Enchytraeids as Indicator Organisms for Chemical Stress in Terrestrial Ecosystems

Ecotoxicology and Environmental Safety 50, 25}43 (2001) Environmental Research, Section B doi:10.1006/eesa.2001.2075, available online at http://www.idealibrary.com on

Enchytraeids as Indicator Organisms for Chemical Stress in Terrestrial Ecosystems Wim Didden* and JoK rg RoK mbke*Wageningen University, Subdepartment of Soil Quality, P.O. Box 8005, 6700 EC Wageningen, The Netherlands; and- ECT Oekotoxicologie GmbH, BoK ttgerstrasse 2-14, D-65439 FloK rsheim/Main, Germany Received June 19, 2000

of soil processes and the organisms involved is still insu$cient to evaluate the hazards of stress on the system level. Moreover, because of the complexity of the soil community and the spatial heterogeneity of soil, it would be extremely time-consuming and expensive to study the system as a whole. This is why increasing attention is being given to the use of indicator organisms: organisms that may be regarded as re#ecting the quality of the soil system. Potential indicator organisms must ful"ll several criteria (mainly after Edwards et al., 1996), in that they should: 1. play a key role in the functioning of the soil ecosystem, so that their response is relevant for conclusions on the system level 2. be present in a wide range of ecosystems, to allow comparisons between systems 3. occur abundantly, so that they are widely available and their response is readily recordable 4. be easy to use both in "eld and laboratory conditions: they should be easily collectable and culturable 5. come into contact with a variety of stress factors, via the soil solution, the solid phase, and the gaseous phase in soil 6. be su$ciently sensitive to a wide range of environmental stresses but not so sensitive that they easily become extinct. A number of taxonomic groups of soil animals have been proposed as indicator organisms, all having speci"c advantages and drawbacks (e.g., Koehler, 1996). Microfauna groups such as Protozoa and Nematoda occur abundantly in all ecosystem types, and generally are sensitive to many stress factors, but only come into contact with the soil solution and are not easy to manipulate. Micro- and macroarthropod species also may exhibit sensitivity to stress factors, but chie#y encounter them via the solid and gas phase in soil. Earthworms are widely used, especially in laboratory tests, and have as main advantages their important role in ecosystems, their ease of culturing, and their exposure to stress factors via all three soil phases. The main disadvantages of earthworms are that the species commonly used in

This review article surveys the available data on enchytraeid sensitivity toward chemical stress, and the e4ects of chemical stress on enchytraeid communities in terrestrial ecosystems. The factors a4ecting bioavailability of stressors to enchytraeids and the nature of direct and indirect e4ects that may occur are discussed, and the importance of laboratory tests and 5eld studies for the evaluation of toxic e4ects is underlined. The existing data on speci5c types of stressors such as pesticides, metals, fertilizers, and industrial and domestic chemicals often show clear responses of enchytraeids, and in many cases interspeci5c di4erences in sensitivity are recorded. It is concluded that, although in several 5elds additional data are required, there are good perspectives for the use of enchytraeids as indicator organisms.  2001 Academic Press Key Words: enchytraeidae; oligochaeta; annelida; toxicity; ecotoxicology; pesticides; metals; fertilizers; laboratory test; 5eld study; bioavailability.

1. INTRODUCTION

There is a growing awareness of the risks that may be involved in the vast amount (both quantitatively and qualitatively) of contaminating substances in the soil. Proper functioning of the soil is essential for terrestrial ecosystems and consequently for human activity, and any risk of impairing this should be taken very seriously. Therefore, information is needed on the e!ect of environmental stresses on the soil system. Here, we will focus on chemical stress factors, de"ned as occurring in those situations where the quantity of a substance exceeds the local background values. In di!erent soils the same level of a substance may have di!erent meanings in this respect. Because of the high complexity and limited accessibility to research of the soil environment, however, our knowledge

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0147-6513/01 $35.00 Copyright  2001 by Academic Press All rights of reproduction in any form reserved.

26

DIDDEN AND ROG MBKE

tests (Eisenia fetida and E. andrei) may not be regarded typical representatives for the majority of ecosystems, and that other species are much more di$cult to handle and to culture (RoK mbke and Moser, 1999). Enchytraeidae (Annelida; Oligochaeta) readily ful"ll the "rst "ve of these criteria (e.g., Didden, 1993; RoK mbke and Moser, 1999), and may therefore be considered suitable candidates as indicator organisms. Recently, much e!ort has been devoted to the development of an enchytraeid laboratory test (RoK mbke and Moser, 2001; Ratte et al., 2001) with which the e!ects of chemical stress on enchytraeid survival and reproduction may be evaluated. It is important to realize, however, that from laboratory tests only direct e!ects can be predicted. Indirect e!ects are more di$cult to establish, because of the inherent unpredictability associated with the way in which such e!ects will occur under "eld conditions. This means that "eld monitoring of enchytraeid communities is indispensable to serve as an early warning system of changes in composition and functioning of the soil system (this probably also holds for sediments; e.g., Giani, 1983, 1984). Although in a number of older "eld investigations no e!ects on enchytraeids were established, this was very likely connected with the resolution of the methods applied (e.g., reliable extraction methods and taxonomic keys were not available until around 1960). The majority of these "eld data concerned enchytraeids as a group, without assessing their species composition; thus shifts in species composition could not be recorded. Studies taking the species composition into account often demonstrated species-related responses leading to a di!erent enchytraeid community. It is very likely that a changed enchytraeid community will result in changed enchytraeid activity, and therefore in changes in ecosystem processes such as decomposition and bioturbation. Although up to now only few studies have addressed this aspect, it certainly may be regarded an important and promising approach. The structure of enchytraeid communities can also be used either alone (Graefe, 1993a) or in combination with other soil organism groups (RoK mbke et al., 1997) for soil quality assessment in the "eld. The basic approach is (analogous to plant sociological methods) that at a site with given environmental factors such as physico-chemical soil properties or climate a certain enchytraeid cenosis can be expected. If the cenosis found at a given site di!ers clearly from the one expected, this is a strong indication that the soil of this site is somehow altered concerning its function as a habitat for soil organisms. The present article concentrates on the e!ect of individual chemicals on enchytraeids, and reviews the available data on the sensitivity of enchytraeids toward chemical stress factors in terrestrial ecosystems, divided over the following categories: pesticides, fertilizers, metals, and other chemicals.

2. RESULTS 2.1. E4ects of Chemical Stress

2.1.1. Laboratory and Field Studies The objectives of ecotoxicological tests are, on the one hand, to be able to understand and predict the e!ects of chemical stress on the ecosystem level and, on the other hand, to be able to interpret "eld data in terms of ecological relevance of xenobiotic substances present. Field studies alone generally are not su$cient to realize these goals: they are often di$cult to interpret because of the inherent complexity of the "eld situation, where multiple physico-chemical and biological factors are involved that also may vary in space and time (e.g., Achazi et al., 1996; Jans et al., 1994). Laboratory tests o!er a possibility to study speci"c e!ects, and may give an indication of the potential e!ects under "eld conditions. For this, however, the complexity of the "eld situation has to be sacri"ced, making the extrapolation of the results to the ecosystem level more di$cult. Thus, laboratory tests may be considered indispensable on the one hand, but need to be as realistic as possible on the other. One possibility to combine the advantages of these two approaches might be the use of microcosms. The use of Terrestrial Model Ecosystems for the study of the e!ects of several pesticides on enchytraeid populations (RoK mbke et al., 1994; Moser et al., 1999) has already produced promising results. For enchytraeids there have been developed several different laboratory test systems, but basically they can be divided in two categories: short-term tests in aqueous or agar medium (e.g., RoK mbke and Knacker, 1989; Westheide et al., 1989) and short-to-medium-term tests in soil. The soil tests may be carried out in homogenized "eld soil (e.g., Salminen et al., 1996; Puurtinen and Martikainen, 1997) or in well-de"ned arti"cial soil (e.g., RoK mbke, 1988) such as the commonly used OECD soil from earthworm acute tests (OECD, 1984). In view of the general considerations discussed in sections 2.1.2 and 2.1.3 it may be expected that the sensitivity found in water will be higher because the substance will be maximally available to the enchytraeids. This was corroborated by RoK mbke and Knacker (1989), who compared toxicity to E. albidus of a number of chemicals in water with that in OECD soil and found that the toxicity in water was in most cases much higher. In soil medium physical characteristics such as solubility and adsorption, and interaction with other components of the food web will be of much more importance. Besides, laboratory tests in soil may be considered more realistic as a representation of "eld situations and therefore to be a better means to predict the e!ect of a substance in the environment. On the other hand it is often more di$cult to interpret the results from soil medium because of the inherent variability

ENCHYTRAEIDS AS INDICATOR ORGANISMS

in physical, chemical, and biological characteristics in soil and the impossibility to mimic all natural factors in the laboratory. Also, tests in water provide a means to establish the sensitivity of enchytraeids toward the substance in question and a possibility to understand the mode of action of the substance in soil. Thus, a substance with no e!ect in water may be expected not to have any direct e!ect in soil; if e!ects in soil tests occur, however, this would probably concern indirect e!ects via the food web or via the physicochemical environment. For example, the herbicide Ustinex (active ingredients Amitrole and Diuron) did not have any e!ect on E. albidus in laboratory tests up to 1000 ppm in soil, whereas in the "eld 4 months after application (maybe due to food shortage or other indirect e!ect) a decrease in enchytraeid numbers and biomass was observed (RoK mbke et al., 1994). Conversely, if a substance produces a direct e!ect in water, tests in soil medium may show to what extent it may be available to enchytraeids under "eld conditions. Thus, although it is necessary (e.g., as part of a registration or noti"cation process) to do standardized tests with soil as a test substrate, both types of tests are important to understand the e!ects of a substance in soil. 2.1.2. Direct and Indirect Ewects In evaluating the e!ects of chemical stress on enchytraeids a number of aspects have to be taken into account. In the "rst place a distinction must be made between direct and indirect e!ects: direct e!ects are caused by toxicants that a!ect the physiology of the animals through chemical interaction, while indirect e!ects occur through changes in the environment. Direct e!ects on enchytraeids occur when two conditions are met: the stressor should be available to the animals, and they should have a measurable sensitivity toward it, which will be expressed by changes in, for example, longevity, growth, or reproduction. To determine the sensitivity in enchytraeids several laboratory tests have been employed, the results of which are discussed in Section 2.2. The total concentration in soil of a potentially toxic substance rarely is a good indication of its actual toxicity for organisms, as the substance may in many ways be unavailable for uptake (for instance, due to strong adsorption onto clay or organic particles, by residence in unaccesible soil pores). This is why the concept of bioavailability is widely being used. Peijnenburg et al. (1997), discussing the behavior of heavy metals in soil, re"ned this concept and proposed a dynamic approach of bioavailability consisting of three distinct aspects: the desorption process, determined by the physico-chemical characteristics of a speci"c soil; the uptake process, determined by physiological characteristics of a speci"c organism; and the toxic e!ects in the organism. Thus, the bioavailability of a toxicant depends on a number of factors, of physico-chemical, physiological, biological, temporal, and spatial nature.

27

Direct e!ects may become manifest in many, often combined, ways, and may be lethal or sublethal in character: * e!ects on metabolism * blocking or overstimulation of enzymes or hormones * e!ects on growth * e!ects on reproduction * e!ects on locomotion * e!ects on cell structure * e!ects on the immunity system * e!ects on behavior * e!ects on sensitivity to infections Not all toxicants are likely to produce direct e!ects on enchytraeids: the available amount may be too low, as in the case of highly adsorbed metals or pesticides. Such substances may nevertheless in#uence the enchytraeid community through secondary poisoning via the food web in soil, or through physico-chemical changes in the environment. A possible example of this was presented by Stockinger et al. (1997) in microcosm experiments with aged soil contaminated by a mixture of various metals (Cu, Zn, Ni, Cd, and V); they suggested that the negative e!ects on introduced individuals of E. buchholzi were at least partly caused indirectly by lack of food (a strong decrease of microorganisms). As is clear from the above, such indirect e!ects may not always be discernable from direct e!ects, yet could be treated also as responses of enchytraeids to such stressors. 2.1.3. Bioavailability The amount of a toxicant that is adsorbed to organic matter or clay particles in soil is to a large extent determined by the adsorption characteristics of the substance itself in combination with the relevant soil characteristics. The absolute amount of a toxicant adsorbed will increase with increasing organic matter or clay content, and generally this may be expected to lower its bioavailability. Yet, it may take some time before the potential adsorption level is reached: Zweers (1996), for instance, found clearly higher EC values V for reproduction in E. crypticus in soil that was contaminated with Zn 2 years before than in freshly contaminated soil, indicating decreasing bioavailability. Likewise, Lokhorst (1997) found lower amounts of Cd in CaCl extracts after 4 weeks of incubation than directly  after the treatment. The factors determining the bioavailability of a toxicant are often interrelated and the e!ects occurring are not always easy to interpret. Some examples may illustrate this point: There are many data supporting the importance of the concentration of a toxicant in the soil solution. Belfroid et al. (1995) argued, based on model calculations, that most organic hydrophobic chemicals are mainly taken up by earthworms from the interstitial water. Only in the case of high organic matter contents (520%) of the soil intake via food might be signi"cant. Concordantly, Zweers (1996),

28

DIDDEN AND ROG MBKE

analyzing a variety of "eld soils, found a good correlation of the Zn concentration in porewater and the concentration in E. crypticus, but no correlation with the total concentration in soil. This relation may not be true for all species and chemical substances, however, due to physiological and behavioral di!erences. SjoK gren et al. (1995), for instance, found that Cognettia sphagnetorum accumulated Zn in linear proportion to the total concentration in the substrate while Cu was accumulated only to a certain level. This indicates that in this species an appreciable part of Zn and Cu intake takes place via food, and that it is able to regulate the accumulation of Cu to a certain extent. In general, this may mean that bioavailability of a substance depends on characteristics not only of the substance itself and of the soil, but also of the species under study. 2.1.4. Environmental Conditions The e!ect of toxic substances may di!er depending on speci"c environmental conditions. The organic matter in soil, for instance, not only a!ects bioavailability through adsorption of toxicants. Augustsson and Rundgren (1998) demonstrated a clear e!ect of food type on the toxicity of Cu to C. sphagnetorum. Likewise, SchoK ne (1971) reported that the tolerance of E. albidus toward high salinity (60) depended on the type of substrate and food and that the development time of cocoons was signi"cantly higher at salinities '30. In analogy to this, Kaufman (1975) found that E. albidus pretreated with the adaptogen Dibasol (2benzylbenzimidazol) survived intoxication with phenol (1400 mg ) L\) in water much better. Interaction with other toxicants may take place on various levels: Posthuma et al. (1997) discerned interaction between toxicants outside the organism (for instance, in the soil solution or with adsorption processes; an example of this is the solubilization of immobilized heavy metals by acidi"cation), during uptake by the organism, and inside the organism. They also found that Cu reduced sorption of Zn, while Zn did not a!ect Cu sorption. This e!ect implies an increase of available Zn in the presence of Cu. S[ ustr et al. (1997) reported a decrease in a-amylase and an increase in xylanase activity in C. sphagnetorum after acidi"cation, while acidi"cation followed by liming produced the opposite e!ect. The importance of temperature for chemical and biological processes is generally acknowledged. KaK hler (1970), for instance, reported a clear interaction of adaptation temperature and salinity on temperature tolerance in E. albidus. Adaptation to increased salinity enhanced the temperature tolerance, especially in cold-adapted specimens, thus in a way enhancing survival. A clear relation with speci"c ions could not be established, however, as the e!ects depended on concentration and adaptation temperature. The moisture content of the soil may in#uence the e!ect of a toxicant in various ways: the available amount of the

substance might increase at higher moisture levels, while the concentration in the soil solution might increase at lower moisture levels. Puurtinen and Martikainen (1997) studied the e!ects of soil moisture on toxicity of Dimethoate and Benomyl to E. crypticus, and reported a negative correlation for Dimethoate, and a positive correlation for Benomyl. Volatile compounds such as chloroform seem to be more toxic in dry soil than in wet soil (Funke and Frank, 1991). In some cases a stress factor like drought can also counteract the e!ects of a toxicant by forcing the animals to move downward in the soil column, where the concentrations of the chemical may be lower than near the soil surface, as in the case of surface-applied pesticides (Martikainen, 1998). 2.1.5. Behavioral and Physiological Ewects The direct e!ects of toxicants under "eld conditions will often be determined by the heterogeneity of the environment, possibly in combination with behavioral responses (Achazi et al., 1996). An indication for the occurrence of an avoidance response in enchytraeids was reported by Salminen and Sulkava (1996) for C. sphagnetorum. This species did not colonize heavily PCP-contaminated patches in a choice experiment. SjoK gren et al. (1995) found indications for avoidance by C. sphagnetorum of Cu#Zn-contaminated patches. A repellent e!ect was also found for Cu by RuK ther and Greven (1990) in E. buchholzi and for PAH in E. crypticus (Achazi et al., 1999). Contaminants, especially metals, may be bound to proteins and accumulate in the chloragogenic tissue, as is commonly found in earthworms and enchytraeids. In this way the toxic e!ect is neutralized but such compounds may subsequently be transferred to a higher trophic level. At the same time a toxicant may a!ect structure and functioning of cells and tissues where it accumulates, as was shown for Enchytraeus doerjesi by Hagens and Westheide (1987). Using ultrahistopathological methods, Purschke et al. (1991) demonstrated that after prolonged exposure cells damaged by chemicals can renew organelles or can undergo complete regeneration. However, as this process requires energy, reproductive capacity and life span of these individuals were signi"cantly lower than in untreated worms. SjoK gren et al. (1995) suggested that autotomy, followed by regeneration, of posterior segments in which metals have accumulated might constitute an e!ective detoxi"cation mechanism in C. sphagnetorum. A clear indication that this may be a more common strategy was presented by Nakamura and Shiraishi (1999) for nickel intoxication in E. buchholzi. E!ects on the immune system have repeatedly been reported in earthworms, and are most probably also of importance in enchytraeids. Yet, to date there are no studies addressing this topic, probably because no adequate techniques are available. Brousseau et al. (1997) developed an elegant technique using #ow cytometry to evaluate

29

ENCHYTRAEIDS AS INDICATOR ORGANISMS

phagocytic activity in coelomocytes of ¸umbricus terrestris. In view of the much smaller size of enchytraeids, it would be necessary to adapt this method concerning the collection of coelomocytes. The e!ect of contaminants on the susceptibility to infections has hardly been subject to study. Yet, this may be an important determinant of the viability of a population. Purrini (1983) reported dramatically higher infection rates with Protozoa (primarily Gregarinida) in enchytraeids in forest sites subject to high SO immissions compared to  more undisturbed forests. The age of the animals under study apparently may also be of importance: RoK mbke and Knacker (1989) reported that young specimens of E. cf. buchholzi were clearly more sensitive to parathion than adult ones. With K Cr O the sensitivity did not di!er.    Interpretation of the e!ects of chemical stress may be complicated due to the fact that chemicals may have a positive e!ect on enchytraeid numbers or biomass. In "eld studies often an extreme compensation reaction (e.g., due to the accumulation of organic matter during the application phase of a herbicide) was observed during the recovery phase (e.g., Beck et al., 1988; Jans et al., 1994), whereas in laboratory tests subtoxic concentrations can cause the same type of e!ect (called hormesis; RoK mbke and Moser, 1999). 2.2. Responses of Enchytraeids to Speci5c Chemical Stresses

2.2.1. Pesticides Direct e!ects of a pesticide on enchytraeids under "eld conditions depend, of course, on its bioavailability. Indirect e!ects may occur via e!ects of a pesticide on the food web in which enchytraeids participate or on the physico-chemical environment and also are dependent on the bioavailability of the substance. Generally, three important parameters determine the bioavailability of pesticides in soil: the persistence, the solubility, and the adsorption to soil particles. For each of these parameters a threshold value may be de"ned beyond which extended exposure is likely to occur. The provisional threshold values presented here are mainly based on BBA (1986, 1990) and van Rijn et al. (1994) and are designed not to be too protective or too unprotective, but are of course liable to change when more relevant data become available. * Pesticides with a half-life of 460 days are not likely to accumulate to an appreciable amount, even when applied repeatedly, while a half-life of '60 days may eventually result in a buildup of the pesticide. * It may be assumed, in analogy to earthworms (Belfroid et al., 1995), that a direct e!ect of pesticides on enchytraeids, for instance on mortality or reproduction, will mainly occur through uptake of the substance via the soil solution. This implies that pesticides with high solubility ('30 mg ) L\) are more likely to come into contact with enchytraeids.

TABLE 1 Tentative Ecologically Relevant Classi5cation of the Bioavailability of Pesticides, Based on Half-Life, Solubility, and Adsorption Class

Half-life

Solubility

Adsorption

A

Short

Low

Weak

B C D

Short Short Short

Low High High

Strong Weak Strong

E

Long

Low

Weak

F G

Long Long

Low High

Strong Weak

H

Long

High

Strong

Bioavailability Temporary, via direct contact or feeding Temporary, via feeding Temporary, via soil solution Temporary, via soil solution and feeding Prolonged, via direct contact or feeding Prolonged, via feeding Prolonged, via soil solution when leaching is weak Prolonged, via soil solution and feeding

* Strong adsorption to soil particles (K '500) will  reduce the bioavailability of a pesticide in the soil solution, but the chance of uptake via ingestion will be relatively increased. The possible combinations of these parameters produce a tentative ecologically relevant classi"cation of pesticides (excepting, of course, formulations with more than one active ingredient), which is presented in Table 1. For a number of pesticides the sensitivity of individual enchytraeid species has been established in the laboratory. Table 2 and Fig. 1 summarize the results of those studies that produced NOEC values on mortality or reproduction or LC values. As may be expected, enchytraeid sensitivity  was generally higher in aqueous medium or agar than in soil, especially soil with a high organic matter content. Where more enchytraeid species have been tested under the same conditions for the same pesticide (with Benomyl, PCP, Parathion, and 2,4,5-T), appreciable di!erences have been found in sensitivity, suggesting this to be a general phenomenon. Surprisingly, the NOECs for reproduction (fecundity or fertility of cocoons) that have been reported for several pesticides were sometimes higher than those for mortality (Table 2). In such cases only short-term e!ects on abundance are more likely to occur under "eld conditions, as the reproductive potential of a population may remain intact. In the following, a number of studies in microcosms or under "eld conditions will be presented by pesticide class: A. Only short-term e!ects may be expected, although irreversible changes in species composition are conceivable in the case of highly sensitive species. Edwards et al. (1968) studied the e!ect of the organophosphorus pesticide Chlorfenvinphos on enchytraeid abundance in a "eld situation and found increased abundance after 160 days. As the half-life of

30

TABLE 2 Sensitivity of Enchytraeids to Miscellanious Chemicals in Laboratory Tests

Substance Pesticide Benomyl

Carbofuran Dimethoate

Cypermethrin Metal Cd

Cr Cu

Zn

E. crypticus

Substrate

OM (%)

Moisture (%)

pH

Agar

NOEC mortality (ppm)

LC (ppm) 

NOEC reproduction (ppm)

21

30

11.8

2.36

Soil

10.1

28

6.2

17

24

16

16

Soil

10.1

39

6.2

17

24

16

16

Soil

10.1

49

6.2

17

24

32

16

21

30

0.118

1.22

E. doerjesi

Agar

E. albidus

Soil Soil

10 10

50 60

6.0 )! 6.0

20 20

42 14

E. 00buchholzi'' Soil E. crypticus Soil F. ratzeli Soil

10 2.5 10

50 60 60

6.0 5.9 6.0

20 21 20

42 7 14

E. doerjesi E. crypticus

Agar Soil

10.1

28

6.2

17

30 24

200

12,000 800

Soil

10.1

39

6.2

17

24

'1600

200

Soil

10.1

49

6.2

17

24

'1600

200

1.2

1.2

E. doerjesi

Agar

E. albidus E. buchholzi E. crypticus

Soil Agar Water Soil Water Soil Water Soil Soil Soil Water

E. E. E. E.

albidus albidus buchholzi crypticus

E. buchholzi


10

50

2.3 10

5.8 )! 5.0  &5.8

50

2.3 2.3 2.3

! ! 

5.8 )! 5.0  &5.0 ! !  5.8  ! ! 7.5  ! ! 5.0 &-

20 21 20 20 20 20 21 20 20 20 21

1.1

'3.6 6.6

2.7 10 EC 

15.2

14 4 1 1 1 14 2 1 1 1 2

726 8.4 0.1 380 20 1277 0.41 250 480 1270 57

Reference

Bethke-Beilfu{ and Westheide, 1987; Westheide et al., 1989, 1991 Puurtinen and Martikainen, 1997 Puurtinen and Martikainen, 1997 Puurtinen and Martikainen, 1997 Bethke-Beilfu{ and Westheide, 1987; Westheide et al., 1989, 1991 RoK mbke and Moser, 1999 RoK mbke and Federschmidt, 1995 Collado et al., 1999 Achazi et al., 1997 RoK mbke and Federschmidt, 1995 Westheide et al., 1989 Puurtinen and Martikainen, 1997 Puurtinen and Martikainen, 1997 Puurtinen and Martikainen, 1997 Westheide et al., 1989

Lokhorst, 1997 Willuhn et al., 1994a,b: 1996a,b Kratz and Brose, 1997 Kratz and Brose, 1997 Lokhorst, 1997 Willuhn et al., 1996a Kratz and Brose, 1997

Willuhn et al., 1996b

Soil

2.5

60

5.9

7

172

Achazi et al., 1997

Soil

2.5

60

5.9

7

1460

Achazi et al., 1997

DIDDEN AND ROG MBKE

Carbendazim

Species

Temperature (3C) Duration (d)

ENCHYTRAEIDS AS INDICATOR ORGANISMS

31

FIG. 1. LC values in water (4 days) and soil (56 days) for various pesticides in laboratory experiments. Test conditions denoted on the X axis. W,  water (data from RoK mbke and Knacker, 1989; Ronday and Houx, 1996; S1, soil, 1% organic matter, pH 5.6 (data from RoK mbke, 1988); S2, soil, 2.5% organic matter, pH 7.4 (data from RoK mbke, 1988); S3, soil, 3% organic matter, pH 4.5 (data from RoK mbke, 1988); S4, soil, 3% organic matter, pH 6.0 (data from RoK mbke, 1988); S5, soil, 5.3% organic matter, pH 5.3 (data from RoK mbke, 1988); S6, soil, 10% organic matter, pH 6.0 (data from RoK mbke, 1988, 1989; RoK mbke et al., 1994).

Chlorfenvinphos is $27 days, it may be assumed that virtually no pesticide was left at that time and that the increase in enchytraeids was an indirect e!ect, due to a decrease in predators or competitors. BaK umler et al. (1978) found no e!ects of repeated applications of the triazine herbicide Simazine for 4 years, but there are no laboratory data on enchytraeid sensitivity toward this pesticide to compare this result with. In a "eld trial in which soil was treated bimonthly with 0.24 ppm of the carbamate Carbendazim for 1 year, reduced abundances were reported in soil with 2.4% organic matter, but no e!ects in soil with 4.4% organic matter (RoK mbke and Federschmidt, 1995). This was supported by data from tests with several Russian "eld soils (Filimonova et al., 1999). These results are in accordance with laboratory results on this pesticide (Table 2), and indicate that the e!ect of Carbendazim is strongly dependent on the soil organic matter content, which is not surprising in view of the relatively low K value of this compound. Since  this chemical has been selected as a reference compound for the Enchytraeid Reproduction Test, in the future more data can be expected. B. It is unlikely that enchytraeids will come into contact with these pesticides through the soil solution but they can possibly be taken up by ingestion, in which case

short-term direct e!ects may occur. Indeed, most data available do not point to long-term e!ects, if any e!ect was found. The low NOECs for mortality and reproduction for Cypermethrin reported by Westheide et al. (1989) (Table 2), however, indicate that uptake via ingestion certainly may not be excluded. In a "eld investigation in a German forest (Jans and Funke, 1989) also a strong e!ect of Cypermethrin was observed: enchytraeid abundances were unaltered in plots treated several months before, but were sharply depressed in plots treated 1 and 2 years earlier. In view of the short half-life of this pesticide this may well have been an indirect e!ect. Praxis-relevant concentrations of the pyrethroid Cy#uthrin did not show any e!ects on the enchytraeids of an acid coniferous forest (Jans et al., 1994). In accordance with the laboratory results presented in Table 2 and Fig. 1 Martikainen et al. (1998) found no e!ects on the enchytraeid community in a 63-day study with the carbamate fungicide Benomyl applied to soil in a quantity of $5 ppm. Yet, as Benomyl degrades to the more toxic carbamate Carbendazim, it is conceivable that in this case e!ects would have been found if the study period had been extended. Despite the relatively low sensitivity of enchytraeid species to the organophosphorus Parathion as found

32

DIDDEN AND ROG MBKE

in laboratory tests (Fig. 1), Weber (1953) reported a short-term (3 weeks) decrease in abundance of enchytraeids in a "eld trial. The same tendency was found in a parallel microcosm and "eld study in a German pasture (RoK mbke et al., 1994). This indicates that Parathion may also be taken up by ingestion, that indirect e!ects may occur, or that avoidance behavior is invoked, resulting in changes in the vertical distribution of the enchytraeid community. For the organophosphorus pesticide Fenithrothion, Martin (1975) found no e!ect on enchytraeid abundance in a New Zealand pasture. Heungens (1970) studied the e!ect of the organochlorine Dicofol on C. sphagnetorum in the "eld 70 days after application and found no e!ects on abundance. This may be explained by the amount applied (1.6 kg ) ha\) and the short half-life of this pesticide: only if C. sphagnetorum is highly sensitive to this pesticide could any e!ects have been expected. The application of the organochlorine Aldrin in an English pasture (Clements et al., 1987) and in coniferous litter (Heungens, 1970) appeared not to have an e!ect on the enchytraeid community. The urea insecticide Dimilin (Di#ubenzuron) did not cause acute e!ects on the enchytraeids of a mixed deciduous forest in South Germany, but there were indications that the worms tried to avoid the chemical by vertical migration (Ruf and RoK mbke, 1996). C. Pesticides from this group may have a strong shortterm e!ect on sensitive species, possibly resulting in loss of species. Occurrence of such strong short-term

e!ects may then entail a long period of recovery of the community. For several pesticides in this group both laboratory and "eld-type data are available. The organochlorine compound PCP has been extensively tested under semi"eld conditions by RoK mbke (1991), Salminen et al. (1995), and Salminen and Haimi (1996, 1997). There is a good agreement between their results and those from laboratory tests, as illustrated in Fig. 2. In a 4-year "eld study (six applications for 2 years followed by 2 years of recovery) the e!ects of very high concentrations of PCP (1 and 5 g ) m\) on the enchytraeids of a moder beech forest were studied in order to identify the general mechanisms of population reactions to stress (Beck et al., 1988; RoK mbke, 1988). The application of PCP had a very drastic and long-lasting e!ect on all species, even in the mineral layer. Recolonization started about 1 year after application of the lower concentration, followed quickly by an overcompensation of several hundred percent (especially in the litter layer; RoK mbke, 1994). However, in the plot treated with the high concentration even at the end of the recovery phase abundance and biomass of enchytraeids were still signi"cantly lower than in the control. Possibly at this concentration the microbial community was a!ected to such extent to inhibit PCP degradation. A number of carbamate pesticides in this group have been investigated under "eld or semi"eld conditions without "nding any adverse e!ects (Heungens, 1969, 1970; Edwards and Lofty, 1971; McColl, 1984). Aldicarb was found to have a lasting negative e!ect on

FIG. 2. Estimated amounts of PCP remaining in soil after application of 500 and 50 ppm (solid lines), as applied in RoK mbke (1991), Salminen et al. (1995), and Salminen and Haimi (1996, 1997). Upper value of NOEC for E. albidus (dotted line) from RoK mbke (1989) and recorded signi"cant di!erences with control (arrows). The estimated degradation is based on a half-life of 48 days, but may have been appreciably slower.

ENCHYTRAEIDS AS INDICATOR ORGANISMS

enchytraeid abundance in both "eld and laboratory experiments (Edwards and Lofty, 1971; Born et al., 1989; Born and Vollmer, 1987), Carbaryl caused effects lasting longer than 1 year on the enchytraeids of a taiga plot in Siberia (Voronova, 1968), and the dithiocarbamate Dazomet reduced enchytraeid abundance in a "eld experiment (Edwards and Lofty, 1971). It would seem therefore that the carbamates in this group are divided in a group to which enchytraeids are sensitive and one to which they are indi!erent. It is unclear, however, what chemical properties of these pesticides are responsible. For the organophosphorus Dimethoate no e!ects were found in "eld trials with low concentrations (Heungens, 1970; Martikainen, 1996; Martikainen et al., 1998). This is in accordance with the high NOECs established in laboratory tests (Table 2). Martin (1975) found no e!ects of Fensulfothion in a "eld study, nor did McColl (1984) with Fenamiphos. Heungens (1970), on the other hand, reported decreased abundance of C. sphagnetorum 70 days after the application of Thionazin. The triazine herbicide Atrazine has been studied in long-term "eld experiments. Popovici et al. (1977) found a decrease in numbers up to 4 months after application of 5 and 8 kg ) ha\, respectively. ChalupskyH (1987, 1989) reported increased enchytraeid abundance with a single application of Atrazine (2.5}20 kg ) ha\), but strongly decreased abundance and diversity in a "eld that was treated yearly. These results may be explained by increased food availability after a single application, but reduced availability with repeated applications. The herbicide 2,4,5-T was applied in a 4-year "eld study (Beck et al., 1988; RoK mbke, 1988). In accordance with the low sensitivity of enchytraeids to this compound found in laboratory tests (Fig. 1) and its relatively quick degradation, enchytraeids were a!ected only at extremely high concentrations and as long as the chemical was applied (12 times bimonthly). Partly even during the application phase but at the latest immediately after the last application, various species began to recolonize the treated plots (preferably the deep litter layer as shown in a litter-bag experiment) (RoK mbke, 1994). As also found in laboratory tests (Fig. 1), 2,4,5-T had species-speci"c e!ects: C. sphagnetorum was clearly less a!ected than mineral dwellers such as Achaeta sp. This difference may have been enhanced by the quick reproduction mode of C. sphagnetorum (fragmentation). D. E!ects of pesticides from this group may be expected to be short-term only, and may occur via the soil solution or via feeding. In accordance with this, the

33

organophosphorus Diazinon was found by Wall and Marganian (1971) to reduce enchytraeid abundance during a 7-day trial along a beach. Van den Brande and Heungens (1969) reported a negative e!ect of Malathion in a Begonia culture, while after about a year the number of enchytraeids was higher than in the control plots. Jans et al. (1994), however, found a strong and long-lasting e!ect of praxis-relevant concentrations of the carbamate Bendiocarb in a "eld study in South Germany, suggesting that enchytraeids are particularly sensitive to this pesticide or that strong indirect e!ects occurred. E. The chance of exposure to this class of pesticides via the soil solution is, as in class A, rather small, but in the case of sensitive species with an indiscriminate mode of feeding an irriversible change in species composition may result. Unfortunately, there are no laboratory or "eld data on pesticides from this group. F. As with class B, any direct e!ect via the soil solution in this group may only occur with species that are highly sensitive to these compounds, but e!ects may also occur via ingestion and the risk of long-term e!ects due to accumulation is apparent. Way and Scopes (1968) found a short-term increase in enchytraeid abundance 2 months after application of the organophosphorus Phorate, possibly an indirect e!ect related with a decrease in other groups, but Clements et al. (1987) reported a negative e!ect of repeated application of Phorate on the enchytraeids of several English grasslands. A number of persistent organochlorine pesticides belong to this group, and have been studied repeatedly in "eld experiments regarding the e!ect on enchytraeids. Although generally no signi"cant e!ects are reported, there also are some exceptions. Byzova (1964) found negative e!ects of DDT in the "rst year after a single application of high doses of 30}50 kg ) ha\. HCH was repeatedly reported to have a negative e!ect on enchytraeid abundances for the "rst weeks after application (Huhta et al., 1967, for C. sphagnetorum; Weber, 1953, for enchytraeids in general) or even for a year after application (Byzova, 1964; Jans and Funke, 1989), while increased abundances are reported for the 2nd and 3rd year after application (Grigorjeva, 1953; Dormidontova, 1973; Jans and Funke, 1989). This increase might be due to an indirect e!ect of the insecticide on predators. No e!ects of c-HCH were found in a microcosm experiment (RoK mbke, 1991) or in laboratory tests with arti"cial or "eld soils (Bruns, personal communication). Edwards and Arnold (1966) found reduced abundances with Heptachlor, and BaK umler et al. (1978) reported decreased abundance after application of

34

DIDDEN AND ROG MBKE

Toxaphene. These results indicate the occurrence of indirect e!ects, exposure via ingestion, or a high sensitivity to even extremely small quantities in solution. G. Pesticides in this group are highly available to enchytraeids for long periods of time, especially in cases where leaching is weak. Unfortunately there are no laboratory data on the sensitivity of enchytraeids for pesticides from this group and only few, short-term, "eld data. The nematicide DBCP has been studied by Heungens (1968, 1969) and Radu et al. (1970) under semi"eld conditions. Heungens (1969) observed an increased abundance of C. sphagnetorum 84 days after application of DBCP, and Heungens (1968) and Radu et al. (1970) found no e!ects on the abundance of C. sphagnetorum and the enchytraeid community, respectively. H. As with class D, pesticides in this group will hardly be available in the soil solution, and any direct e!ect via this route may be expected only in the case of highly toxic compounds. Exposure to these substances will predominantly take place via ingestion, entailing the risk of long-term e!ects due to bioaccumulation. Again, no data on enchytraeid sensitivity are available for pesticides in this group, and only the quaternary

ammonium herbicide Paraquat has been studied in the "eld, by BaK umler et al. (1978), who found no e!ects on enchytraeid abundance in a period of 4 years with repeated applications. Detoxi"cation strategies toward organic chemicals have not been studied. As glutathione S-transferases were found in E. albidus, it might be hypothesized that this enzyme could play a role in metabolizing certain pesticides, as has been shown in lumbricids (Stenersen and "ien, 1981). The investigation of the bioaccumulation of pesticides in enchytraeids started only recently, using Lindane as an example (Amorim et al., 1999; Bruns, personal communication). It is clear that many pesticides have a potential lethal or sublethal e!ect on enchytraeids, even in concentrations far below the conventionally applied dose. As there have also been found clear species-related di!erences in sensitivity, the use of such substances may result in changes in species composition and in the functioning of the enchytraeid community. 2.2.2. Metals. The sensitivity to metals of individual enchytraeid species have repeatedly been investigated in the laboratory. Table 2 and Fig. 3 summarize the results of those studies that

FIG. 3. Results of laboratory tests for metals and various chemicals. Endpoints in the "gure are LC values (open symbols), EC values for   reproduction (gray symbols), and NOEC values for reproduction (black symbols). Test conditions denoted on the X axis. W, 4-day test in water (data from RoK mbke and Knacker, 1989; Kratz and Brose, 1997; Willuhn et al., 1994a,b; 1996a,b); S1, 28-day test in soil, 1.7% organic matter, pH 5.3 (data from Kratz and Brose, 1997); S2, 28-day test in soil, 2.3% organic matter, pH 5.0 (data from Kratz and Brose, 1997); S3, 28-day test in soil, 2.3}2.5% organic matter, pH 5.8}5.9 (data from Kratz and Brose, 1997; Achazi et al., 1997); S4, 28-day test in soil, 2.3% organic matter, pH 7.5 (data from Kratz and Brose, 1997); S5, 28-day test in soil, 10% organic matter, pH 6.0 (data from RoK mbke and Knacker, 1989; Posthuma and Notenboom, 1996; Posthuma et al., 1997); S6, 42-day test in soil, 10% organic matter, pH 5.8 (data from Lokhorst, 1997); S7, 56-day test in soil, 10% organic matter, pH 6.0 (data from RoK mbke, 1991); S8, 40- to 42-day test in soil, 9.6}10% organic matter, pH 7.0}7.3 (data from Elzer, 1995; Lokhorst, 1997).

ENCHYTRAEIDS AS INDICATOR ORGANISMS

produced EC values on reproduction, or LC values.   These data show that, at least for Cd, there exist clear interspeci"c di!erences in sensitivity. As was also found in tests with pesticides, the sensitivity of enchytraeids is clearly higher in watery media than in soil, which is undoubtedly related to reduced availability in soil medium. E. buchholzi was tested in aqueous solutions with several metals and was much more sensitive to Hg and Cu than to Cd and Zn. Yet, in soil tests the sensitivity of E. albidus toward Cd and Cu was of the same order of magnitude as the sensitivity of E. crypticus toward Cd, Cu, and Zn. These results stress the importance of adsorption processes in soil, and indicate strong di!erences in adsorption between the metals involved. Recently, knowledge about the behavior of metals in soil was applied for the prediction of metal bioavailability to E. crypticus in the "eld (Peijnenburg et al., 1999). Apart from bioavailability other environmental characteristics may also play a role in determining the e!ect of metals on enchytraeids. Augustsson and Rundgren (1998) reported clearly lower 70-day EC values toward Cu with  C. sphagnetorum when fed with Pleurococcus (EC $  175 ppm) than when fed with the algae Mortierella isabellina (EC $600 ppm).  Several authors have investigated the e!ects of mixtures of metals in the laboratory. Komulainen and Mikola (1995) tested C. sphagnetorum with 200 ppm Cu#100 ppm Ni in coniferous forest soil for 138 days and found no e!ect of this treatment on the enchytraeids. Regarding Cu, this result is in agreement with those of Augustsson and Rundgren (1998), mentioned above, and with the results of laboratory tests (Fig. 3). Posthuma et al. (1997) used equitoxic mixtures of Cu and Zn with E. crypticus. They found that the joint e!ect of the metals was additive based on the external concentrations but less than additive based on body concentrations, indicating interaction of the metals in sorption and during uptake. RoK mbke (1989) tested E. albidus with brass powder (a mixture of predominantly Zn and Cu used for military purposes) and reported a 28-day LC of  1660 ppm, which is in good agreement with the values for the individual metals as presented in Table 2 and Fig. 3. Concerning the e!ect mechanism of metals, only few studies have been conducted. For example, Siebers and Ehlers (1979) demonstrated that the transintegumentary absorption of glycine, an important additional means of food uptake in many soft-bodied aquatic oligochaetes such as E. albidus, was reduced signi"cantly in the presence of Cd, Hg, Cu, and Ag, whereas other metals such as Al, Cr, Fe, Pb, Ni, Mn, Se, or Zn had no or only negligible e!ects. An important aspect in the e!ect of metals on enchytraeids is their detoxi"cation strategy, which may di!er between metals and between species. Some metals may be accumulated in the animal through complexation in proteins, possibly in speci"c body segments that may be discarded by autotomy (see Section 2.1), but for others no such

35

mechanism may be available. Roth (1993) investigated Pb accumulation in soil animals in a coniferous forest and found the highest concentration in C. sphagnetorum, with a bioconcentration factor (BCF) of up to 1.58; the ambient concentration. For other metals in this same species BCF values up to 40 have been reported for Cd (Heck et al., 1995), up to 3 for Cr (Roth-Holzapfel and Funke, 1989), and up to 4 for zinc (Roth-Holzapfel and Funke, 1989). Comparable values were found for Cd and Zn accumulation in the aquatic species ¸umbricillus rivalis in a heavily polluted stream in South France (Say and Giani, 1981). Much higher BCF values were found in E. buchholzi exposed to metals in agar: after 12 days the BCF values were 262 for Cd, 11.4 for Cu, 9.7 for Pb, and 20 for Zn (RuK ther and Greven, 1990). Only in the case of Cu did the potworms avoid food enriched with metal. In general, the di!erences in bioaccumulation among species seem to be small (RoK mbke et al., 1998), indicating a common detoxi"cation strategy toward metals in enchytraeids. From a "eld investigation in Zn-polluted soils Notenboom et al. (1997) reported that the internal concentrations of Zn in the dominant species Marionina clavata were similar over a wide range of external concentrations, indicating some kind of adaptation. Laboratory reared individuals of E. crypticus tested in the same "eld soil had body burdens of Zn up to a factor of 5 lower. Willuhn et al. (1994a,b; 1996a,b) conducted an interesting series of experiments on the detoxi"cation strategy of E. buchholzi toward Cd and Cu. When exposed to sublethal concentrations of CdCl in water, the worms accumulated Cd to values  60}70; the control value. Treatment with Cd signi"cantly impeded reproduction, but the worms recovered quickly after transfer to uncontaminated medium. Within a few hours after the exposure started, the worms produced a new mRNA species coding for a cystein-rich nonmetallothionein protein that probably was engaged in the binding of Cd. Possibly the reduced reproduction may be considered a trade-o! of this strategy. Exposure to Cu did not invoke the same response in untreated worms, but in Cd-pretreated worms Cu strongly enhanced the production of the detoxi"cation protein. In an experiment with combinations of Zn and Cu (SjoK gren et al., 1995) C. sphagnetorum accumulated Cu only to a certain level, but Zn was accumulated in proportion to the ambient level. This indicates that C. sphagnetorum possesses a mechanism to excrete excess Cu or to block the uptake of Cu. Unfortunately, there are few "eld data on the e!ect of metals on enchytraeids. Bengtsson and Rundgren (1982) reported reduced abundance and diversity of enchytraeids in the vicinity of a brass mill (emitting Cu and Zn), and Salminen and Haimi (1999) reported indications of reduced abundance of C. sphagnetorum near a Cu}Ni smelter. A comparable result was found along a gradient of coniferous forest sites near a zinc smelter in The Netherlands

36

DIDDEN AND ROG MBKE

(Notenboom et al., 1997). Heck et al. (1989) sampled enchytraeid communities along a gradient of lead emissions near a highway and found highest abundances and more K-strategic species in sites receiving less lead. Because of possible interactions with other site-speci"c factors, it is not possible to establish a clear relation with the metals involved in these studies. In naturally lead-contaminated soil C. sphagnetorum was considered to be a less sensitive species in comparison to some microarthropods (Has gvar and Abrahamsen, 1990). Heck et al. (1995) investigated the e!ect of liming with CaCO #K SO in Pb#Cd-polluted forest    sites. Two years after the treatment they found lower concentrations of Pb in the soil solution and in the enchytraeids of the treated plots, but no e!ect with Cd concentrations. Humus-consuming enchytraeids tended to have higher concentrations than litter-consuming species. In general, the enchytraeids of these urban forests showed higher concentrations of Pb and Cd than most other soil organisms, indicating that these worms are good indicators of metal pollution (Weigmann, 1995). 2.2.3. Acid Precipitation and pH Ewects. An environmental issue that attracted much attention in the 1970s and 1980s was the &&acid rain'' problem, which was studied most intensively in temperate European forests. Although many "eld experiments have been carried out in which enchytraeids were involved, few laboratory data are available. Abrahamsen et al. (1980) showed that C. sphagnetorum, a species dominating the acid litter layer in temperate coniferous forests, did not survive in water with H SO at pH values below 4.0. Heungens (1984) conducted   an experiment with the same species in which it was exposed to several chemical substances, including bases, salts, and acids, and concluded that the changes in electrical conductivity were more important for the response than the chemical nature of the substances (the &&salt-e!ect'' reported by Heungens, 1984, and HoK bel et al., 1992). Based on these results it would seem that any dissociating soluble compound could a!ect the enchytraeid community. In several "eld experiments, however, where the pH of coniferous forest plots was manipulated by acidi"cation, liming, or fertilization (e.g., Has gvar and Abrahamsen, 1997; Lohm et al., 1977; Abrahamsen et al., 1980; Bas as th et al., 1980; Abrahamsen, 1983; Huhta et al., 1983; Persson et al., 1987; Hartmann et al., 1989; Heck and RoK mbke, 1990), it was found that C. sphagnetorum reacted positively to slight acidi"cation, sometimes after a temporary reduction in population size, and negatively to liming. Reaction of lumbricids, in these cases mainly Dendrobaena octaedra, was the reverse. However, Pokarshevskii and Persson (1995) could show in a laboratory experiment studying the individual and combined e!ects of di!erent concentrations of lime and oxalic acid on C. sphagnetorum that observed e!ects cannot

be explained by pH changes only. Abrahamsen et al. (1980) reported that Mesenchytraeus pelicensis showed a reaction similar to that of C. sphagnetorum, whereas Enchytronia parva increased with liming. In a survey of the enchytraeid communities in spruce stands that were variously damaged by SO deposition, ChalupskyH (1995) observed no di!er ences in total abundances, C. sphagnetorum clearly dominating at severely damaged sites but partly replaced by Marionina cambrensis at less damaged sites. Graefe (1989) found in a long-term investigation in German forests a decrease in enchytraeid diversity in sites subject to acid deposition; liming decreased total abundance, but increased species diversity in such sites. An accidental immission of lime dust from a cement plant in coniferous forests and moor sites also caused a shift in species composition (Graefe, 1993b). Again, these results indicate a species-speci"c sensitivity toward environmental stress. In German beech woods Heiligenstadt et al. (1993) conducted an acidi"cation experiment with H SO , resulting in decreases of   total enchytraeid abundances, but in a previously limed site the opposite occurred. S[ ustr et al. (1997) studied the e!ects of acidi"cation and subsequent liming on C. sphagnetorum in a mixed forest. Although no e!ects on abundance were found, both acidi"cation and subsequent liming resulted in a decrease of individual biomass and in changes in enzymatic activity. Comparable results were reported for Fridericia sp. after liming (Urbasek and ChalupskyH , 1991). These results corroborate the general picture of enchytraeid communities being adapted to rather narrow pH ranges. This view is substantiated by pH preference laboratory studies with di!erent Enchytraeus species, showing di!erent optima even within one genus (e.g., Dirven-Van Breemen et al., 1994) and by colonization experiments (Has gvar and Abrahamsen, 1980). 2.2.4. Fertilizers. Enchytraeids, like most other soil faunal groups, are dependent on the availability of dead organic matter. It is not surprising therefore that generally higher (up to two or three times) abundances were found in sites where organic fertilizers, such as crop residues, compost, and manures, were applied (e.g., Franz, 1953; Sauerlandt and MarzuschTrappmann, 1959; Alejnikova et al., 1975; Kitazawa and Kitazawa, 1980; AndreH n and LagerloK f, 1983). Organic fertilizer, however, may contain toxic compounds, causing adverse e!ects on enchytraeids, as is suggested by the use of pig slurry sprinkled on a Belgian forest #oor that caused a long-lasting decrease in enchytraeid numbers, perhaps induced by a change of pH in the litter layer (Debry and Monfort, 1978; Debry and Lebrun, 1980). It is unclear whether the application of inorganic fertilizers has any direct e!ect on enchytraeids. The salte!ect mentioned earlier, although occurring rapidly after

ENCHYTRAEIDS AS INDICATOR ORGANISMS

application, is strictly an indirect e!ect. It consists in a decrease in abundance and changes in diversity, probably as a result of changes in the osmotic potential and/or the electrical conductivity of the soil solution. Many studies on inorganic fertilizers reported an initial decrease in abundance, probably attributable to this salt-e!ect, followed by an increase above control values (among others Huhta et al., 1967, 1969, 1986; Utrobina, 1976; Hotanen, 1986; Nakamura, 1988). As the application of inorganic fertilizers generally results in increased primary production, this subsequent increase in abundance may also be regarded an indirect e!ect. This general picture of enchytraeid responses to inorganic fertilizers was not found in all cases, however. In coniferous forest Marshall (1974) reported signi"cantly lower densities and a more downward distribution of enchytraeids after a single application of urea, and Huhta (1984) found similar e!ects of urea and ammonium nitrate on C. sphagnetorum. Similar e!ects, including a change in species composition, were found in Polish meadows fertilized yearly with 680 kg ) ha\ NPK (Makulec, 1976). This author suggested a relation with a change in the soil moisture regime that had occurred in the fertilized meadows. No e!ects on enchytraeid numbers were found after application of relatively low amounts of NP (154/75 kg ) ha\) in a cotton/wheat "eld in Greece (Vavoulidou et al., 1999). Standen (1984) showed that enchytraeid biomass in fertilized hay meadow plots was negatively correlated with phosphorus fertilization, but found no correlation with organic fertilization or nitrate. In an earlier study Standen (1982) reported negative correlations between the number of enchytraeid species and the applied amount of nitrate and sodium. In evaluating the response of enchytraeids to the application of inorganic fertilizers it would seem that the following considerations hold: 1. An initial shock-e!ect may occur through changes in the soil solution and/or in the soil structure and microclimatological regime, resulting in temporarily lower population densities and possibly a changed species composition and depth distribution. In cases where fertilizers are repeatedly applied, this may eventually produce an impoverished community. 2. Although primary production is generally enhanced by fertilization, the soil community mainly bene"ts from it during the growing season, as in many cases the majority of primary production is harvested afterward. This will result in relative food shortage for the communities built up during the growing season. Only in cases where the production is seldomly harvested, as in forests, increased abundances may be expected in the long run. 3. Fertilizer applications often result in less diverse plant communities and hence less diverse dead organic material, reducing the number of niches for enchytraeid species and thus reducing species diversity.

37

Thus, it may be expected that a single application of inorganic fetilizers occasionally results in temporarily higher population densities of enchytraeids. Repeated applications will in many cases reduce both abundance and species diversity. 2.2.5. Other Chemicals 2.2.5.1. Domestic and industrial chemicals. Little research has been directed to the impact of domestic and industrial chemicals on the enchytraeid fauna. As a group, enchytraeids appear to be well able to withstand domestic pollution, as they may thrive in, for instance, sewage beds (Reynoldson, 1939; Williams et al., 1969) or streams polluted with domestic and industrial wastes (e.g., Giani, 1983, 1984). The littoral species ¸umbricillus lineatus and Marionina subterranea have been found to be quite resistant to oil pollution and even to be capable of excreting oil derivatives or accumulating them in the body tissue (Giere, 1979; Giere and Hauschildt, 1979). Results of a microspectro#uorometry study indicate that ¸. lineatus might even possess enzymes to degrade oil products (Zeeck and Matuschek, 1984). This species might be rather exceptional in this respect, as Coates and Ellis (1980) found it to be predominant in the outfall area of a pulp mill, whereas in less contaminated areas the species was replaced by associations of other enchytraeid species (Cross and Ellis, 1981). According to Giere and Pfannkuche (1982), however, high oil concentrations, and even more so when chemical dispersants are added, may a!ect reproduction in ¸. lineatus, an e!ect that may become apparent only after several generations. Pirhonen and Huhta (1984) conducted a "eld experiment in which fuel oil and hydraulic oil was spread on forest soil plots. The enchytraeid population disappeared completely from the fuel oil plots, and started to build up again only after 18 months, but in the plots with hydraulic oil half of the population survived the treatment. Cianciara and Pilarska (1983) reported a decrease in numbers, or even complete disappearance, of enchytraeids in Polish meadows exposed to air pollution from activities of coal industry. In former refuse tips in Germany, Brockmann (1984) found rapid colonization by potworms, which indicates resistance to extreme conditions in at least some species. Similarly, E. crypticus was found to be rather insensitive to enhanced CO concentrations (S[ ustr and S[ imek, 1996), and Yeates  et al. (1997) even reported a concentration-dependent increase of enchytraeid numbers after elevation of CO in the  rhizosphere of a New Zealand pasture, probably due to increased below-ground productivity. The industrial chemical Chloroacetamide (Table 2 and Fig. 3) has repeatedly been used as a reference compound (such as in the earthworm acute test; OECD, 1984). Due to its rapid degradation in soil, even small di!erences in the test performance can lead to strongly di!erent e!ect

38

DIDDEN AND ROG MBKE

concentrations: LC values vary between 4 ppm for E.  albidus (RoK mbke 1989) and 172 ppm for E. crypticus (Achazi et al., 1997) in the same test soil. Due to this same chemical property, di!erences between acute and sublethal e!ects are very small. This chemical therefore cannot be recommended as a reference compound for reproduction tests. The chemical 4-nitrophenol (a metabolite of the insecticide Parathion) was used in an international ringtest to standardize the Enchytraeid Reproduction Test (Fig. 3; RoK mbke and Moser, 1999). LC and NOEC were relatively  close together (55 ppm versus 21.7}46.7 ppm, depending on the design), whereas the EC was clearly lower  (5.1}8.6 ppm). As in the case of Chloroacetamide, these results (including a high variability between the individual test runs) can partly be explained by quick degradation of the compound in soil. Until recently there were few data on the e!ects of ubiquitous chemicals such as dioxines and PAH on enchytraeids. In various tests using di!erent "eld soils contaminated mainly with PAH, Achazi et al. (1997) showed that acute e!ects occurred only in heavily contaminated soils, whereas reproduction was clearly more sensitive. After bioremediation a positive e!ect on the enchytraeids was often observed, but control levels were rarely reached. Faber and Heijmans (1996) conducted a survey of PAH accumulation in river plains, and found that Fridericia ratzeli mainly accumulated the lower-molecular-weight compounds (MW(228). Kratz and Brose (1997), however, reported that benzo[a]pyrene (BaP; MW 252) was accumulated by E. crypticus in aqueous and agar medium, with BCF values ranging from 260 to 1750. Once transposed to PAH-free medium, the body burden decreased quickly. Achazi et al. (1995) reported no direct toxic e!ect of BaP, #uoranthene (FLA), or PCB25 on E. crypticus. They reported low LOEC values for BaP (1 ppm), FLA (10 ppm), and PCB25 (0.2 ppm) for reproduction in agar medium, while in soil the LOEC values were much higher (5100 ppm). Greater sensitivity was found when using avoidance behavior as an endpoint (Achazi et al., 1999). In laboratory tests with aged "eld soils contaminated with heavy metals and PCB and PAH, it was di$cult to di!erentiate between the e!ects of these compounds and the often low pH of the soils on E. crypticus (Achazi et al., 1996). Funke and Frank (1991) conducted "eld tests with four volatile halogenated hydrocarbons in a coniferous forests, using concentrations 10 to 10 higher than ambient. Approximately 100 days after application chloroform and TCA led to signi"cant reductions of the enchytraeid abundance (non-speciesspeci"c). Interestingly, the e!ect of chloroform was signi"cantly higher in dry soil. As most of these substances have high adsorption coe$cients, it is unlikely that distinct toxic e!ects will occur under "eld conditions. The rather high accumulation of PAH by enchytraeids, however, indicates that they may be useful biomonitors for these compounds.

Regarding military chemicals such as TNT (Fig. 3), enchytraeids do not show a high sensitivity. For example, red phosphorous (used as an arti"cial smoke) did not cause any e!ects up to 640 ppm (Knacker et al., 1988). 2.2.5.2. Radioisotopes. Some authors have addressed the e!ects of radioisotopes on Enchytraeidae. Ghilarov and Krivolutzky (1971) did not "nd any e!ect of Sr (1.8}3.4 mCu ) m\) on the population density of the natural population in a birch forest. On the other hand, the application of Pu (1.78 mCu ) m\) reduced the number of enchytraeids slightly in the chernozem soil of a wheat "eld (Krivolutzky and Fedorova, 1973). 2.2.5.3. Pharmaceuticals. Around 1970 several compounds with anticipated antimicrobial and antiparasitic properties were tested in simple water tests using E. albidus. For example, nitrofurfuralacetales (MoK ller et al., 1968), benzylisothiocyanates, and related compounds (Weu!en, 1968; Teubner et al., 1971, 1975), benzoxathiole (Weu!en et al., 1973), and terpenes and terpene derivatives, (GoK ckeritz et al., 1974) were used, showing usually only slight toxicity. Very recently, veterinary drugs (mainly antibiotics) were studied in standardized enchytraeid tests, but the few results gained so far are di$cult to assess (Krogh, personal communication). 3. DISCUSSION

From the data presented above it is apparent that in both laboratory and "eld conditions enchytraeids are sensitive to numerous chemical stressors. It is also clear that the e!ects of chemical stress on enchytraeids are strongly related to bioavailability of the substance involved, which in turn is related to soil and species characteristics and the chemical properties of the substance in question. The e!ect of a toxicant on the enchytraeid community may be mediated by avoidance response and detoxi"cation and accumulation processes, possibly at the expense of other life history traits. In many cases clear interspeci"c di!erences in enchytraeid sensitivity were recorded, which may also be connected with narrow habitat requirements of enchytraeid species such as in the case of pH. A combination of laboratory and "eld studies will generally be indispensible both for prediction of direct e!ects of toxicants and for the interpretation of (indirect) "eld e!ects. Although in recent years much progress has been made in studying the relations of enchytraeids and chemical stress, there still are some areas that deserve further attention: * The laboratory tests that have been used are quite diverse regarding test conditions, complicating the comparability of the data obtained. Therefore, it is imperative that standardized testing methods be adopted in which the focus is on sublethal endpoints.

ENCHYTRAEIDS AS INDICATOR ORGANISMS

* The interaction between environmental factors such as soil properties and chemical stressors needs further study. Especially when using "eld soils, it is often di$cult to distinguish between e!ects of chemicals and those of, for instance, pH, on various enchytraeid species. * For many chemical stressors the potential toxicity to enchytraeids has not yet been established. Notably, information on the e!ects of industrial chemicals (especially PAHs and PCBs) is still de"cient. * In cases where toxicants were studied both in the "eld and in laboratory tests there generally was good agreement between the results. Yet, the number of such studies should be larger to more "rmly sustain their practicability. * The available data mainly focus on some Enchytraeus species, notably E. albidus. As the species from this genus are predominatly opportunistic, it is recommended that ecologically dissimilar genera such as Fridericia, Henlea, and Achaeta also be studied. * It would be helpful for better taxonomic tools to be developed in order to facilitate the identi"cation of enchytraeid species (or species groups), e.g., PC-based expert systems. * There is little information on the e!ects of multiple stress and stress on stress on enchytraeids. Yet, in "eld situations such stress certainly will prevail. It is vital therefore that future research focus on interactions between stressors such as mutual in#uences on bioavailability. * To evaluate e!ects of chemical stress in "eld situations information on the composition of the enchytraeid community in various ecosystem types is needed. Although progress is being made in this respect, there still are many insu$ciently studied ecosystems. In a more general sense, practically all information available comes from "eld sites in the northern hemisphere or from laboratory tests re#ecting their environmental conditions. A general conclusion may be that enchytraeids are very well suited to be used as indicator organisms for chemical stressors. In laboratory soil tests threshold values for e!ects were often recorded around or below the values that were found under "eld conditions. Thus, the results of such tests generally give a good indication of the e!ects that may be expected to occur under "eld conditions, especially when they are used in combination with data on availability in the soil solution and on the sensitivity of enchytraeids in water. This will enable a better understanding of the risks involved with speci"c toxicants. Moreover, it is evident that by their clear interspeci"c di!erences in sensitivity, enchytraeids are well suited for monitoring purposes, as chemical stress under "eld conditions is likely to result in changes in species composition and functioning of the enchytraeid community. REFERENCES Abrahamsen, G. (1983). E!ects of lime and arti"cial acid rain on the enchytraeid (Oligochaeta) fauna in coniferous forest. Holarct. Ecol. 6, 247}254.

39

Abrahamsen, G., Hovland, J., and Has gvar, S. (1980). E!ects of arti"cial acid rain and liming on soil organisms and the decomposition of organic matter. In &&E!ects of Acid Precipitation on Terrestrial Ecosystems'' (T. C. Hutchinson and M. Havas, Eds.), pp. 341}262. Plenum, New York. Achazi, R. K., Chroszcz, G., and Mierke, W. (1997). Standardization of test methods with terrestrial invertebrates for assessing remediation procedures for contaminated soils. ECO-Informa 12. Achazi, R. K., Chroszcz, G., Pilz, B., Rothe, B., Steudel, I., and Throl, C. (1996). Der Ein#uss des pH-Werts und von PCB52 auf Reproduktion und BesiedlungsaktivitaK t von terrestrischen Enchytraeen in PAK-, PCB- und schwermetallbelasteten RieselfeldboK den.
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DIDDEN AND ROG MBKE

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Wall, W. J., and Marganian, V. M. (1971). Control of Culicoides melleus (Coq.) (Diptera: Ceratopogonidae) with granular organophosphorus pesitcides, and the direct e!ect on other fauna. Mosquito News 31, 209}214. Way, M. Y., and Scopes, N. E. A. (1968). Studies on the persistence and e!ects on soil fauna of some soil-applied systemic insecticides. Ann. Appl. Biol. 62, 199}214. Weber, G. (1953). Die Makrofauna leichter und schwerer AckerboK den und ihre Beein#ussung durch P#anzenschutzmittel. Z. P-anzenern. Bodenk. 61, 107}118. Weigmann, G. (1995). Heavy metal burdens in forest soil fauna at polluted sites near Berlin. Acta Zool. Fennica 196, 369}370. Westheide, W., Bethke-Beilfu{, D., and Gebbe, J. (1991). E!ects of Benomyl on reproduction and population structure of enchytraeid oligochaetes (Annelida)*Sublethal tests on agar and soil. Comp. Biochem. Physiol. C 100, 221}224. Westheide, W., Bethke-Beilfu{, D., Hagens, M., and Brockmeyer, V. (1989). Enchytraeiden als Testorganismen*Voraussetzungen fuK r ein terrestrisches Testverfahren und Testergebnisse.