ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 56 (2003) 63–77

Monitoring approaches to assess bioaccessibility and bioavailability

of metals: Matrix issues
W.J.G.M. Peijnenburg and T. Jager
Laboratory for Ecotoxicology, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands
Received 19 March 2003; accepted 19 March 2003

Abstract

Bioavailability and bioaccessibility are complex issues that determine whether or not adverse effects are to be expected when
organisms or plants are exposed to contaminants. Clearly, the determinants of bioavailability and bioaccessibility must be
understood if one is to monitor or, ultimately, predict the effects of metals. On the basis of a dynamic conceptual model, this article
offers an analysis of the physicochemical and biological determinants underlying bioavailability and bioaccessibility. This analysis is
used as the basis for a general monitoring strategy for assessing potentially and actually available and accessible metal fractions in
the environmental matrices of water, soil, and sediment. We conclude that, lack of a universal expression of bioavailable and
bioaccessible metal fractions precludes the presentation of a detailed monitoring strategy that is broadly applicable. Instead, we
recommend that a critical assessment of the endpoints of determination become the basis for a need-specific monitoring strategy.
r 2003 Elsevier Science (USA). All rights reserved.

Keywords: Metal; Bioavailability; Bioaccessibility; Monitoring; Risk assessment; Metal uptake; Equilibrium partitioning; Exposure; Exposure route;
Pore water hypothesis

1. Introduction and this explains why discussions on these topics tend to

1.1. General background
There is growing awareness and concern about the
adverse effects induced by elevated levels of organic and
inorganic compounds not only in the air, water, soil,
and sediment, but also biological and human tissues.
However, there is also increasing recognition that
elevated contaminant levels in themselves are not
necessarily indicative of actually occurring adverse
effects. The issues of bioavailability and bioaccessibility
often determine whether or not the concentration at
which a chemical is present will have effects on
organisms. Nevertheless, the scientific basis for the
adequate use of ‘‘bioavailability’’ and ‘‘bioaccessibility’’
in the assessment of ecological and human risks is weak.
Problems in using the often-mentioned concepts of
bioavailability and bioaccessibility may be a conse-
quence of the absence of clear definitions of these terms,

Corresponding author. Fax: 31-30-274-4413.
E-mail address: wjgm.peijnenburg@rivm.nl
(W.J.G.M. Peijnenburg).
remain unresolved. It is clear, however, that specific
combinations of chemical, biological, and environmen-
tal parameters determine bioavailability and bioaccessi-
bility. Copper is, for examples, more strongly bound to
dissolved organic carbon (DOC) than zinc, and hence
copper is in general less available for uptake by aquatic
organisms. Organisms differ in their efficiency of
digesting sediment, and hence in their capability of
accumulating PCBs and other hydrophobic organics.
Finally, for example, the toxicity of ionizable chlor-
ophenols for soil-dwelling organisms depends on their
speciation, and hence on the magnitude of the environ-
mental parameter pH.
In this article we define ‘bioavailable fraction’ as the
fraction of the total amount of a chemical present in a
specific environmental compartment that, within a given
time span, is either available or can be made available
for uptake by (micro)organisms or plants, from either
the direct surrounding of the organism or the plant, or
by ingestion of food. As proposed before (Hamelink
et al., 1994; Peijnenburg et al., 1997), bioavailability
should be related as a dynamic process. This means that
the relevant endpoints that relate to bioavailability

0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0147-6513(03)00051-4
 ARTICLE IN PRESS
64 W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
should be explicitly taken into consideration, including
the relevant exposure and uptake routes, chemical fluxes
for specific biological species, their time dependency,
and dynamic aspects as well as acclimation or redis-
tribution processes within species. The dynamic ap-
proach of bioavailability should comprise two distinct
and different phases: a physicochemically driven deso-
rption process, and a physiologically driven uptake
process, requiring the identification of specific biotic
species as endpoint. It should be kept in mind that the
quantitative influence of solid-phase constituents on
toxicant binding is considerably larger in the soil and
sediment compartments than in the aqueous compart-
ment. ‘‘Toxicological bioavailability’’ (referring to
redistribution to targets within an individual, and thus
to dose to target tissue) is the third aspect that can be
distinguished as a better defined subdiscipline of the
(often) vague concept of bioavailability. Inherent in the
physiologically driven uptake process is the (genotypic-)
specific demand, the compartmentalization at tissue
and cell levels, and the cellular detoxification. In this
article, toxicological bioavailability will not be further
discussed.
The definition of bioavailability, as just given, implies
that the ‘‘bioaccessible fraction’’ is defined as the
fraction of the total amount of a chemical present in
ingested food, water, or ingested soil and sediment
particles, that at maximum can be released during
digestion. More specifically, the bioaccessible fraction
may also be defined as the fraction that, after ingestion,
may be mobilized into the gut fluids (chyme). This
fraction is considered to represent the maximum amount
of contaminant available for intestinal absorption.
Bioaccessible contaminants can subsequently be ab-
sorbed, in other words, transported across the intestinal
wall and transferred into the blood or lymph stream.
Among others, the compounds may be biotransformed
and excreted in the intestinal epithelium or liver, in what
is referred to as first-pass effect. After these steps, the
contaminants reach the systematic circulation and
thereby the remainder of the body, and may exert
toxicity (Oomen, 2000). Different conditions can affect
bioaccessibility. For example, the bioaccessibility of
metals and ionizable contaminants (acids and bases)
from soil or sediment is expected to be highly dependent
on the pH values in the different compartments of the
gastrointestinal (GT) tract (Castela-Papin et al., 1999,
Hörter and Dressmann, 1997; Serajuddin et al., 1988).
As a consequence, the bioaccessible fraction of ionizable
contaminants and metals present in ingested soil or
sediment is in general larger for mammals than for soil-
dwelling invertebrates, such as earthworms that regulate
gut pH at around neutral.
Another influence that may affect bioaccessibility is
the presence of food. The presence of food increases the
period during which mobilization can take place; this
may be important for compounds for which dissolution
is rate limiting. Also, an increased solubilizing capacity
of the digestive mixture, due to an augumented flow of
the digestive juices or to the presence of food particles,
may increase the mobilization of contaminants from
soil. Bile is known especially to increase the solubilizing
capacity for poorly water-soluble compounds, became
bile salts form micelles that have an apolar interior.
Furthermore, because bile salts have surfactant proper-
ties, they may increase the wetting and thereby the rate
of mobilization from soil (Oomen, 2000).
The focus of this article will be on methodologies for
monitoring bioavailable and bioaccessible fractions in
the environmental matrices of water, soil, and sediment.
Although the focus will be on metals, the bioavailability
and bioaccessibility of organic compounds will also
discussed. Generally, less information is available for
organic compounds than for metals. On the basis of the
equilibrium-partitioning concept (EqP) and a considera-
tion of the most important uptake routes of chemicals
for animals and plants, some general approaches toward
assessing bioavailable and bioaccessible fractions will be
discussed. These dynamic approaches include the
physicochemical and biological fundamentals of bioa-
vailability and bioaccessibility. Subsequently, a general
outline for monitoring is presented.
Bioassays, biomarkers, and biosensors integrate all
aspects of bioavailability and bioaccessibility (exposure,
accumulation, and toxic effects at the receptor level),
and hence they provide an obvious means for monitor-
ing truly bioavailable and bioaccessible fractions. A
large battery of bioassays and biosensors has been
developed. The issue of bioassays and biosensors is,
however, not within the scope of this contribution, and
the reader is referred to specific reviews on this topic.
Finally, it should be noted that monitoring bioavail-
able and bioaccessible fractions in itself is not an
endpoint of assessment; rather, the focus is often on
assessing adverse risks at species, community, popula-
tion, or ecosystem levels. Bioavailable and bioaccessible
fractions, as just defined, can directly be related to
adverse effects on the basis of the critical body burden
concept. A critical internal body burden is the threshold
concentration in the body, above which physiological
functions are irreversibly impaired. Lethal body con-
centrations (LBC) may be estimated from experiments
in which the increase of mortality with exposure time is
observed in conjunction with the concentration of the
metal in the body. Van Straalen (1996) discussed the
possibilities and limitations of estimating ecological
risks from internal residue data of persistent pollutants.
Van Straalen proposed that the residue LBC ratio,
averaged over a number of species, may be used to
assess the risks of toxicants at a certain site. The basic
assumption underlying the critical body burden concept
is that an animal or plant will be adversely affected at a
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W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
fixed body burden, independent of the means by which
that body burden is reached. Therefore, external matrix
effects are integrated in the concept. By dividing the
measured residue in a certain species in the field by the
internal threshold concentration for that particular
species, it is possible to assess in situ risks (Van Wensem
et al., 1994). However, the species with the highest
residue are not necessarily the ones most at risk—a
concept that is obviously related to the physiological
mechanisms underlying the storage and excretion of
metals.
1.2. Equilibrium partitioning—uptake of contaminants by
organisms and plants
The concept of equilibrium partitioning (EqP) is
generally used to describe the uptake of chemicals by
organisms and plants. The EqP concept can be used to
predict body concentrations. It contains the implicit
assumption that bioaccumulation is merely the conse-
quence of thermodynamic equilibrium between chemi-
cals present in pore water and in the body tissues of
plants or organisms. This equilibrium is supposed to be
established by passive diffusion, and any active regula-
tion of internal body concentrations will induce devia-
tions of EqP (see Fig. 1). In terms of monitoring levels of
bioavailable and bioaccessible contaminants in field
situations, the EqP concept implies that it would be
sufficient to monitor contaminant levels in the pore
water, and subsequently predict internal body concen-
trations of the contaminants on the basis of partition
coefficients and bioconcentration factors. For ‘‘soft-
bodied’’ organisms and for plants that do not actively
regulate fluxes of metals and organic compounds, the
EqP concept is able to explain contaminant accumula-
tion in laboratory situations (see, example, Van Gestel
and Ma, 1988; Jager, 1988). However, as indicated by
Belfroid et al. (1996), the general applicability of the
EqP-concept is questionable partly for the following
reasons:
(1) EqP does not consider food as a possible major
route of uptake.
(2) Although the EqP-concept has been shown to be
valid for ‘‘soft-bodied’’ organisms (especially earth-
worms), such evidence is lacking for ‘‘hard-bodied’’
species, such as insects.
(3) In field situations, contaminants are often less
available than expected on the basis of EqP. This
phenomenon is often referred to as aging.
(4) EqP assumes that equilibrium is established under
field conditions. Often, however, this basic require-
ment is not satisfied.
Uptake of chemicals involves the passage of com-
pounds across a biological membrane, mediated by a
carrier or a single solute. Compounds can enter tissues
65
Kp
SOLID
SOLIDPHASE
PHASE SOIL
SOILSOLUTION
SOLUTION
feeding BCF
ORGANISM
ORGANISM-- growth dilution
PLANT
PLANT metabolism
reproduction
Fig. 1. Graphical illustration of the equilibrium partitioning concept.
Kp Partition coefficient; BCF, bioconcentration factor.
through passive or facilitated diffusion, and by active
transport mechanisms. Passive diffusion is the primary
uptake process for many organic chemicals as well as
some metals and organometals. The driving force for
uptake is a fugacity difference between water and the
organism. Although some inorganic and organic metal
complexes may be taken up directly whereas other
ligands may compete with organisms for the metal, it is
the free metal ion that is supposed to be capable of
traversing biological membranes. As a consequence,
metal availability and toxicity are functions of water
chemistry because pore water chemistry (speciation)
determines the free metal ion activity. Formation of
inorganic and organic metal complexes and sorption of
metals to particulate matter have been shown to reduce
toxicity. As a result, the relationship of metal toxicity to
total or dissolved concentrations can be highly variable,
and depends on ambient water chemistry. It is the free
metal ion concentration that often provides a better
indication of availability and toxicity (Sunda and
Guillard, 1976; DiToro et al., 2001). Apart from
speciation, chemical interactions also determine the
partitioning of metals bound to soil surfaces, from the
solid phase to the solution phase. Important influence
include pH, inorganic ligands (e.g., Cl , HCO3 ),
dissolved organic matter, ionic strength (Alloway,
1995), and activities of competing cations such as
Ca2+. Studies with aquatic organisms (Campbell,
1995; Hare and Tessier, 1996), invertebrates (Kiewiet
and Ma, 1991), and plants in nutrient solutions
(Lexmond and Van der Vorm, 1981) revealed that
metal uptake is also influenced by competition of
protons and divalent macro-ions such as Ca2+. The
pH dependency of sorption processes at the soil or
sediment surfaces and at the biological membranes
determines the net effect of a shift in soil or sediment pH
(Plette et al., 1999; Oste et al., 2001). Morel (1983) and
Pagenkopf (1983) introduced models in which the
interactions of chemical species with organisms are used
to predict trace-metal uptake and toxicity. Most
commonly this approach is called the free ion activity
model (FIAM). The FIAM is widely used in aquatic
toxicology and is gaining popularity in studies of
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66 W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
soil–plant relationships (Parker et al., 1995), even
though some exceptions are known to exist (Campbell,
1995). Allen and Hansen (1996) showed how metal
speciation is expected to affect toxicity and, using
chemical equilibration calculations, predicted the range
of effects on copper toxicity resulting from the variation
of site-specific water quality characteristics. Recent
research has advanced the current level of understand-
ing of metal availability to aquatic life by means of
aqueous exposures, and the understanding how to relate
exposure levels to effects. Based on a conceptual model
similar to the gill-site interaction model proposed
by Pagenkopf (1983), biotic ligand models (BLMs) of
acute toxicity have been developed. The BLM considers
not only the effect of the dissolved metal concentrations
on toxicity, but also the metal interactions with organic
and inorganic ligands—interactions that affect metal
speciation and hence availability. Additionally, it
incorporates the competitive interactions of metals
and other cations with the organism at the site of
action of toxicity, the biotic ligand. To date, the BLM
has been used to predict the acute toxicity of copper
and silver to a limited number of aquatic organisms.
BLMs for chronic toxicity are still under development,
whereas for terrestrial plants and soil-dwelling
organisms the relationships between chemical forms of
metals and the bioavailability of the metals are not
sufficiently understood to permit the prediction of
metal toxicity.
The examples given conceal a typical fundamental
difference between exposure in aqueous systems and in
soils or sediments. Terrestrial plants, for example, may
well be exposed in aqueous nutrient solution. As
summarized by McLaughlin (2001), various data have
been reported from which either the validity of the
FIAM becomes clear, or from which additional
chemical species that are actually taken up may be
deduced. These species include complexes of metals and
chelating agents, natural organic ligands (humic materi-
als), or inorganic ligands such as chloride. Studies with
soil-grown plants are scarce, and it is often difficult to
identify the actual ionic species contributing to en-
hanced uptake. One possible explanation is that in the
terrestrial environment, the diffusion layer for plants
and soil-dwelling organism may be much thicker than
for aquatic organisms, because in addition to the
unstirred layer of solution surrounding the root or the
organism, the metal ion has to diffuse through the plant
apoplast and/or the organism mucigel. Additionally, the
soil itself exerts diffusion limitation to metal transport
due to the strong nature of metal binding to the solid
surface and the tortuous nature of soil pores (Nye and
Tinker, 1977; Barber, 1995). In soils and sediments, the
relationship between free metal activity and metal
uptake is therefore not as close a relationship as
previously assumed.
From the foregoing, it may be concluded that dermal
uptake can be described by the EqP theory (Shea, 1988),
presuming a direct relationship between the tissue
concentration and the free metal ion concentration or
the concentration of freely dissolved organic compounds
in pore water. The most notable exception to this
general mechanism is regulated uptake of nutrients and
essential elements. As illustrated in Fig. 2 for soil-
dwelling organisms (Oste, 2001), uptake of chemicals
through the digestive tract may be an important extra
auxillary route. Similar to the dermal route, digestive
uptake is assumed to be the consequence of passive
diffusion (Gobas et al., 1993a, b). Absorption of
chemicals through the digestive tract may result in
higher body concentrations than predicted on the basis
of EqP. This is because of an increase in bioaccessibility
as a consequence of passage through the digestive tract:
reduction of both sorption sites (fats and organic
material) and volume as a result of absorption of food.
The magnitude of the deviation of EqP depends on the
balance between the increase in bioaccessibility on the
one hand, and the rate of elimination via the feces and
other elimination routes on the other. Relatively many
data are available for earthworms, and recently a novel
model describing Eisenia andrei body concentrations for
Cd, Cu, Pb, and Zn as a function of pH, metals, and
soluble organic carbon (SOC) in soil extracts, was
developed and validated by Saxe et al. (2001). The
model quantifies earthworm body concentrations, using
metal partitioning at bulk soil pH and gut pH to
simulate dermal and gut exposure. It has been shown for
these organisms that reduction in the volume of food
during passage through the digestive tract is negligible
(Bolton and Phillipson, 1976; Piearce, 1972), whereas,
dependent on the earthworm species and the type of
food, the efficiency of digestion varies between 2%
(Bolton and Phillipson, 1976) and 40% (Morgan and
Morgan, 1992). For organic substances, this will
produce a maximum deviation of EqP of a factor of
Fig. 2. Schematic view of exposure pathways of metals to soil-dwelling
organisms, showing the dermal and digestive uptake routes.
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B1.5, although a smaller deviation is likely. Similar
information is lacking for most other organisms. To test
the general applicability of the EqP theory it will be
necessary to carryout similar studies focusing on
contaminant levels in food (like litter) and the efficiency
of digestion. It is unlikely that larger deviations from
EqP theory will be found, but additional food intake
may lead to faster equilibration of internal contaminant
levels, whereas ‘‘bound residues’’ may also be made
available (Verma and Pillai, 1991).
In addition to the soil- and organism-related influ-
ences, outlined earlier that control metal uptake,
organisms also respond to other environmental condi-
tions, and these responses may affect metal uptake or
metal toxicity. For example, plants will physically gain
access to more metal in soil when root growth rates are
high, so that temperature and nutrition may affect plant
metal uptake directly through effects on plant growth
(McLaughlin, 2001). Organisms and plants can con-
siderably modify the chemistry of the soil and soil
solution in the rhizosphere for instance (Marschner,
1995; McLaughlin et al., 1998). Among these diverse
effects are the following.
* Movement of additional contaminants to the rhizo-
sphere as a result of convective flow of solution to
plant roots.
* Organism- and plant-induced changes in solution
chemistry that affect sorption (such as changes of pH,
ionic strength, and macronutrient cation concentra-
tions).
* Excretion of organic ligands affecting total metal
concentrations in solution depending on the buffering
capacity of the soil solution.
* Generation of new sorbing surfaces for metals (for
instance by production of dead plant material);
* Stimulation of microbial activity.
The bioavailability of chemicals under field conditions
is often less than predicted by EqP considerations using
laboratory-derived data. For example, decreased bioa-
vailability over time is observed (‘‘aging’’, see, for
example Kelsey and Alexander, 1997). For PAHs it
has, for example, been postulated that part of the total
concentration is not available for uptake, nor can be
made available for EqP (McGroddy et al., 1996),
perhaps because it is strongly bound to soot particles
(Gustafsson et al., 1997). Such deviations from EqP
theory cannot be foreseen on the basis of compound or
soil properties. Even under laboratory conditions,
bioconcentration factors are often lower than expected
on the basis of EqP (Jager, 1998; Peijnenburg et al.,
2000). Limited mass transport from the solid to the
liquid phase (limited replenishment of the liquid phase
after uptake by organisms), combined with lowered
rates of diffusion toward the organisms are probably
67
responsible for a lack of true equilibration (Lord et al.,
1980; Briggs and Lord, 1983). This phenomenon is
generally more manifest in anaerobic sediments than in
soils.
Although the EqP has proved its validity for only a
limited number of species, it may be concluded that EqP
is a suitable methodology for assessing bioavailability of
chemicals from soils and sediments. The most pro-
nounced deviations from EqP (‘‘aging’’ and the lack of
true equilibration) will result in an overestimation of the
risks associated with the presence of chemicals. EqP
may, in this respect, be considered as a worst-case
estimate of the actual and potential risks for organisms
for which uptake via the pore water is the dominant
mode of uptake. Determination of actual pore water
concentrations and desorption kinetics (resupply), com-
bined with the assessment of accumulation patterns for
a suite of organisms, could further refine the risk
assessment. The EqP concept is, however, less suited
for predicting possible adverse effects for organisms
whose predominant exposure route is not the pore
water, or for (an exposure route) in which the
bioavailable fraction is not in equilibrium with the
metal fraction in the pore water. In these cases, EqP may
well lead to an underestimation of accumulation and
effects.
In summary, it may be concluded that the equili-
brium-partitioning concept provides a proper means of
operationalizing the definitions of bioavailability and
bioaccessibility. Each biotic species can be considered as
one of the phases present in the environmental
compartment under consideration, next to the particu-
late and the liquid phases—in other words, the ‘biotic
phase’. Equilibration processes are assumed to take
place among all phases present. The ‘‘biotic phase’’
consists of a variety of species, each with a characteristic
set of exposure routes; this is contrary to common views
that usually consider the biotic species to be a target
rather than a participating phase. The biotic phase,
however, has some specific characteristics that require
special attention. These specific characteristics include,
the generally relatively long equilibration period as
compared with those for most physicochemical pro-
cesses, the concentration-dependent occurrence of toxic
responses, and the role of some equilibria in determining
toxicant uptake (e.g. pore water-exposed organisms
versus those exposed through both the pore water and
the particulate phase).
Although bioavailability and bioaccessibility are
dependent on the metal, the biological species consid-
ered, and the environmental compartment in which the
organism or plant resides, and, although some organ-
isms and plants are capable of actively regulating their
metal fluxes, two major routes of uptake may be
distinguished. The challenge of monitoring bioavailable
and bioaccessible metal fractions therefore ultimately
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68 W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
links metal chemistry in the solid phase and in solution,
to uptake by biotic species.
2. Towards a monitoring strategy for assessing available
and accessible fractions of metals
2.1. General considerations
Any monitoring approach should include the essential
features that dictate metal availability, in other words,
the determinants of metal partitioning and the effect on
biota of such partitioning. Because there is no single
bioavailable or bioaccessible fraction, the terms ‘‘poten-
tially’’ and ‘‘actually available’’ fractions have been
introduced to include the aspect of time in the
assessment of the bioavailable fraction. Given the
species, metal, and matrix dependence of bioavailability
and bioaccessibility, any monitoring strategy to be
developed should be oriented to at providing estimates
of both the potentially and actually available or
accessible fractions. What follows is an overview, the
physicochemical methodologies that are currently avail-
able for application within monitoring strategies for
assessing potentially and actually available and acces-
sible fractions, and approaches to link the physicochem-
ical methodologies to biological uptake. A proposal is
made for a general monitoring strategy.
2.2. Physicochemical tools applicable within monitoring
strategies
As indicated earlier, the dynamic approach of
‘‘bioavailability’’ and ‘‘bioaccessibility’’ should comprise
two distinct phases:
1. A physicochemically driven phase in which partition-
ing and equilibration processes determine the magni-
tude of potentially and actually available metal
fractions, in other words, chemical availability.
2. A physiologically driven uptake phase in which
chemically available metal fractions, dependent on
organism-specific, metal-specific, and environment-
specific conditions, are accumulated by organisms or
plants, in other words, biological availability, which
may subsequently lead to adverse effects on the
organism.
The nature of actually available and accessible
fractions may differ strongly, ranging from the free-
metal activity in solution to the total metal content in
the solid phase of the soil or sediment under considera-
tion. This is the reason for developing a variety of
methods and methodologies to link available and
accessible fractions to labile and nonlabile metal pools.
In most cases an empirical approach is taken, with the
most notable exception being the BLMs, which were
developed for aqueous organisms exposed by means of
the gills, as discussed earlier. Most empirical approaches
start with either single or sequential extraction techni-
ques or digestive methods, and subsequently attempt to
correlate results of extractions or digestions to either
biological uptake or effect data.
Upon release into the aquatic environment, metals
may undergo several fate-determining chemical and
physical processes. Chemical processes are ion exchange,
adsorption, precipitation, dissolution, volatilization,
and complexation.
* Ion exchange is the process in which one positively
charged cation is exchanged for another at a constant
charged surface. Direct exchange at the particulate
surface is an important process for the removal of
trace-metal cations from aquatic systems. Clay
minerals are important natural ion exchange materi-
als. In surficial soils, the clay minerals are
generally coated with metal oxides (Jenne, 1968)
and organic matter (Hart, 1982; Davis, 1984).
These coating substances provide the surface char-
acteristics that are important in the exchange of trace
metals.
* Sorptive phases include clay minerals, iron and
manganese (hydroxy)oxides, and organic matter.
Metal adsorption by soil is highly pH dependent as
a result of the surface chemistry of solid materials.
Soils and sediments have a pH-dependent, or vari-
able, charge associated with the reaction of protons
with oxide and hydroxide minerals, and with certain
functional groups of humic substances (Evans, 1989;
Sposito, 1984).
* Precipitation occurs when the solubility product, Ks ;
for a reaction between a metal ion and ligand in the
system, is exceeded. Some ligands that are important
for the precipitation of metal ions include carbonate,
hydroxide, silicate, phosphate, and (in anoxic envir-
onments) sulfide. In some cases, associated with very
low values of Ks ; precipitation processes fully
determine the activity, and therefore the environ-
mental availability, of the heavy metals in the
aqueous phase. From a thermodynamic point of
view, the most stable precipitates, in other words,
those having the lowest solubility product, will
predominantly be formed. Kinetic restrictions may
in practice however, prevent the most stable pre-
cipitates from being formed. These restrictions may
be related to limited diffusion or dispersion.
* Metals may form complexes with DOC and with
inorganic species such as carbonate, chloride, sulfate,
hydroxide. DOC usually consists of small organic
molecules (e.g. amino acids and sugars) or bigger
fulvic and humic acid molecules. Additionally, strong
complexes may be formed with chelating agents like
EDTA and NTA. Complex formation is strongly
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W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
metal specific. Whereas for instance a large fraction
of copper present in water is usually bound to DOC
(and therefore not available for dermal uptake),
cadmium forms only weak complexes with organic
matter, and the increases of DOC will only slightly
affect cadmium availability.
The chemical processes of ion exchange, adsorption,
precipitation, diffusion, volatilization and complexation
determine the physicochemical form or chemical specia-
tion, of metals in solution. The impact of these processes
on the activities of the free metal ion in solution further
depends on the composition of the aquatic matrix, and
jointly they determine the concentration of the metal ion
in solution. The master variable affecting metal specia-
tion is usually pH. Additionally, ionic strength and
redox conditions are of major importance but the effect
of these parameters is often not straightforward. For
instance, metals tend to be more soluble as pH
decreases. Solubility of metals may, however, increase
at higher pH because of binding with DOC (Allen and
Yin, 1996), because the solubility of organic matter
increases along with pH. As a consequence, it is
necessary that the techniques selected for monitoring
purposes take explicitly into account the potential
impact of pH as well as the other dominant matrix-
related properties affecting metal partitioning. Extrac-
tion techniques may, for example, be carried out at the
pH of the water, soil, or sediment, or at a preset and
fixed pH. Therefore, the pH selected will, in a
quantitative sense, greatly influence the outcome. The
various metal fractions that are present in water are
depicted schematically in Fig. 3.
Physical processes include leaching and uptake by soil
organisms and are closely related to the chemical fate of
the metal. If a metal is adsorbed strongly onto a soil
adsorption phase or onto part of a stable precipitate, the
metal will not be available (or it will have a reduced
availability) to participate in leaching processes or for
uptake by organisms. It is the chemical speciation of
TOTAL METAL
INERT
CONTENT
(MG/L OR FRACTION
MG/KG)
SORBED /
PRECIPITATED
DISSOLVED IN ORGANIC
(PORE) WATER COMPLEXES
INORGANIC
COMPLEXES
FREE METAL
Fig. 3. A simplified schematic overview of the various metal fractions
present in soil, sediment, and water matrices.
69
metals that determines whether they are available for
physical processes. The relationship between metal
speciation and the effect of metals on organisms makes
it an important subject for analysis. Various methodol-
ogies for determining metal speciation in natural
systems have been developed. Attempts at experimental
quantification of metal speciation in natural systems are
hampered by the tendency for any sample manipulation
to lead to a shift in equilibrium with a concomitant
change in speciation. This is obvious in the case of
fractionation processes but somewhat more subtle in the
case of electrochemical methods Amperometric strip-
ping voltammetry (ASV) and polarography exemplify
dynamic techniques that cannot possibly measure the
‘natural’ speciation of a trace element in a water sample,
because the measurement itself disturbs the equilibrium.
All electrochemical speciation results are therefore
operationally defined.
Methods developed for measuring bioavailable and
bioaccessible fractions can be grouped in three cate-
gories: (1) methods for assessing metals in pore waters,
including assessment of metal speciation and the activity
of the free metal ion; (2) single and sequential
extractions; (3) rigorous digestion procedures to deter-
mine total metal concentrations in soils or sediments.
Methods for assessing metals in pore water may
provide an estimate of the actually available and
accessible fraction, whereas extractions and digestions
may provide estimates of potentially available and
accessible fractions.
2.2.1. Assessment in pore waters
Sampling and pretreatment of water samples may at
first glance seem obvious activities. Nevertheless, even in
the case of surface water samples, the (empirical)
expressions obtained for the bioavailable and bioacces-
sible fractions strongly depend on the way the samples
are handled. A large fraction of the metal is associated
with the dissolved organic matter present in the sample.
The size of DOC strongly varies, and filtration of the
sample will especially have a profound effect on the
apparent DOC concentration. Usually a 0.45-mm filter is
selected as the basis for the determination of the total
metal concentration and the bioavailable and bioacces-
sible fractions in water samples.
Soil and sediment solutions are even less defined, are
not impervious to methodological bias, and are, there-
fore for the most part operationally defined. In situ
samples are preferred, because physicochemical and
biological transformation during sampling, storage, and
any further handling of the sample will affect its quality.
This is especially true for anaerobic samples containing
an excess of acid volatile sulfide (AVS). Because AVS
very effectively binds metals and is unstable under
aerobic conditions (oxidation of sulfides and subsequent
release of precipitated metals), it is essential that
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70 W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
anaerobic conditions be maintained during any handling
of anaerobic samples that are used for the determination
of bioavailable or bioaccessible fractions. Furthermore,
the field installations required for obtaining in situ
samples are very resource intensive and impractical from
most bioavailability testing perspectives. Nevertheless,
at some stage field research is needed to validate other
(empirically based) soil extraction procedures aimed at
mimicking ‘‘real’’ soil or sediment solutions (Sauvé,
2001).
Another means of obtaining representative soil or
sediment samples is by centrifugation. This is a
laboratory-intensive procedure and the concentrations
obtained depend on the g force used and the moisture
content of the sample. Simple batch-type extractions
provide a third means of obtaining soil and sediment
solutions. Albeit these extractions basically are part of
the extraction procedures discussed later, in this case the
soil solution is assumed to be represented by the solution
obtained from shaking of either distilled-deionized
water or a dilute salt soil extraction using a small
solid/solution ratio (p1:2). This method allows for the
collection of reasonable sample volumes with a mini-
mum of laboratory manipulations. However, the ab-
sence of counter ions in pure water not only leads to
metal-specific desorption but also affects the solubility
of organic matter as well as of the complexed metal
associated with DOC. Therefore, weak salt extractions
may prove to be better surrogates of soil solutions than
pure water.
Two approaches exist for determining metal specia-
tion in solution: analytical determination and chemical
equilibrium models. As a matter of course, computer
models are based on experimentally derived parameters,
and models cannot be any better than the assumptions
and data on which they are based. Computational
procedures, based on thermodynamic principles, allow
the equilibrium speciation of a system to be calculated
once total component concentrations are known. How-
ever, this approach also presents problems which related
to the reliability of equilibrium constants (or free
energies) employed, the description of the complexation
behavior of naturally occurring organic ligands, and the
description of the adsorption behavior of naturally
occurring adsorption phases. The existence of databases
of critically evaluated equilibrium constants mitigates
the first problem. The efficacy of speciation programs
depends inherently on the inclusion of accurate equili-
brium constants for the species in question. These
equilibrium constants vary depending on their source.
For the complexation behavior of naturally occurring
organic ligands and the adsorption behavior of hydro-
xides and oxides, several modeling approaches have
been advanced (see, for example, Evans, 1989). GEO-
CHEM (Sposito and Mattigod, 1980) and MINEQL
(Westall et al., 1976) are examples of speciation models.
The Windermere Humic Aqueous Model (WHAM) is a
chemical equilibrium model that incorporates discrete
site binding of elements by humic substances with
models for inorganic solution chemistry, precipitation
reactions of oxides, clay binding, and sorption/deso-
rption reactions by fulvic acids (Tipping, 1994). The
NICA–Donnan model, which assumes a continuous
distribution of binding sites, has described metal ion
binding to organic matter. The model is a combination
of the non-Ideal competitive adsorption Model (Bene-
detti et al., 1995), which describes more specific
adsorption on humic substances, and a Donnan
equilibrium model that describes nonspecific adsorption
(Kinniburgh et al., 1996). As indicated by Christensen
et al. (1998), the NICA–Donnan database is limited and
should be expanded.
Various techniques can be used to measure free-metal
activity in solution. The most direct method for
determining free metal species is possibly through ion-
selective electrodes (ISEs), which allow for a direct
measurement of free ions. Furthermore, the manipula-
tions are simple, being similar to pH measurements. In
the case of Cu, the electrode is not prone to excessive
ionic interference, except perhaps high chloride concen-
trations. Cu-ISEs have been used to determine soil
solution activities down to 10 12 M Cu2+ (Sauvé et al.,
1995), and in pure synthetic buffered solutions activities
as low as 10 19 M Cu2+ have been measured (Hansen
et al., 1972). Although other ISEs exist for Cd and Pb,
these ISEs are much less sensitive and will not yield
reliable data for low concentrations of these metals. Free
metal concentrations are commonly well below 10 7 M,
whereas detection limits are B10 4 M.
Voltammetric methods such as differential-pulse
polarography (DPP) and differential-pulse anodic strip-
ping voltammetry (DPASV) are sensitive electrochemi-
cal methods and can be used to determine the
concentrations of ‘‘labile’’ metal. Some studies have
shown that the presence of inorganic or organic colloids
does not interfere with the ASV measurements, but it is
reported that the adsorption of organic matter on the
mercury-drop electrode may hinder metal diffusion and
thus diminish the current readings. Measurements in
solutions containing dissolved organic matter therefore
require the use of calibration solutions with properties
similar to those of the samples used.
Exchange resins are also used to measure the free
metal activity or the relative lability of metals (Beveridge
et al., 1989). Donnan dialysis membranes can also be
used to determine the speciation of solutions. Minnich
and McBride (1987) have, for instance, used this
technique to determine the free-Cu2+ activity in Cu
salts and sewage sludge-amended soils. Recently,
Temminghoff et al. (2000) developed the Wageningen–
Donnan Membrane Technique for directly measuring
metal activities. A separation technique, via Donnan
 ARTICLE IN PRESS
W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
equilibrium across a negatively charged ion-exchange
membrane, was adapted for this purpose. The mem-
brane separates a salt acceptor solution and will
exchange cations in the donor solution (i.e., the solution
in which activities are to be determined) until an
equilibrium is reached between donor and acceptor
solutions. The membrane restricts anion transport, and
at equilibrium the solutions will contain equal amounts
of free ion. Diffusive gradients in thin films (DGT) have
been used to assess bioavailable forms of metals. The
technique utilizes a gel layer to remove ions from
solution, establishing a gradient. A resin at the base of
the gel arrests the diffusion of ions. The DGT technique
is supposed to allow free diffusion of labile species of
metals (complexed or not; Zhang et al., 1998; Davison
et al., 2000). Finally, information on metal speciation
may be gained by separation and/or exchange methods.
For instance, Gregson and Alloway (1984) used gel
permeation chromatography to separate species of Pb
on the basis of molecular weight.
A comparison of measurement techniques for specia-
tion revealed that using synthetic solutions for which the
experimental results could be compared with chemical
equilibrium calculations, ISEs, and ASV gave good
results (McGrath et al., 1986). However, several other
studies showed that some methods give overestimates of
metal lability (Sauvé, 2001). This author concluded that
it is critical to properly compare the performance of
various free ion activity speciation techniques under
identical conditions.
2.2.2. Extractions
Extractions represent an intermediate between ‘‘total’’
digestions and pore water samples. Research concerning
metal behavior in soils and sediments has focused
mainly on the use of various chemical extractions to
describe forms of metal present. An excellent review by
Beckett (1989) described many of these extraction
procedures. Many of the earliest procedures dealt with
metal deficiencies rather than metal contamination.
Grossly speaking, six types of extractions may be
identified:
* (Weak) salt extractions (e.g., CaCl2, Ca(NO3)2,
NH4Ac, NaNO3, Mg salts, BaCl2, in concentrations
from as low as 0.001 M and up to 1 M salt solutions).
* Reductive extractions (e.g., sodium ascorbate, hydro-
xylamine HCl, sodium dithionite).
* Weak acid extractions (e.g., acetic acid, citric acid).
* Strong complexation methods (e.g., DTPA-TEA,
EDTA, NTA).
* Dilute strong acids (e.g., HNO3, HCl, ‘‘double acid’’
(HCl+H2SO4))
* Combined extractants (e.g., Ammonium oxalate–
oxalic acid, Mehlich III (dilute acid, salt, and
EDTA)).
71
Not all methods are useful to study all heavy metals.
For instance, reductive methods are for instance only
useful to study metals that can be reduced, such as iron
and manganese. Additionally, many methods have been
developed with just one element in mind: the ‘‘double
acid’’ extraction has been developed primarily for the
study of zinc availability.
Many extractions are interpreted in several ways. One
common hypothesis is that when some form of
extractant dissolves an adsorbent, the associated metals
are released into solution and can be identified by
analyzing the solution composition. This hypothesis
neglects the relocation of the metal over the remaining
sorption sites. An extraction that, for instance is capable
of destroying the manganese oxides, (such as hydro-
xylamine-HCl), does not give any information about the
amount of copper bound to the manganese oxides as
long as organic matter is present. Almost all of the
copper associated with manganese oxide will immedi-
ately readsorb onto the organic matter of the soil.
The concept of using a chemical extraction to assess
the form of metal was advanced by the development of
sequential extraction procedures. The most common of
which is from Tessier et al. (1979). By using a series of
different chemical extractions increasing in strength,
metals can be classified into certain forms based on their
concentrations in the particular extract, which would
ideally allow the identification of the solid phase to
which the metals are associated. However, an exhaustive
review of sequential fractionation methods is not
warranted because the reagents used cannot truly
identify the suggested metal pools. At best, sequential
extractions identify operationally defined fractions and
none can be consistently related to metal availability or
accessibility. Although it would be important to
standardize extraction protocols, it is important to
realize that specific research needs vary. The ideal
extraction reagents will differ according to the specific
needs. Differences in results for several extractions will
depend partially on soil-type variation used in these
studies. An acid extractant that assesses the ‘‘active’’
form of metal in an organic Canadian soil (Levesque
and Mathur, 1988) may not similarly assess the MgCl2
‘‘exchangeable’’ form in sandy Polish soils (Chlopecka
et al., 1996). From a biological point of view, a certain
extractant may provide a good estimate of metal uptake
by a certain species, whereas another extractant may
provide a better estimate for a different species.
2.2.3. Digestion
The most common approaches to total soil metal
estimates are aimed at determining total recoverable
metal. Many total metal digestions involve hydrofluoric
acid additions to the digestate to break down Si crystal
lattices and extract metal bound or trapped in these
crystal structures. Totally recoverable metal digestions
 ARTICLE IN PRESS
72 W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
are typically designed to stop short of destroying the
crystal structures. Soil digestions typically also employ
elevated temperatures. In recent years microwave
digestion methods have gained popularity because of
increased safety and shorter digestion times. Three US
EPA microwave digestion procedures exist: HNO3,
HNO3-HCl, and HNO3-HCl-HF, (US EPA, 1997).
USEPA states that the first two procedures are designed
to extract metals that can potentially become envir-
onmentally available.
It is important to note that soil metal estimates do not
reflect the totality of the metals present; such a
discrepancy is especially pertinent under low back-
ground concentrations (Sauvé, 2001). The results of
method comparison are also somewhat inconsistent.
Comparing microwave-assisted HNO3 digestion to
hotplate reflux digestion (using an HNO3-HCl-H2O2
reagent) showed almost complete metal-specific biases
(Wei et al., 1997). Because the degree of contamination
is often unknown for environmental purposes, the
harshest method is the most reliable for assessing
residual and true total metals in polluted matrices.
From an ecotoxicological perspective and irrespective of
the methodological details, however, the metal fraction
that is not dissolved in concentrated boiling acid has a
very low bioavailability and bioaccessibility and its
potential toxic contribution correspondingly is very low.
Environmental conditions may, as previously noted,
strongly affect metal binding. Anaerobic soils and
sediments in which an excess of sulfide is present
provide a special case in this respect. Stable metal
sulfides may precipitate under anaerobic conditions.
Formation of metal sulfides will, as a consequence of
their very low solubility products, strongly reduce free
metal activities in solution. Operationally defined
extraction methods are available for determining the
amount of metal bound to sulfides. The general
methodology is extraction with an excess of concen-
trated HCl to liberate the AVS. Hence, the method of
measuring AVS is operationally defined. A measure of
potentially toxic metals in anaerobic sediments is the
amount of metals released during determination of
AVS, which is referred to as simultaneously extracted
metal (SEM). Acute adverse effects are assumed to be
absent so long as AVS exceeds SEM. The AVS-
approach was initially proposed to better assess the
risks for metals in anaerobic sediments; recently it was
slightly modified to include the binding properties of
organic carbon present in the soil or sediment (DiToro
et al., 2000).
2.3. Approaches taken toward assessing bioavailable and
bioaccessible fractions
Even though the relationship between speciation and
toxicity has been illustrated, the identities of the metal
species responsible for the toxic effect are not equivo-
cally known in all cases (Luoma and Davis, 1983).
Nevertheless, the most obvious approach to assessing
available and accessible metal fractions is to relate the
activity of the free metal ion in the water or the pore
water to total uptake. This approach has been success-
fully applied to aquatic organisms. Studies with this
kind of approach are scarce for the soil and the sediment
compartments, because attempts to quantify metal
speciation in natural systems usually lead to a shift in
equilibrium with a concomitant change in speciation
(see earlier). Also technical investments are large,
whereas the techniques are presently not feasible for
large-scale use (Ure and Davidson, 1995). One of the
weaknesses of predicting metal bioavailability using
free-metal activities in solution is the lack of considera-
tion of the dynamic processes responsible for maintain-
ing the free-metal activity, as well as a somewhat
simplified view of biological uptake (Parker and Pedler,
1997). Elemental uptake is controlled by chemical
availability in solution as well as the capacity of the
soil, sediment, or labile fractions present in solution to
supply that element. Most plants and biota will
accumulate many times the amount of metal available
in solution at any given moment. In effect the soil or
sediment solution is emptied and replenished. Metal
uptake is, thus dependent not only on the availability of
the metal in solution (intensity factor) and the uptake
mechanisms, but also on the capacity of the soil or the
sediment solid phases to supply the fluxes of that
particular element (capacity factor). Understanding
bioavailability for species exposed through the pore
water requires a consideration of both aspects: the
intensity of exposure through the FIAM and the
capacity of the soil or the sediment to maintain this
level of free-metal activity in solution (by means of the
techniques discussed later). As a consequence of daily
practice, for example for risk assessment, the approach
in which the activity of the free metal ion in the pore
water is related to total uptake, can on a regular basis
only be applied for the aqueous compartment. At
present, several programs directed at incorporating
further refinements into the BLMs for copper and silver
are in progress to extend the application of these BLMs
to other types of organisms, thus making the BLMs
more suitable for use in the development is so far as of
water quality criteria. Application of the BLM frame-
work to other types of organisms and to other metals, in
both freshwater and marine settings, is in the early stage
of development in so far as studies of the BLM for
cadmium and zinc have begun, whereas efforts for nickel
and aluminum are in the planning stage (DiToro et al.,
2001). An recent approach for measuring effective soil
solution concentrations and metal supplied from the
solid phase is the aforementioned technique of DGT.
This technique of surrogate chemical measurement, first
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W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
reported by Davison and Zhang (1994), has promise as a
quantitative measure of the effective bioavailable metal
for plants, because measured DGT fluxes of Cu relate
well to Cu uptake by Lepidium heterophyllum (Zhang
et al., 2001).
Another second general approach to relating available
and bioavailable fractions to uptake is by means of
multifactorial analyses. In this approach, accumulation
characteristics such as uptake-rate constants, steady-
state concentrations, bioconcentration factors, and
biota to soil (or sediment) accumulation factors are
statistically associated with water, soil, or sediment
characteristics, such as the total metal concentration,
rapidly or slowly desorbing contaminant fractions, pH,
organic matter content, DOC content, and the cation
exchange capacity (CEC). Observations are typically
analyzed by multiple regression analyses, to weigh the
impact of these characteristics against each other (e.g.,
Ma, 1982; Peijnenburg et al., 1999b; Vijver et al., 2001).
It should be noted that this general approach does not
necessarily provide any insight into mechanistic influ-
ences of any of the variables mentioned on metal uptake
by plants or organisms. Nevertheless, such approaches
allow the elimination uptake routes that do not
contribute significantly. Thus, the importance of the
pore water for uptake of chemicals was demonstrated
for oligochaete species (L^kke and Van Gestel, 1998;
Peijnenburg et al., 1999a, b), whereas Vijver et al. (2001)
demonstrated the nonadherence of Folsomia candida to
the pore water hypothesis on the basis of a multivariate
statistical approach. The amount of metal taken up by
this springtail was correlated best to the total metal
concentration in the soil. This might result from metal
uptake via food, directly from the soil solid phase, or
from one or more of the soil constituents. Comparable
relationships were reported for some sediment-dwelling
organisms, and Lee et al. (2000) and Griscom et al.
(2000) showed that dietary uptake may be an important
route for some sediment-dwelling organisms. Unlike in
soils and aqueous systems, reducing conditions often
prevail in sediments. Sediment-dwelling species often
have adapted their physiology to optimize their oxygen
fluxes. This adaptation usually affects metal uptake and
uptake routes, and often hinders the assessment of the
dominant routes of exposure by means of a multivariate
approach.
Instead of total soil concentrations, operationally
defined extraction techniques have been used with the
aim of assessing the pool of solid-phase metal that
buffers the solution metal concentration, thus mimick-
ing the concentrations of labile metal that are available
for uptake by biota or plants, and accounting for the
supply-rate term noted earlier (capacity factor). Exam-
ples include the use of aqueous calcium chloride
solutions to mimic metal uptake by plants (e.g., Gupta
and Aten, 1993), or by earthworms (e.g., Gonzalez et al.,
73
1994), and the relationship reported by Buykx et al.
(1999) between chelex-extractable Cu and metabolic
inhibition of periphyton communities. The latter study
shows that chelex-extractable Cu is an adequate
chemical parameter to describe this biological effect of
copper. In such cases, the influence of water, soil, or
sediment characteristics that affect uptake is measured
directly as a net effect on extractability. The extraction
techniques employed include both sequential extractions
with different media (usually in a series of increasing
extraction efficiency) and pore water collection. Various
successful examples of both approaches have been
published. However, there are often large discrepancies
among extraction techniques with regard to simulating
bioavailable and bioaccessible fractions. Whereas one
specific extraction technique may provide a good
indication of the bioavailable or bioaccessible fraction
for a specific species or plant, there may be no
correlation for other species or plants, using the same
extraction technique. Also, when applied to soils with a
broad spectrum of metal concentrations or soil char-
acteristics, they often fail to predict adequately metal
availability or accessibility. Therefore, it is clear that
there is no single expression for bioavailable or
bioaccessible fractions. Typically, extractions with metal
contents that correlate best with plant metal concentra-
tions are viewed as the best extractions for describing
bioavailability (Brown et al., 1971).
Another condition that needs to be taken into account
in this respect is reported by Korcak and Fanning
(1985), who determined the uptake of zinc and
cadmium, applied as metal salts, by plants grown on
12 different soils. They compared the uptake from these
artificially metal-loaded soils to the uptake from soils
with equivalent concentrations of metals added with
sewage sludge. In the metal salt treated soils, an
increasing CEC correlated with decreased tissue zinc
and cadmium levels. However, in the sludge-treated soil
the zinc and cadmium concentrations in the tissue
increased in direct proportion to the CECs demonstrat-
ing that linking environmental availability and environ-
mental bioavailability may not always be successfully
approached through studies with laboratory-contami-
nated soil. The use of field soils may be crucial to
evaluate the general validity of observational findings
for the field situation.
Extraction procedures have also been applied to
explain differences in metal availability between field
soils from a contaminated field site with artificial soil
substrate amended with metal salts, in this case without
specifying the apparently controlling soil characteristics.
Toxicity of metals for the earthworm Eisenia andrei, for
example, differed strongly between field and artificial
soils when expressed as total metal concentrations
(Spurgeon et al., 1994; Spurgeon and Hopkin, 1995;
Posthuma and Notenboom, 1996). However, when
 ARTICLE IN PRESS
74 W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
exposure was expressed as calcium chloride- or water
extractable metal concentrations, the concentration
response curves were similar in soils with various metal
origin and different soil characteristics. The same
similarity in response was found for an enchytraeid
species (Eracidae crypticus), a springtail (Folsomia
candida), and with various plant species (Notenboom
and Posthuma, 1996). These data support the hypothesis
of uptake through the pore water (or a compartment
with similar equilibration characteristics) of these
taxonomically different species, although the evidence
is circumstantial rather than decisive and mechanisti-
cally clear.
2.4. Toward a monitoring strategy for assessing
bioavailable and bioaccessible metals
These findings illustrate that the consideration of an
EqP approach as the basis for monitoring is an overly
simple approximation of reality. A plethora of simula-
tion approaches for bioavailability and bioaccessibility
are currently available, and validation of the chemical
techniques by biotic uptake and toxicity experiments
yields variable results. Whereas the paradigm of the free
metal ion in solution as being the available species was
confirmed for some organisms or plants (mostly
aquatic), other organisms or plants do not adhere to
the FIAM. Instead, experiments with these biota
validate the suitability of multivariate approaches and
the numerous of extraction techniques developed so far.
Although there is a large variance in species-specific,
metal-specific, and compartment-specific bioavailable
and bioaccessible fractions, determination of the ‘‘most
available or accessible’’ metal pools (though not at all
perfect) will better predict risks than total metal
concentrations or metals removed by strong extractants.
However, one should be aware that especially indices of
actual metal availability or accessibility can change over
time. Such transformations may be due to long-term
changes in such parameters as pH, redox conditions,
organic matter content. Indices of potential availability
or accessibility will respond less, and therefore any
monitoring strategy should take potential available
metal fractions as a starting point. Total metal
concentrations or metal removed by strong extractants
provide a worst-case estimate of possible long-term
changes and should therefore be taken as a starting
point for monitoring, irrespective of the goal of
monitoring. Before designing a monitoring strategy, it
is absolutely critical to have clarity of the purpose of the
monitoring program. Issues to be considered include the
composition of the ecosystem of interest in terms of
environmental properties (e.g., pH and redox condi-
tion), the endpoint of determination (ecosystems versus
specific populations, species, or even individual organ-
isms or plants), the relevant uptake and exposure routes,
< limit
TOTAL METAL
CONTENT
Stop
> limit
PHYSICO CHEMICAL < limit
MATRIX PROPERTIES Stop
> limit
ANALYSIS OTHER ASSAYS
EXTRACTION PORE WATER, LIKE:
MODELLING SEM-AVS, DGT
SPECIATION
Fig. 4. General outline for a monitoring strategy. SEM-AVS,
Simultaneously extracted metal–acid volatile sulfide; DGT, diffusion
gradients in thin film.
and the metals of interest. In common practice such
information will often be lacking, and instead of
focusing on actually available or accessible metal
fractions, the focus emphasis will often be on potentially
available or accessible metal pools. A general monitor-
ing strategy is depicted in Fig. 4. Depending on the
preset endpoint of determination and the critical total
metal levels related to this endpoint, the first step of the
monitoring strategy provides a worst-case approach in
which it is to be deduced whether a worst-case estimate
of the potentially available and accessible metal pool is
likely to be indicative of adverse effects. If so, then it is
to be determined in the next step whether the composi-
tion of the matrix of interest in terms of properties
affecting potential availability or accessibility will
sufficiently reduce the potential adverse impact of the
total metal pool. If this is not the case, then a large
battery of experimental techniques for assessing the
various metal pools may be applied. Depending on the
endpoint under consideration and the metal, matrix-
and species-specific characteristics identified earlier, the
proper set of measurement techniques is to be selected to
make sure that site-specific impacts are adequately taken
into account. The measurement techniques of choice
range from speciation modeling through determination
of SEM-AVS. The scheme depicted in Fig. 4 clearly
depicts that there is no single expression for either the
bioavailable or the bioaccessible fraction.
3. Conclusions
Metal bioavailability and bioaccessibility are complex
phenomena that are dependent on a cascade of matrix-
related, species-related, and metal-related issues. In turn,
the properties affecting bioavailability and bioaccessi-
bility are often interrelated and, as a consequence, the
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W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
effect of these properties is often not straightforward. In
practice, most operationally defined measures of bioa-
vailability or bioaccessibility have limited applicability
among a wide range of matrices, organisms, plants, or
metals. For monitoring purposes, it is therefore neces-
sary to consider adequately the relevant endpoints of
determination. Subsequently, it is possible to select the
most appropriate techniques among the many of
methods so far developed. It is essential that the
methods of choice incorporate expressions of both the
potentially and actually available metal fractions.
Because there is no universal expression of bioavail-
able and bioaccessible metal fractions, it is not possible
at present to develop a detailed monitoring strategy that
is broadly applicable. Clearly, the determinants of
bioavailability and bioaccessibility in a specific situation
must be better understood before one is to monitor or,
ultimately, predict the effects of metals.
References
Allen, H.E., Hansen, D.L., 1996. Water Environ. Res. 68, 42–54.
Allen, H.E., Yin, Y., 1996. The importance of organic matter on the
sorption of cadmium and mercury to soil. The 6th International
Conference on Preservation of Our World in the Wake of Change,
Jerusalem, Israel.
Alloway, B.J., 1995. Heavy Metals in Soils, 2nd Edition. Blackie,
London, UK.
Barber, S.A., 1995. Soil Nutrient Bioavailability. A Mechanistic
Approach. Wiley, New York.
Beckett, P.H.T., 1989. The use of extractants in studies on trace metals
in soils, sewage sludges, and sludge-treated soils. Adv. Soil. Sci. 9,
143–176.
Belfroid, A.C., Sijm, D.T.H.M., Van Gestel, C.A.M., 1996. Environ.
Rev. 4, 276–299.
Benedetti, M.F., Milne, C.J., Kinniburgh, D.G., Van Riemsdijk,
W.H., Koopal, L.H., 1995. Metal-ion binding to humic sub-
stances—Application of the non-Ideal Competitive Adsorption
Model. Environ. Sci. Technol. 29, 446–457.
Beveridge, A., Waller, P., Pickering, W.F., 1989. Evaluation of ‘labile’
metal in sediments using ion exchange resins. Talanta 36, 535–542.
Bolton, P.J., Phillipson, J., 1976. Oecologia 23, 225–245.
Briggs, G.G., Lord, K.A., 1983. Pest Sci. 14, 412–416.
Brown, A.L., Quick, J., Eddings, J.L., 1971. A comparison
of analytical methods for soil zinc. Soil Sci. Am. Proc. 35,
105–107.
Buykx, S.E.J., Cleven, R.F.M.J., Hoegee-Wehmann, A.A., Van den
Hoop, M.A.G.T., 1999. Trace metal speciation in European River
waters. Fresenius J. Anal. Chem. 363, 599–602.
Campbell, P.G.C., 1995. Interactions between trace metals and aquatic
organisms: a critique of the free ion activity model. In: Tessier, A.,
Turner, D.R. (Eds.), Metal Speciation and Bioavailability in
Aquatic Systems. IUPAC Series on Analytical and Physical
Chemistry. Wiley, New York, pp. 45–102.
Castela-Papin, N., Cai, S., Vatier, J., Keller, F., Souleau, C.H.,
Farinotti, R., 1999. Drug interaction with diosmectite: a study
using the artificial stomach-duodenum model. Int. J. Pharm. 182,
111–119.
Chlopecka, A., Bacon, J.R., Wilson, M.J., Kay, J., 1996. Forms of
cadmium, lead, and zinc in contaminated soils from Southwest
Poland. J. Environ. Qual. 27, 1294–1300.
75
Christensen, J.B., Tipping, E., Kinniburgh, D.G., Gron, C., Chris-
tensen, T.H., 1998. Proton binding by groundwater fulvic acids of
different age, origins, and structure modeled with the Model V and
NICA-Donnan model. Environ. Sci. Technol. 32, 3346–3355.
Davis, J.A., 1984. Geochim. Cosmochim. Acta 48, 679–691.
Davison, W., Zhang, H., 1994. Nature 367, 546–548.
Davison, W., Hooda, P.S., Zhang, H., Edwards, A.C., 2000. DG
measured fluxes as surrogates for uptake of metals by plants. Adv.
Envision. Res. 3, 550–555.
DiToro, D.M., Hansen, D.D., McGrath, J., Berry, W.J., 2000.
Predicting the Acute Toxicity of Metals in Sediments Using
Organic Carbon-Normalized SEM and AVS. HydroQual, Inc.,
Mahwah.
DiToro, D.M., Kavvadas, C.D., Mathew, R., Paquin, P.R., Winfield,
R.P., 2001. The Persistence and Availability of Metals in Aquatic
Environments. International Council on Metals and the Environ-
ment, Ottawa, ON, Canada.
Evans, L.J., 1989. Chemistry of metal retention in soils. Environ. Sci.
Technol. 23, 1046–1056.
Gobas, F.A.P.C., McCorquodale, J.R., Haffner, G.D., 1993a.
Environ. Toxicol. Chem. 12, 567–576.
Gobas, F.A.P.C., Zhang, X., Wells, R., 1993b. Environ. Sci. Technol.
27, 2855–2863.
Gonzalez, M.J., Ramos, I., Hernandez, L.M., 1994. Int. J. Environ.
Anal. Chem. 57, 135–150.
Gregson, S.K., Alloway, B.J., 1984. Gel permeation chromatography
studies on the speciation of lead in solutions of heavily polluted
soils. J. Soil Sci. 35, 55–61.
Griscom, S.B., Fischer, N.S., Luoma, S.N., 2000. Geochemical
influences on assimilation of sediment-bound metals in clams and
mussels. Environ. Sci. Technol. 34, 91–99.
Gupta, S.K., Aten, C., 1993. Int. J. Environ. Anal. Chem. 51, 25–46.
Gustafsson, Ö., Haghseta, F., Chan, C., McFarlane, J., Gschwend,
P.M., 1997. Environ. Sci. Technol. 31, 203–209.
Hamelink, J.L., Landrum, P.F., Bergman, H.L., Benson, W.H., 1994.
Bioavailability. Physical, Chemical and Biological Interactions.
CRC Press, Boca Raton, FL.
Hansen, E.H., Lamm, C.G., Rùzicka, J., 1972. Selectrode—the
universal ion-selective electrode. (II) Comparison of copper(II)
electrodes in metal buffers and complexometric titrations. Anal.
Chim. Acta 59, 403–425.
Hare, L., Tessier, A., 1996. Nature 380, 430–432.
Hart, B.T., 1982. In: Sly, P.G. (Ed.), Sediment/Freshwater Interaction.
Dr. W. Junk Publ, Boston.
Hörter, D., Dressmann, J.B., 1997. Adv. Drug Deliv. Res. 25, 3–14.
Jager, T., 1998. Environ. Toxicol. Chem. 17, 2080–2090.
Jenne, E.A., 1968. In: R.A. Baker (Ed.), Trace Inorganics in Water,
Adv. Chem. Ser. 73., American Chemical Society, Washington DC.
Kelsey, J.W., Alexander, M., 1997. Declining bioavailability and
inappropriate estimation of risk of persistent compounds. Envir-
onmental Toxicology and Chemistry 15, 582–585.
Kiewiet, A.T., Ma, W.C., 1991. Ecotoxicol. Environ. Saf. 21,
32–37.
Kinniburgh, D.G., Milne, C.J., Benedetti, M.F., Pinheiro, J.P., Filius,
J., Koopal, L.H., Van Riemsdijk, W.H., 1996. Metal ion binding
by humic acid: application of the NICA-Donnan model. Environ.
Sci. Technol. 30, 1687–1698.
Korcak, R.F., Fanning, D.S., 1985. Soil Sci. 140, 23–34.
Lee, B.-G., Griscom, S.B., Lee, J.-S., Choi, H.J., Koh, C.-H., Luoma,
S.N., Fisher, N.S., 2000. Influences of dietary uptake and reactive
sulfides on metal bioavailability from aquatic sediments. Science
287, 282–284.
Levesque, M., Mathur, S.P., 1988. Soil tests for copper, iron,
manganese, and zinc in histosols: 3. A comparison of eight
extractants for measuring active and reserve forms of elements. Soil
Sci. 145, 215–221.
 ARTICLE IN PRESS
76 W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
Lexmond, T.M., Van der Vorm, P.D.J., 1981. Neth. J. Agric. Sci. 29,
209–230.
L^kke, H., Van Gestel, C.A.M., 1998. Handbook of Soil Invertebrate
Toxicity Tests. Wiley, Chicester, UK.
Lord, K.A., Briggs, G.G., Neale, M.C., Manlove, R., 1980. Pest Sci.
11, 401–408.
Luoma, S.N., Davis, J.A., 1983. Mar. Chem. 12, 159–181.
Ma, W.-C., 1982. Pedobiologia 24, 109–119.
Marschner, H., 1995. Mineral Nutrition of Higher Plants. Academic
Press, London.
McGrath, S.P., Sanders, J.R., Laurie, S.H., Tancock, N.P., 1986.
Analyst 111, 459–465.
McGroddy, S.E., Farrington, J.W., Gschwend, P.M., 1996. Environ.
Sci. Technol. 30, 1172–1177.
McLaughlin, M.J., 2001. Bioavailability of metals to terrestrial plants.
In: Allen, H.E. (Ed.), Bioavailability of Metals in Terrestrial
Ecosystems: Importance of Partitioning for Bioavailability to
Invertebrates, Microbes and Plants. SETAC Press, Pensacola, FL.
McLaughlin, M.J., Smolders, E., Merckx, R., 1998. Soil–root inter-
face: physicochemical processes. In: Huang, P.M. (Ed.), Soil
Chemistry and Ecosystem Health. Soil Science Society of America,
Madison, WI, pp. 233–277.
Minnich, M.M., McBride, M., 1987. Copper activity in soil solution: I.
measurement by ion-selective electrode and Donnan dialysis. Soil
Sci. Soc. Amer. J. 51, 568–572.
Morel, F.M.M., 1983. Principles of Aquatic Chemistry, 1st edition.
Wiley, New York.
Morgan, J.E., Morgan, A.J., 1992. Soil Biol. Biochem. 24, 1691–1697.
Notenboom, J., Posthuma, L., 1996. Report no. 733007003. Dutch
National Institute of Public Health and the Environment.
Bilthoven, The Netherlands.
Nye, P.H., Tinker, P.B., 1977. Solute Movement in the Soil–Root
System. In: Studies in Ecology, Vol. 4. Blackwell, Oxford, UK.
Oomen, A.G., 2000. Determinants of Oral Bioavailability of Soil-
Borne Contaminants. Ph.D. Thesis, University of Utrecht.
Oste, L.A., 2001. Ph.D. Thesis, University of Wageningen, The
Netherlands.
Oste, L.A., Dolfing, J., Ma, W.C., Lexmond, T.M., 2001. Cadmium
uptake by earthworms as related to the availability in the soil and
the intestine. Environ. Toxicol. Chem. 20, 1785–1791.
Pagenkopf, G.K., 1983. Environ. Sci. Technol. 17, 342–347.
Parker, D.R., Pedler, J.F., 1997. Reevaluating the free-ion activity
model of trace metal availability to higher plants. Plant Soil 196,
223–228.
Parker, D.R., Chaney, R.L., Norvell, W.A., 1995. Chemical equili-
brium models: application to plant nutrition research. In: R.H.
Loeppert, A.P. Schwab and S. Goldberg (Eds.), Chemical
equilibrium and reaction models, SSSA Special Publication No.
42, Soil Science Society of America Inc., American Society of
Agronomy, Inc., Madison, WI.
Peijnenburg, W., Posthuma, L., Eijsackers, H., Allen, H., 1997. A
conceptual framework for implementation of bioavailability of
metals for environmental management purposes. Ecotoxicol.
Environ. Saf. 37, 163–172.
Peijnenburg, W.J.G.M., Posthuma, L., Zweers, P.G.P.C., Baerselman,
R., De Groot, A.C., Van Veen, R.P.M., Jager, T., 1999a.
Prediction of metal bioavailability in Dutch field soils for the
Oligochaete Enchytraeus crypticus. Ecotoxicol. Environ. Saf. 43,
170–186.
Peijnenburg, W.J.G.M., Baerselman, R., De Groot, A.C., Jager, T.,
Posthuma, L., Van Veen, R.P.M., 1999b. Relating environmental
availability to bioavailability: soil-type dependent metal accumula-
tion in the oligochaete Eisenia Andrei. Ecotoxicol. Environ. Saf. 44,
294–310.
Peijnenburg, W.J.G.M., Franken, R., de Groot, A., Hogendoorn, E.,
Jager, D.T., Posthuma, L., Ritsema, R., 2000. Ecotoxicological
risks of spreading of contaminate sediments on land: further
research on the ecological meaning of exceeding of risk limits.
Report No. 733007003 (In Dutch). Dutch National Institute of
Public Health and the Environment. Bilthoven, The Netherlands.
Piearce, T.G., 1972. J. Anim. Ecol. 41, 167–188.
Plette, A.C.C., Nederhof, M.M., Temminghoff, E.J.M., Van Riems-
dijk W, H., 1999. Bioavailability of heavy metals in terrestrial and
aquatic systems: a quantitative approach. Environ. Toxicol. Chem.
18, 882–1890.
Posthuma, L., Notenboom, J., 1996. Report No. 719102048. Dutch
National Institute of Public Health and the Environment.
Bilthoven, The Netherlands.
Sauvé, S., 2001. Speciation of metals in soils. In: Allen, H.E. (Ed.),
Bioavailability of Metals in Terrestrial Ecosystems; Importance of
Partitioning for Bioavailability to Invertebrates, microbes and
plants. SETAC Press, Pensacola, FL.
Sauvé, S., McBride, M., Hendershot, W.H., 1995. Ion-selective
electrode measurements of copper(II) activity in contaminated
soils. Arch. Environ. Contam. Toxicol. 29, 373–379.
Saxe, J.K., Impellitteri, C.A., Peijnenburg, W.J.G.M., Allen, H.E.,
2001. Novel model describing trace metal concentrations, in the
earthworm Eisenia andrei. Environ. Sci. Technol. 35, 4522–4529.
Serajuddin, A.T.M., Sheen, P.-C., Mufson, D., Bernstein, D.F.,
Augustine, M.A., 1988. J. Pharm. Sci. 77, 325–329.
Shea, D., 1988. Developing national sediment quality criteria. Environ.
Sci. Technol. 22, 1256–1261.
Sposito, G., 1984. The Surface Chemistry of Soils. Oxford University
Press, New York.
Sposito, G., Mattigod, S.V., 1980. GEOCHEM: A Computer Program
for Calculation of Chemical Equilibria in Soil Solutions and Other
Natural Water Systems. Kearney Foundation of Soil Science,
University of California, Riverside.
Spurgeon, D.J., Hopkin, S.P., 1995. Ecotoxicology 4, 190–205.
Spurgeon, D.J., Hopkin, S.P., Jones, D.T., 1994. Environ. Pollut. 84,
123–130.
Sunda, W.G., Guillard, R.R.L., 1976. J. Marine Res. 34, 511–529.
Temminghoff, E.J.M., Plette, A.C.C., Van Eck, R., Van Riemsdijk,
W.H., 2000. Determination of the chemical speciation of trace
metals in aqueous systems by the Wageningen Donnan Membrane
Technique. Anal. Chim. Acta 417, 149–157.
Tessier, A., Campbell, P.G.C., Bisson, M., 1979. Sequential extraction
procedure for the speciation of particulate trace metals. Anal.
Chem. 51, 844–851.
Tipping, E., 1994. WHAM—A chemical equilibrium model and
computer code for waters, sediments, and soils incorporating a
discrete site/electrostatic model of ion binding by humic sub-
stances. Comp. Geosci. 20, 973–1023.
Ure, A.M., Davidon, C.M., 1995. Chemical Speciation in the
Environment. Chapman and Hall, London.
USEPA, 1997. Microwave-assisted acid dissolution of sediments,
sludges, soils and oils. In: SW846 Test Methods for Evaluating
Solid Waste, Physical/Chemical Methods, 2nd Edition. USEPA
Publication No. 955-001-00000-1. US Government Printing Office,
Washington, DC.
Van Gestel, C.A.M., Ma, W.C., 1988. Ecotoxicol. Environ. Saf. 15,
289–297.
Van Straalen, N.M., 1996. Critical body concentrations: their use in
bioindication. In: Krivolutsky, D.A., van Straalen, N.M. (Eds.),
Bioindicator Systems for Soil Pollutants. Kluwer Academic
Publishers, Dordrecht, The Netherlands, pp. 5–16.
Van Wensem, J., Vegter, J.J., Van Straalen, N.M., 1994. Soil quality
criteria derived from critical body concentrations of metals in soil
invertebrates. Appl. Soil. Ecol. 1, 185–191.
Verma, A., Pillai, M.K.K., 1991. Curr. Sci. 16, 840–843.
Vijver, M., Jager, T., Posthuma, L., Peijnenburg, W., 2001. The impact
of metal pools and soil properties on metal accumulation in
 ARTICLE IN PRESS
W.J.G.M. Peijnenburg, T. Jager / Ecotoxicology and Environmental Safety 56 (2003) 63–77
Folsomia candida (Collembola). Environ. Toxicol. Chem. 20,
712–720.
Wei, Y.-L., Shyu, H.-M., Joehuang, K.-L., 1997. Comparison of
microwave versus hot-plate digestion for nine real-world river
sediments. J. Environ. Qual. 26, 764–768.
Westall, J.C., Zachary, J.L., Morel, F.M.M., 1976. MINEQL: A
Computer Program for the Calculation of the Chemical Equili-
brium Composition of Aqueous Systems, Tech. Note No. 18.
77
Ralph M. Parsons Laboratory, Massachusetts Institute of Tech-
nology, Cambridge, MA.
Zhang, H., Davison, W., Knight, B., McGrath, S., 1998. In situ
measurements of solution concentrations and fluxes of trace metals
in soils using DGT. Environ. Sci. Technol. 32, 704–710.
Zhang, H., Zhao, F.-J., Sun, B., Davidson, W., McGrath, S., 2001. A
new method to measure effective soil solution concentration predicts
copper availability to plants. Environ. Sci. Technol. 35, 2602–2607.