Ca. Beside the well known adverse effect of phosphate treatment on As
stability in soils, Spuller et al. (2007) reported that phosphate amendments
enhanced mobility of Sb and Cu. Moreover, a significant inhibition of
duckweed (Lemna minor) growth by DAP-MCP treatment was observed and
attributed to enhanced Cu and Sb mobility.
Mined phosphate materials, referred to as phosphate rock (PR) in this
chapter, have been extensively used for the immobilization of heavy metals in
aqueous solutions and soils (e.g., Cao et al., 2003a; Knox et al., 2003; Ma et
al., 1995; Ryan et al., 2001; Seaman et al., 2001). Apatite is the main
phosphate mineral occurring in PR as carbonate apatite with substitution of
carbonate for phosphate, F for hydroxyl anion and minor substitution of Ca2+
by Na+ and Mg2+ ions (Knox et al., 2006; Ma et al., 1995). Knox et al. (2003)
evaluated the use of PR from North Carolina (USA) to immobilize aqueous
Co2+, Ba2+, Pb2+, Eu3+ and UO2+. Results showed that PR significantly reduced
the concentrations of each metal. Moreover, the application of the amendment
to a Zn-Pb contaminated soil was effective at increasing plant growth of maize
(Zea mays L.) and oat (Avena sativa L.) and reducing Cd, Pb and Zn
concentration in plant tissues. However, the sequestering agent affected some
essential elements with reduction of P and Fe concentrations in oat tissues. Pb
immobilization efficacy of a North Carolina (USA) PR was tested also by

Geebelen et al. (2003) who showed that it was the most effective amendment
among those they used. However, PR was unable in preventing Pb
phytotoxicity and Pb uptake. In this view, the authors showed that a suite of
bioavailability tests have to be performed for evaluating in situ metal
immobilization. Addition of PR to shooting range soils resulted in the
reduction of water-soluble Pb, although the effect of treatment was minimal
after 11 weeks of incubation (Ma et al., 2007). The effectiveness of PR to
reduce extractability and plant and gastrointestinal bioavailability in Cd-Pb-
Zn-contaminated soils was investigated also by Basta et al. (2001). They found
that PR was slightly effective in reducing Zn and Cd phytoavailability,
whereas it reduced gastrointestinal-available Pb in both gastric and intestinal
solution. Therefore, they concluded that PR treatment can decrease risk
exposure to Pb by the ingestion of contaminated soils. A field demonstration
of the effectiveness of PR, along with PA and DCP, to immobilize Pb at a
contaminated site was conducted by Cao et al. (2002, 2003a), Chen et al.
(2003) and Melamed et al. (2003). The site located in Florida (USA) had been
contaminated by past industrial activities, mainly recycling batteries, and total
Pb concentrations in the surface soil (0-10 cm) ranged from 3762 to 15919
mg/kg. Phosphate treatment transformed up to 60% of total soil Pb from the
non residual to the residual fraction as previously reported by Ma and Rao
(1997). Moreover, in the surface soil TCLP Pb was reduced below EPA’s (US
Environmental Protection Agency) regulatory level by phosphate treatment. In
this view, the authors showed that a mixture of PA and PR was most effective
in immobilizing Pb resulting in less soil pH impact and P leaching. Similar
conclusions were drawn by Yoon et al. (2007) who applied simultaneously PA
and PR in a column experiment for Pb immobilization in a polluted soil. The
results showed that the application of a mixture PA-PR reduced TCLP-Pb and
PBET-Pb in the contaminated soil. Application of PA solubilized Pb minerals
occurring in the soil, facilitating more precipitation of Pb-phosphates.
Moreover, the authors found that the application of layered PR reduced Pb
migration in the soil and minimized soil acidification and P eutrophication
enhanced by PA application. Indeed, Yang et al. (2001) had previously
reported significant reduction in soil pH due to the addition of PA to a soil
from a smelter-contaminated site to immobilize Pb. Reduction of soil pH
increases Pb immobilization by phosphate treatment through the dissolution of
carbonate to which large portion of Pb soil is associated. However, the use of
pure PA may increase leaching of P and other heavy metals (Yoon et al.,
2007). Although phosphate amendment enhanced the accumulation of Pb, Cu
and Zn in the roots of St. Augustine grass (Stenotaphrum secundatum), metal

translocation from roots to shoot was significantly reduced (Cao et al., 2003a).
Similar results were found for maize (Zea mays), sudax (Sorghum bicolor L.
Moench) and barley (Hordeum vulgare) (Chlopecka and Adriano, 1996;
Laperche et al., 1997). This result was attributed to the formation of insoluble
metal phosphate in the roots. In contrast with the above results, Zwonitzer et
al. (2003) added PR or KH2PO4 to a Pb, Cd, Zn contaminated soil and found
that PR application did not result in differences in plant tissue (Sorghum
bicolor L. Moench) concentrations of Cd and Pb, whereas Zn concentrations
were significantly reduced. They concluded that for the soil (from a paint
manufacturing facility) they used, the more soluble phosphate source
(KH2PO4) was more effective in reducing Pb bioavailability than PR. Addition
of phosphate as KH2PO4 to different types of contaminated soils reduced Cd,
Cu, Pb, Zn and U plant uptake (Bolan et al., 2003; Rufyikiri et al., 2006;
Simon, 2005). Zeng et al. (2008) showed the effectiveness of KH2PO4 and PA
also in removing As from a contaminated soil. Both amendments reduced soil
content with removal efficiency up to 20%. However, significant acidification
of the soil and dissolution of Ca or Mg minerals in the soil resulted, especially
using PA. As KH2PO4 almost extracted as much As as PA without serious
damage to soil compared to PA, they concluded that KH2PO4 could be an
environment-friendly extractant. PR application was also effective in
immobilizing industrial solid toxic wastes (e.g., Ioannidis and Zouboulis,
2003; 2005) reducing the release of the metal below the legislative
concentrations limits of leaching test DIN 38414.
Beside high-grade (>30% P2O5) PR, many studies evaluated the
effectiveness in remediation treatment of low-grade (<30% P2O5) PR, which
are generally considered as waste in fertilizer manufacturing. Prasad et al.
(2000a,b), Prasad and Saxena (2004), Saxena and D’Souza (2006) investigated
Pb, Cu, Zn and Co removal capacity of an Indian low-grade PR. They found
that the sorption of heavy metals generally followed the order Pb>Cu>Zn>Co.
The effectiveness of a low-grade PR from Florida (USA) in removing Pb, Cu,
Zn and Cd was evaluated by Corami et al. (2008c). The results showed that the
amount of sorbed metals apparently depended on the initial metal
concentrations and generally followed the order Cu>Pb>Cd>Zn. Similar
results were obtained by Kandah (2004) using a Jordanian low-grade PR for
the removal of Zn and Cd both in single and binary systems. The author
showed that the used PR had higher removal capacity for Zn than for Cd.
Elouar et al. (2008) used an activated PR to remove Pb, Cu, Cd and Zn.
The material was activated with sodium hydroxide and nitric acid in order to
obtain high contact area for the sorption. They found that the removal of heavy
metals by activated PR is slower as compared to non-activated PR; however,
the activated PR proved to be the best adsorbent for the metals compared to
PR. Similar results had been previously obtained by Mouflih et al. (2005)
studying the Pb removal capacity of a Moroccan natural PR activated with
nitric acid. Calcined PR was used for the removal of Pb, Cu and Zn also by
Aklil et al. (2004).
HAP, PR, TSP and DAP application was effective in reducing Cd, Pb and
Zn bioavailability also in an artificially metal-contaminated soil (Chen et al.,
2007). Phosphate treatment reduced metal uptakes in shoots of Brassica
campestris L. and significantly converted amounts of metal from the non
residual to the residual fraction. However, Chen et al. (2007) suggested that
DAP should be applied with caution as it resulted in an increase in metal
availability due to the enhanced soil pH reduction. The results of this study
showed that generally the effectiveness on plant metal uptake of different
phosphates followed the order HAP>PR>DAP>TSP. These results are in
agreement with those reported by Hettiarachchi and Pierzynski (2002) who
found that PR was more effective than TSP or PA in reducing Pb
bioavailability for Zn-Pb contaminated soil. However, these authors showed
that in general TSP reduced Pb, Cd and Zn concentrations in shoot tissues of
sudax (Sorgum vulgare L.) and Swiss chard (Beta vulgaris L.), whereas PR
treatment did not affect metal concentrations in plant tissue.
The results of the above studies clearly demonstrate that PR is effective in
removing heavy metals from polluted media. However, Mavropoulos et al.
(2005) showed that beside apatite other minerals, such as carbonate, occurring
in PR, contribute to some extent to metal immobilization. Although this
improves the overall removal capacity of PR, heavy metals bioavailability in
soils and wastes remediated by PR treatment should increase due to higher
solubility of metal carbonates compared to metal phosphates.
Another phosphate source for remediation treatment of polluted waters
and soils is phosphatic clay, a by-product of the phosphate mining industry.
Phosphatic clay has high content of natural apatite (about 24-32%) along with
phyllosilicate minerals, such as smectite, palygorskite, illite and kaolinite.
Singh et al. (2001; 2006) evaluated the effectiveness of phosphatic clay for the
removal of Pb, Cd and Zn. Phosphatic clay was able to remove the three
metals and the amount of sorbed metal followed the order Pb>Cd>Zn.
Valsami-Jones et al. (1998) suggested that BM may provide a cost-
effective phosphate source for the remediation of metal contaminated media.
Hodson et al. (2000; 2001) showed, in leaching column experiments, that the
addition of BM reduced the availability of Pb, Zn, Cd and Ni in heavy metal-

contaminated soils. However, they found that BM addition might increase the
mobility of Cu in the presence of organic matter. The effectiveness of BM in
immobilizing heavy metals also in sewage sludge has been showed by
Zupančič et al. (2004) who found that BM addition resulted in a significant
immobilization of Ni and Zn. However, they concluded that BM treatment of
sewage sludge cannot be recommended for sanitary hazards arising from
concerns about the safety of skeletal tissue of cattle applied as metal
immobilizing agent in a contaminated soil. Animal bones (AB) are composed
in 65-70% of inorganic matter, mainly hydroxyapatite, and organic substances
mainly fibrous protein collagen. AB were found to be able to remove also Cr3+
from aqueous solutions (Chojnacka, 2005). Key parameters to use AB for the
stabilization of heavy metals are the rate of phosphate release as well as the
total content of phosphate. They have been evaluated for waste-bones of tuna,
pig and cow by Hwang et al. (2007). The authors found that waste-bones are a
source of available phosphorus for the removal of heavy metals. Thomson et
al. (2003) and Choy and McKay (2005) showed that bone char, mainly
produced by pyrolisis of cow bones and containing about 10% carbon and
90% calcium phosphate, has high removal capacity for Cd, Cu and Zn as well
as Am, Pu and U. The use of combustion residues of meat and bone meal
(MBM) to remove Pb and U(VI) from aqueous effluents was investigated by
Deydier et al. (2003) and Krestou et al. (2004). Deydier et al. (2007) continued
their evaluation of MBM efficiency for in situ remediation of lead-
contaminated aqueous solutions and soils through toxicological studies on
amphibian Xenopus laevis which showed that the MBM ashes efficiently
immobilized lead in solution and reduced its bioavailability. Moreover, in the
Nicotiana tabaccum tobacco plant studies they found that the ashes both
neutralize lead toxicity and act as fertilizer. The effectiveness of MBM
addition in the immobilization of Pb, Zn and Cd in a contaminated mine waste
made-ground was investigated by Sneddon et al. (2006). The MBM treatment
resulted in a significant reduction of leachate heavy metal concentrations.
Fish bones-originated phosphates were successfully used to reduce the
mobility of Cd, Co, Hg, Pb and U in a contaminated soil by Kaplan and Knox
(2004) and to immobilize Cr and Pb in aqueous solutions by Ozawa et al.
(2003) and Ozawa and Kanahara (2005). A by-product of the fishing industry
is another important phosphate source for remediation treatment, Apatite II™
having the general composition of Ca10-xNax(PO4)6-x(CO3)x(OH)2 where x<1
(Conca and Wright, 2006). Apatite II™ has low contents of heavy metals and
30-40% residual organics. It has a poorly crystalline structure with respect to
other forms of apatite (Martin et al., 2008), it is highly microporous and

provides a source of phosphate to reduce metal concentrations in aqueous
solution. Apatite II™ was successfully used within a permeable reactive
barrier to reduce the concentrations of Pb and Cd in acid mine drainage
(Conca and Wright, 2006) and as soil amendment to mitigate Cd uptake by
tobacco plants (Keller et al., 2005). The effects of organics associated with
Apatite II™ on its metal removal capacity were evaluated by Martin et al.
(2008). They showed that the organic matter content of this phosphate source
can reduce its Pb removal capacity because of the biological growth, enhanced
by the presence of organic matter, which interferes with the surface sorption of
Pb on the Apatite II™ particles, and the reduction of available PO43- ions in
solution due to their consumption as nutrient source by the biological growth.
Zheng et al. (2007) showed that another phosphate source could be eggshell
waste as carbonate hydroxyapatite synthesized using this waste by-product can
effectively reduce Cd and Cu concentrations.
Finally, various types of phosphate amendment (BM, SSP, TSP and MKP)
have been tested for the immobilization of heavy metals (Pb, Zn, Cu and Cd)
and metalloids (As and Sb) in two mining-impacted soils by means of kinetic
column leaching experiments (Munksgaard and Lottermoser, 2011). MKP
resulted the most effective amendment for Cd immobilization, whereas SSP
and TSP for Pb stabilization. On the contrary, phosphate application did not
significantly reduce the release of As, Cu, Mn, Sb and Zn. The results of this
study highlight the need for detailed soil characterization and trial of
phosphate sources before phosphate-induced immobilization is applied.
4. MECHANISMS OF HEAVY METAL IMMOBILIZATION

Phosphate-based amendments have been widely used to immobilize heavy
metals in contaminated soils. The mechanisms of the metal retention are
different for the various metals and include: dissolution/precipitation reactions,
ion exchange processes, adsorption, substitution of Ca by metals during
crystallization (e.g., Gómez del Río et al., 2004; Jeanjean et al., 1994; Ma et
al., 1994; Vega et al., 2003; Xu et al., 1994). However, limited knowledge on
the relative contribution of each process in immobilizing metals still exists in
the literature (e.g., Cao et al., 2004; Xu et al., 1994).
Phosphate treatment of Pb-contaminated soils reduces the Pb mobility by
the formation of geochemically stable pyromorphite-type minerals
[Pb5(PO4)3X; X = F, Cl, Br or OH] (e.g., Cotter-Howells and Caporn, 1996;
Ma et al., 1993; Mavropoulos et al., 2002; Nriagu, 1974; Xu and Schwartz,

1994; Zhang et al., 1998). The mechanism involves the dissolution of
phosphate amendments and subsequent precipitation of new Pb-phosphates
having very low solubility and bioaccessibility and which are not bioavailable
(Arnich et al., 2003; Hettiarachchi et al., 2001; Scheckel and Ryan, 2003). The
dissolution/precipitation mechanism can be represented by the following
reactions:
Ca5(PO4)3OH + 7H+ → 5Ca2+ + 3H2PO4- + H2O (1)
5Pb2+ + 3H2PO4- + H2O → Pb5(PO4)3OH + 7 H+ (2)
Chloropyromorphite [Pb5(PO4)3Cl] (CP) is the most stable form of Pb-

phosphate that can be formed in soils, followed by hydroxypyromorphite
[Pb5(PO4)3OH] (HP) and fluoropyromorphite [Pb5(PO4)3F] (FP) (Chen et al.,
1997). The precipitation of FP was the main mechanism for Pb immobilization
in soils amended with PR containing F (Cao et al., 2004), whereas CP was the
dominant Pb-bearing phase in port facility soils where a phosphoric acid
production plant was the phosphorus source (Ruby et al. 1994). The formation
of pyromorphite in soil requires dissolution of Pb-bearing minerals and
phosphate amendments being the process strongly pH-dependent. Zhang and
Ryan (1998) showed that complete dissolution of HAP was obtained at pH
between 2 and 5, whereas sorption of Pb on the HAP surface was found at pH
6 to 7.
Although pyromorphite formation was shown to be the main mechanism
for Pb immobilization, the sorption of Pb ions on the HAP surfaces followed
by ion exchange with Ca has been reported by many authors (e.g., Baillez et
al., 2007; Cao et al., 2004; Deydier et al., 2003; Hashimoto and Sato, 2007;
Ma et al., 1993; Mavropoulos et al., 2002; Suzuki et al., 1984; Xu and
Schwartz, 1994; Xu et al., 1994). This mechanism can be represented by the
following reaction:
Ca5(PO4)3OH + xPb2+ → xCa2+ + Ca5-xPbx(PO4)3OH (3)
In particular, Mavropoulos et al. (2002) found that the sorption

mechanism contributed to the immobilization of about 30% of the total Pb.
The mechanism includes two steps: rapid surface complexation of Pb2+ ions on
the ≡POH sites of HAP and secondly ion exchange with Ca2+ ions followed by
the formation of Pb-containing phosphate (e.g., Baillez et al., 2007;

Mavropoulos et al., 2002).
The results of several studies suggest that the mechanisms of
immobilization of other heavy metals are quite more complex and dependent
on factors, such as solution pH and the presence of competitive species (e.g.,
Corami et al., 2007, 2008b; Ma et al., 1994; Xu et al., 1994). The formation of
amorphous Cu phosphate precipitates on HAP surface was suggested by Ma et
al. (1994). Similar findings were reported by Sugiyama et al. (2003) who
proposed the dissolution/precipitation process as the main mechanism for the
removal of Cu. However, Cao et al. (2004) concluded that dissolution/
precipitation reactions were responsible for the removal of only 25% of Cu by
PR, whereas the metal was mainly immobilized by surface adsorption or
complexation. Recently, Liu and Zhao (2007) showed that the application of
nano-sized vivianite [Fe3(PO4)2∙8H2O] in Cu-contaminated soils resulted in the
formation of Cu3(PO4)2 and Cu5(PO4)3OH precipitates. The formation of Cu
phosphate (libenthenite) was reported also by Dybowska et al. (2009);
however, as amongst all the phosphate amendments they tested (synthetic
HAP, natural PR and mammalian meat and bone meal) the precipitation of
metal phosphates occurred only with HAP, they concluded that the
immobilization of the metal occurred mainly through surface sorption and
possibly also through ion exchange rather than dissolution/precipitation
mechanism. In this view, Šljivic et al. (2009) suggested that Cu
immobilization by phosphate treatment includes at least formation of surface
compounds and ion exchange. The results of a combined structural simulation
and EXAFS analysis of the sorption of Cu on HAP confirmed that the fixation
of Cu occurs in the surface sites of the phosphate amendments (Corami et al.,
2008a).
As for Cd, Da Rocha et al. (2002) suggested that Cd immobilization can
be associated to the ion exchange and complexation mechanisms. Similar
results were obtained also in subsequent studies suggesting that Cd fixation by
phosphate amendments involves surface adsorption, ion exchange and
dissolution/precipitation processes (e.g., Corami et al., 2008b,c; Marchat et al.,
2007; Tõnsuaadu et al., 2008; Zhu et al., 2008). In particular, Marchat et al.
(2007) proposed a two steps mechanism: substitution of Ca2+ ions in the HAP
structure by Cd2+ ions sorbed on the surface of HAP grains followed by their
incorporation into the hydroxyapatite bulk to form a solid solution, Ca10-
xCdx(PO4)6(OH)2.
Surface complexation and ion exchange processes rather than
dissolution/precipitation have been suggested as immobilization mechanisms
also for Zn (Chen et al., 1997; Corami et al., 2007; Raicevic et al., 2009; Xu et
al., 1994). Chen et al. (1997) reported that the formation of Zn-phosphate,
hopeite [Zn3(PO4)2∙4H2O], would occur only at pH 2. The formation of hopeite
at the beginning of the reaction of HAP with Zn2+ ions at pH > 4 was reported
by Lusvardi et al. (2002), whereas a mixed Zn-Ca phosphate (scholzite,
CaZn2(PO4)2∙2H2O) occurred as final phase. The formation of a P-Zn complex
in organic and inorganic colloid surface as been identified by Perez-Novo et
al. (2011) as the main mechanism responsible of the observed increase in Zn
sorption in the acidic soils they studied. Lee et al. (2005) showed that the
presence of CO2, due to exchange with air, significantly affects the mechanism
of Zn immobilization by HAP that depends on pH and Zn concentration. At
low [Zn] the immobilization is ascribed to sorption processes that are
accompanied by the precipitation of hydrozincite [Zn5(OH)6(CO3)2] at higher
[Zn]. The increase of Zn concentration results in the precipitation of hopeite as
the dominant mechanism of Zn immobilization. Similar results have been
reported by Sheha (2007) who found that precipitation of hopeite and the
possibly simultaneous adsorption of Zn(OH)+ occurs in the pH range 8-11.5,
whereas at pH 2-8 Zn is removed through surface complexation interactions
and/or ion exchange process.
In contrast to other heavy metals, relatively limited information about Co
immobilization by phosphate amendments has been found in the literature.
According to Gómez del Río et al. (2004) the immobilization of the metal
involves ion exchange and specific sorption over the ≡POH surface groups of
HAP. Similar results have been reported also by Dimović et al. (2009), Pan et
al. (2009), Saxena and D’Souza (2006), Smičiklas et al. (2006) showing that
the ion exchange process acts as the main mechanism for Co2+ removal;
however, the formation of surface complexes contributes to the overall
immobilization mechanism.
The immobilization of Ni by means of phosphate treatment has been
investigated by Elouear et al. (2009), Mobasherpour et al. (2011), Perrone et
al. (2001), Van Nostrand et al. (2007), Zupančič et al. (2006). The results of
these studies suggest that Ni immobilization occurs through both surface
complexation of Ni ions on the ≡POH sites and ion exchange with Ca resulting
in the formation of a compound with formula Ca10-xNix(PO4)2(OH)2.
Compared to other heavy metals, little attention has been paid on Cr
removal by phosphate amendments. Al-Asheh et al. (1999) postulated that the
mechanism for immobilization of Cr3+ by HAP is ion exchange with Ca ions
according to the following reaction:
Ca30[(PO4)6(OH)2]3 + 20Cr3+ → Cr20[(PO4)6(OH)2]3 + 30Ca2+ (4)
Three successive steps have been suggested for the immobilization of the
metal ions: surface complexation of the ions, dissolution of the phosphate
amendments followed by the precipitation of Cr20[(PO4)6(OH)2]3 and slow
metal substitution of Ca (Deydier et al., 2003).
In summary, the mechanisms of the metal immobilization are quite
complex and different for the various metals including dissolution/
precipitation reactions, formation of surface complexes and ion exchange with
Ca. The relative contribution of each process is difficult to be clearly
ascertained and it seems that they could all be involved in the overall
immobilization of heavy metals by phosphate amendments.
5. EFFECT OF PHOSPHATE TREATMENT ON SOIL PH

Mobility of heavy metals in soil environment depends on many factors,
such as metal speciation and total metal content in the soil, type of soil, soil
pH and moisture content. The addition of phosphate-based amendments
changes the soil pH and thereby the mobility of heavy metals. Acidic
conditions favour the dissolution of phosphate amendments to provide
available phosphate ions to immobilize the metals. For example, in alkaline
conditions the availability of phosphate ions released by HAP dissolution is
significantly reduced compared to acidic conditions. This is related to the
solubility of the amendment as it decreases two orders of magnitude for the
increase of each unit of pH in the range 5-8 (Shi and Erickson, 2001). The
application of acidic phosphate salts, such as TSP, DAP, MCP generally
decreases soil pH due to acidification reaction, whereas addition of other
phosphate amendments (e.g., HAP and PR) increases soil pH mainly because
of the ligand exchange phosphate adsorption reaction.
Acidic conditions are essential for Pb immobilization through
dissolution/precipitation mechanism as acidity favours the dissolution of both
Pb sources and phosphate amendments. Inducing acidic conditions is
necessary for efficient Pb immobilization as in these conditions the dissolution
of Pb compounds, such as cerussite, anglesite and galena is enhanced (Cao et
al., 2003a,b; Ryan et al., 2001; Zhang and Ryan, 1998, 1999a,b). The results
reported by Zhang and Ryan (1998) showed that although very low pH values
(pH 2) resulted in a rapid dissolution of HAP, no reduction of Pb solubility
occurred as CP did not precipitate in these pH conditions. Conversely, Pb
concentration was significantly reduced when pH ranged between 4 and 5 as

the formation of CP was optimized (Zhang and Ryan, 1998, 1999a,b). Also,
the formation of HP is favoured at pH 5 following the interaction of HAP with
cerussite, massicot and litharge (Laperche et al., 1997). Therefore, the results
of these studies suggest that initial acidic conditions are necessary for the
dissolution of phosphate sources, while the buffering of pH at almost neutral
conditions is desiderable in order to reduced Pb solubility by precipitation of
pyromorphite.
Addition of natural apatite to a soil contaminated with Cd, Zn and Pb
shifted pH from 5.41 to 5.67 and 5.97 for the two application rates (25 and 50
g/kg), respectively (Knox et al., 2003). Apatite dissolution consumes H+,
thereby reducing soil acidity. Moreover, the impurities of CaCO3 of this
apatite may act as a liming agent increasing soil pH. Chen et al. (2007) also
reported that the addition of HAP and PR increased soil pH (from 5.45 to 5.56
for the larger increase) and in agreement with the results reported by Cheung
et al. (2001) and Hodson et al. (2000) the alkaline properties of HAP were
attributed to its significant content of CaCO3. The treatment of contaminated
sediment with mined apatite also increased the pH: 4.91 in the control
compared to 5.79 of the treated soil (Kaplan and Knox, 2004). This increase
was the result of the H+-consuming reaction of the amendment and ligand
exchange of phosphate for hydrolysis on the surfaces of the minerals. Also
Zhang and Pu (2011) reported pH increase due to the application of PR and
MCP to both calcareous and acidic soils. In another study the application of
PR had negligible effect on soil pH as after the application of the amendment
pH varied from 6.9 to 7.11 and 7.80 to 8.0 in the two studied soils,
respectively (Chen et al., 2006). Similar results after treatment with PR were
reported also by Cao et al. (2003b), Hettiarachchi et al. (2001), Ma and Rao
(1999). Slight increase in soil pH after PR addition was reported by Cui et al.
(2010). Conversely, a strong pH increase was induced by the addition of MCP
and it was related to the much higher solubility of MCP than PR.
Contrary to low soluble phosphate sources, the addition of high soluble
phosphate amendments (DCP, TSP, PA, DAP) decreases soil pH. For
example, Chen et al. (2007) reported a significant reduction (5.56-5.23) of pH
due to the application of DAP. Basta and McGowan (2004) found that DAP
decreased soil pH from 7.1 to 6.5. Thawornchaisit and Polprasertb (2009)
reported that the application of TSP and DAP decreased soil pH with values in
TSP-treated soils lower than those in DAP-treated soils. However, the
decrease was negligible even increasing the amount of phosphate added. The
application of PA reduced pH in all the treated plots of the study reported by
Cao et al. (2003a). Slightly higher pH values were measured when PA was
applied in combination with Ca(H2PO4)2 or PR. These findings were
confirmed by other studies (e.g., Cao et al., 2008; Chen et al., 2003). The
decrease of pH induced by the application of acidic phosphate salts depends on
the application rate. For example, Yang et al. (2001) reported a decrease of
soil pH from 7.22 to 4.34 after the addition of PA at the rate of 5000 mg/kg.
The application of PA at the dose of 2500 mg/kg promoted the reduction of pH
from 7 to 5.2 (Hettiarachchi et al., 2001). Also the buffering capacity of the
soil affects the decrease of pH promoted by the application of PA and a
significant reduction of pH occurs especially in soils with low buffering
capacity (e.g., sandy soils) (Melamed et al., 2003).
The literature review about the effects of phosphate treatment on soil pH
shows that in general the pH conditions in the soil environment heavily affect
the effectiveness of phosphate treatment as slightly acidic conditions are
necessary to optimize the mobility of heavy metals and the release of
phosphate ions from the amendment.
6. EFFECTS OF APPLICATION RATE AND GRAIN SIZE

The amount of phosphate amendments to be applied to immobilize heavy
metals in contaminated soils is a critical factor to obtain effective
immobilization and reduce environmental risks related to the leaching of
excess P and enhanced release of other contaminants in the soil environment.
Generally, molar P/Me ratios have been used as a basis to calculate the
suitable application rate although the amount of phosphate to be added has
also been calculated as a weight percentage of the soil without considering the
molar ratio. In Pb-contaminated soils, as the objective of phosphate treatment
is to induce the immobilization of the metal mainly through the precipitation
of pyromorphite, the application rate P/Pb of 3.5 has been suggested on the
basis of pyromorphite stoichiometry (Ma et al., 1993; Zhang and Ryan,
1999a,b). Zhang and Ryan (1998) used HAP at ratios of 3:5, 6:5 and 9:5 on the
basis of the molar ratio P/Pb. With these application rates the amounts of the
added phosphate were 1, 2 and 3 times the quantity necessary to form
pyromorphite. The increase in the application rate resulted in the increase of
the dissolution of Pb-bearing materials and precipitation of pyromorphite.
Therefore, the availability of phosphate ions is the limiting reactant of the
process. The treatment rates of Ca(H2PO4)2∙H2O for immobilizing Pb in two
soils from shooting ranges were 2.5, 5.0, 7.5 and 10% by soil weight (Cao et
al., 2008). The stoichiometric P/Pb ratio of CP has been used by Hashimoto et
al. (2009) in another Pb-contaminated soil from a shooting range in Japan. In
two pilot-scale field demonstrations in Pb-contaminated sites phosphate
amendments were applied at a molar ratio P/Pb of 4 as among various molar
ratios previously tested it was sufficient to reach the immobilization of Pb in
these sites (Chen et al., 2003; Melamed et al., 2003). In general, the molar
ratios P/Pb range from values (0.1-0.6) below the stoichiometric value of
pyromorphite to values exceeding this ratio by three orders of magnitude (526-
1439) (Cao et al., 2002; Cao et al., 2003a; Cotter-Howells and Caporn, 1996;
Liu et al., 2001; Ma and Rao, 1997; Xenidis et al., 2010; Xu and Schwartz,
1994; Yang et al., 2001; Zhang and Ryan, 1999a). Application rates based on
the stoichiometry of the final products of the immobilization in multi-element
contaminated soils are difficult to be determined as the chemical composition
of the products formed is uncertain and also because in real environments
there is no stoichiometric behaviour. Moreover, the total amount of phosphate
ions applied may not react with insoluble fractions of heavy metals (Basta and
Gradwohl, 2001; Hettiarachchi and Pierzynski, 2002). In these cases an excess
of phosphate is necessary to ensure that enough phosphate ions are available
for immobilizing most heavy metals in soil. For the treatment of a soil
contaminated with Cd, Zn and Pb using natural apatite, application rates of 25
and 50 g/kg have been based on the Pb concentrations (Knox et al., 2003).
Plant toxicity and metal concentrations in the tissues were significantly
reduced by the application of the lowest rate, whereas only minimal
improvement was obtain using the rate of 50 g/kg. Increasing the application
rates of three different phosphate fertilizers from 20 to 80 kg/ha led to an
increase in the extractability of Cd (Lambert et al., 2007). For the behaviour of
Zn during the phosphate treatment no simple relationship could be observed
between the application rate of the amendments and the amounts of metal
extracted. Phosphate rock with an application rate of 5 was used by Chen et al.
(2006). This application rate was based on the total metal content and taking
into account the reduced solubility of PR compared to other phosphate
amendments. A relatively low (1:50) application rate of bone meal resulted
effective in the treatment of a soil contaminated with Zn, Ni, Cu and Pb
(Hodson et al., 2001). Application rates P/Pb ranging from 0:1 to 4:1 of PR
and KH2PO4 were used to evaluate the bioavailability of Pb, Cd and Zn in a
contaminated soil (Zwonitzer et al., 2003). The amount of phosphate added to
the soil did not significantly affect Pb concentrations in sorghum-sudan grass
(Sorghum bicolor L. Moench) tissues. Conversely, high application rates of
the amendments resulted in lower Cd concentrations in plants compared to low
application rates. High amounts of amendments have the same effect also for
Zn when PR was used, whereas in KH2PO4 treated soils the opposite effect
was observed. The authors concluded that this was likely related to the
reduction of pH induced by the application of KH2PO4. The application rate of
phosphate amendments has effect also to the uptake of contaminants by maize
(Zea mays L.) and bean (Phaseolus vulgaris L.). Boisson et al. (1999) applied
HAP on a 0.5, 1 and 5% (w/w) basis to a soil contaminated with Zn, Pb, Cu
and Cd. The addition of 1% HAP resulted in best growth responses whereas
the higher application rate caused growth inhibitions in both species. With the
increase of application rates a decrease in contaminant uptake was observed in
maize leaves. However, also the uptake of some nutrients decreased. In bean
plants the highest application rate caused an increase in the uptake of Cu, Zn
and Ni following the gradual decrease observed increasing the application rate
from 0.5 to 1%. Also, treatment effectiveness increased with phosphate dose in
a synthetic Cd-contaminated soil when TSP, DAP and PR were used at molar
ratios P/Cd of 1:2, 2:3, 1:1 and 2:1 (Thawornchaisit and Polprasertb, 2009).
The concentration of leachable Cd decreased with increasing the application
rate and the most effective dose was 2:1. At this application rate DAP was not
as effective as TSP likely because of the higher solubility of TSP than that of
DAP, providing larger amounts of phosphate to immobilize Cd in soils
compared to DAP. The decrease of Cd concentration in the exchangeable-
soluble fractions of seven contaminated soils with the increase in the
application rate of KH2PO4 was observed also by Bolan et al. (2003).
The results of these studies show that generally the effectiveness of the
immobilization increases with the application rate of the amendments.
However, high application rates should be avoided as they may produce
phytotoxic effects because the same mechanisms that are responsible of metal
immobilization can reduce the uptake of micronutrients by plants. Also, high
application rates involve potential consequences of phosphate leaching in the
environment. Moreover, high phosphate dose involves the alteration of the
properties of the soil and high operation costs. In this view, the results reported
by Montinaro et al. (2008) show that efficient Pb immobilization can be
achieved even at low application rate through the mechanical treatment of the
soil. In particular, immobilization efficiency in soil containing 3% of HAP on
a weight basis and mechanically treated is higher than that obtained in soils
with 5% of HAP without milling. Therefore, the mechanical treatment
drastically reduces the amount of phosphate amendment necessary for efficient
metal immobilization. This avoids significant phytotoxic effects, reduces the
operating costs and also possible input of metals to the soil due to impurities of
the amendments.
The effectiveness of phosphate treatment in soil also depends on the grain
size of the amendment when low soluble sources of phosphate ions such as PR
are used. This aspect has not drawn much attention in the literature as a few
studies addressed this issue. Chen et al. (2006) evaluated the effect of grain
size of PR in two contaminated soils by means of pot experiment. Four
different grain size of PR were applied: <35, 35-72, 72-133 and 133-266 m.
The application rate of PR was 5, based on the P/Me (Pb, Zn, Cd and Cu)
molar ratio. The effectiveness of phosphate treatment was determined
measuring the metal uptake in the shoots of Brassica oleracea L. The results
showed that the concentrations of the metals in the plant shoots decrease with
the decrease of grain size of the amendment. The application of PR having
smaller grain size more effectively reduced the phytoavailability of the metals
and increased the stability of the metals in the soils. This finding is likely
related to the higher specific surface area of the smaller grains compared to
that of larger particles, further suggesting that the overall immobilization
mechanism involves sorption processes on the surface of the phosphate
amendments.
7. PHOSPHATE-INDUCED CONTAMINANTS DESORPTION

Phosphate addition in multi-element contaminated soils may have the
undesirable effect to enhance the mobilization of anionic contaminants, such
as arsenic, selenium and antimony where cationic and anionic contaminants
coexist in soil (Kaplan and Knox, 2004; Mihaljevič et al., 2009; Munksgaard
and Lottermoser, 2011; Smith et al., 2002; Wasay et al., 2000).
Pentavalent oxoanions of As, Sb and P have similar chemical reactivity
and their sorption behaviour follows similar patterns. Therefore, the
replacement of sorbed As and Sb by P anions has been proposed as the main
mechanism contributing in the mobilization of As and Sb in phosphate-
amended soils (Impellitteri, 2005; Liu et al., 2001; Smith et al., 2002; Tighe et
al., 2005; Violante and Pigna, 2002; Xenidis et al., 2010). The competitive
effect of phosphate ions has also been reported for As(III) sorbed on iron
oxides (Dixit and Hering, 2003). However, high contents of Fe oxides in the
soil reduce the competition, although an increase in phosphate addition
improves the competition even in these soils. There is still lack of information
about the behaviour and distribution of Sb in soil as well as about the
competitive effects of phosphate on Sb adsorption. However, significant

release of Sb (and As) from contaminated soils following phosphate treatment
has been reported (e.g., Kilgour et al., 2008). In particular, mobilization of Sb
by phosphate addition seems to depend on the oxidation state being Sb(III)
less extractable than Sb(V).
Arsenic occurs naturally in the geosphere and the major sources of As in
soils are anthropogenic inputs, such as disseccants, fertilizers and pesticides
(Smith et al., 1998). Arsenic compounds accumulate in soil due to slow
depletion by plant uptake, erosion and leaching. In the environment inorganic
As occurs in the oxidation states (V) and (III) and their relative distribution
depends on pH and redox conditions (Lombi et al., 1999; Van Elteren et al.,
2002). In aerobic and natural pH conditions As(V) (arsenate) is predominant
whereas the more toxic form As(III) (arsenite) exists under anoxic conditions
in moderately reducing environments. In soil the mobility of As compounds is
affected by the type and amount of adsorbing soil components, pH and redox
potential (Bissen and Frimmel, 2003; Duker et al., 2005). Arsenic adsorption
in soil is highly correlated to the occurrence of iron oxides suggesting that iron
(hydr)oxides play an important role in the adsorption-desorption reactions
controlling As mobility in soils.
Antimony occurs naturally in most soils at trace levels. Industrial uses of
Sb includes the manufacture of flame retardants, semiconductors, batteries,
automobile brake linings and pigments (Filella et al., 2002a; Oorts et al.,
2008). Antimony compounds are released in the environment mainly by the
incineration of waste, emissions of smelters and combustion of fossil fuels. In
soil Sb exists in the oxidation states (III) and (V) being the pentavalent Sb the
dominant species. Tivalent Sb occurs in natural pH range but in the presence
of Fe and Mn oxides readly oxidizes to Sb(V) (Belzile et al., 2001; Filella et
al., 2002b). In soil environments Sb mobility depends on speciation and
interactions with iron (hydr)oxides and organic matter (Johnson et al., 2005;
Mitsunobu et al., 2006).
Sorption of both As and Sb on iron (hydr)oxides in soil is affected by the
presence of phosphate ions (Hongshao and Stanforth, 2001; Jackson and
Miller, 2000). In particular, the experimental results reported by Liu et al.
(2001) suggest that phosphate and arsenate compete for surface sites on
goethite grains. The sorption mechanism of the two anions on goethite
involves a rapid initial sorption and the formation of surface precipitates
(Hongshao and Stanforth, 2001). The effect of high concentrations of
phosphate ions on the mobilization of As were evaluated by several studies
(e.g., Alam et al., 2001; Boisson et al., 1999; Goh and Lim, 2005; Impellitteri,
2005; Munksgaard and Lottermoser, 2001; Zeng et al., 2008) whereas a

limited number of studies were carried out to examine the effect of low
concentrations of phosphate ions (e.g., Mihaljevič et al., 2009).
The removal of As from naturally contaminated soil by the application of
PA and KH2PO4 was studied in laboratory batch experiments by Zeng et al.
(2008). Both amendments reduced soil As content more than 20% and their
ability to remove As could be attributed to the replacement of As by phosphate
ions. The application of two forms of apatite, biogenic and mined, as
amendment in a sediment contaminated with Cd, Co, Hg, Pb and U
significantly reduced the concentrations of these contaminants (Kaplan and
Knox, 2004). However, phosphate treatment enhanced desorption of As, Se
and Th as the result of phosphate competitive effect. Moreover, the application
of apatite favoured the desorption of organic matter. The increase in As and Sb
mobility induced by the application of different phosphate amendments in
mining-impacted soils has been reported by Munksgaard and Lottermoser
(2011). However, the different phosphate sources have different effects as
SSP, TSP and MKP caused As and Sb release whereas in BM-treated soils no
increase in As and Sb mobility was observed. This was likely due to the lower
content of phosphate dissolved in the experiments with this amendment
compared to the others. Different effects on the mobility of arsenic as a
consequence of the application of various types of amendments have also been
reported by Cui et al. (2010) for a Pb-As contaminated soil from an abandoned
Pb-Zn mine. The addition of PR did not result in an increase in water-soluble
As whereas it increased in the treatments with MCP and SSP. The application
of ferrous sulphate determined a significant decrease in As water-soluble
concentrations suggesting that combined treatment by phosphate and ferrous
sulphate could be an effective approach to reduce metal availability in multi-
element contaminated soils. Enhanced Sb leaching was reported also for
phosphate treated Japanese soils by Nakamaru et al. (2006). However, the
application of NaH2PO4 showed that only 20-40% of soil-sorbed Sb was
phosphate exchangeable suggesting that ligand exchange mechanisms also
contribute to Sb sorption in Japanese soils.
Arsenic release from soils increases its phytoavailability and thereby its
accumulation by plants (Cao et al., 2003b). In a greenhouse experiment TSP
was added to soil at a high application rate (3867 mg/kg) to mimic excessive
application of phosphate as fertilizer and addition of phosphate amendments
for immobilization of heavy metals. The results showed that application of
phosphate to soil increased As accumulation in carrot (Daucus carota L.) and
lettuce (Lactuca sativa L.) by about 5-9 and 2.5-10 times, respectively (Cao
and Ma, 2004). The observed high As uptake by vegetables was mainly
produced by the increase in the water soluble, exchangeable and carbonate As
fraction induced by phosphate ions replacement. This negative effect of
phosphate treatment was significantly minimized through biosolid compost
application which reduced As phytoavailability.
Although enhanced As phytoavailability induced by phosphate application
increases the risk of human exposure to toxic As, it is a key factor for effective
phytoremediation as it can promote adequate pollutant concentrations in the
soil solution for plant uptake and accumulation of contaminants in the
biomass. Chinese brake fern (Pteris vittata L.) is an As hyperaccumulator. It is
an ideal species for phytoremediation because the plant accumulates most of
As in its aboveground biomass and has large biomass. The effects of
phosphate amendments on As uptake by Chinese brake fern have been
investigated by Cao et al. (2003b). The results showed that frond As
concentrations increased up to 265% compared to the control. The authors
concluded that the effectiveness of the remediation of As-contaminated soils
through phytoextraction by Chinese brake fern can be significantly improved
using phosphate amendments to increase As phytoavailability in the soil. In
multi-element contaminated soils phosphate treatment can increase the
accumulation of As in Pteris vittata and also reduce metal uptake by the plant.
In a soil spiked with metals (Pb, Cd and Zn) and As the application of PR
significantly reduced Pb and Cd uptake by the fern and conversely enhanced
As accumulation in the fronds (Fayga and Ma, 2006). The results of this study
further suggest that PR can be an effective amendment for phytoremediation
of soils contaminated with As and heavy metals.
Firing ranges are an important source of Sb contamination because it is
used in the manufacture of bullets as an hardening agent of the Pb-Sb alloy
(Chrysochoou et al., 2007; Griggs et al., 2011). Numerous types of phosphate
amendments have been tested for Pb stabilization in firing range soils (e.g.,
Cao et al., 2003c; Chen et al., 2007; Moseley et al., 2008). However, limited
information about the effect of phosphate treatment on Sb mobility in these
soils is reported in the literature. The effect of phosphate treatment on the
mobility of Sb in firing range soils has been evaluated by Griggs et al. (2011).
Also in these soils the application of four different phosphate-based
amendments enhanced Sb mobility. However, the application of Buffer
Block® amendment did not induce Sb release. This is likely due to the
inclusion of aluminium hydroxide in the amendment composition.
The results of this study further show that Al and Fe (hydr)oxides play an
important role in reducing the mobility of Sb in contaminated soils. Conesa et
al. (2010) observed that phosphate (K2HPO4) addition resulted in an increase

in water and NaNO3-extractable Sb concentrations in two firing range soils
(calcareous with low organic carbon and non-calcareous with elevated organic
carbon content). Also, the addition of phosphate amendments (DAP and MCP)
increased the mobility of Cu and Sb in the soil from a firing range (Spuller et
al., 2007).
Although phosphate treatment effectively immobilizes most of heavy
metals in soils, it may increase environmental risks promoting the mobilization
of other contaminants. In this view, site specific information about soil
mineralogy and physicochemical conditions is necessary to define best
management approach for the remediation of multi-element contaminated
soils.
CONCLUSION
Remediation of contaminated soils using phosphate amendments has
proven effective at reducing the mobility of metals, such as Pb, Cd, Cu, Zn,
Ni, Co, Cr, Sb, etc. Phosphorus-containing materials ranging from readily to
moderately and less soluble along with biogenic phosphates have been used
for soil chemical stabilization. However, the type of phosphate source and the
rate of application should be carefully evaluated to minimize potential
drawbacks of this remediation approach. Indeed, the effect of intensive
phosphate addition on eutrophication of surface water is a main concern. In
this view, the application of mixed phosphate sources could improve the
effectiveness of the treatment reducing the risk of P leaching. Moreover,
enhanced mobility of untargeted contaminants, including mainly As and Sb,
induced by phosphate treatment needs to be addressed. In the remediation of
contaminated soils the best approach can be defined only when specific
information about soil mineralogy and physicochemical conditions of the site
is carefully evaluated.
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Chapter 4
METAL CONTAMINANT SOURCE,

TRANSPORT AND FATE IN
THE ENVIRONMENT AND
PHYTOREMEDIATION METHODS
S. H. Desouki and H. Feng*

Department of Earth and Environmental Studies,
Montclair State University, Montclair, New Jersey, U. S.
ABSTRACT
Heavy metal pollution due to anthropogenic sources is on the rise
causing great environmental and human health problems. Although
remediation of metal contaminants can be via excavation or stabilization
of the soil or sediments, these methods can be quite costly in the long run
and it is not efficient for the small-scale removal of heavy metal
contaminants. Phytoremediation is a fairly new and “green” technology,
and with a focus on heavy metals as the contaminants in question, this
method utilizes plants for in-situ reduction and/or removal of
contaminants from soil, sediments and water. The process of
phytoremediation is quite simple, and with the proper selection of plants
and appropriate site conditions, the risk from these contaminants can be
*
Authors for Correspondence:fengh@mail.montclair.edu (H. Feng). Posting address:Department
of Earth and Environmental Studies, Montclair State University, Montclair, New Jersey
07043, USA.
82 S. H. Desouki and H. Feng
reduced and minimized over time. Phytoremediation is energy efficient,

and an aesthetically pleasing method of remediating sites with low to
moderate levels of contamination. The purpose of this paper is to provide
a general review of metal contaminant source, transport and fate as well
as current phytoremediation methods including their advantages and
disadvantages, and the future directions in research using these methods.
Keywords: Heavy metal pollution;source;transport;phytoremediation.
1. INTRODUCTION
Heavy metal pollution is a widespread problem that has increased
dramatically after the industrial revolution (Goldberg et al. 1979; Trefry and
Shokes 1981; Windom et al. 1989; Bopp and Simpson 1989; Feng et al. 2004,
2008). While it is well known that contamination of heavy metals can be due
to natural sources, the widespread pollution of terrestrial and freshwater
ecosystems is aggravated due to anthropogenic sources (Rai, 2009). After the
advent of the industrial revolution in the early 18th – 19th century, major
advances were made in the way agricultural, mining, transport and
manufacturing activities were done (Crafts, 1985). While the advantage of the
industrial revolution entails an increase in the socioeconomic quality of life for
many humans all around the world – the benefit of such growth isn’t without
costs (Nriagu, 1996). Pollution of the biosphere has increased dramatically due
to the extensive industrial, military, and farming activities that have released
many inorganic pollutants into the environment in the past century (Memon
and Schröder, 2009). Despite the benefits of developing land for human use,
there is an increase in pollutants contaminating our soils and entering our
waterways via surface water runoff. Urbanization due to population growth
and excessive utilization of natural resources, are the main causes for soil,
water and air pollution (Memon and Schröder, 2009).
In order to fully understand the problems and harmful effects of heavy
metal pollution, it is important to define the term “heavy metals”. According to
a technical report published by The International Union of Pure and Applied
Chemistry (IUPAC), and written by John Duffus, the term “heavy metals” is
often misleading and meaningless (Duffus, 2002). The reason for this claim is
due to the fact that the term is often used inconsistently in different situations.
According to this report, the widespread use of the term “heavy metals” in
various publications related to chemical hazards are usually associated with
Metal Contaminant Source, Transport and Fate … 83
the contamination and potential toxicity of these metals. However, according

to legal regulations, there is a list of heavy metals to which this term applies,
and as with most cases involving regulations, this list can differ from one set
to the other – hence the confusion (Duffus, 2002). The tendency to assume that
all heavy metals are highly toxic is unsupported by facts – indeed, this
assumption has no basis in chemical or toxicological data. The importance of
fully understanding the scientific fundamentals of heavy metals and what
constitutes their toxicity in the environment is essential for the advancement of
scientific technologies. In order to rehabilitate and remediate soils from heavy
metal contamination, proper understanding is required in order to effectively
remove the contaminant and it is essential for the future of phytoremediation
processes in the environment.
While it is important for future studies to take into account John Duffus’
IUPAC technical report (Duffus, 2002), for the purposes of this review, we
can generalize the term heavy metals. Heavy metal is a general term which
applies to a group of metals and metalloids that have an atomic density that is
greater than 5 g cm-3 (Nies, 1999). They are inorganic elements that occur
naturally in the Earth’s crust and they are naturally found in soils and rocks
(Duruibe et al., 2007; Wu et al., 2010). Common heavy metals include
cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), lead (Pb), zinc
(Zn), arsenic (As), silver (Ag), iron (Fe), and the platinum group of elements
(Duruibe et al., 2007).
Figure 1. Metals commonly found at Superfund Sites in the US (Data source: US EPA,
1996).
They are widely used in commercial sources such as in electronic

components, machinery and materials. According to the U.S. Agency for
Toxic Substances and Disease Registry (ATSDR), a list ranks lead, mercury,
arsenic, cadmium as first, second, third, and sixth respectively, according to
their prevalence and the severity of their toxicity (McCally, 2002). Figure 1
illustrates the most common contaminants at Superfund Sites within the
United Sites.
Consequently, to supplement the natural geochemical sources of heavy
metals, they can also be released to the environment from a variety of
anthropogenic sources. The most important sources of heavy metals are
combustion processes from incineration, smelting, power generation and the
internal combustion engine.
Table 1. A list of natural and anthropogenic sources of heavy metals as

well as their point and non-point sources of pollution origin. The order of
these processes is listed from the most common to the least common
source (Kennish, 1992)
Natural Sources Anthropogenic Sources

Non-Point Chemical and physical weathering Surface runoff from mining
Sources of metamorphic and igneous rocks operations
Volatilization of heavy metals Combustion of fossil fuels
trapped in sediments and the
subsequent release from soil
Decomposition of plant and animal Urban stormwater runoff that
detritus contains metals from roadways
and atmospheric fallout
Precipitation and atmospheric ______
deposition of airborne particles
from volcanic activity
Wind erosion ______
Forest fire smoke and ash ______
Plant natural accumulation of ______
metals and their subsequent
shedding of above ground portions
Oceanic spray ______
Point ______ Domestic wastewater effluent
Sources from human wastes and
corrosion of water pipes
______ Industrial effluents and waste
sludge
Table 1 provides a summary of the potential sources of heavy metal

pollution. While the number of anthropogenic sources that input heavy metals
into our ecosystem is fairly small, the anthropogenic inputs greatly exceed the
inputs from natural sources.
2. TRANSPORT AND FATE IN ENVIRONMENT

The common definition of dangerous chemicals such as heavy metals, are
those which are toxic, persistent and/or bioaccumulative in the environment
(USEPA, 2006). Since heavy metals cannot be broken down, they are highly
persistent and will tend to accumulate in the environment especially in urban
lake, estuarine or marine sediments (Mudgal et al., 2010). Once they
contaminate the ecosystem, heavy metals can remain a potential threat for
many years (Jabeen et al., 2009). For example, lead (Pb) has been estimated to
have a soil retention time of 150–5000 years and it is reported to maintain high
concentration for as long as 150 years after sludge application to soil (Rai,
2009; Waranusantigul et al., 2008). There is also the risk of formation of
superoxide radicals (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals
(OH-) which can cause severe oxidative damage to lipid, proteins and nucleic
acid molecules (Mudgal et al., 2010). Many heavy metals are toxic to
organisms at low concentrations, however, some heavy metals, such as copper
and zinc are also essential elements to life. The concentration of essential
elements in an organism is homeostatically-controlled under normal conditions
(Babula et al., 2008; Duruibe et al., 2007). Uptake of nutrients is generally
regulated according to nutritional demand - an upset in this balance can create
problems in the organism in question. To illustrate this, enzymes that
synthesize RNA and DNA contain zinc, nickel and copper and it is an integral
part of coenzyme B12 and vitamin B12 (Duruibe et al., 2007). Nonessential
elements such as chromium, lead, and mercury have little or no beneficial role
in the human body and the intake of these metals are often toxic and in
extreme cases lethal (Babula et al., 2008).
Whether the source of heavy metal contamination is natural or
anthropogenic, what determines the concentration in terrestrial and aquatic
organisms depends highly on the size of the source and the extent of
adsorption in the soil and sediment. The degree of adsorption depends on the
type of metal, the absorbent, the physio-chemical characteristics of the
environment (e.g. pH, and redox potential) and the concentrations of other
metals and/or complex chemicals present in the soil, water, and air. Table 2
provides a summary of the known environmental fate and transport of heavy

metals within an ecosystem.
Table 2. Describes the known environmental fate and transport of heavy

metals within an ecosystem
Environmental Fate and Transport
a) Metals that are bound by sediment particles can be

transported via overland flow.
1. Transport
b) Dissolved metals can travel below the land surface and will
by Water
easily sorb to soil particles in the unsaturated zone and the
saturated zone.
a) Metals volatilized into the air can be carried into the land
surface via precipitation and dry fallout.
2. Transport
b) Wind-borne sediment is a potential route for metal transport
by Air
due to the fact that they are readily sorbed to many sediment
types.
Heavy metals accumulate in organisms as a result of direct uptake via

respiration or the consumption of contaminated food. Uptake via food is the
most important method of accumulation in terrestrial organisms and it may
also be important in aquatic environments. Metals exert toxic effects if they
enter the biochemical reactions in an organism. The typical responses caused
by heavy metal interference in biochemical reactions can be: inhibition of
growth, suppression of oxygen consumption, and impairment of reproductive
processes and tissue repair.
3. HEAVY METAL REMEDIATION METHODS

The two most commonly used methods for the removal of heavy metals
are excavation and stabilization of heavy metals. Excavation is one of the
oldest methods used and it is essentially the physical removal of the soil from
the site. The advantage of this method is the complete removal of
contaminants from the area. While this process tends to be quite rapid, the cost
of using this method is also very high. In addition, the transport of the
contaminants to a different area leads to the risk of spreading the contaminated
soil and dust particles during the removal and transport of the contaminated
soil. This method is not cost-effective when large amounts of soil need to be
removed or disposed (Wu et al., 2010).
The second most common method used is the in-situ stabilization
treatment of heavy metals. This method is used to reduce or eliminate the
contaminant’s ability to adversely affect human health and the environment.
Stabilizing heavy metals involves the addition of chemicals to the soil causing
the formation of minerals that contain the heavy metals in a form that is not
easily absorbed by plants, animals, or people. The advantage of using this
method is that it does not disrupt the environment and it does not generate
additional hazardous wastes (Wu et al., 2010). Instead, the heavy metal
combines with the added chemicals to create a less toxic compound so that
they remain in the soil but in a form that is much less harmful to the
environment and humans. While the cost of this method is significantly less
than excavation, it is still quite costly. Other commonly used technologies for
in-situ and ex-situ remediation of heavy metal contaminated soil are pneumatic
fracturing, soil flushing, solidification, vitrification, electrophoresis, chemical
reduction, and soil washing. However, these conventional methods tend to be
limiting in their applicability to small areas and are quite costly (Marques et
al., 2009). The need to develop new and cost-effective technologies has led to
the use of phytoremediation. Phytoremediation is considered an emerging
“green” technology that is effective, non-intrusive and an inexpensive means
of remediating highly contaminated soils (see Table 3). This is due to it being
a more cost-effective alternative to mechanical or chemical methods of
removing hazardous compounds from soil. It is widely popular as each year
new technologies are developing more effective uses of phytoremediation
processes. Not only is it cost-effective and natural, it is also aesthetically
pleasing especially in communities where the technology is being used
(Alkorta and Garbisu, 2001; Jabeen et al., 2009; Rai, 2009).
Table 3. Comparative costs of different heavy metal soil remediation

technologies (Schnoor, 1997)
Type of Remediation Cost/Cubic Meter Time Required

Excavation and Removal $100 - $400 6 – 9 months
In situ Fixation $90 - $200 6 – 9 months
Phytoextraction $15 - $40 18 – 60 months
3.1. Phytoremediation
There are different ways plants can remediate a site. These methods are
subdivided below.
3.1.1. Phytoextraction
The process of phytoextraction involves the uptake of the contaminant by
plant roots and the subsequent accumulation in the aerial parts of the plant.
After sufficient plant growth and accumulation, the aerial plant parts are
harvested and disposed, ensuring the removal of heavy metals (Suthersan,
2002). However, it is applicable only to those sites that contain low to
moderate levels of metal pollution, because plant growth is stunted in heavily
polluted sites (Padmavathiamma and Li, 2007). Chelating agents are added to
the soil to solubilize metals that have a low solubility in the soil solution, thus
increasing the efficiency by which the plants take up heavy metals (Jabeen et
al., 2009).
3.1.2. Phytostabilization
This process is similar to the chemical stabilization process described
earlier. In this technique without the removal of the contaminant from the soil,
plants are used to transform toxic metals to less toxic forms in the soil (Jabeen
et al., 2009). In this process, the stabilization of the waste prevents exposure
through volatilization and water erosions. Plants used in this method should be
able to grow with their roots growing in the contaminated zone. This process
can then alter the biological, chemical or physical conditions in the soil that
convert the toxic forms of metal to less toxic ones. Stabilization of toxic
metals by plants is highly dependent on pH and temperature conditions
(Jabeen et al., 2009). Phytostabilization is an in-situ process and does not
require the removal or disposal of the hazardous biomass.
3.1.3. Rhizofiltration
This method relies on the ability of plant roots to take up and sequester
metal contaminates from aqueous systems such as waste-water streams.
Terrestrial plants are ideal for this method due to the fact that they produce
substantially longer roots with a large surface area. This type of method is
most applicable to aquatic macrophytes (Rai, 2009). It can be done in-situ and
ex-situ. Indian mustard (Brassica juncea) and sunflower (Helianthus annuus)
are the most promising macrophytes for heavy metal removal from water
(Jabeen et al., 2009; Rai, 2009).
3.1.4. Phytovolatization
Phytovolatization is the process where plants uptake water soluble
contaminants and the subsequent release of them into the atmosphere as they
transpire. The contaminant may be modified along the way, as water travels
along the plant's vascular system from the roots to the leaves, whereby the
contaminants evaporate or volatilize into the air surrounding the plant. There
are varying degrees of success with plants as phytovolatilizers with one study
showing poplar trees to volatilize up to 90% of the contaminant they absorb
(Ghosh and Singh, 2005). Phytovolatization has been used primarily for the
removal of mercury where it is transformed into the less toxic gaseous
elemental mercury (Ghosh and Singh, 2005). The disadvantage of using this
technique is that once the contaminants have been volatilized, there can be no
control over the migration of the gaseous end products to other areas (Jabeen
et al., 2009).
3.1.5. Phytodegradation (Phytotransformation)

Phytodegradation is the degradation or breakdown of organic
contaminants by internal and external metabolic processes driven by the plant.
Ex planta metabolic processes hydrolyze organic compounds into smaller
sections that can be absorbed by the plant. Some contaminants can be
absorbed by the plant and are then broken down by plant enzymes. These
smaller pollutant molecules may then be used as metabolites by the plant as it
grows, thus becoming incorporated into the plant tissues. Plant enzymes have
been identified that breakdown ammunition wastes, chlorinated solvents such
as TCE (Trichloroethane), and others which degrade organic herbicides
(Coleman et al., 2002; Meagher 2000; Schroder et al., 2002). Organic
compounds such as explosives, chlorinated solvents, herbicides and
insecticides and inorganic nutrients can be degraded using this method (Jabeen
et al., 2009; Vaněk et al., 2003).
3.2. Advantages and Disadvantages of Phytoremediation
The advantages and disadvantages of phytoremediation have been

discussed in Chaudhry et al. (2000), and are briefly outlined below:
1. Advantages of phytoremediation (Chaudhry et al., 2000)
a) It is more economically viable;

b) It is less disruptive to the environment;

c) Disposal sites are not needed;
d) It is more likely to be accepted by the public as it is more aesthetically
pleasing than traditional methods;
e) It avoids transport of polluted media thus reducing the risk of
spreading the contamination;
f) It has the potential to treat sites polluted with more than one type of
pollutant.
2. Disadvantages of phytoremediation (Chaudhry et al., 2000)
a) It is site specific and the plant selected for a site must be compatible
with the environmental conditions;
b) Success is dependent on the tolerance of the plant to the pollutant,
choosing the wrong plant can thwart remediation efforts;
c) Time taken to remediate sites far exceeds that of other technologies;
d) Contaminant solubility may be increased leading to greater
environmental damage and the possibility of leaching;
e) Currently, this method is effective only in shallow areas as there is a
problem with the roots reaching great depths.
FUTURE CONSIDERATIONS AND CONCLUSIONS

The appropriate remediation treatment approach depends highly on the
physical and chemical forms of the metal pollutants in soil or water. In order
for remediation to be successful, information concerning the physical
characteristics of the site, level of contamination and speciation of metals are
needed in order to obtain an accurate assessment of remedial alternatives (Sas-
Nowosielska et al., 2005). It is important to understand the physiological and
biochemical mechanisms underlying contaminant removal by plants. It is also
important to recognize that phytoremediation is site-specific – in other words,
environmental conditions (climate, substrate, pH, salinity) must be taken into
consideration before a phytoremediation plan is put into place (Jabeen et al.,
2009).
Plants have evolved a great diversity of genetic adaptations to handle the
accumulated pollutants that occur in the environment. Growing and, in some
cases, harvesting plants on a contaminated site as a remediation method is a
passive technique that can be used to clean up sites with shallow and low to
moderate levels of contamination (Alkorta and Garbisu, 2001; Gallagher et al.,

2008; Vaněk et al., 2003). Phytoremediation can be used to clean up metals,
pesticides, solvents, explosives, crude oil, polyaromatic hydrocarbons, and
landfill leachates (Coleman et al., 2002). Some plant species have been shown
to degrade several classes of the highly persistent and resistant organic
compounds such as PCBs (Meagher, 2000). Petroleum polluted soil is another
soil contamination problem that can be remediated using this method (Wang et
al., 2008).
While these promising aspects are in the early stages of development, it is
a highly encouraging and optimistic view on the removal of pollutants from
our environment. Currently, genetic engineering that targets the enhancement
of plant accumulation of contaminants is also in the early stages (Rai, 2009). A
multidisciplinary research effort that integrates the work of plant physiologists,
soil scientists, microbiologists, and environmental managers is an integral part
to making this method work. Phytoremediation has been studied extensively in
research and small-scale demonstrations, but full-scale applications are
currently limited to a small number of projects. Further research and
development is required before it is a widely accepted and a fully efficient
method.
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Chapter 5
POLLUTION SOURCES AND FLUXES IN

THE SOUTH-WESTERN SPANISH LITTORAL
F. Ruiz 1, M. L. González-Regalado1,

J. M. Muñoz2, M. Abad1, M. I. Prudêncio3, M. I. Dias3,
M. I. Carretero4, M. Pozo5 and A. Toscano1
1
Departamento de Geodinámica y Paleontología,
Universidad de Huelva, Huelva, Spain
2
Departamento de Estadística e Investigación Operativa,
Universidad de Sevilla, Sevilla, Spain
3
Instituto Tecnologico e Nuclear, EN-10, 2686-953-Sacavém, Portugal
4
Departamento de Cristalografía, Mineralogía y Química Agrícola,
Universidad de Sevilla, Sevilla, Spain
5
Departamento de Geología y Geoquímica,
Universidad Autónoma de Madrid, Madrid, Spain
ABSTRACT
This paper analyzes the heavy metal distribution (As, Hg and Ni) in
shallow marine sediments of southwestern Spain. The end of the Huelva
bank is the most polluted area of this littoral (Igeo > 3 for As; Hg > 1 mg

Authors for Correspondence: E-mail: ruizmu@dgyp.uhu.es. Posting address: Departamento de
Geodinámica y Paleontología, Universidad de Huelva, Avda. Tres de Marzo, s/n, 21071-
Huelva, Spain.
96 F. Ruiz, M. L. González-Regalado, J. M. Muñoz et al.
kg-1), owing to historical acid mine processes and recent industrial

sewages derived from the catchment areas of the Tinto-Odiel rivers. The
W-E littoral drift currents distribute this pollution towards the east,
mainly in very shallow areas (< 10 m water depth). In addition, pathways
of pollutants transported by the Guadiana river to the inner shelf are
described, with a ‘diffuse’ pollution in the deepest areas studied (10-18
water depth) between Isla Cristina and the Piedras river mouth.
1. INTRODUCTION
Shallow marine areas are the final destination of pollution fluxes derived
from agricultural wastes, industrial sewages, mine processes or urban
effluents. An usual indicator of these environmental impacts is the measure of
heavy metal contents in bottom sediments (Wang et al., 2010; Satpathy et al.,
in press). Some of these elements (e.g. As, Hg, Ni) are dangerous for sea life
and for human food resources (Price, 2008; Hervé et al., 2010; Vishnivetskaya
et al., 2011). Areal extent of pollution may be determined by the analysis of
enrichment factors, pollution indices or the application of several statistical
approaches (Karageorgis et al., 2009; Yalcin, 2009). In these investigations, it
is interesting to obtain the natural background from unpolluted sediments in
order to estimate the pollution levels or the effects of specific pollution sources
with different types of inputs (Fukue et al., 2006; Ozseker et al., 2011). In the
last centuries, the southwestern Spanish littoral has been subjected to an
increasing anthropogenic pressure derived from mining activities or industrial
effluents (Nelson and Lamothe, 1993; Van Geen, 1997). The aims of this
paper are: a) to determine the pollution levels in very shallow sediments of the
adjacent marine areas; b) to delimitate the current state of pollution with the
application of a regional geochemical background; and c) to delineate the
pollution pathways in this zone.
2. STUDY AREA
2.1. General Hydrodynamic Scenario
The hydrodynamic regional scenario of the southwestern Spanish coast is

controlled by the North Atlantic geostrophic current, which flows to the east
within the study zone (Figure 1). Littoral drift currents transport sand-size
Pollution Sources and Fluxes in the South-Western Spanish Littoral 97
sediments from the Portugal coast to the Spanish nearshore zone [(180-300) x
l03 m3/yr, according to C.E.E.P.Y.C. (1979) and Cuena (1991)]. Fluvial
sediment runoffs from the nearby land are important during December and
January when rainfall is highest. Medium-grade, saltation-transported sand
dominates the sediment input of the Guadiana, Tinto and Odiel rivers
(Borrego, 1992). Sediment supply by the Piedras river is restricted due to a
dam upstream.
Fine and very fine sands are the most abundant sediments on the
southwestern Spanish coast from Ayamonte to Matalascañas. Coarser grain
sizes are dominant in samples from down to 11 m water depth from La Antilla
to Punta Umbría and near the mouths of the Guadiana, Tinto, and Odiel rivers.
Clay and silt deposits were found at the junction of the Tinto and Odiel rivers.
The highest content of bioclastic material (mainly Mollusca) is associated with
high percentages of medium-grained sands in the inner part of the Huelva spit
and the deeper samples (> 10 m) in front of the Piedras River mouth (Ruiz et
al., 1997).
2.2. Environmental Framework
The Guadiana, Tinto and Odiel rivers discharge into the Atlantic Ocean
after flowing through the Iberian Pyrite Belt, in which large deposits of
polymetallic massive sulphide are interbedded. Since prehistoric times, large
scale mining and smelting operations have occurred on the river banks and
waters are extremely polluted by sulphide-associated heavy metals (Grande et
al., 2003). Therefore, the Tinto and Odiel rivers transport very high quantities
of these pollutants, which represent 17% of the global flux of dissolved Cu
transported by rivers to the ocean (Olías et al., 2006).
In addition, large amounts of wastes and pollutant effluents have been also
discharged from a variety of chemical factories (phosphate fertilizer plants,
chloro-alkali industries) and petroleum refineries located around the Tinto-
Odiel estuary since the 1960’s. The final consequence of these anthropogenic
inputs is the presence of highly contaminated sediments in the Tinto-Odiel
estuary. This area has been classified among the most polluted zones in the
world, with extremely high concentrations of Cu (1000-3000 mg kg -1), Zn
(400-3400 mg kg -1) or Pb (150-4900 mg kg -1) (Ruiz, 2001; Borrego et al.,
2002).
Impact of this pollution on the adjacent shelf is comparatively poorly
known, with several investigations limited to very shallow areas (< 5 m water
depth; Sáinz and Ruiz, 2006; Usero et al., 2008). This paper attempts to
delimitate the effects of these inputs on deeper areas of this littoral.
3. METHODOLOGY
Surface sediment samples were collected from 43 stations located off the
North Cádiz Gulf at depths ranging from 2.5 to 20 m (Figure 1). These
samples were obtained with a Van Veen modified sediment-grab, with the
collection of the upper 5 cm of sediment from each sampling point. The grain-
size distribution was determined by sieving for particles greater than 63 μm,
owing to the very high proportions of sand-size particles in all samples.
Chemical analysis for heavy metals in sediment were performed on the
bulk samples by X-Ray Assay Laboratories, Toronto (Canada). Metal
concentrations were determined by X-ray fluorescence spectrometry (Ni) and
atomic absorption spectrometry (As and Hg), using the cold-vapor technique
for Hg determination.
Figure 1. Location map of the studied area, with inclusion of the main hydrodynamic
features and sampling points.
Metal pollution assessment from sediment analysis was based on the

geoaccumulation index (Müller, 1981):
Bn
Igeo= log 2 ----------------
1.5 x Cn
where Bn is the measured concentration of the element B in the n sample and

Cn is the regional background of this element in unpolluted sediments. The
factor 1.5 is used becausee of the possible variations of the background data
due to lithogenic effects.
This regional background was obtained for different heavy metals (As, Ni)
and sediment grain sizes by Ruiz et al. (1998) in Holocene, unpolluted marine
sediments of the Tinto-Odiel estuary.
Samples were divided into six groups: unpolluted (Igeo < 1); very low
polluted (1 < Igeo < 2); low polluted (2 < Igeo < 3); moderate polluted (3 < Igeo <
4), highly polluted (4 < Igeo < 5) and very highly polluted (Igeo > 5). This index
has been utilized to assess metal enrichment of riverine (e.g. Herr and Gray,
1997) or marine sediments (e.g. Cukrov et al., 2011).
4. RESULTS
4.1. Heavy Metal Concentrations
Low to very low concentrations of As (< 20 mg kg-1 in most samples)

were found between Isla Cristina and the Piedras River mouth, at water depths
down to 10 m. Nevertheless, these values increase significantly (Table 1: >30
mg kg-1) in the deeper samples collected in this area. The highest
concentrations of this metal have been measured very close to Mazagón, near
the end of the Huelva bank (70-259 mg kg-1). In the Mazagón-Matalascañas
sector, the highest contents (> 35 mg kg-1) were found in the shallowest
samples, with decreasing values eastward and offshore.
The end of the Huelva bank is highly polluted in Hg, with values up to 0.8
mg kg-1. In the sourrounding areas, these values decrease towards the east and
do not exceed 0.25 mg kg-1 near Matalascañas.
In the western sector, moderate contents (0.4-0.66 mg kg-1) occur in the
deeper samples collected near Isla Cristina. Similar concentrations were also
observed between La Antilla and the Piedras river mouth. The Ni content
range from 0.5 to 17 mg kg-1 in the studied area, although only four samples
exceeded 10 mg kg-1 (Table 1).
These samples were collected in very fine to fine sands present near Isla
Cristina (> 10 m water depth) and the adjacent zone to the end of the Huelva
bank.
Table 1. Grain size, heavy metal concentrations (in mg kg-1) and

geoaccumulation indices of samples. MS: medium sand; FS: fine sand;
VFS: very fine sand. Background from Ruiz et al. (1998)
As Hg Ni
Sample Grain size
-1 -1 -1
mg kg Igeo mg kg mg kg Igeo
1 MS 8 <0 0.014 3 <0
2 FS-VFS 33 1.46 0.656 13 0.03
3 MS 11 0.29 0.02 5 0.74
4 MS 10 0.15 0.02 5 0.74
5 FS-VFS 57 2.25 0.031 3 <0
6 MS 12 0.42 0.056 5 0.74
7 FS-VFS 31 1.37 0.53 17 0.42
8 FS-VFS 17 0.5 0.335 7 <0
9 FS-VFS 18 0.58 0.28 12 <0
10 FS-VFS 64 2.42 0.37 6 <0
11 MS 33 1.87 0.119 2 <0
12 FS-VFS 34 1.5 0.262 4 <0
13 FS-VFS 15 0.32 0.137 6 <0
14 FS-VFS 17 0.5 0.55 7 <0
15 FS-VFS 30 1.32 0.443 6 <0
16 FS-VFS 32 1.42 0.069 2 <0
17 FS-VFS 24 1,00 0.23 8 <0
18 FS-VFS 18 0.58 0.174 7 <0
19 FS-VFS 18 0.58 0.2 8 <0
20 FS-VFS 27 1.17 0.241 6 <0
21 FS-VFS 26 1.12 0.19 10 <0
22 MS 10 0.15 0.006 7 1.22
23 FS-VFS 31 1.37 0.106 2 <0
24 FS-VFS 26 1.12 0.081 5 <0
25 FS-VFS 31 1.37 0.097 4 <0
26 MS 12 0.42 0.021 1 <0
27 MS 19 1.08 0.031 0.5 <0
28 FS-VFS 259 4.43 1.27 15 0.23
29 FS-VFS 110 3.2 1.143 8 <0
30 FS-VFS 82 2.77 0.073 2 <0
31 FS-VFS 154 3.68 0.33 9 <0
32 FS-VFS 70 2.54 0.881 7 <0
33 FS-VFS 66 2.46 0.361 6 <0
34 FS-VFS 77 2.68 0.742 7 <0
35 FS-VFS 43 1.84 0.274 5 <0
36 FS-VFS 39 1.7 0.622 9 <0
37 FS-VFS 46 1.94 0.266 6 <0
38 MS 14 0.64 0.035 2 <0
39 FS-VFS 37 1.62 0.221 6 <0
40 FS-VFS 22 0.87 0.276 9 <0
41 FS-VFS 41 1.77 0.236 5 <0
42 FS-VFS 32 1.42 0.186 5 <0
43 FS-VFS 17 0.5 0.099 5 <0
MS 6 2
BACKGROUND No data
FS-VFS 8 8.5
-1
Analitical error (mg kg ) 1 0.005 0.5
4.2. Geoccumulation Indices
The application of the geoaccumulation index permits to contrast the

spatial distribution of As and Ni (Figure 2). In the western sector (La Antilla-
Punta Umbría), a very low to low pollution of the first element (1.37 < Igeo <
2.42) was found between 10 and 20 m water depth, whereas the shallower
areas show unpolluted to very low polluted sediments (0 < Igeo < 1.37).
Figure 2. Pollution levels of the southwestern Spanish littoral, according to the

geoaccumulation indices obtained.
On the contrary, the highest values of this index were observed near the
end of the Huelva bank (2.46 < Igeo < 4.43). In the eastern area (Mazagón-
Matalascañas), the Igeo values are rather constant (1.62-1.94) near the coast and
decreases slightly in the deeper samples (0.5 < Igeo < 1.7).
The distribution of Ni is very different, since almost all samples present

very low enrichment factors. The geoaccumulation index only exceeds 1 in a
single sample located near the Odiel river mouth (Figure 2).
5. DISCUSSION
Heavy metal concentrations measured in shallow marine sediments of
southwestern Spain can be explained by different hydrodynamic and physical-
chemical processes. In the western sector, moderate contents of pollutants (e.g.
As, Hg) are mainly deposited at water depths up to 10 m between Isla Cristina
and the Piedras river mouth. This pollution comes from the contributions of
the Guadiana River, which drains a significant part of the Iberian Pyrite Belt
with a remarkable mining activity since the 19th century. These inputs cause
the contamination by heavy metals in the river mouth and the adjacent zones
(Corredeira et al., 2008). Littoral drift currents will lead this pollutant flow
toward the east in a direction nearly parallel to the coast, mainly between 10
and 18 m water depth.
In contrast, the Isla Cristina-Isla Antilla strech includes unpolluted to low
polluted sediments. This area is partially protected from the Guadiana inputs
by salt marshes and sandy spits that surround Isla Cristina. Pollution levels of
these sedimentary beds are also slightly polluted by heavy metals (Ruiz,
2001).
This pollution decreases near Punta Umbría, despite the proximity of the
Tinto and Odiel rivers. This area is partially protected from the highly polluted
inputs of these rivers by salt marshes bodies (Fig. 1: Saltés Island) and the
Huelva bank, which directs them to the east. These barriers cause a significant
hydrodynamic and sedimentary isolation of this area in relation to the main
channels of both rivers (Borrego, 1992).
The highest polluted sediments of this littoral are located very close to the
end of the Huelva bank (Figure 2; Table 1), clearly related to the Tinto-Odiel
river inputs. In this area, metal deposition is linked to the partial removal of
metals from solution through adsorption, coagulation and flocculation
processes (Braungardt et al., 2003). According to Beckers et al. (2007), this
process is completed at salinities up to 33.5 0/00 and pH up to 7.5, two
conditions that occur together in this area (salinity ~36 0/00; pH ~8) (Spanish
Oceanographical Institute, 1992; Andalusia Board, 1993). This deposition
caused a strong environmental impact in the 1990s, with the presence of
bivalves with very polluted soft tissues by heavy metals, not suitable for
human consumption (Usero et al., 1996). The presence of isolated samples

with lower pollutant concentrations may be due to the periodical dredging of
the main estuarine channel.
In the eastern sector (Mazagón-Matalascañas), pollutant flows are directed
mainly near the coast (< 10 m water depth) by the littoral drift currents (Figure
2). Moderate levels of As and Hg are measured even at 30 km to the east of the
Tinto-Odiel pollution source, whereas lower contents are obtained in deeper
sediments. This pollution pathway controlled is also observed in water models,
with a net transport of heavy metals from the Tinto-Odiel system to the Strait
of Gibraltar (Elbaz-Poulichet et al., 2001). In addition, these data confirm
those reported by González et al. (2007), which found no evidence of such
heavy metal contamination in the middle shelf sediments of the Cádiz Gulf.
CONCLUSIONS
In this paper, a geoaccumulation index is used as tracer of quality
sediment to obtain a general scenario of pollution pathways in shallow marine
sediments of southwestern Spain. The Tinto-Odiel system reveals as the most
important pollution source of this area, with high concentrations of As and Hg
near the end of the Huelva bank. The continuous inputs due to acid mine
drainage processes and industrial wastes cause the presence of moderate levels
of these elements in a wide coastal area owing to the W-E littoral drift
currents.
These elements are also provided, to a lesser extent, by the Guadiana
River, with an additional redistribution to the east by these marine currents.
The main consequence is the presence of moderate geoaccumulation indices
between Isla Cristina and the Piedras river mouth, at 10-18 m water depths.
Spatial distribution of Ni is different, with very low contents of this
element in the study area.
ACKNOWLEDGMENTS
This work was funded by a Spanish DGYCIT Project (CGL2010-
15810/BTE), an Andalusia Excellence Project (SEJ-4770: Ordenación y
gestión integradas del litoral de Andalucía) granted by FEDER and two
Research Groups of the Andalusia Board (RNM-238 and RNM-349). This

paper is a contribution to the IGCP 588 (Preparing for coastal change).
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high spatial resolution trace metal distributions with model simulations
for surface waters of the Gulf of Cadiz. Estuar., Coastal and Shelf Sci., 74,
599-609.
Borrego, J. (1992). Sedimentología del estuario del río Odiel (Huelva, SO
España). Ph. D. thesis, Universidad de Sevilla, unpublished.
Borrego, J., Morales, J. A., De la Torre, M. L. and Grande, J. A. (2002).
Geochemical characteristics of heavy metal pollution in surface sediments
of the Tinto and Odiel river estuary (southwestern Spain). Environ. Geol.,
41, 785-796.
Braungardt, C., Achterberg, E., Elbaz-Poulichet, F. and Morley, N. (2003).
Metal biogeochemistry in an acidic mine polluted estuarine system in
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CEEPYC (1979). Plan de estudio de la dinámica litoral de la provincia de
Huelva. Informe Dirección General de Puertos y Costas, Madrid.
Corredeira, C., Araújo, M. F. and Jouanneau, J. M. (2008). Copper, zinc and
lead impact in SW Iberian shelf sediments: An assessment of recent
historical changes in Guadiana river basin. Geochem. J., 42, 319-329.
Cuena, G. J. (1991). Proyecto de regeneración de las playas de Isla Cristina.
Memoria Ministerio de Obras Públicas y Turismo.
Cukrov, N., Franciskovic-Bilinski, S., Hlaca, B. and Barisic, D. (2011). A
recent history of metal accumulation in the sediments of Rijeka harbor,
Adriatic Sea, Croatia. Mar. Poll. Bull., 63, 154-167.
Elbaz-Poulichet, F., Morley, N. H., Beckers, J. M. and Nomerange, P. (2001).
Metal fluxes through the Strait of Gibraltar: the influence of the Tinto and
Odiel rivers (SW Spain). Mar. Chem., 73, 193-213.
Fukue, M., Yanai, M., Sato, Y., Fujiwara, T., Furukawa, Y. and Tani, S.
(2006). Background values for evaluation of heavy metal contamination in
sediments. J. Hazard. Mat., 136, 111-119.
González, R., Aráujo, M. F., Burloff, D., Cachao, M., Cacalho, J., Corredeira,
C., Dias, J. M. A., Fradique, C., Ferreira, J., Gomez, C., Machado, A.,
Mendes, I. and Rocha, F. (2007). Sediment and pollutant transport in the

Northern Gulf of Cadiz: A multi-proxy approach. J. Mar. Syst., 68, 1-23.
Grande, J. A., Borrego, J., Morales, J. A. and De la Torre, M. L. (2003). A
description of how metal pollution occurs in the Tinto-Odiel rias (Huelva-
Spain) through the application of cluster analysis. Mar. Poll. Bull., 46,
475-480.
Herr, C. and Gray, N. F. (1997). Sampling riverine sediments impacted by acid
mine drainage: problems and solutions. Env. Geol., 29, 37-45.
Hervé, R. P., Andriamalala, R., Yves, M., Marcellin, R., Christine, R. and
Andriamandimbisoa, N. (2010). Assessment of heavy metals
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Karageorgis, A. P., Katsanevakis, S. and Kaberi, H. (2009). Use of Enrichment
Factors for the Assessment of Heavy Metal Contamination in the
Sediments of Koumoundourou Lake, Greece. Water, Air and Soil Poll.,
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Odiel river and estuary system. Estuaries, 16, 496-511.
Olías, M., Cánovas, C. R., Nieto, J. M. and Sarmiento, A. M. (2006).
Evaluation of dissolved contaminant load transported by the Tinto and
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and Muñoz, J. M. (1998). Stratigraphic sequence, elemental
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Tinto-Odiel system on the adjacent littoral sediments of Southwestern
Spain: A statistical approach. Chemosphere, 62, 1612-1622.
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K. (in press). Studies on the variations of heavy metals in the marine
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(1997). A 120-yr record of widespread contamination from mining of the
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3, 106-125.
Chapter 6
CONTROLLED ORGANIC TREATMENT

SYSTEM FOR ACID MINE DRAINAGE (AMD)
AND MUNICIPAL WASTE WATER
O. Pollmann1, N. Pollmann2 and L. van Rensburg1

1
School of Environmental Science and Development,
North-West University, Potchefstroom Campus (PUK), South Africa
2
SCENSO – Scientific Environmental Solutions GbR, Bonn,
Schützeiche 4, 53757 Sankt Augustin, Germany
ABSTRACT
Most landscapes in South Africa are affected by the process of
mining. The environmental results are large amounts of heavy metals and
a low pH in the soil, caused by acid mine drainage (AMD) and in most of
the irrigation water. Additionally, the quantity and quality of water is –
like in most African and developing countries – limited and mostly too
scarce for economical processing to drinking water quality. Mining
processes and the weather conditions affect the whole agricultural sector
in terms of water quantity and water and soil quality.
For this reason, an organic medium was tested to prove of its
function purifying water resulting from mining processes. All results

Authors for Correspondence: 20942737@nwu.ac.za (O. Pollmann). Posting address: School of
Environmental Science and Development, North-West University, Potchefstroom Campus
(PUK),Private Bag X6001 (Internal Box 178), Potchefstroom 2520, South Africa
108 O. Pollmann, N. Pollmann and L. van Rensburg
showed an increase in pH from pH 3.0 of the AMD to more than pH 5.5

and a reduction in heavy metals of about 80%. Acid mine drainage
normally has to be treated with chemicals in order to get an acceptable
quality to release the water into wetlands. The organic treated AMD has
the quality of irrigation water and could be used for irrigation in
agriculture without any negative site effects on the existing soil or water
over the long term. The same organic medium was used to optimize the
efficacy of municipal waste water treatment as a pre-treatment. More than
30% of the full treatment was done by the organic medium with the effect
of decreasing heavy metals and minimizing cytotoxicity and endocrine
disruptors. In controlled samples the cytotoxicity, caused by specific
endocrine disruptors could be significantly reduced by the treatment with
the organic treatment system.
Keywords: Acid mine drainage (AMD), Municipal waste water, Heavy

metals, Biological rehabilitation, Casuarina equisetifolia, Endocrine
disruptors.
1. INTRODUCTION
The aim of the presented research project was to propose a controlled
wetland system which is easy to maintain and to manage, resistant against
external influences and sustainable over long periods of time.
With this in mind a controlled wetland system composed of organic
material compacted in bags was applied for different applications. The first
application was highly contaminated acid mine drainage (AMD), an effluent
from the mining industry, with low pH and high content of heavy metals. The
second application was raw municipal waste water with a high load of
suspended solids (about 30%) and soluble or part-soluble substances (about
70%) which require biological treatment.
The acid mine drainage is usually treated by chemicals such as the
expensive and toxic caustic soda (sodium hydroxide, NaOH) to increase the
pH, in order to reduce corrosion in pipes and pumps (Maree, 2004). The use of
lime (calcium carbonate, CaCO3), is to increase the pH for the precipitation of
metals and some accumulated residues in the cleaned water (Maree, 2004).
This use of chemicals can be reduced by an adapted and optimized controlled
wetland system.
The standard method for treating municipal and industrial sewage water is
a two step treatment – mechanical and biological, where all suspended
Controlled Organic Treatment System ... 109
contaminants are filtered, metabolised by micro-organisms and used as

nutrition for their reproduction. Some of the contaminants are too complex for
the normal micro-organisms to reduce as their enzymes cannot break down the
long chain complex compounds found in some pollutants (Cheung, 1998).
For both applications the controlled wetland system was suitable and
resulted in heavy metal reduction and pH stabilisation. Additionally a
reduction and elimination of cytotoxicity and some of endocrine disrupting
chemicals (EDCs) like estradiol, which are major problems in normal waste
water treatment techniques, could be achieved.
2. MATERIALS AND METHODS

2.1. Organic Medium
The organic medium is a 50% mixture by volume of the pine-bark from

Eliote sp. and leaves from the beech species Casuarina equisetifolia with
various populations of bacteria (e.g. Bacillus licheniformis, Bacillus cereus,
Serratia marcescens, Bacillus pumilus), fungi (e.g. Acremonium sp.,
Aspergillus sp., Geotrichum sp., Phoma sp., Penicillium sp.), yeast and micro
organisms from the semi-decomposed leaves and bark. Casuarina equisetifolia
is also known as iron wood, she wood and beef wood. This wood has a high
amount of iron in the cells and, unlike other plants, can tolerate these
unusually high amounts of iron. If the concentration exceeds the solubility of
the compound it will be precipitated. The solubility varies greatly with the
chemistry and crystallisation of the mineral (Le Bissonnais, 1992). This
reaction is to protect the plant from overdosing the cells with toxic elements.
The wide-open pores allow the leaves to build up a high capillary pressure to
carry the heavy metals to the surface when used for filtration (figure 1 and 2).
The organic medium has the ability to exchange both, positive and
negative ions between the organic material and a fluid. The different strength
of charges is because of the different semi-decomposed organic material such
as pine leaves and bark. When the process of de-composition starts, the charge
of the organic medium changes to low and high negative. Because of the
higher amount of H+-ions from the de-composing processes of the different
organic materials and the humic and organic acids the charge change. Thus
cat-ion and anion exchange can occur in the organic medium at the same time
(figure 3 and 4).
Figure 1. Electron microscope view of Figure 2. Electron microscope view of

the porous structure of the of the vascular tissues organic medium.
organic medium
Figure 3. Microscope picture of precipitation of metal sulphide.
Figure 4. Microscope picture of precipitation of salt (1:500).

The pine-bark in the organic medium contains lignin and tannin, which
raises the pH, so that all heavy metals in the AMD are immobilised. In this pH
range the organic medium binds all heavy metals and salts strongly, even
when the contact time is only about 15 minutes (more contact time will
remove more metals, sulphates and silica).
2.2. Application Acid Mine Drainage (AMD)
To treat acid mine drainage a system of cascades as test beds was designed
with four steps. Each cascade was 25 meters long, 8 meters wide and 0.3
meters deep. The described treatment system was about 75 meters long in
total. Water from flooded mines washes out metals and elements from
explosives mixed with ground water and is forced through vent-shafts and
boreholes to the surface. This AMD water was channelled to the first cascade
filled with bags of the organic medium. The bags were packed against the flow
of the stream to force the water to run through them. The second cascade was
filled with only pine-bark from Eliote sp. and the third filled with only needles
from Casuarina equisetifolia. After each cascade, oxygen was introduced into
the water as it flowed over small waterfalls and then onto a shade net filled
with loose organic material. The last cascade was only filled with dolomitic
rocks, a sedimentary rock, which raised the pH from pH 3.5 to pH 3.8 and also
acted as a medium for the sedimentation of the highly contaminated sludge.
The AMD flowing over the stones was slowed, allowing for the dissipation of
some oxygen. The total process from the inflow of the first cascade to the
outflow after the last cascade took only 20 minutes – this was the contact time
allowed for the material to remove most of the heavy metals and precipitate
them to the surface of the bags. After the pre-treatment of the organic medium
the AMD was channelled to a PVC lined dam to be mixed with other sources
of AMD in order to reduce costs of the treatment with caustic soda and lime as
a post- / final-treatment.
2.3. Application Municipal Waste Water
To optimise and simplify the treatment of sewage water, it is necessary to

decrease the amounts of phosphorous, nitrogen, ammonia, nitrate and nitrite as
well as heavy metals.
For this purpose a closed barrel was prepared through which sewage water
was pumped with pressure from bottom to top through a layer of ~ 25 kg of
organic medium (figure 5 and 6). This allows enough contact time between the
contaminant and the organic medium for ion exchange and to prevent
hydrostatic pressure from collapsing the capillaries of the organic medium and
in turn blocking the whole system.
Figure 5. Treatment of sewage water with the organic medium.
Figure 6. Pre-Treatment with the organic medium at the purification farm.

The water was pumped through the barrel at a rate of 40 litres per minute,
with a contact time of 4 minutes in the barrel. The tests were done twice daily,
in the morning and in the afternoon in order to observe differences in the
compounds of the sewage water at different times of day. The pumping
continued for a period of 30 minutes.
In a mobile lab electrical conductivity (EC) and pH were measured in the
untreated raw sewage water of the inlet of the plant, in the sewage water
treated with the organic medium and in the routinely treated sewage water at
the outlet of the plant.
After a treatment period of 14 days phosphorous, nitrogen, nitrate, nitrite,
iron, magnesium and sulphate, were analysed (table 2) to determine the
effectiveness of the organic medium compared to the routine treatment carried
out in the plant. Therefore samples of the different treated sewage water were
tested for macro-, micro-elements by analysis (straight water analysis,
inductively coupled plasma mass spectrometry (ICP-MS)) at the Eco-
Analytica Laboratory (North-West University).
3. RESULTS AND DISCUSSION

The main difficulty in disposing of polluted acid mine drainage (AMD) is
the large amount of macro-elements and heavy metals it contains, including
calcium, magnesium, potassium, sodium, phosphate, sulphate, ammonium,
chlorine, bicarbonate, iron, manganese, copper, zinc, beryllium it also has a
pH of less than 3.0. This low pH is caused by the release of hydroxide, which
causes free acidity (Neculita, 2007).
A solution for improving the water quality is to raise the pH and
immobilise the heavy metals and remove them from the AMD in a sustainable
manner (table 1). With the use of the organic medium packed in a controlled,
mobile wetland it was shown that by ionic exchange extraction and adsorption,
even in pH ranges of below 3.5, high concentrations of manganese and iron
were removed.
The organic material carries different charges and thus it is possible to
exchange all ions, positive and negative, at the same time. The quality of the
treated AMD after passing through the controlled wetland was under the limits
for irrigation water as specified by the Department of Water Affairs and
Forestry (DWAF, 1996) (table 2).
Table 1. Summary of results before and after treatment with the

organic medium
Table 2. Quality limitations for irrigation (DWAF, 1996) and of AMD

treated by the organic medium
In case of the sewage water treatment the reduction of ammonia (NH4),

nitrite (NO2) and nitrate (NO3) depends on the metabolic activity of bacteria
and the amount of available oxygen. Nitrite concentrations were most
effectively reduced – from 1.15 mg/l to 0.10 mg/l- and nitrate from 85.0 mg/l
to 35.0 mg/l (table 3).
Table 3. Contaminant reduction by the use of the organic medium
Because of the nitrogen fixing micro-organisms (diastrophic organisms) in

the organic medium the amount of nitrogen from nitrite and nitrate was lower
after treatment. Micro-organisms, for example Rhizobium sp. or Frankia sp.,
live in symbiosis with the roots of the plants found in the organic medium and
use ammonia for protein biosynthesis for amino-acids and change the
ammonia against carbon to produce energy as adenosine-triphosphate (ATP)
(Bergmann, 1999).
The roots of the semi-decomposed organic material also produce a high
number of H+-Ions, which are available after ion exchange with other elements
like iron or other metals. This fact explains why ammonium and ammonia
leached out of the organic medium and increased, after contact with the
organic medium, from 24.60 mg/l to 58.20 mg/l of ammonium and from 23.40
mg/l to 54.60 mg/l of ammonia (table 3).
Ion exchange as shown in the table above (table 3), involves the chemical
exchange of ions (charged dissolved molecules or atoms) in solution with ions
in a solid phase. The solid phase (organic medium) was chosen specifically to
adsorb the constituents of interest. To maintain the electrical charge balance,
the organic medium must release an equal amount of charge into solution. For
example, in water softening, the cat-ion exchange within the organic medium
initially holds Na+, but releases them as it adsorbs Ca2+ and Mg2+ from the
water stream moving through or past the organic medium.
For the organic medium, there is a preferential adsorption of either the cat-
ions or the anions if more than one type is present. For example, calcium is
more strongly adsorbed than magnesium, and sulfate is more strongly
adsorbed than nitrate. The organic medium has been developed to maximize
its affinity for obtaining the best binding, related to the lyotrophic series (Al3+
≈ H+ >> Ca2+ > Mg2+ >> K+ ≈ NH4+ > Na+).
Compared to the treatment process used in the sewage plant (total sewage
treatment of 2 - 3 days) the pre-treatment with the organic medium (treatment
time ~ 4 minutes) decreased phosphate by 23.5% as compared to 69.1% in the
plant. The decrease in iron after treatment with the organic medium was 78.6%
and 83.9% in the sewage plant. The best result obtained using the organic
medium was the reduction of nitrite by 91.3%.
The optimal ratio between phosphorous and nitrogen of 1:10 could not be
achieved by either treatment method. The benefits of the organic medium as
an effective pre-treatment method are the reduction of metals and salts in the
sewage water after a very short contact time on small scale.
This natural technique of ion exchange was also effectively tested with
other comparable organic materials. The main mechanism is the semi-
decomposition of the material and its ingredients itself. Therefore other
materials like fertilizers or compost are able to do the same treatment as well.
Additionally sewage water when treated with the organic medium showed
an elimination of cytotoxicity and hereby a reduction of endocrine disruptors
(analysis done by an accredited lab). With reference to endocrine disrupting
activity, the total cytotoxicity of the tested sewage-water could be reduced
from 37% to negative results after treatment with the organic medium. That
implies that substances in the raw sewage-water that interfere with normal
human cells functioning are present in significant amounts before treatment.
The result of the human cell tests (Whitcutt, 2005) was that after treatment
with the organic medium no cytotoxic effect on human cells could be detected.
All of the endocrine disrupting activity including some of the hormones in the
sewage water is reduced, to a concentration lower than detectable trigger
limits, by the treatment of the water. A control sample of 17β-Estradiol
(contraceptive pill) was dissolved in de-ionised water and tested for
cytotoxicity before and after treatment with the organic medium. The results
showed that the organic medium eliminates the cytotoxic effect of 17β-
Estradiol as well.
CONCLUSIONS
The use of the controlled organic treatment system similar to the functions
of a natural wetland demonstrated positive results in acid mine drainage
(AMD) and sewage water treatment. The main factors of concern regarding
the quality of the water could only be effectively treated using organic
material. Other treatment methods for acid mine drainage in particular use
chemicals which cause further environmental problems.
Further research, especially in the field of elimination endocrine
disrupting chemicals, must be done to confirm and substantiate the present
results. The relationship between anthropogenic impacts in form of sewage
water and endocrine disruptors, which find their way back into the genetic
system of all creatures, this method should also be included in Epigenetic
research.
REFERENCES
Aly, Ragaa Aly Taha (2003): Influence of heavy metals zinc, cadmium and
lead on growth and uptake of Acacia saligna, Casuarina equisetifolia and
Cupressus sempervirens. Dissertation, Niedersächsische Staats- und
Universitätsbibliothek Göttingen.
Bergman, J. (1999) ATP: The Perfect Energy Currency for the Cell. Creation
Research Society (CRS) Quarterly, Vol. 36, No 1.
Cheung,K.P. (1998) Sewage treatment and disposal. Department of
Agriculture, Hong Kong University.
Department of Environmental Affairs and Tourism (DEAT) (2007) National
Environmental Management: Waste Management Bill. No. 29487.
Department of Water Affairs and Forestry (DWAF) (2007) South African
National Water Resources Infrastructure Agency Bill. No. 29745.
Department of Water Affairs and Forestry (DWAF) (1996) Water Quality
Guidelines, Volume 7: “Aquatic Ecosystem”. Published by Department of
Water Affairs and Forestry. ISBN 0-7988-5345-X (Volume 7).
Department of Water Affairs and Forestry (DWAF) (1996) Water Quality
Guidelines, Volume 4: “Agricultural Use: Irrigation”. Published by
Department of Water Affairs and Forestry. ISBN 0-7988-5342-5 (Volume
4).
Le Bissonnais, Y., Singer, M.J. (1992): Crusting, runoff, and erosion response
to soil water content and successive rainfalls. Soil Science Society of
America Bulletin, Volume 56, Issue 6, 1992, pages 1898 – 1903.
Maree, J.P., Streydom, W.F., Adlem, C.J.L., De Beer, M., Van Tonder, G.J.,
Van Dijk, B.J. (2004) Neutralization of Acid Mine Water and Sludge
Disposal. WRC Report No. 1057/1/04. Water Research Commission,
Pretoria.
Neculita, C.-M, Zagury, G. J., Bussiere, B. (2007) Passive Treatment of Acid

Mine Drainage in Bioreactors using Sulfate-Reducing Bacteria: Critical
Review and Research Needs. In: Journal of Environmental Quality (J.
Environ. Qual.), Volume 36, pp. 1 – 16, ISSN 0047-2425.
Whitcutt, J.M. (2005) The Weaver human cell test for water quality. South
African Journal of Sciences 101, July/August 2005.
Chapter 7
ASSESSMENT OF TRACE ELEMENT

CONTAMINATION IN THE RIVER AND
ALLUVIAL SEDIMENTS USING A
SEQUENTIAL EXTRACTION TECHNIQUE
AND STATISTICAL ANALYSIS
Sanja M. Sakan and Dragana S. Đorđević

ICTM, Department of Chemistry, University of Belgrade,
Njegoševa 12, Belgrade 11000, Serbia
ABSTRACT
This study presents the results of determination Cu, Cr, Zn, Pb, Cd,
V, Ni, As and Hg content and assessment of their anthropogenic origin,
mobility and contamination level in the river sediment (Tisa River and
tributaries) and alluvial sediments (Danube alluvial formation, Pančevo).
In order to assess mobility and dominate substrates of examined
elements, in this paper was applied sequential extraction method and
analysis of the content of Ca, Mg, Fe, Mn, Ti, K as well as determination
of N, C, H and S content using elemental analysis. The results obtained
by sequential extraction showed a different mobility and dominant

Authors for Correspondence: ssakan@chem.bg.ac.rs (Sanja M. Sakan). Posting address: ICTM,
Department of Chemistry, University of Belgrade, Njegoševa 12, P.O.Box 815, Belgrade
11000, Serbia
120 Sanja M. Sakan and Dragana S. Đorđević
substrate of elements in the Tisa sediments and alluvial sediments in the

Pančevo. There was shown a high mobility of Cu, Zn, Pb and Cd in the
Tisa sediments, which may be an indicator of the existence significant
anthropogenic sources of these elements. The amounts of the studied
elements extracted in the residual fraction were much higher than those in
the non-residual fractions in the Pančevo sediment, except for Cd and V,
indicating that they were mainly from crystalline lattices. Chromium and
nickel were dominantly present in the residual fraction. Vanadium was
extracted in the more mobile fractions from Pančevo sediment, which
may indicate to the existence of contamination with vanadium.
Correlation analysis was performed on total extractable contents of
elements and contents of N, C, H and S in the sediments. The results
showed that manganese oxides and alumosilicates are of great importance
for the binding of Cu, Zn, Cr, Pb and Cd in the Tisa sediment. Vanadium
has a different behaviour, origin and substrate compared to Cu, Cr and
Zn. Hg was positively correlated with Zn and Cd. The correlation of Fe
with Ni is a consequence of their siderofile nature and suggests a
common origin of these elements. Positive correlation between As and
Cr, Zn and Pb is due to their similar geochemical characteristic and
binding with clay minerals.
Results of correlation analysis of element content in the sediment
Pančevo show a similar geochemical origin of Cu, Cr , Zn, Ni and V in
the studied sediments. These elements are bounded to alumosilicates, as
well as oxides of Fe and Mn. There was shown that organic matter is
important for the binding of Zn and Cd. Positive correlation of C with Ca,
As and Hg is the result of the carbonate nature of those elements in the
investigated sediments. S is positively correlated with Hg, which
indicates the presence of Hg in the form of HgS in the studied sediments.
Based on distribution of extracted elements content by localities and
comparison obtained results with Quality Guidelines for sediments, there
was observed the increased contents of some trace elements on
investigated localities.
1. INTRODUCTION
Among the various contaminants, potentially harmful trace elements are
of particular concern due to their environmental persistence, biogeochemical
recycling and ecological risks. Toxic elements are widely distributed in nature,
in places such as water, soil, air and various forms of organisms. Some of
them, like mercury (Hg), cadmium (Cd), lead (Pb), chromium (Cr) and arsenic
(As), general exist at low concentrations, which can bring about huge pollution
and damage to the environment when their accumulations exceed certain
Assessment of Trace Element Contamination in the River … 121
levels [1]. Large uncontrolled metal inputs from agricultural practices, mining
activities, industrial activities, waste disposal, etc. have contributed to increase
pollution in sediments [2-11].
Metal ions are retained on the soil solid phases by different mechanisms
(ion exchange, surface complexation, precipitation, co-precipitation) [12]. The
toxicity and fate of toxic elements in sediments is depended on its chemical
form and therefore quantification of the different forms of element is more
meaningful than the estimation of its total element content. A variety of
procedures based on sorption and desorption experiments have been used to
assess the potential mobility and changes of interaction over time of pollutants
in soils and sediments [13]. An approach that has been widely applied is the
fractionation of elements (or species) of interest into operationally defined
forms under the sequential action of different extractants. Fractionation
schemes used to study this relationship generally evaluate the role of solution,
exchangeable, organically complexed, and oxide (Fe, Mn, Al) bound forms
[14] and allow, in fact, the estimation of heavy metal bioavailability [15].
Sequential extractions were first designed for the selective removal of trace
elements bound to operationally defined sediment fractions [16,17]. The
sequence extraction schemes are successful in distinguishing between metals
originating from anthropogenic sources and metals of geochemical origin. In a
general way, the metals introduced by human activities are considered to be
weakly bound in the exchangeable and carbonate fractions and may become
rapidly available [18].
Although the sequential extraction procedures are not fully specific, they
may provide comparative information on trace-element mobility in soils and
sediments with changing environmental conditions, such as pH or redox
potential, and may help to evaluate the relative contributions of mixed
pollution sources to this mobility. For these reason, the use of sequential
extraction procedures to evaluate the risk in contaminated sediments has
increased. Sequential extraction has been applied usually to soils and
sediments samples [19-25]. Bacon and Davidson [26] concluded that this
method will have a healthy future in the 21st century.
The objectives of this study were: (i) determination of Cu, Cr, Zn, Pb, Cd,
V, Ni, As and Hg content in the river and alluvial sediments, (ii) to determine
the mobility and contamination level of trace elements in the sediments, (iii) to
identify the relationships among the investigated elements in the sediments
and their possible sources using the Pearson’s correlation coefficient analysis
and (iiii) identify risks of toxicity from the metals by comparison with Quality
Guidelines for Sediments and Soils.
2. EXPERIMENTAL
2.1. Study Area
Representative sediments of the study area were taken at two locations.

One part of the samples was sediment of the Tisa River and its tributaries, and
the second part were alluvial sediments from the Danube, which were taken in
the industrial zone of Pančevo. The drainage basin system of the River Danube
covers an area exceeding 800,000 km2 and incorporates 14 countries. This
drainage basin contains a number of major centres of base precious metal
mining with ore deposits associated with volcanic rocks of varying ages and
lithologies [27].
Figure 1. Study area and sampling stations: (1) canal sediments (Danube alluvial
formation)-location of the sampling sites marked as S1 to S14; (2) Tisa River
sediments-samples of tributaries are underlined and the pools are underlined twice.
Through the River Danube drainage basin, the mining and processing of
metal ores lead to point-source release of contaminant metals (Cd, Cu, Pb, Zn)
and As to the local surface drainage network [27]. The investigated alluvial
sediments were taken from the canal located in the industrial zone south-east
of the City of Pančevo, about 15 km east of Belgrade (Figure 1.). Pančevo is
located in South Vojvodina, at the confluence of the Tamiš and the Danube
Rivers. The most important economic industries in Pančevo are crude oil
processing, HIP Petrochemical Complex, fertilizers (Nitrogen Plant), the
textile and fashion industry and agriculture.
The Tisa (Tisza) River is the longest tributary (977 km) of the Danube
River, also having the largest drainage basin (157,200 km2), which is shared
by five countries: Ukraine, Romania, Slovakia, Hungary and Serbia. The upper
Tisa River, flowing through Hungary, has been subjected to severe metal
pollution due to mining activities and major industrial complexes in Romania,
near the border of Romania with Hungary. Heavy floods, which occur
regularly due to snow-melting and intensive precipitation, are a great potential
risk for the enhancement of pollution by metals via the leaching of mine
tailings, which are stocked near to river banks, as well as the spreading of the
contamination from the river bed and further downstream [28]. The Tisa is an
international river and from its confluence with the Danube, the pollution is
transmitted towards the Black Sea.
2.2. Sampling and Sample Preservation
The samples of alluvial sediments were taken in August and September of

2001 along the canal which begins at the Oil Refinery Pančevo and passes
through different industrial complexes. A pipeline for wastewater and
atmospheric water, which begins at the Pančevo Oil Refinery, is placed in this
canal and water from the pipeline flows into a wastewater canal that flows into
the Danube River. The samples were taken from 14 drill-holes (marked as S1–
S14) at different depths, up to 5 m. The drill-hole (DH) denoted with S1 is
nearest to the Refinery and S14 is nearest to the entrance of the canal into the
Danube River (Figure 1). The drill holes are getting under top level of
underground water because the level of underground water is reaching values
up to 3 m under ground surface. Samples from the drill hole for chemical
analyses were collected from the whole depth interval. At each sampling site
was collected approximately 1 kg of sediment.
For the present investigations, the Serbian part of the Tisa watershed was
selected. Sediment samples were collected in the period of July, 2nd − 17th,
2001, from 32 locations of the Tisa River system in Serbia. The locations of
the 24 sampling sites of the River Tisa [18 surface sediments (0−5 cm) and 6
buried sediments (6−30) cm] and 8 sampling sites of tributaries and pools
[surface sediments (0−5 cm)] are presented in Figure 1. The samples of
surface sediments were collected using a plastic scoop, and the samples of
buried sediments were collected by a corer made of PVC tubes of 10 cm in
diameter and 60 cm in length.
After sampling, the sediment samples (river and alluvial) were packed in
pouches and frozen in order to prevent changes in chemical composition of the
sediment.
Since that the same method was applied to the different types of sediment
that had a similar geochemical background, but different sources of
contamination, it was expected that the results would highlight the importance
of the application of sequential extraction to assess risks associated with the
presence of metals in different environments.
2.3. Determination of the Metal Contents
2.3.1. Sequential Extraction

In this paper, sequential extraction procedure was applied for
determination of the metal forms. This method was based on the successive
application of extraction solvents with increasing strength for each subsequent
phase of the extraction. Sorbed heavy metals could be displaced from the
geochemical phases through the use of these extracting agents.
Five extraction steps were applied in this paper (Table 1), in order to
extract the metals from the sediments, following a modified Tessier method
[29].
The main purposes of each extraction phase are described as follows: the
first fraction (F1) of the extraction is defined as the 'Exchangeable fraction', in
which the most mobile portion of metals (adsorbed and ion-exchange bound)
are expected to be dissolved; in the second fraction (F2), the dissolution of
carbonates occurs and the heavy metals, which are precipitated or co-
precipitated, are released. Besides the carbonates, the dissolution of
manganese oxides and the amorphous oxides of iron takes place in the second
phase; metals are specifically adsorbed or co-precipitated.
Table 1. Sequential extraction procedure
Treatment duration Chemical process Sediment fraction

Esa Reagents
and temperature description affected
1M Extraction and
2 hours at room Exchangeable
F1 CH3COO(NH4), dissolution in neutral
temperature (22ºC) species
pH 7 environment
0.6M HCl and Weak reduction and
12 hours at room Carbonates and
F2 0.1M dissolution in acidic
temperature (22ºC) Mn-oxide
NH2OH·HCl environment
0.2M H2C2O4
Strong reduction and
and 10 hours at room
F3 dissolution in acidic Fe-oxides
0.2M temperature (22ºC)
environment
(NH4)2C2O4
30% H2O2
Oxidation and
adjusted to Organic fraction
F4 2 hours at 85ºC dissolution in acidic
pH 2.0 with and sulfides
environment
HNO3
Digestion and
F5 6M HCl 9 hours at 85ºC dissolution in acidic Residual fraction
environment
a
Extraction step.
This phase is called the 'easily reducible phase' or the 'Carbonate and Mn-
oxide fraction'; in the third fraction (F3), the stronger reduction dissolvent is
applied and the dissolution of amorphous and partially crystalline oxides of
iron occurs. This phase is called the 'stronger reducible phase' or 'Fe-oxides
fraction'; the degradation of the organic substances and sulfides occurs in the
fourth fraction (F4). In the organic fraction, the metals are complexed and
adsorbed, and become available through oxidation. This phase is called the
'oxidation phase' or 'Organic fraction and sulfides’; in the final, the fifth
fraction (F5), the degradation of crystalline iron oxides occurs and the metals
bound to them are released. This phase is called the 'Residual fraction'. The
exact extraction procedure has been described elsewhere [30].
For each sequential extraction series, some 10.00g of de-frozen material of
sediment was taken without drying in order to avoid chemical transformations
due to chelating, oxidation processes, etc. Control samples were processed for
each extraction step. The reagents for each sequential extraction step are
described in Table 1. The solid/liquid ratio has been kept as close to 1:45 as
possible during the first, second and third fraction. All the chemicals used in
this paper were of analytical reagent grade.
In the sequential extraction procedure described in this paper, it is
necessary to emphasize that a solid residue, mainly made of silicates, remains
after the application of all five steps. This is insignificant from the
ecochemical aspect and can be considered as insoluble, containing highly

immobile metals bound to the silicate matrix.
Although the sum of metal concentration released in the five fractions is
not always equal to total amounts of certain metals in the sediment sample, we
assumed that this was not valid for Pb, Zn, Cd and Cu, since these elements
were highly chalcophile. Therefore, they show no affinity to the silicate
matrix. Practically, after extraction of heavy metals by sequential extraction,
the concentration of these elements in the silicate matrix was insignificant.
2.3.2. Metal Determination

Flame atomic absorption spectrometry (FAAS) was used to determine the
levels of the trace elements (SpectraAA55 Varian spectrophotometer,
equipped with a hydride vapour system). External standard solutions were
prepared from 1000 mg L–1 stock metal solutions. For minimized
interferences, a multi-element standard stock solution was prepared in which
the ratios of the metals in the multiple element calibration standards were
analogous to their ratios in the samples. These multi-element standards and
blanks were prepared in the same matrix as employed for the extraction to
minimize matrix effects and for background correction [8]. In order to
simulate the composition of the investigated samples, the multi-element
standards also contained elements which were not determined. Possible
interference effects were minimized in this manner. The calibration was
checked every 10–12 samples. As a quality control, duplicate analyses were
performed on all samples and the precision was controlled. The relative
standard deviations of the means of duplicate measurement were less than 10
%. The values of limit of detection (DL) for each determined element,
expressed in g ml–1, were: Cr (0.03), Zn (0.005), Pb (0.05), Cd (0.01), Ni
(0.05), Cu (0.02), V (0.5), As (0.002), and Hg (0.001). All concentrations
values below the detection limit were replaced by half of the DL, as suggested
by Reimann and Filmozer [31] and Relić et al. [8]. The moisture content of
each sample was determined by drying a separate 1 g sample in an oven (105
± 2 °C) to constant weight. From this, a correction to dry mass was obtained,
which was applied to all reported element concentrations.
2.4. Determination of N, C, H and S Content in Sediments
The determination of N, C (total carbon), H and S content in the

investigated sediments was performed by elemental analysis, using Vario EL
III C,H,N,S/O elemental analyzer (Elementar). The total carbon in this

research represents the sum of inorganic and organic carbon.
2.5. Statistical Analysis
To identify the relationship among investigated elements in sediments and

their possible sources, Pearson’s correlation coefficient analysis were
performed using the commercial statistics software package SPSS version 11.5
for Windows.

3.1. Analysis of the Results Obtained by Sequential Extraction
In Table 2 are shown the results obtained by sequential extraction methods

in investigated Tisa river sediment and Danube alluvial sediment (Pančevo).
Content of elements by fractions (1-F1, 2-F2, 3-F3, 4-F4, and 5-F5) is
expressed as the mean content in mg kg-1 (shown in the form of AM ± SD or
mean ± standard deviation) for each fraction individually. Figure 2 shows the
distribution of the content of macroelements (Fe, Mn, Ca, Mg, K, Al, Ti, Li
and Si) by fraction of sequential extraction in sediments of the Tisa and
Pančevo. By analyzing the results of comparative analysis of the contents of
these elements in the studied sediment, it can be observed the following:
Iron (Fe): The largest content of iron was extracted in the fifth, third and
second fraction of the sequential extraction.
In the mobile fractions (2. and 3.), Fe is significantly present in the
sediment of Tisa, while crystalline Fe oxides form are significant occurrence
of Fe in alluvial sediment from the Pančevo. Significant extraction of Fe in the
second, easily reducible fraction is a consequence of dissolution of amorphous
Fe oxyhydroxides [32]. Content of exchangeable and organically bound Fe is
lower in respect to the extracted Fe from Fe oxides, so that organic and
exchangeable fractions have no a great importance as form of iron in the
studied sediments. This distribution indicates that Fe is dominant represent Fe
oxides in the studied sediments.
Figure 2. Distribution of extracted content of macroelements in fractions of sequential

extraction. Explanation: along the x-axis is shown fraction of extraction (marked with
numbers from 1 to 5); along the y-axis is shown the contents of the extracted element,
expressed in %.
Table 2. The distribution of element contents (mg kg-1) by fractions
Tisa sediment
1. fraction 2. fraction 3. fraction 4. fraction 5. fraction
(F1) (F2) (F3) (F4) (F5)
Cu 3.34±2.54 21.7±15.4 33.1±10.4 1.96±1.69 14.8±5.3
Cr 0.44±0.42 2.08±0.95 4.74±1.41 1.42±0.82 7.64±2.70
Zn 19.1±19.1 158±96.5 64.3±21.1 3.75±3.13 52.0±17.2
Pb 1.41±0.72 32.5±20.8 14.4±8.5 0.60±0.36 5.69±4.58
Cd 0.85±0.52 1.04±0.66 0.73±0.58 0.15±0.08 < DLa
Fe 15.6±9.4 5309±2204 13150±3668 94.6±153.2 15227±6297
Mn 365±268 877±441 115±37 14.1±7.3 87.9±39.7
Ni 0.71±0.36 6.09±2.71 11.1±6.1 2.86±1.77 10.4±4.8
Ca 93616±66410 47812±86000 < DL 1110±3350 5090±124321
Mg 1561±1427 4430±5779 999±762 525±435 4413±2162
K 188±132 23.6±14.7 353±222 153±95 1432±796
Al 20.55±28.18 2407±1099 7343±4305 132±342 22204±6587
Ti 7.52±6.51 12.7±9.5 212±128 17.7±17.3 671±577
Li 1.95±1.26 1.59±0.67 10.3±4.4 2.46±1.44 12.5±7.2
Si 1100±329 2924±1495 7187±4687 4711±1430 6875±4904
V 7.20±3.98 17.4±9.0 54.6±16.0 5.90±1.64 93.7±88.7
As < DL 0.68±0.66 0.06±0.10 < DL 0.11±0.14
Hg < DL 0.020±0.012 < DL < DL 0.098±0.067
Pančevo sediment
Cu 0.29±1.91 0.50±1.09 8.80±6.83 0.48±0.58 6.68±11.48
Cr 0.26±0.14 0.31±0.18 2.88±1.54 0.74±0.30 8.32±5.06
Zn 1.52±5.05 4.86±11.46 16.23±11.40 1.76±2.74 31.41±20.10
Pb 0.43±0.23 2.90±8.23 3.25±2.12 0.80±0.87 4.27±4.00
Cd 0.05±0.03 0.06±0.03 < DLa < DL < DL
Fe 2.73±3.21 429±726 5584±2687 374±1171 10765±6316
Mn 16.7±17.2 233±148 51.8±33.1 14.0±20.7 79.3±43.1
Ni 0.31±0.14 2.61±2.08 7.32±3.78 2.63±2.32 8.04±4.81
Ca 48788±19914 77974±38697 < DL 2326±4483 10709±14191
Mg 902±784 6222±5142 1111±957 484±208 4290±2370
K 131±206 11.0±13.7 114±150 113±209 799±669
Al 21.98±28.24 392±471 2949±2460 491±1175 10800±6559
Ti 4.54±2.11 17.5±23.6 48.2±38.4 12.0±39.2 373±209
Li 0.99±0.30 1.77±1.94 3.39±2.55 1.76±1.86 10.8±5.74
Si 175±101 1047±850 5191±3454 2500±1159 4908±4595
V 6.63±4.19 9.88±10.22 35.3±14.2 4.18±2.52 37.4±36.2
As < DL 0.10±0.25 0.07±0.11 < DL 0.07±0.10
Hg < DL 0.004±0.003 < DL < DL 0.057±0.064
a
below than detection limit.
Manganese (Mn): Most of the Mn was extracted in the second fraction in

both types of sediment, indicating that Mn is predominantly present in the
form of oxides. The results of Sánchez et al. [32] show that extracted Mn in
the second fraction is mainly originating from the amorphous Mn
oxyhydroxides. From the Tisa River sediment, a significant part of Mn was
extracted in the first extraction fraction. Mn was also extracted in the other
fractions, but the extracted content was less than 10% for each fraction
separately. In the sediments from Pančevo, Mn is significantly extracted in the
fifth and third fraction, while its content extracted in the first and fourth
fraction is less than 5%. It can be said that the mobile fraction of Mn is
significantly presented in the Tisa sediment, while in the sediment from
Pančevo, Mn is present in imobile, the fifth fraction. These results point to
anthropogenic sources of Mn in the Tisa River basin.
Calcium (Ca): Calcium is in the largest percentage extracted in the first
and the second extraction fraction and slightly in the fourth and the fifth
fraction. In the Tisa sediment, a much larger percentage of Ca is extracted in
exchangeable fraction (1. fraction) in relation to the carbonate bound Ca (2.
fraction). In the sediment of Pančevo, a much higher percentage of Ca was in
form of carbonates compared to the exchangeable fraction. Different
distribution of Ca in the sediment of Pančevo and Tisa could be due to more
intensive interactions that occur between river water and river sediment, and
changes in environmental conditions (pH, Eh) in the Tisa in relation to the
interaction between groundwater and alluvial sediment of Pančevo.
A large percentage of extractable Ca in the first fraction indicates the
significant presence of gypsum in all horizons. Also, the presence of
exchangeable Ca is probably due to desorption and release of Ca from the clay
minerals, since that Ca is an element which is located in the interstitial clay
minerals [32]. A large percentage of extractable Ca in the second fraction
indicates on significant presence of calcite in the studied sediments [32].
A significant amount of extracted Ca in the second fraction of extraction
from alluvial sediments of Pančevo is consistent with results obtained by X-
ray diffraction analysis. Results of diffraction analysis indicated that larger
content of calcite was in the analyzed alluvial sediments of the Danube in
Pančevo in relation to the Tisa sediment. Larger content of calcite in the
alluvial sediments can be explained by fact that samples of sediment from
Pančevo was taken from larger depths, since the carbonates are deposited
mainly in alkaline environments.
Magnesium (Mg): The dominant extraction of Mg in both types of
sediment was in the 2. and 5. fraction, which is due to the presence of Mg in
the sediment mainly in the form of carbonate or silicate. The presence of
extractable Mg in the fifth fraction is the result of partial destruction of clay
mineral using 6M HCl, which is consistent with the results of Sánchez et al.
[32]. The significant part of Mg is present in the form of carbonate in
sediments of Pančevo, while in the sediment of Tisa, Mg is dominantly present
in the form Mg silicate. It should be noted that differences in the content of
extractable Mg in the second and fifth fraction are small. The content of
extractable Mg in other fractions of extraction is significantly lower than in the
second and fifth fraction, and generally, content of extracted elements was less
than 10%.
Potassium (K): The distribution of extractable K by fraction of sequential
extraction shows that this element extracted in the largest percentage in the 5th
fraction, which is due to the partial destruction of silicates in this fraction.
Using 6M HCl for extraction of elements in the fifth fraction, along with
destruction of crystalline Fe oxide, also comes to the partial destruction of
some clay minerals [32]. Some of K was extracted in other extraction fraction,
but significantly less than in the 5th fraction. The distribution of K by fractions
is fairly homogeneous in both types of sediment.
Aluminium (Al): The majority of Al is in the form of silicate in both types
of sediment (the highest percentage of Al was extracted in the 5th fraction of
sequential extraction), while the part of Al is present in the form of oxides.
The presence of extractable Al in the fifth stage is the result of partial
destruction of clay minearals using 6M HCl, which is consistent with the
results of Sánchez et al. [32]. Since the oxalate reagent (ammonium
oxalate/oxalic acid), which was applied in the third extraction fraction a very
effective reagent for the extraction of amorphous aluminum oxide, it is
possible to conclude that the Al in the studied sediments were significantly
present in the form of amorphous Al oxide. In the Tisa River sediments was
some larger content of oxide bound Al in relation to the sediment from
Pančevo.
Titanium (Ti): The highest percentage of Ti is present in the form of
silicates in the studied sediments. Part of Ti is extracted in the third extraction
fraction also, while in other fractions, Ti is extracted in a small percentage.
Dominant extraction of Ti was in the more imobile fraction of extraction,
which is a consequence the stability of Ti minerals. Ti minerals are the most
stable minerals in the land environment.
Lithium (Li): In the studied sediments, Li was the mostly present in the
form of silicate. The presence of extractable Li in other extraction fractions
may be due to partial dissolution of silicates with aplliance of different
reagents. Li distributions by fractions of sequential extraction is very similar
for both types of sediment.
Silicon (Si): Silicon is extracted in all sequential extraction fractions, with

the lowest content of Si extracted in the first stage. It may be noted that Si is
present predominantly in the form of silicates and oxides in the studied
sediments. Extraction of Si in the fourth extraction fraction can indicate on the
existence of associations aluminosilicates-organic matter. In addition to the
building block of silicate minerals, silicon may be present in the form of
colloidal hydrous oxides, which have some importance in the absorptive
process. This fact may confirm the assumption on the partially oxide nature of
Si in the investigated sediments. Fairly homogeneous distribution of Si by
fractions was in both types of sediment. In the sediments of Pančevo, slightly
higher percentage of the element extracted in the 3. and 5. fraction. In the Tisa
sediment, the higher percentage of extractable Si was in the 2. and 5. fraction.
Figure 3 shows the distribution of extracted content of microelements (Cu,
Zn, Cr, Pb, Cd, Ni, V, As and Hg) by fractions of sequential extraction in
sediments of the Tisa and Pančevo. Based on distribution of elements content
by fractions in the studied sediments is evident the following:
Copper (Cu): The largest content of Cu in both types of sediment was
extracted in the 3. extraction fraction, in which dissolve partially crystallized
Fe oxides. Distribution of extracted Cu by the fractions is similar to the
distribution of Fe, indicating the importance of Fe oxides in the bounding of
Cu. In the Tisa sediment is also significant extraction in the second extraction
fraction and some of Cr is extracted in the fifth fraction. In the sediments of
Pančevo, Cu was significantly extracted in the fifth extraction fraction. In
general, it can be concluded that Cu is mainly bounded to the mobile fractions
in the Tisa sediment compared to the sediment from Pančevo. This distribution
by fractions is the result of intensive interactions that occur between river
water and river sediment and changes in environmental conditions (pH, Eh) in
samples of the Tisa in relation to the interaction between groundwater and
alluvial sediment from Pančevo. Also, this distribution points to the existence
of anthropogenic sources of this element in the Tisa sediment. In the literature
[33] is shown that bounding of Cu with carbonate minerals and iron and
manganese oxides indicates pollution of river systems from base mining. The
Tisa River flows through a metallogenic Carpathian province of Romania
where polimetallic ore are mined, which are rich in Zn, Cd, Pb, and Cu, like in
Baia Mare and Baia Borsa.
Zinc (Zn): The largest content of Zn from the Tisa sediment was extracted
in the second extraction fraction (> 50%), followed by extraction in the third
and fifth fraction.
Figure 3. Distribution of extractable trace element contents by fractions of sequential

extraction. Explanation: along the x-axis is shown extraction fractions (marked with
numbers from 1 to 5); along the y-axis is shown the contents of the extracted element,
expressed in %.
From the Pančevo alluvial sediment, more than 50% of Zn were extracted
in the fifth extraction fraction, then in the third and second fraction. This
distribution is consistent with the dominate association of this element in soil
(association with oxides of Fe, Mn, Al and clay minerals). The observed
similarity in distribution of extracted Zn with distribution of Fe, Mn and Mg
by fractions confirming the significance of quoted substrates for Zn bounding.
In the Tisa sediments, Zn was extracted in the more mobile fraction compared
to the alluvial sediment from Pančevo. Fe and Mn oxides are of great
importance in binding of Zn, where the mobile fraction of Fe oxide are
important substrates in the Tisa sediment. The importance of more mobile
fraction was expected, since that most of Zn in river sediments is mainly in
soluble form, bound to Fe and Mn oxides, and carbonates. Also, this
distribution, which is consistent with other results for the polluted river
systems [33], indicates the presence of anthropogenic sources of this element
in the Tisa watershed.
Chromium (Cr): The largest percentage of Cr was extracted in the fifth
and third extraction fraction (Fe oxides, partially crystallized and crystallized,
and silicates), while in the other fractions extracted lesser content of this
element. This distribution is consistent with the literature data, in which shown
that most of Cr in soil is existed in the form of Cr3+ enters in the composition
of minerals and constitute different Cr3+ - and Fe3+ - oxides. Some of Cr is
bounded in the second and fourth fraction, while the content of extractable Cr
is no significant. Over than 60% of Cr in the sediments of Pančevo was
extracted in the fifth fraction, and in Tisa sedimet, the percentage is over 40.
Distribution of extractable chromium content by fractions is similar to the
distribution of Fe, as well as the distribution of Al, Ti, Li, K and Mg. This
distribution shows the importance of oxide and silicate for bounding of
chromium, as well as significant geochemical origin of chromium in the
studied sediments.
Lead (Pb): Most of Pb was extracted in the second, third and fifth
extraction fractions, less content is extracted in the fourth and the first fraction.
The distribution of extracted Pb by fraction is consistent with the most
common form of this element in soil and sediments, such as carbonates of Pb,
binding to clay minerals and oxides of Fe and Mn. On this conclusion suggests
also and similarities in the distribution of Pb with the distributions of Fe, Mn,
Al, Ti, Li, K and Mg. The distribution of Pb by fractions is different in Tisa
sediment and in sediment from Pančevo; in Tisa sediment, the most important
is the binding of lead in the more mobile fractions (about 60% in the second
fraction and about 20% in the third), which indicates that Pb is mostly
bounded to the amorphous and partially crystallized Fe and Mn oxides and

carbonate, and can easily be mobilized with changes of conditions in the
environment. This distribution is characteristic for the lake and river
sediments. In the sediments from Pančevo, most of Pb is extracted in the fifth
fraction (crystalline Fe oxides and silicates), then in the third and second
(oxide and carbonate fractions), indicating that Pb is significantly presented in
the less mobile fractions.
Cadmium (Cd): The majority of Cd from Pančevo sediment was extracted
in mobile fractions of sediment, in the first and second. From the Tisa River
sediment, Cd was mainly extracted in the first and second, then in the third
extraction fraction, while a small percentage of this element extracted in the
fifth stage. These results indicate the great mobility of Cd in sediments of the
Tisa and Pančevo. This distribution by fractions is usually characteristic for Cd
in lake and river sediments. The assumption of dominate substrates of Cd in
the investigated sediments is confirmed with similarities in the distribution of
Cd by fractions with distribution of Fe, Mn and Ca. Also, a large mobility of
Cd may indicate on the existence of significant anthropogenic sources of Cd in
the studied sediments, especially in the Tisa sediments, since that higher
content of Cd was extracted from the Tisa sediment in relation to the alluvial
sediment from Pančevo.
Nickel (Ni): Fairly uniform distribution of Ni by fractions in both types of
sediment, with the largest content of extractable elements in the third, fifth and
second fraction. This suggests that oxides with different degrees of
crystallinity and silicates are the most significant for binding of Ni in studied
sediments. On this conclusion suggests similarities in the distribution of Ni
with the distributions of Fe, Mn, Al, Ti, Li, K and Mg. Some of difference
exists in the extractable contents of elements in the second and fifth fraction of
the studied sediments. Ni is significantly extracted in the second fraction from
the Tisa sediment compared to the sediment from Pančevo. In the fifth
fraction, a higher percentage of Ni was extracted from the sediment of
Pančevo compared to the Tisa sediment. It should also be noted that Ni was
extracted in the fourth extraction fraction, which means that Ni is significantly
bound to the organic matter in sediments of the Tisa and Pančevo.
Vanadium (V): The most important extraction of V was in the third and
fifth fraction, while in other fractions, the extraction was below 10%. A
similar distribution by fractions as vanadium was also for Fe, Mn, Al, Ti, Li, K
and Mg. These elements mainly represent the oxides and silicates in the
sediments. Unlike other elements, V is a more extracted in the mobile fractions
(1-3) from the sediment of Pančevo according to the Tisa River sediment,
while from the Tisa sediment, V is more extracted in the fifth fraction. This
indicates that in alluvial sediments from Pančevo, V has a larger mobility, and
that there are existed significant anthropogenic sources of this element. Since
that alluvial sediments sampled near the Pančevo oil refinery and
petrochemical plants, these distribution of vanadium can point to the existence
of contamination by crude oil. Vanadium is often used as a tracer of pollution
by crude oil in various areas.
Arsenic (As): Arsenic is extracted in the second, third and fifth extraction
fraction, with the largest content extracted in the second fraction. This
distribution indicates that As is significantly present in the form of carbonates,
while the part of As is in the form of oxides. Carbonate nature of As is a result
of the alkaline reaction that has the highest percentage of land in Vojvodina.
Also, there was shown a significant presence of CaCO3 in the investigated
alluvial sediments (mineralogical analysis with X-ray diffractometer), as well
as in Vojvodina soils [34]. In acid soils, the main forms of arsenic are Al and
Fe arsenates (AlAsO4 and FeAsO4), while in alkaline soils, rich limestone, a
major form of As is Ca3 (AsO4)2 [35]. This fact may indicate on the presence
of As in the form of carbonate sediments in Pančevo. The dominant extraction
of As from Tisa sediment was in the second fraction, while a smaller
percentage extracted in the third and fifth. From the sediment of Pančevo,
fairly uniform extracted element content by fractions in the second, third and
fifth fraction, with somewhat higher extraction As content in the second
fraction.
Mercury (Hg): The most important extraction of Hg was in the fifth
fraction, with over 80% of the extracted element from both types of sediment,
indicating a strong association between Hg and crystalline Fe oxides. Some of
the Hg was extracted in the second fraction, with higher percentage of
extracted element from Pančevo sediment in relation to Tisa sediment, which
may suggest the importance of carbonate as a Hg substrate. These results are
consistent with the results of Sánchez et al. [32], where it is shown that the
highest percentage of Hg from soil extracted with 6M HCl (82%). A high
percentage of Hg extracted with 6M HCl can be explained by strong binding
capacity of crystalline Fe oxyhydroxides, as well as solubility of metacinabar
in 6M HCl [32]. The content of extracted Hg in other fractions is negligible,
indicating that Hg is not readily available in the studied sediments.
3.2. The Results of the Total Extracted Element Content
The sum of element contents extracted by sequential extraction, for most

of elements significantlly represents the total element content. In the further
discussion, the term of the total element content will be used for the extracted
elements content in all the five fractions of sequential extraction. The
distribution of the total content of trace elements in sediments at the studied
sites is shown in Figure 4. (Tisa) and Figure 5. (Pančevo).
Figure 4. The total content of elements in sediment samples of Tisa River and its
tributaries. With numbers from 0 to 158 are represented surface sediment of Tisa,
numbers 21 to 158 represents buried Tisa sediment, and the numbers 1 to 8 reperesents
sediment samples of Tisa tributaries and pools.
Figure 4. shows the total content of extracted elements from the Tisa
sediment and its tributaries. The Figure 4. is composed from two parts.
Figure 5. The total content of elements in samples of alluvial sediment of Pančevo.

Location of the sampling sites marked as S1 to S14.
At the top of the image (Figure 4) is shown distributions of the logarithm

values of Cu, Cr, Zn, Pb, V, Cd and Ni content, while on the second, lower
part is shown the distribution of As and Hg content. Since that there is the
great differences in content between most of elements, for their presentation to
an image, there was shown logarithm values of content. Content of Hg in all
investigated sediment samples was under 1 mg kg-1, and in most of sediments
content of As was under 1 mg kg-1. Because of that, content of As and Hg on
Figure 4. is not represent as logarithm.
There was observed a similar distribution by localities of Cu, Cr, Zn, Pb,
Cd, V and Ni in the surface sediments of the Tisa (samples taken at distances
from 0 to 158 km from the mouth), indicating a common origin of these
elements, and geochemical similarities between them. It can be observed that
on localities that are within a distance of 110th km from the mouth, in the
surface sediment was a higher content of V in the Tisa, and by approaching the
border, its content is decreases. Sediments from larger depth contains more of
V according to the surface sediment, while the highest content of V was in
sediment of tributaries compared to the Tisa sediments. This result indicates
that the most important sources of V is related with the Tisa flows through
Serbia (Vojvodina). The increased content of V is associated with the
geochemical enrichment of sediments by this element as well as the existence
of anthropogenic sources on investigated locality. In addition to V, sediment
of tributaries contains more of Ni than the sediment of the Tisa. Increased
content of Ni in the sediments of tributaries can be explained by geochemical
characteristics of parent Pannonian sediments [36], since that on studied
location no significant anthropogenic sources of Ni. Geochemical content of
Ni in sediments and soils is mainly determined by its content in the parent
rock. There was observed a larger content of Cu and Zn in surface sediments
of the Tisa, at 60 km, 110 km, 120 km and 130 km in relation to the
confluence of the Tisa to the Danube. Increased content of Cu and Zn could be
originating from local sources of contamination in Bečej and Senta. Increased
content of Cu, Cr, Zn and Pb in the upper Tisa River flow through Serbia, near
the border with Hungary (part of 90 to 158 km) could be due to entry of these
elements from different countries through which the river flows.
In the studied sediments was observed a low and neatly uniform content of
Hg, and a higher content of element in the surface sediment compared to the
deep sediment, as well as a higher content closer to the mouth of the Danube,
than close to the border. Also, no significant differences in the content of Hg
in sediment of Tisa and its tributaries.
As content was higher in the Tisa sediments compared to the tributary
sediment. Higher content of As was observed in surface sediments at 64, 80
and 120 km and 70 km of the Tisa sediment. Increased content of As can point
to the existence of local sources of contamination on the observed locations.
In general, in the sediments of the Tisa River are bounded more of Cu, Cr,
Zn, Pb, Cd, As and Hg and less of V and Ni than in the sediment of tributaries.
Most of the examined elements in the sediment can be expected as a result of
the Tisa pollution in the countries through which the river flows.
In Figure 5 is shown the results of the total element content in the
investigated alluvial sediments of Pančevo. The values obtained for Cu, Cr,
Zn, Pb, V and Ni are presented in the form of logarithm. Due to low content of
extracted Cd, As, and Hg, contents of these elements is not presented as
logarithm and distribution of these elements is shown in a separate graphic.
Distribution of Cr and Ni content is neatly uniform, in some of sediment
samples was observed slightly higher content of these elements (23-S11/1.5,

24-S1/0.3, 26-S1/2.3, 29-S13/2.7 and 31-S14/2.2 localities), with the increased
content one of element accompanied by the higher content of another element.
Increased Cr and Ni content at some localities is probably due to geochemical
enrichment. For zinc was observed fairly uniform content of the extracted
elements in the research areas, with slightly larger values in sediment samples
with labels 10-S4/1.0 (164 mg kg-1) and 24-S12/0.3 (111 mg kg-1). These
values may indicate on existence of anthropogenic Zn sources, taking into
account fact that concentrations of elements at these sites significantly larger
than for other sediment samples. For lead, the highest content was observed in
sediments from drill holes that are located closest to the refinery (samples with
labels 1-S1/1.0 and 2-S1/2.4), indicating to the impact of the refinery on the Pb
content. There was observed a decrease in lead content with the increase
distance from the refinery, with a significant decrease was observed with
moving away from wells labeled as S13. The largest content of cadmium was
observed in sediment samples, which were marked as 14-S6/0.9, 20-S10/0.8,
22-S11/0.0 and 24-S12/0.3.
These samples were taken from a depth less than 1m. Generally, the value
of extractable Cd on some localities was larger than 1 mg kg-1, which indicate
on the anthropogenic origin of Cd. The content of Cu in sediment sample
marked as 3-S1/3.2 is significantly larger than in other sediment samples.
These data indicate on the existence of some local anthropogenic sources of
this element. The content of vanadium in the sediments marked as 2-S1/2.4,
29-S13/2.7 and 30-S14/0.7 was significantly larger than V content in the other
sediments. Generally, the content of V in all sediments may indicate on the
origin of this element from crude oil, since that investigated sediments was
sampled near to the refinery. When considering the distribution of As and Hg
in the investigated alluvial sediments, it should be noted that the values of the
extracted elements content is quite low. For As was observed increased
content in the sediment sample labeled with 20 (S10/0.8). For Hg it is possible
to notice that in the sediments marked as 23 (S11/1.5) to the sediment sample
31 (S14/2.2), element content is much lower than on other sites.
3.3. Comparison of Element Content with Quality Guidelines for

Sediments and Soils
The contents of the elements in the investigated sediments (as min and
max), values for the MAQ (Maximum Allowed Quantity) for soil in Serbia
[37], the legislative limits of the EU (Council Directive 86/278/EEC) [38], the
Canadian Sediment Quality Guidelines for elements in freshwater sediments
[39] and the CSST-sediment quality guidelines [40] are given in Table 3.
The results of a comparison of the metal contents in the Tisa river
sediments with the Quality Guidelines for Sediments and Soils show that at
some localities, the contents of Cd, Cu, Pb, Ni and Zn exceeded the criteria
(PEL and PEC), above which harmful effects are likely to be observed and the
concentration levels of these elements are unsafe when compared with the
Serbian MAQ (Maximum Allowed Quantity). Also, results of the comparison
of the metal content with the legislative limits of the EU indicated that in some
of the sediments elevated levels of Zn, Cd, and Cu, which were above the
maximum permitted limits according to European law (Council Directive
86/278/EEC). These results indicate the existence of anthropogenic sources of
Cd, Cu, Pb, Ni and Zn in the investigated area of the Tisa river. Results of
comparison element content from Pančevo alluvial sediment with Quality
Guidelines indicated that on this locality no significant contamination with
investigated toxic elements.
3.4. Correlation Analysis of the Total Elements Content
Inter-element relationships can provide interesting information on element

sources and pathways. Results of correlation analysis and values of Pearson's
correlation coefficient and levels of significance are shown in Tables 4, 5, and
6. Correlation analysis was performed for the total content of extracted
elements and total N, C, H and S content in sediment of Tisa and its
tributaries, and alluvial sediments (Pančevo). For samples of Tisa sediment,
the results of correlation analysis between the total content of elements and N,
C, H and S are determined and presented for 18 sediment samples (Table 6).
Due to technical problems, there was not possible to determine the content of
N, C, H, and in the remaining 14 samples.
3.4.1. Correlation Analysis of Examined Elements in the Tisa Sediments

Results of correlation analysis indicate that mostly manganese oxides and
aluminosilicates are of great importance for the bounding of Cu, Zn, Cr, Pb
and Cd. On importance of clay minerals to bound elements show the positive
correlation of Zn and Cd with hydrogen content. Vanadium has a different
behavior, origin and substrates in respect to Cu, Cr and Zn.
Table 3. Element content in the investigated sediments (mg kg–1) and Quality Guidelines for soil and sediment
Pančevo Tisa EU Canada SQGc CSSTf

MAQa
Min Max Min Max limitb ISQd PELe TECg MECh PECi
Cd 0.60 1.88 2.04 4.97 2 1-3 0.6 3.5 0.99 3.0 5.0
Cr 5.52 26.7 6.99 24.44 100 nd 37.3 90.0 43 76.5 110
Cu 1.72 87.1 31.6 162 100 50-140 35.7 197 32 91 150
Pb 2.99 54.4 11.2 123 100 50-300 35.0 91.3 36 83 130
Ni 6.67 39.5 17.3 54.6 50 30-75 nd nd 23 36 49
Zn 23.5 164 53.9 567 300 150-300 123 315 120 290 460
V 46.9 239 75.4 508 ndk nd nd nd nd nd nd
As < DLj 1.801 < DL 3.07 25 nd 5.9 17.0 9.8 21.4 33
Hg < DL 0.30 < DL 0.35 2 1-1.5 0.17 0.486 0.18 0.64 1.2
a
MAQ-Maximum Allowed Quantity, Official Gazette of Serbia [37].
b
Council Directive 86/278/EEC [38].
c
Summary of existing Canadian Sediment Quality Guidelines (freshwater sediment) [39].
d
ISQG-Interim sediment quality guideline-below which harmful effects are unlikely to be observed.
e
PEL-Probable effect level-above which harmful effects are likely to be observed.
f
Consensus-Based Sediment Quality Guidelines Recommendations for Use and Application [40].
g
TEC-Treshold Effect Concentrations.
h
MEC-Midpoint Effect Concentration-concentration midway between the TEC and PEC concentrations (TEC+PEC/2=MEC) .
i
PEC-Probable Effect Concentrations.
j
below detection limit.
k
no data.
Table 4. Pearson correlation coefficients between extracted elements content and N, C, H and S - sediment Pančevoa
Cu Cr Zn Pb Cd Fe Mn Ni Ca Mg K Al Ti Li Si V As Hg N C H S
Cu 0.36 0.37 0.60 0.55 0.64 0.52 0.61 0.60 0.42 0.43 0.71
Cr 0.41 0.59 0.85 0.66 0.68 0.56 0.73 0.36 0.63 0.39 0.72
Zn 0.53 0.43 0.50 -0.43 0.37 0.51 0.55 0.47
Pb 0.36 -0.46 0.71 0.37
Cd 0.50 0.42 0.46 0.47 0.40
Fe 0.84 0.79 -0.50 0.38 0.64 0.90 0.70 0.53 0.52 0.46 0.90
Mn 0.69 -0.39 0.65 0.80 0.62 0.50 0.79
Ni -0.48 0.69 0.77 0.61 0.43 0.80
Ca 0.37 -0.57 -0.53 -0.61 0.62 -0.50
Mg 0.53 -0.38 -0.51
K 0.74 0.57 0.59 0.38 0.37 0.75
Al 0.38 0.67 0.69 0.55 0.49 0.90
Ti 0.49
Li 0.46 0.54 0.72
Si 0.37 0.38 0.50
V 0.54
As 0.64 0.77
Hg 0.68 0.46
N 0.47
C
H
S
a
a Correlation is significant at 0.01 level (values are shown in bold) Correlation is significant at 0.05 level (values are no shown in bold)
Correlation is significant at 0.01 level (values are shown in bold) Correlation is significant at 0.05 level (values are no shown in
bold).
Table 5. Pearson correlation coefficients between extracted elements content-Tisa sedimenta
Cu Cr Zn Pb Cd As Hg Fe Mn Ni Ca Mg K Al Ti Li Si V
Cu 0.86 0.92 0.90 0.62 0.74 -0.41 -0.43 0.40 -0.39 -0.51
Cr 0.82 0.79 0.46 0.42 0.65 -0.36 -0.40 -0.44 -0.56
Zn 0.92 0.66 0.46 0.42 0.80 -0.58 -0.59 -0.41 0.50 -0.48 -0.60
Pb 0.70 0.36 0.69 -0.55 -0.57 -0.42 0.40 -0.47 -0.57
Cd 0.38 0.67 0.43
As -0.44 -0.43 -0.40
Hg 0.51 0.74 0.58
Fe 0.41 0.85 0.45 0.54 0.69 0.64 0.82
Mn 0.53 -0.50 -0.53
Ni 0.44 0.53 0.64 0.48 0.78
Ca 0.97 0.77 0.50 0.56
Mg 0.82 0.56 0.69 0.40
K 0.59 0.85 0.49
Al 0.62
Ti 0.55 0.57
Li 0.49
Si
V
a
Correlation is significant at 0.01 level (values are shown in bold) Correlation is significant at 0.05 level (values are no shown in bold)
a Correlation is significant at 0.01 level (values are shown in bold) Correlation is significant at 0.05 level (values are no
shown in bold).
Table 6. Pearson correlation coefficientsa between extracted elements content and N, C, H and S content –
Tisa sediment
Cu Cr Zn Pb Cd Fe Mn Ni Ca Mg K Al Ti Li Si V Hg As
N
C 0.50b 0.56
H 0.52 0.50
S
a
Results of correlation analysis for 18 samples of Tisa sediments in which determined N, C, H and S content.
b
Correlation is significant at 0.01 level.
Negative correlation with Ca and Mg suggest that carbonates are not

important for bounding of Cu, Cr, Zn and Pb in the studied sediments. Silicate
minerals with Ti are also not important for the bounding of these elements. Hg
was positively correlated with Zn and Cd, while As have a similarities in
behaviour with the Zn and Pb.
Positive correlation of Fe with Mn, Ni, Ca, K, Mg, Al and Li indicate that
the behavior of Fe is significantly determined with clay minerals content, as a
consequence of bounding Fe with clay minerals, as well as association of Fe
oxide with clay minerals. The correlation of Fe with Ni is a consequence of
their siderofile nature and suggests a common origin of these two elements.
The positive correlation of Cu, Zn, Cd, Pb and Hg with Mn are a consequence
of the binding of these elements with Mn oxides and their geochemical
similarities. Positive correlations of Mn with Al indicate on association of Mn
oxides with silicates.
Ni is positively correlated with Fe, which indicating the importance of Fe
oxides in the bounding of this element. Ni is positively correlated with the
content of Ca, K, Mg, Al and Li, indicating on the importance of clay minerals
in the Ni bounding.
The positive correlation of Ca with Fe, Ni, K, Mg, Ti and Li suggest that a
significant percentage of Ca is desorbed from clay minerals. Negative
correlation with Ca, Cu, Cr, Zn, Pb and As showed that carbonates are not
significant as substrates of the elements in investigated sediment, since that Ca
is also represented carbonates.
Mg in the investigated sediments significantly represents clay minerals, as
indicated by the positive correlation of this element with Fe, Ni, Ca, K, Ti, Li
and V. The negative correlation of this element with the content of Cu, Cr, Zn,
Pb and As showed that carbonates are not significant substrates of these
elements, since that Mg in the studied sediments among the clay minerals also
represents and carbonates.
K in the studied sediments represent clay minerals, as indicated by the
positive correlation of this element with Fe, Ni, Ca, Mg, Ti, Li and V.
Negative correlation of K content with Zn and Pb content may be due to
competitive binding of K, Zn and Pb, since K is a competitor of many heavy
metals in the sorption process.
Al in the investigated sediment is positively correlated with Zn, Pb, Cd,
Fe, Mn, Ni, Li and Hg, suggesting that this elements significantly represents
alumosilicates in the investigated sediments. Also, some of Al is present in the
form of oxides. Correlation of Al with Fe and Mn can point out on the
association between Fe and Mn oxides and clay minerals.
Positive correlations of Li with Fe, Ni, Ca, K, Mg, Al, Ti and V indicate
on origin of Li from aluminosilicates, mainly from clay minerals.
Si is positively correlated with Hg, which indicates that the silicates are
important in controlling of Hg content.
V is positively correlated with K, Mg, Ti and Li, as a result litofile nature
of this element. Negative correlation of V with Cu, Cr, Zn, Pb and Mn show
different behavior and the origin of V compared to other elements.
Positive correlation between As and Cr, Zn and Pb is due to their similar
origin and bounding with clay minerals. In the studied sediments, As has a
different properties and behavior with regard to Ca, Mg and Ti, as indicated by
the negative correlation between these elements and As.
Hg was positively correlated with Zn, Cd, Mn, Al and Si, indicating the
importance of alumosilicates in the Hg bounding. Significant correlation
between Hg with Zn and Cd is due to halcofile nature of these elements.
The C content was positively correlated with Fe and Ni, which may
suggest on the association of organic matter with Fe oxides. The C is
significantly represents organic matter in the studied Tisa sediments.
H is positively correlated with Zn and Pb, confirming the importance of
alumosilicates for bounding of these elements.
3.4.2. Correlation Analysis of Examined Elements in the Pančevo

Alluvial Sediments
Results of correlation analysis for Pančevo alluvial sediments indicated
that Cu is positively correlated with Cr, Zn, Ni, V, Fe, Mn, K, Al, Li, Si, and
H, which may point out on similar geochemical origin of Cu, Cr, Zn, Ni and V
in the investigated sediments. These elements are bounded to alumosilicates,
but there is a possibility also for their bounding with Fe and Mn oxides.
Positive correlation of Cr with Cu, Zn, Cd, V, Ni, Fe, Mn, Mg, Al, Ti, Li
and H show the importance of clay minerals and oxide fractions in bounding
of Cr, Cu, Zn, Cd, V and Ni. Negative correlation of Cr with Ca indicates that
carbonates are not significant substrates of Cr in the studied sediments.
Zn was positively correlated with Cu, Cr, Ni, Fe, Mn, K, Al, N and H, and
negatively correlated with Ca content. These correlations indicate that: clay
minerals and organic matter are important for bounding of Zn, carbonates have
no significance for the bounding of Zn, as well as on similar geochemical
behavior of Zn, Cu, Cr and Ni.
Pb is positively correlated with Ni, K and H, and negatively correlated
with Ca. These results indicate that Pb and Ni are associated with clay
minerals and have a common origin. Negative correlation of Pb with Ca

content indicates that carbonates are no important Pb substrate.
Cd was positively correlated with Cr, Fe, Mn, Al, N and H, which
indicates a similar origin of Cd and Cr, as well as existence of common
substrate of these elements. Also, correlation with N indicates that organic
matter is important for the bounding of Cd.
Positive correlation of Fe with Cu, Cr, Zn, Cd, Ni, V, Mn, K, Mg, Al, Ti,
Li Si and H show that Fe oxides and clay minerals are of great importance in
binding most of the elements in the studied sediments. Iron is negatively
correlated with Ca.
Mn is positively correlated with Cu, Cr, Zn, Cd, Ni, Fe, K, Al, Li, Si and
H, which indicates on the importance of Mn oxides in the bounding of Cu, Cr,
Zn, Cd and Ni. Also, these correlations indicate on existence of associations of
oxide Fe and Mn, as well as association of Mn oxide with clay minerals.
Manganese is also negatively correlated with Ca.
Ni is positively correlated with Cu, Cr, Zn, Pb, V, Fe, Mn, K, Al, Li and
H, suggesting the common substrates of Cu, Cr, Zn, Pb and V (clay minerals
and oxides of Fe and Mn). Ni was negatively correlated with Ca.
Positive correlation of Ca with Mg and total C indicates that dominant
nature of Ca in the sediment of Pančevo is carbonate. Positive correlation of
Mg with Cr, Fe and Li indicate that Mg in the studied sediments represents
predominantly silicates, as evidenced by negative correlation between Mg with
As content. Some of the extracted Mg originates from carbonates, which
indicated a positive correlation of Mg with Ca.
Positive correlation of K with Fe, Mn, Ni, Al, Li, Si, V and H is a
confirmation of the origin of K, which is desorbed from clay minerals. K was
positively correlated with Cu, Zn and Pb, indicating that clay minerals are
important for bounding of these elements. The negative correlation between K,
which represented clay minerals and calcium, which represents carbonate in
the investigated sediments is a consequence of the competitiveness of these
two substrates for the bounding of trace elements.
Al is positively correlated with elements that significant represent
alumosilicates in the investigated sediments, as follows: K, Ti, Li, Si and H.
Clay minerals are important for bounding: Cu, Cr, Zn, Cd, Ni and V. Positive
correlation with N indicating on the existence of association of organic matter
with clay minerals, and negative correlation of Al with Ca, which is a
consequence of the competitiveness of carbonate and clay minerals as a
substrates.
Ti is positively correlated with Cr, Al and Si, which indicates that

alumosilicates with Ti are of great importance for the bounding of Cr.
Li in the investigated sediments dominantly represents alumosilicates.
Alumosilicates are significant substrate for bounding of Cu, Cr, Ni and V, as
indicated by positive correlations of Li with the following elements: Cu, Cr,
Fe, Mn, Ni, K, Mg, Al, Si, V and H.
Si is represented silicates in the studied sediments (positive correlation
with K, Al, Ti, Li, Fe and Mn) that are most important for bounding of Cu
(positive correlation Si with Cu).
Geochemistry and behavior of V in the studied sediments is significantly
determined by the presence of clay minerals, as indicated by positive
correlations of V with the elements that represent clay minerals.
In investigated sediments, As is positively correlated with Hg and C, and
negatively correlated with Mg. These correlations suggesting that the As in the
studied sediments significantly associated with carbonates, which are also
important substrates of Hg. Negative correlation with Mg indicate that clay
minerals are no significant for the bounding of As in alluvial sediments of
Pančevo.
Hg is positive correlated with As, C and S, which indicates that Hg is
predominantly present in the form of carbonates and sulphides.
N is positively correlated with Zn, Cd, Fe, Mn, K and Al, and negatively
correlated with Ca. These results suggest on associations of organic matter
with clay minerals, which is important in the binding of Zn and Cd.
Positive correlations of C with Ca, As and Hg are the result of the
carbonate nature of these elements in the investigated alluvial sediments.
H is an indicator of clay mineral content, as indicated by positive
correlations H with: Cu, Cr, Zn, Pb, Cd, Fe, Mn, Ni, K, Al, Si and V. Positive
correlation with N indicate on the association of clay minerals and organic
matter, and negative correlation with calcium is a consequence of competition
carbonates and clay minerals as a substrates of elements.
S is positively correlated with Hg, which indicates the presence of Hg in
the form of HgS in the investigated sediments.
CONCLUSION
The purpose of this study was to determine Cu, Cr, Zn, Pb, Cd, V, Ni, As
and Hg content and assessment of their anthropogenic origin, mobility and
contamination level in the river sediment (Tisa River and tributaries) and
alluvial sediments (Danube alluvial formation, Pančevo). The results of

sequential extraction, correlation analysis and comparison of element content
with Quality Guidelines for sediments and soils showed that these methods
were useful in interpreting the large data set, and for extracting structural
information. The conclusions drawn from this study are as follows:
1) In the studied sediments, Fe and Mn are dominantly representing

oxides, Ca is in form of carbonates, Mg is mainly in the form of
carbonates and silicates, Ti and Li are the mostly present in the form
of silicates, and Al and Si are present predominantly in the form
silicates and oxides.
2) The results obtained by sequential extraction showed a different
mobility and dominant substrate of elements in the Tisa sediments and
alluvial sediments in the Pančevo. There was shown a high mobility
of Cu, Zn, Pb and Cd in the Tisa sediments, which may be an
indicator of the existence significant anthropogenic sources of these
elements. The amounts of the studied elements extracted in the
residual fraction were much higher than those in the non-residual
fractions in the Pančevo sediment, except for Cd and V, indicating
that they were mainly from crystalline lattices.
3) Chromium and nickel were dominantly present in the residual
fraction. Vanadium was extracted in the more mobile fractions from
Pančevo sediment, which may indicate to the existence of
contamination with vanadium. Since that alluvial sediments sampled
near the Pančevo oil refinery and petrochemical plants, these
distribution of vanadium can point to the existence of contamination
by crude oil. As is significantly present in the form of carbonates,
while the part of As is in the form of oxides. The most important
extraction of Hg was in the fifth fraction, indicating a strong
association between Hg and crystalline Fe oxides.
4) There was observed a similar distribution by localities of Cu, Cr, Zn,
Pb, Cd, V and Ni in the surface sediments of the Tisa (samples taken
at distances from 0 to 158 km from the mouth), indicating a common
origin of these elements, and geochemical similarities between them.
In general, in the sediments of the Tisa River bounded more of Cu,
Cr, Zn, Pb, Cd, As and Hg and less of V and Ni than in the sediment
of tributaries. Most of the examined elements in the sediment it can be
expected as a result of the Tisa pollution in the countries through
which the river flows.
5) Distribution of Cr, Ni and Zn content is neatly uniform in Danube

alluvial sediments, with slightly larger values of Zn in some sediment
samples. For lead, the highest content was observed in sediments from
drill holes that are located closest to the refinery, indicating to the
impact of the refinery on the Pb content. The value of extractable Cd
content on some localities indicate on the anthropogenic origin of this
element. Generally, the content of V in all sediments may indicate on
the origin of this element from crude oil. When considering the
distribution of As and Hg in the investigated alluvial sediments, it
should be noted that the values of the extracted elements content is
quite low.
6) Results of correlation analysis indicate that mostly manganese oxides
and alumosilicates are of great importance for the bounding of Cu, Zn,
Cr, Pb and Cd. Carbonates are not important for bounding of Cu, Cr,
Zn and Pb in the studied Tisa sediments. Hg was positively correlated
with Zn and Cd, while As have a similarities in behaviour with the Zn
and Pb. The positive correlation of Cu, Zn, Cd, Pb and Hg with Mn
are consequence of the binding of these elements with Mn oxides and
their geochemical similarities. Positive correlations of Mn with Al
indicate association of Mn oxides with silicates. H is positively
correlated with Zn and Pb, confirming the importance of
alumosilicates for bounding of these elements.
7) Results of correlation analysis for Pančevo alluvial sediments
indicated that Cu is positively correlated with Cr, Zn, Ni, V, Fe, Mn,
K, Al, Li, Si, and H, which may point out on similar geochemical
origin of Cu, Cr, Zn, Ni and V in the investigated sediments. Fe
oxides and clay minerals are of great importance in the binding most
of the elements in the studied Pančevo alluvial sediments. Positive
correlation of Cr with Cu, Zn, Cd, V, Ni, Fe, Mn, Mg, Al, Ti, Li and
H show the importance of clay minerals and oxide fractions in
bounding of Cr, Cu, Zn, Cd, V and Ni. There was shown the
importance of Mn oxides in the bounding of Cu, Cr, Zn, Cd and Ni,
and the existence of associations of oxide Fe and Mn, as well as
association of Mn oxide with clay minerals. As is in the studied
sediments significantly associated with carbonates. Hg is
predominantly present in the form of carbonates and sulphides.
8) The results of a comparison of the metal contents in the Tisa river
sediments with the Quality Guidelines for Sediments and Soils
indicate the existence of anthropogenic sources of Cd, Cu, Pb, Ni and
Zn in the investigated area of the Tisa River. Results of comparison

element content from Pančevo alluvial sediment with Quality
Guidelines indicated that on this locality no significant contamination
with investigated toxic elements.
ACKNOWLEDGMENTS
This research was supported by the Ministry of Science and Technological
Development of the Republic of Serbia, Grant No. 172001.
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Chapter 8
METAL CONCENTRATIONS IN
MARINE SEDIMENTS INFLUENCED BY
ANTHROPOGENIC ACTIVITIES IN
BAHRAIN, ARABIAN GULF
Humood Naser
Department of Biology, College of Science,
University of Bahrain, Kingdom of Bahrain
ABSTRACT
The Arabian Gulf is considered among the highest anthropogenically
impacted regions in the world. Heavy metals are considered a major
anthropogenic contaminant of coastal and marine environments leading to
degradation of natural habitats and posing threats to human health.
Concentrations of cadmium, copper, lead and zinc in marine sediments
influenced by industrial and sewage effluents, discharges from major
power and desalination plants, and reclamation and coastal modification
in Bahrain were analyzed using voltammetry technique. The overall mean
values in sediments for Cd, Cu, Pb and Zn were 1.37, 14.67, 17.43 and
40.99 mg kg-1, respectively. Elevated concentrations of Zn and Cu were
detected in areas influenced by brine wastewater discharges, and
navigational activities. Higher concentrations of Cd and Pb that attributed
 
Author for Correspondence: hnaser@sci.uob.bh, Posting address: Department of Biology,
College of Science, University of Bahrain, P. O. Box: 32038, Kingdom of Bahrain.
158 Humood Naser
to petroleum industries and effluents from variety of factories and

industrial facilities in that area were observed off the eastern coastline of
Bahrain. Despite a localized increase in Cd, heavy metal concentrations
in Bahrain are within the range of variations that are reported in
sediments from the Arabian Gulf.
1. INTRODUCTION
Heavy metals are considered a major anthropogenic contaminant of
coastal and subtidal marine habitats that may lead to degradation of
ecosystems (Ansari et al., 2004). Heavy metals in seawater and marine
sediments are incorporated into the aquatic food webs and then biomagnified
at higher trophic levels (Hosono et al., 2011). Therefore, heavy metal pollution
is a major concern for both environment and human health (Joksimovic et al.,
2011).
Naturally, heavy metals could be introduced to the marine environments
through atmospheric deposition, leaching of soils, and erosion (Kennish,
2001). Anthropologically, coastal and marine environments are susceptible to
the input of heavy metals due to industrial and sewage effluents, discharges
from desalination plants, ports and shipping activities, and mobilization of
sediments associated with dredging and reclamation (Akhter, 1990; Fowler et
al., 1993; Nayar et al., 2004; Hashim and Hajjaj, 2005; Miri and Chouikhi,
2005).
Heavy metal contamination in coastal and marine environments is
becoming an increasingly serious threat in the Arabian Gulf (De Mora et al.,
2010), which is considered among the highest anthropogenically impacted
regions in the world (Halpern et al., 2008).
The Arabian Gulf is naturally stressed due to marked fluctuations in sea
temperatures and high salinities, and additional anthropogenic input of heavy
metals could arguably be critical for its marine ecosystems (Akhter and Al-
Jowder, 1997). The coasts of the Arabian Gulf are subject to intensive
dredging and reclamation activities, and pollution from various land-based
sources (Sheppard et al., 2010).
Likewise, Bahraini coastal and marine environments are subject to
anthropogenic activates that may contribute significantly to the overall load of
heavy metal pollution (Figure 1).
Metal Concentrations in Marine Sediments Influenced … 159
Figure 1. Maps showing the Arabian Gulf and Bahrain, and locations of major land-
based anthropogenic activities contributing to heavy metals enrichment in coastal and
marine environments in Bahrain.
Dredging and disposal activities induce changes in environmental

conditions in the impacted sites, which may affect the mobility of associated
heavy metals (Tack et al., 1998; Berg et al., 2001; Guevara-Riba et al., 2004).
160 Humood Naser
Table 1. Major industrial facilities that discharge effluents into the

marine environment in Bahrain (Naser, 2010)
Factory/Company Activity Effluent

m3day-1
Bahrain Petroleum Company Oil refining 700 000
Aluminium Bahrain Aluminium smelter 4 000
Aluminium Extrusion Company Aluminium extrusions 350
Gulf Aluminium Rolling Mills Aluminium sheets and coils 150 000
Gulf Petrochemical Industry Ammonia, methanol, urea 850 000
Gulf International Company Iron industry 60
Arab Ship building and Ship repair dry dock 10 000
Repair Yard
Total 1 714 410
Bahraini coastal and marine environments are the prime target for most of
the major housing, recreational, and economic projects, which typically
associated with intensive dredging and reclamation activities (Naser, 2011).
Industrial and sewage effluents are widely recognized as major sources of
heavy metals inputs to aquatic environments (Nyamangara et al., 2008).
Bahrain has witnessed a rapid industrial growth, mainly in the sectors of oil
refining, aluminum and petrochemical industries. Several companies and
industrial factories are producing effluents that may contain hydrocarbons, and
heavy metals. It is estimated that around 1, 714, 410 m3 day-1 of industrial
effluents are discharged to the shallow subtidal areas (Table 1).
Sewage effluents are major source of coastal pollution in Bahrain. Several
sewage treatment plants varying in size and the degree of treatment are
discharging effluents to the coastal and subtidal areas in Bahrain. The main
one is Tubli Water Pollution Control Centre (TWPCC), which discharges
around 160 000 m3 day-1 of treated effluents into the shallow water of Tubli
Bay. Nuidrat sewage treatment plant contains a series of aeration pounds and
discharges around 7 000 m3 day-1 of effluents directly into the mangrove
swamps at Ras-Sand area in Tubli Bay.
Bahrain, like most of the Arabian Gulf countries, depends mainly on
desalination seawater as a source of potable water. In Bahrain, there are
currently four major desalination plants producing fresh water and energy.
Sitra Power and Water Station (SPWS) is the largest plant in Bahrain, with a
capacity of 125 megawatts of electrical power and 25 million gallons per day
of desalinated water using multi-stage flash technology (Khalaf and Redha,
2001). SPWS produces around 66000 m3 day-1 of effluent to the marine
environment of which 12 000 m3 are brine water. This brine water causes an
average increase in temperature of 7.5 oC above the natural water temperatures
of summer and winter (Altayaran and Madany, 1992). Bujarjor desalination
plant produces 46 000 m3 day-1 of drinking quality water by desalinating
brackish groundwater based on the reverse osmosis technique. The quantity of
effluents being discharged into the marine environment is estimated to be
around 1134 m3 day-1 (Khalaf and Redha, 2001).
The Arabian Gulf is considered the largest reserve of oil in the world.
Consequently, Bahrain is under a permanent threat from oil related pollution.
The sources of oil spills are offshore oil wells, underwater pipelines, oil tanker
incidents, oil terminals, loading and handling operations, weathered oil and tar
balls, and illegal dumping of ballast water (Literathy et al., 2002).
Sediments act as important sinks of heavy metals in coastal and marine
environments (Berg et al., 2001). Sediments have frequently been analyzed for
monitoring purposes and for detecting sources of pollution in aquatic
environments (Nyamangara et al., 2008). This study, therefore, aims to
determine the concentrations of selected heavy metals, namely lead (Pb), zinc
(Zn), copper (Cu), and cadmium (Cd) in marine sediments influenced by
anthropogenic activities in Bahrain.

2.1. Site Selection and Sampling Design
Tubli Bay and subtidal areas off the eastern coastline of Bahrain were
selected to investigate heavy metal concentrations due to land-based
anthropogenic activities such as reclamation and dredging, industrial and
sewage effluents, brine water discharges from desalination plants, and oil
pollution. The marine area of Tubli Bay has been reduced during the last
decade due to extensive reclamation activities. Two major sewage treatment
plants are discharging treated and partially treated sewage into the bay. The
eastern coastline of Bahrain is heavily occupied by industrial facilities
including an oil refinery, aluminum smelter, and desalination plants.
Samples were collected subtidally in a transect design in order to detected
the inshore-offshore gradient of heavy metal concentrations from the land-
based activities (Figure 2). Sediment samples were collected from five
transects (hereinafter T1, T2, T3, T4 and T5). Transects were divided into ten
stations with equal distance between each one (250 m).
162 Humood Naser
Similar distance between each

station Transect
Land-based
activities
and/or
pollution
Sediment sampling for physical and chemical analyses
Figure 2. Sampling design of sediments in an inshore-offshore orientation based on the

source of activity or pollution, namely sewage and industrial effluents and brine
wastewater discharge.
The selected sampling transects may be divided into three types based on
sources of impacts:
1. Sites affected by sewage effluents from Tubli and Nudirat sewage

treatment plants, reclamation impacts and coastal modification (T1
and T2).
2. Site affected by a major power and desalination plant (Sitra plant),
port, navigational channel, and ship building and repairing yard (T3).
3. Sites affected by industrial effluents from major industries such as oil
refinery, petrochemical and aluminum factories, and desalination
plants (T4 and T5).
2.2. Sediment Sampling and Analyses
Environmental factors such as depth (m), salinity (PSU), and seawater

temperature (oC) were measured at each station using GARMN FishFinder
240, ATAGO refractometer, and a thermometer, respectively. Sediment
samples were collected from a boat using hand operator Van Veen grab,
transferred into polyethylene and stored under ice. At the laboratory, samples
were stored at -10 oC until the treatment phase. Sediment samples were
analyzed physically (organic content and grain size analysis), and chemically
(sediment digestion and analysis of the selected heavy metals).
Approximately, 50 g of homogenized sediment were sieved on a
mechanical sieve shaker (KARL KOLB) through six sieves (mesh sizes from
0.038–2 mm). Weight of sediment fraction in each sieve was obtained.
Another portion of the homogenized sample was used to obtain the organic
content of the sediment by incinerating a known weight at a temperature of
450 oC for 12 hours. Difference in weight before and after ashing was
obtained, and percentage of organic content (ash-free dry weight) was
determined.
Microwave-assisted digestion was conducted using a MARS5 microwave
(CEM Corporation, USA). The digestion was performed according to US-EPA
3051 methods. Harbour sediment (LGC6156) from the Laboratory of the
Government Chemist, UK was used as a reference material for quality
assurance (metals recovery 93.1 - 98.3 %). The chemical reagents used in the
digestion were analytical grade ‘AnalaR’ from VWR Chemicals. The samples
were analyzed for zinc, cadmium, lead, and copper using the voltammetry
technique (Metrohm® 757 VA computrace) and following Metrohm
Application Bulletin No. 231/2e.
3. RESULTS
3.1. Physical Conditions and Sediment Characteristics
Generally, salinity levels ranged between 44-46 PSU reflecting typical

high salinities in the Arabian Gulf. However, localized reduction in salinity
levels were recorded in stations 1 and 2 of T1 (37 and 42 PSU, respectively)
due to the low salinity of wastewater associated with domestic sewage
effluents. Similarly, an increase in salinity was observed in station 1 of T3,
which is likely due to the brine wastewater discharge from the power and
desalination plant.
A substantial increase in surface seawater temperature was recorded in
station 1 of T3. A difference of 14 oC was measured at a distance of 250 m
from the outlet of the power and desalination plant. It is evident that this
increase in seawater temperature around the outlet is attributed to the brine
wastewater that associated with high temperatures.
The first five stations and station 7 of T1 were characterized by
accumulated organic matter due to continued discharge of sewage effluents.
Additionally, the last three stations of the same transect were composed of
hard substrate. Only station 6 was physically and chemically analyzed.
Consequently T1 was eliminated from the overall analyses of heavy metal
concentrations. The substrates of stations 1-4 of T3 were mainly coarse sand
mixed with patches of gravels and small stones. Conversely, most of the
164 Humood Naser
remaining stations of T3 were composed of fine or very fine sand, which are
associated with the navigational channel leading to the nearby port. Transect
T4 was mostly composed of fine sand, while T5 ranged from medium to fine
sand.
Table 2. Environmental measurements and sediment characteristics of

T1, T2 and stations 1-5 of T3
Mean gain size (Ø)
% Organic content
Temperature (oC)
Salinity (PSU)
Description
Depth (m)
Transect
Station
1 37 17 0.8 - Organic matter 100

5 45 14 10.8 - Organic matter 100
1 44 14 4.2
6 2.40 Fine sand 2.23
8 45 14 2.5 - hard substrate -
1 44 13 1.5 2.00 Medium sand 2.38
2 44 14 1.6 2.09 Fine sand 3.84
3 44 14 2.9 2.54 Fine sand 2.99
4 44 14 2.6 3.55 Very fine sand 8.59
5 44 14 2.5 3.73 Very fine sand 4.01
2 6 45 15 2.5 3.40 Very fine sand 3.06
7 45 15 2.4 3.30 Very fine sand 4.95
8 45 15 2.5 1.79 Medium sand 2.68
9 45 15 3.0 2.26 Fine sand 2.77
3 3 45 16 0.9 - hard substrate -
4 45 17 3.3 0.23 Coarse sand 2.67
5 45 16 7.5 2.66 Fine sand 4.03
Table 3. Environmental measurements and sediment characteristics of

stations 6-10 of T3, T4, and T5
Mean gain size (Ø)
% Organic content
Temperature (oC)
Salinity (PSU)
Description
Depth (m)
Transect
Station
6 45 16 10.8 2.77 Fine sand 4.70

7 45 16 9.9 3.19 Very fine sand 6.36
3 8 45 16 8.3 2.10 Fine sand 4.71
9 45 15 8.5 2.47 Fine sand 4.52
10 45 15 7.9 3.40 Very fine sand 4.96
1 45 15 1.3 2.23 Fine sand 3.96
2 45 15 1.9 2.24 Fine sand 5.90
3 45 15 4.0 2.25 Fine sand 4.24
4 45 15 5.0 1.08 Medium sand 3.55
5 45 15 5.5 1.68 Medium sand 2.19
4 6 46 15 5.3 2.07 Fine sand 2.93
7 46 15 5.9 2.78 Fine sand 3.82
8 46 15 6.4 2.71 Fine sand 3.91
9 46 15 6.8 2.88 Fine sand 4.81
10 46 15 5.9 2.40 Fine sand 3.68
1 45 15 1.4 1.38 Medium sand 2.21
2 45 15 2.6 1.38 Medium sand 2.00
3 45 15 3.0 2.45 Fine sand 4.52
4 45 15 2.5 0.82 Coarse sand 2.99
5 45 14 4.5 1.27 Medium sand 2.28
5 6 45 14 6.0 3.26 Very fine sand 2.46
7 45 14 5.9 3.35 Very fine sand 2.34
8 45 14 5.9 3.36 Very fine sand 2.60
9 45 14 5.9 3.42 Very fine sand 3.82
10 45 14 5.6 3.45 Very fine sand 4.33
Organic content in the sediment ranged between 2.00 – 8.59 % with an

overall mean of 3.79 %. However, variation among transects were observed
T2, T3, T4, and T5 ranged between 2.38-8.59, 2.67-6.36, 2.19-5.90 and 2.00-
166 Humood Naser
4.52 % with means of 3.90, 4.56, 3.89 and 2.99, respectively. Details of
environmental measurements and sediment characteristics are presented in
Tables 2 and 3.
3.2. Spatial Gradient of Heavy Metal Contaminations
Transect sampling design was adopted to detect onshore-offshore spatial

gradient of heavy metals. Generally, concentrations of zinc and copper showed
fluctuations through stations, and no clear trends of onshore-offshore gradient
were clearly detected (Figures 3 and 4).
Zn
100
80
-1
60
mg kg
40
20
0
1 2 3 4 5 6 7 8 9 10
Stations
T2 T3 T4 T5
Figure 3. Concentration of zinc in stations of T2, T3, T4 and T5, with no clear
onshore-offshore gradient.
Cu
35
30
25
-1
20
mg kg
15
10
5
0
1 2 3 4 5 6 7 8 9 10
Stations
T2 T3 T4 T5
Figure 4. Concentration of copper in stations of T2, T3, T4 and T5, with no clear
onshore-offshore gradient.
Pb
40
35
30
25
-1
mg kg
20
15
10
5
0
1 2 3 4 5 6 7 8 9 10
Stations
T2 T3 T4 T5
Figure 5. Concentration of lead in stations of T2, T3, T4 and T5, with a clear seaward
decrease in stations of T4.
Cd
20
18
16
14
12
-1
mg kg
10
8
6
4
2
0
1 2 3 4 5 6 7 8 9 10
Stations
T2 T3 T4 T5
Figure 6. Concentration of cadmium in stations of T2, T3, T4 and T5, with significant
increase in stations 1, 9 and 10 of T4.
Conversely, T4 showed a clear seaward decrease in lead concentrations

(Figure 5). A localized increase in cadmium concentration was recorded in
station 1 of T4 adjacent to the outlet of Bujarjor desalination plant with a
concentration of 19.14 mg kg-1. Similarly, higher concentrations of cadmium
were detected in stations 9 and 10 of the same transect (3.35 and 7.64 mg kg-1,
respectively) (Figure 6).
168 Humood Naser
3.3. MEAN CONCENTRATIONS OF HEAVY METALS IN

SAMPLING SITES
Substantial increases in the mean concentrations of zinc and copper in T3
were observed reflecting the anthropogenic inputs associated with brine
wastewater discharge and navigational activities in the nearby port.
Mean concentrations of metals in sampling transects
80
70
60
50 Zn
mg kg-1
Cd
40
Pb
30 Cu
20
10
0
T2 T3 T4 T5
Transects
Figure 7. Heavy metal concentrations in sampling transects; error bar (T2: n = 9, T3: n
= 7, T4 and T5: n = 10).
Mean concentrations of cadmium and lead were higher off the eastern
coastline compared with Tubli Bay (T1 and T2) reflecting industrial activities
along the eastern coastal areas (Figure 7).
3.4. Variation of Metal Concentrations in Comparison with

Regional and International Guidelines
Comparison between metal concentrations in Bahrain and the Canadian

sediment quality guidelines revealed that the overall mean concentration of
cadmium significantly exceeded the interim sediment quality guideline.
Indeed, the maximum concentration of cadmium (19.14 mg kg-1) was three
times higher than the probable effect level, above which adverse biological
effects are usually or always observed.
Table 4. Comparison between metal concentrations in Bahrain and the

Canadian sediment guidelines, 1 (ISQG): interim sediment quality
guideline, 2 (PEL): probable effect level, above which adverse biological
effects are usually or always observed (CCME, 2001); 3 range of metal
concentrations reported in sediments from the Arabian Gulf
(ROPME, 2003)
Metal Bahrain Canadian Standards Arabian Gulf

mg kg -1
Mean Range ISQG1 PEL2 Range3
Pb 17.43 8.59 – 34.58 30.2 112 0.20 – 64.30

Cu 14.67 4.25 – 31.98 18.7 108 1.30 – 142.0
Zn 40.99 20.94 – 78.90 124 271 0.70 – 410.3
Cd 1.37 0.083 – 19.14 0.7 4.2 0.01 – 4.5
Apart from cadmium, heavy metal concentrations of this study were

within the range of variations that are reported in sediments from the Arabian
Gulf (Table 4).
4. DISCUSSION
Although some trace metals are essential biological elements for aquatic
organisms, all metals could be potentially toxic when certain threshold
concentrations are exceeded (Ansari et al., 2004). The metals that were
analyzed in the present study could be broadly arranged according to
increasing level of toxicity from lead, zinc, copper, to cadmium (Abel, 1996).
Lead is toxic to animals and humans. It can accumulate in individual
organisms as well as the entire food chain. Lead can physiologically and
neurologically affect humans. Zinc is essential for plants, animals and humans.
However, excessive quantities of zinc may lead to acute or chronic toxicity.
Copper is essential micronutrient required in the growth of plants and animals.
In humans it contributes to the production of blood hemoglobin. In large
doses, copper is toxic and excessive ingestion can destroy red blood cells.
170 Humood Naser
Cadmium is a very toxic metal and has been responsible for a number of
deaths (Abel, 1996; Kennish, 2001).
After entering the marine environment either naturally or
anthropogenically, heavy metals are distributed among water, biotic and
sediment compartments. Sediment distribution is influenced by biological,
chemical and physical properties of the sediment (De Gregori et al., 1996). It
is widely recognized that fine sediments often contain higher metal levels than
the coarser grained sediments (Al-Abdali et al., 1996; Belzunce et al., 2001;
Garcia et al., 2008). Similarly, this study showed a correlation between the
heavy metal concentrations and the particle size of the sediment and its
organic content.
It is recognized that heavy metals are by-product from desalination
process due to corrosion (Mohamed et al., 2005; Lattemann and Hopner,
2008). Mean concentrations of Zn and Cu were elevated nearby the main
desalination plant in Bahrain (SPWS). Similarly, a significant increase in Cd
concentration that exceeded the limits of national and international standard
guidelines was detected nearby the outlet of Bujarjor desalination plant. Such
localized increase in Cd due to desalination plants was similarly reported in
the Arabian Gulf with a maximum concentration of 31.59 mg kg-1 (Sadiq,
2002).
Heavy metals Pb, Cu, Zn, and Cd are associated with oil industry, and are
toxic to organisms due to bioaccumulation (Munawar et al., 2002). Adeniyi
and Afolabi (2002) reported elevated concentrations of heavy metals in soil
within the vicinity of facilities handling refined petroleum products. The
Bahraini eastern coastline is recognized as a hotspot for high concentrations of
hydrocarbons and heavy metals (De Mora et al, 2004). Indeed, the shallow
subtidal area off the oil refinery is considered to be chronically polluted (De
Mora, 2010). Localized hotspots have been reported for Cu, Pb and Zn in
Bahrain, which were mainly attributed to petroleum industries (De Mora et al.,
2004, 2010). The effect of industrial effluents was reflected in the present
study by the increase in the mean concentrations of Cd and Pb off the eastern
coastline in Bahrain (T4 and T5).
Concentrations of heavy metals in the Arabian Gulf were generally found
comparable to uncontaminated offshore continental shelf areas, reflecting
natural background levels (Basaham and El-Sayed, 1998). However, ROPME
(2003) indicated that elevated concentrations of metals in the Arabian Gulf are
mainly associated with sediments from impacted areas adjacent to harbours
and industrial facilities. Likewise, this study detected a localized
contamination of Cd that attributed to the industrial land-based effluents.
Sources of heavy metal pollution in Bahrain are not limited to sewage and
industrial effluents, but extend to other activities such as dredging and
reclamation. It is widely documented that mobilization and concentration of
heavy metals increase after dredging and reclamation activities (Singh et al.,
1998; Guerra et al., 2009; Hedge et al., 2009). In the light of the current
escalation in coastal reclamation and development in Bahrain, such activities
are considered a main source for mobilizing heavy metals.
CONCLUSION
Heavy metals are anthropologically introduced to the Bahraini coastal and
marine environments through a variety of sources and activities, including
sewage and industrial effluents, brine discharges and coastal modifications.
This study detected localized hotspots of metal contamination that could be
attributed to industrial pollution. Metal contamination in Bahrain is a major
threat to the environment and human health. Therefore, continuous spatial and
temporal monitoring is critically needed for better management of coastal and
marine environments in Bahrain.
ACKNOWLEDGMENT
Thanks are due to the staff at the Department of Biology, College of
Science, University of Bahrain, Technical assistance provided by F.
Al-Ammari is greatly appreciated.
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Chapter 9
COMPARATIVE STUDY IN THE BIVALVE

CAPACITY FOR METAL BIOACCUMULATION
IN THE BALEARIC ISLANDS (SPAIN).
MYTILUS GALLOPROVINCIALIS AS AN
ADEQUATE SENTINEL SPECIES FOR
MONITORING COASTAL WATERS
A. Sureda1, S. Tejada2, A. Box1,

J. Benedicto3 and S. Deudero4
1
G.rup de Nutrició Comunitària i Estrès Oxidatiu (IUNICS), Departament
de Biologia Fonamental i Ciències de la Salut, Universitat de les Illes
Balears, Ctra. Valldemossa Km 7.5.E-07122, Palma de Mallorca,
Balearic Islands, Spain
2.
Experimental Laboratory. Research Unit. Son Llàtzer Hospital. IUNICS.
Ctra. Manacor km 4, CP 07198, Palma de Mallorca,
Balearic Islands, Spain
3
Centro Oceanográfico de Murcia, Instituto Español de Oceanografía (IEO)
22, CP 30740, San Pedro del Pinatar, Murcia, Spain
 
Authors for Correspondence: E-mail: tosugo@hotmail.com (A. Sureda) Posting address: Grup
de Nutrició Comunitària i Estrès Oxidatiu (IUNICS), Departament de Biologia Fonamental
i Ciències de la Salut, Universitat de les Illes Balears, Ctra. Valldemossa Km 7.5. E-07122,
Palma de Mallorca, Balearic Islands, Spain
178 A. Sureda, S. Tejada, A. Box et al.
ABSTRACT
Metals are major pollutants in the industrial world causing long-term
effects on marine ecosystems. Bivalves are widely used as sentinel
species for monitoring coastal environments because they have a large
capacity to accumulate pollutants in their tissues to a degree that is
suitable for measurement. Our aim was to compare the metal
bioaccumulation capacity of four different bivalves –Mytilus
galloprovincialis, Venus verrucosa, Lithophaga litophaga, Chamalea
gallina- and the echinoderm Paracentrotus lividus. Samples were
collected from aquaculture farms and Pb, Cd, Cu and Hg were
determined in soft tissues. All bivalves presented higher levels of metals
than P. lividus indicating that they are good markers of bioaccumulation.
The mussel M. galloprovincialis was the bivalve that accumulated more
quantity of metals. As consequence, a second study was performed with
caged mussels from four areas around Mallorca Island (Alcudia, Porto
Colom, Santa Ponsa and Sóller) to validate the use of the caged mussels
as biomarker of metal contamination. Metal concentration was
determined in the whole mussel and antioxidant enzymes (catalase,
superoxide dismutase and glutathione peroxidase) were determined in the
digestive gland. Although there were not significant differences between
the selected stations in antioxidant enzyme activities, a direct correlation
between metal concentration and antioxidant enzymes were evidenced.
These results indicate that mussels presented increased stressful states in
the areas with the highest metal levels. M. galloprovincialis was the
species with the highest metal concentration and this characteristic
together with its easy collection and manipulation indicates that mussels
are useful biomarkers of metal contamination in coastal waters.
Keywords: Pollution, heavy metals, mussel, oxidative stress.
1. INTRODUCTION
Pollutants from industrial and agricultural sources, that can cause adverse
effects, are continuously increasing [18, 28]. Persistent pollutants such as
PCBs, PAHs, and toxic metals (Cd, Hg, Ag, Co, Cr, Ni, Pb, Zn, Cu, etc.) in
different compartments of the marine environment have become a major threat
to the health of the marine ecosystem. Metals are major pollutants in the
industrial world [22]. These pollutants can cause long-term effects since they
continuously accumulate and concentrate metals in their tissues [21].
Accumulation in living organisms leads to concentrations several orders of
Comparative Study in the Bivalve Capacity … 179
magnitude higher than those of the surrounding water [7]. Moreover, the
ingested metals cause toxicity at the base of food webs but also in top
consumers if the assimilated metals build up through the food web [4].
Classically, marine pollution evaluation is based on direct measurements of
the abiotic components and on measurements of the abundance and
bioaccumulation of metals in selected marine organisms [9].
Nowadays, the use of biomarkers to analyze the effects of exposure to
chemical pollutants in the aquatic environment is more extended [10, 36, 12].
Measuring the same biomarkers in different localities simultaneously provides
information about the pollution status; it also allows bio-monitoring the
alterations due to environmental changes and allows a better comprehension of
the mechanistic mode of action of the environmental pollutants on the
organisms [17]. Biomarkers also allow an early detection of the effects of
pollutants at sublethal concentration which could be important to activate
bioremediation strategies [39]. The oxidative metabolism of cells is a
continuous source of reactive oxygen species (ROS), resulting from univalent
reduction of O2, that can damage most cellular components [26, 37]. Under
physiological conditions, cells contain a complex network of antioxidant
defense that scavenges the generation of ROS and avoids the damages related
to their high reactivity [20]. Antioxidant defense includes both enzymatic and
non-enzymatic antioxidants. The antioxidant system involves enzymes such as
superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase
(GPx) that act by detoxifying the generated ROS. In polluted environments
and especially in coastal waters, living organisms are most often exposed to
chemical contaminants [26]. Organisms have developed mechanisms to
protect themselves from the toxic effects of the increased ROS production,
activating the antioxidant system. The levels or activities of antioxidants are
potential biomarkers revealing a contaminant-mediated biological effect on the
organism [41, 5, 42]. Benthic organisms, and especially bivalve molluscs, are
widely used as sentinel species for monitoring coastal environments [12, 19,
38]. Bivalves are filter-feeding organisms which may be exposed to large
amounts of chemical pollutants. Bivalves constitute interesting indicators of
coastal pollution because they exhibit the following characteristics: a
sedentary nature, a wide geographical distribution, they can be sampled easily
and they highly accumulate pollutants. The caging techniques have been
highly developed in recent years because they have various advantages, such
as the genetic uniformity of individuals from the same cohort (limiting natural
variability) and the possibility of placing caged mussels at selected sites [3].
Caged mussels also avoid the interferences in the antioxidant response of the
mussel adaptation to the studied sites [2]. Despite caged mussels presented
better characteristics for the evaluation of contaminants and the antioxidant
response, wild mussel presents the advantage of its natural and permanent
availability which allows to perform rapid pollutant determinations in sites
with natural populations.
The aim of the present work was divided in two different assays. In the
first assay, several bivalves and an echinoderm were used to determine the
degree of metal bioaccumulation in soft tissues. In the second assay, mussels
caged in several areas along Balearic Islands were used to validate the utility
of this species as sentinel organism by determining the antioxidant enzyme
activities, metal concentration and the possible correlations between them.

2.1. Bivalves and Echinoderm Sampling for Metal Analysis
Sample collection was carried out at two locations of the island of

Mallorca. The study sites are production zones for aquaculture of molluscs and
other marine invertebrates located in the Bay of Palma (Ministerio de
Agricultura, Pesca y Alimentación. Orden 22/09/2005). The studied species
compressed four mollusc bivalve species (Mytilus galloprovincialis, Venus
verrucosa, Lithophaga lithophaga, Chamelea gallina) and one echinoderm,
the sea urchin Paracentrotus lividus, for comparative pourposes. Once the
specimens had been collected, they were rinsed, measured, weighed and deep
frozen at -20ºC until analysis. To minimize size-related variation, each bivalve
species was selected with similar shell length and the echinoderm with similar
carapace diameter. A shell length of 50 ± 5 mm was selected for M.
galloprovincialis, 40 ± 5 for V. verrucosa, 66 ± 7 mm for L. lithophaga, 20 ± 5
mm for C. gallina and 43 ± 4 mm carapace diameter for P. lividus.
2.2. Studied Areas for Bioaccumulation of M. Galloprovincialis
Four stations (Alcudia, Porto Colom, Santa Ponsa and Sóller) were
selected along Mallorca Island waters (Figure 1). These locations were
selected on the basis of different impact degree of the human activities (Table
1).
Table 1. Contaminations sources in the locations studied
Site Human impact source Degree of impact

Alcudia Bay Commercial and mercancy harbour HIGH
1 marina
Touristic use (high number of hotel)
Agricultural residues arrive to sea
through Alcudia Albufera
Porto Colom Beach use LOW/MODERATE
Traditional fishery harbour
Santa Ponsa Touristic use (moderate degree of MODERATE/HIGH
urbanisation)
Commercial harbour
Sóller Touristic use (moderate degree of MODERATE/HIGH
urbanisation)
Commercial harbour
1 marina
Locations selected around the Mallorca Island on the basis of different impact degree
of the human activities.
Figure 1. Stations selected around the Mallorca Island characterized by a different

human impact degree.
Alcudia Harbour, considered as reference of polluted station, is affected

by a highly ship transport activities and also receives high quantities of
agricultural residues from an albufera. Santa Ponsa and Sóller are two
locations with moderate/high human activities, whereas Porto Colom has
low/moderate impact of human activities.
2.3. Experimental Procedure
Mediterranean mussels (M. galloprovincialis) were harvested in the Sète

region (South East France) under pristine conditions and maintained in the
Thau lagoon (France) for one week before transplantation. Mussels were
characterized by similar genetic origin and by the same age (18-24 month) and
the same length (50 mm of shell) in order to minimize the variability. These
characteristics would make possible to analyse the different responses
depending on the transplantation site.
M. galloprovincialis were actively transplanted to different areas of the
Balearic Islands at depths of 20–35 meters. Mussels were placed with a buoy 5
meters depth, connected to the bottom with a rope and lasted with 30-40 kg
iron chain on the bottom in order to immobilize the caged M.
galloprovincialis.
Mussels were caged for three months, time enough to adapt to the new
environmental conditions, and were transplanted to the selected stations on
March and collected before the spawning period. Mussels were collected,
cleaned of epiphytes and immediately frozen with liquid nitrogen and
maintained at -20ºC on board until arrival to the laboratory where mussels
were stored at -70ºC. All samples were manipulated and stored in the same
conditions in order to avoid differences related to the storage conditions.
2.4. Sample Processing
For metal determination, the whole soft tissue of bivalves (n = 8) was

taken for analysis, triturated with Ultraturrax and freeze-dried. The full body
tissues of P. lividus (n = 8) were used for analyses. After the freeze-drying
processes, samples were homogenised with a mixer mill and stored in acid-
washed glass vials at room temperature until analysis.
Fresh and dry weight measurements of the soft tissues were performed for
each sample in order to refer the results to the dry weight. Mineralization of
samples was carried out at 130ºC for 5 h with nitric acid in a microwave oven
using high pressure Teflon reactors.
For biochemical analysis, digestive glands from each specimen of mussel
(n=8 for each station) were dissected out and immediately homogenized in ten
volumes (w/v) of 100 mM Tris-HCl buffer pH 7.5. Each homogenate was
briefly sonicated (2-3 s) using ultrasonic processor and centrifuged at 9000 g
at 4ºC for 15 min (Sureda et al., 2006). After centrifugation, supernatants were
collected and immediately used for the determination of enzymatic activities.
All assays were performed in duplicate. All results were referred to the total
protein content of the samples (Biorad Protein Assay) using bovine serum
albumin as standard.
2.5. Antioxidant Enzyme Activities
All antioxidant enzyme activities were determined with a Shimadzu UV-

2100 spectrophotometer at 37ºC. CAT activity (K / mg protein) was measured
by a method of based on the decomposition of H2O2 [1]. Glutathione
peroxidase (GP) activity (nmol / min / mg protein) was measured using an
adaptation of the method of Flohé and Gunzler [16].
This activity was determined with H2O2 as substrate and Glutathione
reductase (GR) and NADPH as enzyme and non-enzymatic indicators,
respectively. SOD activity (pmol / min / mg protein) was evaluated with the
xanthine oxidase-cytochrome C method as described by McCord and
Fridovich [30].
2.6. Metal Determinations
Determination of trace metal content followed the specifications of the

National (Ministerio de Sanidad y Consumo. Orden 2/08/91) and European
legislation (Directive 2001/22/EC, for mercury levels). All chemical analyses
were performed following the guidelines recommended for chemical
contaminant monitoring in marine organisms (UNEP/FAO/IOC/IAEA, 1993).
The tissue samples were analyzed for the following metals: lead, cadmium,
copper and mercury. The quantitative analyses were carried out by atomic
absorption spectrophotometry (Perking–Elmer 4110-ZL) using graphite
furnace or flame. Mercury was determined by flow injection cold-vapour
atomic absorption spectrometry (FIMS, Perking–Elmer).
The internal quality control was assured by testing control, blanks and
duplicated samples in each analytical series. The external accuracy of the
analytical procedures was tested by using certified reference material (Mytilus
edulis tissue; CRM no. 278, Community Bureau of Reference) and by
participation in international intercomparison exercises, such as the
QUASIMEME programme.
2.7. Statistical Analysis
Statistical analysis was carried out using a statistical package (SPSS 16.0
for Windows®). The statistical significance of the data was compared by one-
way analysis of variance (ANOVA). Least significant differences t-test (LSD)
post hoc paired comparison was further made to recognize deviant groups.
Results are expressed as mean ± S.E.M. and p<0.05 was considered
statistically significant. The possible bivariate correlations between the
different parameters were also analysed. Correlations between the metal
concentrations and biomarker responses were computed with the two-tailed
Pearson’s correlation analyses.
3. RESULTS
Concentrations levels of the metals lead, cadmium, copper and mercury in
the different bivalves studied in the current work and the sea ursine
Paracentrotus lividus is showed in Figure 2. Mytilus galloprovincialis had the
highest levels or all metals except for cadmium, whereas P. lividus
accumulated the lowest levels in all determined metall. When total metal
concentration was analysed no significant differences were evidenced between
bivalves (M. galloprovincialis 35.4 ± 6.7 g/g DW; V. verrucosa 31.5 ± 6.9
g/g DW; L. litophaga 26.5 ± 4.8 g/g DW; C. gallina 26.6 ± 8.1 g/g DW),
whereas P. Lividus metal concentration was significantly lower with respect to
all bivalves (8.70 ± 1.32 g/g DW).
Table 2 showed the activity of the antioxidant enzymes CAT, SOD and
GPx at the four studied locations (Alcudia, Porto Colom, Santa Ponsa and
Sóller) determined in the digestive gland of M. galloprovincialis. Alcudia
station was the location with the highest enzyme activity in any case while
Porto Colom showed the lowest values. Also, in table 2, the concentration of
the different studied metals is showed for every station.
Table 2. Antioxidant enzymes and metal concentration
Alcudia Porto Colom Santa Ponsa Sóller

Catalase
K/mg prot 313  9 291  5 300  8 298  11
SOD
pmol/min/
14.9  0.7 13.0  0.5 13.6  0.7 13.2  0.9
mg prot
GPx
nmol/min/
13.0  7.5 11.9  4.1 12.3  58 12.4  8.4
mg prot
Hg
g/g DW 0.256  0.006 0.196  0.013* 0.189  0.010* 0.186  0.009*
Cd
g/g DW 3.05  0.06 2.69  0.08* 2.52  0.11* 2.49  0.08*
Pb
g/g DW 2.23  0.06 1.93  0.14* 2.09  0.07 2.29  0.06 #
Ni
g/g DW 1.22  0.05 0.73  0.05* 1.09  0.03 0.82  0.06*
Total metal
g/g DW 6.84  0.16 5.59  0.26* 5.86  0.20* 5.83  0.14*
Antioxidant enzyme activities determined the digestive gland of M. galloprovincialis
and metal concentration in the whole individuals in four locations studied along
Mallorca island waters characterised by a different anthropogenic impact. One-
way ANOVA, p<0.05 was considered as statistically significant. (*) Significant
differences respect to Alcudia. (#) Significant differences respect to Porto Colom.
Values represent mean ± S.E.M.
Again, the highest values were observed at Alcudia location, except for
the nickel; the lowest levels were reached in Porto Colom and Sóller. In
general, the impact of tracer metals was higher in Alcudia than in the other
studied locations around the Mallorca Island. Additionally, the high levels
corresponded to lead and cadmium at any station.
The bivariate correlations between antioxidant enzyme activities and
metal concentrations are represented in Table 3 for the mussel M.
galloprovincialis. The higher positive correlations were found between CAT
and mercury and cadmium, and between SOD and cadmium.
Table 3. Bivariate correlations
CAT SOD GPx

Hg 0.67 ** 0.52 ** 0.44 *
Cd 0.68 ** 0.64 ** 0.48 **
Pb 0.49 ** 0.36 0.44 *
Ni 0.48 ** 0.58 ** 0.41 *
Bivariate correlations between metal concentrations (mercury (Hg), cadmium (Cd),
lead (Pb), nickel (Ni)), and the antioxidant enzymes (CAT, catalase, SOD,
superoxide dismutasa, GPx, gluthatione peroxidasa) in four locations studied
along Mallorca island waters characterised by a different anthropogenic impact in
the mussel Mytilus galloprovincialis. * indicates p < 0.05 and ** indicates p <
0.01.
Figure 2. Concentrations levels of the metals lead, cadmium, copper and mercury in
the studied bivalves and the sea ursine P. lividus. Note that, in general, M.
galloprovincialis has the highest metals levels while P. lividus showed the lowest
levels in any case. One-way ANOWA with p<0.05 considered as statistically
significant. * Significant differences respect to M. galloprovincialis. # Significant
differences respect to V. verrucosa. $ Significant differences respect to L. litophaga.
Data represent mean ± S.E.M.
4. DISCUSSION
In the present work, the pollution levels in waters of the Balearic Islands
were assessed by evaluating metal concentrations and enzymes activities in the
mussel Mytilus galloprovincialis and other bivalves and the echinoderm

Paracentrotus lividus. Moreover, correlations between the studied parameters
showed that mussel M. galloprovinciales is a good indicator of lead and
cadmium concentration, while the P. lividus showed not significant amounts of
the studied metal levels.
Pollutants from industrial and agricultural sources that can cause adverse
effects are increasing in marine ecosystems [18, 28]. These pollutants can
cause long-term effects on marine ecosystems even if their impacts have no
visible influence. The accumulation of metals in organisms is used as a
bioindicator for environmental quality assessment [24, 31]. In the current
work, the highest metal concentrations were found in the mussels M.
galloprovincialis, indicating that the mussels are a good bioindicator for the
quality of the waters and surroundings. Possible explanations for variability
between the concentration of metals in the tissues of the different molluscs and
the echinoderm P. lividus could be due to the species-specific differences in
bioaccumulation dynamics [11]. Metal concentrations were similar to those
previously reported in the same region [14], although other works showed
higher levels in M. galloprovincialis than the ones observed in this work [2, 8].
Lead and cadmium exhibited the highest values in the mussels, which can be
considered as indicative of pollution in the organisms since these both metals
are potential immuno-suppressants [6, 23]. Taking altogether, metal
concentrations in the mussel M. galloprovincialis can provide a measure of the
effects of potential pollutants in the marine environment, since these metals
have the highest toxic potential. However, little data are available on the
pollutant content in mussels [13, 8, 14] in the localities studied in the current
work.
The study of pollution has been based on the analysis of the metal levels
in water and in living organisms [9]. Most recently, the study of the
antioxidant enzyme activities is being used to assess the quality of the aquatic
environment [12]. In fact, the objective of most studies is to examine the use
of biomarkers as a measure of pollution-induced stress in aquatic organisms
[32, 12, 19]. In the present work, although the selected stations presented
different degree of anthropogenic impact, evidenced by the different human
uses and infrastructures, no significant manifestations were reported in the
antioxidant enzymes. In fact, the increased levels of the metals, observed in
the different areas with moderate or high impact, were not marked in the
antioxidant enzyme responses. These results indicated that the degree of
pollution is not different enough to induce an evident antioxidant response.
However, many studies have studied changes in biomarkers in areas highly
exposed to several pollutants, which are capable to induce very significant

changes in antioxidant enzymes and markers of oxidative damage [43]. The
present results show no significant differences between the studied stations,
indicating the same activity level of the antioxidant system and a good
adaptability of mussels to the anthropogenic activity. In a review of over 900
papers of biomarker responses in the literature, Wu et al. [46] highlighted the
uses and limitations of biomarkers as well as the importance of understanding
temporal changes in responses, especially in reference to the time for
induction, adaptation or recovery of particular biomarker responses. In the
present chapter, seasonal differences were avoided to not have this influence in
the results.
GPx and CAT protect cells against the deleterious effects of oxyradical
generation by maintaining endogenous reactive oxygen species at relatively
low levels and attenuating the damages related to their high reactivity [25].
GPx catalyzes the reduction of hydrogen peroxide into water or organic
peroxides to their corresponding stable alcohols by oxidizing the reduced
glutathione (GSH) into its oxidized form (GSSG). CAT is responsible for the
breakdown of hydrogen peroxide into water and oxygen, which may be
produced during basal aerobic metabolism or after a pollution-enhanced
oxyradical generation [27]. Both GPx and CAT activities have been used as
indicators of oxidative stress in aquatic organisms, including bivalves, caused
by exposure to pollutants [35]. SOD is one of the main enzymes of the cell's
antioxidant defence mechanism that detoxified the superoxide anion and that is
indicative of the toxicity for the marine species [40]. The present results show
that the toxic properties of the studied metals have not promoted the induction
of GPx, CAT or SOD activities, suggestive of an oxidative stress state,
indicating that the antioxidant system was protecting cells from the deleterious
effects.
Many studies have shown positive correlations between levels of
antioxidant defenses and the presence (levels, concentration) of xenobiotics
[34, 44, 33]. In fact, the induced biomarker response can be related to the
metal concentrations which the mussel is exposed to. In the current work,
several significant positive correlations were found between enzyme
concentrations and metal concentrations in the mussel M. galloprovincialis
indicating an effect of metal bioaccumulation on mussel physiology. The
results of the correlation analyses presented here indicate that the antioxidant
enzyme activities in mussels may be affected by trace metal pollution. Indeed,
it has been described that the exposure to contaminants tends to rapidly induce
antioxidant enzymes activity [15]. The positive correlations found between
biomarkers and trace metal concentrations in the mussels confirm the

dependence of the antioxidant system from the environmental bioavailability
of metals, as it has been previously reported [35, 45]. Several studies have
reported direct correlations between catalase and glutathione peroxidase and
liver polycyclic aromatic hydrocarbons (PAHs) in fish [33], between
glutathione S-transferase and PAHs and metals in mussels [45] or between
metallothionein like proteins and cadmium in the bivalve Cerastoderma
glaucum [29].
CONCLUSION
The results presented in this chapter support the use of the antioxidant
enzymatic activities together with metal concentration, measured in the mussel
M. galloprovincialis in waters of the Meditarrean Sea, as an appropriate
biomarkers for monitoring chemical pollution in marine ecosystems;
reinforced by the positive correlations between all activities measured and the
most environmental relevant pollutants. These correlations allow
differentiating areas characterised by moderate differences in the degree of
pollution, which was not possible to evidence only with biochemical assays.
The use of biomarkers are an useful tool for monitoring stressful situations
such as pollution in marine organisms providing a temporally and spatially
integrated measure of pollutants and allowing to early detect of potential
environmental damage.
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Chapter 10
RISK ASSESSMENT FOR THE ENVIRONMENT,

POPULATION AND INFRASTRUCTURES OF
SOME ABANDONED TAILING PONDS IN
THE MINING DISTRICT OF
CARTAGENA-LA UNION (SE SPAIN)
À. Faz Cano1, J. A. Acosta1, S. Martínez-Martínez1,

P. Martínez-Pagán2, D. M. Carmona1,3,
R. Zornoza1 and S. Kabas1
1
Sustainable Use, Management and Reclamation of Soil and Water
Research Group, Agrarian Science and Technology Department,
Technical University of Cartagena, Cartagena, Murcia, Spain
2
Geotechnics and Explotation Methods Research Group. Department of
Mining, Geology, and Cartographic Engineering, Cartagena, Murcia, Spain
3
Grupo de Investigaciones Ambientales, Universidad Pontificia
Bolivariana. Circular 1ª No. 70-01. Medellín, Colombia
 
Authors for Correspondence: angel.fazcano@upct.e (FazCano, À). Posting address:
Sustainable Use, Management and Reclamation of Soil and Water Research Group.
Agrarian Science and Technology Department. Technical University of Cartagena, Paseo
Alfonso XIII, 52. 30203 Cartagena, Murcia, Spain
196 À. Faz Cano, J. A. Acosta, S. Martínez-Martínez et al.
ABSTRACT
The mining district of Cartagena-La Unión (South-East of Spain)
has been among the most important mining centers in Spain in obtaining
lead, silver and zinc. For more than two thousand years, different
civilizations exploited mineral resources in this area. More than eighty
structures of mining wastes (tailing ponds) coming from old mineral
washing facilities were built, being currently one of the main sources of
pollution. The goal of this chapter is to provide a geophysical and
geochemical assessment of eleven tailing ponds selected in the mining
district of Cartagena-La Unión, giving a description of the risks for the
environment, population and close infrastructures. To achieve this
objective, surface soil samples were taken in these tailing ponds where
pH, texture, electrical conductivity, and total, bioavailable and water
soluble heavy metals were obtained. Geophysics was based on laying out
a series of profiles in which physical parameters of the materials in depth
were obtained. The equipment used provides 2D and/or 3D sections of
electrical resistivity changes (Ohm/m).
The geochemical and geophysical studies showed that only “Brunita”
tailing pond was structurally instable with a volume of 1000000 m3. The
pH values showed that the most acidic tailing ponds were “Descargador”
(< 3.5), “Regente” (< 3.5) and “Brunita” (3.5-4.4); oppositely,
“Encontrada C. Ponce” was the most alkaline. Regarding electrical
conductivity, most ponds showed a low salinity. Brunita was the tailing
pond with highest content of salts (> 16 dS m-1). The predominant
textures in the selected tailing ponds were coarse. All tailing ponds had
high total concentrations of Pb (maximum values: 21308 mg kg-1 in “ El
Lirio” and 21272 mg kg-1 in “Encontrada- C. Ponce”), Zn (maximum
values: 23575 mg kg-1 in “El Lirio” and 20971 mg kg-1 in “Cabezo Rajao-
La Unión”) and Cu (maximum values:1823 mg kg-1 in “Peña del Águila-
La Unión” and 1470 mg kg-1 in “Cabezo Rajao-La Unión”) which are
above the limit values for all legislation used in this study.
In general trends, the highest fractions of bioavailable and water
soluble metals were found in tailing ponds with ultra acidic conditions
(pH<3.5), such as “Regente” with 72%, 4%, 17% and 14% of
bioavailable Cd, Cu, Pb and Zn, respectively; and with 2.7%, 1.4%; 0.2%
and 24% of water soluble Cd, Cu, Pb and Zn, respectively. Therefore,
significant risks can arise in these tailing ponds, because the soil particles
can reach natural or populated areas by erosion or collapse by structural
instability, adversely affecting soil, surface and underground waters,
plants, animals and human populations as well as infrastructures close to
these tailing ponds. It is important to highlight that in the acidic tailing
ponds the problems of mobility of heavy metals are increased and so,
Risk Assessment for the Environment … 197
higher environmental risks may exist. For the reasons above,

rehabilitation plans should be carried out in these tailing ponds.
Keywords: tailing pond, environmental risks, geophysical and geochemical

analysis.
1. INTRODUCTION
In southeast of Spain, the mining districts of Cartagena-La Unión and
Mazarrón, have been among the most important mining centers in the country
in obtaining lead, silver and zinc. For more than two thousand years, different
civilizations such as the Iberians, Greeks, Phoenicians, Carthaginians,
Romans, Visigoths, Arabs and Spanish, among others, exploited mineral
resources in these areas. However, this mining activity had varying degrees of
importance in the period of mining operation until 1962 in the Mazarrón
Mining District and until 1991 in Cartagena-La Unión Mining District..
Thus, from the late nineteenth century to the early twentieth century the
mining district of Cartagena-La Unión became one of the largest producers of
lead. The landscape degradation has come out leaving a total desolation
appearance. Nowadays, a large area of the Mining District of Cartagena-La
Unión is dominated by areas where natural soils have been lost, because they
have been collected in the pit-mining opening or operating hollows, covered
by mining waste or contaminated by the discharge of effluents.
Also, sludge from the numerous gravimetric washing plants was dumped
on land and into the sea. Eighty structures of mining residuals coming from
old mineral washing facilities were built and currently these structures are one
of the main sources of pollution.These are storing structures coming from
sulphur floating processes (Ayala and Rodríguez, 1986). The sulphur waste
deposit was poured beside a dike which would act as a barrier and favour the
formation of a sand beach. The volume of water that composed the waste was
poured through decanting towers or concrete decanting pipes installed on the
slope. When the waste tailing pond was full, the pond was enlarged by
building a new dike (Martínez-Pagán, 2006).
Although these abandoned tailing ponds have heterogeneous physical and
chemical properties, the generation of acidic waters and high levels of heavy
metals are generally common characteristics (Chambers and Sidle, 1991;
Michelutti and Wiseman, 1995; Dudka and Adriano, 1997).
These tailings can be dangerous because they can cause problems of

instability or collapse slip favored by the phenomena of subsidence of the
deposit, due to the accumulation of water on its surface, seismic situations or
adverse weather condition (Fundación Sierra Minera, 2001). Most tailings
present gullies, landslides and a high degree of erosion, since they do not have
a proper drainage system. Besides, they provide solid or thin materials to
runoff which can produce acidic waters, altering the landscape. The lack of
vegetation on the surface of most tailing ponds increase the risk of wind and
water erosion and transportation of contaminated material to the surroundings
(Ortega et al., 1993).
Thus, the environmental impact caused by the waste produced by mining
and metallurgical activities, has resulted in marked negative effects on soil,
water resources (surface, groundwater and marine water), landscape,
atmosphere and biota (Zanuzzi, 2007).
The effects on vegetation and wildlife are the direct consequences of the
occupation and destruction of soil structure, which is seen in the realization of
open pits and in the construction of mining ponds and tailings, which
eradicates the natural plant cover, destroying the habitats for fauna and
ultimately causing its demise. The risk of erosion, by water and wind, is the
most important environmental issue due to its implications. The effects of the
erosion of mineral tailings are manifested in the contamination of soil, the
riverbeds of streams (watercourses), the marine environment and the loss of
stability of these structures. The intensity of the winds is a factor that
determines the risk of wind erosion, which impacts all areas without
vegetation. It mainly affects tailing ponds because of its low stability of soil
aggregates, the lack of vegetation and the oxidation of sulfides to sulfate.
These compounds form a very low-density crust which is eroded by wind and,
sometimes, can be long distances transported (Ros, 1997).
Similarly, water erosion is the main contribution of sediment highly
polluting surface waters and the marine environment, mainly the Mar Menor
lagoon and the Mediterranean coast (CAAMA, 1999, García et al., 2003; Faz
et al., 2004). Runoff not only produces the erosion of materials but also favors
the formation of acid waters. These waters are generated when sulfide-bearing
rocks in the study area, pyrite, sphalerite and galena, are exposed to the action
of water, oxygen and bacteria, also known this process as acid mine drainage.
Its high corrosive power, aggravated by the heavy metal content has different
effects such as degradation of aquatic systems (especially the Mar Menor
laggon), removal of vegetation, introduction of heavy metals in the food chain,
the toxicity of the soil and loss of agricultural capacity, and so forth.
With regards to groundwater, one of the effects of mining is the alteration

of the piezometric levels by disruption of phreatic levels in the open pits and
appearance of “upwelling” or seepage of acid water.
Finally, it is important to mention the visual impact associated with the
surface mining operations especialy brought out by open pits and tailing
ponds, which changes the visual characteristics of the area, makes
physiographic modifications and alterations in the landscape quality, and
causes to the discordance of colors, textures and shapes discordant with the
natural environment (Ros, 1997; Ros, 1999, García et al., 2004).
The goal of this study is to provide a geophysical and geochemical
assessment of the current tailing ponds in the Mining District of Cartagena-La
Unión, making a prediagnosis of the risks for the environment, population and
infrastructures. These structures constitute a source of pollution by heavy
metals, acid materials and acid mine drainage, which can reach other natural or
populated areas by erosion or collapse by structural instability, adversely
affecting soil, water, plants, animals and human populations as well as
infrastructures close to the tailing ponds.
2. MATERIAL AND METHODS

2.1. Study Area
Eleven tailing ponds were studied in the Mining District of Cartagena-La

Unión that is located in the South-East of the Murcia Region (Figure 1.).
The Mining District of Cartagena-La Unión has semiarid Mediterranean
climate, with a quite irregular distribution of rainfall in time and space, whose
annual average is inferior to 400 mm. Mean monthly precipitation in the study
area ranges between 36 mm in January, and 5 mm in July. Rainfall usually
concentrates on a few periods of time, having torrential character, mainly in
September-October. This fact is very important in the area because rain
intervenes in erosion processes and relief modelling. Average annual
temperatures in the area are very regular, with an average of 18 ºC. January is
the coldest month, with an average temperature of 10 ºC, while August is the
warmest month, with an average temperature of 26 ºC. The most important
winds come from East and Northeast.
The Mining District of Cartagena-La Unión occupies an area of
approximately 100 km2 and extends in a range of 23 km long and about 4 km
width NS. From a hydrological perspective, the Mining District of Cartagena-
La Unión is characterized by exhibiting a lot of small aquifers. These aquifers

are placed both in Triassic dolomites and marbles, and in neogenous
quaternary and detritic filling materials. However, due to the high structural
complexity in the area, together with the mining activity effects, it is difficult
to identify and establish the hydrogeological units (Robles-Arenas et al.,
2004).
Figure 1. Location of studied tailing ponds in Cartagena-La Unión Mining District.
The soils of this study area have the characteristics of the semiarid areas of
Mediterranean climate, where the irregularity and torrential rains, with a high
erosive power, remove the surface layer due to the absence of vegetation and
therefore impedes the soil development. The dominant soil types in the study
area according to the USDA Soil Taxonomy are: Calcic Paleargid; Typic,
Lithic and Xeric Haplocalcid (USDA, 2010).
2.2. Methodology
2.2.1. Geophysical Study

At mining tailing ponds resistivity measurements were carried out in
several locations to guarantee sufficient depth of investigation to reach the
interface between the substratum and tailings with suitable resolution
(Bernard, 2003).
We used a SYSCAL R1 switch 72 resistivity-meter (IRIS Instruments,
2001) with multicore cable designed to connect simultaneously up to 72
electrodes. Each stainless-steel electrode was 30-cm long and connected to the
resistivity meter through take out clips. The coordinates of each end electrode
placement were recorded using a GPS unit to properly locate them on the map.
In the mining tailing ponds, several 2D ERI profiles were liid out. An ERI
profile had either 54 or 36 electrodes with 5-m electrode spacing depending on
the profile length. Electrical data were acquired using a Schlumberger-Wenner
array due to its sensitiveness to vertical and horizontal electrical resistivity
variations and it is a good compromise between Wenner and Dipole-dipole
arrays (Loke, 2010). In addition, we selected a small area at tailing ponds for
carrying out a quasi-3D ERI survey.
Electrical measurements collected from each profile were analyzed in a
two-stage process. First, we used PROSYS II software (IRIS Instruments,
France) for initial data processing to remove bad electrical values and make
the necessary topographical correction to the resistivity measurements.
Following this stage, the corrected data were used in an inversion process with
RES2DINV and RESEDINV software (Loke, 2010) for 2D and 3D ERI
analyses, respectively. The 3D ERI data was presented in blocks using Slicer
Dicer software (Pixotec, USA).
We used Surfer 8 software (Golden Software, Colorado USA) to calculate
the volume of tailings stored in the mining tailing ponds. The volume of each
tailing pond was solved using a gridding method, specifically the kriging
method (Orche, 1999), that led to the computation of the volume by employing
the Surfer software, using depth to bedrock calculated from the resistivity
values as an input parameter in Surfer. Surfer calculated the total volume in
the pond using three mathematical manipulations (or rules): trapezoidal,
Simpson and Simpson 3/8.
2.2.2. Geochemical Study

Two types of samplings are carried out in each pond, a surface sampling
(0-15 cm) and a depth sampling (mechanical drill from 0 - to bedrock). The
analyses for this study were: pH measured in 1:1 water-soil ratio using the
method of Peech (1965) referred to water (to pH-put Basic 20 CRISON),
electrical conductivity analysed according to Bower and Wilcox (1965),
particle size analyzes carried out using the FAO-ISRIC system (1990). For the
quantification of the total metals an acid digestion (nitric-perchloric) was used
(Risser and Baker, 1990), for the bioavailable metals we utilized DTPA in the
ratio of 1:2. soil-extractant (Norwell, 1978), and for soluble metals we used
nano-pure water extraction (1:5 soil-water ratio). Measurements of metals
were carried out using atomic absorption spectrophotometer (AAnalyst 800,
Perkin Elmer).

3.1. Geophysics Analysis
Table 1 clearly shows that there is a high variability in the extension of the
tailing ponds, ranging from 12330 m2 to 159812 m2. There are two of them
that according to their size are the highest, these are Cabezo Rajao-La Unión
tailing pond (159812 m2) and Encontrada C. Ponce tailing pond (146364 m2).
These tailing ponds also have the longest perimeters (1868 m and 1406 m,
respectively). These parameters are of especial interest for reclamation
activities since they indicate how much extension should be reclaimed.
Table 1. Analysed variables by geophysics in the Mining District of

Cartagena-La Unión
PARAMETERS FROM GEOPHYSICS ANALYSIS

TAILING PONDS Area Perimeter Volume Tailing layer
(m2) (m) (m3) thickness (m)
Lirio 72952 1625 740325 38
Brunita 98373 1276 925050 38
Las Lajas 18300 575 109712 14
Descargador 22500 590 184146 32
Encontrada - C. Ponce 146364 1406 1132186 38
Encontrada-Cartagena 43500 935 94600 29
Cabezo Rajao-La Unión 159 812 1868 423233 15-24
Peña del Águila-La Unión 12330 520 161644 11-19
San Lázaro-La Unión 18100 725 46799 15
Regente-Cartagena 76443 1061 832893 27
Loma del Beal-Cartagena 84637 1215 1809355 34
(a)
(b)
Figure 2. a) 3D model from the of Peña del Águila tailing pond, and b) 3D model from
the El Lirio tailing pond.
However, to the largest tailing pond in volumen is the Loma del Beal-
Cartagena tailing pond, which has a volume of about 1809355 m3 of waste
materials. The next important tailing pond, in tern of volumen, is La
Encontrada C. Ponce tailings pond, which shows a volume of 1132186 m3.
Other tailings ponds which must be regarded according their volume are
Brunita and Regente-Cartagena. These tailings ponds have a volume of nearly
1000000 m3. The high amount of waste, which is storage into theses mining
ponds, suggests that the planned reclamation tasks should be in situ, since the
final cost of any ex situ treatment will be very expensive.
According to the ERI method, the tailing layer thickness ranges from 14 m
(e.g.: Las Lajas tailings pond) to 38 m, such as in El Lirio, Brunita, and La
Encontrada C. Ponce tailing ponds. Figure 2 shows the 3D model which
depicts the depth of the interface tailings-bedrock obtained by the ERI method
from Peña del Aguila and El Lirio, which were used to calculate the final
volume of the tailing ponds. In general, the structural and material
composition of the tailing ponds can be generally categorized into three groups
based on empirical electrical resistivity (ER) values and physical and chemical
properties of materials sampled at various depths. The materials with low
resistivity (<8 Ohm.m) correspond to the fine-grain tailings while the materials
associated with coarse waste rocks have intermediate ER (8-150 Ohm.m). The
third type of materials exhibits ER >150 Ohm.m and it is related to the hard
bedrock underlying the pond. The correlations between ER and the different
types of materials in the tailing ponds were corroborated by the electrical
conductivity analysis from mechanical drilling-core samples. For example, we
measured EC values > 338 mSm-1 in fine-grained tailing materials with ER <
3.0 Ohm.m; this relationship is expected because 1 Ohm.m correspond to
1000/mSm-1 (Parasnis, 1997). The ER in fine tailings can be attributed to
several factors including fine particle size distribution, presence of metals,
high water content, bulk density, high cation exchange capacity that
contributed to the high conductivity (or low ER) (Samouelian et al., 2005;
Corwin and Lesch, 2005). Only in one of the ponds (Brunita) signals of
structural instability, such as gullies, fractures or subsidence were observed.
High metal content is known to aid conduction of electricity in soils,
explaining the low ER values in mine tailings. The high amounts of Cd, Cu,
Pb and Zn in mine tailings in the study area have been reported earlier (e.g.,
Conesa et al., 2007). In addition, soil electrical conductivity is known to
increase with the total concentrations of soluble salts in soils (US Salinity
Laboratory Staff, 1954). The presence of continuous porosity (or connected
pores) in fine materials contributes to the low ER of tailing materials. The high
surface area of fine materials in tailings holds high amount of water and
metallic ions capable of conducting electricity, thus decreasing the ER values
in the tailing ponds. High electrical resistivity values usually found in the
substratum are characteristics of the bedrock; in fact, electrical resistivity >150
Ohm.m were reported for limonitic bedrock in the study area (Faz et al.,
2003). The physical and chemical properties of the coarse waste rock are
intermediate between the fine tailings and bedrock, hence exhibited ER is
expected to be lower than bedrock but higher than fine tailings.
The ERI resistivity sections previously corroborated held knowledge that
the electrical resistivity in the first few meters of the tailing ponds are lower
than in substratum (Campbell and Fitterman, 2000; Shemang et al., 2003;
Persson et al., 2006; Aracil et al., 2007). For metals, low ER values coincided,
in general, with the high values of Pb and Zn. The inverse relationship
between ER and metal contents is qualitatively illustrated by the superimposed
EC data given on a 2D resistivity sections. The mean contents of selected
metals in three categories of materials based on ER values strongly support our
claim on inverse relationships between ER values and metal content.
However, correlations of ER values with materials in mining tailing ponds
should be interpreted with care because ER values from field measurements
are affected by several factors including particle size distribution,
arrangements of voids, moisture content and ionic composition (e.g.,
Samouelian et al., 2005; Corwin and Lesch, 2005).
Hence, we can state that the ERI method has proved to be very suitable to
identify the position of the interface tailings-bedrock. Because of the high
electrical resistivity contrast between tailings and bedrock this let us obtain the
interface geometry which is necessary to solve the volume of the dumped
tailing in the ponds using the ERT method and the Surfer software.
3.2. Geochemical Characterization
The different categories established by Soil Survey Division Staff (1993)

in terms of pH values are shown in Table 2. There are two tailing ponds that
have an ultra acid average value of pH. In depth, most ponds have slightly
alkaline pH. El Lirio and Peña del Águila-La Unión show both neutral mean
values of pH in surface and depth. The ponds with a low pH are depleted in
bases (Cobertera, 1993), corroborated by the absence of calcium carbonate on
most of the studied ponds (Conesa et al., 2006). In addition, in these ponds the
heavy metal mobility is enhanced owing to the pH. Oppositely, alkalinity in
some of the tailing ponds is likely due to the presence of high amount of
sodium coming from sea water used in the mineral treatment; in these cases
the mobility of metals is prevented.
The classification of the different tailing ponds in terms of mean values of
electrical conductivity according to USDA (2006) is given in Table 3.
Regarding surface samples, most ponds are non saline, very slightly saline or
slightly saline.
Table 2. Characterization of ponds in terms of pH in surface and depth
pH Range Pond (surface) Pond (depth)

Descargador,
Descargador, Regente-
Ultra acid (< 3.5) Regente-
Cartagena
Cartagena
Extremely acid (3.5- 4.4) Brunita --
Strongly acid (5.1-5.5) -- --
Moderately acid (5.6 - 6.5) -- Brunita
San Lázaro-La
Slightly acid (6.1-6.5)
Unión Las Lajas
El Lirio, Cabezo
Rajao-La Unión,
El Lirio, Peña del Águila-
Neutral (6.6 - 7.3) Peña del Águila-La
La Unión
Unión
Encontrada C. Ponce,
Las Lajas,
Encontrada-Cartagena,
Encontrada-
Slightly alkaline (7.4-7.8) Cabezo Rajao-La Unión,
Cartagena, Loma del
San Lázaro-La Unión,
Beal-Cartagena
Loma del Beal-Cartagena
Moderately
(7.4 - 8.4) Encontrada C. Ponce --
alkaline
Brunita is the pond with highest content of salts. Regarding depth

samples, values are similar to those observed in surface samples, highlighting
the absence of strongly saline ponds in depth. The textural class of the
different ponds is shown in Table 4. Results of soil samples permit the
characterization of the studied ponds as sandy loam, silty loam, loam, silty
clay loam and sand-sandy loam, which makes the wastes moderately
permeable, with low water retention capacity. The two revegetated ponds
(Encontrada C. Ponce and Encontrada Cartagena) have loam and silty-clay
loam textural class, respectively. In depth, results of soil samples permit the
characterization of the studied ponds as sandy loam, silty loam, loam and
sand-sandy loam.
Table 3. Characterization of the tailing ponds in terms of mean values of

Electrical Conductivity (EC) in surface and depth
Electrical
Range (dS m-1) Pond (surface) Pond (depth)
conductivity
Las Lajas, Encontrada C.
Non saline (0 - 2) El Lirio
Ponce
Encontrada C. Ponce,
Cabezo Rajao-La
Encontrada-Cartagena, San Unión, Peña del
Very slightly Lázaro-La Unión, Regente- Águila-La Unión, San
(2 - 4)
saline Cartagena, Loma del Beal- Lázaro-La Unión,
Cartagena Regente-Cartagena,
Loma del Beal-
Cartagena
El Lirio, Cabezo Rajao-La
Slightly saline (4 - 8) Unión, Peña del Águila-La Brunita, Las Lajas
Unión
Moderately Descargador,
(8 - 16) Descargador
saline Encontrada-Cartagena
Strongly saline (> 16) Brunita --
With regards to total heavy metals (Table 5), El Lirio shows the highest
values of Cd and Zn, while Encontrada-Cartagena and Peña del Águila-La
Unión show the highest values of Pb and Cu, respectively. After comparing
the maximum concentration of metals in each tailing pond with the thresholds
established by different legislations from countries such as Germany, Canada,
Denmark or Italy (Kabata-Pendias, 1995; Gon and Andersen, 2003;
Ministerial Decree, 1999 a,b), among others, we can conclude that all ponds
are above the limit values for all legislations.
The same conclusion arises when considering the generic reference levels
(GRL) established for soil from the Murcia Region (Martínez-Sánchez and
Pérez-Sirvent, 2007). Therefore, remediation actions should be carried out in
order to reduce the risk for human and ecosystems.
Table 4. Characterization of tailing ponds in terms of mean values of

textural class in surface and depth
Textural class Pond (surface) Pond (depth)

El Lirio, Brunita, Lajas,
Brunita, Descargador, Encontrada
Cabezo Rajao-La Unión,
C. Ponce,
Peña del Águila-La
Cabezo Rajao-La Unión,
Sandy loam Unión, San Lázaro-La
San Lázaro-La Unión, Regente-
Unión, Regente
Cartagena
Cartagena, Loma del
Beal-Cartagena
El Lirio, Lajas, Encontrada-
Slity loam Descargador Cartagena, Loma del Beal-
Cartagena
Loam Encontrada C. Ponce --
Silty clay loam Encontrada Cartagena --
Sand-sandy loam -- Peña del Águila-La Unión
Figures 3 and 4 show the concentrations of the bioavailable fraction for

each studied metal (Cd, Cu, Pb, Zn) in each tailing pond. In general trends, the
highest fractions of bioavailable metals are found in ponds with ultra acid
conditions (pH< 3.5), such as Regente pond with 72%, 4%, 17% and 14%
compared with their total concentration for Cd, Cu, Pb and Zn, respectively. In
all tailing ponds, the concentrations of the bioavailable fractions of Zn and Pb
were found to be present in very high levels compared to other metals (Figure
3 and 4), which suggest that high concentrations of these metals can be
uptaken by plants with the subsequent risk for the food chain (Pastor et al.,
2007).
However, the highest percentages were reported for cadmium. Therefore, Cd
is the metal with highest availability to be absorbed by plants and, as
consequence, with the highest risk to be mobilized through the food chain.
These high contents of the bioavailable fraction of these metals pose
impedance for the establishment of vegetation communities. Thus, for the
reclamation of these ponds, an effort should be made to decrease the
bioavailability of metals in the surface of the tailing ponds. According to the
soil plant toxicity levels proposed by Kataba-Pendias and Pendias (2001), all
tailing ponds have concentrations of total Cd, Pb and Zn higher than those
levels (>3 mg Cd kg-1, >500 mg Pb kg-1 and >400 mg Pb kg-1). Even for some
of the ponds, the concentrations of available metals are higher than those
limits, therefore the risk of mobility of these metals to the food chain is an
important aspect that must be taken into account in future reclamation actions.
Table 5. Concentration of heavy metals in the studied tailing ponds and in

different regulations
Mean and range of concentrations in heavy metals on the surface tailing ponds (mg kg-
1
)
Cd Cu Pb Zn
35.3(10.1- 129 (88- 4517 (3597- 11992 (5645-
El Lirio
68.0) 282) 7181) 23575)
4.43(1.98- 185(94- 1769(692- 1989(901-
Brunita
6.51) 286) 3487) 2589)
7.15(1.94- 113(53- 5267(749- 3761(759-
Las Lajas
7.92) 219) 13058) 9438)
31.3(12.2- 235(65- 6532(2713- 6907 (4101-
Descargador
48.7) 475) 11710) 12661)
17.4 (9.06- 159 (38- 5578 (2839- 3861(2166-
Encontrada - C. Ponce
30.47) 464) 21272) 9813)
21.8 (10.6- 62.5 (155- 8073 (2304- 5892 (2407-
Encontrada Cartagena
30.5) 32) 21608) 11489)
8.73 (0.86- 104 (18- 1035 (48- 2005 (161-
Cabezo Rajao-La Unión
60.12) 1469) 7737) 20971)
Peña del Águila-La 28.9 (0.4- 235 (37- 5985 (363- 7401 (2017-
Unión 65.4) 1823) 15834) 15578)
12.5 (1.6- 29.6 (6.6- 2753 (772- 4901 (1537-
San Lázaro-La Unión
27.2) 68.5) 7213) 18428)
5.12 (0.53- 180 (47- 1696 (301- 5204 (5107-
Regente-Cartagena
22.41) 467) 3542) 22312)
Loma del Beal- 31.4 (9.5- 72.3 (37.8- 4650 (2080- 12772 (5107-
Cartagena 64.1) 113) 9345) 22312)
Levels of Cd, Cu, Pb and Zn (mg kg-1) allowed in different countries
Cd Cu Pb Zn
Germany (1) 2 50 500 300
Austria (1) 5 100 100 300
Canada (1) 8 100 200 400
Japan (1) - 125 400 250
United Kingdom (1) 3 100 100 300
Denmark (2) 5 500 400 1000
Italy (3) 10 600 1000 1500
Netherlands (4) 12 190 530 720
France (5) 60 - 2000 -
Spain (Murcia) (6) 0,55 26,5 50 93
(1) Maximum permitted values (Kabata-Pendias, 1995)
(2) Values for cleaning-up (Gon and Andersen, 2003)
(3) Limits for commercial or industrial use (Ministerial Decree, 1999a,b)
(4) Intervention values (Ministry of Housing, Netherlands, 1994)
(5) Limits for commercial or industrial use (Darmendrail, 2001)
(6) GRL: Generic reference levels (Martínez-Sánchez and Pérez-Sirvent, 2007)
800
Available concentration, mg kg-1 Pb-available Zn-available
700
600
500
400
300
200
100
0
Lirio Brunita Las Lajas Descargador Encont C. Encont Cabezo Peña del San Lázaro Regente Loma del
Ponce Ctgna Rajao Águila Beal
Figure 3. Mean values of bioavailable Pb and Zn in surface samples of each tailing

pond.
7
Available concentration, mg kg-1
Cd-available Cu-available
6
0
Figure 4. Mean values of bioavailable Cd and Cu in surface samples of each tailing

pond.
In fact, one of the possible reasons for the lack of vegetation in the tailing
ponds is the high concentration of bioavailable metals. To guarantee the
development of vegetation cover which mitigates erosion risks and create
aesthetic value to the land, bioavailable metals should be decreased below
toxicity limits. Nonetheless, once vegetation is developed a thorough
assessment of metal accumulation in tissues is crucial to avoid risks for the
food chain. Figures 5 and 6 show the fraction of soluble metals for each
studied metal (Cd, Cu, Pb, Zn) and pond.
Water soluble concentration, mg kg -1
8
Cd-soluble Cu-soluble Pb-soluble
7
0
Figure 5. Mean values of soluble Cd, Cu and Pb in surface samples of each tailing
pond.
Water soluble concentration, mg kg -1
1400
Zn-soluble
1200
1000
800
600
400
200
0
Figure 6. Mean values of Zn in surface samples of each tailing pond.
Water soluble metals follow the same pattern than bioavailable

concentrations. The highest fraction of soluble metals is found in ponds with
ultra acid conditions (pH< 3.5), such as Regente with 54%, 1.4%, 0.2% and
14% for Cd, Cu, Pb and Zn, respectively. Cd is the metal with highest mobility
to be leached by water, followed by Zn, Cu and Pb. Autier and White (2004)
concluded that cadmium is more mobile in soil than most of other heavy
metals, and Liu et al. (2005) also reported that cadmium and zinc are more
mobile than copper in an area affected by lead/zinc mine spill.
The high concentrations of soluble Zn and the high percentages of soluble
Cd pose high risks of pollution of superficial and ground waters by solution
and leaching. However, from previous hydrological studies in the study zone
we know that the groundwater piezometric levels are between 30 and 150 m
depth in the area were tailings are located. In addition, we need to highlight
that tailing ponds are formed by cementing agents such as oxides and
hydroxides of iron, which provoke the formation of crusting, very hardened,
forming a coherent mass or strongly cemented, causing difficulties in water
infiltration. Thus, the water infiltration and leaching of metals into
groundwater is minimal.
In the case of lead, an apposite trend was observed. In spite of the high
total and bioavailable concentration of lead in most of the ponds, the soluble
fraction was very low, even lower than cadmium in some ponds; this shows
the low solubility of this metal, also reported by other authors (e.g. Conesa et
al., 2008, Burgos et al., 2006). This demonstrates that lead is not highly
mobilized under the current waste conditions, and it is not an environmental
risk in its soluble fraction. Thus, for the reclamation of these ponds, and effort
should be made to decrease the mobility of Zn and Cd in the ponds.
CONCLUSIONS
Based on the results of 2D and 3D ERI and intrusive sampling, we
classified materials in the tailing ponds into three categories: fine tailings –
low ER (< 8 Ohm.m), coarse waste rock – intermediate ER (8 – 150 Ohm.m),
and bedrock – high ER (>150 Ohm.m). The low ER strongly correlates with
fine particle size, pH, high electrical conductivity and elevated contents of Cd,
Cu, Pb and Zn in fine tailings.
According to the geochemical analysis, pH values of the studied tailing
ponds ranged from neutral to extremely acid. This property is responsible for
the highest soluble and bioavailable concentrations of Cd, Zn and Pb in acid
ponds, showing a higher risk of heavy metals transfer to the surrounding areas.
Oppositely, total concentrations of lead, zinc and cadmium in all ponds were
markedly higher than the intervention values, indicating that the
concentrations of heavy metals in the ponds pose a high risk for environment
and human health. Thus, reclamation programs should be carry out in all
ponds.
Concentrations of bioavailable Zn and Pb, and percentages of bioavailable

Cd were very high, which suggests high risk of mobility via plants and
microorganisms uptake with the subsequent risk for the food chain. In
addition, mean concentration of soluble zinc and percentage of soluble
cadmium were the highest in most of the ponds. These metals could be
mobilized by runoff water and reach streams. Thus, the immobilization of
these metals should be a priority in reclamation actions.
Taking into account these results, the futures actions should be addressed
to improve the fertility of these ponds and reduce potential heavy metal
mobility in order to promote the establishment of vegetation and reduction of
risks.
In a general way, the application of the geophysical and geochemical
studies are of enough relevance to know the grade of stability of the materials,
the erosive capacity of the superficial waters, the dynamics and analysis of
risks of the pollutants to the biotic and abiotic media, and finally, the capacity
of the vegetation to adapt to this soil type. The development of a vegetation
cover would impede the negative effects of erosion, which propitiates the
dispersion of pollutants to the adjacent soils, to the superficial and
underground waters, as well as to the components of the food chain.
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INDEX
algae, 4
A alkalinity, 205
allopolyploid, 31
acetic acid, 24
aluminium, 65
acid, ix, 22, 24, 27, 28, 29, 36, 45, 46, 50,
aluminum oxide, 131
53, 54, 75, 76, 77, 78, 79, 96, 103, 105,
amino, 24, 26, 115
107, 108, 111, 113, 116, 131, 136, 182,
amino acids, 24, 26
183, 198, 199, 202, 205, 206, 208, 211,
ammonia, 111, 114, 115
212, 217
ammonium, 9, 113, 115, 131
acid mine drainage (AMD), ix, 107, 108,
amphibia, 52
113, 116
animal feeding operations, 8, 12
acidic, xii, xiii, 23, 48, 56, 57, 58, 59, 76,
ANOVA, 184, 185
104, 125, 196, 197, 198
antimony, 62, 67, 71, 72, 74, 75, 77
acidity, 34, 57, 58, 113
antioxidant, xii, 40, 178, 179, 180, 183, 184,
activated carbon, 72
185, 186, 187, 188, 189, 190, 192, 193
activity level, 188
appraisals, 3
adaptability, 188
aquaculture, xi, 178, 180
adaptation, 90, 180, 183, 188
aquatic systems, 191, 198
additives, 74
aqueous solutions, 48, 52, 69, 70, 73, 74,
adenosine, 115
75, 77, 78, 79
adsorption, viii, 43, 53, 55, 56, 57, 63, 71,
aquifers, 200
72, 73, 75, 85, 102, 113, 115
Arabian Gulf, vi, xi, 157, 158, 159, 160,
adverse effects, viii, 44, 178, 187
161, 163, 169, 170, 172, 173, 174
adverse weather, 198
Arabidopsis thaliana, 38
agricultural NPS pollution, vii, 2, 8, 13
arsenic, 46, 62, 64, 67, 68, 69, 70, 72, 74,
agricultural sector, ix, 107
77, 78, 83, 84, 105, 120, 136
agriculture, x, 2, 3, 4, 6, 7, 8, 9, 13, 15, 16,
Asian development bank, 12
40, 45, 108, 123, 214
assessment, x, xii, 3, 16, 34, 35, 74, 76, 90,
airborne particles, 84
98, 104, 106, 119, 149, 173, 187, 196,
Alaska, 213
199, 210, 215
alcohols, 27, 188
atmosphere, 89, 198
220 Index
atmospheric deposition, 84, 158 breeding, 14

atoms, 115 brine wastewater discharges, xi, 157
ATP, 25, 39, 115, 117
Austria, 209
C
B Ca2+, 48, 54, 55, 115

cadmium, xi, 39, 40, 44, 67, 68, 69, 71, 72,
bacteria, 12, 27, 28, 29, 30, 31, 32, 35, 38, 77, 79, 83, 84, 117, 120, 140, 157, 161,
109, 114, 174, 198 163, 167, 168, 169, 183, 184, 185, 186,
bacterium, 38 187, 189, 191, 192, 193, 208, 211, 212,
Bahrain, xi, 157, 159, 160, 161, 168, 169, 213
170, 171, 172, 173 caged mussels, xii, 178, 179
banks, 97, 123 calcium, 52, 71, 72, 74, 77, 78, 108, 113,
barriers, 102 115, 148, 149, 205
base, 122, 132, 179 calcium carbonate, 108, 205
batteries, 49, 63 calibration, 126
Beijing, 1, 3, 19 canals, 106
benefits, 29, 82, 116 capillary, 109
beryllium, 113 carapace, 180
bicarbonate, 113 carbon, 52, 66, 115, 126
bioaccumulation, xi, 170, 173, 178, 179, carboxyl, 27
180, 187, 188 cascades, 111
bioavailability, viii, 22, 23, 28, 43, 46, 49, case studies, vii
51, 52, 60, 67, 68, 69, 71, 121, 189, 191, case study, 152, 153, 172
208 catchments, 15
bioenergy, 94 cation, 27, 77, 204
biomarkers, xii, 178, 179, 187, 189, 190, cattle, 12, 52
191, 192, 193 cell fusion, 30
biomass, 29, 35, 65, 88 changing environment, 121
biomonitoring, 192 chelates, 22
bioremediation, 33, 35, 36, 38, 75, 179 chemical, vii, viii, 8, 14, 19, 21, 22, 34, 43,
biosafety, 31, 34, 35 44, 60, 62, 66, 67, 69, 74, 76, 82, 85, 87,
biosphere, 82 88, 90, 97, 102, 115, 121, 123, 124, 125,
biosynthesis, 115 154, 163, 170, 172, 179, 183, 189, 197,
biotic, 170, 213 204, 205, 214
bonds, 22 chemical characteristics, 85, 214
bone, viii, 44, 46, 52, 55, 60, 68, 69, 70, 71, chemical degradation, 44
75, 77 chemical properties, 76, 172, 197, 204, 205
bone char, viii, 44, 46, 52, 68, 69, 75 chemical reactivity, 62
bone meal, viii, 44, 46, 52, 55, 60, 70, 71, chemicals, x, 85, 87, 108, 109, 117, 125
77 Chile, 172
bones, 52, 67, 69, 70, 71 China, vii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
boreholes, 111 13, 14, 15, 16, 17, 19, 20, 35, 36, 40, 41,
Botswana, 217 92, 106, 152, 153, 191
breakdown, 89, 188 Chinese farmland, vii, 1, 11, 12, 13
Index 221
chlorine, 113 contaminant, ix, xi, 61, 72, 77, 82, 83, 87,
chloroplast, 39 88, 89, 90, 105, 112, 123, 154, 157, 158,
chromium, 44, 69, 75, 83, 85, 120, 134 179, 183
cities, 12, 105 contaminated food, 86
classes, 24, 34, 91 contaminated sites, 60
classification, 206 contaminated soil, viii, 41, 44, 45, 46, 48,
clay minerals, x, 78, 120, 130, 131, 134, 51, 52, 53, 55, 59, 62, 63, 64, 65, 66, 67,
141, 146, 147, 148, 149, 151 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79,
cleaning, 209 87, 92, 93, 94, 153, 214
climate, 14, 15, 32, 90 contaminated soils, viii, 41, 44, 45, 48, 49,
climate change, 15 52, 53, 55, 59, 62, 63, 64, 65, 66, 67, 68,
cluster analysis, 105 69, 71, 72, 73, 74, 75, 76, 77, 87, 92, 94,
CO2, 56 153
coastal region, 92, 192 contamination, vii, viii, ix, x, xii, 8, 13, 43,
cobalt, 44, 75 44, 46, 65, 71, 82, 83, 85, 90, 91, 92,
coding, 38 102, 103, 104, 106, 119, 121, 123, 124,
coenzyme, 85 136, 139, 141, 149, 150, 152, 153, 154,
collagen, 52 155, 158, 170, 171, 173, 174, 178, 189,
Colombia, 195 190, 191, 192, 198
combustion, 52, 63, 70, 84 Continental, 173
combustion processes, 84 COOH, 23
commercial, 78, 84, 127, 209 copper, xi, 24, 44, 67, 68, 69, 83, 85, 113,
communities, 87, 208 157, 161, 163, 166, 168, 169, 183, 184,
comparative analysis, 127 186, 190, 191, 212
compatibility, 34, 35 correlation analysis, xi, 120, 141, 145, 147,
competition, 62, 149 150, 151
competitiveness, 148 correlation coefficient, 121, 127, 141, 143,
complexity, 200 144, 145
composition, 52, 60, 65, 109, 124, 126, 134, correlations, 146, 147, 148, 149, 151, 180,
204, 205 184, 185, 186, 187, 188, 189, 204, 205
compost, 38, 65, 68, 116 corrosion, 84, 108, 170
compounds, 39, 44, 47, 55, 57, 63, 87, 89, cost, vii, 20, 28, 35, 46, 51, 70, 75, 86, 87,
109, 113, 198 204
comprehension, 179 Council of Ministers, 155
computation, 201 CRM, 184
conduction, 204 Croatia, 104
conductivity, 204, 207 crop, viii, 8, 9, 11, 20, 21, 32, 36
Congress, 155, 216 crop production, 11
consensus, 12, 142, 155 crop residue, 8
constituents, 115 crops, 9, 14, 32
construction, 13, 198 crude oil, 91, 123, 136, 140, 150, 151
consumers, 179 crust, 83, 198
consumption, 9, 53, 86, 103, 173 crystalline, x, 46, 52, 71, 74, 78, 120, 125,
contact time, 111, 112, 113, 116 127, 131, 135, 136, 150
crystallinity, 135
222 Index
crystallisation, 109 digestion, 153, 162, 163, 202

crystallization, 53 direct measure, 179
Cuba, 152 discharges, xi, 157, 158, 160, 161, 171
cultivation, 26 discordance, 199
cycling, vii, 16, 105 diseases, 44
cystathionine, 39 dispersion, 152, 213
cytochrome, 183 dissolved oxygen, 4, 193
cytoplasm, 27 distribution, ix, xi, 13, 25, 26, 31, 34, 40,
cytotoxicity, x, 108, 109, 116 62, 63, 71, 72, 95, 98, 101, 102, 103,
105, 120, 127, 129, 130, 131, 132, 134,
135, 136, 137, 138, 139, 140, 150, 151,
D 170, 172, 179, 199, 204, 205
diversity, 90
damages, 179, 188 DNA, 85, 193
danger, viii, 20 DOI, 106
data distribution, 155 drainage, ix, x, 45, 53, 103, 105, 107, 108,
data processing, 201 111, 113, 116, 122, 123, 154, 198, 199,
data set, 150 217
deaths, 170 drinking water, ix, 2, 46, 107
decomposition, 116, 183 drying, 125, 126, 182
decontamination, 69, 78 dumping, 161
defence, 188
deficiency, 21, 48
deficit, 40 E
degradation, xi, 11, 78, 89, 125, 157, 158,
197, 198 earthworms, 29
Delta, 93 ecology, 22, 34, 38
demonstrations, 60, 91 economic growth, 4
Denmark, 207, 209 economic losses, 4
Department of Agriculture, 117, 217 economics, 16
deposition, 102 ecosystem, vii, 4, 85, 86, 174, 178
deposits, 97, 122 ecotoxicological, 70, 106
depth, ix, xii, 26, 96, 97, 98, 99, 101, 102, effluents, xi, 52, 70, 71, 84, 96, 97, 108,
103, 123, 139, 140, 162, 182, 196, 201, 157, 158, 160, 161, 162, 163, 170, 171,
204, 205, 206, 207, 208, 212, 213 173, 174, 197
desalination plants, xi, 157, 158, 160, 161, egg, 12
162, 170, 173, 174 electrical conductivity, xii, 113, 196, 202,
desorption, 63, 64, 72, 73, 76, 121, 130 204, 206, 212, 214
destruction, 130, 131, 198 electricity, 204
detectable, 116 electrochemical treatment, viii, 43
detection, 126, 129, 142, 179 electrodes, 201
detoxification, 25, 38 electrophoresis, 87
developed countries, 2, 13 encoding, 26, 29
developing countries, ix, 107 endocrine, x, 108, 109, 116, 117
diffraction, 130 energy, ix, 45, 82, 115, 160
diffusion, 68, 69 enforcement, 2
Index 223
engineering, viii, 20, 30, 31, 33 farmers, 12, 13, 14

England, 191 farmland, vii, 1, 2, 8, 11, 12, 13, 20, 32, 40
environment, xii, 9, 11, 12, 14, 20, 29, 31, farms, xi, 12, 178
34, 40, 45, 50, 57, 59, 61, 63, 70, 71, 75, fashion industry, 123
77, 82, 83, 84, 85, 87, 90, 91, 120, 125, fauna, 198
131, 135, 158, 161, 171, 179, 187, 191, fertility, 28, 34, 213
196, 199, 212 fertilization, 9, 15, 31, 76
environmental aspects, 175 fertilizers, 8, 11, 13, 45, 60, 63, 72, 73, 77,
environmental change, 179 116, 123
environmental conditions, viii, 43, 90, 130, field crops, 32
132, 159, 182 field tests, 46
environmental effects, 13, 68 filling materials, 200
environmental factors, vii filtration, viii, 43, 109
environmental impact, ix, 15, 44, 96, 102, fish, 16, 75, 172, 189, 193
153, 155, 198 fisheries, 4
Environmental Protection Agency ( EPA), fishing, 2, 52
2, 8, 12, 17, 49, 83, 93, 163 fixation, 21, 55, 74
environmental quality, 3, 187 flame, 63, 183
environmental regulations, 173 flame retardants, 63
environments, xi, 60, 63, 86, 124, 130, 158, flocculation, 102
160, 161, 171, 178, 179 floods, 123
enzymes, xii, 26, 27, 39, 85, 89, 109, 178, fluctuations, 158, 166
179, 184, 185, 186, 187, 188, 190, 192, fluid, 109, 173
193 fluorescence, 98
erosion, xiii, 63, 84, 117, 158, 196, 198, food, viii, 20, 43, 77, 86, 96, 158, 169, 179,
199, 210, 213 190, 198, 208, 211, 213
ethylene glycol, 24 food chain, viii, 20, 43, 77, 169, 190, 198,
EU, 141, 142, 154 208, 211, 213
Europe, 3, 93 food web, 158, 179
eutrophication of major lakes, vii, 1 force, 111
EXAFS, 55, 69, 73 formation, viii, x, 43, 44, 50, 53, 54, 55, 56,
explosives, 89, 91, 111 57, 58, 63, 69, 76, 85, 87, 119, 122, 150,
exposure, 49, 88, 179, 188, 193 197, 198, 212
external influences, 108 formula, 56
extraction, x, 21, 67, 74, 113, 119, 121, 124, fractures, 204
125, 126, 127, 128, 130, 131, 132, 133, France, 152, 153, 182, 190, 201, 209, 213,
134, 135, 136, 137, 150, 153, 154, 155, 215
202 freshwater, 82, 141, 142, 190
extracts, 73, 175 fungi, 27, 37, 109
F G
factories, xi, 97, 158, 160, 162 gel, 23

Fairbanks, 213 genes, 21, 25, 29, 31, 32, 34, 35
famine, 32
224 Index
genetic engineering, viii, 20, 26, 29, 36, 39, Huelva bank, ix, 95, 99, 101, 102, 103
91 human, ix, xi, xiii, 2, 11, 20, 22, 26, 39, 65,
genus, 32 81, 82, 84, 85, 87, 92, 96, 103, 116, 118,
geometry, 205 121, 157, 158, 171, 173, 180, 181, 182,
Germany, 107, 207, 209 187, 196, 199, 207, 212
gland, xii, 178, 184, 185, 191, 192, 193 human body, 20, 85
glutathione, xii, 178, 179, 188, 189, 191 human exposure, 65
GPS, 201 human health, ix, xi, 11, 22, 81, 87, 157,
grades, 14 158, 171, 212
grain size, viii, 44, 62, 68, 97, 99, 162 Hungary, 123, 139
graphite, 183 husbandry, 10
grass, 50, 60 hybrid, 30, 31
Greece, 105 hybridization, 30
Greeks, 197 hydrocarbons, 91, 160, 170, 171, 172
greenhouse, 64 hydrogen, 77, 85, 141, 188
groundwater, 45, 46, 69, 130, 132, 161, 174, hydrogen peroxide, 85, 188
198, 199, 212, 217 hydrolysis, 58
growth, 4, 12, 35, 48, 53, 61, 76, 82, 86, 88, hydrothermal system, 105
117, 160, 169 hydroxide, 65, 72, 77, 113
growth rate, 35 hydroxyapatite, 45, 46, 52, 53, 55, 67, 69,
Guadiana river, ix, 96, 104 71, 72, 73, 74, 75, 76, 77, 78, 79
guidelines, 13, 141, 155, 168, 169, 170, 172, hydroxyl, 48, 85
183 hydroxylapatite, 79
Guinea, 105 hypersensitivity, 39, 40
Gulf of Mexico, 14
I
H
immobilization, 44, 47, 48, 51, 52, 53, 54,
habitats, 158, 173, 198 55, 56, 57, 59, 61, 62, 64, 67, 68, 69, 71,
HAP, 46, 47, 51, 54, 55, 56, 57, 58, 59, 61 73, 74, 77, 78, 213
harmful effects, 82, 141, 142 immune response, 193
harvesting, 90 immune system, 191
hazardous waste, 87 immunity, 33
hazards, 52, 82 impact assessment, 173, 174
health, 70, 155, 174, 178 Impact Assessment, 174
health effects, 155 impairments, 8
hemoglobin, 169 impurities, 58, 62
histidine, 25, 37 in vitro, 46
Holocene, 99, 105 in vivo, 67
Hong Kong, 117, 190 India, 93, 106, 154, 155
hormones, 33, 116 individuals, 179, 185
hot spots, vii, 2 Indonesia, 173
hotel, 181 induction, 188
hotspots, 170, 171 industrial revolution, 20, 82
housing, 160 industrial wastes, 103
Index 225
industries, xi, 97, 123, 158, 160, 162, 170

industry, 4, 13, 16, 51, 52, 108, 160, 170, L
174
lakes, vii, 1, 2, 3, 4, 7, 8, 15, 16, 46
infertile polyploid species, viii, 20, 31, 36
land disposal, 174
infrastructure, 2, 12, 13
landscape, 197, 198, 199
ingestion, 49, 169
landscapes, ix, 107
ingredients, 116
lattices, x, 120, 150
inhibition, 48, 86
leaching, viii, 9, 13, 21, 22, 28, 29, 44, 49,
inoculation, 23
51, 53, 59, 61, 63, 64, 66, 71, 77, 90,
in-situ reduction, ix, 81
123, 158, 212
integrity, 173
lead, xi, xii, 29, 40, 44, 52, 67, 68, 69, 70,
interface, 201, 204, 205
71, 72, 73, 74, 75, 76, 77, 78, 79, 83, 84,
interference, 86, 126
85, 102, 104, 117, 120, 123, 134, 140,
intervention, 212
151, 157, 158, 161, 163, 167, 168, 169,
inversion, 201
183, 184, 185, 186, 187, 196, 197, 212,
invertebrates, 180, 191
214
investment, 2
lead content, 140
ion exchange, viii, 43, 53, 54, 55, 56, 57,
legislation, xiii, 31, 35, 183, 196
112, 115, 116, 121
ligand, 57, 58, 64
ion-exchange, 124
light, 171
ions, 22, 23, 24, 25, 26, 27, 29, 45, 48, 53,
lignin, 111
54, 55, 56, 57, 59, 62, 63, 64, 65, 67, 68,
limestone, 136
69, 72, 74, 76, 77, 109, 113, 115, 121,
liver, 189, 191, 193
205
livestock, 3, 12
Ireland, 16
localization, 38
iron, 24, 37, 48, 62, 63, 67, 69, 70, 72, 73,
LSD, 184
77, 78, 83, 109, 113, 115, 116, 124, 125,
127, 132, 182, 212
irrigation, ix, x, 8, 9, 107, 108, 113, 114 M
irrigation water, ix, x, 8, 107, 108, 113
isolation, 102 machinery, 84
Italy, 43, 190, 207, 209 macrobenthos, 174
magnesium, 113, 115
magnitude, 57, 60, 179
J
majority, 27, 131, 135
mammals, 24, 191
Japan, 60, 209
management, 2, 13, 14, 45, 66, 171, 217
manganese, x, 44, 67, 71, 113, 120, 124,
K 132, 141, 151, 155
manipulation, xii, 178
K+, 115 manufacturing, 50, 82
kinetic model, 69 manure, 2, 8, 12, 13, 23, 38
kinetic studies, 76 marine environment, xi, 157, 158, 159, 160,
kinetics, 73 161, 170, 171, 174, 178, 187, 198
Kuwait, 172, 174 marine species, 188
226 Index
mass, 113, 126, 212 moisture content, 57, 126, 205

mass spectrometry, 113 molar ratios, 60
materials, xii, 45, 46, 48, 59, 66, 71, 79, 84, molecules, 85, 89, 115
109, 116, 192, 196, 198, 199, 204, 205, mollusks, 192
212, 213 multivariate analysis, 74
matrix, 126 mussels, xii, 173, 178, 179, 180, 182, 187,
matter, 27, 32, 52, 53, 147, 164 188, 190, 191, 193
MCP, 46, 47, 48, 57, 58, 64, 66 mutant, 31
measurement, xi, 35, 126, 178
measurements, 164, 165, 166, 179, 182,
201, 205, 214 N
meat, 12, 52, 55, 70
media, viii, 43, 48, 51, 90, 213 Na+, 48, 115
Mediterranean, 74, 152, 173, 182, 189, 190, nanoparticles, 73
192, 193, 198, 199, 200 natural apatites, viii, 44, 70
Mediterranean climate, 199, 200 natural habitats, xi, 157
melting, 123 natural resources, 82
mercury, 27, 39, 44, 83, 84, 85, 89, 120, negative consequences, 72
183, 184, 185, 186, 191 negative effects, 9, 45, 46, 198, 213
Mercury, 106, 136, 155, 183 Netherlands, 2, 172, 209, 216
metabolism, 40, 91, 179, 188, 191 neural network, 15
metabolites, 89 neutral, 48, 58, 125, 205, 212
metal ion, 20, 22, 23, 25, 26, 27, 28, 29, 35, next generation, 31, 34
57, 66, 75 nickel, x, 37, 38, 67, 74, 75, 76, 78, 85, 120,
metal ions, 20, 22, 23, 25, 26, 27, 28, 29, 150, 155, 185, 186
57, 66, 75 nitrite, 111, 113, 114, 115, 116
metallurgy, 45 nitrogen, vii, 1, 2, 8, 14, 15, 111, 113, 115,
meter, 201 116, 182, 190
methanol, 160 nodules, 28, 32
Mg2+, 48, 115 non-enzymatic antioxidants, 179
microbes, vii, 20, 23, 26, 27, 28, 31, 32, 36 non-point source (NPS), vii, 2
microelectronics, 45 North America, 3, 93
micronutrients, 61 Norway, 2, 16
microorganisms, 37, 213 NPS, vii, 2, 3, 8, 9, 12, 13, 17
microremediation, vii, 19, 20, 21, 28 NRCS, 217
microscope, 110 nucleic acid, 85
migration, 49, 77, 89 nutrients, 2, 8, 11, 12, 15, 16, 33, 34, 53, 61,
military, 82 76, 85, 89, 152
mineral resources, xii, 196, 197 nutrition, 23, 44, 109
mineral water, 78
Mississippi River, 14 O
modelling, 199
models, 13, 103 oceans, 3, 4
modifications, 171, 199 OH, 52, 53, 55, 56, 57, 85
moisture, 57, 126, 205
Index 227
oil, 136, 150, 153, 160, 161, 162, 170, 172, phosphates, viii, 43, 46, 49, 51, 52, 54, 55,
193 66, 69, 72, 76, 77, 78
oil spill, 161, 193 phosphorous, 111, 113, 116
operating costs, 62 phosphorus, vii, 1, 2, 8, 11, 12, 14, 15, 16,
operations, 12, 84, 97, 161, 199 52, 54, 71, 72, 76, 77, 78, 79
opportunities, 33, 41, 94 physical characteristics, 90
optimization, 45 physical properties, 170
ores, 123 Physiological, 37
organic compounds, 89, 91, 172 physiology, 37, 38, 188, 190
organic matter, xi, 52, 53, 63, 64, 120, 132, phytoplankton, 174
135, 147, 148, 149, 153, 163, 172 phytoremediation, vii, ix, 19, 21, 28, 32, 38,
organic peroxides, 188 39, 40, 41, 65, 81, 82, 83, 87, 89, 90, 91,
organism, 44, 85, 86, 179, 180, 191 92, 93
overgrazing, 8 pipeline, 123
oxalate, 73, 131 plankton, 16
oxidation, 38, 46, 63, 125, 198 plant growth, 33, 38, 48, 67, 71, 88
oxidative damage, 85, 188, 191 plants, vii, ix, xi, xiii, 2, 4, 19, 20, 21, 22,
oxidative stress, 178, 188, 192, 193 23, 24, 25, 26, 28, 30, 31, 32, 34, 35, 36,
oxygen, 12, 86, 111, 114, 188, 198 37, 38, 39, 40, 53, 60, 61, 64, 72, 76, 81,
oxygen consumption, 86 87, 88, 89, 90, 91, 93, 97, 109, 115, 136,
oysters, 173 150, 152, 157, 158, 160, 161, 162, 169,
170, 173, 174, 196, 197, 199, 208, 213,
214
P plasma membrane, 40
plasmid, 29, 31
parallel, 102 platinum, 83
partition, 154 poison, 28
pathogens, 12 policy, 13, 14, 15
pathways, ix, 96, 103, 141 pollen, 31
PCBs, 91, 178 pollination, 31
Pearl River Delta, 191 pollutants, ix, xi, 2, 3, 5, 14, 20, 39, 82, 90,
peptides, 26 91, 93, 96, 97, 102, 109, 121, 154, 178,
peroxide, 188 179, 187, 188, 189, 191, 193, 213
petroleum, xi, 94, 97, 158, 170, 171, 172, pollution, vii, viii, ix, xii, 2, 3, 8, 9, 14, 15,
174 16, 20, 21, 29, 31, 34, 35, 36, 38, 76, 78,
Petroleum, 91, 94, 160, 172 81, 82, 84, 85, 88, 92, 93, 96, 97, 98,
pH, viii, ix, xii, xiii, 44, 49, 51, 54, 55, 56, 101, 102, 103, 104, 105, 120, 121, 123,
57, 58, 59, 61, 63, 68, 71, 72, 73, 78, 79, 132, 136, 139, 150, 158, 160, 161, 162,
85, 88, 90, 102, 107, 108, 109, 111, 113, 171, 172, 179, 186, 187, 188, 189, 191,
121, 125, 130, 132, 183, 196, 202, 205, 193, 196, 197, 199, 212
206, 208, 211, 212 polycyclic aromatic hydrocarbon, 189, 192
phosphate, viii, 27, 43, 44, 45, 46, 47, 48, polypeptides, 24
51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, polyploid, viii, 20, 30, 31, 36
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, polysaccharides, 27
74, 75, 76, 77, 78, 79, 97, 113, 116
phosphate rocks, viii, 44, 73, 74
228 Index
ponds, xii, xiii, 92, 196, 197, 198, 199, 200, reagents, 22, 24, 29, 125, 131, 163
201, 202, 204, 205, 206, 207, 208, 209, recovery, 9, 163, 188
210, 211, 212, 213, 215 recreational, 160
pools, 122, 124, 137 recycling, 49, 120
population, xii, 31, 32, 82, 196, 199 red blood cells, 169
population growth, 82 redistribution, 103
porosity, 204 registry, 84, 91
Portugal, 95, 97 regulations, 14, 83, 209
positive correlation, 141, 146, 148, 149, rehabilitation, xiii, 108, 197, 215
151, 185, 188, 189 remediation, vii, viii, ix, 19, 20, 21, 32, 40,
potassium, 8, 46, 113 43, 44, 45, 46, 48, 50, 51, 52, 65, 66, 67,
Potchefstroom, 107 70, 71, 72, 81, 87, 90, 91, 207, 214, 217
power generation, 84 repair, 86, 160
power plants, 45 reproduction, 109
precipitation, viii, 27, 43, 46, 49, 53, 54, 55, residuals, 197
57, 59, 86, 108, 110, 121, 123, 199 residues, 24, 52, 108, 181, 182
principal component analysis, 154 resistance, 37, 38, 93
producers, 197 resolution, 104, 201
project, 108 resources, 15, 96
protection, 14, 15, 38, 155, 172 respiration, 86
proteins, 23, 27, 29, 36, 85, 189, 191 response, 15, 117, 179, 187, 188, 190, 193
protons, 23 restoration, 215
public health, viii, 43 reverse osmosis, 161
pumps, 108 risk, ix, 2, 11, 13, 21, 35, 36, 49, 65, 66, 77,
pure water, 202 81, 85, 86, 90, 121, 123, 153, 198, 207,
purification, 112 208, 212, 213
PVC, 111, 124 risk assessment, 35, 36, 153
pyrite, 106, 198 risks, xii, xiii, 59, 66, 120, 121, 124, 152,
153, 173, 196, 197, 199, 210, 212, 213
river basins, 3
Q river flows, 139, 150
river systems, 132, 134
quality assurance, 163 RNA, 85
quality control, 126, 184 Romania, 123, 132
quality of life, 82 room temperature, 125, 182
quantification, 121, 202 roots, 23, 24, 25, 28, 30, 31, 37, 38, 50, 88,
89, 90, 115
R rules, 201
runoff, 2, 3, 7, 12, 13, 16, 82, 84, 117, 198,
radicals, 85, 191 213
rainfall, 9, 97, 199
reactant, 59
S
reactions, 53, 54, 55, 57, 63, 73, 86
reactive oxygen, 179, 188, 191 safety, 25, 52
reactivity, 179, 188 salinity, xii, 90, 102, 162, 163, 196
Index 229
salinity levels, 163 soil type, 32, 200, 213

salts, xii, 57, 59, 111, 116, 196, 204, 206 Solea senegalensis, 192
samplings, 201 solid phase, 115, 121
saturation, 175 solid waste, 72
science, 173, 217 solidification, 87
seasonality, 16 solubility, 22, 28, 51, 54, 57, 58, 60, 69, 79,
secretion, 23 88, 90, 109, 136, 212
sedimentation, 111 solution, 44, 46, 49, 52, 53, 55, 65, 70, 73,
sediments, ix, x, xi, 76, 81, 84, 85, 93, 94, 74, 77, 78, 79, 88, 102, 113, 115, 121,
95, 96, 97, 99, 101, 102, 103, 104, 105, 126, 212
106, 119, 120, 121, 122, 123, 124, 126, solvents, 89, 91, 124
127, 130, 131, 132, 134, 135, 136, 137, sorption, 50, 51, 53, 54, 55, 56, 62, 63, 64,
138, 139, 140, 141, 142, 145, 146, 147, 68, 69, 70, 72, 74, 75, 76, 77, 78, 121,
148, 149, 150, 151, 152, 153, 154, 155, 146, 213
157, 158, 161, 162, 169, 170, 172, 173, sorption process, 56, 62, 146
174, 175, 190, 198 South Africa, ix, 107, 117, 118
seed, 34 Spain, ix, xii, 95, 102, 103, 104, 105, 106,
selectivity, 26, 29 172, 173, 177, 192, 193, 195, 196, 197,
selenium, 28, 38, 39, 40, 62, 72, 191 209, 214
self-pollinated plants, viii, 20, 31, 36 speciation, viii, 38, 44, 57, 63, 67, 72, 90,
semiconductors, 63 153, 191
Serbia, 119, 123, 124, 139, 140, 142, 152, species, viii, xi, 20, 24, 26, 31, 32, 34, 35,
153, 155 36, 37, 38, 55, 61, 63, 65, 72, 91, 109,
serum albumin, 183 121, 125, 178, 179, 180, 187, 188, 190,
sewage, xi, 2, 7, 8, 12, 52, 77, 79, 108, 111, 191
112, 113, 114, 116, 117, 153, 157, 158, specific surface, 21, 23, 28, 29, 36, 62
160, 161, 162, 163, 171, 174 specifications, 183
shade, 111 spectrophotometry, 183
shoot, 24, 25, 30, 50, 51 spectroscopy, 73
shoots, 37, 51, 62 stability, 48, 62, 75, 131, 198, 213
short supply, 11 stabilization, viii, ix, 44, 52, 53, 65, 66, 69,
showing, 56, 89, 159, 212 71, 72, 76, 77, 81, 86, 87, 88, 153
side effects, viii, 20 standard deviation, 126, 127
signaling pathway, 40 state, 3, 4, 12, 15, 16, 26, 27, 63, 96, 188,
signals, 204 205, 217
silica, 111 states, xii, 12, 63, 178
silicon, 132 statistics, 127
silver, xii, 83, 191, 196, 197 steel, 201
simulation, 55, 76 stimulation, 38
simulations, 104 stock, 126
Slovakia, 123 stoichiometry, 59
sludge, 8, 52, 79, 84, 85, 111, 153, 171, 197 storage, 25, 182, 204
sodium, 50, 77, 108, 113, 206 stormwater, 84
sodium hydroxide, 50, 108 stress, 25, 27, 35, 39, 40, 187, 188, 192
soil particles, xiii, 86, 196 structure, 28, 52, 55, 92, 110, 198
230 Index
substitution, 46, 48, 53, 55, 57 TPA, 24

substrates, x, 90, 119, 120, 134, 135, 136, trace elements, xi, 120, 121, 126, 137, 148,
141, 146, 147, 148, 149, 150, 155, 163, 172
164, 183 transformations, 125
sulfate, 115, 198 translocation, 29, 37, 50, 191
sulfur, 39 transpiration, 25, 30, 34, 35
sulphur, 197 transplantation, 182
Superfund, 83, 84 transport, ix, 25, 37, 67, 82, 86, 90, 96, 97,
suppression, 86 103, 105, 182
surface area, 88, 205 transportation, 4, 23, 24, 25, 26, 30, 34, 198
surface layer, 200 treatment, viii, x, 2, 13, 43, 44, 46, 48, 49,
surface mining, 199 50, 51, 52, 53, 55, 56, 58, 59, 61, 62, 63,
surplus, 3, 9 64, 65, 66, 71, 78, 87, 90, 108, 109, 111,
suspensions, 79 113, 114, 115, 116, 117, 153, 155, 160,
sustainable development, 4 161, 162, 204, 206
Sweden, 16, 216 treatment methods, 117
symbiosis, 38, 115 trial, 53
synthesis, 24, 25 triploid, 31
Turkey, 106, 152, 190
T
U
tar, 161
target, 22, 30, 160 U.S. Department of Agriculture (USDA),
TCE, 89 200, 206, 217
techniques, vii, 2, 109, 179 Ukraine, 123
technologies, viii, 13, 14, 43, 44, 87 ultrasound, 154
technology, ix, 21, 28, 29, 30, 31, 33, 35, United Kingdom (UK), 2, 16, 163, 209
36, 81, 87, 92, 93, 160 United States (USA), 2, 12, 37, 38, 48, 50,
temperature, 34, 88, 125, 161, 162, 163, 199 81, 94, 163, 190, 201, 217
terminals, 161 uranium, 73, 76, 78
texture, xii, 23, 28, 196 urban, 3, 4, 12, 78, 79, 85, 92, 96
thermal treatment, 70 urbanisation, 181
thermodynamics, 70 urbanization, 92
threats, xi, 11, 157 urea, 9, 160
tides, 9 UV, 183
tissue, 50, 51, 52, 86, 182, 183, 184, 192
tobacco, 39, 40, 52, 53, 72
tourism, 4 V
toxic effect, 44, 86, 179
toxic metals, 88, 178, 216 vacuole, 25
toxic waste, 50 valence, 26, 29
toxicity, vii, 22, 26, 52, 60, 67, 75, 78, 83, Valencia, 190
84, 91, 121, 152, 153, 169, 173, 179, validation, 190
188, 191, 192, 198, 208, 210 vanadium, x, 120, 135, 140, 150
Toyota, 173 vapor, 98
Index 231
variables, 15, 202 wells, 140, 161

variations, xi, 99, 106, 158, 169, 189, 201 wetlands, x, 2, 8, 108, 153, 215
vascular system, 89 wild type, 26
vegetables, 65 wildlife, 198
vegetation, 198, 200, 208, 210, 213, 216 Wisconsin, 155, 216
Venezuela, 173 wood, 68, 109
Venus, xi, 178, 180 World Bank, 12
viruses, 12
vitamin B1, 85
vitamin B12, 85 X
volatilization, 27, 28, 38, 39, 88
X-ray diffraction, 130
xylem, 25, 28, 29
W
Washington, 217 Y
waste, x, 8, 12, 45, 46, 50, 52, 53, 63, 71,
72, 75, 79, 84, 88, 108, 109, 121, 197, yeast, 109
198, 204, 205, 212, 216 yield, 11
waste disposal, 45, 121
wastewater, xi, 7, 84, 92, 108, 109, 123, Z
157, 162, 163, 168
water quality, vii, 2, 3, 4, 5, 8, 12, 15, 78, Zimbabwe, 174
113, 118 zinc, xi, xii, 44, 67, 68, 69, 71, 73, 79, 83,
water resources, 198 85, 104, 113, 117, 140, 155, 157, 161,
water supplies, 4 163, 166, 168, 169, 191, 196, 197, 212,
water systems, vii, 1, 4, 5 213, 214
watershed, 6, 7, 15, 16, 124, 134
waterways, 82

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ENVIRONMENTAL HEALTH - PHYSICAL, CHEMICAL

AND BIOLOGICAL FACTORS

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NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. † New York

Chapter 6 Controlled Organic Treatment System for Acid Mine

This book is written by internationally-famous soil biologists and

soil of contaminants, such as As, Se and W, have to be addressed for

Results of correlation analysis of element content in the sediment Pančevo

accumulated more quantity of metals. As consequence, a second study was

Rajao-La Unión”) and Cu (maximum values:1823 mg kg-1 in “Peña del

THE NON-POINT POLLUTION OF

Gang Xu 1, Hongbo Shao.1,2* ,Yingchun Lv1,

(NPS) pollution from agricultural is the leading source of water pollution.

Keywords: eutrophication; non-point source pollution; farmland

2. THE N AND P POLLUTION IN MAJOR WATER SYSTEM

Water system 1960s 1970s 1980s 1990s Current Main pollutants

TN and TP in watershed N and P2O5 loads from agriculture

doubled from 1.11mg/L to 2.54 mg/L and from 0.052mg/L to 0.1mg/L,

Figure.1. The sources of TN and TP in three lakes in China.

3. THE MAIN CAUSES OF NPP FROM AGRICULTURE IN

3.1 Fertilizer in Farmland

Nutrients such as phosphorus, nitrogen, and potassium in the form of

Figure.2. Indicators of NPP from Agriculture, China (FAOSTAT).

1960s 1980s Present

Figure.3. Change of soil available P in Chinese farmland from 1980 to 2006.

As is shown in table 4, the remained phosphorus in Chinese farmland has

Table 4. The phosphorus added and remained in Chinese farmland

1980 1366000 11 4.8

3.2 Animal Feeding Operation

By confining animals in small areas or lots, farmers and ranchers can

4. FUTURE WORKS ABOUT AGRICULTURAL NPP IN CHINA

4.1 The Runoff Mechanism of NPS from Chinese Agriculture

In order to reduce NPS from Chinese agriculture, much more detailed

4.2 Rapid Assessment Techniques

4.3 Best Agriculture Management

As is shown in Figure 3, larger amount of fertilizer accumulated in Chinese

Arheimer, B., Liden, R. (2000) Nitrogen and phosphorus concentrations from

Zhang WL, Wu SX, Ji HJ, H., K. (2004) Estimation of Agricultural Non-Point

SOME ADVANCES IN BIO-REMOVING

Liye Chu*1,2, Hongbo Shao1,2*, Junna Sun2,3,

microbes, are preferred because of their cost-effectiveness, environmental

Keywords: Heavy metals; Soil; Phytoremediation; Microremediation;

more than 10,000mg/kg (or 10,000ppm)of Mn or Zn, it is defined as a

2. MECHANISMS OF FOUR METAL-REMOVING-METHODS

2.1. Mechanism of Chemical or Physical Remediation

Chemical or physical method is early used and even endemically

Figure1 EDTA-Pb complexes. Dotted bonds to Pb are coordinate.

By adding synthetic chelators such as EDTA (ethylenediamine-tetracetic

2.2. Mechanism of Animal Remediation

Animal here mainly refers to earthworm because it is one of the most

quality [15-18]. By their feeding, burrowing, excreting and metabolic redox

2.3. Mechanism of Phytoremediation

2.3.1. Accumulation and Transport

Table 1. Chelators that are commonly used or of importance

Nature helators Phytochelatin (PC) Metallothionein (MT) Organic acids

Among nature chelators, Phytochelatin (PC) and Metallothionein (MT)

Figure 2. MerB converting organic Hg to Hg(2).

Liye Chu1,2, Hongbo Shao1,2, Junna Sun2,3,