Search

Sulfonated and hydroxy-sulfonated PCBs were recently discovered by our group as new PCB soil contaminants, constituting about 1% of their parent compounds in soil. Here we investigate for the first time the bioaccumulation of these metabolites as well as hydroxy-PCBs and native PCBs in earthworms. A sequence of three experiments, at increasing complexity and ecological realism, were performed with four different earthworm species (Eisenia foetida Savigny, Lumbricus terrestris L, Allolobophora chlorotica Savigny and Aporrectodea caliginosa Savigny) exposed to contaminated soils. The first experiment confirmed that when exposing earthworms to soil contaminated with a single hexa-chlorinated congener (PCB 155), no formation of polar metabolites in earthworms could be detected. This allowed to plan the following two experiments, using a soil from a PCB contaminated site and rich in relatively high levels (10–130 μg kg−1) of hydroxy-, sulfonated-, and hydroxy-sulfonated-PCBs. Bioaccumulation factors (BAFs) and bioconcentration factors (BCFs) were then obtained in the second and third experiments, to compare the accumulation behavior of these chemicals in laboratory and natural conditions. Regressions between BAF/BCF and Log Kow/Log D, produced a variety of results, being generally significant between BCF and PCBs and not significant in the other cases. In general, the metabolites accumulated in earthworms with detectable concentrations in their tissues (8–115 μg kg−1), although sulfonated and hydroxy-sulfonated PCBs showed BAF and BCF values lower (up to two orders of magnitude) than those calculated for the parent PCBs, given their lower lipophilicity.

It is recognised that the establishment and function of soil biota is critical for successful mine residue rehabilitation. Bauxite residues are alkaline, saline and sodic and, whilst methods for establishing vegetation are well studied, little is known about key soil fauna such as earthworms. At a bauxite residue disposal area in Ireland, a 12-year-old rehabilitated residue was examined for evidence of earthworm populations. Five species of earthworm, dominated by Allolobophora chlorotica, were recorded in the rehabilitated residue representing the endogeic, epigeic and epi-anecic ecological groups. To further understand the potential for rehabilitated residues to support earthworm communities, a series of exposure tests was conducted. Whilst unamended residues (pH 10.2, EC 0.629 mS cm−1, ESP 54) was hostile to A. chlorotica survival, 100% survival was observed after 90 days for gypsum and organic-amended residue at salinity of up to 2.9 mS cm−1, possibly due to calcium becoming the dominant cation. Survival of earthworms at salinities higher than anticipated tolerance levels suggests that specific ion dominance plays a role in earthworm survival in saline soils. Percent mass change was negatively correlated with pH, EC and sodium content of the residues. Residue from the 12-year-old site also supported the anecic species Aporrectodea longa over 100 days. Percent mass change in residue samples retrieved from the 12-year-old site was significantly greater (p

International audience; Earthworms are common organisms in soil toxicity-testing framework, and endogeic species are currently recommended due to their ecological role in agroecosystem. However, little is known on their pesticide metabolic capacities. We firstly compared the baseline activity of B-esterases and glutathione-5-transferase in Allolobophora chlorotica and Aporrectodea caliginosa. Secondly, vulnerability of these species to pesticide exposure was assessed by in vitro trials using the organophosphate (OP) chlorpyrifosethyl-oxon (CPOx) and ethyl-paraoxon (POx), and by short-term (7 days) in vivo metabolic responses in soil contaminated with pesticides. Among B-esterases, acetylcholinesterase (AChE) activity was abundant in the microsomal fraction (80% and 70% of total activity for A. caliginosa and A. chlorotica, respectively). Carboxylesterase (CbE) activities were measured using three substrates to examine species differences in isoenzyme and sensitivity to both in vitro and in vivo exposure. CbEs were mainly found in the cytosolic fraction (80% and 60% for A. caliginosa and A. chlorotica respectively). GST was exclusively found in the soluble fraction for both species. Both OPs inhibited B-esterases in a concentration-dependent manner. In vitro trials revealed a pesticide-specific response, being A. chlorotica AChE more sensitive to CPOx compared to POx. CbE activity was inhibited at the same extent in both species. The 7d exposure showed A. chlorotica less sensitive to both OPs, which contrasted with outcomes from in vitro experiments. This non-related functional between both approaches for assessing pesticide toxicity suggests that other mechanisms linked with in vivo OP bioactivation and excretion could have a significant role in the OP toxicity in endogeic earthworms.

Increasing commercial application of silver nanoparticles (Ag NP) and subsequent presence in wastewater and sewage sludge has raised concerns regarding their effects in the aquatic and terrestrial environment. Several studies have employed standardised acute and chronic earthworm-based tests to establish the toxicological effects of Ag NP within soil. These studies have relied heavily on the use of epigiec earthworm species which may have limited ecological relevance in mineral soil. This study assessed the influence of Ag NP (uncoated 80 nm powder) and AgNO3 on survival, change in biomass and avoidance behaviour in a soil dwelling (endogiec) species, Allolobophora chlorotica. Earthworms were exposed for 14 days to soils spiked with Ag NP or AgNO3 at 0, 12.5, 25, 50 and 100 mg kg-1 either separately for survival and biomass measurement, or combined within a linear gradient to assess avoidance. Avoidance behaviour was shown to provide the most sensitive endpoint with an observable effect at an Ag NP / AgNO3 concentration of 12.5 mg kg-1 compared with 50 mg kg-1 for biomass change and 100 mg kg-1 for survival. Greater mortality was observed in AgNO3 (66.7%) compared with Ag NP-spiked soils (12.5%) at 100 mg kg-1, attributed to increased presence of silver ions. Although comparison of results with studies employing Eisenia fetida and Eisenia andrei suggest that the A. chlorotica response to Ag NP is more sensitive, further research employing both epigeic and endogeic earthworms under similar experimental conditions is required to confirm this observation.

Soil dwelling earthworms are now adopted more widely in ecotoxicology, so it is vital to establish if standardised test parameters remain applicable. The main aim of this study was to determine the influence of OECD artificial soil on selected soil-dwelling, endogeic earthworm species. In an initial experiment, biomass change in mature Allolobophora chlorotica was recorded in Standard OECD Artificial Soil (AS) and also in Kettering Loam (KL). In a second experiment, avoidance behaviour was recorded in a linear gradient with varying proportions of AS and KL (100% AS, 75% AS + 25% KL, 50% KS + 50% KL, 25% AS + 75% KL, 100% KL) with either A. chlorotica or Octolasion cyaneum. Results showed a significant decrease in A. chlorotica biomass in AS relative to KL, and in the linear gradient, both earthworm species preferentially occupied sections containing higher proportions of KL over AS. Soil texture and specifically % composition and particle size of sand are proposed as key factors that influenced observed results. This research suggests that more suitable substrates are required for ecotoxicology tests with soil dwelling earthworms.

By means of a literature survey, earthworm species of significant relevance for soil functions in different biogeographical regions of Europe (Atlantic, Boreal, Mediterranean) were identified. These focal earthworm species, defined here according to the EFSA Guidance Document on the environmental risk assessment (ERA) of genetically modified plants, are typical for arable soils under crop rotations with maize and/or potatoes within the three regions represented by Ireland, Sweden and Spain, respectively. Focal earthworm species were selected following a matrix of four steps: Identification of functional groups, categorization of non-target species, ranking species on ecological criteria, and final selection of focal species. They are recommended as appropriate non-target organisms to assess environmental risks of genetically modified (GM) crops; in this case maize and potatoes. In total, 44 literature sources on earthworms in arable cropping systems including maize or potato from Ireland, Sweden and Spain were collected, which present information on species diversity, individual density and specific relevance for soil functions. By means of condensed literature data, those species were identified which (i) play an important functional role in respective soil systems, (ii) are well adapted to the biogeographical regions, (iii) are expected to occur in high abundances under cultivation of maize or potato and (iv) fulfill the requirements for an ERA test system based on life-history traits. First, primary and secondary decomposers were identified as functional groups being exposed to the GM crops. In a second step, anecic and endogeic species were categorized as potential species. In step three, eight anecic and endogeic earthworm species belonging to the family Lumbricidae were ranked as relevant species: Aporrectodea caliginosa, Aporrectodea rosea, Aporrectodea longa, Allolobophora chlorotica, Lumbricus terrestris, Lumbricus friendi, Octodrilus complanatus and Octolasion cyaneum. Five out of these eight species are relevant for each biogeographical region with an overlap in the species. Finally, the earthworm species Ap. caliginosa (endogeic, secondary decomposer) and L. terrestris (anecic, primary decomposer) were selected as focal species. In the Mediterranean region L. terrestris may be substituted by the more relevant anecic species L. friendi. The selected focal species are recommended to be included in a standardized laboratory ERA test system based on life-history traits. © 2016 Elsevier B.V.

International audience; Recent EFSA (European Food Safety Authority) reports highlighted that the ecological risk assessment of pesticides needed to go further by taking more into account the impacts of chemicals on biodiversity under field conditions. We assessed the effects of two commercial formulations of fungicides separately and in mixture, i.e., Cuprafor Micro® (containing 500 g kg−1 copper oxychloride) at 4 (C1, corresponding to 3.1 mg kg−1 dry soil of copper) and 40 kg ha−1 (C10), and Swing® Gold (50 g L−1 epoxiconazole EPX and 133 g L−1 dimoxystrobin DMX) at one (D1, 5.81 10−2 and 1.55 10−1 mg kg−1 dry soil of EPX and DMX, respectively) and ten times (D10) the recommended field rate, on earthworms at 1, 6, 12, 18 and 24 months after the application following the international ISO standard no. 11268-3 to determine the effects on earthworms in field situations. The D10 treatment significantly reduced the species diversity (Shannon diversity index, 54% of the control), anecic abundance (29% of the control), and total biomass (49% of the control) over the first 18 months of experiment. The Shannon diversity index also decreased in the mixture treatment (both fungicides at the recommended dose) at 1 and 6 months after the first application (68% of the control at both sampling dates), and in C10 (78% of the control) at 18 months compared with the control. Lumbricus terrestris, Aporrectodea caliginosa, Aporrectodea giardi, Aporrectodea longa, and Allolobophora chlorotica were (in decreasing order) the most sensitive species to the tested fungicides. This study not only addressed field ecotoxicological effects of fungicides at the community level and ecological recovery, but it also pinpointed some methodological weaknesses (e.g., regarding fungicide concentrations in soil and statistics) of the guideline to determine the effects on earthworms in field situations.

Earthworms are widely known to impact soil health, having a keyrole in nutrient cycling and are often referred to as soil engineers.They are vital for soil microbial assemblages particularly through theirfeeding and burrowing activity in soil. Earthworms feed on soil organicmatter and litter, and the resulting casts alter the soil microbialcommunity. However, the gut microbiome of earthworms remains less known.In this study, we used amplicon sequencing of the 16S rRNA gene forbacteria and 18S rRNA gene for eukaryotes to assess the gut communityassemblages of earthworm species within three genera Aporrectodea,Allolobophora and Lumbricus that represent different life forms sharingthe same habitat. The objective was to compare the gut microbiomeprofiles of eukaryotic and prokaryotic organisms to assess significanceof earthworm life forms, and to explore the cross kingdom networks in anattempt to identify keystone species. We found a high eukaryoticdiversity with a dominance of the SAR supergroup along with fungi andmetazoan in the earthworm gut. The bacterial community were dominated bymembers of Proteobacteria, Acidobacteria, Actinobacteria, Firmicutes, andVerrucomicrobia. The eukaryotic and prokaryotic communities showedsimilar differences in alpha diversity, being lowest in Lumbricusherculeus. The beta diversity showed earthworm species as a key factor inshaping gut microbiomes with L. herculeus harboring distinct microbiomescompared to species of Aporrectodea caliginosa, A. longa, A. tuberculataand Allolobophora chlorotica. Cross kingdom networks showed highinteractions between several protist and bacterial OTUs. In conclusion,this study suggested that the community assemblages of gut microbiomeswas shaped by earthworm species and life form, and such assemblageconsist of cross kingdom interactions among eukaryotes and prokaryotes.

International audience; Landscape features are known to alter the spatial genetic variation of aboveground organisms. Here, we tested the hypothesis that the genetic structure of belowground organisms also responds to landscape structure. Microsatellite markers were used to carry out a landscape genetic study of two endogeic earthworm species, Allolobophora chlorotica (N = 440, eight microsatellites) and Aporrectodea icterica (N = 519, seven microsatellites), in an agricultural landscape in the North of France, where landscape features were characterized with high accuracy. We found that habitat fragmentation impacted genetic variation of earthworm populations at the local scale. A significant relationship was observed between genetic diversity (He, Ar) and several landscape features in A. icterica populations and A. chlorotica. Moreover, a strong genetic differentiation between sites was observed in both species, with a low degree of genetic admixture and high Fst values. The landscape connectivity analysis at the regional scale, including isolation by distance, least-cost path and cost-weighted distance approaches, showed that genetic distances were linked to landscape connectivity in A. chlorotica. This indicates that the fragmentation of natural habitats has shaped their dispersal patterns and local effective population sizes. Landscape connectivity analysis confirmed that a priori favourable habitats such as grasslands may constitute dispersal corridors for these species.

We tested experimentally if photoautotrophic microorganisms are a carbon source for invertebrates in temperate soils. We exposed forest or arable soils to a 13 CO 2 -enriched atmosphere and quantified 13 C assimilation by three common animal groups: earthworms (Oligochaeta), springtails (Hexapoda) and slugs (Gastropoda). Endogeic earthworms ( Allolobophora chlorotica ) and hemiedaphic springtails ( Ceratophysella denticulata ) were highly 13 C enriched when incubated under light, deriving up to 3.0 and 17.0%, respectively, of their body carbon from the microbial source in 7 days. Earthworms assimilated more 13 C in undisturbed soil than when the microbial material was mixed into the soil, presumably reflecting selective surface grazing. By contrast, neither adult nor newly hatched terrestrial slugs ( Deroceras reticulatum ) grazed on algal mats. Non-photosynthetic 13 CO 2 fixation in the dark was negligible. We conclude from these preliminary laboratory experiments that, in addition to litter and root-derived carbon from vascular plants, photoautotrophic soil surface microorganisms (cyanobacteria, algae) may be an ecologically important carbon input route for temperate soil animals that are traditionally assigned to the decomposer channel in soil food web models and carbon cycling studies.

The introduction of earthworms into soils contaminated with metals and metalloids has been suggested\ud to aid restoration practices. Eisenia veneta (epigeic), Lumbricus terrestris (anecic) and Allolobophora\ud chlorotica (endogeic) earthworms were cultivated in columns containing 900 g soil with 1130, 345, 113\ud and 131 mg kg1 of As, Cu, Pb and Zn, respectively, for up to 112 days, in parallel with earthworm-free\ud columns. Leachate was produced by pouring water on the soil surface to saturate the soil and generate\ud downflow. Ryegrass was grown on the top of columns to assess metal uptake into biota. Different\ud ecological groups affected metals in the same way by increasing concentrations and free ion activities in\ud leachate, but anecic L. terrestris had the greatest effect by increasing leachate concentrations of As by\ud 267%, Cu by 393%, Pb by 190%, and Zn by 429% compared to earthworm-free columns. Ryegrass\ud grown in earthworm-bearing soil accumulated more metal and the soil microbial community exhibited\ud greater stress. Results are consistent with earthworm enhanced degradation of organic matter leading\ud to release of organically bound elements. The degradation of organic matter also releases organic acids\ud which decrease the soil pH. The earthworms do not appear to carry out a unique process, but increase\ud the rate of a process that is already occurring. The impact of earthworms on metal mobility and\ud availability should therefore be considered when inoculating earthworms into contaminated soils as\ud new pathways to receptors may be created or the flow of metals and metalloids to receptors may be\ud elevated.

Metal pollution affects earthworm coelomocytes, including their differential counts, riboflavin content and metallothioneins (MT) involved in metal homoeostasis and detoxification. The present work shows effects of Ni, Cu, Zn, Cd, and Pb at the same molarity (1 mM) on coelomocytes of Allolobophora chlorotica after 2-day worm dermal exposure to metal chlorides. Numbers of coelomocytes/eleocytes extruded by electric shock and amounts of riboflavin in coelomocyte lysates were significantly decreased in Cu-exposed worms, less diminished in response to Ni, Zn, Cd, and unaffected by Pb. In sharp contrast, real-time PCR revealed a very strong (272 fold) MT-mRNA induction in response to Cd only. The induction was very low in response to Zn, Cu, Pb, and Ni ions (2.6, 2.1, 1.4, and 1.3-fold, respectively). In conclusion, decreased cell counts and riboflavin content are molecular biomarkers of Cu exposure while induction of MT-mRNA is a molecular biomarker of worm Cd exposure.

Bioturbation of metal contaminated soils contributes considerably to redistribution and surfacing of contaminated soil from deeper layers. To experimentally measure the contribution of Allolobophora chlorotica, Aporrectodea caliginosa, Lumbricus rubellus and L. terrestris to soil surface casting, a time-course experiment was performed under laboratory conditions. Earthworms were incubated in perspex columns filled with sandy soil (2% organic matter, 2.9% clay) or loamy clay soil (15% organic matter, 20% clay), and surface casts were collected after up to 80 days. On the sandy soil, A. caliginosa and L. rubellus brought approximately 7.1–16 g dry wt. casts/g fresh wt. earthworm to the surface, which is significantly more than A. chlorotica and L. terrestris (2.5–5.0 g dry wt./g fresh wt.). A. caliginosa was the only species that produced significantly more surface casts in the sandy soil than in the loamy clay soil. In the loamy clay soil, no differences in biomass-corrected casting rates were found among the species. Surface casting rates tended to decrease after 20 days. Considering the densities of the different species in a Dutch floodplain area Afferdensche and Deestsche Waarden, surface cast production is estimated to amount to 2.0 kg dry soil/m2 after 80 days, which could be extrapolated to 2.7–9.1 kg/m2 per year. These amounts correspond to a surface deposition of a layer of approximately 1.9–6.5 mm/year, which is of the same order or even slightly higher than the sedimentation rate and much higher than the amount of soil brought to the soil surface by bioturbating small mammals.

To get a better understanding of earthworm' responses towards flooding, three laboratory experiments were performed with the species Allolobophora chlorotica, Aporrectodea caliginosa and Lumbricus rubellus. Flooding response was determined in a pot experiment, in which the earthworms were incubated for 42 days in flooded or non-flooded soil, with or without heavy metal pollution. To determine moisture preference, earthworms were incubated for 9 days in aquaria with five compartments, containing soil with a moisture gradient (%, w/w), ranging from 35%, 45% (field capacity), 55%, 65% (saturated) to 65%+ (saturated and an extra water layer). Effects on earthworm health were studied by incubating earthworms of each species for 42 days in soil with the same range of moisture contents and determining the dry/wet weight ratio and dry weight gain as an indication of earthworm health. A. chlorotica was tolerant to water, although the worms tended to escape from flooded soil. Their health was significantly lower in the flooded soils (P

The levels of the enzymes, glutathione S -transferase, catalase, NAD(P)H-cytochrome c reductases, and DT-diaphorase were determined and compared in the tissues of three invertebrates commonly used in monitoring environmental quality: a freshwater mussel, Dreissena polymorpha , the earthworm Allolobophora chlorotica and the fourth instar of Chironomus riparius . It was found that the activities of GST, catalase, and NAD(P)-cytochrome c reductases were comparable in A. chlorotica and C. riparius , whereas comparatively a higher GST and a lower catalase activity was determined in the mussel tissues. DT-diaphorase was not detectable in A. chlorotica and the C. riparius larvae tissues, whereas this enzyme is present in the gills and the rest of soft mussel tissues (soft mussel tissues minus gills). It is suggested that the relatively low catalase activity observed in the tissues of the latter organism might be compensated by the presence of the antixidant role of DT-diaphorase. In addition, the inducibility of DT-diaphorase in D. polymorpha , by butylated hydroxyanisole (BHA) and lead (Pb) was investigated. Despite the bioaccumulation of both BHA (5.2±0.14 μg g −1 wet weight) and Pb (233.7±0.95 mg kg −1 dry weight) in the soft mussel tissues, the mussel DT-diaphorase was not induced. Although the activity of NADPH-cytochrome c (P-450) reductase was also not affected by these reagents, its activity was 2-fold higher in the gills than the rest of soft mussel tissues.

Earthworms dominate the animal biomass in moist floodplain soils. They are known to survive long periods in aerated water, but little is known about earthworm population dynamics in floodplain systems with changing inundation frequencies. This study determined earthworm population dynamics in a floodplain system, in relation to frequency and duration of flooding events. From October 2000 to May 2003 earthworms were hand sorted in the 'Afferdensche en Deestsche Waarden', a floodplain on the south bank of the river Rhine, near Druten, The Netherlands. Earthworm numbers and biomasses per age class (adult, subadult, juvenile) were recorded. Numbers and biomasses tend to decrease during flooding. Lumbricus terrestris was found in high numbers (>10/m2) only at the end of a flooding period. Allolobophora chlorotica was hardly affected by flooding; their biomass remained stable during the year. Aporrectodea caliginosa showed fluctuating numbers and biomasses during the sampling period that did not correlate with flooding frequency. Numbers and biomasses of Lumbricus rubellus were strongly reduced at the end of each flooding event, but their population densities fully recovered until next flooding event. Earthworm populations in floodplains fluctuate in time, depending on the season and on the time, duration and frequency of flooding. Different earthworm species react differently towards these flooding dynamics. © 2004 Elsevier GmbH. All rights reserved.

The objective of this study was to determine the impact of earthworm bioturbation on the distribution and availability of zinc in the soil profile. Experiments were carried out with Allolobophora chlorotica and Aporrectodea caliginosa in 24 perspex columns (0 10 cm), filled with 20-23 cm non-polluted soil (OM 2%, clay 2.9%, pH 0.01 M CaCl2 6.4), that was covered by a 3-5 cm layer of aged zinc spiked soil (500 mg Zn/kg dry soil) and another 2 cm non-polluted soil on top. After 80 and 175 days, columns were sacrificed and each cm from the top down to a depth of 15 cm was sampled. Earthworm casts, placed on top of the soil, were collected. Each sample was analyzed for total and CaCl2-exchangeable zinc concentrations. Effects of earthworm bioturbation were most pronounced after 1,75 days. For A. chlorotica, total and CaCl2-exchangeable zinc concentrations in the polluted layers were lower with than without earthworms. Total zinc concentrations in the non-polluted layers were higher in columns with earthworms. Casts of A. chlorotica collected on the soil surface showed slightly higher total zinc concentrations than non-polluted soil. Casts were found throughout the whole column. For A. caliginosa there were no differences in total zinc concentration between columns with and without earthworms. CaCl2-exchangeable zinc concentrations in the polluted layers were lower for columns with earthworms. Casts were mainly placed on top of the soil and contained total zinc concentrations intermediate between those in non-polluted and polluted soil layers. This study shows that different endogeic earthworm species have different effects on zinc distribution and availability in soils. A. chlorotica transfers soil throughout the whole column, effectively mixing it, while A. caliginosa decreases metal availability and transfers polluted soil to the soil surface. (c) 2004 Elsevier Ltd. All rights reserved.

The behaviour of earthworms belonging to two different species and ecological types (A porrectodea nocturna and A llolobophora chlorotica) was studied using two-dimensional (2D) terraria. Two experiments were set up to gain insight into the nature of interactions between these earthworms. Firstly, the evolution of the burrow systems was analysed with the density of the earthworms varying from one to five individuals. Secondly, a burrow system was first established by using one earthworm which was then removed before the introduction of a second earthworm. This second earthworm therefore encountered a burrow system created either by a conspecific earthworm or by an earthworm of the other species. These experiments showed that: (1) intra- and interspecific interactions occur between earthworms, (2) these interactions are dependent on the physical presence of the earthworms, and (3) spatial avoidance can occur (A. chlorotica avoiding burrows created by A. nocturna). The results suggest that earthworm burrow systems are "individual structures", rarely used by other earthworms when inhabited. When abandoned, the burrows may be recolonised depending on the ecological type of the earthworm under consideration.

Burrow systems of earthworms contained in artificial cores were analysed through X-ray computed tomography and 3D skeleton reconstructions. Gas diffusion experiments were carried out on these cores to characterize soil transfer properties associated with the different burrow systems. Three types of cores were studied: cores in which Aporrectodea nocturna, an anecic earthworm was introduced (treatment 1), cores in which Allolobophora chlorotica, an endogeic earthworm was introduced (treatment 2) and cores that contained both species (treatment 3). Comparisons of the characteristics of the burrow systems of treatment 1 and 2 show important differences: the burrow system of A. nocturna comprises fewer burrows, which are longer, less branched, more vertical and have a lower sinuosity. The burrow system of A. chlorotica is characterized by lower continuity, which however did not result in a lower soil diffusivity. To study the burrow systems made by the two species in the same core (treatment 3), a separation that was based on differences in pore diameter between the two species and that takes into account the burrow orientation was designed. This separation was proven to be efficient since it resulted in low percentages of errors (around 10%) when applied to the burrow systems of treatments 1 and 2. Comparison of the burrow systems from treatments 1 and 3 demonstrated that the burrow system of A. nocturna was influenced by the presence of A. chlorotica: in treatment 3, A. nocturna made burrows that were smaller, more vertical and less branched. However, these interactions have to be confirmed under natural conditions. © 2001 Elsevier Science B.V. All rights reserved.

The aim of this study was to measure the pressures exerted by earthworms during burrowing. For this purpose we developed two methods with which to quantify the axial and radial pressure. The data were recorded with an electronic balance that was connected to a PC. Artificial earthworm burrows were used to standardize the measurements. Plexiglas tubes with diameters ranging from 2 to 6.3 mm which corresponded to the diameter of the earthworms were used. A pin was placed inside the tubes, on which the earthworms exerted a pressure by peristaltic locomotion. Only the maximum values of the pressure measurement were taken into account for evaluation, and the arithmetic mean was calculated. The measurements were conducted with Aporrectodea longa, Lumbricus terrestris, Aporrectodea caliginosa, Octolasion cyaneum, Allolobophora chlorotica, Aporrectodea rosea, Lumbricus rubellus and Dendrobaena octaedra. The species examined were classified into ecological groups. The mean axial pressures exerted by each group were in the order: epigeic (14–25 kPa), endogeic (27–39 kPa) and anecic (46–65 kPa). For the mean radial pressure the order was: epigeic (39–63 kPa), anecic (72–93 kPa) and endogeic (59–195 kPa). It was apparent from the results that radial pressure is the most important pressure with respect to the burrowing activity of earthworms.

Predation on three species of earthworms by the terrestrial planarian, Artioposthia triangulata, was compared in columns of artificial soil (10 cm dia, 1 m depth) at four different temperatures (5, 10, 12 and 16°C). The planarian significantly reduced the number of earthworms and affected the vertical distribution of all species by reducing prey numbers in the top 20 cm. Greater earthworm mortality occurred at higher temperatures but there was no difference in the numbers of each species that were killed.

Earthworm cocoons are mostly found in the uppermost soil layers and are therefore often exposed to low temperatures during winter. In the present study, cocoons of five taxa of earthworms were investigated for their tolerance to freezing, melting points of cocoon fluids and dehydration of cocoons when exposed to a frozen environment. Embryos of the taxa investigated were freeze intolerant. The melting points of fully hydrated cocoon fluids were high (above −0.3°C) and thermal hysteresis factors were absent. Exposure to a frozen environment caused the cocoons to dehydrate drastically and dehydrated cocoons showed significantly lower super-cooling points than fully hydrated cocoons, reducing the risk of freezing for dehydrated cocoons. It is proposed therefore that the cold-hardiness strategy of the earthworm cocoons is based on dehydration upon exposure to subzero temperatures in the frozen environment. Cocoons of three surface-dwelling taxa, Dendrobaena octaedra, Dendrodrilus rubidus tenuis and Dendrodrilus rubidus norvegicus had lower supercooling points and survived frost exposure better than cocoons of two deeper-dwelling taxa, Aporrectodea caliginosa and Allolobophora chlorotica. One of the investigated taxa, D. r. norvegicus, was collected from a cold alpine habitat. However, it was not more cold hardy than the closely related D. r. tenuis collected from a lowland temperate habitat. D. octaedra was the most cold hardy taxon, its cocoons being able to withstand −8°C for 3 months and −13.5°C for 2 weeks in frozen soil.

We have shown previously that stubby worms Allolobophora chlorotica are sensitive to environmental stress, including metal-polluted soil. In order to discern the mechanisms of this sensitivity, adult (clitellate) Al. chlorotica were exposed in the laboratory to soil samples soaked with water (control) or Ni (1 and 2 mg/kg), Zn (1.25 and 2.5 g/kg) or Pb (5 and 10 g/kg) chlorides. Worms avoided contact with metal contaminants by prolonging burrowing time in metal-soaked samples, especially in the case of lead. Higher concentrations of the investigated metals were lethal for worms. During a 3 week exposure to lower metal concentrations, nickel and lead readily accumulated in the bodies of worms while zinc was efficiently regulated. However, body weights and numbers of non-invasively retrieved free coelomocytes (consisting of amoebocytes and riboflavin-loaded eleocytes) were significantly lower only in zinc-exposed worms. We assume that zinc regulation in worm bodies is more energy-demanding than nickel or lead bioaccumulation, thus this might be responsible for inhibition of the body gain and diminution of immunocompetent cells in zinc-exposed earthworms. Alternatively, missing free coelomocytes may actually be involved in Zn trafficking and removal through nephridia and/or in the formation of multicellular brown bodies, since metal can unbalance host/bacteria relationships.

Carbendazim-amended soil was placed above or below unamended soil. Control tests comprised two layers of unamended soil. Allolobophora chlorotica earthworms were added to either the upper or the unamended soil. After 72 h vertical distributions of earthworms were compared between control and carbendazim-amended experiments. Earthworm distributions in the carbendazim-amended test containers differed significantly from the ‘normal’ distribution observed in the control tests. In the majority of the experiments, earthworms significantly altered their burrowing behaviour to avoid carbendazim. However, when earthworms were added to an upper layer of carbendazim-amended soil they remained in this layer. This non-avoidance is attributed to (1) the earthworms’ inability to sense the lower layer of unamended soil and (2) the toxic effect of carbendazim inhibiting burrowing. Earthworms modified their burrowing behaviour in response to carbendazim in the soil. This may explain anomalous results observed in pesticide field trials when carbendazim is used as a control substance.

The effects of wastewater sludge amendments on nitrogen forms and urease activity in soil and earthworm (Lumbricus terrestris) casts were investigated. Two different wastewater sludges (municipal and canned food industry) were added to clay loam agricultural soil samples at doses of 20, 40 and 80 g dry raw sludge kg(-1). Following sludge amendment, an incubation study was performed and mineral nitrogen (NH(4)(+)-N+NO(3)(-)-N), total-N and urease activity were determined in soil and earthworm casts. Results indicated that the addition of wastewater sludge significantly increased urease activity, mineral-N and total-N contents of soil and casts. Furthermore, urease activity, mineral-N and total-N contents of casts were found to be significantly higher than that of the surrounding soil. Application of canned food industry sludge at a dose of 80 g kg(-1)(200 t ha(-1)) was the most effective application on nitrogen and urease contents. On the other hand, 40 g kg(-1) (100 t ha(-1)) treatment was found to be the most suitable dose for municipal wastewater sludge.

A total of 20 earthworm species were identified. Two of them, Metaphire californica and Pontoscolex corethrurus , are recorded for the first time on Tenerife. The discovery of the latter species constitutes also a new record for the Canary Islands, and thus the geographical area of its distribution in Macaronesia is enlarged. Furthermore, two species groups are recognized, the first comprises exotic earthworms clearly associated with tropical plantations ( Amynthas conicis, A. gracilis, A. morrisi, A. rodericensis and Pithemera bicincta ), introduced by man from the 15th century onwards, and thus forming part of modern Canarian fauna. The second group includes a considerable number of lumbricids which are widely distributed throughout the subhumid thermo-canarian zone ( Allolobophora chlorotica, Eisenia andrei and E. eiseni , among others) which were able to Actapt themselves to the prevailing conditions in areas of banana production. The present article also includes information concerning the chemical characteristics of the soils where the studied species live. The soils of the South-Southwest slopes of Tenerife are principally alkaline and harbour a considerably poorer earthworm fauna.

During the past 25 yr, research by the authors at Rothamsted Experimental Station investigated many aspects of the utilization of earthworms in land improvement and environmental management. Results of some of these investigations are summarized in this paper with the aim of illustrating the general principles of how earthworm populations can be manipulated and managed for environmental improvement. The use of earthworms in land improvement and reclamation: we investigated the effects of inoculating earthworms of the species Lumbricus terrestris L., Aporrectodea longa (Ude), Aporrectodea caliginosa (Sav.) and Allolobophora chlorotica (Sav.) into intact soil profiles in the laboratory, plots on direct-drilled, arable land in the field and newly-capped waste disposal sites that had few or no earthworms. In all these studies the earthworms increased significantly in number and rate of growth and yield of plants growing on the inoculated sites. Earthworms for inoculation were obtained by field collection after watering soil with dilute formaldehyde solution. The use of earthworms in organic waste management: the life cycles and productivity of Eisenia fetida (Sav.), Eudrilus eugeniae (Kinberg), Perionyx excavatus (Michaelsen) and Dendrobaena veneta (Rosa), and their potential in processing animal and plant wastes, from sewage, agricultural, domestic, urban and industrial sources are summarized. The preprocessing of wastes, their population ecology, optimum stocking rates, the mechanization of processing and utilization of the product are discussed. Results of experiments on the effects of temperatures of 10, 15, 20 and 25°C and a range of soil moisture contents of 70, 75, 80, 85 and 90% on the growth, cocoon production and cocoon hatching of the four species are summarized. The use of earthworms in assessment of the environmental effects of chemicals: earthworms can be used as key indicators to predict the effects of chemicals on other soil invertebrates. Methods of testing chemicals against earthworms in field and laboratory are reviewed. Two standardized laboratory test methods, one exposing earthworms to chemicals on filter paper and one to chemicals in artificial soils are described, and the median lethal concentration (LC 50 ) for chloracetamide, pentachlorophenol, chlordane, carbaryl, potassium bromide, copper sulfate and trichloracetic acid calculated, based on assays done in 34 laboratories. The relevancy of the two tests in environmental toxicity testing is reviewed.

Earthworms play a major role in many aspects of soil fertility, food web ecology and ecosystem functioning, and hence are frequently the subjects of, for example, ecological and toxicological research. Our aim was to examine the genetic structure of common earthworm species, to identify cryptic lineages or species that may be distinct ecotypes or biotypes (and hence confound current research based upon morphotypes) and to try to explain the massive cryptic diversity that eventually emerged. We demonstrated that species such as Allolobophora chlorotica, Aporrectodea longa, Aporrectodea rosea and Lumbricus rubellus all comprise highly divergent lineages with species-level divergence at the mitochondrial cytochrome oxidase I (COI) gene. In Allo. chlorotica alone, we found 55 haplotypes for COI, with 35 of these being found in pink and 20 in green morph worms. There were no cases of the two colour morphs sharing COI haplotypes. Phylogenetic analyses of mitochondrial COI and 16S genes showed the presence of five highly divergent lineages, suggesting the presence of multiple cryptic species within Allo. chlorotica. There was no clear geographical pattern to lineage distribution and many populations were polymorphic for both mitochondrial DNA lineage and colour morph. Amplified fragment length polymorphism results, based on two primer combinations, were broadly congruent with mitochondrial DNA results with one significant exception. Despite showing over 14% divergence at COI, amplified fragment length polymorphism markers showed that the two green morph lineages may be interbreeding and therefore represent a single taxon. The cryptic diversity revealed by these results has profound consequences for all areas of earthworm research.

Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.

To validate cholinesterase activity as a biomarker of pesticide exposure, we characterized the tissue distribution (whole body, nervous tissue and crop/gizzard), activity at two seasons of cholinesterase in six different species of earthworms collected in an unpolluted field: Lumbricus terrestris, Lumbricus castaneus, Aporrectodea nocturna, Aporrectodea caliginosa, Allolobophora chlorotica and Aporrectodea rosea. The major part of total cholinesterase activity was found in the nervous tissue while activity in crop/gizzard was weak. The level of the total cholinesterase activity was stable for each species considered throughout the year (spring and autumn). Lumbricus species exhibited three-fold higher specific activity than the others (0.086+/-0.015 U mg(-1) and 0.235+/-0.036 U mg(-1) for Allolobophora or Aporrectodea, and Lumbricus species respectively). This stability of the base level makes cholinesterase activity a useful biomarker for monitoring effects of pesticide under natural conditions. Cholinesterase activity was characterized using different substrates and inhibitors. It seems likely that the cholinesterases are acetylcholinesterases in most species investigated as they preferentially hydrolyzed acetylthiocholine and were inhibited by eserine, but not by tetraisopropyl pyrophoramide (iso-OMPA). Characterization of cholinesterase from Allolobophora chlorotica is uncertain and it cannot be classified as a true AChE.

The present study used cholinesterase (ChE) activity in earthworms as a biomarker of pesticide exposure at 17 apple orchards using different pest protection strategies (organic, integrated pest management [IPM], conventional, and abandoned) located within a 300-km2 subregion near Avignon in southeastern France). The most common earthworm species in the 17 orchards was Allolobophora chlorotica. We examined inherent variability in ChE activity that might be attributable to soil characteristics and found that differences in soil structure or type did not significantly influence ChE activity. Furthermore, there was no relation between ChE specific activity and earthworm weight, and thus activity does not require correction for weight. Ten earthworms were collected in two successive months (April and May 2003) from each of the 17 orchards. Compared to the activity in worms from the control abandoned orchards, ChE activity was significantly decreased in earthworms from half the IPM and conventional orchards in April and all these orchards in May. Notably, ChE activity was also lower in earthworms from three organic orchards during May. No relation was observed between ChE decrease and the number of treatments (total or only organophosphorous and carbamate pesticides). Cholinesterase activity in earthworms from abandoned orchards varied between the two collecting periods, illustrating the difficulty in obtaining reference values for the use of ChE as a biomarker in field studies.

The relationships between the 13 most common species of Por tuguese earthworms and 20 soil characteristics have been investi gated by analysis of ecological profiles, correspondence analysis and canonical correlation. Two groups emerged: one comprising basi cally the epigeic species Allolobophora oliveirae, Dendrobaena madeiren‐sis, Dendrobaena octaedra and Eisenia eiseni, which tend to prefer organic acid soils with little calcium or magnesium and large quan tities of aluminium in the exchange complex, and the other made up of the endogeic species Allolobophora caliginosa, Allolobophora mol‐leri, Allolobophora rosea and Allolobophora chlorotica, which exhibit the opposite tendencies.

1. 1. The biological distribution of guanidine derivatives has been studied by paper chromatography in terrestrial and freshwater annelids (Oligochaeta, Hirudinea) and platyhelminthes (Turbellarea) from Hungary. 2. 2. Arginine, though present in all animals studied, was found in appreciable amounts only in platyhelminthes, whose tissues did not contain any other detectable guanidino derivative. 3. 3. All Oligochaeta (Lumbricidae, Tubificidae) contain free and phosphorylated lombricine (guanidoethylserylphosphoric acid) as the major guanidino derivative of the tissues; two of them ( Allolobophora chlorotica and A. dubiosa ) contain also hirudonine (diamidinospermidine), previously identified in the leech, Hirudo medicinalis (L.). The quantitative distribution of arginine, hirudonine and lombricine in these Allolobophora was established by specific titration. 4. 4. From all the Hirudinea (Erpobdellidae, Glossiphoniidae, Hirudidae) studied here, only the Hirudidae contain hirudonine, always associated with small amounts of arcaine (diamidinoputrescine) and of arginine; only minute amounts of arginine were detected in the other families. The nature and presence of a phosphagen in the hirudinea is discussed. 5. 5. The number, kind and distribution of guanidine derivatives in terrestrial and freshwater annelids seem to be less diverse than in the marine annelids previously studied. The pattern of distribution seems to be unrelated to location and environment.

Earthworms serve as good indicators of heavy metal contamination due to their innate sensitivity towards soil pollution. However, to date, not many studies have focused on endogeic earthworms, such as the omnipresent Allolobophora chlorotica. The current study was designed to verify whether this earthworm could serve as a novel distinctively susceptible species for environmental contamination studies. We show that the dermal exposure to Cu, Ni, and Cd affected the mortality and morphology of A. chlorotica, and the number and functioning of coelomocytes. These features particularly were pronounced in animals treated with Ni and Cu and interestingly to a lesser extend with Cd. In contrast, Cd induced a strong expression of metallothioneins (MT-2) and heat shock proteins (HSP72). The presence of MT-2 was detected not only in coelomocytes but also in the intestine, blood vessels, and epidermis. In conclusion, Allolobophora chlorotica coelomocytes are adopted to respond differentially to various heavy metals, generating powerful response towards potentially most dangerous exogenous non-essential elements.

Surface cast production was investigated for several species of earthworms, including some not normally considered to produce surface casts. In single-species culture, the amount of cast material deposited on the surface by different species varied, with Allolobophora chlorotica < Lumbricus rubellus < Aporrectodea caliginosa < Aporrectodea longa. In field communities, results indicated that a single species dominated surface casting activity. The order of species dominance was consistent with the above findings from single-species cultures, if Lumbricus terrestris was considered to be equivalent to Ap. longa. Surface casting is an essential function within earthworm communities which maintains their living space. However, it involves an energy cost and carries a risk of predation. Therefore, there are disadvantages associated with this activity. All of the species studied produced surface casts unless other, usually larger, species were present.

Glutathione S-transferase activity (EC 2.5.1.18) was demonstrated in six species of earthworms of the family Lumbricidae: Eisenia foetida, Lumbricus terrestris, Lumbricus rebellus, Allolobophora longa, Allolobophora caliginosa and Allolobophora chlorotica. Considerable activity was obtained with 1-chlorl-2,4-dinitrobenzene and low activity with 3,4-dichloro-1-nitrobenzene, but no enzymic reaction was detectable with sulphobromophthalein 1,2-epoxy-3-(p-nitrophenoxy)propane of trans-4-phenylbut-3-en-2-one as substrates. Enzyme prepartations from L. rubellus and A. longa were the most active, whereas A. chlorotica gave the lowest activity. The ratio of the activities obtained with 1-chloro-2,4-dinitrobenzene and 3,4-cichloro-1-nitrobenzene was very different in the various species, but no phylogenetic pattern was evident. Isoelectric focusing gave rise to various activity peaks as measured with 1-chloro-2,4-dinitrobenzene as a substrate, and the activity profiles of the species examined appeared to follow a taxonomic pattern. The activity of Allolobophora had the highest peak in the alkaline region, whereas that of Lumbricus had the highest peak in the acid region. Eisenia showed a very complex activity profile, with the highest peak ne pH 7. As determined by an enzymic assay, all the species contained glutathione, on an average about 0.5 mumol/g wet wt. Conjugation with glutathione catalysed by glutathione S-transferases may consequently be an important detoxification mechanism in earthworms.

The carabid beetle Pterostichus melanarius is a major natural enemy of pests, such as aphids and slugs in agricultural systems. Earthworms are a dominant non-pest component of the diet of P. melanarius which help sustain the beetles during periods when the pest population is low or absent. In this study we wanted to test whether this predator exercises prey choice among different earthworm species or ecological groups. High levels of genetic diversity within morphological species of earthworm necessitated the development of primers that were specific not just to species but lineages and sub-lineages within species as well. Gut samples from beetles were analysed using multiplex-PCR and fluorescent-labelled primers. Calibratory feeding trials were undertaken to calculate median detection times for prey DNA following ingestion. Extensive testing demonstrated that the primers were species-specific, that detection periods were negatively related to amplicon size and that meal size had a highly significant effect on detection periods. Monte Carlo simulations showed that, in general, worms were being predated in proportion to their densities in the field with little evidence of prey choice, other than probable avoidance of the larger, deep-living species. There was no evidence that epigeic species were being taken preferentially in comparison with endogeic species. There was also no evidence that defensive secretions by Allolobophora chlorotica reduced predation pressure on this species by P. melanarius. We concluded that any management system that increases earthworm densities generally, regardless of component species, is likely to be optimal for increasing numbers of this beneficial beetle predator.