Jérôme Silvestre

Lead-induced genotoxicity to Vicia faba L.roots in relation with metal cell uptake and initial speciation

Formation of organometallic complexes in soil solution strongly influence metals phytoavailability. However, only few studies deal with the influence of metal speciation both on plant uptake and genotoxicity. In the present study, Vicia faba seedlings were exposed for 6h in controlled hydroponic conditions to 5 μM of lead nitrate alone and chelated to varying degrees by different organic ligands. Ethylenediaminetetraacetic acid and citric acid were, respectively, chosen as models of humic substances and low weight organic acids present in natural soil solutions. Visual Minteq software was used to estimate free lead cations concentration and ultimately to design the experimental layout. For all experimental conditions, both micronucleus test and measure of lead uptake by plants were finally performed. Chelation of Pb by EDTA, a strong chelator, dose-dependently increased the uptake in V. faba roots while its genotoxicity was significantly reduced, suggesting a protective role of EDTA. A weak correlation was observed between total lead concentration absorbed by roots and genotoxicity (r(2)=0.65). In contrast, a strong relationship (r(2)=0.93) exists between Pb(2+) concentration in exposure media and genotoxicity in the experiment performed with EDTA. Citric acid induced labile organometallic complexes did not demonstrate any significant changes in lead genotoxicity or uptake. These results demonstrate that metal speciation knowledge could improve the interpretation of V. faba genotoxicity test performed to test soil quality.

A field study of lead phytoextraction by various scented Pelargonium cultivars.

Phytoremediation appears to be a promising technique for metal soil clean up, although its successful application on a large scale still remains a challenge. Field experiments for six scented Pelargonium cultivars, conducted on two Pb-contaminated calcareous and acidic soils, revealed vigorous plant growth, with no symptoms of morpho-phytotoxicity in spite of high Pb accumulation levels. Lead contents in the harvestable parts of all plants grown on the acidic and more contaminated soil were significantly higher than those grown on the calcareous soil. Three cultivars (Attar of Roses, Clorinda and Atomic Snowflake) are Pb-hyperaccumulator plants: they accumulated more than 1,000 mg Pb kg(-1)DW, with high biomass produced.

Environmentally relevant approaches to assess nanoparticles ecotoxicity: A review

Despite the increasing production and use of nanoparticles (NPs), there is a lack of knowledge about their environmental fate and ecotoxicity. Studies in environmentally relevant conditions are necessary to better assess these parameters, but such studies are rather rare. The present work represents first time that studies on engineered NPs using environmentally relevant exposure methods have been reviewed. These exposure methods differ from standardized protocols and can be classified into three groups: experimental trophic chains that allow study of the trophic route, multi-species exposures under laboratory conditions that allow for complex but controlled exposure and outdoor exposures that are more similar to environmentally realistic conditions. The majority of studies of micro- or mesocosms have focused on NP partitioning and bioaccumulation. The other major parameter that has been studied is NP ecotoxicity, which has been assessed in single species, in single species via the trophic route, and at the community level. The induction of biochemical defense systems, immunomodulation, effects on growth and reproduction, behavioral alterations and mortality have been used as indicators of major toxicity, depending on the species studied. The major effects of NPs on both microbial and algal communities include modifications of community compositions and diversities, decreased biomass and changes in community activities.

Sensitivity of freshwater periphytic diatoms to agricultural herbicides

The biomonitoring of pesticide pollution in streams and rivers using algae such as diatoms remains difficult. The responses of diatom communities to toxic stress in stream water are disturbed by the variations of environmental parameters. In this study, periphytic algae collected in situ were exposed under controlled conditions to two major herbicides used in French agriculture (isoproturon and s-metolachlor). Three exposure regimes were tested: 5 and 30 microg L(-1) for 6 days and 30 microg L(-1) for 3 days followed by a recovery period of 3 days. The algal biomasses were assessed from pigment concentrations (chlorophyll a and c) and from live cell density. The highest concentration (30 microg L(-1)) of isoproturon inhibited the biomass increase statistically significantly. In periphyton exposed to 5 and 30 microg L(-1) of s-metolachlor, chlorophyll c concentration and live cell density were also statistically significantly lower than in the control. Periphyton left to recover after reduced exposure duration (3 days) showed higher growth rates after treatment with s-metolachlor than with isoproturon. Taxonomic identifications showed that species like Melosira varians, Nitzschia dissipata and Cocconeis placentula were not affected by the herbicide exposure. Other species like Eolimna minima and Navicula reichardtiana were more sensitive. Studying diatoms according to their trophic mode showed that facultative heterotroph species were statistically significantly favoured by isoproturon exposure at the highest concentration. Results obtained with s-metolachlor exposure showed a disturbance of cell multiplication rather than that of photosynthesis. These results suggest that photosynthesis inhibitors like isoproturon favour species able to survive when the autotroph mode is inhibited.

Lead-induced DNA damage in Vicia faba root cells: Potential involvement of oxidative stress

Genotoxic effects of lead (0-20μM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10μM. In addition, at this concentration, DNA damage time-dependently increased until 12h. Then, a decrease in DNA damages was recorded. The significant induction of micronucleus formation also reinforced the genotoxic character of this metal. Direct interaction of lead with DNA was also evaluated with the a-cellular comet assay. The data showed that DNA breakages were not associated with a direct effect of lead on DNA. In order to investigate the relationship between lead genotoxicity and oxidative stress, V. faba were exposed to lead in the presence or absence of the antioxidant Vitamin E, or the NADPH-oxidase inhibitor dephenylene iodonium (DPI). The total inhibition of the genotoxic effects of lead (DNA breakage and micronucleus formation) by these compounds reveals the major role of reactive oxygen species (ROS) in the genotoxicity of lead. These results highlight, for the first time in vivo and in whole-plant roots, the relationship between ROS, DNA strand-breaks and chromosome aberrations induced by lead.

Herbicide effects on freshwater benthic diatoms: induction of nucleus alterations and silica cell wall abnormalities.

Benthic diatoms are well known bio-indicators of river pollution by nutrients (nitrogen and phosphorus). Biological indexes, based on diatom sensitivity for non-toxic pollution, have been developed to assess the water quality. Nevertheless, they are not reliable tools to detect pollution by pesticides. Many authors have suggested that toxic agents, like pesticides, induce abnormalities of the diatom cell wall (frustule). High abnormal frustule abundances have been reported in natural diatom communities sampled in streams contaminated by pesticides. However, no direct link was found between the abundances of abnormal frustules in these communities and the pesticide concentrations in stream water. In the present study, a freshwater benthic diatom community, isolated from natural biofilm and cultured under controlled conditions, was treated with a known genotoxic herbicide, maleic hydrazide (MH). Cells were exposed to three concentrations of MH (5x10(-6), 10(-6), 10(-7)M) for 6h followed by a 24h-recovery time. After MH treatments, nucleus alterations were observed: abnormal nucleus location, micronucleus, multinuclear cell or disruption of the nuclear membrane. A dose-dependent increase of nuclear alterations was observed. The difference between the control (9.65 nuclear alterations per 1000 cells observed (9.65 per thousand), S.D.=4.23) and the highest concentrations (29.40 per thousand, S.D.=8.49 for 10(-6)M and 35.96 per thousand, S.D.=3.71 for 5x10(-6)M) was statistically significant (Tukey test, P<0.05). Diatoms also exhibited frustules with deformed morphology and abnormal ornamentation. Significantly increased abundances of abnormal frustules were observed for the highest concentrations (10(-6) and 5x10(-6)M; Tukey test, P<0.05). These two parameters tended to increase together (Pearson correlation=0.702, P<0.05). The results suggest that the induction of abnormal frustules could be associated with the genotoxic effects of MH. The alterations observed could be related to the effects of MH on the synthesis of the proteins involved in frustule formation or in the regulation of the cytoskeleton of the diatom cells.

Assessment of the genotoxicity of Cu and Zn in raw and anaerobically digested slurry with the Vicia faba micronucleus test.

Genotoxicity of Cu and Zn was assessed by use of the micronucleus (MN) test on Vicia faba roots. Plants were exposed to various leachates of raw and anaerobically digested pig slurry, with maximum total concentrations of 200microM Cu and 600microM Zn. The results indicated stabilisation of the organic matter during anaerobic digestion of the slurry and bioconversion of some phytotoxic organic compounds (e.g. phenols or p-cresol), but did not show a relationship between Cu and Zn concentrations and MN frequency. Exposure of Vicia plants to binary inorganic solutions of Cu and Zn (CuSO(4)/ZnSO(4), 1:3) showed a significant micronucleus induction at concentrations of 40microM Cu and 120microM Zn and higher. When MN frequency was plotted against dissolved Cu (<0.45microm), applied as slurry or as CuSO(4), a single curve was obtained. At concentrations lower than 10microM, modulation of the genotoxic effect of Cu was found. At concentrations up to 150microM, MN induction increased significantly, while phytotoxic symptoms appeared at higher concentrations.

Toxicity of CeO2 nanoparticles at different trophic levels--effects on diatoms, chironomids and amphibians.

The aim of the present work is to provide wider information on the toxicity of cerium dioxide nanoparticles (CeO2 NPs) in aquatic environments, by studying the toxicity of two types of CeO2 NPs for four species (diatoms Nitzschia palea, the sediment-dwelling invertebrate Chironomus riparius, and the amphibian larvae Xenopus laevis and Pleurodeles waltl.). The two types of CeO2 NPs have different intrinsic properties: some of them are small citrate-coated spheres (2-5 nm), and the others are larger uncoated plates (20-60 nm). Acute toxicity (mortality at 48 or 96 h, depending on the test-organism) was assessed for the four species, from 0.1 to 100 mg L(-1) of NPs. Sub-lethal effects were assessed on chironomids exposed between 0.01 and 1 mg L(-1) of NPs. Mortality, growth inhibition and genotoxic effects were evaluated on amphibian larvae from 0.1 to 10 mg L(-1). Results reveal that no acute toxicity occurs on any species after short exposures, even at the highest concentrations. Mortality (35%) is observed on Xenopus larvae after 12d of exposure at the highest concentration of one type of NPs. No significant effects were observed on chironomids during chronic exposure. Xenopus larvae growth was inhibited from 1 mg L(-1) of both NPs while growth inhibition is observed on Pleurodeles only at the highest concentration of one type of NPs. No genotoxicity was observed on Xenopus but Pleurodeles exhibited dose-dependent genotoxic effects when exposed to one type of NPs. Observed differences in toxicity are discussed focusing on the studied compartment, routes of exposure, species and NPs.

Multi-walled carbon nanotubes, natural organic matter, and the benthic diatom Nitzschia palea: “A sticky story”

Abstract Different effects of multi-walled carbon nanotubes (MWCNTs) on the freshwater diatom Nitzschia palea were examined. MWCNTs used in this study (MWCNT) were dispersed either by sonication without (MWCNTsonicated) or with a realistic concentration (10 mg L−1) of Natural Organic Matter (MWCNT+NOM). A pocket-size device was designed to distinguish shading effect (using MWCNT suspensions as external filters) from total exposure effect of MWCNTsonicated and MWCNT+NOM on benthic algae. This study demonstrates that cell division was strongly inhibited after a 48 h exposure to MWCNT+NOM compared to MWCNTsonicated. This device did not yield a quantifiable contribution of shading to growth inhibition of MWCNTsonicated and below 10 mg L−1 of MWCNT+NOM. In all cases, neither lethal effects nor drops in photosynthetic quantum yield were observed. After a 6-d exposure, a complete growth recovery was observed for all conditions except at the highest concentration of MWCNT+NOM. Different microscopic approaches using carbohydrates markers revealed the strong affinity between MWCNT and extracellular polymeric substances (EPS) produced by N. palea. These seem to constitute a defensive mechanism against MWCNT.

DIRECT EFFECTS OF HUMIC-LIKE SUBSTANCE ON GROWTH, WATER, AND MINERAL NUTRITION OF VARIOUS SPECIES

The influence of humic-like substances was studied on various crop plants (cucumber, maize, pelargonium, and wheat, for example) cultivated under hydroponic conditions. The humic-like product, called SHB®, was extracted from poplar sawdust by a thermical and mechanical process and applied diluted into nutrient solutions of soilless culture at concentrations ranging between 100 to 400 mg SHB® L−1. The application of the humic-like substance induced a biostimulating effect on plant growth of each species studied. Treated plants present a faster development and reach reproductive stage three to five days earlier than control. SHB® also provoked a better efficiency of plant water uptake and they improved the mineral nutrition. It is assumed that most of direct effects on plant metabolisms are related to the low molecular size fractions (LMS) because it is the only part of SHB® which can penetrate into root tissues. But, it is also suggested that high molecular size fractions (HMS) could participate in plant water saving by slowing down its passage in roots.