Annette de Vaufleury

Spatial distribution of metals in smelter-impacted soils of woody habitats: Influence of landscape and soil properties, and risk for wildlife

The spatial distribution of total and CaCl(2)-extracted Cd, Pb and Zn concentrations in smelter-impacted soils was investigated over a polluted site (40 km(2)) in Northern France. The study was conducted on 262 soils sampled in woody habitats. Total and extracted concentrations of trace metals (TMs) rose up to 2402 and 59.5 mg kg(-1) for Cd, 41 960 and 13.7 mg kg(-1) for Pb, 38 760 and 143.0 mg kg(-1) for Zn, respectively. The spatial dependence of both total and extracted concentrations showed a high spatial auto-correlation and ordinary kriging was used to predict soil concentrations. Investigating which variables influenced metal concentrations and their spatial distribution, we found that total concentrations mostly depended on the distance from the smelter, the wind and the organic carbon, while extracted concentrations were mainly modulated by the pH. Conditionally to those variables, other soil properties and landscape characteristics influenced both total and extracted concentrations. We conclude that total and extracted TM concentrations are governed by different processes which act at various spatial ranges: total concentrations are mainly related to input and retention of metals (large scale) whereas extracted concentrations were mainly explained by factors controlling metal solubility in soils (local scale). Spatial distributions of total and extracted TMs differed over the area, which should be considered for risk assessment. Maps of risk based on the US EPAs Eco-SSLs (for plants, invertebrates and vertebrates) were realized, showing that wildlife may be at risk but that the relevance of Eco-SSL values is questionable.

Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail Helix aspersa (Gastropoda, Pulmonata)†

The aim of the present work was to study the effect of Cd2+ exposure on metallothionein (MT) induction and on the distribution of metals (Cd, Cu, and Zn) in the terrestrial pulmonate Helix aspersa. In particular, the soluble and nonsoluble pools of the accumulated metals and their tissue distribution in uncontaminated and contaminated edible snails were investigated after a two-week exposure to Cd2+. In the soluble cytosolic pool of the midgut gland of H. aspersa, three metal-specific putative MT isoforms were separated following a fractionation protocol with diethylaminoethyl cellulose, size-exclusion chromatography, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Interestingly, one of the above isoforms seems to bind both Cd and Cu, which may in addition mobilize, after induction by Cd2+, some of the intracellular Cu and, thus, perhaps increase the Cu pool in the cytosolic fraction. The cDNA and its translated amino acid sequence of a Cd2+-binding MT isoform from the snail midgut gland was characterized and attributed to one of the putative MT isoforms obtained by RP-HPLC. The amino acid sequence of this Cd-MT isoform of H. aspersa differed from similar sequences described in other terrestrial pulmonates, such as Helix pomatia or Arianta arbustorum, by only a few amino acids (n = 4 and 8, respectively). That the identified Cd-MT from H. aspersa is inducible by Cd2+ also was shown, chromatographic evidence aside, by a specific polymerase chain reaction protocol on a cDNA basis, which included a noninducible housekeeping gene as a control.

How terrestrial snails can be used in risk assessment of soils.

Among soil invertebrates, terrestrial snails are herbivorous and detritivorous organisms exposed to polluted soils by both digestive and cutaneous routes. Using laboratory-reared snails (Helix aspersa aspersa), we describe how the effects of contaminants on survival and growth of snails can be evaluated in laboratory bioassays. A national ring test was performed to assess the effect of Cd added to the soil or to the food. The ecotoxicity of sewage sludge also was evaluated. The present results demonstrate that toxicity depends on both the pollutants and the exposure route. Cadmium was sixfold more toxic for snails exposed via food contamination (median effective concentration [EC50], 68-139 microg/g) than via soil contamination (EC50, 534-877 microg/g), whereas the opposite occurred with the sewage sludge (EC50, 55% of sludge in the food and 10% of waste in the soil). A logistic relationship linked growth inhibition and internal Cd concentrations, which can reach 2,000 microg/g in the viscera of snails exposed to 626 microg/g in the food. No clear trend was found between Cu, Zn, Pb, Cr, and Ni concentrations in the sludge and in snail tissues. These data enabled the development of an international standard, which should enhance the use of terrestrial gastropods for both fundamental research and routine risk assessment in the terrestrial environment.

Responses of wild small mammals to a pollution gradient: host factors influence metal and metallothionein levels.

We investigated how host factors (species, age, gender) modulated Cd, Pb, Zn, and Cu concentrations, metallothionein levels (MTs) and their relationships in 7 sympatric small mammal species along a pollution gradient. Cd concentrations in liver and kidneys increased with age in all species. Age effect on other metals and MTs differs among species. Gender did not influence metal and MT levels except in the bank vole. Three patterns linking internal metal concentrations and MTs were observed along the gradient: a low metal accumulation with a (i) high (wood mouse) or (ii) low (bank vole) level of MTs accompanied by a slight or no increase of MTs with Cd accumulation; (iii) an elevated metal accumulation with a sharp increase of MTs (common and pygmy shrews). In risk assessment and biomonitoring perspectives, we conclude that measurements of MTs and metals might be associated because they cannot be interpreted properly when considered separately.

How subcellular partitioning can help to understand heavy metal accumulation and elimination kinetics in snails

To understand bioaccumulation kinetics of metals within biota inhabiting industrially contaminated soils, toxicokinetic dynamics and subcellular fractionation were carried out with the terrestrial snail Helix aspersa in a long-term (six-month) laboratory experiment. Accumulation and elimination kinetics were determined for Cd, Pb, and Zn in both viscera and foot of snails and were described accurately by one-compartment models. The subcellular fractions were obtained by sequential centrifugations and were analyzed by isolating metal-rich granules, tissue fragments, and cytosolic fractions. Different fractions showed metal-specific binding capacities that might be useful in identifying the biological significance of accumulated metal levels in snails. Cadmium was retrieved mainly from the cytosolic fraction, where it was stored in the long term and not excreted, thus explaining the linear accumulation patterns. Most of the accumulated Pb was found in the granular fraction, and snails appeared able to excrete these concretions, leading to achievement of a steady state in internal Pb body burdens. Significant levels of Pb, however, were retrieved at the end of the depuration phase and retained in the cell debris fraction. Zinc showed affinities for both cytosolic and granular fractions, leading to intermediate uptake and excretion patterns. The dynamics of the different sequestration forms at the subcellular level support the observed kinetics of metal body burdens and, in association with the determination of uptake fluxes, allow precise assessment of metal accumulation in snails.

Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus

Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C.aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80–90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30–50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

Transfer of Cd, Cu, Ni, Pb, and Zn in a soil-plant-invertebrate food chain: a microcosm study.

The transfer of Cd, Cu, Ni, Pb, and Zn was evaluated in a soil-plant (lettuce, Lactuca sativa)-invertebrate (snail, Helix aspersa) food chain during a microcosm experiment. Two agricultural soils, polluted and unpolluted, were studied. Lettuce was cultivated for eight weeks before introduction of snails into the microcosms (M-snails). In a parallel experiment, snails were exposed to lettuce only (i.e., without soil) in simpler exposure devices called containers (C-snails). Snail exposure duration was eight weeks for both M- and C-snails. No effects on snail survival were found. Both M- and C-snails exposed to polluted soil showed a growth reduction, but only after two weeks of exposure. Time-dependent accumulation in M-snails exposed to the polluted environment showed a regular increase of Cd and Zn concentrations over time and a rapid increase of Pb concentrations within the first two weeks, which then remained stable. Copper and Ni concentrations did not increase during any of the experiments. Concentrations in M- and C-snails were compared to estimate the relative contribution of soil and plant to the total bioaccumulation. The results suggest that the soil contribution may be higher than 80% for Pb, from 30 to 60% for Zn, and from 2 to 40% for Cd.

Spatially Explicit Analysis of Metal Transfer to Biota: Influence of Soil Contamination and Landscape

Concepts and developments for a new field in ecotoxicology, referred to as “landscape ecotoxicology,” were proposed in the 1990s; however, to date, few studies have been developed in this emergent field. In fact, there is a strong interest in developing this area, both for renewing the concepts and tools used in ecotoxicology as well as for responding to practical issues, such as risk assessment. The aim of this study was to investigate the spatial heterogeneity of metal bioaccumulation in animals in order to identify the role of spatially explicit factors, such as landscape as well as total and extractable metal concentrations in soils. Over a smelter-impacted area, we studied the accumulation of trace metals (TMs: Cd, Pb and Zn) in invertebrates (the grove snail Cepaea sp and the glass snail Oxychilus draparnaudi) and vertebrates (the bank vole Myodes glareolus and the greater white-toothed shrew Crocidura russula). Total and CaCl2-extractable concentrations of TMs were measured in soils from woody patches where the animals were captured. TM concentrations in animals exhibited a high spatial heterogeneity. They increased with soil pollution and were better explained by total rather than CaCl2-extractable TM concentrations, except in Cepaea sp. TM levels in animals and their variations along the pollution gradient were modulated by the landscape, and this influence was species and metal specific. Median soil metal concentrations (predicted by universal kriging) were calculated in buffers of increasing size and were related to bioaccumulation. The spatial scale at which TM concentrations in animals and soils showed the strongest correlations varied between metals, species and landscapes. The potential underlying mechanisms of landscape influence (community functioning, behaviour, etc.) are discussed. Present results highlight the need for the further development of landscape ecotoxicology and multi-scale approaches, which would enhance our understanding of pollutant transfer and effects in ecosystems.

Increased response to cadmium and Bacillus thuringiensis maize toxicity in the snail Helix aspersa infected by the nematode Phasmarhabditis hermaphrodita

To determine the effect of nematode infection on the response of snails to selected toxins, we infected Helix aspersa with 0-, 0.25-, 1-, or 4-fold the recommended field dose of a commercial nematode application for agricultural use. In the first experiment, the snails also were exposed to cadmium via food and soil at concentrations of 0, 30, 60, 120, or 240 mg/kg in a full-factorial design. In the second experiment, snails were infected with nematodes and also fed either Bt (expressing Bacillus thuringiensis toxin) maize or non-Bt maize. The snails were weighed at the beginning and end (after four weeks) of the experiments, and mortality was checked daily. Neither exposure of snails to nematodes nor exposure of snails to cadmium or Bt toxin affected the survival rates of snails. The number of dead snails was highest for combinations of nematode treatments with cadmium concentrations of 120 and 240 mg/kg. In both experiments (Bt and cadmium), the growth rate decreased with increasing nematode dose. The Bt maize was not harmful to the snails in the absence of nematodes, but infected snails grew faster when fed non-Bt maize. The growth rate of snails exposed to cadmium decreased with exposure to increasing Cd concentrations and differed significantly between the no-nematode treatment and the treatments with nematode doses of one- and fourfold the recommended field dose. Snails treated with the highest dose of nematodes accumulated the highest cadmium concentrations.

Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene.