Adverse effects of fly ashes used as immobilizing agents for highly metal-contaminated soils on Xenopus laevis oocytes survival and maturation-a study performed in the north of France with field soil extracts.

Amphibians are now recognized as the most endangered group. One of this decline causes is the degradation of their habitat through direct contamination of water, soil leaching, or runoff from surrounding contaminated soils and environments. In the North of France, the extensive industrial activities resulted in massive soil contamination by metal compounds. Mineral amendments were added to soils to decrease trace metal mobility. Because of the large areas to be treated, the use of inexpensive industrial by-products was favored. Two types of fly ashes were both tested in an experimental site with the plantation of trees in 2000. Aim of the present work was to investigate the effects of extracts from metal-contaminated soils treated or not for 10 years with fly ashes on Xenopus laevis oocyte using cell biology approaches. Indeed, our previous studies have shown that the Xenopus oocyte is a relevant model to study the metal ion toxicity. Survival and maturation of oocyte exposed to the soil extracts were evaluated by phenotypic approaches and electrophysiological recordings. An extract derived from a metal-contaminated soil treated for 10 years with sulfo-calcic ashes induced the largest effects. Membrane integrity appeared affected and ion fluxes in exposed oocytes were changed. Thus, it appeared that extracted elements from certain mineral amendments used to prevent the mobility of metals in the case of highly metal-contaminated soils could have a negative impact on X. laevis oocytes.

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.

Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage.

Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

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.

Xenopus laevis oocyte maturation is affected by metal chlorides.

Few studies have been conducted using Xenopus laevis germ cells as oocytes, though these cells offer many advantages allowing both electrophysiological studies and morphological examination. Our aim was to investigate the effects of metal (cadmium, lead, cobalt and zinc) exposures using cell biology approaches. First, cell survival was evaluated with both phenotypical and electrophysiological approaches. Secondly, the effect of metals on oocyte maturation was assessed with morphological observations and electrophysiological recordings. From survival experiments, our results showed that metal chlorides did not affect cell morphology but strongly depolarized X. laevis oocyte resting potential. In addition, cadmium chloride was able to inhibit progesterone-induced oocyte maturation. By contrast, zinc, but also to a lesser extent cadmium, cobalt and lead, were able to enhance spontaneous oocyte maturation in the absence of progesterone stimulation. Finally, electrophysiological recordings revealed that some metal chlorides (lead, cadmium) exposures could disturb calcium signaling in X. laevis oocyte by modifying calcium-activated chloride currents. Our results demonstrated the high sensitivity of X. laevis oocytes toward exogenous metals such as lead and cadmium. In addition, the cellular events recorded might have a predictive value of effects occurring later on the ability of oocytes to be fertilized. Together, these results suggest a potential use of this cellular lab model as a tool for ecotoxicological assessment of contaminated fresh waters.

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.

Growth and metal accumulation in Porcellio scaber exposed to poplar litter from Cd-, Pb-, and Zn-contaminated sites.

This study aimed at determining, in the laboratory, the effects of poplar litter collected in woody habitats contaminated by heavy metals on growth and metal accumulation in the terrestrial isopod Porcellio scaber. Cd, Pb, and Zn pseudototal and CaCl(2)-extractable concentrations in litter types were determined using AAS. Juveniles were fed ad libitum, individually, for 28 days with four litter types presenting an increasing gradient of metal contamination. Individuals were weighed every week and metal body burdens were determined at the end of the experiment. From the first week until the end of the experiment, a decrease in P. scaber growth related to the increase of metal concentration in litter types was recorded. Significant correlations were observed between metal body burdens and metal concentrations in litter types. However, Cd accumulation in woodlice appeared to be related to the Cd/Zn concentration ratio in litter types. All these results showed the potential of weight gain in P. scaber as a suitable indicator for litter quality assessment with ecological relevance.

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.

Metallic trace element body burdens and gene expression analysis of biomarker candidates in Eisenia fetida, using an "exposure/depuration" experimental scheme with field soils.

Smelting plant activities lead to the accumulation of Metal Trace Elements (MTEs) in soils. The presence of high concentrations of MTEs can generate an environmental stress likely to affect macroinvertebrates living in close soil contact such as the Annelida Oligochaeta. Eisenia fetida, an ecotoxicologically important test species, was successively exposed to two field soils: (1) a highly contaminated agricultural topsoil collected near the former smelter Metaleurop Nord (Noyelles-Godault, France) which contaminated surrounding soils by its atmospheric emissions [exposure phase], and then (2) a slightly contaminated topsoil from an urban garden located in the conurbation of Lille (Wambrechies) [depuration phase]. Two analyses were performed during each phase. Firstly, the gene expression levels of four biomarker candidates identified in previous studies were analyzed in E. fetida coelomocytes. These candidates are Cd-metallothionein, phytochelatin synthase, coactosin-like protein and lysenin. Secondly, the body burdens of the following elements Cd, Pb, Zn, Cu, Fe, Ca, and P were measured. Moreover, both analyses were also performed in Lumbricus rubellus, an Annelid species collected from the two tested soil-originating sites. Analysis of gene expression and MTE body burdens in both species are discussed to: (1) evaluate expression biomarkers; (2) gain insight the detoxification processes and the long-term response to a metallic stress and (3) compare the responses observed in a test species (E. fetida) with the responses of a field species (L. rubellus).

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.