Print
Terrestrial isopods as model organisms in soil ecotoxicology: a review
expand article infoCornelis A.M. van Gestel, Susana Loureiro§, Promoz Zidar|
‡ Vrije University, Amsterdam, Netherlands
§ University of Aveiro, Aveiro, Portugal
| University of Ljubljana, Ljubljana, Slovenia
Open Access

Abstract

Isopods play an important role in the decomposition of leaf litter and therefore are making a significant contribution to nutrient cycling and soil ecosystem services. As a consequence, isopods are relevant models in soil ecotoxicology, both in laboratory toxicity tests and in field monitoring and bioindication studies. This paper aims at reviewing the use of isopods as test organisms in soil ecotoxicology. It provides an overview of the use of isopods in laboratory toxicity tests, with special focus on comparing different exposure methods, test durations, and ecotoxicological endpoints. A brief overview of toxicity data suggests that chemicals are more toxic to isopods when exposed through soil compared to food. The potential of isopods to be used in bioindication and biomonitoring is discussed. Based on the overview of toxicity data and test methods, recommendations are given for the use of isopods in standardized laboratory toxicity tests as well as in situ monitoring studies.

Keywords

Bioaccumulation, biomonitoring, indicator organisms, Isopoda, toxicity tests

Introduction

Increasing human activities have caused serious effects on man and the environment. Since the industrial revolution in the 19th century, pollution from industries and metal contamination from mining activities has increased. In the 20th century, after the Second World War, the massive use of pesticides resulted in widespread environmental pollution. The first evidence of chemical pollution was shown by air pollution, for instance leading to smog episodes in cities. Air pollution was a major problem already in the 19th century and still is one of the major factors threatening human health, especially in rapidly developing industrial regions like China (see e.g., Kan et al. 2012). The quality of surface waters also became seriously affected, with many incidences of fish killings all over the world (see e.g., Langer 1964); but reports on soil pollution for some time seemed to lag behind those on air and water pollution. Since the 1980s, however, soil pollution has also become highlighted as a major issue. It was realised that polluted soils are generally more difficult and considerably more expensive to remediate than cleaning or preventing water and air pollution (see e.g., Swartjes 2011).

Apart from threatening human health, air, water and soil pollution may also affect ecosystems. By affecting major functions of natural systems, pollution can alter the so-called ecosystem services (Hunt and Wall 2002; Faber and Van Wensem 2012), which include 1. provisioning services: material outputs from ecosystems, such as food, water, and other resources, 2. regulating services: ecosystems acting as regulators e.g., of the quality of air and soil or by providing flood and disease control, 3. habitat or supporting services: providing living space for plants or animals, maintaining a diversity of different breeds of plants and animals, etc., and 4. cultural services: non-material benefits people obtain from contact with ecosystems, such as aesthetic, spiritual and psychological benefits (The Millennium Ecosystem Assessment 2005; TEEB 2010). For maintaining ecosystem services, a high biodiversity is essential, as was recently shown by Soliveres et al. (2016).

Pollution may damage ecosystems and by that the support ecosystems provide to (the quality of) human life. Protection of ecosystem services therefore is essential not only for safeguarding the health of our ecosystems but also for our own benefit (Faber and Van Wensem 2012; TEEB 2010), and should thus receive more attention in the risk assessment of chemicals (see e.g., Nienstedt et al. 2012; Forbes and Calow 2013; Maltby 2013). For that reason, ecotoxicologists and ecologists are aiming at protecting ecosystems, including all key-organisms that together contribute to its functioning.

Isopods play an important role in the functioning of soil ecosystems and therefore also in the ecosystem services provided by soils. They act mainly on the first processes of litter fragmentation, contributing to the input of high quality organic matter, and increasing the microbiome for further nutrient cycling in soil (Hassall et al. 1987). This paper will discuss the possible role of isopods as model organisms in ecotoxicology. For that purpose, before focusing on isopods, we will first briefly describe some ecotoxicological concepts and the general principles of ecotoxicological risk assessment. Next we will discuss the use of isopods as model organisms in predictive (and diagnostic) ecotoxicological approaches. This will include the use of different endpoints, the relevance of different routes of exposure and the importance of bioavailability, and will be supported by a review of the literature on the toxicity and bioaccumulation of different chemicals in isopods. Finally we will discuss the use of isopods as diagnostic tools in monitoring soil quality.

Ecotoxicology and ecotoxicological risk assessment

After the publication of the book Silent Spring by Rachel Carson (1962), awareness of the side effects of pesticides and other chemicals on the environment increased. The term ecotoxicology was coined only in 1977 by Truhaut. The aim of ecotoxicology is to protect ecosystems from adverse effects of chemical pollution, alone or in combination with other anthropogenic stressors. To realise its aims, ecotoxicology makes use of knowledge from different disciplines, like environmental chemistry, toxicology, and ecology (Van Leeuwen and Vermeire 2007).

Environmental chemistry provides insight into the way chemicals interact with components of the environment, determining their fate, and therefore also the exposure of organisms and ecosystems. From knowledge of persistence and partitioning, environmental chemistry enables estimating Predicted Environmental Concentrations (PEC), which are the starting point for the risk assessment of chemicals. One important issue is bioavailability: the notion that total concentration of a chemical in the environment is not indicative of its risk, because only a fraction of it may be available for uptake and therefore causing effects (Van Gestel 2008, 2012). Especially in soils, due to their large capacity of complexing chemicals, bioavailability is an important issue. Therefore, and besides the distribution of chemicals in environmental compartments, bioavailability will focus also on the fate of chemicals in the organisms, bridging the gap between what is in the environment and the compartmentalisation of chemicals inside biota (external versus internal bioavailability) (Vijver et al. 2004).

Toxicology provides insight into the interaction of chemical pollutants with molecules, tissues, and organs resulting in effects at the molecular and individual level (Van Leeuwen and Vermeire 2007). From (laboratory) tests, dose-response relationships are derived to provide quantitative measures of the toxicity of chemicals, like LC50 (lethal concentration killing 50 % of the tested population), EC10 or EC50 (concentrations causing 10 % or 50 % reduction in a measured parameter, e.g., growth or reproduction, compared with the response in the untreated control) or NOEC (No-Observed Effect Concentration, the highest concentration tested causing no significant reduction in a measured parameter compared to the untreated control). LC50 values usually are determined in short-term or acute toxicity tests, with mortality as the main endpoint. EC10, EC50, and NOEC values are derived in sublethal toxicity tests, using growth, reproduction or another sublethal endpoint. Such tests usually require longer exposure times, and are therefore sometimes indicated as chronic tests. The term chronic however, should only be used when test duration includes a substantial part of the life span of the test organism. From the available toxicity data, a safe concentration of a pollutant can be derived, as a so-called Predicted No-Effect Concentration (PNEC). Different approaches may be followed to derive a PNEC, extrapolating effects from acute (LC50) to sublethal effects (EC10, EC50 or NOEC), from one or few species to many species and from the laboratory to the field. The resulting PNEC should be protective of effects not only at the individual level but at higher levels of biological organisation, like populations and communities.

Ecology provides the knowledge for the extrapolation from individual-level effects to effects on populations, communities, and ecosystems. Ecology adds knowledge about life histories of organisms, their functioning in different processes or their interactions with other species or the abiotic environment. Knowledge on life histories and other ecological properties or traits of species also helps understanding how organisms will be exposed to chemicals in the environment and how this may affect populations. The behaviour of an organism may for instance differ depending on its life stage, while it may have great influence on exposure. Again, this seems even more important in soils, where pollution is only rarely distributed homogenously. Finally, knowledge about ecological interactions between species may help translating effects to the community and ecosystem level.

Ecotoxicology may follow predictive/prospective and diagnostic/retrospective approaches (Van Gestel 2012). Predictive approaches aim at preventing possible effects of chemicals before they are introduced onto the market or to limit side effects after introduction. Predictive ecotoxicological risk assessment of chemicals is based on a comparison of the hazard of a chemical, expressed as its toxicity or safe levels derived from that (PNEC values), with predicted or measured exposure levels (PEC or MEC). This will provide an assessment of the possible risk predicted or diagnosed for exposed ecosystems. Standards or risk limits for chemicals in the environment generally are based on PNEC values, sometimes lowered by application of a safety or uncertainty factor, depending for instance on the availability of toxicity data (number of data, representation of different species (see also below), endpoints etc.).

Diagnostic approaches are applied to monitor possible effects of chemicals after introduction onto the market, e.g., as post-registration monitoring of pesticides, or to assess the actual risk of contaminated soils. Diagnostic risk assessment, for instance of contaminated soils, relies on a combination of ecological, toxicological and chemical approaches. Basically, toxicity tests (in this case usually called bioassays) are used as diagnosis tools to assess the toxicity of soil samples from the contaminated site. Results of the bioassays are considered together with those of measurements of total or available concentrations of a selected number of chemicals and ecological field observations. The added value of bioassays is that they provide information on the actual risk of bioavailable concentrations of all chemicals present in the contaminated samples; such information cannot be obtained from chemical analyses (see e.g., van Gestel et al. 2001; Loureiro et al. 2005, 2006). Together these approaches result in an assessment of the potential risk of soil contamination, for instance in the TRIAD approach, which combines three lines of evidence: chemistry (total or available concentrations of pollutants), toxicity of the polluted soil and on site ecological observations (Jensen and Mesman 2006).

Toxicity tests

One key issue in ecotoxicological risk assessment is the selection of test species for generating the required toxicity data. For a proper risk assessment it is crucial that test species are representative of the community or ecosystem to be protected. Criteria for selection of tests and therefore also for organisms to be used in toxicity tests have been summarised by Van Gestel et al. (1997). They include 1. Practical arguments, including issues like feasibility, cost-effectiveness and rapidity of the test, 2. Acceptability of tests, like the need to be reproducible and standardised, and 3. Ecological significance, including sensitivity, biological validity etc.

There is no species that is most sensitive to all pollutants. Which species is most sensitive depends on the mode of action and possibly also other properties of the chemical, and the properties of the organism (e.g., presence of specific targets, physiology, etc.). It is therefore important to always test a number of species, with different life traits, functions, and position within a trophic chain. Such a battery of test species should be (according to Van Gestel et al. 1997):

  1. Representative of the ecosystem to protect, so including organisms having different life-histories, representing different functional groups, different taxonomic groups and different routes of exposure;
  2. Representative of responses relevant for the protection of populations and communities; and
  3. Uniform, so all tests in a battery should be applicable to the same test media and applying the same test conditions.

Once tests have been developed, accepted, and validated, they may be standardised by international organisations like the International Standardization Organization (ISO) or the Organization for Economic Co-operation and Development (OECD). Chemical registration authorities usually only accept results of tests standardised by these organisations. For the soil environment different OECD and ISO standardised tests are available (for an overview see e.g., Van Gestel 2012), including earthworms, enchytraeids, springtails, insect larvae and molluscs as test species. ISO tests are primarily focusing on diagnostic/retrospective risk assessment while OECD tests are required for predictive/prospective risk assessment. Some tests determine short-term or acute exposures, usually with mortality as the endpoint, while others focus on sublethal endpoints like reproduction. Also tests on avoidance behaviour with earthworms (ISO 2008) and springtails (ISO 2011) have been developed as a fast method of assessing another relevant sublethal endpoint.

The ecological relevance of isopods, their typical routes of exposure (soil, food) and life history characteristics, the possibility to determine different endpoints (see below), and the fact that they have already been used for testing for more than 30 years, make them highly suitable test organisms (Drobne 1997; Van Gestel 2012). Unfortunately, so far, no toxicity tests on isopods have reached the level of standardisation, although several methods have been applied in the literature. One reason for the lack of a standardised test with isopods could be their relatively long and more complex life cycle compared to springtails or earthworms. This makes it more difficult, at least for a number of species, to culture them in the lab. For many isopod species it takes several months or more from hatching to reproductive age. Another problem is the rather large variation in test results, which could in part be attributed to the difficulty of obtaining age-synchronised groups of test animals but could also be an intrinsic property of isopods, like the moulting process or their daily excretion process within the hepatopancreas. Both the long life span and the large variation, together with the ability of females to store sperm, make it more difficult to standardise a reproduction toxicity test. Another point which may hamper the standardisation of toxicity tests with isopods may be related to the uncertainty about the main route of exposure (soil vs. food; see below). Nevertheless, there are possibilities of getting around these problems, as will become clear from the following sections.

Main sources of isopod exposure in soil

In terrestrial systems, several pollutants can reach the soils from diverse sources and can also be found in decaying organic matter. These sources can be considered as point and/or diffuse (non-point) and they will include different forms of contamination: gaseous (atmospheric), solid or liquid hazardous substances that will be mixed with soil.

Industry and commerce are two of the major economic sectors responsible for soil contamination (36 %), either by negligence or by accident (European Environment Agency (EEA); www.eea.europa.eu). This includes atmospheric emissions from production, spilling or burying chemical substances directly in the soil or through runoff from surrounding areas. Such polluted sites are common in Europe and worldwide, and are commonly named as historically-contaminated sites, where metals (37.3 %) and mineral oils (33.7 %) are the main harmful substances to be considered, followed by Polycyclic Aromatic Hydrocarbons (PAHs) (13.3 %), Aromatic Hydrocarbons (BTEX) (6 %), phenols (3.6 %), chlorinated hydrocarbons (2.4 %), and other chemical compounds (3.6 %) (EIONET priority data, EEA). Along with industrial and commercial sources, waste treatment and disposal is another main source of soil contamination (EEA; www.eea.europa.eu). Sewage sludge is often applied to agricultural fields as fertiliser. This provides a considerable input of hazardous substances to soils, some of them considered as emerging pollutants, like those in daily care products, pharmaceuticals, or nanomaterials. In addition, Phthalates (e.g., diethylhexylphthalate (DEHP), dibutylphthalate (DBP)), Octylphenols, Nonylphenols, Linear alkylbenzene sulfonates (LAS), Polychlorinated biphenyls (PCBs), PAHs and metals are amongst the substances most commonly found in sewage sludge that is applied to soils for agricultural purposes. Considering this cocktail of chemicals along with pesticides and fertilisers, agricultural soils are a major sink for contaminants. Pesticides have been introduced by man in ecosystems initially as natural compounds, by using poisonous plants or extracting chemical substances from other natural sources. Later in the 20th century, especially after the 2nd World war, anthropogenically modified or synthesised compounds have been introduced at a large scale, that nowadays represent a wide range of organic chemical groups, including organophosphates, carbamates, triazines, organochlorines, pyrethroids, neonicotinoids, sulfonylurea and biopesticides (EEA; www.eea.europa.eu).

Along with agricultural areas, urban areas have been identified as hotspots for soil contamination (Santorufo et al. 2012), with sources like petrol stations and mechanical workshops where PAHs can be found (Din et al. 2013), traffic and urban roads as sources of metals (e.g., copper, zinc, lead) or changing soil pH (see e.g., Falkengren-Grerup 1986; Løkke et al. 1996; Blake et al. 1999), and agricultural practices in small gardens leading to increasing levels of several contaminants like metals and organic chemical substances. One of these case studies was performed in China where agricultural plastic film was spotted as a possible important source of soil phthalate ester contamination in a suburban area (Wang et al. 2013).

Lately emerging chemicals like nanomaterials (e.g., nanoparticles) are showing a potential risk to aquatic and terrestrial organisms as they are being used for different applications and are expected to appear in the environment, mainly through sewage sludge discharges. Recently also microplastics have been added to this list of pollutants potentially threatening the soil environment (Rillig et al. 2017), although some biodegradable plastics can also be used by isopods as a food source facilitating the degradation of these materials (Wood and Zimmer 2014).

In addition to chemical stress, natural stress can also affect the performance of soil organisms, including isopods. Considering their evolution from water to land, terrestrial isopods have acquired several features to succeed their appearance in terrestrial environments. One limiting habitat property is moisture content, while temperature, salinity, and UV radiation increase are also of importance. These factors can become stressors on their own when tending to extremes but they can also act as joint stressors in addition to chemicals. This joint effect can be caused by the interaction of stressors in exposure media but most interestingly also inside the organisms. It is known that several environmental conditions like temperature fluctuation patterns will influence an organism’s physiology and behaviour, changing therefore the metabolism of chemicals upon exposure or affecting its behaviour, e.g., its aggregation behaviour (Hassall et al. 2012). Donker et al. (1998) showed that zinc was highly toxic to Porcellio scaber at higher temperatures mainly due to an increase in isopod metabolism rather than due to the increase of zinc body burdens. UV radiation was also reported to harm isopods (Porcellionides pruinosus) by itself, showing that juveniles and pre-adults were more affected than adults, mainly for energy-related parameters (Morgado et al. 2013). Besides temperature, there are only few studies investigating the effects of exposure conditions on the toxicity of chemicals to terrestrial isopods.

Routes of exposure

In soil, chemicals may be distributed over different compartments, the soil solid phase, pore water, and air. Isopods living on and in the soil may be exposed to all three compartments, with food acting as another compartment from which chemicals may be taken up. Very little data is available on the way isopods are exposed and on what the relative importance of each route of exposure is. Likely, the relative importance of either route of exposure is dependent on the properties of the chemical, including its volatility, water solubility and sorption or soil/water partition constant, and the properties of the soil, like organic matter content, clay content, and pH. As a consequence of these factors, bioavailability and therefore exposure may be quite different for soil and food. For volatile chemicals inhalation may present another route of uptake, which again will be hard to quantify. It therefore remains hard to predict the role of either route of exposure, and this will also require knowledge of several factors related to isopod behaviour (see Løkke and Van Gestel 1998 for some considerations on routes of exposure).

In the toxicity tests described in the literature, generally two routes of exposure have been tested, usually separately: exposure to food only or to soil only (see Table 1 for a summary of literature data on the toxicity of selected chemicals to isopods; Suppl. material 1: Table S1 in the Supporting Information provides a more detailed but not exhaustive overview of toxicity data). In few cases, however, animals were exposed to both treated food and treated soil. In the latter case, it should be noted that it will be very hard to realise the same exposure in terms of bioavailable concentrations in food and soil as properties may also be quite distinct with food generally containing much higher organic matter contents than soil (but with different properties). As a consequence, Fischer et al. (1997) found that dimethoate was more toxic for Porcellio scaber when mixed in with soil than with food. This finding was supported by Hornung et al. (1998b) with data for copper and LAS, using the same species, by Sousa et al. (2000) studying the accumulation of lindane in Porcellionides pruinosus and by Vink et al. (1995) determining toxicity of benomyl, carbofuran, and diazinon to the latter species.

Summary of data on the toxicity of chemicals to isopods in different tests with exposures in soil or through food. For each chemical, species and endpoint, the lowest value is reported. For a more complete overview of data, it is referred to the Supporting Information.

Test compound Species Soil/food Time (d) Criterion Endpoint Result (mg/kg dry soil or food) Reference
2-phenylethyl isothiocyanate Porcellio scaber food 28 LC50 survival >1000 Van Ommen Kloeke et al. 2012
Lufa2.2 28 LC50 survival 65.3
3-phenylpropionitrile Porcellio scaber food 28 LC50 survival >1000 Van Ommen Kloeke et al. 2012
Lufa2.2 28 LC50 survival 155
abamectin Porcellio scaber Lufa 2.2 21 LC50 survival 69 Kolar et al. 2008
Lufa 2.2 21 NOEC weight loss 3 Kolar et al. 2010
AgNO3 Porcellionides pruinosus food 14 EC50 growth 233 Tourinho et al. 2015
Lufa 2.2 14 LC50 survival 396
14 EC50 consumption 56.7
2 EC50 avoidance 13.9
AgNPs Porcellionides pruinosus food 14 EC50 growth >1500 Tourinho et al. 2015
Lufa 2.2 14 LC50 survival >455
14 EC50 growth 114
2 EC50 avoidance 15.8
benomyl Porcellionides pruinosus 2 Soils 14 LC50 survival >1000 Jänsch et al. 2005
benzo[a]anthracene Oniscus asellus food 329 NOEC growth 3 Van Brummelen et al. 1996a
Porcellio scaber food 112 NOEC growth >9.6
benzo[a]pyrene Oniscus asellus food 63 NOEC growth 10.6 Van Brummelen et al. 1996a
Porcellio scaber food 63 NOEC growth 10.6
bisphenol A Porcellio scaber sandy soil 112 NOEC growth ≤10 Lemos et al. 2010
70 LC50 survival 910 Lemos et al. 2009
carbendazim Porcellionides pruinosus 2 Soils 14 LC50 survival >1000 Jänsch et al. 2005
Cd Armadillidium vulgare food 21 NOEC MT/ HSP70 expression 43.14 Mazzei et al. 2015
Oniscus asellus food 91 LC50 survival ~1600 Crommentuijn et al. 1994
Porcellio scaber food 308 LC50 survival 86 Crommentuijn et al. 1995
70 EC10 growth/biomass 1.35 Abdel-Lateif et al. 1998
21 LOEC food selection 20 Zidar et al. 2005
21 NOEC moulting/survival >200
Porcellionides pruinosus food 28 EC50 egestion ratio 370 Loureiro et al. 2006
28 LOEC assimilation efficiency 19850
chloranthraniliprole Porcellio scaber Lufa 2.2 32 LC50 survival >1000 Lavtizar et al. 2016
32 NOEC growth >1000
chlorpyrifos Porcellionides pruinosus Lufa 2.2 14 NOEC biomass ≥3 Morgado et al. 2016
Cu Porcellio scaber food 28 EC10 growth 45 Farkas et al. 1996
28 LC50 survival 1117
Lufa 2.2 28 NOEC growth 500 Hornung et al. 1998a
28 LC50 survival 3755
Porcellionides pruinosus food 28 EC50 consumption ratio 1038 Loureiro et al. 2006
28 EC50 egestion ratio 483
28 LOEC assimilation efficiency >10500
Lufa 2.2 2 EC50 avoidance behavior 802 Loureiro et al. 2005
dimethoate Porcellio scaber food 28 LC50 survival >75 Fischer et al. 1997
28 NOEC growth >75 Hornung et al. 1998
2 soils 28 EC10 female gravidity 3.8 Fischer et al. 1997
Lufa 2.2 28 NOEC growth 10 Hornung et al. 1998a
28 NOEC food consumption 10
Porcellio dilatatus black silt 2 NOEC active time <5 Engenheiro et al. 2005
Porcellionides pruinosus Lufa 2.2 2 EC50 avoidance behavior 28.7-39.7 Loureiro et al. 2009; Santos et al. 2010
doramectin Porcellio scaber Lufa 2.2 21 LC50 survival >300 Kolar et al. 2008
endosulfan Porcellio dilatatus food 21 NOEC glycogen / lipids <0.1 Ribeiro et al. 2001
fluoranthene Oniscus asellus food 329 NOEC growth, reproduction >267 Van Brummelen et al. 1996a
fluorene Oniscus asellus food 329 NOEC protein (females) 7 Van Brummelen et al. 1996a
Porcellio scaber 112 NOEC growth >219
glyphosate Porcellionides pruinosus Lufa2.2 2 EC50 avoidance behavior 39.7 Santos et al. 2010
imidacloprid Porcellio scaber food 14 NOEC growth 5 Drobne et al. 2008
Lufa 2.2 28 LC50 survival 7.6 De Lima e Silva et al. 2017
lambda-cyhalothrin Porcellionides pruinosus 2 Soils 14 LC50 survival 0.5-1.4 Jänsch et al. 2005
14 EC50 reproduction 0.13-0.4
lasalocid Porcellio scaber Lufa 2.2 28 NOEC growth 202 Žižek and Zidar 2013
2 NOEC avoidance behavior <4.51
mancozeb Porcellionides pruinosus Lufa 2.2 14 NOEC biomass 176 Morgado et al. 2016
Ni Porcellionides pruinosus Lufa2.2 1-8 NOEC integrated biomarkers 50 Ferreira et al. 2015
parathion Porcellio dilatatus food 21 NOEC AChE <0.1 Ribeiro et al. 1999
Pb Armadillidium vulgare food 21 NOEC MT/ HSP70 expression 478 Mazzei et al. 2015
Porcellio scaber food 80 NOEC oxygen consumption 1178 Knigge and Köhler 2000
Porcellionides pruinosus food 28 EC50 egestion ratio 14050 Loureiro et al. 2006
28 LOEC assimilation efficiency >42070
28 LOEC growth efficiency >31790
phenanthrene Oniscus asellus food 329 NOEC growth, reproduction >235 Van Brummelen et al. 1996a
Porcellionides pruinosus Lufa 2.2 14 LC50 survival 110-143 Tourinho et al. 2015
14 EC50 biomass 16.6-31.6
spirodiclofen Porcellionides pruinosus Lufa2.2 2 EC50 avoidance behavior 0.9 Santos et al. 2010
thiacloprid Porcellio scaber Lufa 2.2 28 LC50 survival >32 De Lima e Silva et al. 2017
28 EC50 consumption >32
TiO2 NPs Porcellio scaber food 3 NOEC CAT/GST >3000 Jemec et al. 2008
tributyltin Porcellionides pruinosus food 14 NOEC consumption rate 1 Silva et al. 2014
soil 14 LC50 survival 99.2
2 EC50 avoidance behavior <0.2
vinclozolin Porcellio scaber sandy soil 70 LC50 survival 298 Lemos et al. 2009
35 NOEC molt delay 10
Zn Porcellio scaber food 72 EC50 growth 1916 Van Straalen et al. 2005
35 NOEC fecal production 1000 Drobne and Hopkin 1995
Porcellionides pruinosus food 28 EC50 consumption ratio 11100 Loureiro et al. 2006
28 EC50 assimilation efficiency 3650
28 EC50 egestion ratio 3520
4 Soils 14 LC50 survival 1792-2352 Tourinho et al. 2013
14 EC50 biomass 312-1400
ZnO non-nano Porcellionides pruinosus 4 Soils 14 LC50 survival 2169-2894 Tourinho et al. 2013
14 EC50 biomass 119-1951
ZnO NPs (3–8 nm) Porcellionides pruinosus 4 Soils 14 LC50 survival 1757->3369 Tourinho et al. 2013
14 EC50 biomass 713-1479

Food can be an important route of exposure, especially in case of input from the air with freshly fallen decaying leaves containing high levels of pollution. Food exposure may also be more uncertain and more difficult to quantify due to the possibility of isopods avoiding contaminated food. It is known that isopods may be able to survive for very long time without food (Donker 1992), but starvation will of course affect sublethal endpoints like reproduction by affecting energy reserves.

Vijver et al. (2005) demonstrated that surface adsorption of metals is negligible compared to uptake, so dermal uptake seems less relevant than oral uptake unless metals may pass the skin to be internalised rapidly. As mentioned above, soil exposure was shown to be more effective in causing toxicity and bioaccumulation of chemicals in isopods, so it seems that in spite of the low surface sorption, dermal uptake may be an important route of exposure of isopods.

In addition, it should be noted that organisms may affect exposure by their behaviour. Feeding behaviour affects the dietary exposure, while mobility may play a role in determining the degree of contact with soil and therefore may affect soil exposure.

It will be difficult to construct an experiment that completely separates the different exposure routes. This also is not necessary when the focus is not on the mechanisms behind uptake and accumulation but rather on the consequences of exposure in terms of the toxicity. The amount of chemical accumulated instead of the concentration in the environment might provide a suitable measure of exposure and integrates aspects of bioavailability and route of exposure (Escher and Hermens 2004).

It is also not self-evident that the route of exposure will be the same under laboratory and field test conditions. In most standard laboratory tests, the test animal is kept on a relatively thin, homogeneous soil layer and is (if food is provided) forced to feed on a single food item (Hornung et al. 1998a). The only possibility to avoid the polluted food item is to stop feeding, which will only occur when the pollutant makes the food item distasteful or when it is affecting the health of the organism (see e.g., Zidar et al. 2004). In field or mesocosm studies, various food items may be available and test animals may have the ability to escape to less contaminated, deeper layers or to safe micro sites in the heterogeneous soil environment, or simply switch to less toxic food.

Toxicity testing with isopods: species selection

The first record on relating isopods to terrestrial contamination was a study from Martin et al., in 1976, where the availability and uptake of several metals from woodland litter were recorded and described in the woodlouse Oniscus asellus. In 1977, this species was mentioned as a biomonitor of environmental cadmium (Coughtrey et al. 1977) and later, in 1982, Oniscus asellus was used by Hopkin and Martin to study the distribution of several metals present in soil. Isopods were mentioned as bioindicators of zinc pollution in England (Hopkin et al. 1986), using species like Porcellio scaber and Oniscus asellus (Hopkin et al. 1989). Porcellio scaber was used as test species in ecotoxicology for the first time in 1978, by Beeby, where the combined uptake of lead and calcium was studied and discussed, continuing later studying the effects of lead assimilation on the brood size of this species (Beeby 1980).

In 1991, Armadillidium vulgare appeared as a test species in a study on the sequestration of copper and zinc in the hepatopancreas, and its relation with previous exposure to lead (Tomita et al. 1991). Vink et al. (1995) discussed the importance of exposure routes and for that used the saprotrophic species Porcellionides pruinosus. In 1998, Porcellio laevis appeared as a test species to be used in laboratory experiments where mortality, body mass and behaviour were recorded upon lead exposure (Odendaal and Reinecke 1998). One year later, Ribeiro et al. (1999) described the effects of parathion-ethyl and endosulfan-sulfate on enzyme activities (AChE and LDH) of Porcellio dilatatus, also showing that this species was a good candidate for biomarkers in ecotoxicity tests.

Porcellionides pruinosus has more recently been proposed as a suitable test species for ecotoxicity testing. The advantage of this species is its somewhat shorter life cycle making it easier to culture, and therefore it is more suitable for performing reproduction toxicity tests (e.g., Jänsch et al. 2005). Although this species seems less abundant in temperate zones, and is more representative of Mediterranean and tropical regions, it can easily be tested at 20 °C, which is the standard temperature for most soil toxicity tests.

Based on the above, it remains difficult to recommend one or the other species for toxicity isopod testing. A toxicity test with isopods may therefore use different species, but its duration and design may differ depending on the species chosen.

Toxicity testing with isopods: exposure set-up and endpoints

Although no standard test guidelines are available for assessing chemical toxicity to isopods, they are used as test organisms, applying different routes of exposure (food, soil), different test durations, and different endpoints. Table 2 provides an overview of different isopod toxicity tests and the endpoints determined as described in the literature. Figures 1–2 show some typical experimental setups employed for toxicity testing with isopods.

Figures 1–2. 

1 Design of feeding inhibition tests with isopods, applying exposure through food (left) or to contaminated soil with contaminated or uncontaminated food (right). In the test with contaminated food only, the animals are kept on a net or gauze allowing also for collecting faeces produced; this will enable estimating food assimilation efficiency. By offering the animals pre-weighed disks or pieces of leaf, food consumption can easily be determined. 2 Design of an avoidance test with isopods. The test uses containers with two compartments. One compartment is filled with contaminated soil, the other one with clean soil. After two days of exposure, the position of the animals in the container is checked. By testing a range of concentrations, including a control (clean soil in both compartments), a dose-response relationship for avoidance may be obtained. The test may also be used to assess avoidance responses to field-contaminated soils, but in that case it might be more difficult to find a proper control soil. Drawing made by Paula Tourinho.

Toxicity tests with isopods in soil use either artificial soil, prepared following the methods described by OECD (1984), or natural soils. Since the work of Hornung et al. (1998a), the use of the German standard soil Lufa 2.2 seems most preferred. This soil has a fairly constant composition, with properties appearing less variable than those of artificial soils prepared by different laboratories (see e.g., Bielska et al. 2012). Generally chemicals are mixed in with the soil at different concentrations to assess toxicity. Food exposure uses different matrices, ranging from intact tree leaves (or pieces of leaf) to finely ground leaf material or pellets prepared from tree leaves, sometimes mixed with some other materials. In case intact pieces of leaf are used, they are either soaked, smeared or sprayed with solutions containing different concentrations of the test chemical to achieve a range of exposure. Ground leaves of pelleted food may be spiked by mixing in the test chemicals at different concentrations. In all cases, both soil and food, a control without test chemical is included. In case the chemical has low water solubility and spiking the chemical required a solvent, a solvent control is included as well. Another methodology has been also applied by mixing chemical powder directly in soil and afterwards adjusting its moisture content, which has proved to be a good and reliable technique (Loureiro et al. 2005).

Toxicity tests with isopods have been carried out with several endpoints (Tables 1, 2). Along with the systems biology approach, exposures have been carried out using endpoints at the individual, organ, tissue, and cell or even at the molecular level. Tests focusing on whole organism responses (as shown in Table 2) usually range from 7 to 28 days and evaluate parameters like growth, feeding activity, moulting, mortality or behaviour and > 28 days when reproduction is the endpoint (e.g., Hornung et al. 1998a; Drobne and Hopkin 1995; Drobne and Štrus 1996; Loureiro et al. 2005). These parameters are considered ecologically relevant and usually related to the specific ecological properties of isopods or/and their role in soil function. As examples, feeding inhibition tests are closely related with the isopods’ function as detritivores, and reduced feeding rate as a consequence of either avoidance or intoxication by ingestion of contaminated food is related to the soil habitat function. Feeding inhibition tests were first developed using contaminated food as exposure route. This could be somehow difficult to transpose to real contamination scenarios and therefore new strategies for using this ecologically relevant parameter were developed. New studies on the effects of soil contamination on the feeding activity of isopods have been carried out, along with effects on their biomass (Tourinho et al. 2013).

Overview of isopod toxicity and bioaccumulation test methods described in the literature. References are just given as an example; in many cases several papers are available describing a more or less similar method of testing. For an overview of toxicity data generated using these methods, see Table 1 and also Table S1 in the supporting information.

Toxicity test Species Age Exposure time (days) Route of exposure Endpoints Test validity criteria References
acute toxicity Porcellionides pruinosus adult 14 soil survival Tourinho et al. 2013
growth toxicity Porcellio scaber juvenile 28 soil (artificial and natural) survival, biomass change control mortality <20% Hornung et al. 1998a
food pellets, leaf litter survival, biomass change control mortality <20%
reproduction toxicity Porcellio scaber adult up to 70 soil (artificial and natural) survival, oosorption, gravid females, offspring control mortality <20% Hornung et al. 1998a
food pellets, leaf litter survival, oosorption, gravid females, offspring control mortality <20%
Porcellio dilatatus adult 54 food (lettuce incorporated in gelatine) survival, time to pregnancy, pregnancy duration, abortions, juveniles Calhôa et al. 2012
feeding inhibition Porcellio scaber adult 21 food (pellets) food consumption rate, chemical assimilation, growth, moulting and survival Zidar et al. 2005
Porcellionides pruinosus pre-adult 14 soil consumption rate, assimilation rate, biomass change Tourinho et al. 2013; Silva et al. 2014
food (leaf litter) consumption rate, assimilation rate, excretion rate, biomass change Loureiro et al. 2006
feeding inhibition Porcellio scaber
Oniscus asellus
adult 35 food (leaf litter) feeding rate, excretion rate, assimilation efficiency, accumulation, chemical ingestion Drobne and Hopkin 1995
Porcellio scaber adult 28 food (leaf litter or pellets) body mass gain, food consumption, gravid females, juveniles Farkas et al. 1996
84 food (leaf litter or pellets) survival
avoidance behaviour Porcellionides pruinosus adult 2 soil % avoidance, habitat function no avoidance in control vs control Loureiro et al. 2005
foraging behaviour Porcellio scaber adult 2 food preference (video tracking) Zidar et al. 2005
bioaccumulation Porcellionides pruinosus pre-adult 40 (21 uptake; 19 elimination) soil bioaccumulation, kinetics Sousa et al. 2000
pre-adult 41 (21 uptake; 20 elimination) food (leaf litter) bioaccumulation, kinetics
bioaccumulation Porcellio scaber adult 32 (16 uptake; 16 elimination) Leaf powder bioaccumulation kinetics Kampe and Schlechtriem 2016

Avoidance response behaviour to contaminated food or soil is also the fastest endpoint, with test durations of no more than two days. No standard test guideline for avoidance tests with isopods is available, but such tests have been done by e.g., Loureiro et al. (2005) and Zidar et al. (2005) using Porcellionides pruinosus and Porcellio scaber, respectively. The latter test did not only focus on avoidance but also included foraging behaviour as an endpoint. Avoidance tests may be as sensitive as reproduction for some chemicals, while for others it is at least as sensitive as survival. Behavioural response studies with isopods have been developed due to organisms’ ability to detect chemicals, by using their chemoreceptors located on the second antenna, as a major advantage for establishing avoidance behaviour tests (Loureiro et al. 2005). The methodology proposed by Loureiro et al. (2005) was adapted from the earthworm avoidance behaviour test (ISO 2008). It is easy to perform and suitable to evaluate laboratory soils spiked with organic chemicals, metals, single and as binary mixtures (Loureiro et al. 2009), but also with natural contaminated soils from an abandoned mine (Loureiro et al. 2005).

Another interesting endpoint that is directly related to decomposition is the microbiome in the isopod gut (Drobne et al. 2002). This may also provide information on how colonisation and further decomposition of isopod faeces in the environment is influenced by the presence of chemicals.

In addition, mortality, growth, and reproduction are more related to the population level and bridge the gap to higher levels of biological organisation. These studies are more difficult to perform, as mentioned above, due to the life-span of isopods and their moulting behaviour. Reproduction tests with isopod have been performed in a 48-week exposure test via food contaminated with PAHs using Oniscus asellus (Van Brummelen et al. 1996a), and by Lemos et al. (2010) using Porcellio scaber exposed to bisphenol A and vinclozolin.

Looking at lower organisational levels, biomarkers are defined as any measurable biochemical, cellular, histological, physiological or behavioural change that can provide evidence of exposure and/or effects from one to more contaminants (Van Gestel and Van Brummelen 1996). Different kinds of biomarkers have been studied in isopods and some were successfully implemented in field studies. The isopods’ hepatopancreas has been used as a key organ to evaluate deleterious effects due to chemical bioaccumulation. The hepatopancreas is a storage organ where detoxified, sequestered chemicals (metals) can be stored but it also plays an important role in the animal’s metabolism. The hepatopancreas contains two cell types, the Big cells (B cells) and the Small (S cells), that differ in their excretion behaviour. The S cells are known to accumulate large amounts of metals, mainly related to metal storage, and B cells are renewed frequently, therefore playing the main role in excretion (Hopkin and Martin 1982). The histopathological changes of isopods’ digestive glands have also been characterised and studied (Drobne and Strus 1996; Odendaal and Reinecke 2003, 2004; Lapanje et al. 2008; Lešer et al. 2008). The neonicotinoid insecticide imidacloprid showed to affect the digestive gland epithelial thickness in Porcellio scaber (Drobne et al. 2008). In addition, and for the species Porcellionides pruinosus the mean percentage of cellular area (thickness) of digestive gland epithelia was also significantly lower in animals from the smelting work compared to animals from unpolluted sites. Later studies revealed that epithelial thickness can be significantly affected by starvation (Lešer et al. 2008).

In order to study endocrine disruptor effects, Lemos et al. (2009) studied moulting behaviour and total ecdysteroid (20E) concentration of Porcellio scaber upon exposure to bisphenol A and vinclozolin. At the molecular or enzymatic level, isopods have also been used to evaluate the response to oxidative stress and neurologic effects. Several biomarker methodologies have been adapted from other organisms’ protocols to be used in isopods. Stress proteins (e.g., heat-shock proteins; HSPs) were studied in Oniscus asellus as a molecular marker of multiple metal exposure, while cholinesterase activity was measured in Porcellio dilatatus upon exposure to dimethoate and related with locomotor activity (Engenheiro et al. 2005). Other biomarkers have also been used to evaluate chemical exposure, such as lysosomal membrane stability (Nolde et al. 2006), glutathione S-transferase (GST) or catalase (Jemec et al. 2008), or energy reserve contents, by studying the effects of chemicals on the lipid, protein and glycogen contents (Donker 1992). To improve biomarker methodologies in isopods and also to use basal levels as foundations for isopod exposure or just for isopod health status in cultures, Ferreira et al. (2010) characterised the basal levels of several biomarkers and energy reserves in Porcellionides pruinosus.

These biomarkers have been used to detect effects of individual chemicals but also to unravel modes of actions of chemicals and explain their effects when present in the environment as mixtures. In the study of Santos et al. (2010a), molluscicide baits induced extreme effects to the isopod Porcellionides pruinosus. In this study glutathione S-transferase (GST), acetylcholinesterase (AChE) and catalase (CAT) were analysed upon exposure to single chemicals and to binary mixtures. Although the carbamate methiocarb significantly inhibited AChE activity, no oxidative stress was detected (by using CAT and GST levels). On the other hand, metaldehyde showed a completely different mode of action with no effects on AChE, but inducing a decrease in GST activity as well as a general increase in CAT activity. The combined exposure to the two molluscicides resulted in a general decrease in AChE and CAT activity, but no visible effects were observed in terms of GST activity.

HSPs, initially discovered in salivary glands of Drosophila exposed to heat (Ritossa 1962), were intensively investigated in isopods in the 1990s (Köhler et al. 1992, 2000; Köhler and Eckwert 1997; Eckwert and Köhler 1997; Knigge and Köhler 2000; Arts et al. 2004; Weeks et al. 2004). HSPs in Porcellio scaber and Oniscus asellus from metal-polluted and unpolluted sites were analysed and compared with semi-field (microcosm) and laboratory studies. In field populations a high inter-site variability of the hsp70 level was detected. The stress response level was positively correlated with metal solubility, C/N ratio and negatively correlated with soil pH and with site-specific pollution history-adaptation (Köhler et al. 2000; Arts et al. 2004; Weeks et al. 2004). Potential adaptation in HSP responses was demonstrated previously in a laboratory study where Oniscus asellus from unpolluted and polluted sites were exposed to metals. In non-adapted populations the level of stress proteins increased after exposure to metals, while in metal adapted populations the induction of stress proteins was less prominent or even decreased (Eckwert and Köhler 1997). Some interspecies variation in HSP level was reported as well (Arts et al. 2004; Weeks et al. 2004). In Porcellio scaber collected from the vicinity of smelter, the HSP level was comparable to control animals while in Oniscus asellus from the same location it was much higher compared to Porcellio scaber and control animals (Arts et al. 2004). Arts et al. (2004) concluded that hsp70 level is a suitable biomarker of effect in non-adapted and adapted populations but of exposure only in non-adapted individuals.

In addition to HSPs, several other biomarkers were studied in situ in relation to predominantly metal(s) contamination. Metal storage granules and energy reserves were investigated in Oniscus asellus and Porcellio scaber in relation to distance to the smelter at Avonmount, UK (Schill and Köhler 2004). Porcellio scaber and Oniscus asellus showed different response to metal pollution, which was in accordance to what was achieved by Arts et al. (2004). In Oniscus asellus the number and size of metal granules increased with decreasing distance to the smelter while the amount of lipids and glycogen decreased. None of this was found in Porcellio scaber. Nolde et al. (2006) and Lapanje et al. (2008) investigated lysosomal membrane stability (LMS) of digestive gland cells in Porcellio scaber as a biomarker of effect. LMS in animals from highly mercury polluted environments was less affected compared to animals from moderately or unpolluted environments when exposed to mercury. Besides LMS, gut bacterial structure was less affected as well (Lapanje et al. 2008).

Bioaccumulation of chemicals in isopods

Considering the extremely high metal concentrations found in animals from contaminated areas (see e.g., Hopkin 1989), metal bioaccumulation in isopods has been studied to a great extent. These studies focused on several elements. Initial studies were mainly restricted to assessing whole body metal concentrations in isopods exposed for a fixed time to contaminated soil or food. Later studies also included metal uptake and elimination kinetics, sometimes also in relation to the internal distribution of metals inside the isopod’s body (see e.g., Figure 3). Finally, several studies focused more in detail on the mechanisms of metal sequestration in hepatopancreas tissues.

Figure 3. 

Schematic overview of the routes of uptake and internal processing of chemical pollutants in isopods. Adapted from Donker et al. (1996).

Isopods have been shown to have a tremendous capacity of storing metals in the hepatopancreas. As mentioned above, the hepatopancreas plays an important role in metal sequestration. Due to the high storage capacity of the hepatopancreas, metal uptake kinetics tends to be fairly slow in isopods. Basically, this means that it takes a long time to reach equilibrium, as was shown for cadmium (Vijver et al. 2006). This high capability of sequestering metals is strongly related to storage of the metal in inert fractions, as was shown for zinc and cadmium (Vijver et al. 2006). As a consequence, the lethal body concentration for cadmium is very high in isopods (Porcellio scaber, Oniscus asellus) compared to other soil arthropods (Crommentuijn et al. 1994). This also explains why metal concentrations in isopods from contaminated sites can reach very high levels without causing the population to go extinct. But it also means that static tests with fixed exposure times will never be able to provide insight into body concentrations to be expected in field-exposed animals. At best, such tests may provide Bioaccumulation Factors (BAFs), relating body concentrations to soil or litter concentrations. Since kinetics is slow, BAFs for metals in isopods will be time-dependent. For that reason, it is preferred to estimate BAFs on the basis of uptake and elimination kinetics parameters derived from toxicokinetics tests rather than from static tests. In addition, it is well known that BAFs for metals are also concentration-dependent (McGeer et al. 2003).

Metal speciation in food may have an effect on metal uptake in isopods, as was for instance shown by Calhôa et al. (2006) and Monteiro et al. (2008). These studies demonstrate that Cd speciation and subcellular distribution in plants influence the assimilation efficiency of Cd in the isopod Porcellio dilatatus. Only few studies have addressed the potential for food chain transfer of the inert metal fractions in the hepatopancreas of isopods. Bioaccumulation of organic compounds in isopods has been determined in several studies, but only few studies were performed to assess uptake and elimination kinetics of organic compounds in isopods (e.g., Van Brummelen and Van Straalen 1996; Sousa et al. 2000; Loureiro et al. 2002; Santos et al. 2003; Stroomberg et al. 2004; Kampe and Schlechtriem 2016). From the available literature, it is clear that isopods do show high uptake of metals like Cd, but less of Pb and uptake of organic chemicals may be quite variable. The studies of Van Brummelen and Van Straalen (1996) and Stroomberg et al. (2004) demonstrated that isopods (Porcellio scaber) have a high capacity of biotransforming PAHs. As a result these compounds are rapidly eliminated from the isopod’s body and PAH concentrations tend to be fairly low in animals from polluted sites when compared to other soil invertebrates like earthworms (Van Brummelen et al. 1996b).

Test methods for determining the uptake and elimination kinetics of organic chemicals in isopods are summarised in Table 2. The methods use either soil or food exposure, and include an uptake phase during which the animals are exposed to contaminated media followed by an elimination phase on clean media. In addition to the species mentioned in Table 2, Porcellionides pruinosus, also other isopod species could be used. Bioaccumulation kinetics approaches may also be used to assess the bioavailability of contaminants in soils. As proposed by Van Straalen et al. (2005) the uptake rates (or initial slope of the uptake curve) determined in uptake and elimination tests may provide an indication of metal bioavailability. Animals can be exposed to soil from the field under controlled laboratory conditions, but such an approach may also be applied to freshly spiked soils. A similar approach was used by Udovič et al. (2013) to assess the remediation efficiency. The disadvantage of such approach is that it neglects the impact of natural environmental conditions. This disadvantage could however, also be seen as an advantage: the bioassay provides an integrated assessment of the situation at the time of sampling, but it could be much more expensive, time- and labour-consuming than just performing chemical measurements.

The use of isopods in microcosm/mesocosm-based toxicity tests

Single species toxicity tests have several shortcomings (adapted from Van Leeuwen and Vermeire 2007) among others:

  1. they are performed under stable and controlled laboratory conditions that are not similar to natural conditions;
  2. interactions between different species and interaction with natural stressors are not taken into account;
  3. the distribution and degradation of chemicals is ignored;
  4. usually genetically homogeneous laboratory raised animals are used.

In contrast to single species toxicity tests microcosms, mesocosms or macrocosms are small, medium, or large multispecies systems that simulate natural situations to a certain degree (Walker et al. 2012). They are more ecologically relevant and compared to full-scale field tests less complex and less expensive. They can be performed in a controlled laboratory environment or under the field conditions. Microcosms with isopods were designed from small containers filled with soil (Eckwert and Köhler 1997, Köhler et al. 2000; Arts et al. 2004; Engenheiro et al. 2005) or sand (Van Wensem 1989) with partly decayed leaves on the surface to complex systems with several plant and animal species (Gunderson et al. 1997; Foerster et al. 2006; Domene et al. 2010; Santos et al. 2011). Uncontaminated and artificially contaminated soils (Santos et al. 2011) or field-contaminated soils (Köhler and Eckwert 1997; Köhler et al. 2000; Arts et al. 2004) were used. Microcosms were exposed to the field conditions (Eckwert and Köhler 1997, Köhler et al. 2000; Arts et al. 2004), to greenhouse semi-field conditions (Gunderson et al. 1997; Engenheiro et al. 2005) or performed in the laboratory (Foerster et al. 2006; Santos et al. 2011). Chemicals were applied by spraying (Santos et al. 2011) or introduced into the soil (Domene et al. 2010). Besides mortality, growth, and reproduction (Gunderson et al. 1997; Foerster et al. 2006), carbon dioxide production (Van Wensem 1989), locomotor behaviour (Engenheiro et al. 2005) and diverse molecular biomarkers (Köhler and Eckwert 1997; Köhler et al. 2000; Arts et al. 2004; Engenheiro et al. 2005; Santos et al. 2011) were measured.

The main disadvantage of such multispecies tests is that the more they imitate a natural environment the more difficult they are to replicate and to standardise. Microcosm tests are higher tier tests, usually designed to test a specific hypothesis, and not to be used routinely.

Isopods in field studies and biomonitoring

Field studies may take both a predictive and a diagnostic approach. In case of a predictive approach, field studies are just the next step after micro- or mesocosm-based toxicity tests described above. Such tests have rarely been done with isopods. Diagnostic field studies are mainly performed within the framework of monitoring, in order to assess the occurrence of effects at (contaminated) field sites. This section will mainly focus on the latter approaches.

Field (in situ) studies on the effects of pollutants on biota bridge the gap between laboratory-conducted toxicity studies and abiotic measuring of pollution. An increased concentration of a pollutant in the environment does not necessarily mean disruptive effects to biota. To cause toxic effects, a chemical needs to be sensed or taken up by the organism; therefore, bioavailability is crucial for toxicity. Moreover, in the field organisms simultaneously respond to a variety of anthropogenic and also natural stressors with antagonistic and synergistic actions among them. Therefore, biological monitoring is important to measure the disruptive effects of pollutants to biota. There are four main approaches to biological monitoring of pollution (adapted from Hopkin 1993; see also Walker et al. 2012):

  1. Monitoring the changes in community structure; absence or presence of a particular species indicates particular pollution.
  2. Measuring the concentration of pollutants in a tolerant indicator species; body concentrations indicate bioavailability of pollutants and also indicate the toxicity level to other more sensitive related organisms.
  3. Measuring the effects of pollutants on organisms; physiological, biochemical, cellular and other markers at the organism level can be used as a screening tool in monitoring.
  4. The detection of genetically different populations of species that have evolved resistance in response to a pollutant.

In 1975 a marine monitoring scheme ‘The mussel watch’ was proposed to follow the level of marine contamination with metals, artificial radionuclides, petroleum and chlorinated hydrocarbons (Goldberg 1975). This has led to a considerable effort to find a terrestrial invertebrate group equivalent to mussels to monitor contamination and bioavailability of metals on land. Attributes of mussels that also terrestrial invertebrates had to fulfil were: common and widespread, large populations, resistant to pollutants, bioaccumulation, and long half-lives of pollutants once accumulated in the body. Isopods, already known at that time by their remarkable ability to accumulate Cu (Wieser 1961, 1968; Wieser and Makart 1961), were soon suggested as a useful tool for monitoring available Cu (Wieser et al. 1976), as well as Cd and Pb (Martin et al. 1976) in contaminated ecosystems. This suggestion was supported by further work of Wieser et al. (1977) and Coughtrey et al. (1977) on Cu and Cd, respectively. The authors analysed soil and litter metal concentrations in several polluted regions (around smelters and mines) in Austria and England and compared them with concentrations in isopods from the same locations. Mainly three species of isopods were examined: Tracheoniscus ratkei, Oniscus asellus and Porcellio scaber. Total body concentrations of metals and concentrations in different body parts (hepatopancreas, gut, ovaries, and exoskeleton) were analysed. The main findings were:

  1. concentrations of Cu and Cd in isopods increase with increasing metal concentrations in litter (Martin et al. 1976; Wieser et al. 1976, 1977; Coughtrey et al. 1977);
  2. the Bioaccumulation Factor isopods/litter (BAF) for Cu (Wieser et al. 1977) and Cd (Martin et al. 1976) is around 6, the highest among terrestrial invertebrates;
  3. mean Cu content in isopods is highly correlated with Cu concentration in litter but less with Cu concentration in soil (Wieser et al. 1977);
  4. Cu and Cd content in isopods is related to body weight (Wieser et al. 1976, 1977; Coughtrey et al. 1977); and
  5. Cu content in isopods fluctuates with season and it is temperature-dependent (Wieser et al. 1977).

Almost ten years later, results from a large field study were published (Hopkin et al. 1986). Concentrations of Zn, Cd, Pb and Cu in litter from 89 sites around smelting works in the Avonmouth area, south-west England were mapped together with metal concentrations in Porcellio scaber (hepatopancreas and whole animal concentrations) (Hopkin et al. 1986). In the next study by Hopkin et al. (1993) Zn, Cd, Pb, and Cu concentrations in Porcellio scaber were compared with concentrations in Oniscus asellus from the same sites and correlated with soil concentrations. Main findings of these studies were:

  1. concentrations in isopods also correlate with soil or litter concentrations of Zn and Pb, like Cd and Cu (Hopkin et al. 1986, 1993);
  2. Zn, Pb, Cd, and Cu concentrations in the hepatopancreas correlate with whole animal concentrations at all sites (Hopkin et al. 1986), thus analyses of separate body parts are not necessary;
  3. BAFs for Zn and Pb are much lower compared to Cu and Cd (Hopkin et al. 1986);
  4. BAFs related to litter are much higher compared to soil and vary greatly between locations; and
  5. 5. correlation between Porcellio scaber and Oniscus asellus is closer than between isopods and soil, therefore concentrations in Oniscus asellus and probably also in other invertebrate groups can be predicted from those in Porcellio scaber (Hopkin et al. 1993).

Isopods were also studied in urban areas as bioindicators for Zn (Hopkin et al. 1989) as well as Pb and Cd pollution (Dallinger et al. 1992). Porcellio scaber and Oniscus asellus were sampled from 63 sites in Reading, England (Hopkin et al. 1989) and 356 points over the city of Innsbruck, Austria (Dallinger et al. 1992). As leaves were not present in most of the urban sites, soil was sampled at the same locations. The main findings were:

  1. Zn concentrations in Porcellio scaber were about two times higher than those in Oniscus asellus at each site (Hopkin et al. 1989);
  2. sources of Zn pollution can be identified according to soil or isopod samples on a small scale (0.5 km apart) but not on a large scale (5 km apart) (Hopkin et al. 1989);
  3. Pb concentrations in isopods correlate with traffic density in individual districts of the city (Dallinger et al. 1992).

Field studies where isopods were used in monitoring of contamination with organic chemicals are very rare compared to metal contamination. Some laboratory studies showed that isopods may also accumulate organic chemicals, like veterinary pharmaceuticals (Kolar et al. 2010) or rodenticides (Brooke et al. 2013). The antiparasitic abamectin accumulated in Porcellio scaber in a dose-dependent manner (Kolar et al. 2010) but no data about its retention time were provided. The anticoagulant rodenticide brodifacoum that accumulates in isopods (different species) was measurable even after 45 days after exposure (Brooke et al. 2013). Bioaccumulation of PAHs was also shown (Van Brummelen et al. 1996b), but concentrations were relatively low compared to e.g., earthworms, which might be attributed to the high capability of isopods to biotransform and eliminate these compounds (Van Brummelen and Van Straalen 1996; Stroomberg et al. 2004).

All these studies showed that isopods, particularly Porcellio scaber, have favourable attributes to become a leading organism in terrestrial biomonitoring (see e.g., Paoletti and Hassall 1999), especially of metal contamination. Nonetheless, they never reached the status of Mytilus edulis in marine biomonitoring, just as no other terrestrial invertebrate group has. There are several reasons for that. All the studies mentioned above showed a certain discrepancy between metal concentrations in isopods and in soil or litter from different locations with comparable metal contamination. As the digestive system is the main route for metal intake in terrestrial isopods (reviewed by Hopkin 1989), their internal metal concentration is actually a measure of metal availability in their food source and not in the soil. Metal concentrations in isopods were in fact more significantly correlated with metal concentrations in litter than in soil. In the field, the isopod diet consists mainly of leaf litter, but the exact food source is often hard to define and in some urban locations also hard to collect. Besides, animals are mobile and their resting locations are not necessarily their feeding locations. To make things even more complex, isopods also discriminate food sources due to different plant species, plant defences (e.g., tannins) and state of microbial decay (Hassall and Rushton 1984; Gunnarsson 1987; Szlávecz and Maiorana 1990) and can probably also differentiate certain contaminants (Loureiro et al. 2005; Zidar et al. 2012; Žižek and Zidar 2013), including metals (Zidar et al. 2005).

The previously mentioned studies also demonstrated a strong correlation between body metal concentrations and isopod body mass. It was therefore suggested to compare animals from the same weight class (Wieser et al. 1977) or to use the slope of regression lines (weight – metal concentration) rather than mean metal concentration for comparison between locations, as population-size structure from different locations may vary (Coughtrey et al. 1977). But size and weight are no reliable markers for age, which indicates time of exposure. The overlap of age classes could occur when the weight of younger but better fed isopods exceeds the weight of individuals born in the previous year (Hopkin 1989). Overlap of age classes might also explain the seasonal fluctuation of copper content in isopods with the maximum in winter and minimum in summer (Wieser et al. 1977), when this year’s generation of isopods already starts to reproduce. Witzel (1998) reported that accumulation of Pb and Cd in Porcellio scaber shows two different phases. Up to the age of 2–3 months assimilation exceeds the rate of growth and leads to rapidly increasing concentrations. After 3 months (at a mass of around 5 mg fresh weight) the rate of accumulation is proportional to the rate of growth and the heavy metal concentrations remain on a stabilised level. Besides, size of isopods varies between polluted and unpolluted sites. Donker et al. (1993) reported that females from Porcellio scaber populations near a Pb mine and a Zn smelter reproduced at lower weight. This might indicate slower growth or earlier maturity compared to females from clean environments. A significant difference in the mean and maximum size of Porcellio scaber has also been reported among sites in the Avonmouth area in England (Jones and Hopkin 1998). At the most polluted sites populations with the smallest maximum size were found, which might be related to higher energy expenditure for detoxification but also to a genetic differentiation of populations in polluted environments.

In the terrestrial environment metal contamination on one side and isopod distribution on the other are influenced more prominently by local environmental conditions compared to marine ecosystems. Amount and distribution of rainfall during the year, wind directions and relief of the landscape together with soil chemistry and vegetation influence the deposition, retention, and availability of metals to biomonitoring organisms on one side and appropriate conditions for their living on the other. All this makes ‘a global woodlouse watch scheme’ (Hopkin et al. 1993) even more difficult. Nevertheless, attempts to use isopods as indicators of metal pollution are still being made, as for instance shown by a study on the use of isopods as an indicator of mercury pollution (Pedrini-Martha et al. 2012; Longo et al. 2013).

Faber et al. (2013) proposed a conceptual approach for implementing ecosystem services in monitoring frameworks. They showed that isopods can also be included in monitoring soil quality and in that way contribute to the assessment of possible effects of land use and other human activities on ecosystem services.

Conclusions

Isopods are important organisms in terrestrial ecosystems. For that reason they should be considered as test organisms in soil ecotoxicology. A standardised test with isopods could be a relevant and important addition to the existing battery of toxicity tests with soil invertebrates. The difficulties in culturing and testing could be overcome by selecting species with shorter life cycles, like Porcellionides pruinosus, and by putting more effort in optimising culture conditions for species like Porcellio scaber. This may also help developing standardised toxicity tests that include more relevant endpoints like reproduction, in addition to growth and feeding activity. Little insight exists in the difference in sensitivity of isopod species to different chemicals. The harmonisation between exposure time, the existence of validation criteria based on basal levels for optimum exposure (considering temperature and time) and common endpoints could be a step forward for the accuracy improvement and comparison between studies. Isopods are also relevant and useful organisms for use in field monitoring approaches, for instance to assess the bioavailability of metals, possible (post-registration) effects of pesticide use, exposure to (mixtures of) chemicals and in biological soil quality networks aimed at protecting ecosystem services.

Acknowledgements

The authors would like to thank Paula Tourinho for the isopod test drawings. Susana Loureiro acknowledges FCT/MEC through national funds, and the co-funding by the FEDER (POCI-01-0145-FEDER-00763), within the PT2020 Partnership Agreement and Compete 2020, to CESAM.

References

  • Beeby A (1978) Interaction of lead and calcium-uptake by woodlouse, Porcellio scaber (Isopoda, Porcellionidae). Oecologia 32: 255–262. https://doi.org/10.1007/BF00366076
  • Beeby A (1980) Lead assimilation and brood-size in the woodlouse Porcellio scaber (Crustacea, Isopoda) following oviposition Pedobiologia 20: 360–365.
  • Blake L, Goulding KWT, Mott CJB, Johnston AE (1999) Changes in soil chemistry accompanying acidification over more than 100 years under woodland and grass at Rothamsted Experimental Station, UK. European Journal of Soil Science 50: 401–412. https://doi.org/10.1046/j.1365-2389.1999.00253.x
  • Bielska L, Hovorkova I, Komprdova K, Hofman J (2012) Variability of standard artificial soils: physico-chemical properties and phenanthrene desorption measured by means of supercritical fluid extraction. Environmental Pollution 16: 1–7. https://doi.org/10.1016/j.envpol.2011.12.009
  • Brooke M de L, Cuthbert RJ, Harrison G, Gordon C, Taggart MA (2013) Persistence of brodifacoum in cockroach and woodlice: Implications for secondary poisoning during rodent eradications. Ecotoxicology and Environmental Safety 97: 183–188. https://doi.org/10.1016/j.ecoenv.2013.08.007
  • Calhôa CF, Soares AMVM, Mann RM (2006) Cadmium assimilation in the terrestrial isopod, Porcellio dilatatus - Is trophic transfer important? Science of the Total Environment 371: 206–213. https://doi.org/10.1016/j.scitotenv.2006.09.013
  • Calhôa CF, Soares AMVM, Loureiro S (2012) Effects on survival and reproduction of Porcellio dilatatus exposed to different Cd species. Ecotoxicology 21: 48–55. https://doi.org/10.1007/s10646-011-0762-6
  • 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. https://doi.org/10.1897/1552-8618(1994)13[1781:LBCAAP]2.0.CO;2
  • Crommentuijn T, Doodeman C, van der Pol JJC, Doornekamp A, Rademaker MCJ, van Gestel CAM (1995) Sublethal sensitivity index as an ecotoxicity parameter measuring energy allocation under toxicant stress – application to cadmium in soil arthropods. Ecotoxicology and Environmental Safety 31: 192–200. https://doi.org/10.1006/eesa.1995.1062
  • Arts MSJS, Schill RO, Knigge T, Eckwert H, Kammenga JE, Köhler HR (2004) Stress proteins (hsp70, hsp60) induced in isopods and nematodes by field exposure to metals in a gradient near Avonmouth, UK. Ecotoxicology 13: 739–755. https://doi.org/10.1007/s10646-003-4473-5
  • Dallinger R, Berger B, Birkel S (1992) Terrestrial isopods: useful biological indicators of urban metal pollution. Oecologia 89: 32–41. https://doi.org/10.1007/BF00319012
  • De Lima e Silva C, Brennan N, Brouwer JM, Commandeur D, Verweij RA, van Gestel CAM (2017) Comparative toxicity of imidacloprid and thiacloprid to different species of soil invertebrates. Ecotoxicology 26: 555–564. https://doi.org/10.1007/s10646-017-1790-7
  • Din IU, Rashid A, Mahmood T, Khalid A (2013) Effect of land use activities on PAH contamination in urban soils of Rawalpindi and Islamabad, Pakistan. Environmental Monitoring and Assessment 185: 8685–8694. https://doi.org/10.1007/s10661-013-3204-5
  • Donker MH (1992) Energy reserves and distribution of metals in populations of the isopod Porcellio scaber from metal-contaminated sites. Functional Ecology 6: 445–454. https://doi.org/10.2307/2389282
  • Donker MH, Raedecker MH, van Straalen NM (1996) The role of zinc regulation in the zinc tolerance mechanism of the terrestrial isopod Porcellio scaber. Journal of Applied Ecology 33: 955–964. https://doi.org/10.2307/2404677
  • Donker MH, Zonneveld C, van Straalen NM (1993) Early reproduction and increased reproductive allocation in metal-adapted populations of the terrestrial isopod Porcellio scaber. Oecologia 96: 316–323. https://doi.org/10.1007/BF00317500
  • Drobne D, Hopkin SP (1995) The toxicity of zinc to terrestrial isopods in a “standard” laboratory test. Ecotoxicology and Environmental Safety 31: 1–6. https://doi.org/10.1006/eesa.1995.1037
  • 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. https://doi.org/10.1002/etc.5620210320
  • 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. https://doi.org/10.1016/j.chemosphere.2007.11.042
  • Eckwert H, Köhler HR (1997) The indicative value of the hsp70 stress response as a marker for metal effects in Oniscus asellus (Isopoda) field populations: variability between populations from metal-polluted and uncontaminated sites. Applied Soil Ecology 6: 275–282. https://doi.org/10.1016/s0929-1393(97)00020-6
  • Engenheiro EL, Hankard PK, Sousa JP, Lemos MF, Weeks JM, Soares AMVM (2005) Influence of dimethoate on acetylcholinesterase activity and locomotor function in terrestrial isopods. Environmental Toxicology and Chemistry 24: 603–609. https://doi.org/10.1897/04-131R.1
  • Escher BI, Hermens JLM (2004) Internal exposure: linking bioavailability to effects. Environmental Science and Technology 38: 455A–462A. https://doi.org/10.1021/es0406740
  • Faber JH, van Wensem J (2012) Elaborations on the use of the ecosystem services concept for application in ecological risk assessment for soils. Science of the Total Environment 415: 3–8. https://doi.org/10.1016/j.scitotenv.2011.05.059
  • Faber JH, Creamer RE, Mulder C, Römbke J, Rutgers M, Sousa JP, Stone D, Griffiths BS (2013) The practicalities and pitfalls of establishing a policy-relevant and cost-effective soil biological monitoring scheme. Integrated Environmental Assessment and Management 9: 276–284. https://doi.org/10.1002/ieam.1398
  • Falkengren-Grerup U (1986) Soil acidification and vegetation changes in deciduous forest in southern Sweden. Oecologia 70: 339–347. https://doi.org/10.1007/BF00379494
  • Farkas S, Hornung E, Fischer E (1996) Toxicity of copper to Porcellio scaber Latr. (Isopoda) under different nutritional conditions. Bulletin of Environmental Contamination and Toxicology 57: 582–588.
  • Ferreira NGC, Santos MJG, Domingues I, Calhôa CF, Monteiro M, Amorim MJB, Soares AMVM, Loureiro S (2010) Basal levels of enzymatic biomarkers and energy reserves in Porcellionides pruinosus. Soil Biology and Biochemistry 42: 2128–2136. https://doi.org/10.1016/j.soilbio.2010.08.008
  • Ferreira NGC, Morgado R, Santos MJG, Soares AMVM, Loureiro S (2015) Biomarkers and energy reserves in the isopod Porcellionides pruinosus: The effects of long-term exposure to dimethoate. Science of the Total Environment 502: 91–102. https://doi.org/10.1016/j.scitotenv.2014.08.062
  • Fischer E, Farkas S, Hornung E, Past T (1997) Sublethal effects of an organophosphorous insecticide, dimethoate, on the isopod Porcellio scaber Latr. Comparative Biochemistry and Physiology 116C: 161–166. https://doi.org/10.1016/s0742-8413(96)00164-8
  • Foerster B, Garcia M, Francimari O, Römbke J (2006) Effects of carbendazim and lambda-cyhalothrin on soil invertebrates and leaf litter decomposition in semi-field and field tests under tropical conditions (Amazônia, Brazil). European Journal of Soil Biology 42: S171–S179. https://doi.org/10.1016/j.ejsobi.2006.07.011
  • Forbes V, Calow P (2013) Use of the ecosystem services concept in ecological risk assessment of chemicals. Integrated Environmental Assessment and Management 9: 269–275. https://doi.org/10.1002/ieam.1368
  • Gunderson CA, Kostuk JM, Gibbs MH, Napolitano GE, Wicker LF, Richmond JE, Stewart A J (1997) Multispecies toxicity assessment of compost produced in bioremediation of an explosives-contaminated sediment. Environmental Toxicology and Chemistry 16: 2529–2537. https://doi.org/10.1897/1551-5028(1997)016<2529:MTAOCP>2.3.CO;2
  • Gunnarsson T (1987) Selective feeding on a maple leaf by Oniscus asellus (Isopoda). Pedobiologia 30: 161–165.
  • Hassall M, Rushton SP (1984) Feeding behaviour of terrestrial isopods in relation to plant defences and microbial activity. Symposia of the Zoological Society of London 53: 487–505.
  • Hassall M, Turner JG, Rands MRW (1987) Effects of terrestrial isopods on the decomposition of woodland leaf litter. Oecologia 72: 597–604. https://doi.org/10.1007/BF00378988
  • Hopkin SP, Hardisty GN, Martin MH (1986) The woodlouse Porcellio scaber as a biological indicator of zinc, cadmium, lead and copper pollution. Environmental Pollution (series B) 11: 271–290. https://doi.org/10.1016/0143-148X(86)90045-5
  • Hopkin SP (1989) Ecophysiology of Metals in Terrestrial Invertebrates. Elsevier Applied Science, London, 366 pp.
  • Hopkin SP, Hames CAC, Bragg S (1989) Terrestrial isopods as biological indicators of zinc pollution in the Reading area, South East England. Monitore zoologico italiano (N.S. ) Monografia 4: 477–488.
  • Hopkin SP, Jones DT, Dietrich D (1993) The isopod Porcellio scaber as a monitor of the bioavailability of metals in terrestrial ecosystems: towards a global ‘woodlouse watch’ scheme. Science of the Total Environment Supplement: 357–365. https://doi.org/10.1016/S0048-9697(05)80036-1
  • Hopkin SP, Martin MH (1982) The distribution of zinc, cadmium, lead and copper within the woodlouse Oniscus asellus (Crustacea, Isopoda). Oecologia 54: 227–232. https://doi.org/10.1007/BF00378396
  • Hopkin SP (1993) In situ biological monitoring of pollution in terrestrial and aquatic ecosystems. In: Calow P (Ed.) Handbook of Ecotoxicology. Blackwell Scientific Publications, Oxford, 397–427.
  • Hornung E, Farkas S, Fischer E (1998a) Tests on the isopod Porcellio scaber. In: Løkke H, van Gestel CAM (Eds) Handbook of Soil Invertebrate Toxicity Tests. John Wiley & Sons, Chichester, 207–226.
  • Hornung E, Fischer E, Farkas S (1998b) Isopod reproduction as a tool for sublethal-toxicity tests. Israel Journal of Zoology 44: 445–450.
  • ISO (2008) Soil quality – Avoidance test for testing the quality of soils and effects of chemicals on behaviour - Part 1: Test with earthworms (Eisenia fetida and Eisenia andrei). ISO 17512-1. International Organization for Standardization, Geneva, Switzerland.
  • ISO (2011) Soil quality – Avoidance test for testing the quality of soils and effects of chemicals on behaviour – Part 2: Test with collembolans (Folsomia candida). ISO 17512-2. International Organization for Standardization, Geneva, Switzerland.
  • Jänsch S, Garcia M, Römbke J (2005) Acute and chronic isopod testing using tropical Porcellionides pruinosus and three model pesticides. European Journal of Soil Biology 41: 143–152. https://doi.org/10.1016/j.ejsobi.2005.09.010
  • Jemec A, Drobne D, Remškar M, Sepčić K, Tišler T (2008) Effects of ingested nano-sized titanium dioxide on terrestrial isopods (Porcellio scaber). Environmental Toxicology and Chemistry 27: 1904–1914. https://doi.org/10.1897/08-036.1
  • Jensen J, Mesman M (Eds) (2006) Ecological Risk Assessment of Contaminated Land. Decision Support for Site Specific Investigations. National Institute for Public Health and the Environment, RIVM, Bilthoven, 136 pp.
  • Kampe S, Schlechtriem C (2016) Bioaccumulation of Hexachlorobenzene in the terrestrial isopod Porcellio scaber. Environmental Toxicology and Chemistry 35: 2867–2873. https://doi.org/10.1002/etc.3473
  • Knigge T, Köhler HR (2000) Lead impact on nutrition, energy reserves, respiration and stress protein (Hsp 70) level in Porcellio scaber (Isopoda) populations differently preconditioned in their habitats. Environmental Pollution 108: 209–217. https://doi.org/10.1016/S0269-7491(99)00188-8
  • Köhler HR, Triebskorn R, Stocker W, Kloetzel PM, Alberti G (1992) The 70 kD shock protein (HSP-70) in soil invertebrates – a possible tool for monitoring environmental toxicants. Archives of Environmental Contamination and Toxicology 22: 334–338. https://doi.org/10.1007/BF00212095
  • Köhler HR, Eckwert H (1997) The induction of stress proteins (hsp) in Oniscus asellus (Isopoda) as a molecular marker of multiple metal exposure: 2. Joint toxicity and transfer to field situations. Ecotoxicology 6: 263–274. https://doi.org/10.1023/A:1018635012910
  • Kolar L, Jemec A, van Gestel CAM, Valant J, Hrzenjak R, Kozuh Erzen N, Zidar P (2010) Toxicity of abamectin to the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). Ecotoxicology 19: 917–927. https://doi.org/10.1007/s10646-010-0473-4
  • Lapanje A, Drobne D, Nolde N, Valant J, Muscet B, Leser V, Rupnik M (2008) Long-term Hg pollution induced Hg tolerance in the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). Environmental Pollution 153: 537–547. https://doi.org/10.1016/j.envpol.2007.09.016
  • Lemos MFL, van Gestel CAM, Soares AMVM (2009) Endocrine disruption in a terrestrial isopod under exposure to bisphenol A and vinclozolin. Journal of Soils and Sediments 9: 492–500. https://doi.org/10.1007/s11368-009-0104-y
  • Lemos MFL, van Gestel CAM, Soares AMVM (2010) Reproductive toxicity of the endocrine disrupters vinclozolin and bisphenol A in the terrestrial isopod Porcellio scaber (Latreille, 1804). Chemosphere 78: 907–913. https://doi.org/10.1016/j.chemosphere.2009.10.063
  • Lešer V, Drobne D, Vilhar B, Kladnik A, Znidarsic N, Štrus J (2008) Epithelial thickness and lipid droplets in the hepatopancreas of Porcellio scaber (Crustacea: Isopoda) in different physiological conditions. Zoology 111: 419–432. https://doi.org/10.1016/j.zool.2007.10.007
  • Løkke H, Bak J, Falkengren-Grerup U, Finlay R D, Ilvesniemi H, Holm Nygaard P, Starr M (1996) Critical loads of acidic deposition for forest soils: is the current approach adequate? Ambio 25: 510–516.
  • Løkke H, van Gestel CAM (Eds) (1998) Handbook on soil invertebrate testing. West Sussex, John Wiley & Sons, Chichester, 281 pp.
  • Longo G, Trovato M, Mazzei V, Ferrante M, Oliveri Conti G (2013) Ligia italica (Isopoda, Oniscidea) as bioindicator of mercury pollution of marine rocky coasts. Plos ONE 8: e58548. https://doi.org/10.1371/journal.pone.0058548
  • Loureiro S, Sousa JP, Nogueira AJA, Soares AMVM (2002) Assimilation efficiency and toxicokinetics of 14C-lindane in the terrestrial isopod Porcellionides pruinosus: the role of isopods in degradation of persistent soil pollutants. Ecotoxicology 11: 481–490. https://doi.org/10.1023/A:1021013519330
  • Loureiro S, Santos C, Pinto G, Costa A, Monteiro M, Nogueira AJA, Soares AMVM (2006) Toxicity assessment of two soils from Jales Mine (Portugal) using plants: growth and biochemical parameters. Archives of Environmental Contamination and Toxicology 50: 182–190. https://doi.org/10.1007/s00244-004-0261-3
  • Loureiro S, Amorim MJB, Campos B, Rodrigues SMG, Soares AMVM (2009) Assessing joint toxicity of chemicals in Enchytraeus albidus (Enchytraeidae) and Porcellionides pruinosus (Isopoda) using avoidance behaviour as an endpoint. Environmental Pollution 157: 625–636. https://doi.org/10.1016/j.envpol.2008.08.010
  • Maltby L (2013) Ecosystem services and the protection, restoration, and management of ecosystems exposed to chemical stressors. Environmental Toxicology and Chemistry 32: 974–983. https://doi.org/10.1002/etc.2212
  • Martin MH, Coughtrey PJ, Young EW (1976) Observations on the availability of lead, zinc, cadmium and copper in woodland litter and the uptake of lead, zinc and cadmium by the woodlouse Oniscus asellus. Chemosphere 5: 313–318. https://doi.org/10.1016/0045-6535(76)90005-9
  • Mazzei V, Giannetto A, Brundo MV, Maisano M, Ferrante M, Copat C, Mauceri A, Longo G (2015) Metallothioneins and heat shock proteins 70 in Armadillidium vulgare (Isopoda, Oniscidea) exposed to cadmium and lead. Ecotoxicology and Environmental Safety 116: 99–106. https://doi.org/10.1016/j.ecoenv.2015.03.007
  • McGeer JC, Brix KV, Skeaff JM, DeForest DK, Brigham SI, Adams WJ, Green A (2003) Inverse relationship between bioconcentration factor and exposure concentration for metals: Implications for hazard assessment of metals in the aquatic environment. Environmental Toxicology and Chemistry 22: 1017–1037. https://doi.org/10.1897/1551-5028(2003)022<1017:IRBBFA>2.0.CO;2
  • Millennium Ecosystem Assessment (2005) Ecosystems and Human Wellbeing: Synthesis. Island Press, Washington, DC, USA, 137 pp.
  • Monteiro MS, Santos C, Soares AMVM, Mann RM (2008) Does subcellular distribution in plants dictate the trophic bioavailability of cadmium to Porcellio dilatatus (Crustacea, Isopoda)? Environmental Toxicology and Chemistry 12: 2548–2556. https://doi.org/10.1897/08-154.1
  • Morgado R, Ferreira NGC, Tourinho P, Ribeiro F, Soares AMVM, Loureiro S (2013) Environmental- and growth stage-related differences in the susceptibility of terrestrial isopods to UV radiation. Journal of Photochemistry and Photobiology B: Biology 126: 60–71. https://doi.org/10.1016/j.jphotobiol.2013.07.002
  • Morgado RG, Gomes PAD, Ferreira NGC, Cardoso DN, Santos MJG, Soares AMVM, Loureiro S (2016) Toxicity interaction between chlorpyrifos, mancozeb and soil moisture to the terrestrial isopod Porcellionides pruinosus. Chemosphere 144: 1845–1853. https://doi.org/10.1016/j.chemosphere.2015.10.034
  • Nienstedt KM, Brock TCM, van Wensem J, Montforts M, Hart A, Aagaard A, Alix A, Boesten J, Bopp SK, Brown C, Capri E, Forbes V, Köpp H, Liess M, Luttik R, Maltby L, Sousa JP, Streissl F, Hardy AR (2012) Development of a framework based on an ecosystem services approach for deriving specific protection goals for environmental risk assessment of pesticides. Science of the Total Environment 415: 31–38. https://doi.org/10.1016/j.scitotenv.2011.05.057
  • Nolde N, Drobne D, Valant J, Padovan I, Horvat M (2006) Lysosomal membrane stability in laboratory- and field-exposed terrestrial isopods Porcellio scaber (Isopoda, Crustacea). Environmental Toxicology and Chemistry 25: 2114–2122. https://doi.org/10.1897/05-593R1.1
  • Odendaal JP, Reinecke AJ (1998) The effect of high lead concentrations on the mortality, mass and behaviour of Porcellio laevis Latr. (Crustacea, Isopoda) in laboratory tests. South African Journal of Zoology 33: 143–146. https://doi.org/10.1080/02541858.1998.11448464
  • Odendaal JP, Reinecke AJ (2003) Quantifying histopathological alterations in the hepatopancreas of the woodlouse Porcellio laevis (Isopoda) as a biomarker of cadmium exposure. Ecotoxicology and Environmental Safety 56: 319–325. https://doi.org/10.1016/S0147-6513(02)00163-X
  • Odendaal JP, Reinecke AJ (2004) Bioaccumulation of cadmium and zinc, and field validation of a histological biomarker in terrestrial isopods. Bulletin of Environmental Contamination and Toxicology 72: 769–776. https://doi.org/10.1007/s00OO464-004-0311-y
  • OECD (1984) OECD Guidelines for the Testing of Chemicals No. 207. Earthworm, Acute toxicity tests. Organisation for Economic Co-operation and Development, Paris.
  • Paoletti MG, Hassall M (1999) : Woodlice (Isopoda: Oniscidea): their potential for assessing sustainability and use as bioindicators. Agriculture, Ecosystems and Environment 74: 157–165. https://doi.org/10.1016/S0167-8809(99)00035-3
  • Pedrini-Martha V, Sager M, Werner R, Dallinger R (2012) Patterns of urban mercury contamination detected by bioindication with terrestrial isopods. Archives of Environmental Contamination and Toxicology 63: 209–219. https://doi.org/10.1007/s00244-012-9766-3
  • Ribeiro S, Guilhermino L, Sousa JP, Soares AMVM (1999) Novel bioassay based on acetylcholinesterase and lactate dehydrogenase activities to evaluate the toxicity of chemicals to soil isopods. Ecotoxicology and Environmental Safety 44: 287–293. https://doi.org/10.1006/eesa.1999.1837
  • Ribeiro S, Sousa JP, Nogueira AJA, Soares AMVM (2001) Effect of endosulfan and parathion on energy reserves and physiological parameters of the terrestrial isopod Porcellio dilatatus. Ecotoxicology and Environmental Safety 49: 131–138. https://doi.org/10.1006/eesa.2001.2045
  • Santos MJG, Ferreira NGC, Soares AMVM, Loureiro S (2010a) Toxic effects of molluscicidal baits to the terrestrial isopod Porcellionides pruinosus (Brandt, 1833). Journal of Soils and Sediments 10: 1335–1343. https://doi.org/10.1007/s11368-010-0246-y
  • Santos MJG, Soares AMVM, Loureiro S (2010b) Joint effects of three plant protection products to the terrestrial isopod Porcellionides pruinosus and the collembolan Folsomia candida. Chemosphere 80: 1021–1030. https://doi.org/10.1016/j.chemosphere.2010.05.031
  • Santos MJG, Morgado R, Ferreira NGC, Soares AMVM, Loureiro S (2011) Evaluation of the joint effect of glyphosate and dimethoate using a small-scale terrestrial ecosystem. Ecotoxicology and Environmental Safety 74: 1994–2001. https://doi.org/10.1016/j.ecoenv.2011.06.003
  • Santos SAP, Sousa JP, Frost M, Soares AMVM (2003) Time-dependent toxicokinetics of [14C]Lindane in the terrestrial isopod Porcellionides pruinosis. Environmental Toxicology and Chemistry 22: 2221–2227. https://doi.org/10.1897/02-458
  • Schill RO, Köhler HR (2004) Energy reserves and metal-storage granules in the hepatopancreas of Oniscus asellus and Porcellio scaber (Isopoda) from a metal gradient at Avonmouth, UK. Ecotoxicology 13: 787–796. https://doi.org/10.1007/s10646-003-4476-2
  • Silva PV, Silva ARR, Mendo S, Loureiro S (2014) Toxicity of tributyltin (TBT) to terrestrial organisms and its species sensitivity distribution. Science of the Total Environment 466: 1037–1046. https://doi.org/10.1016/j.scitotenv.2013.08.002
  • Soliveres S, van der Plas F, Manning P, Prati D, Gossner MM et al. (2016) Biodiversity at multiple trophic levels is needed for ecosystem multifunctionality. Nature 536: 456–459. https://doi.org/10.1038/nature19092
  • Stroomberg GJ, Ariese F, van Gestel CAM, van Hattum B, Velthorst NH, van Straalen NM (2004) Pyrene biotransformation and kinetics in the hepatopancreas of the isopod Porcellio scaber. Archives of Environmental Contamination and Toxicology 47: 324–331. https://doi.org/10.1007/s00244-004-3097-y
  • Szlávecz K, Maiorana VC (1990) Food selection by isopods in paired choice tests. In: Juchault P, Mocquard JP (Eds) Proceedings Third Symposium on the Biology of Terrestrial Isopods, Poitiers, 115–121.
  • TEEB (2010) The Economics of Ecosystems and Biodiversity: Mainstreaming the Economics of Nature: A synthesis of the approach, conclusions and recommendations of TEEB. United Nations Environment Programme. Progress Press, Malta, 36 pp.
  • Tomita M, Heisey R, Witkus R, Vernon GM (1991) Sequestration of copper and zinc in the hepatopancreas of Armadillidium vulgare Latreille following exposure to lead. Bulletin of Environmental Contamination and Toxicology 46: 894–900. https://doi.org/10.1007/BF01689735
  • Tourinho PS, van Gestel CAM, Lofts S, Soares AMVM, Loureiro S (2013) Influence of soil pH on the toxicity of zinc oxide nanoparticles to the terrestrial isopod Porcellionides pruinosus. Environmental Toxicology and Chemistry 32: 2808–2815. https://doi.org/10.1002/etc.2369
  • Tourinho PS, van Gestel CAM, Jurkschat K, Soares AMVM, Loureiro S (2015) Effects of soil and dietary exposures to Ag nanoparticles and AgNO3 in the terrestrial isopod Porcellionides pruinosus. Environmental Pollution 205: 170–177. https://doi.org/10.1016/j.envpol.2015.05.044
  • Tourinho PS, Waalewijn-Kool PL, Zantkuijl I, Jurkschat K, Svendsen C, Soares AMVM, Loureiro S, van Gestel CAM (2015) CeO2 nanoparticles induce no changes in phenanthrene toxicity to the soil organisms Porcellionides pruinosus and Folsomia candida. Ecotoxicology and Environmental Safety 113: 201–206. https://doi.org/10.1016/j.ecoenv.2014.12.006
  • Van Brummelen TC, Verweij RA, Wedzinga SA, van Gestel CAM (1996b) Polycyclic aromatic hydrocarbons in earthworms and isopods from contaminated forest soils. Chemosphere 32: 315–341. https://doi.org/10.1016/0045-6535(95)00340-1
  • Van Brummelen TC, van Straalen NM (1996) Uptake and elimination of benzo[a]pyrene in the terrestrial isopod Porcellio scaber. Archives of Environmental Contamination and Toxicology 31: 277–285. https://doi.org/10.1007/BF00212378
  • Van Gestel CAM, van Brummelen TC (1996) Incorporation of the biomarker concept in ecotoxicology calls for a redefinition of terms. Ecotoxicology 5: 217–225. https://doi.org/10.1007/BF00118992
  • Van Gestel CAM, Léon CD, van Straalen NM (1997) Evaluation of soil fauna ecotoxicity tests regarding their use in risk assessment. In: Tarradellas J, Bitton G, Rossel D (Eds) Soil Ecotoxicology. CRC Press, Inc., Boca Raton, 291–317.
  • Van Gestel CAM, van der Waarde JJ, Derksen JGM, van der Hoek EE, Veul MFXW, Bouwens S, Rusch B, Kronenburg R, Stokman GNM (2001) The use of acute and chronic bioassays to determine the ecological risk and bioremediation efficiency of oil-polluted soils. Environmental Toxicology and Chemistry 20: 1438–1449. https://doi.org/10.1897/1551-5028(2001)020<1438:TUOAAC>2.0.CO;2
  • Van Ommen Kloeke AEE, Jager T, van Gestel CAM, Ellers J, van Pomeren M, Krommenhoek T, Styrishave B, Hansen M, Roelofs D (2012) Time-related survival effects of two gluconasturtiin hydrolysis products on the terrestrial isopod Porcellio scaber. Chemosphere 89: 1084–1090. https://doi.org/10.1016/j.chemosphere.2012.05.074
  • Van Straalen NM, Donker M, Vijver MG, van Gestel CAM (2005) Bioavailability of contaminants estimated from uptake rates into soil invertebrates. Environmental Pollution 136: 409–417. https://doi.org/10.1016/j.envpol.2005.01.019
  • Van Wensem J (1989) A terrestrial micro-ecosystem for measuring effects of pollutants on isopod-mediated litter decomposition. Hydrobiologia 188/189: 507–516. https://doi.org/10.1007/BF00027818
  • Vijver MG, van Gestel CAM, Lanno RP, van Straalen NM, Peijnenburg WJGM (2004) Internal metal sequestration and its ecotoxicological relevance: A review. Environmental Science and Technology 38: 4705–4712. https://doi.org/10.1021/es040354g
  • Vijver MG, Wolterbeek HT, Vink JPM, van Gestel CAM (2005) Surface adsorption of metals onto the earthworm Lumbricus rubellus and the isopod Porcellio scaber is negligible compared to absorption, in the body. Science of the Total Environment 340: 271–280. https://doi.org/10.1016/j.scitotenv.2004.12.018
  • Vijver MG, Vink JPM, Jager T, van Straalen NM, Wolterbeek HT, van Gestel CAM (2006) Kinetics of Zn and Cd accumulation in the isopod Porcellio scaber exposed to contaminated soil and/or food. Soil Biology and Biochemistry 38: 1554–1563. https://doi.org/10.1016/j.soilbio.2005.11.006
  • Walker CH, Hopkin SP, Sibly RM, Peakall DB (2012) Principles of Ecotoxicology. Fourth Edition. CRC Press, Taylor & Francis Group, Boca Raton, Florida, 360 pp.
  • Wang J, Luo YM, Teng Y, Ma WT, Christie P, Li ZG (2013) Soil contamination by phthalate esters in Chinese intensive vegetable production systems with different modes of use of plastic film. Environmental Pollution 180: 265–273. https://doi.org/10.1016/j.envpol.2013.05.036
  • Weeks JM, Spurgeon D, Svendsen C, Hankard PK, Kammenga JE, Dallinger R, Köhler HR, Simonsen V, Scott-Fordsmand J (2004) Critical analysis of soil invertebrate biomarkers: a field case study in Avonmouth, UK. Ecotoxicology 13: 817–822. https://doi.org/10.1007/s10646-003-4479-z
  • Wieser W, Makart H (1961) Der Sauerstroffverbrauch und der Gehalt an Ca, Cu und Einigen Anderen Spurelementen bei Terrestrische Asseln. Zeitschrift für Naturfoschung Part B – Chemie, Biochemie, Biophysik, Biologie und Verwandten Gebiete B 16: 816.
  • Wieser W, Dallinger R, Busch G (1977) The flow of copper through a terrestrial food chain. II. Factors influencing the copper content of isopods. Oecologia (Berlin) 30: 265–272. https://doi.org/10.1007/BF01833633
  • Witzel B (1998) Uptake, storage and loss of cadmium and lead in the woodlouse Porcellio scaber (Crustacea, Isopoda). Water, Air, and Soil Pollution 108: 51–68. https://doi.org/10.1023/A:1005086123969
  • Zidar P, Drobne D, Štrus J, van Gestel CAM, Donker M (2004) Food selection as a means of Cu intake reduction in the terrestrial isopod Porcellio scaber (Crustacea, Isopoda). Applied Soil Ecology 25: 257–265. https://doi.org/10.1016/j.apsoil.2003.09.005
  • Zidar P, Bozic J, Strus J (2005) Behavioral response in the terrestrial isopod Porcellio scaber (Crustacea) offered a choice of uncontaminated and cadmium-contaminated food. Ecotoxicology 14: 493–502. https://doi.org/10.1007/s10646-005-0005-9
  • Zidar P, Hribar M, Zizek S, Strus J (2012) Behavioural response of terrestrial isopods (Crustacea: Isopoda) to pyrethrins in soil or food. European Journal of Soil Biology 51: 51–55. https://doi.org/10.1016/j.ejsobi.2012.03.010