Evaluation of Exposure Metrics for Effect Assessment of Soil Invertebrates

Critical Reviews in Environmental Science and Technology, 42:1862–1893, 2012Copyright © Taylor & Francis Group, LLCISSN: 1064-3389 print / 1547-6537 onlineDOI: 10.1080/10643389.2011.574100

Evaluation of Exposure Metrics for EffectAssessment of Soil Invertebrates

WILLIE PEIJNENBURG,1,2 ETTORE CAPRI,3 CHRISTINE KULA,4

MATTHIAS LIESS,5 ROBERT LUTTIK,5 MARK MONTFORTS,6

KARIN NIENSTEDT,7 JORG ROMBKE,8 JOSE PAULO SOUSA,9

and JOHN JENSEN10

1Laboratory for Ecological Risk Assessment, RIVM, Bilthoven, the Netherlands2Institute of Environmental Sciences, Leiden University, Leiden, the Netherlands

3Universita Cattolica del Sacro Cuore, Piacenza, Italy4Biologische Bundesanstalt fur Land- und Forstwirtschaft, Fachgruppe Biologische

Mittelprufung, Braunschweig, Germany5Department System Ecotoxicology, Helmholtz Centre for Environmental Research–UFZ,

Leipzig, Germany6Substance Expertise Centre, RIVM, Bilthoven, the Netherlands

7European Food Safety Agency, Parma, Italy8ECT Oekotoxikologie GmbH, Florsheim, Germany

9Department of Life Sciences, Institute of Marine Research, University of Coimbra,Coimbra, Portugal

10National Environmental Research Institute, Aarhus University, Roskilde, Denmark

Risk and hazard assessments for the soil environment are performedon the basis of the total content of a contaminant in the dry bulksoil. Presently, scientific evidence is emerging and indicating thatpore water may be a more relevant exposure medium for uptakeof chemicals by biota and plants in soil. To deduce the degreeto which pore water concentrations are indeed a better metrics forquantifying uptake of organic chemicals by terrestrial biota (mostlyinvertebrates), a literature search was performed and the availableevidence in favor of any metrics was gathered in the context of amandate of the European Food Safety Authority. It is concluded thatknowledge on uptake routes of contaminants by soil invertebrates isfar from complete. Overall it is clear that uptake of organic contam-inants depends on species, soil type, and the chemical properties.The mode of exposure of soil invertebrates is determined by the way

Address correspondence to Willie Peijnenburg, RIVM, Laboratory for Ecological Risk As-sessment, PO Box 1, 3720 BA, Bilthoven, the Netherlands. E-mail: [email protected]

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Effect Assessment of Soil Invertebrates 1863

animals are in contact with their local environment. Morphology,physiology, and behavior are important factors in this respect, as isthe mode of uptake of food, water, and oxygen. The contribution oforal uptake may vary within a specific taxon but for soil organismsin close contact with the soil solution, pore water–mediated uptakeis in general the dominant pathway and it is commonly modifiedby soil specific ageing and speciation, and by specific factors ofthe organisms, such as nutrition status. Residual uptake appears tobe the most important uptake route following pore water–mediateduptake. It is likely that in this case, too, pore water is involved ascarrier in or at the surface of the soil in which the chemicals aredissolved. Intraspecies (especially between different life stages) andinterspecies variances (e.g., size and ecological preferences) willmost likely modify the actual contribution of potential exposurepathways, and a distinction must be made between hard-bodiedand soft-bodied organisms. Hard-bodied organisms rely for uptakeof oxygen and water on specialized organs, whereas water (porewater) and oxygen are mainly taken up via the skin in soft-bodiedorganisms. Hard-bodied animals are nevertheless in contact withpore water, as shown for spiders, woodlice, and collembolans. Up-take of nutrients and chemicals is possible for all invertebrates viatheir food, and this may be an important route in case of foodsources in which high concentrations of chemicals are present.The assimilation efficiency will however depend on species-specificproperties of the digestive tract and no general conclusions are tobe generated in this respect.

KEY WORDS: effect assessment, exposure, exposure pathways,hazard assessment, invertebrates, organics, pesticides, plants, riskassessment, soil, uptake routes

INTRODUCTION

Risk assessment of hazardous chemicals is traditionally conducted by eithercomparing a generically derived effect concentration with a generically de-rived exposure concentration (Toxicity-Exposure Ratio [TER] in the case ofplant protection products [PPPs]) or by comparing the Predicted Environmen-tal Concentration (PEC) to the Predicted No-observed Effect Concentration(PNEC) in the case of chemicals that are not in use as PPPs. In all cases,the risk assessment is based on the expression of TER, PEC, and PNEC interms of the total content of the chemical in the exposure medium. The totalcontent of pesticide in the top 5 cm of soil has been used as the generic ex-posure level in the terrestrial risk assessment (European and Mediterranean

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1864 W. Peijnenburg et al.

Plant Protection Organization, 2003; European Commission, 2002; ScientificCommittee on Toxicology, Ecotoxicology and the Environment, 2000).

However, it has for a long time been accepted that pore water is the mainroute of exposure and uptake of metals and persistent organic pollutants forin-soil organisms such as enchytraeids and earthworms. In an earlier reviewthe pore water concept has already been supported quantitatively for plantsand nematodes (Boesten, 1993), after which the concept has been reviewedseveral times to include additional soil organisms (see for example Belfroidet al., 1996; Herrchen et al., 1997). Hermens et al. (2007) postulated thattotal or nominal concentrations are not appropriate for risk assessment, andrecently a comparison between the total content approach and the porewater concept has been presented for PPPs (Linden et al., 2008).

As terrestrial risk assessment is still based on the total content of thechemical despite the latest developments introduced previously, there is aneed to evaluate the scientific relevance of this metric for exposure and,therefore, for risk assessment. The aim of this contribution is to present theresults of a review carried out in the context of a mandate of the EuropeanFood Safety Authority (EFSA) on the available scientific evidence on totalconcentration and soil pore water concentration as metrics for the predictionof toxicological effects on soil invertebrates (European Food Safety Authority[EFSA], 2010).

A two-stage literature review was conducted, supplemented by literaturealready known. The contribution is structured as follows: after a descriptionof the methods used to perform the literature study, three conceptual frame-works for describing the equilibration of chemical concentrations and/or ac-tivities between the various soil constituents and biota are presented. Theseframeworks were used to interpret literature data on the issues determiningthe way biota are effectively exposed to PPPs in their local environment.Subsequently the relative importance of the various uptake routes of organiccontaminants for different species are discussed and the major conclusionsregarding the most suited metrics for describing uptake are presented.

Methods Used in the Literature Review

A two-stage literature research on Web of Science (http://www.isiwebofknowledge.com/) focusing on soil invertebrates was conducted forliterature published during the period January 2000 to April 2008. The liter-ature search resulted in 137 hits that needed further consideration and wassupplemented by literature (older than 2000) already known. The abstractsgenerated in this literature search were inspected and expert judgment wasapplied to select the references considered relevant for further consideration.

Plants are not fully considered in this search because the pore waterconcept has already been supported quantitatively for them, as Boesten(1993) concluded that he concentration of the dissolved chemical in the

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http://www.isiwebofknowledge.com/

http://www.isiwebofknowledge.com/


liquid phase in soil is directly linked to adverse effects of the chemical onplants. Thereupon, exposure of plants via the aqueous phase is obviousbecause (apart from air-deposited particles that enter the stomata) plantsare not capable of taking up solid particles. Actually, hydroponic culturesare increasingly used to generate uptake and toxicity profiles of chemicalswithout the interference of the soil matrix, thus allowing for the control andreproduction of the experimental conditions such as homogeneous exposureof the root system and avoiding typical local variations encountered in a soilsystem (Bouldin et al., 2006; Flocco et al., 2002; Narayanan et al., 1995).

With respect to soil invertebrates, it should be noted that ingestionof food items from the litter layer that may be contaminated with sprayresidues may provide a significant additional mode of uptake besides ex-posure to the moistened surface of the litter particles (EFSA, 2010). Thisuptake can be modeled neither by pore water exposure nor by exposurevia the soil solid phase. Hence, effects following significant uptake via thisroute might not be supportive of either pore water–dominated or soil solidphase–dominated uptake, while also not falsifying either exposure route. As aconsequence, exposure routes of other than purely in soil-dwelling terrestrialorganisms may include more uptake routes than via pore water or soil solidphase.

This review includes an assessment of parameters that are considered(most) important in determining the ecotoxicologically relevant concentra-tion (ERC) of organic chemicals (Boesten et al., 2007). Detailed suggestionson how to include the parameters affecting the exposure concentration (e.g.,organic matter [OM], cation exchange capacity [CEC], pH, ageing) in modelestimates for cases where measurements are not available are not presentedin this contribution.

Results

The response of test organisms to exposure to chemical stressors is a functionof the concentration at the receptors within the organisms. This internalconcentration is a function of all exposure routes as related to the activityof the organism in the soil, and is schematically depicted in Figure 1 for thetop layer of soil and the overlying litter layer. The relative importance of theroutes varies between species, between ecological traits (i.e., a measurablemorphological, ecological, or physiological characteristic of an individual or,in this case, a species), and between chemical compounds. Subsequently,the theoretical framework is presented and the main drivers are discussed.Given the width of the topic, an overview of the methods used so far tomeasure concentrations of chemicals in soil is not included although thechoice of the method defines the context for which these observations areapplicable.

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FIGURE 1. Schematic representation of the functional relationships between earthworms andtheir external environment (Drawing based on a figure by G. G. Brown [Doube and Brown,1998, Figure 1, p. 180]).

Three conceptual frameworks provide the basic concepts for the inter-pretation of the results of studies retrieved with the literature search. It shouldbe noted on forehand that the three concepts bare considerable similarities.

The first conceptual framework is the concept of chemical equilibriumin which chemical activities (or fugacities) are the driving factor for trans-port and distribution processes, including passive uptake of chemicals bybiota (MacKay et al., 1992). The fugacity concept dictates chemical fugacitiesto be similar across biological membranes and explains observed variabil-ity in uptake patterns for organisms for which active uptake (e.g., feedingand ingestion of solid soil particles) is of importance. Similarly, the con-cept explains why deviations from pore water uptake are often observed forhighly hydrophobic contaminants (i.e., with log-transformed values of theoctanol-water partition coefficient, Kow, approximately >5). Within the fu-gacity concept, Reichenberg and Mayer (2006) identified two complementaryaspects of bioavailability of chemicals, these being the accessible quantityand the chemical activity that is to be deduced from this quantity as relatedto the physicochemical conditions. Both aspects are dealt with explicitly inthis review, realizing that chemical bioavailability for organisms living in con-taminated soil may be more complex than just a matter of simple measuresof contaminants in collected pore water content.

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SOIL SOLIDS SOIL SOLUTION

BCFfeeding

Kp

Effects on:growth metabolism reproduction

BIOTA

FIGURE 2. Schematic representation of the equilibrium partitioning concept. BCF = biocon-centration factor; Kp = partitioning coefficient.

The second conceptual framework is the concept of equilibrium parti-tioning in which chemical activities in the pore water are assumed to driveuptake and effects (Van Gestel, 1997). The equilibrium partitioning conceptis schematically given in Figure 2. Unlike the basic assumptions within theconcept of chemical equilibrium, it is within this basic concept explicitlyrealized that the morphology, physiology, and behavior of biota dominateactual uptake and effects. It should be noted that the fugacity concept andthe concept of equilibrium partitioning share many communalities and infact, Reichenberger and Mayer (2006) added another concept: the thermo-dynamic concept of the chemical potential.

The third and most general concept is the concept of bioavailability.An International Organization for Standardization (ISO) working group onbioavailability (Harmsen, 2007; International Organization for Standardiza-tion [ISO], 2008a) established a general bioavailability scheme that is in partgiven in Figure 3. It should be noted that various definitions of bioavailabil-ity have been used so far. In this review, the definition of bioavailability aspresented by Peijnenburg and Jager (2003) was used: The fraction of a totalamount of a chemical present in a specific environmental compartment that,within a given time span, is either available or can be made available foruptake by (micro)organisms or plants, from either the direct surroundings ofthe organisms or the plant or by ingestion of food.

On top of these three concepts, the concept of bioaccessibility was pos-tulated (Naidu et al., 2008; Semple et al., 2004). The bioaccessible fractionmay be defined as the fraction of the total amount of a chemical present iningested food, water, or ingested soil and sediment particles that at maxi-mum can be released during digestion. More specifically, the bioaccessiblefraction may also be defined as the fraction that, after ingestion, may bemobilized into the gut fluids (chyme). This fraction is considered to repre-sent the maximum amount of contaminant available for intestinal absorption.Bioaccessible contaminants can subsequently be absorbed, in other words,

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FIGURE 3. Schematic generalized depiction of the bioavailability concept, modified fromISO/DIS 17402 (International Organization for Standardization, 2008a).

transported across the intestinal wall and transferred into the blood or lymphstream. The emphasis of this study is on routes of exposure. Additional pro-cesses within organisms such as digestion were therefore not considered andonly indirect attention was paid to the concept of bioaccessibility.

In practical terms, the three concepts have in common that it is theconcentration of the chemical in the pore water that is to be used as thebasis for modeling uptake and effects of chemicals. Moreover, no conceptsbased on total concentration have been defended so far.

The three concepts do not restrict the mathematics to be applied, in thesense that real-time modeling of dynamic situations is possible. The Equilib-rium Partitioning Method derives its name from the concept that chemicalactivities strive for equilibrium, or for the lowest thermodynamic potential.With respect to the actual application of these concepts it should be noted

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that in controlled laboratory conditions steady-state conditions are targeted.Most test results of effect studies, but also soil sorption isotherm studies,hence pertain to these steady-state conditions. In the actual field situationfor which the risk of chemicals needs to be assessed, steady-state conditions,if any exist, are short-lived. This kinetic aspect should be included in the ex-posure assessment but it is a challenge to combine this consistently with theeffect assessment.

EXPOSURE PATHWAYS OF SOIL ORGANISMS

In the broadest context, three issues determine the way biota are exposedto their local environment: (a) the morphology of the organism, (b) thephysiology of the organism, and (c) the behavior of the organism.

Consequently, soil-dwelling organisms are exposed to chemicals by avariety of pathways. Most organisms share the feature that the relative con-tribution of each pathway varies. On top of ecological impacts, these contri-butions depend on factors such as the hydrophobicity of the chemical andvariations in environmental conditions such as soil type and climate.

It is the aim of this section to overview the main exposure pathways ofsoil-dwelling invertebrates, taking account of their ecological preferences. Asit is nowadays considered self-evident that plants are exposed via the aque-ous phase of the soil compartment, the hypothesis of pore water–mediatedexposure of soil invertebrates is given a central place.

Most of the information provided here originates from experiments car-ried out in a laboratory setting, as real field data are scarce. Therefore itis anticipated that most data on adverse effects of plant protection prod-ucts will become available for invertebrates for which standardized testingguidelines have been developed (Table 1). Information on these species isconsidered especially valuable in this respect. Standardized tests protocolswith other organism groups such as nematodes or isopods are presentlyunder development.

Terrestrial species may be exposed along various uptake routes to po-tentially toxic pollutants. These routes include (a) pore water, (b) ingestionof food and soil particles, and (c) inhalation of air present in the soil pores.

The relative importance of each of these uptake routes is determined bymorphological (e.g., structure of the epidermis), physiological (e.g., modeof uptake of water [drinking vs. uptake via the skin], mode of uptake ofoxygen, feeding habits), and behavioral properties. A general subdivisionmay be made between so-called soft-bodied organisms (e.g., nematodes,earthworms, enchytraeids, some insect larvae) and hard-bodied invertebrates(arthropods such as spiders, mites, insects, collembolans, millipedes, cen-tipedes, harvestman, isopods; some other terrestrial crustaceans such as some

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TABLE 1. Available guidelines for testing effects of chemicals on soil invertebrates

Species Guideline Test type

Eisenia fetida,Eisenia andrei

Oligochaeta:Lumbricidae

OECD 207 (Organization forEconomic Cooperation andDevelopment, 1984) ISO 11268-1(International Organization forStandardization, 1993)

Acute

Eisenia fetida,EiseniaandreiAndrei


OECD 222 (Organization forEconomic Cooperation andDevelopment, 2004a) ISO 11268-2(International Organization forStandardization, 1998)

reproduction

Eisenia fetida,Eisenia andrei


ISO 17512 (InternationalOrganization for Standardization,2008a, 2008b)

Avoidance

Enchytraeusalbidus,Enchytraeuscrypticus

Oligochaeta:Enchytraeidae

OECD 220 (Organization forEconomic Cooperation andDevelopment, 2004b) ISO 16387(International Organization forStandardization, 2004)

reproduction

Caenorhabditiselegans

Rhabditida:Rhabiditidae

ISO 10872 (InternationalOrganization for Standardization,2010b)

reproduction

Folsomia candida Collembola,Isotomidae

ISO 11267 (InternationalOrganization for Standardization,1999b)

reproduction

Hypoaspis aculeifer Acari:Laelapidae

OECD 226 (Organization forEconomic Cooperation andDevelopment, 2008)

Reproduction

Terrestrial ModelEcosystems

— PERAS workshop (Schaeffer et al.2010)

Semi-fieldstudies

crab species). Hard-bodied organisms have evolved special organs for as-similation of oxygen and water, although for soft-bodied biota uptake viathe skin is the most important route of uptake of water and oxygen. Contam-inants and nutrients may also be taken up via these distinct exposure routeswhile uptake of contaminants via food is possible for all biota.

Some insight in the physiology and ecology of terrestrial organisms isneeded to enable evaluation of the importance of the various uptake routes.The structure of the skin, mode of uptake of water, mode of uptake ofoxygen, and feeding habit are important variables in this respect. Thereupon,the behavior of the organisms is of importance.

MorphologySTRUCTURE OF THE SKIN

Soft-Bodied Organisms. The skin of earthworms and enchytraeids con-sists of a layer of muscle tissue covered by a thin hypodermis and a cuticle.

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The skin is covered by a mucus layer produced by a dorsal pore, and/or byliquid produced by the renal tubules.

The cuticle of nematodes consists mainly of collagen (proteins), thecomposition of which is species-dependent and dependent on the stage ofdevelopment of the nematode. The cuticle is usually covered with a thinlayer (glycocalyx) consisting of proteins, fats, and sugars, the compositionof which is species dependent and dependent on the stage of development.

Hard-Bodied Organisms. A characteristic feature of arthropods is theirfirm cuticle, which due to its relative nonpermeability allows arthropods toregulate the osmotic pressure of the body liquids and to reduce loss of water.

The cuticle consists of the following layers: the epicuticle consists mainlyof lipids and provides the main barrier against loss of water. No chitine ispresent in the epicuticle and in crustaceans calcium is present. The exocuticleof arthropods consists mainly of chitine and protein-chitine complexes andis tanned with quinones. It is therefore quite firm. The endocuticle is nottanned and therefore soft. The epidermis has a thickness of one cell layerand is responsible for the production (excretion) of the cuticle. A waxy layermainly consisting of fats covers the epicuticle of most arthropods. The waxylayer is lacking in case of primitive land-dwelling crustaceans whereas it isjust partly developed for species better acclimated to terrestrial habitats (suchas woodlice). This implies that the cuticle of crustaceans is more permeablefor water and gases than the cuticle of for instance insects. Hadley andQuinlan (1989) showed that removal of the waxy layer for spiders inducesa drastic increase of the permeability of the cuticle as the evaporation ofwater increased by as much as a factor of 200. Permeability of the cuticlesis impacted by various factors such as temperature and air humidity. Theepidermis is fully permeable to water.

WATER UPTAKE

Soft-Bodied Organisms. Earthworms and other soft-bodied species in-gest water mainly via the skin. Excretion of water by earthworms occurs viathe dorsal pores, renal tubules, and gastrointestinal tract. Earthworms are ca-pable of accumulating chemicals via the skin, in some cases (e.g., chloride,even against a concentration gradient [Wallwork, 1983]).

Hard-Bodied Organisms. Arthropods can ingest water via variousroutes: (a) by drinking or by absorbing from wet or humid surfaces, forinstance via the cuticle (e.g., for some species of isopods); (b) via consump-tion of (living or dead) food; and (c) by absorption from air.

Either the mouth or the rectum provides in all cases the main route ofuptake, whilst for insect larvae uptake via the skin has been claimed (Edney,1977).

For uptake of water via drinking, spiders are capable of taking up waterfrom a substrate against the water tension and some species are able towithdraw water at a water tension of the soil of 400 mm Hg (i.e., a water

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tension well below field capacity). Centipedes and various arthropods absorbwater via special structures in the rectum, whereas in case of collembolansthe ventral tube is used for drinking. In isopods this is done via the mouthand anus (Warburg, 1993).

For uptake of water via food, isopods and many other arthropods meettheir water requirements mainly by consumption of humid food; insect larvaeconsume humid sand for this purpose (Hadley, 1994). In a quantitative sense,to our knowledge hardly anything is known about the amounts of watertaken up by arthropods via consumption of food.

For uptake of water via air, absorption of water via the air is possibleonly at conditions of high humidity. According to Carefoot (1993) isopodsare capable of absorption of water from air at air humidity exceeding 90%,at conditions of very high humidity, such as is often the case in soil pores.In that situation water uptake via the cuticle is even possible. Eggs of arthro-pods are able to take up water from their surroundings via the cuticle andvia specially developed structures (hydropyles); in most cases this concernswater in air, although uptake of pore water usually cannot be ruled out.

PhysiologyUPTAKE OF OXYGEN

Although most chemicals in view of their volatility and sorption propertiesare typically not taken up via the gas phase, insight in the way soil biotainteract with (pore) air enhances the understanding of the modes of uptakeof nutrients, water, and contaminants. Exchange of gases such as oxygenand carbon dioxide usually takes place via diffusion:

1. Direct diffusion between water and cells is relevant for organisms thatretrieve oxygen from the surrounding (pore) water, such as protozoa,nematodes, rotifers, earthworms, and enchytraeids.

2. Direct diffusion between air and cells is relevant for many insects breath-ing via tracheas.

3. Diffusion between air and blood is usually via a wet surface such asthe skin of soft-bodied invertebrates, the booklungs of spiders, and thediffusion lungs of snails.

It should be noted that the difference between the first and the thirdprocess is not clear yet. Earthworms for instance excrete mucus and liquidvia their dorsal pores to keep the skin humid and thus to promote uptakeof oxygen and release of CO2, as well as to avoid dehydration (Wallwork,1983). Small invertebrates with a mass of up to a few milligrams benefit fromdirect exchange of O2 and CO2 with their surroundings as this minimizesthe diffusion length and as it allows all parts of the body to participate in

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this process. According to Wells (1980) this explains why many species ofnematodes, flatworms, and other invertebrates have a long and thin body.

Uptake of oxygen by woodlice takes place via the pleopods and viathe skin, with an increasing contribution of the pleopods for species thatare less dependent on a humid environment. For some woodlouse speciesan increase of oxygen uptake was found on decreasing air humidity, andoxygen is mostly taken up on the basis of the legs and via the belly, wherethe cuticle is thinnest.

UPTAKE OF FOOD AND SOIL PARTICLES

Soil organisms represent a plethora of trophic levels: detritivores, fungivores,herbivores, carnivores, and omnivores. Within some taxa all species belongto the same trophic level, although other organism groups contain a largediversity of trophic levels. For instance, all spiders are carnivores, whereasthe group of oribatid mites contains species feeding on fungi, algae, lichens,bacteria, protozoas, and nematodes. This implies that it is not possible torelate exclusively food uptake to phylogenetic considerations. Saprophagicspecies do not feed solely on nutrients released from dead organic substrateas these organisms are often also microbivores and consume bacteria andfungi jointly with the dead material. Additionally, within a species severalmodes of feeding can be present as for instance larvae need different nutri-tion than animals in their adult stages.

Uptake of nutrients from the food ingested takes place in the gastroin-testinal tract. Most invertebrates are somehow capable of regulating andbuffering (e.g., by excretion of Ca, and of carboanhydrase to bind CO2 toCa) the pH of the gastrointestinal tract. For many species, pH is around neu-tral (6–7), but species exist with very high (9–10) or low pH in their guts tostimulate digestion of the food and uptake of nutrients (Hopkin, 1989).

Earthworms and enchytraeids feed mainly on organic substrates and,dependent on the species, to a greater or lesser extent also on soil particles.Some species eat all types of organic material, other species select food richin nitrogen and sugars and low on polyphenols. This implies that the relativeamount of mineral soil found in the gastrointestinal tract of earthwormsmay vary greatly across earthworm species (Wallwork, 1983). Thereupon adifferentiation with regard to particle size is notable. Various enzymes arepresent in the gastrointestinal tract of earthworms.

The gut of arthropods contains cuticles that allow only for passageof water, ions, and small molecules (< 1000 Da). Absorption of chemi-cals/nutrients is possible in the midgut and in the first part of the hindgut.Some groups, such as oribatid mites use special digestive enzymes, amongothers produced in the gastrointestinal tract by the microflora present. Otherspecies such as flies and spiders consume semiliquid externally predigestedfood.

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Woodlice feed on detritus and decaying wood, and the foregut and thehindgut serve as a semipermeable membrane mainly taking up water. Othersubstances such as glucose are barely taken up in the gut. The hepatopan-creas is for woodlice of importance as this is the organ in which the finaldigestion and absorption of nutrients and other compounds takes place. Forthese organisms too, a high population of active microorganisms is presentin the gastrointestinal tract, stimulating digestion (Belfroid and Van Gestel,1999).

Overall, uptake of nutrients and chemicals is possible for all inverte-brates via their food, and this may be an important route in case of foodsources in which high concentrations of chemicals are present. A strikingexample was provided in this respect by Bengtsson et al. (1983), as theseauthors showed that Collembola may take up significant quantities of metalswhen fed metal-containing fungi.

Behavior

Earthworms are a class of organisms for which typical behavioral patternsstrongly affect effective exposure. The various species are classified as epige-ics (surface dwelling and surface feeding), endogeics (topsoil dwellers feed-ing on mineral soil), and anecics (deep burrowing species often feeding onfresh surface litter), although several additional subgroups have been pro-posed (e.g., for the endogeics and epigeics). Some earthworm species donot seem to fit into any particular category or, rather, fit in between pro-posed categories (e.g., epi-endogeic, endo-anecic). Ecological preferencesand behavior of earthworms (and soil invertebrates in general) are affectedby external factors such as soil and weather conditions: earthworms will forinstance surface less in winter time, and rainfall will enhance surfacing toavoid drowning in the burrows. Given the inhomogeneous (vertical) distri-bution of plant protection products across the soil profile, effective exposureof soil invertebrates will thus be modified by feeding and burrowing strate-gies. In addition, Bruns et al. (2001), Hendriks et al. (2001), and Hendriksand Heikens (2001) showed that body size is an important parameter ininterspecies variability, with smaller animals (e.g., enchytraeids) in generalaccumulating higher levels of chemicals at similar exposure conditions thanlarger animals (e.g., earthworms). Finally, avoidance behavior is an impor-tant parameter affecting effective exposure (Rombke, 2008). Yeardley et al.(1996), Stephenson et al. (1998), and Hund-Rinke et al. (2003), among oth-ers, developed and validated a test useful for assessing typical avoidancebehavior of earthworms that has been presently standardized (ISO, 2008b).Almost the same stage has been reached with a collembolan avoidance test(ISO, 2010a; Natal-da-Luz et al., 2008). For organisms that are active in thesoil layer and in the litter layer, the correct modeling of the exposure in both

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layers is important because this determines the relative importance of activefeeding and residual contact versus passive contact.

EVALUATION OF TOTAL CONTENT AND PORE WATERAS EXPOSURE METRICS FOR THE EFFECT ASSESSMENT

OF SOIL ORGANISMS

The starting point in the basic concepts presented in the previous sectionis that the dose at the target site is central in explaining the dose-responsephenomena observed in the field and in the laboratory. However, because itis impossible to measure the dose at the target site, the free concentration inpore water is considered the useful alternative over the total concentrationin the soil.

Starting from this perspective, the research reviewed subsequently aimsat understanding the basic concepts in more detail. It is often demonstratedthat the variability in results obtained from different soils or from soils withdifferent ageing time is reduced when the free concentration in the porewater is taken as a metric compared to the use of total concentration. Furtherresearch often focuses on factors adding additional variability, such as theuptake via food, nonequilibrium situations, speciation, or saturation of thepore water.

Definition of Pore Water

Pore water is the water in a soil. In the context of this review it is definedas the water found in the unsaturated zone above the water table. Theunsaturated zone contains solid particles (soil particles and organic matter),water (pore water), and air (pore air) located in the pores between the soilparticles. The pore water may contain dissolved organic matter to variousdegrees. The amount of pore water in soil also varies according to spatialand temporal scales. Both the dissolved organic matter as well as the amountof pore water available may influence the uptake in invertebrates.

Definition of Total Content

The total concentration is typically defined as the concentration (i.e., mass ofsubstance per mass of dry soil) that is extractable from the soil matrix usinga well defined relative harsh extraction technique using organic solvents andinput of energy by shaking and/or elevated temperature (often the boilingtemperature of the extractants) and pressure.

Total concentrations may be either expressed on a dry or wet massbasis of the soil. As wet mass in general is ill-defined, it is to be preferred toexpress total concentrations on the basis of soil dry mass. It should be noted

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in this respect that, although this is not justified for scientific reasons, it isstill common to express total concentrations as nominal concentrations (i.e.,the total mass of chemical added to a certain mass amount of dry or wetsoil). In the latter handling procedures of the spiked soils, possible lossesdue to volatilization, degradation, and sorption to the glass matrix of vesselstypically used in laboratory handling of soils may occur.

What is not extractable is often referred to as nonextractable residue(NER) or bound residue. It was defined by Roberts (1984) and later adoptedby the International Union of Pure and Applied Chemistry as chemicalspecies originating from pesticides used according to good agricultural prac-tices that are nonextracted by methods, which do not significantly change thechemical structure of these residues. Barriuso et al. (2008) reviewed the datafor plant protection products and found NER in the range of 5–100%. Dithio-carbamates generally had the highest NER and organophosphates the lowest.Only 12% of the plant protection products had an NER exceeding 70%.

Evidence on the Relative Importance of Pore Wateras an Exposure Pathway

Two pragmatic approaches have regularly been used to support that theconcentration in pore water is in most circumstances a more precise expo-sure metric than the total concentration in soil: changes in effects over time(ageing) and normalization of effects observed in various soil types. Theseare described in more details subsequently. Additionally, in this section theexposure via ingestion and bioconcentration are discussed.

TOXICITY IN AGED SOIL

The degradation of a toxicant over time will obviously lead to a reduced toxi-city. However, a reduced toxicity over time may be seen even in cases whereno or only a limited degradation is observed. It is now a well-establishedfact that ageing over time (i.e., an increased contact time between the chem-icals and the soil matrix, often named sequestration), typically leads to adecrease in pore water concentration and, as a consequence, to a decreaseof the amount of toxicant available for uptake in organisms (Alexander 1995,Cornelissen et al. 1998). Morrison et al. (2000) and Robertson and Alexander(1998), for example, demonstrated significantly reduced toxicity of DDT anddieldrin for earthworms in aged soil although the total concentrations werealmost unchanged. Styrishave et al. (2008) observed a similar response ofcollembolans when exposed to pyrene in aged soils. These authors showeda parallel reduction in the pore water concentration of pyrene as measuredwith solid-phase microextraction (SPME), performed with a fiber coated witha liquid (polymer), a solid (sorbent), or a combination of both. The fiber isexposed to a sample of a contaminated soil under shaking until equilibrium

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is reached. The amount sampled by the fiber is negligibly small, so the orig-inal freely dissolved concentration in the soil is not affected), but the EC50

based on pore water did not change. The fact that the toxicity of the contam-inated soil sample reduced over time without a similar reduction in the totalconcentration is a clear indication of the limitations in the total concentrationconcept. Additionally it supports the pore water concept as a more precisemeasure of the bioavailable fraction.

TOXICITY ACROSS VARIOUS SOIL TYPES

Comparing observed effects in different soil types of varying properties andhence differing sorption characteristics is an alternative, but yet comparable,approach to elucidate the accuracy of total concentration as parameter. Ifthe total concentration metric is a suitable concept for exposure and/or risk,equal total concentrations should result in the same response, no matterthe properties of the soil type. Amorim et al. (2005) found, however, afactor of 30 regarding differences in the chronic toxicity of the herbicidephenmedipham to soil-dwelling enchytraeids, when exposed in 18 differentsoil types. Almost the same factor (27) was found when comparing theEC50 values of tributyltin-oxide in chronic earthworm tests performed in 10different soils, although the toxicity of the same chemical differed only bya factor of 8 in collembolan tests with the same soils (Rombke et al., 2007).Under the same conditions, the factor was even higher (50) in plant testswith Brassica rapa.

Styrishave et al. (2008) found an effect of organic matter (OM) on thebioavailability of pyrene when they compared the reproductive effect con-centrations (EC50) for the euedaphic collembolan, Folsomia candida, undervarious test conditions. The soil used was a sandy loam soil with naturalorganic matter content of 2.6% (Askov soil). It was enriched with increasingorganic matter concentrations of 5%, 10%, and 20% and was aged for 0, 56,and 112 days. The EC50 values of the collembolans increased with increas-ing organic matter content of the soil and also with ageing, resulting in upto threefold differences between low organic matter content soil and highorganic matter content soil. Pore water concentrations determined by SPMEdecreased with increasing organic matter content. The EC50 values based onpore water concentrations also varied by a factor 3 at approximately 23 ±9 µg.L!1, but without correlation to ageing or organic matter content. Thesefindings support the equilibrium-partitioning theory.

The effects of chlorophenols on plants in soils and in nutrient solutionshas been investigated by Van Gestel et al. (1996) and Hulzebos et al. (1993).They found that pore water corrections based only on sorption did notimprove the results. Only after a correction for speciation, did the porewater results based on the nondissociated fraction converge between soils.Comparison with the results of nutrient solutions indicates that there is no

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additional action through the solid phase. Moreover, the results based onpore water are much closer to those based on nutrient solutions than thetotal content results are, also for the nondissociated species. Especially fordissociating compounds, actual concentrations in the pore water have to bemeasured and full understanding of the test conditions would be necessaryin order to perform risk assessment.

Van Gestel and Ma (1988, 1990) showed that the calculated LC50 values(mg.Lpore water

!1) for the earthworm species Lumbricus rubellus and Eiseniaandrei exposed to chlorophenols and chlorobenzenes in four different soiltypes were comparable (within a factor of 2), when normalized for organicmatter content, using the Freundlich partitioning coefficient Kf and its expo-nent 1/n, determined in a shake-flask study. In contrast, the nonnormalizedvalues (total concentration in soil: mg.kgsoil

!1) varied within a factor of 6.Pore water and hence uptake via pore water was concluded to be the de-terminant of toxicity. However, concentrations were not measured and thespeciation of the dissociating substances was not considered in the article.Based on the fraction nondissociated (calculated at pKa 4.74 and the givenpH-KCl), the EC50 values for pentachlorophenol (PCP) differ a factor of upto 10. Where for plants accounting for the nondissociating fraction reducedthe variability between results for PCP; for the earthworms the opposite istrue.

Lock et al. (2002) tested the toxicity of lindane (log Kow 3.85) againstcollemboles, earthworms, and enchytraeids. It was hypothesized that toxic-ity should decrease with increasing organic matter content. The LC50 and noobserved effect concentrations (NOECs) based on total content in dry massvaried between soils, without apparent correlation to the soil organic mat-ter content. Looking at the standard endpoints, the authors concluded thatthe equilibrium partitioning theory (EPT) was not valid for all species, be-cause the expected relationship with organic matter (and hence pore water)was not consistently found. However, the results were based on nominaldry mass concentrations, neither the soil pore water concentrations weremeasured (nor calculated) nor were the sorption kinetics in the differentsoils characterized and accounted for. The expected relationship has thusnot been adequately described, which makes it difficult to draw conclusionsfrom the experiment.

Sverdrup et al. (2002) studied the effect of polycyclic aromatic hydrocar-bons (PAHs) on the collembolan Folsomia fimetaria and showed that onlyPAHs with reported log Kow values <5.2 (i.e., naphthalene, acenaphthene,acenaphthylene, anthracene, phenanthrene, fluorene, pyrene, and fluoran-thene) significantly affected the survival or reproduction of the test organ-isms. Threshold values for the toxicity of the individual PAHs could be ex-pressed as pore water concentrations by the use of reported Koc values. Forthe PAHs with a log Kow <5.2, toxicity significantly increased with increas-ing lipophilicity of the substances (r2 = 0.67; p = 0.012; n = 8), suggesting

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a narcotic mode of toxic action for most substances. Using this quantita-tive structure-activity relationship (QSAR) to calculate threshold values forthe toxicity of the remaining nontoxic substances (benz[a]anthracene, chry-sene, benzo[b]fluoranthene, benzo[k]fluoranthene, dibenz[a,h]anthracene,benzo[a]pyrene, perylene, and indeno[1,2,3-cd]pyrene), the absence of toxic-ity could, in most cases, be explained by a limited water solubility, indicatingthat these substances do act by narcosis as the mode of toxic action and thattheir toxicity is governed by concentrations in the pore water.

Based on the previous it is evident that toxicity does frequently deviateacross various soil types. The most likely explanation for this is changesin adsorption and hence pore water exposure, although this has not beenvalidated by measurement (or models) in all cases.

CIRCUMSTANCES WHERE EXPOSURE VIA FOOD AND SOIL INGESTION MAY PLAY

A ROLE

The contribution of oral uptake of chemicals depends on the rate of con-sumption of the matrix being ingested by the animal and the uptake ef-ficiency. Both parameters depend on the matrix being eaten: uptake byingestion of soil will differ from uptake by ingestion of food sources such asleaves and dung.

Uptake processes are physiologically driven and affected by speciesspecific parameters (traits) such as morphology, surface-volume relationship,feeding strategy and preferences, and individual behavior. The relative im-portance of the various exposure routes may vary between groups of speciesaccording to their traits (e.g., soft- and hard bodied species) and vertical dis-tribution. Furthermore, when first taken up the toxicological availability at thetarget organ is related to internal processes controlled by organism specificparameters such as allocation, metabolism, detoxification, storage capacity,excretion, and energy resources.

Gyldenkærne and Jørgensen (2000) modeled the exposure and effectfor soil dwelling arthropods accounting for soil, pore water, food, and aircontact. The model gave generally good correlations between predictionsand test results based on results for pyrethroids (high Koc) and dimethoate(low Koc). They concluded, therefore, that the most important route ofuptake is from the soil. Furthermore, they predicted that the bioavailableamount of a pesticide in the soil is a function of the pore water concentration.For the very lipophilic pyrethroids, however, predictions are not reliable. Thismodeling assessment confirms that also for soil-dwelling organisms, soil porewater concentrations are an accurate measure for effects of pesticides withlow to high Kow. Deviating effects for highly lipophilic compounds may bedue to the strong sorption and hence other exposure routes or processes thanthose presently understood, in combination with the specific toxicokineticsof the compounds (in this case pyrethroids), could be possible.

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Sousa et al. (2000) exposed Porcellionides pruinouis (a species commonin compost heaps with the ability to dig and to be in closer contact with thesurrounding medium) to 14C-labeled lindane via either food (0.2 mg.kg!1)or soil (0.1 mg.kg!1) and found that internal concentrations in the isopodsfollowing exposure via soil exceeded internal concentrations after food ex-posure by a factor of 25. Also, Santos et al. (2003) found that internal 14C-lindane body burdens in Porcellionides pruinosus were better related tolindane concentrations in soil extracts than to the total lindane content insoil. These results suggest that uptake via pore water is of importance forisopods and this species is a good candidate from the saprophagous groupsupporting the EPT (Sousa et al., 2000).

Although in contact with soil, isopods live mostly on the soil surface anddietary uptake seems to dominate; according to van Brummelen et al. (1996)several nonequilibrium situations occur. These are related to the structureof the litter layer (instability of the leaf surface and water-film interface)and to the isopod adaptation to avoid water losses by reducing contactwith the surrounding medium to a minimum. Furthermore, the contributionof oral uptake by isopods increases upon increasing hydrophobicity, andespecially at log Kow values larger than around 6, oral uptake may becomethe dominant uptake route. Given the present trend toward developing plantprotection products with typical cutoff limits for log Kow of around 4–5, porewater mediated uptake seems the most common uptake route.

Ma et al.’s (1995) study with earthworms illustrates other complicatingfactors in interpreting exposure routes as they observed that the availabilityof noncontaminated food induces a significantly lower uptake (factor of 2–3)of PAHs from soil by the earthworm Lumbricus rubellus. This indicates thatthe amount of soil passing the gastrointestinal tract may vary and dependson the amount of nutrients in the soil as well as in other food sources. Thisfinding may, however, also be explained by reduced mobility of the wormson increasing food availability, thus increasing local depletion of PAHs inthe pore water.

According to Jager (1998) a distinction can be made between earthworm(and other) species predominantly feeding on leaf material and species feed-ing on humic material. The latter category includes the compost worms Eise-nia fetida and Eisenia andrei, whereas the former group includes Lumbricusterrestris and Lumbricus rubellus. L. rubellus is supposed to ingest significantquantities of soil only when digging new burrows and when actively search-ing for food. Beyer (1996) indeed showed a temporary increase of internalhexachlorobenzene concentrations following transfer of L. rubellus to a newsoil and subsequent creation of new burrows. Thereupon, Belfroid et al.(1994) found that accumulation factors are lower for L. terrestris than for E.andrei, which could imply that soil uptake for anecic species is lower thanfor epigeic species. E. andrei would thus be a worst-case species within the

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class of oligochaetes. Bruns et al. (2001) showed in this respect that small-bodied organisms in general have higher bioaccumulation factors (BAFs)than larger animals as Bruns et al. compared uptake for various enchytraeidsspecies including earthworms.

Accumulation factors for uptake of DDT by slugs from soil exceed ac-cumulation factors from food by a factor of 10. This is most likely due todiffusion of DDT from the soil to the outer skin of the slugs (Belfroid andVan Gestel, 1999), which shows that uptake via soil is more important forslugs than update via food. It should be noted that Legierse (1998) found thatthe kinetics and the steady-state conditions of hydrophobic organic contam-inants in aquatic snails are not related to the fat content of the animals as isthe case in many other organisms such as fish and worms. Scaling of internalconcentrations of hydrophobic organic contaminants to the lipid content asperformed with the EPT is therefore not possible for snails. Instead, uptakekinetics and steady-state conditions for aquatic snails are determined by ei-ther protein or carbohydrate-driven metabolism. This is most likely also thecase for terrestrial snails.

COMPARING BIOCONCENTRATION ACROSS SPECIES, MATRIXES AND CHEMICAL

PROPERTIES

If pore water were the only significant exposure route, comparable bio-concentration of contaminants from water and from soil would be expectedwhen assuming similar uptake and elimination processes at equilibrium con-ditions. This implies similarity of bioconcentration from water and from soilfollowing normalization for the pollutant concentration in the soil pore water.It is implicitly assumed that uptake is concentration independent. Belfroidand Van Gestel (1999) showed that in a number of cases bioconcentrationfactor (BCF) and BAF values are similar, whereas differences increase by afactor of greater than 2 for hydrophobic chemicals such as hexachlorbenzene(BAF exceed BCF by a factor 2.1–2.7) and especially benzo[a]pyrene (factor4.3). Especially for hydrophobic chemicals, bioaccumulation in soil exceedsbioconcentration from water in freshly spiked and aged soils.

It should, however, be noted that these findings are based on normaliza-tion by means of the soil sorption constant, Kd, implicitly assuming linearityof sorption (also the partitioning coefficient, Kp, is often used to indicate theextent of soil sorption of chemicals). In a field setting, however, actual porewater concentrations may deviate by over a factor of 10 from concentrationspredicted on the basis of Kd because of a number of reasons (e.g., vary-ing daily weather conditions and day/night climatic variations). Thereupon,local depletion may play a role (albeit that this would reduce bioaccumula-tion) because it cannot be ruled out that replenishment of the soil solutionfollowing depletion by biota has lower kinetics than uptake, and hence israte limiting for actual uptake. This would also imply that longer times areneeded to reach steady-state conditions. Briggs and Lord (1983) showed for

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instance that initial uptake of aldicarb at 22% humidity exceeds uptake at 8%humidity (i.e., conditions in which depletion is more likely). Finally, elim-ination and elimination kinetics may play an important role in the amountof chemical accumulated at steady state. Belfroid and Sijm (1998) showedfor instance that elimination is affected by the amount of organic material inthe vicinity of worms. This will affect the kinetics of elimination and hencethe final amount accumulated in the organism, thus hindering the basic re-quirement of similarity of uptake and elimination kinetics in water and insoil.

Hickman and Reid (2005) found no correlation between the extractabil-ity of phenanthrene, a hydrophobic organic contaminant, from four differentsoil types and the accumulation in earthworms (Lumbricus rubellus). Theorganic matter content ranged from 2.5% to almost 70% (peat), but yet onlylimited difference in accumulation was observed between the different soils.They used aqueous-based cyclodextrin and CO2 equilibrated water to ex-tract phenanthrene from the soil. Both extraction methods (but especiallycyclodextrin) showed very good correlation with microbial mineralization,indicating that the bioavailability of contaminants to microorganisms andearthworms, as expected, is not comparable. One hypothetical reason for thiscould be that microorganisms are only exposed through pore water whereasingestion of particle-associated substances may occur for earthworms. Thisis supported with a study with the soil nematode Caenorhabditis elegans,which is believed to be solely exposed through pore water (Sochova et al.,2007). Testing seven different pollutants, including the pesticides toxapheneand hexachlorobenzene, in soil tests as well as aquatic tests, Sochova et al.showed that C. elegans frequently was less sensitive than other invertebratespecies in soil but had comparable sensitivity to other aquatic species inwater tests.

The choice of extracting method is of course much dependent on thephysicochemical properties of the molecule studied: cyclodextrins proved tobe a good estimation of the bioavailability of hydrophobic compounds suchas PAHs (Puglisi et al., 2007a; Semple et al., 2004) and PCBs (Puglisi et al.,2007b). For more ionic compounds such as the weak acid pentachlorophe-nol, it was instead found that earthworm body tissue accumulation correlatesbetter with the amount of contaminant extracted in water (Spagnuolo et al.,2010).

Although hypothetical this could indicate that for some soil-dwellingorganisms, ingestion of polluted soil material may add to the overall toxicity,whereas others solely are exposed through pore water.

A model developed by Belfroid et al. (1995) in which estimates of ac-cumulation of organics by earthworms were generated (inputs were biocon-centration in water, sorption and elimination constants, rate of soil ingestion,and uptake efficiency) showed that in most cases uptake from pore water isthe dominant process. However, for chemicals with log Kow exceeding 5,

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oral uptake may contribute significantly: about 10% for soils with an averageorganic matter content of 3%, and about 50% for soils with an organic mattercontent of 20%. This implies that at maximum, the modeled concentrationof organic contaminants in earthworms exceeds the EPT prediction by afactor of 2. This is similar to the difference found in general when com-paring BAF and BCF values, as reported by Jager (2003). Further support,although not for a soil-inhabiting species, is obtained from experiments withLumbriculus variegatus. Sormunen et al. (2008) showed that these sediment-ingesting worms have access to an additional bioavailable chemical fractionof polychlorinated biphenyls (PCBs) on top of the pore water pool that wasespecially evident when pore water concentrations of PCB 77 approachedthe solubility limit. Thus, feeding may modify the bioavailable fraction thatcannot be explained by simple equilibrium partitioning models.

Yu et al. (2006) studied the relationship between bioavailability, mea-sured as bioconcentration factors, to earthworms (Aporrectodea caliginosa)and the absorption or desorption of three pesticides in five soils. They foundthat the sorption processes were mainly controlled by the organic mattercontent. Furthermore, Yu et al. (2006) found a good relationship betweensorption and concentration in worms for myclobutanil and butachlor withlog Kow values of 2.94 and 3.71, but not for chlorpyrifos with a log Kow of4.70.

Not only the quantity of organic matter, but also its quality influencesthe availability of hydrophobic organic pollutants. Special constituents in soil,often referred to as carbonaceous carbon phases (e.g., black carbon, coal,kerogen), can be responsible for 90–99% of total sorption of organic com-pounds in soil (Cornelissen and Gustafsson, 2005; Koelmans et al., 2006).This leads to 10–1000 times stronger sorption than on the basis of amorphoussoft organic matter only and as a consequence to similar reductions in bioac-cumulation and actual risk. The relative amount of black carbon or similarconstituents is, however, not expected to be very high in agricultural soils,and therefore more likely to play a role when assessing risk of contaminatedland than risk of plant protection products.

Uptake in Sediment-Dwelling Organisms. It should be noted that thenext examples deal with sediment-dwelling organisms instead of with soilinhabiting taxa. The general findings reported for sediment organisms are,however, illustrative for the uptake routes for soil organisms. Therefore,caution to extrapolate these results with sediment dwelling deposit feedersto soil organisms should be used.

In a systematic approach, Kraaij et al. (2003) deduced that sedimentbioaccumulation of compounds up to log Kow 7.5 in Tubificidae can bedescribed as bioconcentration from pore water. In addition, the pore waterconcentrations of hydrophobic organic chemicals (4.5 < log Kow < 7.5) areestablished by equilibrium partitioning between the rapidly desorbing frac-tion of hydrophobic organic chemicals in the sediment and the pore-water.

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Taken together, these findings indicate that EPT is a conceptually correctrepresentation of sediment bioaccumulation, provided that sequestration isaccounted for. This implies that the risk assessment of sediment associ-ated hydrophobic organic chemicals can be significantly simplified: With amethod at hand for measuring freely dissolved pore water concentrationsof hydrophobic organic chemicals, body residues of hydrophobic organicchemicals in soft-bodied sediment organisms can be estimated on the basisof concentrations in pore water and bioconcentration factors.

Lu et al. (2004) found that substance specific properties significantlychanged the relative importance of uptake routes. They measured theuptake of PAHs to a sediment-dwelling and deposit-feeding tubificidoligochaete (Ilyodrilus templetoni). Whereas only 5% of the uptake ofbenzo(a)pyrene (log Kow of 6.13) originated from the pore water, morethan 80% of the phenanthrene (log Kow of 4.57) was taken up via porewater. Leppanen and Kukkonen (2000) used the benefit of the feature of thefresh water oligochaete Lumbriculus variegatus to fragmentate as a repro-duction method to elucidate the importance of feeding as an uptake routeof PAHs. The parent animal (front fragment) continues to feed, whereas theoffspring (the posterior fragment) does not ingest sediment for 5–6 weeks.Ingestion of sediment considerably increased accumulation of as well pyreneas well as benzo(a)pyrene showing the importance of exposure to particlebound residues in deposit feeders.

Dermal and Topical Uptake. Basically, residual and topical uptake in-duce significant deviations from EPT, and it is therefore not surprising thatEverts (1990) found that residual uptake of deltamethrin by the spider Oe-dothorax apicatus was the most important pathway. Additional researchconfirmed that direct exposure and oral uptake are of limited importance inthe field. However, the availability of the residue was dependent on the hu-midity and a decrease of humidity to 66% of field capacity caused a decreaseof toxicity of 75%. Additional studies of Akkerhuis (1993) with this spiderdisplayed a negative correlation between uptake of deltamethrin and thesorption capacity of the soil cover. It may thus be hypothesized that uptakeis facilitated under conditions in which the contaminant is loosely boundto a substrate and in which the organism is in close contact with water inorder to allow for exchange of the contaminant. Although these parametersare different from the basic properties underlying the EPT concept, they doshare some communality.

CONCLUSIONS

Pore water concentration is the first explaining factor, explaining most of thevariance in uptake data. Above uptake via pore water, there are other routesof uptake and part of the fast desorbing fraction of chemicals present in the

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soil need to be taken into account in assessing risks of organic contaminantsto specific species present in terrestrial ecosystems. The extent in whichnonpore fractions are taken up by biota depends on the organism.

The mode of exposure of soil invertebrates is determined by the wayanimals are in contact with their local environment. Morphology, physiology,and behavior are important factors in this respect, as is the mode of uptakeof food, water and oxygen. Intraspecies (especially between different lifestages) and interspecies variances (e.g., size and ecological preferences) willmost likely modify the actual contribution of potential exposure pathways.

A distinction must be made between hard-bodied and soft-bodied or-ganisms. Hard-bodied organisms rely for uptake of oxygen and water onspecialized organs, whilst uptake of water (pore water) via the skin is themain route of uptake for soft-bodied organisms. Hard-bodied animals arenevertheless in contact with pore water, as shown for spiders, woodlice andcollembolans. Thereupon, these organisms will satisfy their need of waterby consumption of humid food and possibly soil. Uptake of nutrients andchemicals is possible for all invertebrates via their food, and this may bean important route in case of food sources in which high concentrations ofchemicals are present. The assimilation efficiency will however depend onspecies-specific properties of the digestive tract and no general conclusionsare to be generated in this respect.

Knowledge on uptake routes of organic contaminants and of metalsby soil invertebrates is far from complete. Most information is available forearthworms, collembolans and isopods. The EPT appears to be valid forearthworms and collembolans in laboratory settings, although some specificuncertainties such as food type need further investigation.

Overall it is clear that uptake of organic contaminants depends onspecies, soil type, and the chemical properties. The contribution of oraluptake may vary within a specific taxon but for soil organisms in close con-tact with the soil solution, pore water–mediated uptake is in general thedominant pathway and it is commonly modified by soil-specific ageing andspeciation, and by specific factors of the organisms, such as nutrition status.

To estimate pore water concentrations, adequate sorption coefficientsare needed. Sorption coefficients are found to range more than an order ofmagnitude. The actual pore water concentrations of dissociating compoundsare subject to even more influencing parameters that are difficult to pin-point. As a result, an incorrect characterization of the sorption properties ofthe soil-compound combination may cause differences of several orders ofmagnitude in the pore water concentration estimations.

Insufficient field studies are available for allowing a confirmation of thevalidity of the equilibrium partitioning theory. Residual uptake appears to bethe most important uptake route following pore water mediated uptake. Itis likely that in this case too, pore water is involved as carrier in or at thesurface of the soil in which the chemicals are dissolved.

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It will be also important to carry out future research at multispecieslevel, by comparing exposure and toxicity at different trophic levels: becausebioavailability is indeed changing from one species to the other integratedassessments will become very useful for a more realistic risk approach ofchemicals in soils and in order to promote a better recognition of this processat regulatory level.

ACKNOWLEDGMENTS

Peter Melis (RIVM) is kindly acknowledged for performing the literaturesearch. Part of the work reported here is included in a scientific opinionof the Panel on Plant Protection Products and their Residues (PPR Panel)of the European Food Safety Authority (2009), which is freely available onhttp://www.efsa.europa.eu/.

REFERENCES

Akkerhuis, G.A.J.M. (1993). Physical conditions affecting pyrethroid toxicity inarthropods. PhD thesis, University of Wageningen, Wageningen, the Nether-lands.

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Evaluation of Exposure Metrics for Effect Assessment of Soil Invertebrates

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