The effects of polystyrene microparticles on the environmental availability and bioavailability of As, Cd and Hg in soil for the land snail Cantareus aspersus.

The combined contamination of terrestrial environments by metal(loid)s (MEs) and microplastics (MPs) is a major environmental issue. Once MPs enter soils, they can interact with MEs and modify their environmental availability, environmental bioavailability, and potential toxic effects on biota. Although research efforts have been made to describe the underlying mechanisms driving MP and ME interactions, the effects of MPs on ME bioavailability in terrestrial Mollusca have not yet been documented. To fill this gap, we exposed the terrestrial snail Cantareus aspersus to different combinations of polystyrene (PS) and arsenic (As), cadmium (Cd), or mercury (Hg) concentrations. Using kinetic approaches, we then assessed the variations in the environmental availability of As, Cd or Hg after three weeks of equilibration and in the environmental bioavailability of As, Cd or Hg to snails after four weeks of exposure. We showed that while environmental availability was influenced by the total ME concentration, the effects of PS were limited. Although an increase in As availability was observed for the highest exposure concentrations at the beginning of the experiment, the soil ageing processes led to rapid adsorption in the soil regardless of the PS particle concentration. Concerning transfers to snail, ME bioaccumulation was ME concentration-dependent but not modified by the PS concentration in the soils. Nevertheless, the kinetic approaches evidenced an increase in As (2- to 2.6-fold) and Cd (1.6-fold), but not Hg, environmental bioavailability or excretion (2.3- to 3.6-fold for As, 1.8-fold for Cd) at low PS concentrations. However, these impacts were no longer observable at the highest PS exposure concentrations because of the increase in the bioaccessibility of MEs in the snail digestive tract. The generalization of such hormetic responses and the identification of the precise mechanisms involved necessitate further research to deepen our understanding of the MP-mediated behaviour of MEs in co-occurring scenarios.

Ecogenotoxicity assessment with land snails: A mini-review.

In the context of the increasing environmental and sanitary crisis, it is accepted that soil pollution can cause health alterations and disturb natural population dynamics. Consequently, the assessment of the genotoxic potential of compounds found in contaminated soils is important. Indeed, the alteration of genomic integrity may increase the risk of cancer development and may impair reproduction and long-term population dynamics. Among the methodologies to assess terrestrial genotoxic potential, there has been growing interest during the last decade in monitoring alterations of the genome in bioindicators of soil quality. As some land snail species are recognized bioindicators of soil quality, especially to assess the environmental and toxicological bioavailability of compounds, this review focuses on current knowledge regarding the genotoxicology of land snails. Classical biomarkers to assess genotoxic effects have been used (e.g., DNA breakage, micronuclei, random amplification polymorphic DNA) at various stages of the life cycle, including embryos. The studies were performed in vitro, in vivo, in situ and ex situ and covered a diverse set of contaminants (nanoparticles, metal(loid)s, pesticides, polycyclic aromatic hydrocarbons) and snail species (Cantareus aspersus, Eobania vermiculata, Theba pisana, Helix lucorum). Based on recent studies reviewed here, the use of land snails to map soil genotoxic potential is promising due to their ability to reveal pollution and subsequent environmental risks. Moreover, the position of snails in the trophic chain and the existing bridges between contaminant bioavailability to snails and bioaccessibility to humans reinforce the value of land snail-based ecotoxicological assessment.

In situ and ex situ bioassays with Cantareus aspersus for environmental risk assessment of metal(loid) and PAH-contaminated soils.

Environmental risk assessment of contaminated soils requires bioindicators that allow the assessment of bioavailability and toxicity of chemicals. Although many bioassays can determine the ecotoxicity of soil samples in the laboratory, few are available and standardized for on-site application. Bioassays based on specific threshold values that assess the in situ and ex situ bioavailability and risk of metal(loid)s and polycyclic aromatic hydrocarbons (PAHs) in soils to the land snail Cantareus aspersus have never been simultaneously applied to the same soils. The aims of this study were to compare the results provided by in situ and ex situ bioassays and to determine their respective importance for environmental risk assessment. The feasibility and reproducibility of the in situ bioassay were assessed using an international ring test. This study used five plots located at a former industrial site and six laboratories participated in the ring test. The results revealed the impact of environmental parameters on the bioavailability of metal(loid)s and PAHs to snails exposed in the field to structured soils and vegetation compared to those exposed under laboratory conditions to soil collected from the same field site (excavated soils). The risk coefficients were generally higher ex situ than in situ, with some exceptions (mainly due to Cd and Mo), which might be explained by the in situ contribution of plants and humus layer as sources of exposure of snails to contaminants and by climatic parameters. The ring test showed good agreement among laboratories, which determined the same levels of risk in most of the plots. Comparison of the bioavailability to land snails and the subsequent risk estimated in situ or ex situ highlighted the complementarity between both approaches in the environmental risk assessment of contaminated soils, namely, to guide decisions on the fate and future use of the sites (e.g., excavation, embankments, and land restoration). Integr Environ Assess Manag 2022;18:539-554. © 2021 SETAC.

Polyethylene microplastic toxicity to the terrestrial snail Cantareus aspersus: size matters.

Plastic has become the most widespread human-made material and small fragments (< 5mm, so called microplastics, MPs) accumulate in all the ecosystems. It is now admitted that the terrestrial environment represents an important sink for MPs and it has only recently become the focus of research, notably in ecotoxicology. In spite of a growing body of evidence regarding the potential effects of MPs on soil biota, more efforts are needed to address issues in this field. The aim of our study was to measure, at different levels of biological organization, the responses of Cantareus aspersus snail to low-density polyethylene (LDPE) particles dispersed in their food. Juvenile snails were exposed to a range of LDPE concentrations (10, 25, and 50% v/v) and sizes (median particle size (d50) of 120, 292, 340, and 560 µm). This study showed no snail feeding avoidance toward LDPE. The ingestion and digestion processes along the snail digestive tract did not lead to a measurable fragmentation of the MP particles. At the individual scale, big sized particles improved growth at the lowest exposure concentration tested, whereas at the molecular level, only small sized particles triggered oxidative stress but without causing quantifiable cyto- or genotoxic effects. The underlying mechanisms remain to be elucidated which strengthens the necessity to improve our knowledge on the effects of MPs on various biological models to better evaluate their environmental risks in terrestrial environments.

From environmental bioavailability of metal(loid)s to their ecogenotoxicological effects in land snails.

To date, no study has linked the environmental and the ecogenotoxicological bioavailability of contaminants to land snails. Yet, understanding the specific ecotoxicological mechanisms from bioaccumulation to genotoxicity is necessary e.g., to build an adverse outcome pathway relevant to risk assessment. Consequently, the aim of our study is to look for relationships between accumulated concentrations of As, Cd and Hg in sub-adult snails and ecotoxicological effects at the individual (survival and growth) and molecular (genomic stability) levels. This study combines random amplified polymorphic DNA (RAPD) coupled with high-resolution capillary electrophoresis system (HRS) and micronucleus (MN) assay on haemocytes to consider various types of cytogenomic damage, such as chromosomal aberrations, breakages, adducts and mutations. The results showed alteration of the individual endpoints at higher accumulation quotients (AQs) that reflect the excess of transfers to snails, especially with decreased survival for As. In addition, genotoxic effects were observed with an increased occurrence of MN in haemocytes for the three meta(loid)s considered (R2 from 0.57 to 0.61 as a function of the meta(loid)s). No concentration-dependent decrease in genome stability was highlighted by RAPD-HRS in snails exposed to As and Cd but not Hg. Our results demonstrate the complementarity of the RAPD-HRS and the MN assay for understanding the different genotoxic mechanisms of the three metal(loid)s studied in land snails. They show a way to better assess environmental risks of contaminated soils by associating ecotoxicity, genotoxicity and bioaccumulation assays (ISO 24032), i.e., ecogenotoxicological bioavailability. Convergences highlighted here between the bioaccumulation of metal(loid)s in viscera and genotoxic effects in haemocytes constitute a way to better assess the bioavailability of contaminants in soils to the land snail and the subsequent environmental risk.

Origin, fate and ecotoxicity of manganese from legacy metallurgical wastes.

Over the course of history, mining and metallurgical activities have influenced the socioeconomic development of human populations. However, these past and current activities can also lead to substantial environmental contamination by various metals. Here, we used an interdisciplinary approach (incorporating archaeology, mineralogy, environmental chemistry and ecotoxicology) to investigate the origin, fate and potential ecotoxicity of anomalous manganese (Mn) concentrations detected in the ancient mining district of Berthelange (medieval period, eastern France). Mineralogical investigations of slag samples showed that smelting temperature conditions in medieval bloomeries led to the production of slags mainly composed of Fe- and Mn-rich olivine, i.e., fayalites. Further mineralogical analyses of bulk soil and clay fractions allowed us to identify the presence of serpentine. This evidence of olivine weathering can account for the release of Mn from slags into the soil. In addition, chemical analyses of total and available (exchangeable and reducible) Mn concentrations in soil samples clearly showed the contribution of slags deposited 1000 years ago to soil contamination. A complementary ecotoxicity bioassay performed on soils from a slag heap using the land snail Cantareus aspersus confirmed that a significant fraction of the Mn detected in soils remains available for partitioning with the soil solution and transfer to soil organisms. Although no growth inhibition of snails was observed after 28 days of exposure, the animals accumulated quite elevated Mn concentrations in their tissues. Our study emphasizes the environmental availability and bioavailability of Mn from ancient metallurgical wastes to soil-dwelling invertebrates, i.e., snails, even one millennium after their deposition. Hence, as for more recent industrial sites, past mining ecosystems must be a cause of concern for the scientific community and public authorities.

Transfer of sulfidized silver from silver nanoparticles, in sewage sludge, to plants and primary consumers in agricultural soil environment.

Consumer products containing silver nanoparticles (AgNPs) release silver (Ag) to the environment, particularly wastewater. Sewage sludge (SS), which contains numerous contaminants including Ag, is recycled by spreading on agricultural land. Although slight impacts and bioaccumulation of Ag sulfide (Ag2S, the main species found in SS) in terrestrial organisms have been demonstrated, possible trophic transfer into plants and subsequently animal species has not been examined. Accordingly, the present study experimentally measured the transfer of Ag from AgNPs and sulfidized Ag into plants and primary consumers and compared their bioavailability. Nine plant cultivars were grown in soil mixed with SS containing Ag, which revealed that bioaccumulation of Ag by plants is species-dependent. Ryegrass (the plant species with the greatest accumulation - up to 0.2 mg kg-1) was then cultivated on a larger scale to expose snails and locusts for several weeks. While locusts did not accumulate Ag after two weeks of exposure, snails exhibited Ag bioaccumulation after 5 weeks when soil was accessible. Sulfidized Ag derived from AgNPs were less available (bioaccumulation up to 2.5 mg kg-1) than the Ag from the original AgNPs (bioaccumulation up to 15 mg kg-1). This transfer potential of Ag could have consequences for food webs due to chronic exposure linked to SS spreading practices. This study shows that transformations of AgNPs in treatment plants attenuate but do not completely eliminate the risk of Ag to plant and animal species SS.

From bioavailability to risk assessment of polluted soil using snails: link between excess transfer and inhibition of sexual maturation.

An accurate assessment of the environmental risk of soils contaminated by metal(loid)s (MEs) requires quantifying exposure and knowing the toxicity of contaminants transferred to biota. For this purpose, two indices have been developed with the bioindicator Cantareus aspersus to assess exposure (SET: sum of the excess of transfer) and risk (ERITME: evaluation of the risk of the transferred metal elements) of multi-contaminated soils. If the SET and ERITME indices allow characterization of exposure and risk based on unspecific toxicity points, then the link between these indices and real effects on some toxicological endpoints, such as growth or sexual maturation, remains to be demonstrated. For this purpose, sub-adult snails were exposed for 28 days to 38 ME-contaminated soils. Relationships between the SET and/or ERITME indices and health alterations in C. aspersus were determined using Spearman correlations, linear regressions, univariate regression trees, and kinetic models. Relationships were determined between the values of the SET and ERITME indices, bioaccumulation as an indicator of ME bioavailability, and the alteration in physiological endpoints, such as the shell development used as a non-invasive indicator of sexual maturation. The results enabled the determination of three levels of risk according to the differences in reaching sexual maturity: no risk, uncertain, and proven risk depended on whether the value of ERITME was below, in, or beyond the interval [2574-22720], respectively. This study provides the first benchmarks with the SET and ERITME indices to interpret the risk of contaminated soils to snails and to relate the environmental and toxicological bioavailability of ME mixtures.

Impact of ageing and soil contaminants on telomere length in the land snail.

Telomeres (TLs) are non-coding DNA sequences that are usually shortened with ageing and/or chemical exposure. Bioindicators such as the land snail can be used to assess the environmental risk of contaminated soils. As for most invertebrates, the evolution of TLs with ageing or exposure to contaminants is unknown in this mollusc. The aims of this study were to explore the relationships between ageing, contaminant exposure, sublethal effects and TL length in the terrestrial gastropod Cantareus aspersus. TL length was investigated in haemocytes from five age classes of C. aspersus. The impact of contaminants on sub-adult snails exposed to Cd, Hg or a mixture of polycyclic aromatic hydrocarbons (PAHs) in soils for one or two months was studied. Bioaccumulation, growth, sexual maturity and TLs were measured. TL attrition was significant for the juvenile and sub-adult stages, but not later. Exposure to Cd increased the mortality (around 30%). Exposure to polluted soils inhibited growth (19-40%) and sexual maturity (6-100%). Although the health of the snails exposed to Cd, Hg and PAHs was altered, TL length in haemocytes was not disturbed, suggesting a high capacity of this snail species to maintain its TLs in haemocytes under chemical stress. These results first address TL length in snails and reveal that the relationship commonly proposed for vertebrates between TL shortening and ageing or exposure to contaminants cannot be generalized.

Urban soil phytomanagement for Zn and Cd in situ removal, greening, and Zn-rich biomass production taking care of snail exposure.

The phytoextraction potential of Arabidopsis halleri (L.) O'Kane & Al Shehbaz and Salix viminalis L. to partially remove Zn and Cd in soil was investigated. In an urban field site, a very short rotation coppice of willows was implemented, and growth parameters were monitored for 3 years. A. halleri was cultivated in the same site with or without fertilizer to improve biomass yield and/or Zn and Cd aerial part concentrations. Effects of harvest and co-cultivation on these two parameters were measured. To determine if willows and A. halleri leaves were risky in case of consumption by a herbivorous invertebrate like the landsnail Cantareus aspersus, metal concentrations of snails fed with Zn- and Cd-enriched and low enriched leaves were compared. Willows and A. halleri grew well on the metal-contaminated soil (1.7 and 616 mg kg-1 Cd and Zn, respectively). The A. halleri Zn foliar concentration reached the Zn hyperaccumulation threshold (> 10,000 mg kg-1 DW) in the presence of NPK fertilizer and although the soil was alkaline (pH > 8.2). Cd concentration increased with harvest and fertilizer. Cd and Zn foliar concentrations of willows were far above baseline values. Laboratory snails exposure revealed that willow leaves ingestion caused a moderate increase of Cd, Pb, and Zn bioaccumulation in snails compared to the one caused by A. halleri ingestion. The soil and plant metal concentrations were reflected by field snail biomonitoring. This study confirmed the interest of selecting A. halleri and willows to partially remove Zn and Cd in the soil and emphasized their potential usefulness in greening urban contaminated area and producing raw materials for green chemistry while paying attention to the environmental pollutant transfer.

Assessing natural clays of a contaminated site to stabilize and reduce the ecotoxicity of a coal tar.

Stabilization/solidification is widely used for the immobilization of pollutants in matrices. This work addresses the effect of illite amendment to a liquid coal tar on organic compounds (OCs) immobilization, especially PAHs and BTEX. For practical purpose, illite was selected as raw clay material available on the coal tar contaminated site. Contaminants availability and ecotoxicity of clay/tar matrices at various ratios and considering several treatments were assessed. Varying the tar mass fraction from 1 to 0.12, strongly viscous pastes, pellets and powders were obtained successively, with minimal contaminant mobility observed for tar fractions ranging from 0.28 to 0.80. Pellets obtained for the tar fraction of 0.33 were particularly studied for toxicity tests, because of their ease of handling. Using the land snail Cantareus aspersus as an ecotoxicity probe, mechanisms of PAHs bioavailability considering several treatments were studied. Considering either land snails for direct contact or exposed to vapors, or Lymnaea stagnalis for contact with leachates, toxicity of matrices decreased with ageing or even better with incineration.

Telomere dynamic in humans and animals: Review and perspectives in environmental toxicology.

Telomeres (TLs) play major roles in stabilizing the genome and are usually shortened with ageing. The maintenance of TLs is ensured by two mechanisms involving telomerase (TA) enzyme and alternative lengthening telomeres (ALT). TL shortening and/or TA inhibition have been related to health effects on organisms (leading to reduced reproductive lifespan and survival), suggesting that they could be key processes in toxicity mechanisms (at molecular and cellular levels) and relevant as an early warning of exposure and effect of chemicals on human health and animal population dynamics. Consequently, a critical analysis of knowledge about relationships between TL dynamic and environmental pollution is essential to highlight the relevance of TL measurement in environmental toxicology. The first objective of this review is to provide a survey on the basic knowledge about TL structure, roles, maintenance mechanisms and causes of shortening in both vertebrates (including humans) and invertebrates. Overall, TL length decreases with ageing but some unexpected exceptions are reported (e.g., in species with different lifespans, such as the nematode Caenorhabditis elegans or the crustacean Homarus americanus). Inconsistent results reported in various biological groups or even between species of the same genus (e.g., the microcrustacean Daphnia sp.) indicate that the relation usually proposed between TL shortening and a decrease in TA activity cannot be generalized and depends on the species, stage of development or lifespan. Although the scientific literature provides evidence of the effect of ageing on TL shortening, much less information on the relationships between shortening, maintenance of TLs, influence of other endogenous and environmental drivers, including exposure to chemical pollutants, is available, especially in invertebrates. The second objective of this review is to connect knowledge on TL dynamic and exposure to contaminants. Most of the studies published on humans rely on correlative epidemiological approaches and few in vitro experiments. They have shown TL attrition when exposed to contaminants, such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), pesticides and metallic elements (ME). In other vertebrates, the studies we found deals mainly with birds and, overall, report a disturbance of TL dynamic consecutively to exposure to chemicals, including metals and organic compounds. In invertebrates, no data are available and the potential of TL dynamic in environmental risk assessment remains to be explored. On the basis of the main gaps identified some research perspectives (e.g., impact of endogenous and environmental drivers, dose response effects, link between TL length, TA activity, longevity and ageing) are proposed to better understand the potential of TL and TA measurements in humans and animals in environmental toxicology.

A full life-cycle bioassay with Cantareus aspersus shows reproductive effects of a glyphosate-based herbicide suggesting potential endocrine disruption.

A full life-cycle (240 days) bioassay using the terrestrial snail, Cantareus aspersus, allowing exposure during embryogenesis and/or the growth and reproduction phases, was used to assess the effects of Bypass®, a glyphosate-based herbicide (GlyBH), on a range of endpoints, including parameters under endocrine control. As a positive control, a mixture (R-A) made of diquat (Reglone®) and nonylphenols (NP, Agral®), known for its endocrine disrupting effects in other organisms, was tested. At environmental concentrations, both pesticides (R-A mixture and GlyBH) enhanced growth but reduced reproduction. The R-A mixture acted mainly on the fecundity through a delay in egg-laying of approximately 20 days and a strongly reduced number of clutches. This latter dysfunction may be caused by a permanent eversion of the penis, suggesting a disrupting effect at the neuro-endocrine level, which prevented normal mating. GlyBH acted on fertility, possibly due to a decrease in the fertilization of eggs laid by adults exposed during their embryonic development. These results, associated with the absence of observed effects on gonad histology of GlyBH exposed snails, suggested that the underlying mechanisms are neuro-endocrine.

Palaeo-pollution from mining activities in the Vosges Mountains: 1000 years and still bioavailable.

Mining and smelting activities have contaminated the environment with trace metals (TMs) at a worldwide scale for at least two millennia. A combination of chemical approaches and active biomonitoring was performed to analyse the environmental availability and bioavailability of TM palaeo-pollution in a former PbAg mining district in the Vosges Mountains, France. Along a soil TM contamination gradient that covered eight stations, including two archaeological mining sites, the toxicokinetics of six TMs (Pb, Cd, As, Ag, Co, Sb) in the snail Cantareus aspersus revealed that palaeo-pollution from the studied sites remains bioavailable. This study provides the first data on the accumulation kinetics of Ag and Co for C. aspersus. The environmental availability of the TMs was estimated with three chemical extraction methods (aqua regia, EDTA 50 mM, CaCl2 10 mM). Univariate regression analyses showed that EDTA extraction is the best method for estimating the bioavailability of Pb, As, Ag, Co and Sb to snails. None of the three extractants was efficient for Cd. A multivariate analysis of bioaccumulation data revealed that TM bioavailability and transfer were modulated by exposure sources (soil, humus and vegetation) rather than by soil physico-chemical characteristics. Hence, although the deposition of mining wastes dates back several centuries, these wastes still represent a source of contamination that must be considered to develop relevant site management and environmental risk assessment.

How contamination sources and soil properties can influence the Cd and Pb bioavailability to snails.

To better understand the fate of metals in the environment, numerous parameters must be studied, such as the soil properties and the different sources of contamination for the organisms. Among bioindicators of soil quality, the garden snail (Cantareus aspersus) integrates multiple sources (e.g. soil, plant) and routes (e.g. digestive, cutaneous) of contamination. However, the contribution of each source on metal bioavailability and how soil properties influence these contributions have never been studied when considering the dynamic process of bioavailability. Using accumulation kinetics, this study showed that the main assimilation source of Cd was lettuce (68%), whereas the main source of Pb was the soil (90%). The plant contribution increased in response to a 2-unit soil pH decrease. Unexpectedly, an increase in the soil contribution to metal assimilation accompanied an increase in the organic matter (OM) content of the soil. For both metals, no significant excretion and influence of source on excretion have been modelled either during exposure or depuration. This study highlights how the contribution of different sources to metal bioavailability changes based on changes in soil parameters, such as pH and OM, and the complexity of the processes that modulate metal bioavailability.

Metallothionein gene expression in embryos of the terrestrial snail (Cantareus aspersus) exposed to cadmium and copper in the Bordeaux mixture.

The response specificity of three metallothionein (MT) genes (CdMT, CuMT and Cd/CuMT) was assessed after long-term exposure (20 days) of Cantareus aspersus eggs to cadmium (Cd) (2 to 6 mg/L) or to the fungicide Bordeaux mixture (BM) (2.5 and 7.5 g/L). MT gene expression measured by quantitative real-time PCR (qRT-PCR) revealed that in the unexposed embryos, the transcript levels of the three MT genes decreased significantly through embryonic development. However, the CdMT gene was strongly upregulated with increasing Cd exposure concentration, whereas the transcript levels of the other two genes increased less pronouncedly, but significantly above an exposure concentration of 4 mg Cd/L. Upon exposure to BM, all three MT genes were significantly upregulated above a BM concentration of 2.5 g/L. It is concluded that long-term Cd exposure in hatched snails induced patterns of MT gene expression that differed from those obtained after short-term exposure (24 h).

Mercury toxicity to terrestrial snails in a partial life cycle experiment.

Despite growing concerns about the potential adverse effects of elevated mercury (Hg) concentrations in the terrestrial environment, only a few toxicity data are available for soil invertebrates. The chronic toxicity of inorganic Hg-Hg(II)--through food or soil contaminations was therefore assessed for the snail Cantareus aspersus, a well-recognized soil quality bioindicator. The 28-day EC50s (the concentrations causing 50% effect) for the snail growth were 600 and 5048 mg Hg kg(-1) for food and soil, respectively. A survey of growth over its entire duration (91 days) allowed to show that the effects took place rapidly after the beginning of exposure and persisted in the long term. Reproduction was also impaired, and we established 28-day EC50s for sexual maturation and fecundity of 831 and 339 mg Hg kg(-1), respectively, for food and 1719 and 53 mg Hg kg(-1), respectively, for soil. Total Hg analyses in snails exposed to contaminated matrices revealed important bioaccumulation capacities up to 2000 mg Hg kg(-1) viscera. Critical limits in internal Hg concentration of about 500 and 1000 mg Hg kg(-1) were determined as thresholds for the induction of growth toxicity through food and soil exposure, respectively. These different values underlined differences in the uptake and toxicological dynamics of Hg according to its bioavailability in the source of exposure.

Differential sensitivity of snail embryos to cadmium: relation to age and metallothionein gene expression.

The aim of this study was to determine whether cadmium (Cd) sensitivity of Cantareus aspersus embryos is age-dependent and influenced by metallothionein (MT) gene expression. Hatching success and the expression of three MT isoform genes (Ca-CdMT, Ca-CuMT and Ca-Cd/CuMT) were measured in embryos exposed to increasing Cd concentrations for 24 h starting on the sixth day of development. Isoform gene expression was quantified on days 7 and 12 after exposure. Results were compared to those of embryos exposed to the same conditions as above, but from the beginning of embryogenesis (day 0). Transcription of the Cd-specific MT gene (Ca-CdMT) was observed from the first day of development, whereas the two other genes did not respond to Cd at all. Overall, Cd sensitivity of embryos decreased with increasing age of development, as assessed by age-dependent increase of EC50 values for hatching rate, and increasing Cd threshold concentrations for Ca-CdMT expression.

Genotoxicity assessment of pesticides on terrestrial snail embryos by analysis of random amplified polymorphic DNA profiles.

The study explores the relevance of coupling Random Amplified Polymorphic DNA (RAPD) and a High-Resolution capillary electrophoresis System (HRS) method for assessing the genotoxic potential of the wide variety commercial formulations of pesticides. Using this technique, the genotoxic potential of a glyphosate-based herbicide (Roundup Flash(®) (RU)) and two fungicide formulations based on tebuconazole and copper (Corail(®) and Bordeaux mixture (BM), respectively) was evaluated on terrestrial snail embryos. Clutches of Cantareus aspersus were exposed during their entire embryonic development to a range of concentration around the EC50 values (based on hatching success) for each compound tested. Three primers were used for the RAPD amplifications of pesticides samples. RAPD-HRS revealed concentration-dependent modifications in profiles generated with the three primers in RU(®)-exposed embryos from 30 mg/L glyphosate. For Corail(®)-exposed embryos, only two of the three primers were able to show alterations in profiles from 0.05 mg/L tebuconazole. For BM-exposed embryos, no signs of genotoxicity were observed. All changes observed in amplification profiles have been detected at concentrations lower than the recommended doses for vineyard field applications. Our study demonstrates the efficiency of coupling RAPD and HRS to efficiently screen the effect of pesticide formulations on DNA.