Impact of cadmium on forage kale (Brassica oleracea var. viridis cv "Prover") after 3-,10- and 56-day exposure to a Cd-spiked field soil.

Cadmium (Cd) is a highly toxic element for living organisms and is widespread in metal-contaminated soils. As organisms which can grow up on these polluted areas, plants have some protection mechanisms against Cd issues. Among the plant kingdom, the Brassicaceae family includes species which are known to be able to tolerate and accumulate Cd in their tissues. In this study, Brassica oleracea var. viridis cv "Prover" was exposed to a range of artificially Cd-contaminated soils (from 2.5 up to 20 mg kg-1) during 3, 10, and 56 days and the effects on life traits, photosynthesis activity, antioxidant enzymatic activities were studied. Metal accumulation was quantified, as well as DNA damage, by means of the comet assay and immunodetection of 8-OHdG levels. Globally, B. oleracea was relatively tolerant to those Cd exposures. However, comet assay and detection of 8-OHdG revealed some DNA damage but which are not significant. According to metal accumulation analysis, B. oleracea var. viridis cv Prover could be a good candidate for alternative growing in contaminated areas.

Accumulation, speciation and localization of silver nanoparticles in the earthworm Eisenia fetida.

The use of silver nanoparticles (AgNPs) in agriculture and many consumer products has led to a significant release of Ag in the environment. Although Ag toxicity in terrestrial organisms has been studied extensively, very little is known about the accumulation capacity and coping mechanisms of organisms in Ag-contaminated soil. In this context, we exposed Eisenia fetida earthworms to artificial OECD soil spiked with a range of concentrations of Ag (AgNPs or AgNO3). The main aims were to (1) identify the location and form of accumulation of Ag in the exposed earthworms and (2) better understand the physiological mechanisms involved in Ag detoxification. The results showed that similar doses of AgNPs or AgNO3 did not have the same effect on E. fetida survival. The two forms of Ag added to soil exhibited substantial differences in speciation at the end of exposure, but the Ag speciation and content of Ag in earthworms were similar, suggesting that biotransformation of Ag occurred. Finally, 3D images of intact earthworms obtained by X-ray micro-computed tomography revealed that Ag accumulated preferentially in the chloragogen tissue, coelomocytes, and nephridial epithelium. Thus, E. fetida bioaccumulates Ag, but a regulation mechanism limits its impact in a very efficient manner. The location of Ag in the organism, the competition between Ag and Cu, and the speciation of internal Ag suggest a link between Ag and the thiol-rich proteins that are widely present in these tissues, most probably metallothioneins, which are key proteins in the sequestration and detoxification of metals.

Medium-term effects of Ag supplied directly or via sewage sludge to an agricultural soil on Eisenia fetida earthworm and soil microbial communities.

The widespread use of silver nanoparticles (AgNPs) in consumer products that release Ag throughout their life cycle has raised potential environmental concerns. AgNPs primarily accumulate in soil through the spreading of sewage sludge (SS). In this study, the effects of direct exposure to AgNPs or indirect exposure via SS contaminated with AgNPs on the earthworm Eisenia fetida and soil microbial communities were compared, through 3 scenarios offering increasing exposure concentrations. The effects of Ag speciation were analyzed by spiking SS with AgNPs or AgNO3 before application to soil. SS treatment strongly impacted Ag speciation due to the formation of Ag2S species that remained sulfided after mixing in the soil. The life traits and expression of lysenin, superoxide dismutase, cd-metallothionein genes in earthworms were not impacted by Ag after 5 weeks of exposure, but direct exposure to Ag without SS led to bioaccumulation of Ag, suggesting transfer in the food chain. Ag exposure led to a decrease in potential carbon respiration only when directly added to the soil. The addition of SS had a greater effect on soil microbial diversity than the form of Ag, and the formation of Ag sulfides in SS reduced the impact of AgNPs on E. fetida and soil microorganisms compared with direct addition.

Effects of glyphosate and a commercial formulation Roundup® exposures on maturation of Xenopus laevis oocytes.

Pesticides are often found at high concentrations in small ponds near agricultural field where amphibians are used to live and reproduce. Even if there are many studies on the impacts of phytopharmaceutical active ingredients in amphibian toxicology, only a few are interested in the earlier steps of their life cycle. While their populations are highly threatened with extinction. The aim of this work is to characterize the effects of glyphosate and its commercial formulation Roundup® GT Max on the Xenopus laevis oocyte maturation which is an essential preparation for the laying and the fertilization. Glyphosate is an extensively used herbicide, not only known for its effectiveness but also for its indirect impacts on non-target organisms. Our results showed that exposures to both forms of glyphosate delayed this hormone-dependent process and were responsible for spontaneous maturation. Severe and particular morphogenesis abnormalities of the meiotic spindle were also observed. The MAPK pathway and the MPF did not seem to be affected by exposures. The xenopus oocyte is particularly affected by the exposures and appears as a relevant model for assessing the effects of environmental contamination.

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.

Ecotoxicology of silver nanoparticles and their derivatives introduced in soil with or without sewage sludge: A review of effects on microorganisms, plants and animals.

Silver nanoparticles (AgNPs) are widely incorporated in many products, partly due to their antimicrobial properties. The subsequent discharge of this form of silver into wastewater leads to an accumulation of silver species (AgNPs and derivatives resulting from their chemical transformation), in sewage sludge. As a result of the land application of sewage sludge for agricultural or remediation purposes, soils are the primary receiver media of silver contamination. Research on the long-term impact of AgNPs on the environment is ongoing, and this paper is the first review that summarizes the existing state of scientific knowledge on the potential impact of silver species introduced into the soil via sewage sludge, from microorganisms to earthworms and plants. Silver species can easily enter cells through biological membranes and affect the physiology of organisms, resulting in toxic effects. In soils, exposure to AgNPs may change microbial biomass and diversity, decrease plant growth and inhibit soil invertebrate reproduction. Physiological, biochemical and molecular effects have been documented in various soil organisms and microorganisms. Negative effects on organisms of the dominant form of silver in sewage sludge, silver sulfide (Ag2S), have been observed, although these effects are attenuated compared to the effects of metallic AgNPs. However, silver toxicity is complex to evaluate and much remains unknown about the ecotoxicology of silver species in soils, especially with respect to the possibility of transfer along the trophic chain via accumulation in plant and animal tissues. Critical points related to the hazards associated with the presence of silver species in the environment are described, and important issues concerning the ecotoxicity of sewage sludge applied to soil are discussed to highlight gaps in existing scientific knowledge and essential research directions for improving risk assessment.

Exposures to chemical contaminants: What can we learn from reproduction and development endpoints in the amphibian toxicology literature?

Environmental contamination is one of the major factors or cofactors affecting amphibian populations. Since 2000, the number of studies conducted in laboratory conditions to understand impacts of chemical exposures increased. They aimed to characterize biological effects on amphibians. This review proposes an overview of biological responses reported after exposures to metals, phytopharmaceuticals or emerging organic contaminants and focuses on endpoints relating to reproduction and development. Due to amphibian peculiar features, these periods of their life cycle are especially critical to pollutant exposures. Despite the large range of tested compounds, the same model species are often used as biological models and morphological alterations are the most studied observations. From the results, the laboratory-to-field extrapolation remained uneasy and exposure designs have to be more elaborated to be closer to environmental conditions. Few studies proposed such experimental approaches. Lastly, gametes, embryos and larvae constitute key stages of amphibian life cycle that can be harmed by exposures to freshwater pollutants. Specific efforts have to be intensified on the earliest stages and notably germ cells.

Ferroquine, the next generation antimalarial drug, has antitumor activity.

Despite the tremendous progress in medicine, cancer remains one of the most serious global health problems awaiting new effective therapies. Here we present ferroquine (FQ), the next generation antimalarial drug, as a promising candidate for repositioning as cancer therapeutics. We report that FQ potently inhibits autophagy, perturbs lysosomal function and impairs prostate tumor growth in vivo. We demonstrate that FQ negatively regulates Akt kinase and hypoxia-inducible factor-1α (HIF-1α) and is particularly effective in starved and hypoxic conditions frequently observed in advanced solid cancers. FQ enhances the anticancer activity of several chemotherapeutics suggesting its potential application as an adjuvant to existing anticancer therapy. Alike its parent compound chloroquine (CQ), FQ accumulates within and deacidifies lysosomes. Further, FQ induces lysosomal membrane permeabilization, mitochondrial depolarization and caspase-independent cancer cell death. Overall, our work identifies ferroquine as a promising new drug with a potent anticancer activity.

Maturation of Xenopus laevis oocytes under cadmium and lead exposures: Cell biology investigations.

Since amphibians are recognised as good models to assess the quality of environments, only few studies have dealt with the impacts of chemical contaminants on their gametes, while toxic effects at this stage will alter all the next steps of their life cycle. Therefore, we propose to investigate the oocyte maturation of Xenopus laevis in cadmium- and lead-contaminated conditions. The impacts of cadmium and lead ions were explored on events involved in the hormone-dependent process of maturation. In time-course experiments, cadmium, at the highest concentration, delayed and prevented the germinal vesicle breakdown. Even in the absence of progesterone this ion could also induce it. No such spontaneous maturation was observed after lead exposures. An acceleration of the process at the highest tested concentration of lead (90µM), in presence of progesterone, was recorded. Cytological observations highlighted that cadmium exposures drove severe disturbances of meiotic spindle morphogenesis. At last, cadmium exposures altered the MAPK pathway, regarding the activation of ERK2 and RSK, but also the activation and the activity of the MPF, by disturbing the state of phosphorylation of Cdc2 and histone H3. Xenopus laevis oocytes were affected by these metal ion exposures, notably by Cd2+. Signatures of these metal exposures on the oocyte maturation were detected. This germ cell appeared to be a relevant model to assess the effects of environmental contaminants such as metals.

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.

Mycosubtilin and surfactin are efficient, low ecotoxicity molecules for the biocontrol of lettuce downy mildew.

The use of surfactin and mycosubtilin as an eco-friendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50 % response by the exposed test organisms) was about 100 mg L(-1) in Microtox assays and 6 mg L(-1) in Daphnia magna immobilization tests for mycosubtilin and 125 mg L(-1) and 25 mg L(-1) for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100 mg L(-1) of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/surfactin (50:50 mg L(-1)) gave the same result on B. lactucae development as 100 mg L(-1) of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin.

Gene expression analysis of 4 biomarker candidates in Eisenia fetida exposed to an environmental metallic trace elements gradient: a microcosm study.

Past activities of 2 smelters (Metaleurop Nord and Nyrstar) led to the accumulation of high amounts of Metal Trace Elements (TEs) in top soils of the Noyelles-Godault/Auby area, Northern France. Earthworms were exposed to polluted soils collected in this area to study and better understand the physiological changes, the mechanisms of acclimation, and detoxification resulting from TE exposure. Previously we have cloned and transcriptionally characterized potential biomarkers from immune cells of the ecotoxicologically important earthworm species Eisenia fetida exposed in vivo to TE-spiked standard soils. In the present study, analysis of expression kinetics of four candidate indicator genes (Cadmium-metallothionein, coactosin like protein, phytochelatin synthase and lysenin) was performed in E. fetida after microcosm exposures to natural soils exhibiting an environmental cadmium (Cd) gradient in a kinetic manner. TE body burdens were also measured. This microcosm study provided insights into: (1) the ability of the 4 tested genes to serve as expression biomarkers, (2) detoxification processes through the expression analysis of selected genes, and (3) influence of land uses on the response of potential biomarkers (gene expression or TE uptake).

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.

Improved single-cell gel electrophoresis assay for detecting DNA damage in Eisenia foetida.

The earthworm (Eisenia foetida) is an attractive sentinel species for detecting genotoxicity in soil. In this study, an improved single-cell gel electrophoresis (SCGE) assay was developed for detecting DNA damage in the coelomocytes (lymphocytes) of earthworms. Coelomocytes were obtained from the coelomic fluid using a modified extrusion medium that did not include the mucolytic agent guaiacol. The extruded coelomocytes contained at least three types of cells: eleocytes (75% of the total), amoebocytes, and granulocytes. The DNA migration parameters were determined for untreated cells of each type in order that the assay could be performed with minimum inter- and intra-individual variation. In addition, lysis time was reduced to 10 min, and only one neutralization step was used. DNA damage was detected in isolated eleocytes treated with hydrogen peroxide and cadmium, and in eleocytes from earthworms exposed for up to 21 days to soil containing polycyclic aromatic hydrocarbons. The SCGE assay using earthworm eleocytes appears to be a sensitive biomarker for evaluating exposure to genotoxic compounds.