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.

Mass balance approach to assess the impact of cadmium decrease in mineral phosphate fertilizers on health risk: The case-study of French agricultural soils.

Cadmium is a ubiquitous and highly toxic contaminant that can cause serious adverse effects. The European Food Safety Authority (EFSA) and the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) have shown that the risk related to food contamination by cadmium cannot be ruled out in Europe and France. Fertilizing material is one of the main sources of cadmium contamination in the food chain on which regulators can play to reduce cadmium exposure in the population. The aim of this work was to develop a mass-balance approach integrating the various environmental sources of cadmium to estimate the effects of a decrease in cadmium concentrations in crop fertilizers on dietary exposure and on the health risk. This approach led to a predictive model that can be used as a decision-making tool. Representative and protective fertilization scenarios associated with controlled cadmium levels in mineral phosphate fertilizers were simulated and converted into cadmium fluxes. Cadmium inputs from industrial mineral phosphate fertilizers were then compared with cadmium brought by the application of manure, sewage sludge and farm anaerobic digest, at the levels typical of French agricultural practices. Regardless of the fertilizer and scenario used, a flux lower than 2 g Cd.ha-1.year-1 reduces both the accumulation in soils and the transfer of cadmium in the food chain. It corresponds to a cadmium content of 20 mg.kg P2O5-1 or less in mineral phosphate fertilizers. Modelling the transfer of cadmium from the soil to consumed food made it possible to propose cadmium limits in fertilizers applied in France. In a global context of ecological transition to promote human health, this research will help risk managers and public authorities in the regulatory decision-making process for the reduction of environmental cadmium contamination and human exposure.

Variations in the transfer of radiocesium (137Cs) and radiostrontium (90Sr) from milk to cheese.

This study aimed to compare the transfer of 2 manmade radionuclides, radiocesium (137Cs) and radiostrontium (90Sr), from cow milk to whey and cheese in 3 different types of French cheese production with rennet coagulation. Most of the 137Cs was present in the aqueous phase and became concentrated in the whey. For 137Cs transfer to whey, the processing factor (Pf; i.e., the ratio of the activity concentrations) ranged between 0.86 and 1.30 (n = 12). The food processing retention factor (Fr), calculated using the processing efficiency, ranged between 0.85 and 1.19 (n = 9). No statistical difference of Pf and Fr to whey is identified for 137Cs and the cheese products. The Pf calculated for 90Sr transfer to cheese ranged between 3.95 and 12.16, with significant differences depending on the type of cheese. In addition, a linear correlation is observed between 90Sr Pf to cheese and the Ca level in the cheese (r2 = 0.57). Thus, the Pf is enhanced in hard cheeses that are enriched in calcium. This is confirmed by nearly constant Fr values, ranging between 0.66 and 0.83.

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.

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.

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.

Tritium levels in milk in the vicinity of chronic tritium releases.

Tritium is the radioactive isotope of hydrogen. It can be integrated into most biological molecules. Even though its radiotoxicity is weak, the effects of tritium can be increased following concentration in critical compartments of living organisms. For a better understanding of tritium circulation in the environment and to highlight transfer constants between compartments, we studied the tritiation of different agricultural matrices chronically exposed to tritium. Milk is one of the most frequently monitored foodstuffs in the vicinity of points known for chronic release of radionuclides firstly because dairy products find their way into most homes but also because it integrates deposition over large areas at a local scale. It is a food which contains all the main nutrients, especially proteins, carbohydrates and lipids. We thus studied the tritium levels of milk in chronic exposure conditions by comparing the tritiation of the main hydrogenated components of milk, first, component by component, then, sample by sample. Significant correlations were found between the specific activities of drinking water and free water of milk as well as between the tritium levels of cattle feed dry matter and of the main organic components of milk. Our findings stress the importance of the metabolism on the distribution of tritium in the different compartments. Overall, dilution of hydrogen in the environmental compartments was found to play an important role dimming possible isotopic effects even in a food chain chronically exposed to tritium.

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.

137Cs availability for soil to understory transfer in different types of forest ecosystems.

A quantitative analysis of 137Cs bioavailability in forest soils in the long term after the Chernobyl NPP accident based on a 3-year (1996-1998) investigation is presented. Five forest sites with different trees, composition and properties of soil were studied to identify factors determining radiocaesium transfer to different understory species. The following parameters were investigated: 137Cs activity concentrations and its speciation in various horizons of forest soil, accumulation of this radionuclide by different species of understory vegetation and distribution of root biomass in the soil profile. It has been shown that one decade after the deposition maximum 137Cs activity in soil of the experimental sites considered is located in different soil layers dependent on moisture regime, characteristics of litter and soil properties. A linear dependence between aggregated transfer factors for different species and groups of species of understory vegetation and exchangeable and available fractions of radiocaesium in soil was found. The vertical distribution of 137Cs activity, percentage of exchangeable radiocaesium in each horizon of litter and soil, as well as distribution of root systems (mycelia) over the soil profile are key factors governing variations in the availability of 137Cs for transfer to all forest understory components.

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.

137Cs distribution among annual rings of different tree species contaminated after the Chernobyl accident.

The distributions of 137Cs among annual rings of Pinus sylvestris and Betula pendula at four experimental sites located in the most contaminated areas in the Russian territory after the Chernobyl accident in 1986 were studied. Trees of different ages were sampled from four forest sites with different tree compositions and soil properties. The data analysis shows that 137Cs is very mobile in wood and the 1986 rings do not show the highest contamination. The difference between pine and birch in the pattern of radial 137Cs distribution can be satisfactorily explained by the difference in radial ray composition. 137Cs radial distribution in the wood can be described as the sum of two exponential functions for both species. The function parameters are height, age and species dependent. The distribution of 137Cs in birch wood reveals much more pronounced dependence on site characteristics and/or the age of trees than pines. The data obtained can be used to assess 137Cs content in wood.

Effect of lead pollution on testate amoebae communities living in Sphagnum fallax: an experimental study.

We studied the effects of lead pollution on testate amoebae communities living on Sphagnum fallax by growing this moss under controlled conditions. A progressive series of lead (Pb) concentration was used in the growing solution of the mosses: 0 (control), 625 and 2,500 microgL(-1). The mosses were sampled and analysed for accumulated Pb and testate amoeba communities after 0, 6, 12, and 20 weeks. Species richness, total density and total biomass of testate amoebae declined in response to the Pb treatment and changed over time. The Pb x Time cross-effect was significant for species richness, and total density but not for the total biomass and Shannon diversity. Furthermore, the testate amoebae species richness and the total density were negatively correlated to the Pb concentrations actually accumulated in the moss at the end of the experiment. Species-specific responses of testate amoebae to Pb pollution were identified. Our results thus confirm the sensitivity of testate amoebae to lead pollution.

Effects of experimental lead pollution on the microbial communities associated with Sphagnum fallax (Bryophyta).

Ecotoxicological studies usually focus on single microbial species under controlled conditions. As a result, little is known about the responses of different microbial functional groups or individual species to stresses. In an aim to assess the response of complex microbial communities to pollution in their natural habitat, we studied the effect of a simulated lead pollution on the microbial community (bacteria, cyanobacteria, protists, fungi, and micrometazoa) living on Sphagnum fallax. Mosses were grown in the laboratory with 0 (control), 625, and 2,500 microg L(-1) of Pb(2+) diluted in a standard nutrient solution and were sampled after 0, 6, 12, and 20 weeks. The biomasses of bacteria, microalgae, testate amoebae, and ciliates were dramatically and significantly decreased in both Pb addition treatments after 6, 12, and 20 weeks in comparison with the control. The biomass of cyanobacteria declined after 6 and 12 weeks in the highest Pb treatment. The biomasses of fungi, rotifers, and nematodes decreased along the duration of the experiment but were not significantly affected by lead addition. Consequently, the total microbial biomass was lower for both Pb addition treatments after 12 and 20 weeks than in the controls. The community structure was strongly modified due to changes in the densities of testate amoebae and ciliates, whereas the relative contribution of bacteria to the microbial biomass was stable. Differences in responses among the microbial groups suggest changes in the trophic links among them. The correlation between the biomass of bacteria and that of ciliates or testate amoebae increased with increasing Pb loading. We interpret this result as an effect on the grazing pathways of these predators and by the Pb effect on other potential prey (i.e., smaller protists). The community approach used here complements classical ecotoxicological studies by providing clues to the complex effect of pollutant-affecting organisms both directly and indirectly through trophic effects and could potentially find applications for pollution monitoring.

Relationship between testate amoeba (protist) communities and atmospheric heavy metals accumulated in Barbula indica (bryophyta) in Vietnam.

We studied the relationships between testate amoeba communities and heavy metal (Pb, Cd, Zn, Ni, Cu, Mn, and Fe) concentrations in the moss Barbula indica sampled at 29 sites in and around the city of Hanoi (Vietnam). Our first approach was to compare the heavy metal concentrations and testate amoeba variables between the city (zone 1) and the surrounding (zone 2). Mean moss concentrations of Pb, Cd, Zn, Ni, and Cu were significantly higher and testate amoeba species richness and abundance were significantly lower in zone 1 and the abundance of eight taxa differed significantly between the two zones. We then studied the correlation between heavy metals and testate amoebae. Species richness and abundance were correlated negatively to Pb concentration. Shannon H' was negatively correlated to both Pb and Cd. The abundance of several species was negatively correlated with Pb, Cd, Zn, and Ni; however, at the community level, Pb emerged as the only significant variable in a redundancy analysis. Our results suggest that testate amoebae are sensitive to and may be good bioindicators for heavy metal pollution, especially lead. Further research is needed to understand the causal relationships underlying the observed patterns.

Characterization and localization of a cheese georegion using edaphic criteria (Jura mountains, France).

Comté cheese, made from raw whole milk with local starter lactic acid bacteria, has been manufactured in the traditional manner in the French part of the Jura mountains since the 13th century. Its flavor vary not only by factors such as grass and hay, but also by geographical production sites. Connoisseurs are able to identify the origin of a certain number of cheeses, which raises the question of whether one can speak of georegions (terroirs) or specific geographical areas as in the case of wine. A new method of large-scale cartography (agro-pedological units) covering 20 cheese cooperatives provided a statistical comparison showing the different edaphic sectors. A sensory analysis of 106 cheeses from these 20 cooperatives (summer or winter cheese with 3 or 6 mo of ripening) has demonstrated Comté georegions corresponding to 85% of the edaphic sectors. Thus, these sectors are equivalent to cheese georegions that are natural entities represented synthetically by agropedological units that have their own vegetation and their own specific type of Comté cheese.

Water permeability and revolving movement in Phaseolus vulgaris L. twining shoots.

Osmotic water permeability (Pos) was measured in protoplasts isolated from different tissues of Phaseolus vulgaris twining shoot. Parenchyma protoplasts exhibited more frequently high Pos values than epidermis protoplasts did. Water channels could facilitate water movement between parenchyma cells whereas cell-to-cell water transport mostly occurs through plasmodesmata in epidermis.

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.

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.