Comparative avoidance behaviour of the earthworm Eisenia fetida towards chloride, nitrate and sulphate salts of Cd, Cu and Zn using filter paper and extruded water agar gels as exposure media.

We studied the avoidance behaviour of the earthworm Eisenia fetida towards Cd, Cu, and Zn, trace elements (TEs) tested as chloride, nitrate and sulphate salts. Sub adults were exposed individually using dual-cell chambers at 20+2°C in the dark. Recordings were realised at different dates from 2h to 32h. We used filter paper and extruded water agar gel as exposure media to evaluate the contribution of the dermal and the digestive exposure routes on the avoidance reactions. Exposures to Cu or Cd (10mgmetal ionL(-1)) resulted in highly significant avoidance reactions through the exposure duration. Worms avoided Zn poorly and reactions towards Zn salts varied along the exposure. Worm sensitivity towards TEs differed between salts and this could result from differential toxicity or accessibility of these TE salts to earthworms. The anion in itself was not the determinant of the avoidance reactions since exposures to similar concentrations of these anions using calcium salts did not result in significant avoidance worm behaviour. Avoidance responses towards TEs were higher in the case of water agar exposures than in filter paper exposures. Thus, dermal contacts with TE solutions would elicit worm avoidance but signals from receptors located inside the digestive tract could reinforce this behaviour. The use of extruded water agar gels as the substrate allows checking the real sensitivity of earthworm species towards TEs since the TE concentrations leading to significant avoidance reactions were below those reported in the literature when using TE-spiked soils.

Earthworm populations of highly metal-contaminated soils restored by fly ash-aided phytostabilisation.

Highly metal contaminated soils found in the North of France are the result of intense industrial past. These soils are now unfit for the cultivation of agricultural products for human consumption. Solutions have to be found to improve the quality of these soils, and especially to reduce the availability of trace elements (TEs). Phytostabilisation and ash-aided phytostabilisation applied since 2000 to an experimental site located near a former metallurgical site (Metaleurop-Nord) was shown previously as efficacious in reducing TEs mobility in soils. The aim of the study was to check whether this ten years trial had influenced earthworm communities. This experimental site was compared to plots located in the surroundings and differing by the use of soils. Main results are that: (1) whatever the use of soils, earthworm communities are composed of few species with moderate abundance in comparison with communities found in similar habitats outside the TEs-contaminated area, (2) the highest abundance and specific richness (4-5 species) were observed in afforested plots with various tree species, (3) ash amendments in afforested plots did not increase the species richness and modified the communities favoring anecic worms but disfavoring epigeic ones. These findings raised the questions of when and how to perform the addition of ashes firstly, to avoid negative effects on soil fauna and secondly, to keep positive effects on metal immobilization.

Effects of field metal-contaminated soils submitted to phytostabilisation and fly ash-aided phytostabilisation on the avoidance behaviour of the earthworm Eisenia fetida.

The earthworm Eisenia fetida avoidance behaviour test was used to assess the quality recovery of metal-contaminated soils from lands submitted for 10 years to remediation. Soils were from plots located in the surroundings of a former lead smelter plant of Northern France. Metal concentrations in the soils ranged from 93 to 1231, 56 to 1424, 0.3 to 20 and 15 to 45.5mg metal/kg dry soil for Pb, Zn, Cd and Cu, respectively. Several former agricultural plots were treated either by a single phytostabilisation process involving the plantation of a tree mix or by fly ash aided-phytostabilisation. Silico-aluminous or sulfo-calcic ashes used were ploughed up to a 25- to 30-cm soil depth at a rate of 23.3kg/m(2) (i.e., 6 percent W/W). E. fetida was shown to avoid significantly the 10 years ash-treated soils whose habitat function has to be considered as limited. This avoidance would relate to a change of the texture of soils induced by the addition of ashes and consisting in an increased level of fine silts together with a decreased level of clays. By contrast, afforested metal-contaminated soils appeared for E. fetida as more attractive than unplanted ones. Regarding the influence of the metal contamination of the soils on E. fetida, none of the soils tested even the highest contaminated one was significantly avoided by worms. This lack of reaction would result from the low bioavailability of the metals in the soils tested. At the lights of our results and those previously published on both these ashes and these ash-treated soils, the usefulness of these soil treatments is discussed.

Litter breakdown as a tool for assessment of the efficiency of afforestation and ash-aided phytostabilization on metal-contaminated soils functioning in Northern France.

The main objective of the study was to assess the efficiency of phytoremediation methods implemented for 14 years on highly metal-contaminated soils. The different experimental strategies were plots planted with a tree mix or with a single tree species coupled or not with the use of fly-ashes as an amendment to limit metals mobility in soil. The breakdown of poplar litter on the four plots was monitored during 10 months. In parallel, colonization of litter bags by functional groups of mesofauna (Collembola and Acari) was followed. Two mesh-sized litter bags were used to allow distinguishing microbial and mesofaunal actions on the litter breakdown. We observed the breakdown of litter in four studied plots. Litter breakdown occurred faster in 3-mm litter bags than 250 µm ones during summer demonstrating the importance of mesofauna. Mixed plantation allowed faster litter breakdown than mono-specific plantation. A higher abundance of mesofauna and/or better abiotic conditions (moisture, shading…) could explain this result. Regarding litter breakdown and mesofauna, no significant difference was observed between the amended plots and those subjected to soil phytomanagement. However, communities of the studied area are disturbed since a low abundance of detritivores was observed. This could explain also the slower litter breakdown than expected in our study. To conclude, among the phytomanagement methods tested, mixed plantations could provide a benefit for the restoration of degraded soils. By contrast, the use of fly-ashes does not seem to have any effect on the functionality of ecosystem neither on the litter breakdown process nor on the abundance of mesofauna.

A thesaurus for soil invertebrate trait-based approaches.

Soil invertebrates are known to be much involved in soil behaviour and therefore in the provision of ecosystem services. Functional trait-based approaches are methodologies which can be used to understand soil invertebrates' responses to their environment. They (i) improve the predictions and (ii) are less dependent on space and time. The way traits have been used recently has led to misunderstandings in the integration and interpretation of data. Trait semantics are especially concerned. The aim of this paper is to propose a thesaurus for soil invertebrate trait-based approaches. T-SITA, an Internet platform, is the first initiative to deal with the semantics of traits and ecological preferences for soil invertebrates. It reflects the agreement of a scientific expert community to fix semantic properties (e.g. definition) of approximately 100 traits and ecological preferences. In addition, T-SITA has been successfully linked with a fully operational database of soil invertebrate traits. Such a link enhances data integration and improves the scientific integrity of data.

Assessing the effects of FBC ash treatments of metal-contaminated soils using life history traits and metal bioaccumulation analysis of the earthworm Eisenia andrei.

Earthworms (Eisenia andrei) were exposed, in controlled conditions, to metal-contaminated soils previously treated in situ with two types of fluidized bed combustion ashes. Effects on this species were determined by life history traits analysis. Metal immobilizing efficiency of ashes was indicated by metal bioaccumulation. Ashes-treated soils reduced worm mortality compared to the untreated soil. However, these ashes reduced both cocoon hatching success and hatchlings numbers compared to the untreated soil. In addition, sulfo-calcical ashes reduced or delayed worm maturity and lowered cocoon production compared to silico-alumineous ones. Metal immobilizing efficiency of ashes was demonstrated for Zn, Cu and to a lesser extent Pb. Only silico-alumineous ashes reduced Cd bioaccumulation, although Cd was still bioconcentrated. Thus, although ash additions to metal-contaminated soils may help in immobilizing metals, their use might result, depending on the chemical nature of ashes, to severe detrimental effects on earthworm reproduction with possible long term consequences to populations.

Metallothionein response following cadmium exposure in the oligochaete Eisenia fetida.

We studied the metallothionein (MT) response in cadmium-exposed worms (Eisenia fetida) both at the protein level by Dot Immunobinding Assay (DIA) with a polyclonal antibody raised against the most immunogenic part of this protein and at the expression level by Northern blotting using a specific probe. MT appeared as two close isoforms. DIA results clearly demonstrated significant differences in MT level of whole worm heat-treated supernatants between E. fetida exposed to Cd concentrations as low as 8 mg Cd kg(-1) of dry soil compared to controls. Northern blotting analysis performed on whole bodies of worms revealed that a single exposure to 8 mg Cd kg(-1) of dry soil for 1 day resulted in the production of MT mRNA. This response was maintained for exposure of at least 1 month. Clear differences of MT gene expression were also observed between worms exposed to different Cd concentrations (8, 80 or 800 mg Cd kg(-1) of dry soil). Immunocytochemistry demonstrated that MT was located in the chloragogenous tissue surrounding the gut where metals are known to be accumulated. This work revealed that E. fetida MT is a sensitive and relevant biomarker of Cd exposure and especially when considering gene expression response. Further experiments have now to prove its usefulness in natural metal-contaminated soil toxicity assessments.

Stimulation by cadmium of myohemerythrin-like cells in the gut of the annelid Nereis diversicolor.

Isolated guts of Nereis diversicolor revealed the existence of a cadmium-binding protein, the MPII, belonging to the group of hemerythrins and myohemerythrins. The presence of MPII in the cells of the intestine was demonstrated by immunocytochemistry, using anti-MPII, a monoclonal antibody. In addition, using in situ hybridization and northern blotting, it was shown that MPII-cells are the site of synthesis of this molecule. Exposure of the worms to cadmium led to the cellular activation process of MPII-cells (i.e. transformation of the nucleolus, development of the endoplasmic reticulum and the Golgi apparatus), although MPII mRNA transcript levels were unchanged. Enzyme-linked immunosorbent assay (ELISA) of gut extracts revealed that MPII levels were increased after exposure to Cd, so it appears that this protein is synthesized as a response to Cd exposure without any new synthesis of mRNA. This mechanism of regulation is quite similar to that reported in the case of mammalian ferritin and may be involved in the regulation of Cd levels in this worm.