American Journal of Analytical Chemistry, 2013, 4, 633-641
Published Online November 2013 (http://www.scirp.org/journal/ajac)
http://dx.doi.org/10.4236/ajac.2013.411075
Open Access AJAC
Analytical Relevance of Trace Metal Speciation in
Environmental and Biophysicochemical Systems
Nsikak U. Benson*, Winifred U. Anake, Ifedolapo O. Olanrewaju
Environmental Chemistry Research Group, Department of Industrial Chemistry, Covenant University, Ota, Nigeria
Received August 24, 2013; revised September 26, 2013; accepted October 14, 2013
Copyright © 2013 Nsikak U. Benson et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
This article presents a review of the analytical relevance of trace metal speciation analysis, which must be considered in
environmental and biophysicochemical systems for reliable and efficient assessment and monitoring of trace metals.
Examples are given of methodological approaches used for speciation analysis. An overview of speciation analysis in
sediments, aquatic ecosystems and agrosystems is also presented.
Keywords: Trace Metals; Speciation; Speciation Analysis; Sediments; Water; Agrosystems
1. Introduction
Trace metals are introduced anthropogenically as micro-
pollutants into our environment from several sources
such as industrial, agricultural and domestic wastewater/
effluents [1]. Over the years, their fate, transport and
pollution in the environment especially the aquatic eco-
systems are becoming an environmental problem of con-
cern owing to their ecotoxic properties. The biological
activity and availability, biogeological fate, transport and
eventual effect of trace metals in the environment and
biological systems are a function of the chemical species
in which they occur [2].
In trace metal chemistry, it is a common practice to
simply quantify trace metal contents in samples of in-
terest as total level or concentration. In recent times, in-
creased attention has been given to the determination and
quantification of trace metals when assessing their im-
pacts in environmental systems (air, water, soil and bi-
ota) in this form. Growing evidence indicates that the
determination of the total trace metal concentration in
soil [3,4], sediments [5,6], marine and fresh waters [6,7],
biota [8,9], and consumer products [10] principally high-
lights simplified ways of expressing measures of metal
pollution in matrix of interest. Although these proce-
dural assessments are scientifically recognized, they have
proved deficient in presenting trace metals in their phys-
icochemical forms as well as predicting their toxicity
[11]. The analysis and quantification of trace metals in
environmental and biological systems of interest as total
concentration are seemingly tenable but misleading, and
therefore requires complementary partitioning informa-
tion, which will characteristically elucidate the different
elemental coexisting forms.
However, considering the enhanced understanding of
biological, metabolic and toxicological effects of these
trace metals, it has become necessary to measure trace
metals as “total” as well as determine quantitatively the
different chemical forms of these trace metals. This is
necessary since toxicity of trace elements especially al-
kylated and organometals is a function of their speciation
coefficient [2]. Thus, chemical speciation analysis has
become an efficient and reliable tool for assessment of
environmental and ecotoxicological risks posed by trace
metals. Partitioning studies of heavy metals in water and
sediments have emerged as an important instrument in
environmental toxicological researches. In recent times,
it has become the core of metal pollution studies through
which heavy metal species have been harnessed in deter-
mining their potential bioavailability and remobilization
within human, sedimentary, biotic and aquatic systems.
The greatest interest in metal speciation in natural envi-
ronments and biosystems is probably explained by their
influence on the bioavailability and toxicity of metals.
1.1. A Review of Current Usage of the Terms
“Speciation” and “Speciation Analysis”
Although there is a discernable difference between the
*Corresponding author.

N. U. BENSON ET AL. 634
terms “speciation” and “speciation analysis”, there still
exists some form of confusion on their usage by envi-
ronmental and analytical chemists, biologists, geochem-
ists, and in evolutionary concept. However, in order to
avoid confusion, an attempt is made to clearly define
these terms. According to IUPAC, speciation in chemis-
try simply refers to the distribution of an element among-
st defined chemical species, while speciation analysis
describes the analytical activities of identifying and/or
measuring the quantities of one or more individual che-
mical species or forms in an environmental or biosystems
[12].
Furthermore, for chemical elements, the chemical spe-
cies refers to the specific form(s) of the element present
in terms of oxidation or electronic state, isotopic compo-
sition and molecular structure. The distribution and bio-
availability of trace metals in environmental samples
such as soil, sediments, water, atmospheric particulate
and biospecimens can be considered to obtain a better
understanding of environment-organism interactions.
Thus, bioactivity and bioavailability of trace metals
strictly depend on their chemical coexisting forms, and
therefore their speciation [13]. The determination of total
metal concentration in living systems, environmental
substances and biospecimens is not sufficient to assess
the environmental impact of polluted sediments since
heavy metals may have different chemical forms and
only a fraction can be remobilized easily [2,14]. Studies
on the distribution and speciation of heavy metals in
sediments can provide not only information on the de-
gree of pollution, but especially the actual environmental
impact on metal bioavailability as well as their origin
[14].
There are many possible approaches to trace metal
speciation. These include the spectral characterization,
kinetic, direct species-specific, computational and hybrid
techniques, among others. Several contemporary tech-
niques are being developed or improved upon earlier
procedures. However, the choice of any of these ap-
proaches is subject to the nature and physical properties
of the elemental species to be determined. To date, it has
generally been accepted that the most appropriate method
to evaluate heavy metals is the selective sequential ex-
traction procedures [15]. Selective extractions are widely
used in soil and sediment analysis to evaluate long-term
potential emission of pollutants and to study the distribu-
tion of pollutants among the geochemical phases [16].
They are also used to determine the metals associated
with source constituents in sedimentary deposits. Ac-
cording to [17], metals with an anthropogenic origin are
mainly extracted in the first step of the procedure, while
lithogenic metals are found in the last step of the process
corresponding to the residual fraction [14].
In environmental and biophysicochemical systems, the
geochemistry, bioavailability and toxicity of trace metals
are a function of the physicochemical characteristics of
the forms in which it is present. Additionally, the physio-
logical characteristics of an organism constitute impor-
tant factors that influence its speciation. However, bio-
logical availability of a trace metal is not a function of
total metal concentration, but rather of particular species
of the metal that can either interact directly with an or-
ganism or can convert readily to species which can in-
teract (the kinetically labile metal concentration) [2,14].
1.2. Analytical Significance of Trace Metal
Speciation
Analytical measurements expressed as total content of
specific metal in an environmental and biological mate-
rial are insufficient. Therefore, one of the most important
significance of speciation analysis is the qualitative and
quantitative signature it has given to specific metal spe-
cies, which could be employed in the assessment of the
index of toxicity impacts of elements. Speciation analysis
is an important present-day analytical tool particularly
used for the elucidation of the chemical form(s) as well
as the quantitative estimation of a specific element when
conducting toxicological and biochemical investigations.
After years of considerable researches on metals pol-
lution, it is now widely held that the distribution, mobil-
ity, bioavailability and toxicity of trace metals in envi-
ronmental and biological systems depend not simply on
their concentrations, but critically on their chemical
forms. It is also known that individual metal species
possesses a different chemical activity and ability to
transform. Therefore, for proper estimation of the degree
of the toxic effect of metals, their distribution among
coexisting forms in aquatic environment must be known.
This is realized through the use of metal speciation
analysis. Thus, speciation analysis can increase the in-
formation capacity of collected results via characterizing
in detail some of the most important chemical forms of
an element in order to understand the transformations
between forms that are likely to occur, and to infer from
such information the probable environmental and health
consequences.
Trace metal speciation analysis is an important envi-
ronmental analytical tool for forecasting metal fate in
aquatic ecosystems and developing effective methods for
water quality monitoring. Chemical compounds that dif-
fer in isotopic composition, conformation, oxidation or
electronic state, or in the nature of their complexed or
covalently bound substituents can be regarded as distinct
chemical species [12]. In the light of this, a systematic
approach highlighting identifiable species and distinct
transient forms of an element, its coordinated atoms or
excited states structurally could be categorized into nu-
clear (isotopic) composition, electronic or oxidation state,
Open Access AJAC

N. U. BENSON ET AL. 635
inorganic, organic and macromolecular compounds and
complexes.
1.3. Techniques, Types of Speciation Analysis
and Their Applications
The toxicity and bioavailability of heavy metals are not
only a function of their total concentration in water, but
also a function of the concentration and ratio of the vari-
ous coexisting forms [18,19]. However, estimation and
elaborate quantification of these free metal species can
be achieved through one of the predominant trends of
heavy metal analysis-elemental speciation, which em-
ploys various speciation techniques. Although the vari-
ous schemes developed by several researchers are capa-
ble of quantifying the amount of free and bound metal, it
has been noted that only the most sensitive techniques
are suitable for speciation analysis. According to [20],
speciation techniques using inductively coupled plasma
mass spectrophotometry (ICP-MS), inductively coupled
plasma atomic emission spectrophotometry (ICP-AES),
and electrothermal atomic absorption spectrometry (ET-
AAS) could be considered as the most sensitive and se-
lective techniques.
However, continuing developments in analytical che-
mistry have provided a platform for the proliferation of
investigations that have now seen the coupling of versa-
tile separation techniques, such as high performance liq-
uid chromatography, gas chromatography and capillary
electrophoresis (CE) to a highly sensitive detector, such
as ICP-MS, which has generated substantial attractive
analytical tools for ultra-trace elemental speciation analy-
sis [21].
The extraction, detection and ultratrace quantitative
and qualitative determinations of elements through spe-
ciation analysis can be carried out in five different ways
[15]. However, the choice of any of the five types is a
function of the aim and scope of the analytical investiga-
tion. A concise summary of the basic types of speciation
analysis commonly encountered in chemical analysis,
their characteristics, areas and examples of where the
speciation analysis principles could be applied is pre-
sented in Table 1.
2. Speciation Studies in Different
Environmental Strata
2.1. Speciation Studies in Sediment
Phase-selective chemical extractions or fractionation
schemes involving multistep extraction procedures is one
of the approaches employed for understanding metal
speciation especially in sediment analysis [22-24]. A
typical fractionation scheme is the procedure developed
by [22], which delineates the metal species sequentially
as exchangeable, carbonate-bound, iron and manganese
oxide-bound, organically-bound and residual. Soil, sedi-
ments or precipitates are known reservoirs or sinks of
trace metals in the environments, and the metals may be
present in several different physicochemical forms/pha-
ses [25-27]. These phases are water soluble, exchange-
able; specifically adsorbed; carbonate; secondary Fe and
Mn oxides; organic matter; sulphides and silicates [28].
This procedure that was originally developed as a pivotal
heavy metal speciation scheme using extracting agents
has evolved as the foundation for recent advances in
fractionation speciation schemes. The sequential extrac-
tion or fractionation schemes are a very useful method,
for characterizing solid phases associated trace elements
in soils, sediments or particulates [29-33]. It must be
pointed out that the extracting reagents employed in
speciation analysis are chosen based on their selectivity
and specificity towards a particular physicochemical
species of trace metal [28]. The reagents cocktail for re-
spective extraction steps are capable of disrupting the
binding agents between individual element and the sedi-
ments thus allowing possible release of metal species
into the solution. A typical multistep sequential extrac-
tion scheme is shown in Table 2.
More so, a modified sequential chemical extraction
procedure developed by [36] for partitioning studies of
particulate bound cadmium in soil was conveniently
classified into eight fractions vis-à-vis: exchangeable,
carbonate-bound, metal-organic complex-bound, easily
reducible metal oxide-bound, organic-bound, amorphous
mineral colloid-bound, crystalline Fe oxide-bound, and
residual fractions (Table 3).
However, following the pioneering research by [22], a
relatively large number of fractionation schemes have
been developed, which employ series of reagents to
separate individual fractions of trace metals. In general,
sequential extraction procedures have not been standard-
ized therefore compelling individual researchers to use
schemes developed through their effort. However, in
order to streamline speciation analysis for repeatability
and reproducibility through harmonization and stan-
dardization of extraction protocol, a sequential extraction
method has been developed by the Standards, Measure-
ments and Testing Programme (formerly Community
Bureau of Reference, BCR) of the Commission of Euro-
pean Communities. In this three-stage sequential extrac-
tion procedure, trace metals are divided into acid-solu-
ble/exchangeable, reducible and oxidisable fractions,
which are leached with reagents. A summary of this ap-
proach is presented in Table 4 [37-39].
A modification of this method into a four-stage se-
quential leaching procedure has been developed and ap-
plied to assess the bioavailability and environmental mo-
Open Access AJAC

N. U. BENSON ET AL.
Open Access AJAC
636
Table 1. Basic types of speciation analysis and application in chemical analysis [15].
Type of speciation Characteristics Areas of applicationExample(s) of application
1. Physical Speciation Involves the determination of the different
forms of the same chemical species.
Air, water and soil
pollution analyses.
a) Trace metals analysis
(soluble and suspended fraction).
b) Trace metals analysis of different
forms present in soil and sediment after
sequential extraction.
2. Chemical Speciation Involves determination of chemical species.
A type of chemical speciation that involves
the extraction, detection and determination
of a specific chemical species or analyte.
a) Air, water and soil
pollution analyses.
b) Food contamination
studies.
a) Determination of tributyltin (TBT)
or triphenyltin (TPhT) in environmental
biota, sediments, seawater, etc.
b) Determination of methylmercury
in fish tissue or lead in food products.
2.1 Screening Speciation
2.1.1 Distribution Speciation A type of screening speciation that involves
the detection and determination selected chemical
individual in particular elements of analyzed
sample. Usually employed in analyses of
biological samples.
a) Air, water and soil
pollution analyses.
b) Ecotoxicological
studies.
a) Trace metals analyses especially
in blood serum and cells.
b) Determination of trace metals
in plants samples.
This is a type of chemical speciation that leads
to the extraction, detection and determination of
a set or group of analytes that possess a definite
set of characteristics; or the specific group of
compounds or trace metals existing in different
compounds and forms and at the specific
oxidation level.
a) Air, water and soil
pollution analyses.
b) Ecotoxicological
studies.
c) Food contamination
studies.
a) Determination of redox forms of
chromium, Cr(VI) in environmental
pollution analyses.
b) Determination of elementary, inor-
ganic and organic forms of mercury in
the environment and food products.
2.2 Group Speciation
2.2.1 Individual Speciation
A type of group speciation that involves the extrac-
tion, detection and determination of all chemical
species in analyzed sample.
a) Air, water and soil
pollution analyses.
b) Ecotoxicological
studies.
c) Food contamination
studies.
a) Identification and determination
of chemical species defined as to
molecular, complex, electronic or
nuclear structure.
Table 2. Multistep phase-selective extraction schemes for metal speciation [34,35].
Steps Species Reagent Extraction time/temp.
I Exchangeable 20 ml 1 M MgCl2 (pH = 7),
1 M ammonium acetate (pH = 7)
30 min
10 min
II Carbonates or specifically adsorbed 1 M sodium acetate (pH = 5) 300 min
III Mn oxide-bound 0.1 M NH4OH·HCl in 0.01 M HNO3 30 min (Room temp.)
IV Fe-Mn oxide-bound 0.04 M NH4OH·HCl in 25% (v/v) acetic acid 360 min (96˚C)
V Organically- and
sulphides-bound 30% H2O2 (pH = 2 with HNO3), then 3.2 M sodium acetate in 20% (v/v) HNO3 300 min (85˚C) Room temp.
VI Residual Digestion with HF-HClO4 (5:1) ratio
Table 3. Multistep sequential extraction schemes for metal speciation [36].
Steps Species Reagent Extraction time/temp.
I Exchangeable 10 ml Mg(NO3)2 (pH = 7), 1 M ammonium acetate (pH = 7) 4 h at 25˚C
II Carbonate-bound 25 ml 1 M sodium acetate (pH = 5) 6 h at 25˚C
III Metallic organic complex-bound 30 ml 0.1 M sodium pyrophosphate (Na4P2O7·10H2O) (pH = 10) 20 h at 25˚C
IV Easily reducible metal oxide-bound 20 ml 0.01M NH2OH·HCl in 0.01 M HNO3 30 min at 25˚C
V H2O2 extractable organic-bound
5 ml 30% H2O2 (pH = 2 with HNO3), then 3 ml 0.02 M HNO3
Add 3 ml 30% H2O2 (pH = 2 with HNO3), cool and add 10 ml
2.0 M Mg(NO3)2 in 20% HNO3
2 h at 85˚C
4 h at 25˚C (dark)
VI Amorphous mineral colloid-bound 10 ml 0.2 M (NH4)2C2O4 (pH = 3). 4 h at 25˚C (dark)
VII Crystalline Fe oxide bound 25 ml 0.2 M (NH4)2C2O4 (pH = 3) in 0.1 M ascorbic acid 30 min at 95˚C
VII Residual Digestion with HF-HClO4 (5:1) ratio

N. U. BENSON ET AL. 637
Table 4. Three-step sequential extraction proce dur e de veloped by BCR [37-39].
Species Reagent Extraction time/temp.
Exchangeable,
Water- and acid-soluble 40 cm3 0.11 M CH3COOH per 1.0g of sample Shake using mechanical shaker
overnight at 25˚C
Reducible species (metal
oxides- and hydroxides-bound) 40 cm3 0.1 M NH2OH.HCl (adjusted to pH = 2 with HNO3) added to residue. 300 min at 25˚C
Oxidisable species (organic
matter- and sulphides-bound)
10 cm3 8.8 M H2O2 added to residue in water bath.
Evaporate solution to few cm3.
After cooling, add 50 cm3 1 M CH3COONH4
(adjusted to pH = 2 with HNO3) to residue.
60 min at room temperature
60 min at 85˚C
360 min (25˚C)
bility of some heavy metals in total suspended particu-
lates [40].
2.2. Speciation Studies in Aquatic Ecosystems
It is widely known that pollutants such as trace metals
are introduced into aquatic ecosystems mainly through
natural and anthropogenic sources. The weathering of
rocks and volcanic eruptions are among the natural
sources, while aerial deposition from automotive traffic
and power plants, mining, industrial activities, urbaniza-
tion, and agricultural activities constitute human-induced
sources [41-43]. Human-induced emissions and inputs
into freshwater, estuarine, seawater or ocean systems can
give rise to higher concentrations of the metals relative to
the natural threshold concentrations. Once these con-
taminants enter into freshwater or marine ecosystems,
they are capable of distributing into water, sediment, and
biota compartments. In most cases, the overabundance of
heavy metals even in trace levels in surface or mixed
layer water could develop a metal pollution ecological
footprint capable of posing serious health risks to sea
fauna and flora, humans, and in general to the environ-
ment. In aquatic ecosystems such as estuaries, rivers,
streams, and oceans, the determination of the total con-
centrations of heavy metals in surface water and sedi-
ment is a useful analytical tool in identifying pollution
hotspots as well as the identification of human-mediated
sources of metal inputs, whereas speciation studies is a
multipurpose useful tool in determining the bioavailabil-
ity and toxicity of heavy metals, and in understanding the
pollution regimes and metal-sediment diffusive fluxes or
interactions in aquatic systems [44,45].
All heavy metals exist in surface waters in colloidal,
particulate, and dissolved phases, although dissolved
concentrations are generally low [46]. The colloidal and
particulate metal may be found in 1) hydroxides, oxides,
silicates, or sulfides; or 2) adsorbed to clay, silica, or
organic matter. The soluble forms are generally ions or
unionized organometallic chelates or complexes. The
solubility of trace metals in surface waters is predomi-
nately controlled by the water pH, the type and concen-
tration of ligands on which the metal could adsorb, and
the oxidation state of the mineral components and the
redox environment of the system.
The distribution of trace metals in aquatic substrates
such as sediment, surface water, and microorganisms has
been identified as exchangeable, carbonates, oxidizing,
organic matter and residual fractions [22,47,48]. Studies
have shown that the physicochemical forms of trace met-
als determine their potential bioavailability and remo-
bilization in aquatic systems [1,11]. However, the che-
mical speciation of metals could be made possible by
changing ecohydrological perturbations, or as the organic
particulates binding the metals ultimately decompose [1,
49]. Trace metals in different sequential extraction aside
reflecting the metal component or integrity of an ecosys-
tem could be used to realize the chemical behavior with
respect to remobilization.
In the estuaries, partitioning studies are particularly
important because metal speciation is influenced by the
constantly changing environmental conditions including
salinity, pH and sediment redox potential [43,50,51]. The
speciation of dissolved metals in seawater is relatively
well understood. It is known, for example, that free ions
of Cu and Cd are the most bioavailable inorganic forms
and account for only a small proportion of the total dis-
solved metal concentration [52].
2.3. Speciation Studies in Agrosystems
Heavy metals are found naturally in undisturbed soils
and, in fact, small amounts of many metals are required
by plants as micro- and macronutrients to remain healthy.
The sources of heavy metals in soils are primarily an-
thropogenic, although some are known to occur naturally,
but rarely at toxic levels. Human induced activities have
dramatically modified the composition and organization
of soils. Non-regulated large-scale disposal of municipal
sludge, domestic, urban and industrial wastes, agricul-
tural application, manufacturing and mining activities
have resulted in increased heavy metal contamination of
urban and agricultural soils. Other potential sources of
human-induced soil contamination with heavy metals
include industrial and traffic dust emitted into the at-
mosphere, land application of domestic or industrial
sewage sludge, mineral, mainly phosphorous, fertilizers
and pesticides [38,53,54]. However, these sources are
Open Access AJAC

N. U. BENSON ET AL. 638
capable of creating patchy hotspots of metal contamina-
tion, which could be of high concern and pose possible
dangers to human and animals in contact with the con-
taminated soils [55].
Excess heavy metal accumulation in the agrosystems
is toxic to humans and other animals. Unlike organic
contaminants, most metals in the soil environment do not
undergo breakdown by microbial organisms or chemical
degradation, and therefore concentrations of metals per-
sist in soils for a long time after their input [56]. Once
metals find their way into the soil, they could remain in
the soil environment, or bioaccumulate and biotransform
in plants and food chain, and as well as get seeped into
groundwater [57]. These biopersistence, bioaccumulative
and biotransformative properties could lead to enhanced
levels of heavy metals in soils, and subsequent bioavail-
ability and uptake by plants. However, this depends not
only on heavy metal contents in soils but is also gov-
erned by factors such as soil pH, organic matter and clay
contents [58].
Trace metal pollution of soils is a pervasive problem
that often constitutes serious short- and long-term risks
for humans, plants, groundwater quality and ecosystem
health. Several researches have been reported on heavy
metal contamination in agrosystems especially arising
from human-mediated sources such as industrial waste,
automobile emission, mining or processing activities, and
agricultural practice [59]. Majority of these researches
are based on analytical quantifications that reports heavy
metal concentrations as the total content of metals in
analyzed matrices. Fewer attempts have been made to
evaluate and report the speciation of heavy metals in par-
ticulate species [15,22]. Moreover, the expression of
heavy metal contents as “total” concentration to assess
the quality of metal pollution in agrosystems is mislead-
ing and analytically uninformative. Information about the
fate and toxicity of heavy metals in a contaminated soil
could be sufficiently highlighted through speciation
analyses of soil samples [60,61]. In the light of the fore-
going, it is imperative that in monitoring studies and risk
assessments of metal contaminated soils, not only the
total or extractable contents should be considered, but
also the chemical forms (species) of the metal contami-
nants must be known. Such studies may help to minimize
human health risks associated with trace metals con-
tamination and aid in the evaluation of their bioavailabil-
ity [62].
Metal bioavailability in different soils, however, de-
pends on soil properties such as the pH, metal contents,
particle size, organic matter, and wetness. A research
report has indicated that the mobility, bioavailability,
storage, retention and toxicity of trace metals in living
organisms, food and the environment is a function of the
chemical forms in which they enter the ecosystems and
the final forms in which they are present therein [63].
Therefore, in order to determine the binding forms of
heavy metals in soil, it is imperative that chemical ex-
traction procedures should be employed. A large number
of sequential extraction procedures, which utilizes series
of reagents to separate individual fractions of heavy met-
als have been developed, majority of which are derived
from the pioneering studies by [22].
Speciation analysis of trace elements in soils may be
performed using either physical or chemical methods, but
the latter offers a reliable and more sensitive approach.
The chemical protocols basically employ chemical solu-
tions of varying, but specific, strengths and reactivities to
release heavy metals from the different fractions of soil
samples of interest as a means of quantifying the coex-
isting metal species [64]. Elemental quantification in
soils can be achieved through single reagent leaching, ion
exchange resins, and sequential extraction procedures.
The theory involved in the latter is that the most mobile
metals are leached in the first fraction and continue in
order of decreasing of mobility. Common examples of
the sequential extraction techniques are the Tessier Pro-
cedure, the Community Bureau of Reference (BCR)
Procedure, the Maiz Short Extraction Procedure, the
Galán Procedure, and the Geological Society of Canada
Procedure [24,65-67]. These sequential extraction pro-
cedures promote fractionation. However, despite that a
number of extraction schemes have been proposed and
developed by several researchers, there abound contro-
versies regarding some of the sequential techniques.
Nevertheless, the Tessier Procedure is generally accepted
as the most commonly used protocol followed closely by
the Community Bureau of Reference Procedure, although
it is still plagued by limitations.
3. Conclusion and Future Perspectives
Although studies on trace metal speciation has been ac-
tive for decades, the field has been instrument and
method limited. However, in recent times, the number of
research carried out on trace metal speciation has in-
creased considerably with the improvement of existing
methodologies, recent advances on hyphenated systems
and the development of in situ automated monitoring
profilers that are capable of monitoring specific fractions
of trace metals. Nevertheless, new and universally ac-
cepted, reliable and cost effective analytical tools capable
of performing in situ, real-time monitoring with mini-
mum perturbation of the environmental matrix should be
developed. This paper examined the analytical relevance
and the need for trace metal speciation in environmental
and biochemical systems in order to determine the dis-
tribution of specific species present for a better under-
standing of their degree of toxicity, mobility and stability.
Judging from the foregoing, the use of speciation of ele-
Open Access AJAC

N. U. BENSON ET AL. 639
ments to assess the bioavailability and mobility of heavy
metals in environmental and biophysicochemical systems
remains a sine qua non for a better understanding of the
different chemical forms of a particular element or its
compounds and associated patterns of toxicity. It is there-
fore suggested that researchers should adopt the speci-
ation analysis while conducting the assessment of trace
metals in order to be able to obtain and proffer additional
useful information on species of differing carcinogenic
potential in biosystems and environmental matrices in
preference to total metal determination.
REFERENCES
[1] N. Benson, E. D. Udosen and O. Akpabio, “Interseasonal
Distribution and Partitioning of Heavy Metals in Subtidal
Sediment of Qua Iboe Estuary and Associated Creeks,
Niger Delta (Nigeria),” Environmental Monitoring and
Assessment, Vol. 146, No. 1-3, 2008, pp. 253-265.
http://dx.doi.org/10.1007/s10661-007-0077-5
[2] A. Sanz-Medel, “Toxic Trace Metal Speciation: Impor-
tance and Tools for Environmental and Biological Analy-
sis,” Pure and Applied Chemistry, Vol. 70, No. 12, 1998,
pp. 2281-2285.
http://dx.doi.org/10.1351/pac199870122281
[3] N. U. Benson, “Lead, Nickel, Vanadium, Cobalt, Copper
and Manganese Distributions in Intensely Cultivated
Floodplain Ultisol of Cross River, Nigeria,” International
Journal of Soil Science, Vol. 1, No. 2, 2006, pp. 140-145.
http://dx.doi.org/10.3923/ijss.2006.140.145
[4] M. I. Yahaya, G. C. Ezeh, Y. F. Musa and S. Y. Moham-
mad, “Analysis of Heavy Metals Concentration in Road
Sides Soil in Yauri, Nigeria,” African Journal of Pure
and Applied Chemistry, Vol. 4, No. 3, 2010, pp. 22-30.
[5] E. D. Udosen, N. U. Benson and J. P. Essien, “Trends in
Heavy Metals and Hydrocarbon Burdens in Stubbs Creek,
A Tributary of the Qua Iboe River Estuary, Nigeria,”
Trends in Applied Sciences and Research, Vol. 2, No. 4,
2007, pp. 312-319.
http://dx.doi.org/10.3923/tasr.2007.312.319
[6] J. P. Essien, S. P. Antai and A. A. Olajire, “Distribution,
Seasonal Variations and Ecotoxicological Significance of
Heavy Metals in Sediments of Cross River Estuary Man-
grove Swamp,” Water, Air and Soil Pollution, Vol. 197,
No. 1-4, 2009, pp. 91-105.
http://dx.doi.org/10.1007/s11270-008-9793-x
[7] N. U. Benson, J. P. Essien and D. E. Bassey, “Hydrobi-
ological Constraints of Trace Metals in Surface Water,
Coastal Sediments and Water Lily of Calabar River, Ni-
geria,” African Journal of Biotechnology, Vol. 6, No. 20,
2007, pp. 2358-2362.
[8] O. O. Ayejuyo, O. O. Tovide and B. O. Moronkola,
“Heavy Metal Distribution in Crab (Callinectes amnicola)
Living on the Shores of Ojo Rivers, Lagos, Nigeria,” En-
vironmentalist, Vol. 29, No. 1, 2009, pp. 33-36.
http://dx.doi.org/10.1007/s10669-008-9177-1
[9] D. S. Abolude, O. A. Davies and D. W. Avong, “Level of
Heavy Metals in Freshwater Crab (Cardisoma guahumi)
Obtained from Ahmadu Bello University Reservoir, Zaria
Nigeria,” International Journal of Animal and Veterinary
Advances, Vol. 1, No. 2, 2009, pp. 54-58.
[10] J. A. Omolaoye, A. Uzairu and C. Gimba, “Heavy Metal
Assessment of Some Soft Plastic Toys Imported into Ni-
geria from China,” Journal of Environmental Chemistry
and Ecotoxicology, Vol. 2, No. 8, 2010, pp. 126-130.
[11] A. O. Ibhadon, P. Wright and R. Daniels, “Trace Metal
Speciation and Contamination in an Intertidal Estuary,”
Journal of Environmental Monitoring, Vol. 6, No. 8,
2004, pp. 679-683. http://dx.doi.org/10.1039/b315954j
[12] D. M. Templeton, F. Ariese, R. Cornelis, L-G. Danielsson,
H. Muntau, H. P. Van Leeuwen and R. Łobiński, “Guide-
lines for Terms Related to Chemical Speciation and Frac-
tionation of Elements: Definitions, Structural Aspects,
and Methodological Approaches,” Pure and Applied Che-
mistry, Vol. 72, No. 8, 2000, pp. 1453-1470.
http://dx.doi.org/10.1351/pac200072081453
[13] B. Jones and A. Turki, “Distribution and Speciation of
Heavy Metals in Surficial Sediments from Tees Estuary,
Northeast England,” Marine Pollution Bulletin, Vol. 34,
No. 10, 1997, pp. 768-779.
http://dx.doi.org/10.1016/S0025-326X(97)00047-7
[14] M. Ramirez, S. Massolo, R. Frache and J. Correa, “Metal
Speciation and Environmental Impact on Sandy Beaches
Due to El Salvador Copper Mines, Chile,” Marine Pollu-
tion Bulletin, Vol. 50, No. 1, 2005, pp. 62-72.
http://dx.doi.org/10.1016/j.marpolbul.2004.08.010
[15] A. Kot and J. Namiesnik, “The Role of Speciation in
Analytical Chemistry, “Trends in Analytical Chemistry,
Vol. 19, No. 2-3, 2000, pp. 69-79.
http://dx.doi.org/10.1016/S0165-9936(99)00195-8
[16] G. Rauret, J. López-Sánchez, A. Sahuquillo, R. Rubio, C.
Davidson, A. Ure and P. Quevauviller, “Improvement of
the BCR three Step Sequential Extraction Procedure Prior
to the Certification of New Sediment and Soil Reference
Materials,” Journal of Environmental Monitoring, Vol. 1,
No. 1, 1999, pp. 57-61.
http://dx.doi.org/10.1039/a807854h
[17] R. Rubio, J. Lopez-Sanchez and G. Ruaret, “La Especia-
cion Solida de Trazas de Metales en Sedimentos. Aplica-
cion a Sedimentos muy Contaminados,” Anales de Quí-
mica, Vol. 87, 1991, pp. 599-605.
[18] D. M. Templeton, “Biomedical Aspects of Trace Element
Speciation,” Fresenius’ Journal of Analytical Chemistry,
Vol. 363, No. 5-6, 1999, pp. 505-511.
http://dx.doi.org/10.1007/s002160051234
[19] R. P. Linnik and O. A. Zaporozhets, “Solid-Phase Rea-
gent for Molecular Spectroscopic Determination of Heavy
Metal Speciation in Natural Water,” Analytical and Bio-
analytical Chemistry, Vol. 375, No. 8, 2003, pp. 1083-
1088.
[20] O. Zaporozhets and R. Linnik, “Hyphenated Techniques
in Speciation Analysis of Metals in Natural Waters,”
2004.
http://www.ecotest.univ.kiev.ua/nauka/speciation_e.htm
[21] A. L. Rosen and G. M. Hieftje, “Inductively Coupled
Plasma Mass Spectrometry and Electrospray Mass Spec-
trometry for Speciation Analysis: Applications and In-
Open Access AJAC

N. U. BENSON ET AL. 640
strumentation,” Spectrochimica Acta Part B: Atomic
Spectroscopy, Vol. 59, No. 2, 2004, pp. 135-146.
http://dx.doi.org/10.1016/j.sab.2003.09.004
[22] A. Tessier, P. Campell and M. Bison, “Sequential Extrac-
tion Procedure for the Speciation of Particulate Trace
Metals,” Analytical Chemistry, Vol. 51, No. 7, 1979, pp.
844-850. http://dx.doi.org/10.1021/ac50043a017
[23] L. M. Shuman, “Fractionation Method for Soil Micronu-
trients,” Soil Science, Vol. 140, No. 1, 1985, pp. 11-22.
http://dx.doi.org/10.1097/00010694-198507000-00003
[24] A. Ure, P. Quevaullier, H. Muntau and B. Griepink,
“Speciation of Heavy Metals in Soils and Sediments. An
Account of the Improvement and Harmonization of Ex-
traction Techniques Undertaken under the Auspices of the
BCR of the CEC,” International Journal of Environ-
mental Analytical Chemistry, Vol. 51, No. 1-4, 1993, pp.
135-151. http://dx.doi.org/10.1080/03067319308027619
[25] G. Sposito, “The Chemical Forms of Trace Metals in
Soils,” In: I. Thornton, Ed., Applied Environmental Geo-
chemistry, Academic Press, London, 1983, pp. 123-170.
[26] P. H. T. Beckett, “The Use of Extractants in Studies on
the Trace Metals in Soils, Sewage Sludges and Sludge-
Treated Soils,” Advances in Soil Sciences, Vol. 9, No. 5,
1988, pp. 144-175.
[27] A. H. Mahvi, “Application of Agricultural Fibers in Pol-
lution Removal from Aqueous Solution,” International
Journal of Environmental Science and Technology, Vol. 5,
No. 2, 2008, pp. 275-285.
http://dx.doi.org/10.1007/BF03326022
[28] W. M. Gitari, L. F. Petrik, D. L. Key and C. Okujeni,
“Partitioning of Major and Trace Inorganic Contaminants
in Fly Ash Acid Mine Drainage Derived Solid Residues,”
International Journal of Environmental Science and Te-
chnology, Vol. 7, No. 3, 2010, pp. 519-534.
http://dx.doi.org/10.1007/BF03326161
[29] P. Adamo, S. Dudka, M. Wilson and W. McHardy, “Che-
mical and Mineralogical Forms of Cu and Ni in Con-
taminated Soils from the Sudbury Mining and Smelting
Region, Canada,” Environmental Pollution, Vol. 91, No.
1, 1996, pp. 11-19.
http://dx.doi.org/10.1016/0269-7491(95)00035-P
[30] L. Q. Ma and G. N. Rao, “Chemical Fractionation of
Cadmium, Copper, Nickel and Zinc Contaminated Soils,”
Journal of Environmental Quality, Vol. 26, No. 1, 1997,
pp. 259-264.
[31] F. Mohsenzadeh, J. Nouri, A. Ranjbar, F. Mohammadian
and A. Babaie, “Air Pollution Control through Kiln Re-
cycling By-Pass Dust in a Cement Factory: Iran,” Journal
of Environmental Health Science and Engineering, Vol. 3,
No. 1, 2006, pp. 5-8.
http://dx.doi.org/10.2134/jeq1997.0047242500260001003
6x
[32] A. R. Karbassi, S. M. Monavari, G. R. Bidhendi, J. Nouri
and K. Nematpour, “Metal Pollution Assessment of Sedi-
ment and Water in the Shur River,” Environmental Mo-
nitoring and Assessment, Vol. 147, No. 1-3, 2008, pp.
107-116. http://dx.doi.org/10.1007/s10661-007-0102-8
[33] X. D. Feng, Z. Dang, W. Huang and C. Yang, “Chemical
Speciation of Fine Particle Bound Trace Metals,” Inter-
national Journal of Environmental Science and Technol-
ogy, Vol. 6, No. 3, 2009, pp. 337-346.
[34] B. Nowak, “Sequential Extraction of Metals Forms in the
Soil near a Roadway in Southern Poland,” Analyst, Vol.
120, No. 3, 1995, pp. 737-739.
http://dx.doi.org/10.1039/an9952000737
[35] R. E. Jervis, S. S. Krishna, M. M. Ko, L. D. Vela, T. G.
Pringle, A. C. Chan and L. Xing, “Biological Incinerator
emission of Toxic Inorganics, Their Residues and Their
Availability,” Analyst, Vol. 120, No. 3, 1995, pp. 651-658.
http://dx.doi.org/10.1039/an9952000651
[36] G. S. R. Krishnamurti, P. M. Huang, K. C. J. Van Rees, L.
M. Kozak and H. P. W. Rostad, “Speciation of Particu-
late-Bound Cadmium of Soils and Its Bioavailability,”
Analyst, Vol. 120, No. 3, 1995, pp. 659-665.
http://dx.doi.org/10.1039/an9952000659
[37] C. M. Davidson, P. C. S. Ferreira and A. M. Ure, “Some
Sources of Variability in Application of the Three-Stage
Sequential Extraction Procedure Recommended by BCR
to Industrially-Contaminated Soil,” Fresenius’ Journal of
Analytical Chemistry, Vol. 363, No. 5-6, 1999, pp. 446-
451. http://dx.doi.org/10.1007/s002160051220
[38] A. Moc’ko and W. Wacławek, “Three-Step Extraction Pro-
cedure for Determination of Heavy Metals Availability to
Vegetables,” Analytical and Bioanalytical Chemistry, Vol.
380, No. 5-6, 2004, pp. 813-817.
http://dx.doi.org/10.1007/s00216-004-2832-6
[39] H.-S. Jung, S.-T. Yun, B.-Y. Choi, H.-M. Kim, M. Jung,
S.-O. Kim and K.-H. Kim, “Geochemical Studies on the
Contamination and Dispersion of Trace Metals in Inter-
tidal Sediments Around a Military Air Weapons Shooting
Range,” Journal of Soils and Sediments, Vol. 10, No. 6,
2010, pp. 1142-1158.
http://dx.doi.org/10.1007/s11368-010-0248-9
[40] R. Pöykiö, P. Perämäki, I. Välimäki and T. Kuokkanen,
“Estimation of Environmental Mobility of Heavy Metals
Using a Sequential Leaching of Particulate Material Emit-
ted from an Opencast Chrome Mine Complex,” Analytical
and Bioanalytical Chemistry, Vol. 373, No. 3, 2002, pp.
190-194. http://dx.doi.org/10.1007/s00216-002-1299-6
[41] T. Peytoni, A. Mcintosh, V. Anderson and K. Yost, “Aerial
Input of Heavy Metals into an Aquatic Ecosystem,” Water,
Air and Soil Pollution, Vol. 5, No. 4, 1976, pp. 443-451.
http://dx.doi.org/10.1007/BF00280845
[42] E. Z. Ochieng, J. O. Lalah and S. O. Wandiga, “Anthropo-
nic Sources of Heavy Metals in the Indian Ocean Coast of
Kenya,” Bulletin of Environmental Contamination and
Toxicology, Vol. 83, No. 4, 2009, pp. 600-607.
http://dx.doi.org/10.1007/s00128-009-9807-4
[43] J. O. Lalah, E. Z. Ochieng and S. O. Wandiga, “Sources
of Heavy Metal Input into Winam Gulf, Kenya,” Bulletin
of Environmental Contamination and Toxicology, Vol. 81,
No. 3, 2008, pp. 277-284.
http://dx.doi.org/10.1007/s00128-008-9452-3
[44] C. K. Jain, “Metal Fractionation Study on Bed Sediments
of River Yamuna, India,” Water Research, Vol. 38, No. 3,
2004, pp. 569-578.
http://dx.doi.org/10.1016/j.watres.2003.10.042
[45] J. L. Trujillo-Cárdenas, N. P. Saucedo-Torres, P. F.
Open Access AJAC

N. U. BENSON ET AL.
Open Access AJAC
641
Zárate del Valle, N. Ríos-Donato, E. Mendizábal and S.
Gómez-Salazarb, “Speciation and Sources of Toxic Met-
als in Sediments of Lake Chapala, Mexico,” Journal of
the Mexican Chemical Society, Vol. 54, No. 2, 2010, pp.
79-87.
[46] M. J. Kennish, “Ecology of Estuaries: Anthropogenic Ef-
fects,” CRC, Boca Raton, 1992.
[47] S. R. Stephens, B. J. Alloway, J. E. Carter and A. Parker,
“Towards the Characterization of Heavy Metals in Dre-
dged Canal Sediments and an Appreciation of Availabi-
lity: Two Examples from the UK,” Environmental Pollu-
tion, Vol. 113, No. 2, 2001, pp. 395-401.
http://dx.doi.org/10.1016/S0269-7491(00)00178-0
[48] J. N. Egila and D. N. Nimyel, “Determination of Trace
Metal Speciation in Sediments from Some Dams,” Jour-
nal of Chemical. Society of Nigeria, Vol. 27, No. 1, 2002,
pp. 71-77.
[49] S. V. Matagi, D. Swai and R. Mugabe, “Heavy Metal
Remove/Mechanisms in Wetlands,” African Journal of
Tropical Hydrobiology and Fisheries, Vol. 8, 1998, pp.
23-35.
[50] M. Kersten and U. Forstner, “Chemical Fractionation of
Heavy Metals in Anoxic Estuarine and Coastal Sedi-
ments,” Water Science and Technology, Vol. 18, No. 4-5,
1986, pp. 121-130.
[51] W. Calmano, J. Hong and U. Forstner, “Binding and Mo-
bilisation of Heavy Metals in Contaminated Sediments
Affected by pH and Redox Potential,” Water Science and
Technology, Vol. 28, No. 8-9, 1993, pp. 223-235.
[52] D. W. Engel, W. G. Sunda and B. A. Fowler, “Factors
Affecting Trace Metal Uptake and Toxicity to Estuarine
Organisms. I. Environmental Parameters,” In: J. F. Vern-
berg, A. Calabrese, F. P. Thurberg and W. B. Vernberg,
Eds., Biological Monitoring of Marine Pollutants, Aca-
demic Press, New York, 1981, pp. 127-143.
http://dx.doi.org/10.1016/B978-0-12-718450-0.50011-0
[53] D. C. Adriano, “Trace Elements in Terrestrial Environ-
ment—Biogeochemistry, Bioavailability, and Risks of Me-
tals,” 2nd Edition, Springer, New York, 2001.
http://dx.doi.org/10.1007/978-0-387-21510-5
[54] J. E. Fergusson, “The Heavy Elements: Chemistry of En-
vironmental Impact and Health Effects,” Pergamon Press,
Oxford, 1990.
[55] Q. X. Zhou and Y. F. Song, “Remediation of Contaminated
Soils: Principles and Methods,” Science Press, Beijing,
2004, pp. 135-141.
[56] G. L. Guo, Q. X. Zhou and L. Q. Ma, “Availability and
Assessment of Fixing Additives for the in Situ Remedia-
tion of Heavy Metal Contaminated Soils: A Review,” En-
vironmental Monitoring and Assessment, Vol. 116, No. 1-
3, 2006, pp. 513-528.
http://dx.doi.org/10.1007/s10661-006-7668-4
[57] C. F. Lin, S. S. Lo, H. Y. Lin and Y. C. Lee, “Stabiliza-
tion of Cadmium Contaminated Soils Using Synthesized
Zeolite,” Journal of Hazardous Materials, Vol. 60, No. 3,
1998, pp. 217-226.
[58] K. Fytianos, G. Katsianis, P. Triantafyllou and G. Zacha-
riadis, “Accumulation of Heavy Metals in Vegetables
Grown in an Industrial Area in Relation to Soil,” Bulletin
Environmental Contamination and Toxicology, Vol. 67,
No. 3, 2001, pp. 423-430.
http://dx.doi.org/10.1007/s001280141
[59] Q. X. Zhou and G. H. Huan, “Environmental Biogeo-
chemistry and Global Environmental Changes,” Science
Press, Beijing, 2001.
[60] I. M. C. Lo and X. Y. Yang, “Removal and Distribution
of Metals from Contaminated Soils by a Sequential Ex-
traction Method,” Waste Management, Vol. 18, No. 1,
1998, pp. 1-7.
http://dx.doi.org/10.1016/S0956-053X(97)10005-8
[61] D. Peakall and J. Burger, “Methodologies for Assessing
Exposure to Metals: Speciation, Bioavailability of Metals,
and Ecological Host Factors,” Ecotoxicology and Envi-
ronmental Safety, Vol. 56, No. 1, 2003, pp. 110-121.
http://dx.doi.org/10.1016/S0147-6513(03)00055-1
[62] J. M. Scheyer, “Estimating Dietary Risk from Soils in
Urban Gardens,” In: W. Burghardt and C. Dornauf, Eds.,
Proceedings of the First International Conference on
Soils of Urban, Industrial, Traffic, and Mining Areas,
Essen, 2000, pp. 479-484.
[63] R. Cornelis, “Speciation of Trace Elements: A Way to a
Safer World,” Analytical and Bioanalytical Chemistry,
Vol. 373, No. 3, 2002, pp. 123-124.
[64] P. C. Ryan, S. Hillier and A. J. Wall, “Stepwise Effects of
the BCR Sequential Chemical Extraction Procedure on
Dissolution and Metal Release from Common Ferromag-
nesian Clay Minerals: A Combined Solution Chemistry
and X-Ray Powder Diffraction Study,” Science of the
Total Environment, Vol. 407, No. 1, 2008, pp. 603-614.
http://dx.doi.org/10.1016/j.scitotenv.2008.09.019
[65] E. Galán, J. L. Gómez Ariza, I. González, J. C. Fernández
Caliani, E. Morales and I. Giráldez, “Utility of the Tech-
niques of Sequential Extraction in the Improvement of the
Mineralogica Characterization by DRX of Grounds and
Sediments with High Contents of Iron Oxides,” In: Book
of Lectures and Summaries of XV the Scientific Meeting
of the Spanish Clay Society, 1999, pp. 68-69.
[66] I. Maiz, I. Arambarri, R. Garcia and E. Millán, “Evalua-
tion of Heavy Metal Availability in Polluted Soils by Two
Sequential Extraction Procedures Using Factor Analysis,”
Environmental Pollution, Vol. 110, No. 1, 2000, pp. 3-9.
http://dx.doi.org/10.1016/S0269-7491(99)00287-0
[67] A. J. Zimmerman and D. C. Weindorf, “Heavy Metal and
Trace Metal Analysis in Soil by Sequential Extraction: A
Review of Procedures,” International Journal of Analyti-
cal Chemistry, Vol. 2010, No. 2010, 2010, Article ID:
387803. http://dx.doi.org/10.1155/2010/387803