Soil ecotoxicology: state of the art and future directions.

Abstract

Developments in soil ecotoxicology started with observations on pesticide effects on soil invertebrates in the 1960s. To support the risk assessment of chemicals, in the 1980s and 1990s development of toxicity tests was the main issue, including single species tests and also more realistic test systems like model ecosystems and field tests focusing on structural and functional endpoints. In the mean time, awareness grew about issues like bioavailability and routes of exposure, while biochemical endpoints (biomarkers) were proposed as sensitive and potential early-warning tools. In recent years, interactions between different chemicals (mixture toxicity) and between chemical and other stressors attracted scientific interest. With the development of molecular biology, omics tools are gaining increasing interest, while the ecological relevance of exposure and effects is translating into concepts like (chemical) stress ecology, ecological vulnerability and trait-based approaches. This contribution addresses historical developments and focuses on current issues in soil ecotoxicology. It is concluded that soil ecotoxicological risk assessment would benefit from extending the available battery of toxicity tests by including e.g. isopods, by paying more attention to exposure, bioavailability and toxicokinetics, and by developing more insight into the ecology of soil organisms to support better understanding of exposure and long-term consequences of chemical exposure at the individual, population and community level. Ecotoxicogenomics tools may also be helpful in this, but will require considerable further research before they can be applied in the practice of soil ecotoxicological risk assessment.

Full text

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Soil ecotoxicology: state of the art and future directions
Review ARtiCleS
275
Soil ecotoxicology: state of the art and future directions
Cornelis A.M. van Gestel1
1 Department of Ecological Science, Faculty of Earth and Life Sciences, VU University, De Boelelaan 1085,
1081 HV Amsterdam, The Netherlands
Corresponding author: Cornelis A.M. van Gestel (kees.van.gestel@falw.vu.nl)
Academic editor: J. Štrus  |  Received 25 November 2011  |  Accepted 26 January 2012  |  Published 20 March 2012
Citation: van Gestel CAM (2012) Soil ecotoxicology: state of the art and future directions. In: Štrus J, Taiti S, Sf-
enthourakis S (Eds) Advances in Terrestrial Isopod Biology. ZooKeys 176: 275–296. doi: 10.3897/zookeys.176.2275
Abstract
Developments in soil ecotoxicology started with observations on pesticide effects on soil invertebrates in
the 1960s. To support the risk assessment of chemicals, in the 1980s and 1990s development of toxic-
ity tests was the main issue, including single species tests and also more realistic test systems like model
ecosystems and field tests focusing on structural and functional endpoints. In the mean time, awareness
grew about issues like bioavailability and routes of exposure, while biochemical endpoints (biomarkers)
were proposed as sensitive and potential early-warning tools. In recent years, interactions between dif-
ferent chemicals (mixture toxicity) and between chemical and other stressors attracted scientific interest.
With the development of molecular biology, omics tools are gaining increasing interest, while the ecologi-
cal relevance of exposure and effects is translating into concepts like (chemical) stress ecology, ecological
vulnerability and trait-based approaches. This contribution addresses historical developments and focuses
on current issues in soil ecotoxicology. It is concluded that soil ecotoxicological risk assessment would
benefit from extending the available battery of toxicity tests by including e.g. isopods, by paying more
attention to exposure, bioavailability and toxicokinetics, and by developing more insight into the ecology
of soil organisms to support better understanding of exposure and long-term consequences of chemical
exposure at the individual, population and community level. Ecotoxicogenomics tools may also be help-
ful in this, but will require considerable further research before they can be applied in the practice of soil
ecotoxicological risk assessment.
Keywords
Toxicity tests, bioavailability, ecological effects, biomarkers, soil organisms, isopods
ZooKeys 176: 275–296 (2012)
doi: 10.3897/zookeys.176.2275
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Copyright Cornelis A.M. van Gestel. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Cornelis A.M. van Gestel / ZooKeys 176: 275–296 (2012)
276
introduction
Ecotoxicology studies the effects of chemicals on organisms in the environment, with
the final aim to protect the structure and functioning of ecosystems. This aim generally
is achieved by assessing effects on single species of selected test organisms and trying
to extrapolate the obtained (no) effect concentrations to safe levels for populations and
communities. In the ecotoxicological risk assessment of chemicals, such safe levels are
then compared with predicted or measured exposure levels to assess the possible risk
for exposed ecosystems.
This paper will give an overview of developments in soil ecotoxicology, focusing on
soil invertebrates, starting with a historical overview. Based on that, the state-of-the-art
of current soil ecotoxicology will be depicted. This is done by first describing the way
soil ecotoxicological data are used in the risk assessment of new and existing chemicals
or the assessment of the risks of soil contamination. Next, the development of soil
toxicity tests is outlined, followed by considerations on the inclusion of bioavailability,
and the use of multiple species, model ecosystem and field tests. Then tools of assessing
the possible risk of contaminated soils are described. Finally, some thoughts are given
on the future of soil ecotoxicology. As this paper was written on the basis of a presenta-
tion at an isopod meeting, special attention will be given to the use of isopods in soil
ecotoxicological testing.
Historical perspective of soil ecotoxicology
When thinking of ecotoxicological effects, it is often referred to Rachel Carson, pub-
lishing her book ‘Silent Spring’ in 1962. This book was among the first describing the
negative side-effects of the increasing use of synthetic pesticides that started from the
second World war onwards. The book mainly focused on pesticide effects on birds,
especially singing birds that apparently became silent due to the effects of chlorinated
pesticides accumulating in the food chain. This book however, was not unique in ring-
ing the alarm bell, although other bells did not sound that loud.
The first soil ecotoxicological papers date back to the 1960s, reporting observa-
tions on the negative effects of pesticides on soil invertebrates (e.g., Fox 1964, Edwards
1969). Similar to developments in aquatic ecotoxicology, these observations triggered
the performance of toxicity tests with selected organisms to enable prediction of such
side-effects in the field. First results of such toxicity tests, using Collembola and earth-
worms, were published by the end of the 1960s, also on pesticides (e.g., Ghabbour and
Imman 1967, Scopes and Liechtenstein 1967). In the mean time, the Organization
for Economic Co-operation and Development (OECD) started developing Guidelines
for the testing of chemicals, to support the chemical risk assessment and pesticide
registration procedures developed in most Western countries. It took another 15 years
before the first toxicity test with soil invertebrates was internationally standardized by
OECD, using earthworms and only focusing on short-term (acute) responses like sur-

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Soil ecotoxicology: state of the art and future directions 277
vival (OECD 1984). In the 1980s and 1990s, the development of soil ecotoxicological
tests received more attention, e.g. in the SECOFASE project funded by the European
Union, that explored the possibilities of developing toxicity tests with different soil
invertebrates, including earthworms, enchytraeids, nematodes, Collembola, staphyli-
nid beetles, mites, centipedes, millipedes and isopods (Løkke and Van Gestel 1998).
Several of the methods developed in SECOFASE never made it to standardization, but
the project laid a basis for testing new species, using sub-lethal endpoints and includ-
ing interactions between species.
During the last ten years, there has been a renewed attention for effects of mixtures
of chemicals in soil (Van Gestel et al. 2011), while the interaction of chemicals with
other stress factors, like temperature and soil moisture content, also came into focus
(Holmstrup et al. 2010). In addition, the available test methods outline below are
nowadays applied to new and emerging chemicals, especially to determine the toxicity
of nanoparticles using earthworms (e.g. Shoults-Wilson et al. 2011a,b; Heckmann et
al. 2011; Hooper et al. 2011), Collembola (Kool et al. 2011), and isopods (e.g. Jemec
et al. 2008; Drobne et al. 2009; Pipan-Tkalec et al. 2010).
ecotoxicological risk assessment
In ecotoxicological risk assessment, two approaches can be distinguished. One ap-
proach aims at predicting possible effects of (new) chemicals in order to regulate their
use or prevent their introduction onto the market. This predictive approach (prognosis)
uses laboratory toxicity tests to obtain toxicity data that are used to derive safe levels of
chemicals in the environment. The second approach is assessing the actual ecological
risk or damage in case of pollution. This diagnostic approach (diagnosis) enables setting
priorities for remediation and risk reduction, and may provide triggers for the manage-
ment of contaminated land.
Prognosis starts from the paradigms also used in human toxicology. It assumes
that the risk of a chemical for ecosystems can be estimated from its toxicity to a
number of surrogate test or indicator species, exposed in standard laboratory toxicity
tests. These tests aim at assessing toxicity, which is expressed in terms of dose-re-
sponse relationships for effects on selected endpoints like survival, growth and repro-
duction. Toxicity is quantified by parameters like LC10 and LC50 (the concentrations
killing 10% and 50% of the exposed test organisms, respectively), EC10 and EC50
(the concentrations causing 10% and 50% reduction, respectively in a measured
endpoint, e.g. growth or number of juveniles produced), and NOEC and LOEC
(no-observable and lowest-observable effect concentration, respectively). Since there
is no ‘most sensitive species’ a battery of toxicity tests is needed to obtain proper
insight into the potential hazard of a chemical for the ecosystem. In prognosis, the
outcome of toxicity tests is used to establish thresholds or safe levels of chemicals in
soil, which can be compared with measured or predicted exposure data (soil concen-
trations) to assess the (potential) risk.

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A critical part of this procedure is the derivation of safe levels of chemicals on
the basis of available toxicity data. When only short-term (acute) toxicity data are
available (usually focusing on survival) or data for a limited number of species, some-
what arbitrary application factors are applied to derive safe levels that should protect
ecosystems. For example, when only one or two LC50 values are available, a factor of
1000 is applied to the lowest LC50 value; this factor should be sufficient to extrapolate
from acute to chronic effects (factor of 10), from one or few species to many species
(factor of 10), and from laboratory to field (factor of 10). When sublethal toxicity
data (usually NOEC or EC10 values for effects on e.g. reproduction) are available for
3 or more species, application of a factor of 10 to the lowest value is considered suf-
ficiently protective. When many toxicity data are available (preferably ≥ 8) for species
representative of different taxonomic groups (see below), a statistical method may be
applied. Such a statistical method is used to construct a species-sensitivity distribution
(SSD), which assumes a log-normal or log-logistic distribution of the sensitivities of
species in an ecosystem. From such an SSD the 95% lower limit is selected as the safe
level. At this level, at least 95% of the species in the ecosystem are supposed to be safe
(Posthuma et al. 2002).
Diagnosis may use the same tools as applied for prognoses, but it more heavily
relies on ecological methods and environmental chemistry. Basically, toxicity tests or
bioassays are used as diagnosis tools to assess toxicity of soil samples from a contami-
nated site. Results of the bioassays, together with those of chemical measurements and
ecological field observations, are used to assess the potential risk of soil contamination.
The three tools together form the TRIAD approach (Jensen and Mesman 2006).
toxicity tests
Both prognosis and diagnosis use toxicity tests, and in both cases a battery of tests is
recommended. Criteria to select tests for such a battery have been formulated e.g. by
Van Gestel et al. (1997). These criteria among others include:
1. Practicability, referring to the feasibility and cost-effectiveness of a test;
2. Acceptability, including aspects like standardization, reproducibility and statis-
tical validity of a test method as well as its broad chemical responsiveness;
3. Ecological meaning, including sensitivity and ecological realism of the test
method.
To obtain a balanced battery of tests, in addition the following criteria need to be
taken into account (Van Gestel et al. 1997):
1. Representativeness for the ecosystem to protect: this includes e.g. the represen-
tation of organisms having different life-histories, representing different func-
tional groups, different taxonomic groups and different routes of exposure;

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Soil ecotoxicology: state of the art and future directions279
2. Representativeness of responses, to make sure responses measured really are
relevant for the protection of populations and communities;
3. Uniformity, which refers to the possibility to apply all tests in a battery to the
same test media.
By the end of the 1990s and early 2000 toxicity tests, using sub-lethal endpoints
like reproduction, were standardized for enchytraeids, earthworms and Collembola by
both the OECD and the International Standardization Organization (ISO). But also
Environment Canada, the United States Environmental Protection Agency (EPA) and
ASTM International (formerly known as the American Society for Testing and Ma-
terials) have described similar methods. Recently, for the same organisms, avoidance
behaviour tests have been described, while for earthworms and enchytraeids a bioac-
cumulation test is available. Table 1 provides an overview of the toxicity tests with soil
invertebrates available at the moment.
The oldest standardized toxicity test guideline with soil invertebrates, OECD 207
(OECD 1984), describes two short-term toxicity tests, one using 14 days exposure
in soil and the other one exposing the worms for 2 days to treated filter paper. Both
methods use survival as the only endpoint. The test on filter paper is only rarely applied
nowadays, as it does not have any relevance for exposure in soil. It may however, be a
table 1. A selection of available toxicity tests with soil invertebrates.
Test
organism
Species
Duration
(days)
EndpointGuidelineReference
Earthworms
Eisenia fetida/
Eisenia andrei
14Survival
OECD 207
ISO 11268-1
ISO 11268-2
OECD 222
ISO 17512-1 ISO (2008a)
OECD (1984)
ISO (1993)
ISO (1998)
OECD (2004b)
28 (+28) Reproduction
2 Avoidance
Species
diversity;
abundance
Survival,
Reproduction
Field test, different
species
Up to 1
year
ISO 11268-3 ISO (1999b)
Enchytraeids
Enchytraeus
albidus, other
Enchytraeus species
21 (+21)
ISO 16387
OECD 220
No standard
guidelines
ISO (2004)
OECD (2004a)
Amorim et al.
(2008a,b)
2Avoidance
MolluscaHelix aspersa 28
Survival,
Growth
Survival,
Reproduction
Survival
Reproduction
ISO 15952 ISO (2006)
Mites
Hypoaspis aculeifer 14OECD 226 OECD (2008)
Platynothrus
peltifer
14
70
No standard
guideline
No standard
guideline
No standard
guideline
Van Gestel and
Doornekamp (1998)
Oppia nitens
28ReproductionPrincz et al. (2010)
2AvoidanceOwojori et al. (2011)

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useful exposure method for the rapid screening of chemicals, assessing the uptake and/
or biotransformation of chemicals or other types of mechanistic research.
Compared to survival, reproduction is a more relevant endpoint when translating
effects to the population level. For that reason, an earthworm toxicity test focusing
on reproduction has been developed (ISO 1998, OECD 2004b). Although focus is
on reproduction, it is essential in this test to also include weight change of the earth-
worms, since there is evidence for a trade off between reproduction and growth, which
may affect their response to toxicants (Van Gestel et al. 1992, Van Gestel et al. 1995).
Like for earthworms, the tests with enchytraeids (ISO 2004, OECD 2004a) also focus
on reproduction and survival as the endpoint, but for these organisms no separate
short-term (acute) and sub-lethal tests were developed. All the reproduction toxicity
tests available with oligochaetes typically have test durations of 21-28 days. In case of
the earthworm test with Eisenia fetida or Eisenia andrei and the enchytraeid test with
Enchytraeus albidus, this means exposing adult worms for 28 and 21 days, respectively,
after which they are collected from the soil; the cocoons are incubated for another
28 or 21 days, respectively to enable determining the number of juveniles produced.
Nowadays, Enchytraeus crypticus seems to be more commonly used for toxicity testing
than Enchytraeus albidus. Since that species is smaller, adults are not removed from the
soil and reproduction is determined after 28 days of exposure. Considering the shorter
life cycle of Enchytraeus crypticus and its high reproductive output, limiting test dura-
tion to 21 days has recently been advocated (Van Gestel et al. 2011).
A standardized test with snails has been developed by ISO (2006), focusing on
survival and growth of juveniles snails (Helix aspersa aspersa) after 28 days exposure.
Standardized toxicity tests with soil arthropods include the collembolan species
Folsomia candida (ISO 1999a) and Folsomia fimetaria (OECD 2009), the predatory
mite Hypoaspis (Geolaelaps) aculeifer (OECD 2008), and larvae of the insect Oxythyrea
funesta (ISO 2005). The tests with the collembolans and the predatory mite typically
Test
organism
Species
Duration
(days)
EndpointGuidelineReference
Isopods
Porcellio scaber28
Survival,
growth
Survival,
reproduction
No standard
guideline
No standard
guidelines
No standard
guidelines
ISO 11267
OECD 232
ISO 17512-2 ISO (2011)
ISO 20963
Hornung et al.
(1998a,b)
Porcellionides
pruinosis
14
Jänsch et al. (2005)
2Avoidance
Loureiro et al. (2005)
Collembola
Folsomia candida
Folsomia fimetaria
28
Survival,
Reproduction
Avoidance
Survival
Adult
or larval
survival; adult
behaviour,
respiration
ISO (1999a);
OECD (2009)
2
14 InsectsOxythyrea funesta ISO (2005)
Carabid
beetles
Pterostichus
oblongopunctatus;
Poecilus cupreus
Different
durations
No standard
guidelines
Schrader et al.
(1998); Bednarska et
al. (2010)

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Soil ecotoxicology: state of the art and future directions 281
focus on survival and reproduction after 28, 21 and 14 days exposure, respectively
for the parthenogenetic Folsomia candida, the sexually reproducing Folsomia fimetaria
and the predatory mite Hypoaspis aculeifer. The test with insect larvae only focuses on
survival.
Tests with carabid beetles have been performed using adult Pterostichus oblon-
gopunctatus or larvae of Poecilus cupreus, but these tests have not been standardized and
use different life stages (larvae, adults), endpoints (survival, mobility, respiration) and
test durations (from few weeks to several months) depending on the aims of the study
(e.g. Schrader et al. 1998, Bednarska and Laskowski 2008, Bednarska et al. 2010).
Also the toxicity tests with the oribatid mites Oppia nitens and Platynothrus peltifer
described in the literature (Van Gestel and Doornekamp 1998, Princz et al. 2010) are
not yet standardized. These tests focus on survival and reproduction.
For assessing chemical toxicity to isopods, also no standard test guidelines are avail-
able. Nevertheless, isopods are used as test organisms, using different test durations,
different routes of exposure (food, soil) and different endpoints. Drobne and Hopkin
(1995) described a test exposing Porcellio scaber via food and determining effects of
zinc on feeding rates. Hornung et al. (1998a) developed a draft test guideline, allow-
ing for exposure both via food and in soil, also using Porcellio scaber as the test spe-
cies. Several different endpoints have been proposed, but considering the difficulty
of culturing Porcellio scaber in the laboratory survival, growth and food consumption
rate so far have been used most frequently (see e.g. Odendaal and Reinecke 2004,
Nolde et al. 2006, Kolar et al. 2010). Another interesting endpoint may be related to
the composition of the gut microflora of isopods (Drobne et al. 2002). Nevertheless,
isopod (Porcellio scaber) reproduction also has been proposed as a test endpoint (Hor-
nung et al. 1998b). Successful reproduction isopod experiments have been performed,
e.g. by Van Brummelen et al. (1996a), in a 48-week test with Oniscus asellus, applying
dietary exposure to Polycyclic Aromatic Hydrocarbons (PAHs), and by Lemos et al.
(2010) assessing the reproduction toxicity to Porcellio scaber of some chemicals with
suspected endocrine-disrupting effects. Other isopod species, like Porcellionides prui-
nosis, are more easily cultured in the laboratory, and therefore may be more suitable for
performing reproduction toxicity tests (e.g., Jänsch et al. 2005).
Recently, avoidance response was introduced as an easy, fast and sensitive end-
point. For some chemicals avoidance response may be as sensitive as reproduction,
while for others it is at least as sensitive as survival. Great advantage of avoidance tests is
that they are fast, with test durations of no more than 2 days. Standard test guidelines
for avoidance tests have been developed for earthworms (ISO 2008a) and Collembola
(ISO 2011), but similar tests have also been performed with enchytraeids (Amorim
et al. 2008a,b, Novais et al. 2010), oribatid mites (Owojori et al. 2011) and isopods
(Loureiro et al. 2005, Zidar et al. 2005).
In addition to these tests with soil invertebrates, ISO and OECD have also devel-
oped a number of toxicity tests with plants, which are important in soil ecosystems as
primary producers. Also, several tests are available focusing on the effects of microbial
communities or processes performed by microorganisms, like nitrification.

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Considering the fact that for a proper risk assessment a battery of tests is desirable,
it is important to consider the currently available test methods. The current set of avail-
able tests (Table 1) shows an underrepresentation of arthropods in comparison with
their abundance in the field when compared with other species like Oligochaetes. And
of the available or suggested tests with arthropods, only the one with Collembola has
been standardized. Development and international standardization of more toxicity
tests with representative arthropod species therefore is highly needed (see criteria for
the selection of test species outlined above). The ecological relevance of isopods, their
typical routes of exposure (soil, food) and life history characteristics, the possibility to
determine different endpoint, and the fact that they have already been used for testing
for more than 30 years, make them highly suitable test organisms. Standardization of
toxicity tests with isopods therefore is highly recommended.
Bioavailability
For reasons of standardization and to facilitate comparison of results, all standardized
tests use a standard soil type: the so-called OECD artificial soil, first introduced in
the earthworm acute toxicity test developed by OECD (1984) This artificial soil is
composed by mixing readily available materials like sphagnum peat (10%), kaolin clay
(20%) and quartz sand (70%); by adding some CaCO3 pH is adjusted to approx. 6.0.
The properties of this soil resemble those of a sandy loam soil. Recently, within OECD
the use of 5% peat has been advocated when testing pesticides, in order to increase
the ‘worst case’ realism of the artificial test soil for low organic (agricultural) field soils.
Also with the aim to increase realism, the SECOFASE project started using the natural
LUFA 2.2 soil (Løkke and Van Gestel 1998). The LUFA soils are commercially avail-
able from the Landwirtschaftliche Untersuchungs und Forschungsanstalt (LUFA) in
Speyer, Germany. Since that time, LUFA 2.2 standard soil seems to become more com-
monly used for toxicity tests with soil invertebrates, also because several test species like
the collembolan Folsomia candida and the enchytraeid Enchytraeus crypticus seem to
perform as good in this natural soil as they do in artificial soil.
The notion that soil type was important when determining the toxicity of chemi-
cals went along with the increasing insight into the concept of bioavailability: only a
fraction of the total amount of chemical in the soil is available for uptake by organisms
and therefore of relevance for risk assessment. This was, for instance, demonstrated by
Bradham et al. (2006), exposing the earthworm Eisenia andrei for 28 days to different
soil types spiked with one concentration (2000 mg/kg) of lead (Pb). While in some
soils all worms died, in other soils no mortality was seen and in the remainder of the
soils only part of the earthworms died. This finding could be explained from the differ-
ences in soil properties, especially pH, clay and organic matter content that affected the
availability of lead for the earthworms. The pore-water hypothesis or equilibrium par-
titioning theory was developed to enable linking toxicity of organic chemicals to the
concentration available in the pore water (Van Gestel and Ma 1988, 1990). For metals,

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Soil ecotoxicology: state of the art and future directions 283
application of this approach turned out to be less straightforward because metal specia-
tion in soils is much more complex (Van Gestel 1997) and many factors may affect
metal bioavailability in soils (e.g., Allen 2002, Lanno et al. 2004). It nowadays is real-
ized that bioavailability is not a static but rather a dynamic concept (Luoma and Rain-
bow 2005, Van Gestel 2008). This was demonstrated by Van Straalen et al. (2005),
exposing isopods (Porcellio scaber) from a metal-contaminated and a non-polluted site
to zinc via the food. Both groups showed the same EC50 expressed on the basis of zinc
concentrations in the diet, but contrary to the expectation effects could not be ex-
plained from zinc concentrations in the animals. So, it seems that flux of zinc through
the animals rather than total zinc concentration was determining their sensitivity.
These findings also suggest that when considering bioavailability, not only chemi-
cal partitioning of chemicals in the exposure medium (soil, food) and pore-water
concentrations have to be considered. The biology of bioavailability also needs atten-
tion. One aspect of this is the way organisms deal with chemicals. For metals, internal
compartmentalization has been shown to be an important aspect (Rainbow 2002), as
it determines what fraction of the metal is present in the body in a metabolically avail-
able form. Most soil invertebrates have the capacity to sequester at least part of their
metal burden in such a way that it does no longer pose a risk. Isopods use the hepato-
pancreas for a very efficient storage of excess metals (Hopkin and Martin 1982), while
earthworms have their chloragogenous tissue that serves the same purpose (Morgan
and Morgan 1998, Morgan et al. 1999). As a consequence, both isopods and earth-
worms show a huge capacity of storing metals like cadmium, which after uptake
are hardly eliminated. Other soil invertebrates, like Collembola and beetles, use the
midgut epithelium for metal storage. Upon moulting, also the midgut epithelium is
renewed enabling these organisms to excrete excess metal (Hopkin 1989). Internal
sequestration determines what fraction of the total metal burden in an organism may
contribute to its toxicity or is available for trophic transfer to its predators (Vijver et
al. 2004). This was for instance demonstrated by Crommentuijn et al. (1994), who
found very high Lethal Body Concentrations for cadmium in isopods compared to
other arthropods that could be attributed to the highly efficient storage of the metal
in an inert form.
Another way organisms may deal with potentially toxic chemicals is by biotrans-
formation. The process of biotransformation aims at making chemicals more hydro-
philic and in this way facilitating their excretion. Isopods and Collembola have been
shown to be extremely efficient in biotransforming organic chemicals like Polycyclic
Aromatic Hydrocarbons (PAHs), which are excreted by these organisms with half lives
of approximately 1 day (Van Brummelen and Van Straalen 1996, Howsam and Van
Straalen 2003, Stroomberg et al. 2004). Earthworms seem less efficient in doing so.
Possible consequence of this rapid biotransformation is that potentially toxic metabo-
lites may be produced. This has been shown in isopods, with even DNA adducts being
formed upon exposure to PAHs (Van Brummelen et al. 1996a). This may also lead to
long-term damage and possible effects on subsequent generations as has been shown
for phenanthrene in the collembolan Folsomia candida (Leon Paumen et al. 2008). As

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very little is known about such multi-generation effects, further research on the long-
term effects of chemicals is urgently needed.
An important biological aspect that may affect the exposure of soil invertebrates
to chemicals is their behaviour. Soil by definition is a heterogeneous environment. As
a consequence, also the distribution of chemicals in soil is heterogeneous. Chemicals
reaching soil by areal deposition for instance accumulate in the topsoil layer, leading
to a depth-related concentration gradient as was shown for PAHs in forest soils (Van
Brummelen et al. 1996b). Depending on the habitat and mobility, organisms may be
more or less exposed to chemicals present in the topsoil layer. Similarly, the effect of
pesticides on earthworms was shown to be highly related to their mobility with epigeic
and anecic species being much more vulnerable compared to endogeic species. Espe-
cially anecic species, like Lumbricus terrestris, which come to the soil surface to forage
and mate at night, may experience a very high exposure shortly after pesticide spray-
ing (Edwards and Brown 1982). Also in case of spatially heterogeneous soil pollution,
behaviour may affect exposure, as was shown for earthworm exposure to copper in
a heterogeneously polluted soil by Marinussen and Van der Zee (1996) In the latter
study, knowledge of the uptake and elimination kinetics showed to be very helpful in
predicting metal concentrations in the earthworms living in a heterogeneously pol-
luted environment. Also in case of isopods, behaviour is an important factor determin-
ing exposure. Unfortunately, no research has been done on the chemical exposure of
isopods and consequent effects in relation to their behaviour in the field.
Multiple species, model ecosystem (microcosm) and field tests
All standardized toxicity tests with soil invertebrates focus on assessing the effects of
chemicals on single species of organisms. To enable assessment of toxic effects in a
more realistic setting, micro- or model ecosystems have been developed, ranging from
artificially composed set-ups with a number of selected different species introduced
in a well homogenized soil (e.g., Burrows and Edwards 2004) to intact soil columns
extracted from the field and incubated in the laboratory (e.g., Knacker et al. 2004).
Such model ecosystems or microcosms allow assessing effects at the community level,
taking into account the interactions between species. Although basically considered
single-species tests, earthworm and isopod tests focusing on decomposition or feed-
ing activity in fact also are multispecies tests as in these tests also the interaction with
microorganisms in the gut and in the soil or food are important. In addition, the end-
point (decomposition) has high ecological relevance for assessing potential effects on
the functioning of the soil ecosystem (e.g. Hobbelen et al. 2006). The only field test
available aims at assessing pesticide effects on earthworms (ISO 1999b), but can be
combined with a decomposition or litter bag test (Römbke et al. 2003, OECD 2006,
Dinter et al. 2008).
Since the introduction of the term Ecotoxicology, the question for “putting more
eco into ecotoxicology” has been raised. Some authors even argued that ecotoxicology

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Soil ecotoxicology: state of the art and future directions 285
should not be seen as a sub-discipline of toxicology but rather as a case of stress ecology
(Van Straalen 2003). This notion has triggered the focus on more ecologically relevant
test designs, integrated approaches including responses at different levels of biological
organization, and taking into account the normal operating range of parameters de-
scribing the structure and functioning of soil ecosystems.
Since early 2000, with the notion of stress ecology, more complex issues have been
receiving attention, with ecological vulnerability, trait-based analysis and effects on
functional endpoints (so-called ecosystem services) being key items (e.g., De Lange et
al. 2009, Saad et al. 2011). The application of these trait-based approaches in soil
ecotoxicology on one hand offers promising perspectives, on the other hand it also
demonstrates an enormous lack of knowledge on the traits represented by different
species and groups of soil invertebrates.
Diagnosis
Many of the tests initially developed for assessing the toxicity of single chemicals are
also used for assessing the toxicity of field samples. In addition to the tests mentioned
above, a bioassay using the nematode Caenorhabditis elegans has been developed by
ISO (2010) Such bioassays may be applied together with chemical analysis and field
observations. The resulting TRIAD approach is a useful tool for the actual risk assess-
ment of contaminated sites (Jensen and Mesman 2006). ISO (2008b) gives guidance
on the choice of different bioassays, depending on the purpose of the risk assessment
and taking into account aspects like land use.
Other diagnostic tools include effects at the biochemical level. Such biomarkers
may act as a sensitive, early warning indicator of possible effects at higher levels of
biological organization (Spurgeon et al. 2005), and also may provide information on
the mode of action of a chemical (Kammenga et al. 2000). Biomarkers may be applied
both to organisms captured from the field and to test organisms exposed to field sam-
ples under controlled laboratory conditions (see e.g. Van Gestel et al. 2009). Isopods
may be used for such biomarker studies (Köhler et al. 1999, Stroomberg et al. 1999,
Stanek et al. 2006, Drobne et al. 2008, Lemos et al. 2009), while also their potential
of accumulating metals has been proposed as a suitable monitoring tool (‘woodlouse
watch’ scheme) especially in metal-contaminated areas (Hopkin et al. 1993).
Spurgeon et al. (2005), comparing different biochemical endpoints, demonstrated
that responses at the gene level were most sensitive. This notion also plays a role in the
recent developments of genetic tools (genomics, proteomics and transcriptomics etc.),
which has resulted in a vast extension of the ecotoxicological tool box. Ecotoxicogenom-
ics nowadays is seen as a tool to enable better understanding of molecular mechanisms
of action of chemicals (Snape et al. 2004), while it may provide insight into the way
soil invertebrates are able to develop resistance to pollution, e.g. metal or pesticide
tolerance (Van Straalen et al. 2011). Ecotoxicogenomics may also help unravelling
the mechanisms by which (metal-based) nanoparticles affect organisms, as e.g. was

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determined for the nematode Caenorhabditis elegans (Roh et al. 2009, 2010). Ecotoxi-
cogenomics may also open the way for developing new diagnostic tools for assessing
possible effects of soil pollution (Van Straalen and Roelofs 2008, Nota et al. 2010).
Some authors have advocated that ecotoxicogenomics may enable bridging the gap be-
tween genes and populations (Fedorenkova et al. 2010). It remains however, uncertain
whether time is ready for such ‘from gene to population extrapolations’ (Van Straalen
et al. 2010). In a recent review, Van Straalen and Feder (2012) discuss the possible
use of environmental genomics in the ecotoxicological risk assessment of chemicals.
Community and population genomics may provide insight into the species composi-
tion at different sites and the possible relationship with pollution. Genome scans may
also provide information on genetic changes in specific species that have been exposed
to contaminated soils over many generations. Gene expression profiling may provide
information on toxicant-induced changes in gene expression. The meaning of these
changes however, remains unclear as the linkage between gene expression (transcrip-
tomics) and the functioning of the genes (proteomics) often is not straightforward.
At the moment, gene expression analysis is applied to only few species for which the
genome has more or less completely been described, like the nematode Caenorhab-
ditis elegans, the springtail Folsomia candida and the earthworms Lumbricus rubellus
and Eisenia fetida, thus limiting wider application. Information on background gene
expression is lacking, hampering a proper interpretation of responses under stressed
condition. Van Straalen and Feder (2012) therefore conclude that more research is
needed before genomics tools can make a sound contribution to the risk assessment
of chemicals.
Outlook
Final aim of (soil) ecotoxicology is the understanding of the long-term effects of chem-
icals on ecosystems. As such, focus on long-term sub-lethal effects is essential, but
it also requires detailed understanding of the processes of exposure, uptake, internal
processing (metabolism, sequestration) and intoxication in individual organisms as
well as the translation of effects to higher levels of biological organization. From the
overview presented in this paper, it may have become clear that soil ecotoxicology has
shown a tremendous development in the past 40 years. From the initial realization that
chemicals may affect soil organisms, through the development of standardized toxicity
tests and the use of soil chemistry to develop the concept of bioavailability, soil ecotoxi-
cology has grown to a mature field of science. The incorporation of biochemical and
omics tools on one hand and the link with ecology on the other hand, does guarantee
that soil ecotoxicology remains an important player in the field of stress ecology. In
spite of the promising developments outlined above, the following aspects need further
attention in the near future:

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Soil ecotoxicology: state of the art and future directions 287
Toxicity tests
Although several toxicity tests are available for soil organisms (Table 1), it is obvious
that the current battery is not complete and also not well balanced. As mentioned
above, it seems there is an under-representation of arthropods. Isopod toxicity testing
seems most advanced, while these organisms also represent an ecologically important
and relevant group of soil arthropods. In addition, they offer the possibility of expo-
sure via soil and food, while effects may be determined at different levels including
biochemical and genomics, individual (growth, behaviour) as well as ecological (feed-
ing activity). It therefore is recommended to put more effort on standardizing isopod
toxicity tests for sublethal endpoints. Finally, it has to be noted that the currently avail-
able toxicity tests may need adjustment to make them applicable for determining the
toxicity of new and emerging chemicals, like nanoparticles.
Bioavailability
For better enabling extrapolation from laboratory tests to the field and among soil
types, it is essential to get better understanding of the routes of uptake of chemicals
in organisms. This not only requires attention for the chemical aspects, but also needs
a greater emphasis of the ‘biological’ aspects of bioavailability. This may also require
paying closer attention to the way organisms are exposed in the field, and attention for
the dynamics of exposure and bioavailability.
Kinetics
For a better understanding of bioavailability but also of the toxicity of single chemicals
and mixtures, it is essential to increase our understanding of toxicokinetics and toxico-
dynamics. Such understanding will also enhance the possibilities to extrapolate effects
in time and to higher levels of biological organization, like the population level. Ki-
netics also is of great importance when considering the toxicity of new chemicals, like
nanoparticles, that may show changing properties with time as a consequence of aggre-
gation, agglomeration and dissolution processes. Finally, kinetics should not only ad-
dress whole organisms but should also include the way organisms deal with chemicals
internally (biotransformation, sequestration, internal distribution and translocation).
Ecology
For better understanding exposure in the field and predicting ecosystem effects, our
knowledge on the ecology of soil invertebrates needs much better development. Such
knowledge also is crucial for the description of the normal operating range of structural

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and functional endpoints and for the application of trait-based approaches to under-
stand and predict effects of chemicals on soil invertebrate communities and ecosystem
services provided by these communities.
Ecotoxicogenomics
For the application of genomics tools in the diagnosis of soil pollution it is essential
to better understand the link between gene expression level responses and ecologically
relevant endpoints. A better understanding of gene expression responses may also help
unraveling the mechanisms of action of chemicals, single and in mixtures, and as such
be helpful in predicting toxicity. In the long run, a better understanding of responses at
the genomics level may even provide tools for cross-species extrapolation and the devel-
opment of completely new models for mixture toxicity, especially when combined with
toxicokinetics and toxicodynamics data. Genomics tools may also help unraveling the
causes of long-term effects of chemicals, e.g. multi-generation effects as a consequence of
accumulation of damage in earlier generations. But all these applications will require an
enormous amount of information on the meaning of gene expression profiles in relation
to background conditions, in relation to chemical exposure both outside and inside the
body and related to ecologically relevant endpoints like growth and reproduction.
References
Allen HE (Ed) (2002) Bioavailability of Metals in Terrestrial Ecosystems: Importance of Parti-
tioning for Bioavailability to Invertebrates, Microbes, and Plants. Society of Environmen-
tal Toxicology and Chemistry (SETAC), Pensacola, Florida, USA.
Amorim MJB, Novais S, Römbke J, Soares AMVM (2008a) Avoidance test with Enchytraeus
albidus (Enchytraeidae): Effects of different exposure time and soil properties. Environ-
mental Pollution 155: 112–116. doi: 10.1016/j.envpol.2007.10.028
Amorim MJB, Novais S, Römbke J, Soares AMVM (2008b) Enchytraeus albidus (Enchytraei-
dae): A test organism in a standard avoidance test? Effects of different chemical substances.
Environment International 34: 363–371. doi: 10.1016/j.envint.2007.08.010
Bednarska AJ, Gerhardt A, Laskowski R (2010) Locomotor activity and respiration rate of the
ground beetle, Pterostichus oblongopunctatus (Coleoptera: Carabidae), exposed to elevated
nickel concentration at different temperatures: novel application of Multispecies Freshwa-
ter Biomonitor®. Ecotoxicology 19: 864–871. doi: 10.1007/s10646-010-0467-2
Bednarska AJ, Laskowski R (2008) Effects of nickel and temperature on the ground beetle
Pterostichus oblongopunctatus (Coleoptera : Carabidae). Ecotoxicology 17: 189–198. doi:
10.1007/s10646-007-0183-8
Bradham K, Dayton EA, Basta NT, Schroder J, Payton M, Lanno RP (2006) Effect of soil
properties on lead bioavailability and toxicity to earthworms. Environmental Toxicology
and Chemistry 25: 769–775. doi: 10.1897/04-552R.1

Page 15

Soil ecotoxicology: state of the art and future directions289
Burrows LA, Edwards CA (2004) The use of soil microcosms to assess the impact of carbendazim
on soil ecosystems. Ecotoxicology 13: 143-161. doi: 10.1016/S1164-5563(02)01153-6
Carson R (1962) Silent Spring. Houghton Mifflin Company, New York.
Crommentuijn T, Doodeman CJAM, Doornekamp A, Van der Pol JJC, Bedaux JJM, Van
Gestel CAM (1994) Lethal body concentrations and accumulation patterns determine
time-dependent toxicity of cadmium in soil arthropods. Environmental Toxicology and
Chemistry 13: 1781–1789.
De Lange HJ, Lahr J, Van der Pol JJC, Wessels Y, Faber JH (2009) Ecological vulnerability in
wildlife: an expert judgment and multicriteria analysis tool using ecological traits to assess
relative impact of pollutants. Environmental Toxicology and Chemistry 28: 2233–2240.
doi: 10.1897/08-626.S1
Dinter A, Coulson M, Heimbach F, Keppler J, Krieg W, Kölzer U (2008) Technical experiences
made with the litter bag test as required for the risk assessment of plant protection products
in soil. Journal of Soils and Sediments 8: 333–339. doi 10.1007/s11368-008-0028-y
Drobne D, Hopkin SP (1995) The toxicity of zinc to terrestrial isopods in a “standard” labora-
tory test. Ecotoxicology and Environmental Safety 31: 1–6. doi: 10.1006/eesa.1995.1037
Drobne D, Jemec A, Pipan Tkalec Z (2009) In vivo screening to determine hazards of na-
noparticles: Nanosized TiO2. Environmental Pollution 157: 1157–1164. doi: 10.1016/j.
envpol.2008.10.018
Drobne D, Blazic M, Van Gestel CAM, Leser V, Zidar P, Jemec A, Trebse P (2008) Toxicity of
imidacloprid to the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). Chemosphere
71:1326–1334. doi: 10.1016/j.chemosphere.2007.11.042
Drobne D, Rupnik M, Lapanje A, Štrus J, Janc M (2002). Isopod gut microflora parameters as
endpoints in toxicity studies. Environmental Toxicology and Chemistry 21: 604-609. doi:
10.1897/1551-5028(2002)021<0604:IGMPAE>2.0.CO;2
Edwards CA (1969) Soil pollutants and soil animals. Scientific American 220: 88-99. doi:
10.1038/scientificamerican0469-88
Edwards PJ, Brown SM (1982) Use of grassland plots to study the effect of pesticides on earth-
worms. Pedobiologia 24: 145–150.
Fedorenkova A, Vonk JA, Lenders HJR, Ouborg NJ, Breure AM, Hendriks AJ (2010) Ecotoxi-
cogenomics: Bridging the gap between genes and populations. Environmental Science and
Technology 44: 4328–4333. doi: 10.1021/es9037287
Fox CJS (1964) The effect of five herbicides on the number of certain invertebrate animals in
grassland soil. Canadian Journal of Plant Sciences 44: 405–409. doi: 10.4141/cjps64-080
Ghabbour SI, Imam M (1967) The effect of five herbicides on three Oligochaete species. Revue
d’Ecologie et du Biologie du Sol 4: 119–122.
Heckmann L-H, Hovgaard M, Sutherland D, Autrup H, Besenbacher F, Scott-Fordsmand J
(2011) Limit-test toxicity screening of selected inorganic nanoparticles to the earthworm
Eisenia fetida. Ecotoxicology 20: 226–233. doi: 10.1007/s10646-010-0574-0
Hobbelen PHF, Koolhaas JE, van Gestel CAM (2006) Effects of heavy metals on the litter
consumption by the earthworm Lumbricus rubellus in field soils. Pedobiologia 50:51–60.
doi: 10.1016/j.pedobi.2005.10.004