Combining human and snail indicators for an integrative risk assessment of metal(loid)-contaminated soils.

With the new soil uses such as land restoration and to protect wilderness, the human health risk assessment (HHRA) and environmental risk assessment (ERA) should be combined. Based on the relationships demonstrated between an indicator of soil quality, the land snail, and human exposure, the aim of this study is to examine the snail and human risk indicators for twenty-nine soils contaminated by metal(loid)s. HHRA was evaluated by both hazard quotient and carcinogenic risk. When the human health indicators were ranked as uncertain, they were weighted by bioaccessibility to refine the risk assessment. The ERA was performed with risk coefficient after ex situ snail exposure. The results showed strong and novel relationships between human health and environmental risk indicators that had never been found before. For 62% of the soils, both indicators revealed either a confirmed risk or an uncertain level of risk. Overall pollutants present greater risk for human than for environment, with 55 vs 28% of the studied soils classified in the proven risk, respectively. An original integrative risk assessment of polluted soils has been proposed, that shall help setting up relevant strategies to manage contaminated soils considering not only human but also environmental indicators of risk.

Ex situ environmental risk assessment of polluted soils using threshold guide values for the land snail Cantareus aspersus.

Environmental risk assessment of contaminated soils should ideally be carried out with complementary approaches (chemical and biological) conducted in situ and ex situ. While biological methods based on the assessment of effect and bioaccumulation in bioindicators exist for soil fauna organisms, such as land snails, the methodology is currently limited in the field to 14 metallic elements (MEs). To provide new relevant tools to the stakeholders of polluted fields, the aim of this work is to determine ex situ threshold guide values (ex situ TGVs), for 15 MEs, 16 polycyclic aromatic hydrocarbons (PAHs) and 7 polychlorinated biphenyls (PCBs). These ex situ TGVs are the usual concentration of contaminants found in the viscera of the bioindicator Cantareus aspersus after 28 days of exposure to uncontaminated soils. The second objective was to assess and validate the relevance of these ex situ TGVs for the interpretation of contamination levels in various European contaminated soils based on global index calculations: i) The sum of the excess of transfers (SETs) and ii) the weighted SETs based on the general toxicity points of each contaminant used to evaluate the risk of transferred MEs, PAHs and PCBs (ERITMEs, ERITPAHs and ERITPCBs, respectively). In addition, the influence of soil physico-chemical properties on accumulation was modelled to better understand their roles in bioavailability. The presented ex situ TGV and the associated indicators (the global sum of the excess of transfers and global ecotoxicological risk) provide a basis by which stakeholders can prioritize the management of polluted soils depending on the risk they may represent. The determination of ex situ TGVs for organic and inorganic compounds provides new tools to characterize excess contaminant transfers, and it will also allow the use of snails for ERAs, notably for common pollutants, such as PAHs and PCBs for which guide values are not available.

Bioaccessibility of metal(loid)s in soils to humans and their bioavailability to snails: A way to associate human health and ecotoxicological risk assessment?

Human health risk assessment (HHRA) and ecotoxicological risk assessment (ERA) of contaminated soils are frequently performed separately and based on total soil concentrations without considering the concepts of mobility, bioaccessibility and bioavailability. However, some chemical and biological assays rarely used in combination can be applied to more accurately assess the exposure of organisms to metal(loid)s and thus to better estimate the links between soil contamination and effects. For humans, the unified bioaccessibility method (UBM) assesses oral bioaccessibility, while for soil fauna such as land snails, the bioaccumulation test reflects the bioavailability of contaminants. The aim of this study is to explore the relationship between oral bioaccessibility and the bioavailability of arsenic, cadmium and lead in twenty-nine contaminated soils. The results show a modulation of bioaccumulation and bioaccessibility of metal(loid)s by soil physicochemical parameters (organic matter especially). For the three metal(loid)s studied, strong relationships were modelled between the UBM and snail tests (0.77 < r²adj.<0.95), depending on the parameters of the linear regressions (contaminant and phases of the UBM test). The original models proposed demonstrate the feasibility of linking bioaccessibility to humans and bioavailability to snails and the relevance of their association for an integrative risk assessment of contaminated soils.

Assessment of the bioavailability and depuration of uranium, cesium and thorium in snails (Cantareus aspersus) using kinetics models.

Uranium ore waste has led to soil contamination that may affect both environmental and soil health. To analyze the risk of metal transfer, metal bioavailability must be estimated by measuring biological parameters. Kinetic studies allow taking into account the dynamic mechanisms of bioavailability, as well as the steady state concentration in organisms necessary to take into account for relevant risk assessment. In this way, this work aims to model the snail accumulation and excretion kinetics of uranium (U), cesium (Cs) and thorium (Th). Results indicate an absence of Cs and Th accumulation showing the low bioavailability of these two elements and a strong uranium accumulation in snails related to the levels of soil contamination. During the depuration phase, most of the uranium ingested was excreted by the snails. After removing the source of uranium by soil remediation, continued snails excretion of accumulated uranium would lead to the return of their initial internal concentration, thus the potential trophic transfer of this hazardous element would stop.

Soil parameters are key factors to predict metal bioavailability to snails based on chemical extractant data.

Although soil characteristics modulate metal mobility and bioavailability to organisms, they are often ignored in the risk assessment of metal transfer. This paper aims to determine the ability of chemical methods to assess and predict cadmium (Cd), lead (Pb) and zinc (Zn) environmental bioavailability to the land snail Cantareus aspersus. Snails were exposed in the laboratory for 28 days to 17 soils from around a former smelter. The soils were selected for their range of pH, organic matter, clay content, and Cd, Pb and Zn concentrations. The influence of soil properties on environmental availability (estimated using HF-HClO(4), EDTA, CaCl(2), NH(4)NO(3), NaNO(3), free ion activity and total dissolved metal concentration in soil solution) and on environmental bioavailability (modelled using accumulation kinetics) was identified. Among the seven chemical methods, only the EDTA and the total soil concentration can be used to assess Cd and Pb environmental bioavailability to snails (r²(adj)=0.67 and 0.77, respectively). For Zn, none of the chemical methods were suitable. Taking into account the influence of the soil characteristics (pH and CEC) allows a better prediction of Cd and Pb environmental bioavailability (r²(adj)=0.82 and 0.83, respectively). Even though alone none of the chemical methods tested could assess Zn environmental bioavailability to snails, the addition of pH, iron and aluminium oxides allowed the variation of assimilation fluxes to be predicted. A conceptual and practical method to use soil characteristics for risk assessment is proposed based on these results. We conclude that as yet there is no universal chemical method to predict metal environmental bioavailability to snails, and that the soil factors having the greatest impact depend on the metal considered.

Use of chemical methods to assess Cd and Pb bioavailability to the snail Cantareus aspersus: a first attempt taking into account soil characteristics.

Bioavailability is a key parameter in conditioning contaminant transfer to biota. However, in risk assessment of terrestrial contamination, insufficient attention is being paid to the influence of soil type on trace metal bioavailability. This paper addresses the influence of soil properties on the chemical availability of cadmium (Cd) and lead (Pb) (CaCl(2) extraction and ionic activity) and bioavailability (accumulation kinetics) to the land snail Cantareus aspersus. Snails were exposed to nine contaminated soils differing by a single characteristic (pH or organic matter content or clay content) for 28 days. Toxicokinetic models were applied to determine metal uptake and excretion rates in snails and multivariate regression was used to relate uptake parameters to soil properties. The results showed that alkalinisation of soil and an increase of the organic matter content decreased Pb and Cd bioavailability to snails whereas kaolin clay had no significant influence. The CaCl(2)-extractable concentrations tended to overestimate the effects of pH when used to explain metal uptake rate. We conclude that factors other than those controlling the extractable fraction affect metal bioavailability to snails, confirming the requirement of biota measurements in risk assessment procedures.

Differential liver proteome mapping of control and cadmium-fed rats.

A comparative study of proteome maps from control and Cd-exposed rat liver was performed using a new technology of two-dimensional liquid chromatography separation method (PF-2D system, Beckman Coulter). Rats were fed for one month 0 or 100 µg Cd g(-1). The between-replicate and between-sample variations showed good repeatability and suitable reproducibility for the two dimensions of separation of proteins. In this complex mixture, PF-2D led to the separation of two major peaks which differed between control and Cd-exposed rat livers, one being identified by mass spectrometry as Cu/Zn superoxide dismutase (SOD), a well-known biomarker of Cd exposure, the other as phosphatidylethanolamine binding protein (PEBP). SOD content was decreased in Cd-exposed rat liver, compared to the control group which was corroborated by a significant decrease of SOD activity. PEBP content also tended to be decreased after Cd exposure. Present results demonstrate interest but also limitations of proteomic approach using PF-2D system to analyze effects of chemicals on organisms.

Arsenic transfer and impacts on snails exposed to stabilized and untreated As-contaminated soils.

An As-contaminated soil (Unt) was amended with either iron grit (Z), a coal fly ash (beringite, B) or B + Z (BZ) and placed in lysimeters in 1997. An uncontaminated soil (R) was also studied. In summer and autumn 2003, lettuces were cultivated in the lysimeters and snails were caged for one month. Lettuce As concentrations were higher during the summer, while no differences occurred in snails between seasons. Snail As concentrations (microg g(-1) DW) ranged from 2.5 to 7.0 in B, Z and BZ, and peaked at 17.5 in Unt. In summer, snail survival was affected in Unt and Z compared to R and B while no mortality was noticed in autumn. Snail growth decreased only in B, BZ and Unt in autumn. Snail As concentrations suggest a risk for their predators even on the remediated soils.

Coping with cadmium exposure in various ways: the two helicid snails Helix pomatia and Cantareus aspersus share the metal transcription factor-2, but differ in promoter organization and transcription of their Cd-metallothionein genes.

Gastropods are able to withstand fluctuating availabilities of nonessential trace elements such as cadmium by induction of Cd-specific metallothionein isoform (Cd-MT) expression. As in other species, the induction mechanism involves the binding of metal-regulatory transcription factors (MTF-1 or MTF-2) to metal responsive elements (MREs) in the MT promoter regions. Cd-dependent transcription of Cd-MT genes was assessed by quantitative real time PCR in two helicid gastropods, Helix pomatia and Cantareus aspersus, over a period of eight days. The promoter regions of the Cd-MT genes of the two species were sequenced and compared regarding the position of MREs and other relevant potential transcription factor binding sites (TFBs). Cd-MT gene transcription is induced after Cd exposure in Helix pomatia and Cantareus aspersus, showing a transient peak in Helix pomatia, contrasting with a persistent induction rate in Cantareus aspersus. Since the existence of MTF-2 was verified in both species, differing transcription patterns of Cd-MT genes must be due to functional differences in their metal-responsive promoter regions. Both promoters contain a proximal cluster of three MREs overlapping with TFBs for the transcriptional regulator Sp1. In contrast to Cantareus aspersus, however, the Cd-MT gene of Helix pomatia hosts an additional distal MRE overlapping with a Sp1 binding site and a CACCC box. Inhibitory effects of MRE overlapping Sp1 binding sites were observed in other MT genes. We therefore suggest that transient Cd-MT transcription upon Cd(2+) exposure in Helix pomatia may be the result of an inhibitory action of the distal MRE cluster.

Contamination of woody habitat soils around a former lead smelter in the North of France.

The contamination of the topsoil of 262 woody habitats around a former lead smelter in the North of France was assessed. In this urbanized and industrialized area, these kinds of habitats comprise of hedges, groves, small woods, anthropogenic creations and one large forest. Except for the latter, which is 3 km away, these woody habitat soils often present a high anthropization degree (a significant amount of pebbles and stones related to human activities) with a high metal contamination. In the studied woody habitat topsoils, Cd, Pb and Zn concentrations largely exceeded those of agricultural topsoils located in the same environmental context. Therefore, atmospheric emissions from the smelter are not the only cause of the high contamination of the woody habitat soils. This last one is related to the nature and the contamination level of deposit in relation with human activities (rubbles, slag, soils, etc). With regard to the results obtained with chemical extractions, the mobility of Cd, Pb and Zn in these soils is also greater than in agricultural soils. In the forest, pollutant solubility is increased by soil acidic pH. The variability of the physico-chemical parameters and the high metal contamination of the topsoils are the main characteristics of the woody habitats located around the former smelter. Although never taken into account during risk assessment, the disturbance of these environmental components could have important biogeochemical impacts (nutrients and metal cycles). Moreover, any modification of the soils' use could potentially cause mobilization and transfer of the pollutants to the biosphere. Six years after the closure of the smelter, and as social and economic pressures considerably increase in this area, the study of these peculiar ecosystems is necessary to understand and predict the bioavailability, transfer, bioaccumulation and effects of pollutants in food chains.

Effects of subchronic digestive exposure to organic or inorganic cadmium on biomarkers in rat tissues.

In an experimental food chain, Wistar rats were fed cadmium (Cd) in an inorganic (CdCl(2)) or organic (mainly associated with metallothionein from Helix aspersa snail viscera) form. After 1 month of exposure to 100 microg inorganic Cd g(-1) in food, an induction of metallothionein was observed in all target tissues. In liver, glutathione peroxidase (GSH-Px) activity decreased and alanine aminotransferase (ALAT) activity increased, suggesting that Cd causes hepatotoxicity. However, lipid peroxidation as well as catalase and caspase 3 (a marker of apoptosis) activities were not modified. At a rather low exposure (2.5 microg Cd g(-1)), metallothionein level in the kidney was found to be the most sensitive biomarker of exposure for both Cd forms. In the small intestine of rats ingesting inorganic Cd, metallothionein expression was significantly higher than that observed for rats fed organic Cd. Present results allowed proposing a simple design to assess the effect of a chemical in a trophic transfer approach.

Comparison of transfer and effects of Cd on rats exposed in a short experimental snail-rat food chain or to CdCl2 dosed food.

Transfer and toxic effects of two cadmium (Cd) forms, inorganic (CdCl2 dosed rat food) or organic (contaminated snail-based rat food) were studied in Wistar rat. Cd concentrations in rat food were 0 and 2.5 microg Cd g(-1) for both inorganic and organic forms and a high concentration of 100 microg Cd g(-1) was also tested for the inorganic form. Rats were exposed for four weeks to contaminated food. Both forms of Cd were bioavailable to rats, with a percentage of transfer from food to rats of around 1% for all contaminated groups. Cd concentrations in rat tissues increased with increasing Cd concentrations in the food. Rats fed with organic form of Cd accumulated significantly more Cd in the main organ for Cd toxicity, the kidney, than those eating the inorganic form. Survival was not affected for any rat group but a decrease in growth and food consumption was observed for the inorganic form. As a defence system against Cd toxicity, rats increased their metallothionein (MT) synthesis at the highest Cd concentration in the target organs (kidney, liver and small intestine) and even did the same at low Cd concentrations (2.5 microg Cd g(-1)) in the kidney. At this low Cd concentration, MT induction was lower in the small intestine of rats ingesting organic Cd than those ingesting inorganic Cd. Bioavailability of organic and inorganic forms of Cd was similar, but subsequent Cd distribution within organs was different. This quantification of the trophic transfer of both inorganic and organic forms of a toxicant is a basis for a better assessment of the fate and effects of chemicals in food webs.

Lead concentrations in feathers and blood of common blackbirds (Turdus merula) and in earthworms inhabiting unpolluted and moderately polluted urban areas.

Despite the dramatic decrease of atmospheric lead (Pb) concentrations in urban areas of most industrialised countries, we hypothesised that urban common blackbirds (Turdus merula) may still be contaminated by Pb concentrations of toxicological concern due to transfer from soil through the food chain. We sampled blackbirds and earthworms, one of their main preys, in Besançon, a middle-size city of Eastern France (where atmospheric Pb concentrations decreased from 0.5 microg/m(3) in 1987 to nearly 0 in 2002) and in a rural reference site. Lead concentrations were determined in the tissues of the different functional groups of earthworms (anecic, epigeous and endogeous) and in blood, washed and unwashed outermost tail feathers and breast feathers of blackbirds. Fresh masses and an index of individual body condition were measured in the two blackbird populations as biomarkers of possible toxic effects. Lead concentrations in earthworms did not differ among functional groups but were significantly higher in urban individuals than in rural ones. Concentrations in outermost tail feathers, breast feathers and blood were significantly higher in urban blackbirds (7.75+/-4.50, 3.15+/-1.77 and 0.15+/-0.09 microg/g, respectively) than in rural individuals. In urban blackbirds, concentrations in washed and unwashed outermost tail feathers allowed estimating the external contamination (probably due to deposition of dusts and/or to excretion of the uropygial gland) at 37% of the total Pb concentration of the unwashed feathers. Remaining 63% should be linked to food chain transfer of persistent Pb from urban soils. Among the 23 sampled blackbirds, 4 of them (3 in the urban site and 1 in the rural site) exhibited blood Pb concentrations higher than the benchmark value (0.20 microg/g) related to subclinical and physiological effects in birds. Variations in body condition index were not correlated to Pb concentrations in blackbird tissues. Present results suggest that Pb may still be of environmental concern for blackbirds in urban areas because of the persistence of Pb in soils and its transfer through the food chain.

Bioconcentration of cadmium and toxic effects on life-history traits of pond snails (Lymnaea palustris and Lymnaea stagnalis) in laboratory bioassays.

We studied the bioconcentration and the individual effects of Cd on life-history traits of Lymnaea stagnalis and Lymnaea palustris exposed to increasing Cd concentrations for 4 weeks in controlled conditions. Internal Cd concentrations were linearly correlated to Cd concentrations of exposure in both foot and viscera of L. stagnalis and in the foot of L. palustris, and they were linked by a logistic relationship with environmental contamination in the viscera of L. palustris. If LC50S were higher than the highest Cd concentrations of exposure (L. stagnalis: 160 microg L(-1); L. palustris: 320 microg L(-1)), other dose-dependent responses affecting life-history traits were noted in both species. In L. stagnalis, EC50 for growth was evaluated at 142 microg L(-1) and growth inhibition was correlated with internal Cd concentrations by a linear relationship. L. palustris was more sensitive to Cd than L. stagnalis because its EC50-growth was three times lower (58 microg L(-1) after 28 days). Dose-dependent responses were obtained for several parameters of fecundity of L. palustris. EC50 for the mean number of egg masses or mean number of eggs per individual were close to 60 microg L(-1), whereas for the mean number of eggs per egg mass, the EC50 was higher, with a value of 124 microg L(-1). The percentage of hatching versus the total number of eggs was 60% in controls, and no embryos were able to hatch at the lowest tested Cd concentration, 40 microg L(-1). The high sensitivity of fertility was due to Cd blocking embryo development, particularly for the latest embryonic stages just before hatching. The present results constitute useful data to develop laboratory tests using pond snails for freshwater risk assessment.

A field method using microcosms to evaluate transfer of Cd, Cu, Ni, Pb and Zn from sewage sludge amended forest soils to Helix aspersa snails.

Juvenile Helix aspersa snails exposed in field microcosms were used to assess the transfer of Cd, Cu, Ni, Pb and Zn from forest soils amended with liquid and composted sewage sludge. Zn concentrations and contents were significantly higher in snails exposed to liquid and composted sludge after 5 and 7 weeks of exposure, when compared with control. Trends were less clear for the other metals. Present results show that Zn, among the cocktail of metallic trace elements (MTE) coming from sewage sludge disposal, represents the principal concern for food chain transfer and secondary poisoning risks. The microcosm design used in this experiment was well suited for relatively long-term (about 2 months) active biomonitoring with H. aspersa snails. The snails quickly indicated the variations of MTE concentrations in their immediate environment. Therefore, the present study provides a simple but efficient field tool to evaluate MTE bioavailability and transfer.

Transfer of cadmium from plant leaves and vegetable flour to the snail Helix aspersa: bioaccumulation and effects.

Juvenile Helix aspersa snails were exposed for 4 weeks to fresh rape leaves (Brassica napus), contaminated by simulating superficial deposits of increasing concentrations of cadmium (Cd). The Cd concentration in leaves was 0.1 in control and 38.4, 93.1, and 177.2 microg x g(-1) (dry mass) in Cd-treated food. The concentration in snail tissues increased with increasing Cd concentrations in the food. The bioaccumulation factors ranged from 4.8 (control) to 2.4 (highest exposed group), indicating a biomagnification of Cd in this food chain. The growth of the snails treated with Cd was reduced by 17, 24, and 43% respectively, compared to the control group. Comparison of these results with those obtained with snails exposed to similar Cd concentrations in a vegetable flour revealed that accumulation and effects were relatively consistent, demonstrating a comparable bioavailability of Cd in the two diets. Tests using growing H. aspersa snails exposed to metals in flour or fresh leaves can be useful for risk assessment purposes.

Effects of dimethoate on snail B-esterase and growth as a function of dose, time and exposure route in a laboratory bioassay.

The aim was to study the effects of dimethoate on enzymatic targets and on the growth of Helix aspersa for different times and modes of exposure under laboratory conditions. Young snails were exposed to increasing dimethoate concentrations in the food (D.exp) or in an artificial substrate (S.exp) for 1, 2, 7 and 14 days. Both acetylcholinesterase (AChE) and carboxylesterase (CaE) activities were measured in the foot of the snails for each concentration and exposure time tested. Growth was evaluated after 7 days of exposure. AChE inhibition, dose-dependent for all lengths of exposure, was stronger in S.exp. AChE was more sensitive than CaE for both modes of exposure. IC50(-7) days was 38.3 micrograms g-1 in D.exp and 11.7 micrograms g-1 in S.exp for AChE and was higher than 150 micrograms g-1 in two exposure modes for CaE. AChE activity decreased from the first day to reach maximum inhibition after 7 days of exposure. As noted for B-esterase activities, growth inhibition was stronger in S.exp and was only significant for AChE inhibition of > 90%. The present results show that AChE activity could be used to give early warning of toxic effects of dimethoate in terrestrial gastropods.

Transfer and effects of cadmium in an experimental food chain involving the snail Helix aspersa and the predatory carabid beetle Chrysocarabus splendens.

The transfer and the toxic effects of Cd were studied in an experimental food chain involving the snail Helix aspersa as prey organism and one of its natural predators, the carabid beetle Chrysocarabus splendens. Juvenile snails were fed plant-based food enriched with 0, 10, 50 and 100 microg g(-1) of Cd, then were offered as prey to beetle larvae from egg hatching to pupation stage. Cd concentrations in snail tissues increased with increasing Cd concentration in food and with duration of exposure. Bioaccumulation factors ranged from 1.87 to 3.39, showing that H. aspersa snails, even in their early life stages, belong to macroconcentrator species for Cd. No significant reduction of snail consumption by beetles was found in exposed groups. Cd concentrations in beetle larvae remained very low (lower than 1 microg g(-1) for all groups), demonstrating a very effective regulation capacity in beetle larvae. However, Cd concentrations in highest exposed groups were higher than those found in control groups. Cd contents in adult beetles were lower than in larvae, showing a loss of Cd during metamorphosis. Despite the low Cd concentrations found in beetles, their exposure to Cd contaminated snails led to 31% of mortality, which occurred only during pupation and for the highest exposure level. No clear sublethal effects were found. These results showed that snails inhabiting heavily polluted areas may represent a risk of secondary poisoning for predatory invertebrates and provided quantitative data on the transfer of Cd between two compartments of a terrestrial food chain.

The garden snail (Helix aspersa) as a bioindicator of organophosphorus exposure: effects of dimethoate on survival, growth, and acetylcholinesterase activity.

The garden snail (Helix aspersa) is currently used as bioindicator of metallic pollution. Our objective was to extend its use to organic chemicals by studying the effects and tissue concentrations of the organophosphorus pesticide dimethoate following dietary uptake. After exposure for four weeks to increasing doses of pesticide in the diet, the median lethal concentration (LC50) was determined to be 3,700 microg/g food. Clinical signs indicated a no-observed-effect concentration of 100 microg/g and a lowest-observed-effect concentration of 250 microg/g. The growth parameters were decreased with increasing exposure to the pesticide. The median effective concentration (EC50), which was evaluated based on both shell diameter and dry weight inhibitions, was 665 and 424 microg/g, respectively, and the EC10 was 180 and 145 microg/g, respectively. Accumulation in the viscera was related to the amount of dimethoate in the food. The bioconcentration factors were low (>6 x 10(-3)). Acetylcholinesterase (AChE) activity was strongly decreased (80% from 250 microg/g). In conclusion, we demonstrated that the species H. aspersa could be a useful sentinel organism for organophosphorus contamination surveys. Among the effects measured, the inhibition of AChE activities and clinical signs were the most sensitive, followed by the growth parameters. These results confirm the suitability of the garden snail for development of sublethal toxicity tests using primary consumers and aboveground organisms.