Polymetallic Gradient Research Articles

Obtaining of Copper- and Nickel-Based Polymetallic Gradient Products by Wire-Feed Electron Beam Additive Manufacturing

The structure and mechanical properties of gradient transition zones of the copper-nickel system formed by additive electron beam technology have been investigated. Pure copper and nickel alloy Ni80Cr20 were used for printing. The data obtained testify to the complex and heterogeneous nature of structure formation when printing both by single-wire method and using double-wire controlled feeding of material into the melt bath. In the samples, the formation of defects of different scale from local inhomogeneities of the structure to pores and cracks is possible. The mechanical properties of the structural gradient zone are at a sufficiently high level and depend on the ratio of the system components.

Biomarker responses of Eisenia andrei to a polymetallic gradient near a lead mining site in North Tunisia

Eisenia andrei earthworms were exposed for 7 and 14 days to six samples of soil taken from around an abandoned lead (Pb) mine and characterized by different levels of metal contamination (S6-S1, this latter being the most contaminated soil). The organisms were analyzed for metal bioaccumulation and for biological parameters as biomarkers of stress (lysosomal membrane stability; lipofuscin lysosomal content; lysosomal/cytoplasmic volume ratio) and genotoxicity (Micronucleus frequency). Chemical analysis showed the loads of Pb, Cd, Zn, and Cu in the worms following exposure. Among the stress biomarkers, lysosomal membrane stability was significantly affected in the coelomocytes of the earthworms exposed already 7 days to different contaminated soils. Organisms exposed for 14 days to S1 showed in the cells of the chloragogenous tissue, a particularly relevant increase in lipofuscin, a biomarker of oxidative stress, and an increase in the lysosome/cytoplasm volume ratio, indicating stressful condition at the tissue level. Moreover, in the same conditions, a decrease in total body weight was observed. At the longer exposure time, the coelomocytes of worms exposed to S1, S2, and S3 (soils with higher metal concentrations) showed a significant increase in micronuclei (MNi) frequency. Expressions of the P21 and topoisomerase genes, which are involved in DNA repair, showed significant up-regulation in the cells of worms exposed to S1, S2, S3, S4 and to a less extend S6. This may indicate that the worms were only able to successfully reduce the level of DNA damage in S4 and S5 if considering MN frequency data. The biomarker data was integrated by the Earthworm Expert System, allowing an objective interpretation of the complex biological data and clearly defining the areas in which the presence of chemicals is toxic for the edaphic organisms.

Parallel changes in the taxonomical structure of bacterial communities exposed to a similar environmental disturbance

Bacterial communities play a central role in ecosystems, by regulating biogeochemical fluxes. Therefore, understanding how multiple functional interactions between species face environmental perturbations is a major concern in conservation biology. Because bacteria can use several strategies, including horizontal gene transfers (HGT), to cope with rapidly changing environmental conditions, potential decoupling between function and taxonomy makes the use of a given species as a general bioindicator problematic. The present work is a first step to characterize the impact of a recent polymetallic gradient over the taxonomical networks of five lacustrine bacterial communities. Given that evolutionary convergence represents one of the best illustration of natural selection, we focused on a system composed of two pairs of impacted and clean lakes in order to test whether similar perturbation exerts a comparable impact on the taxonomical networks of independent bacterial communities. First, we showed that similar environmental stress drove parallel structural changes at the taxonomic level on two independent bacterial communities. Second, we showed that a long-term exposure to contaminant gradients drove significant taxonomic structure changes within three interconnected bacterial communities. Thus, this model lake system is relevant to characterize the strategies, namely acclimation and/or adaptation, of bacterial communities facing environmental perturbations, such as metal contamination.

Effects of chronic metal exposure on wild fish populations revealed by high-throughput cDNA sequencing

Given the inherent variability of aquatic systems, predicting the in situ effects of contaminants on such ecosystems still represents a major challenge for ecotoxicology. In this context, transcriptomic tools can help identify and investigate the mechanisms of toxicity beyond the traditional morphometric, physiological and population-level endpoints. In this study, we used the 454 sequencing technology to examine the in situ effects of chronic metal (Cd, Cu) exposure on the yellow perch (Perca flavescens) transcriptome. Total hepatic mRNA from fish sampled along a polymetallic gradient was extracted, reverse transcribed, labeled with unique barcode sequences and sequenced. This approach allowed us to identify correlations between the transcription level of single genes and the hepatic concentrations of individual metals; 71% of the correlations established were negative. Chronic metal exposure was thus associated with a decrease in the transcription levels of numerous genes involved in protein biosynthesis, in the immune system, and in lipid and energy metabolism. Our results suggest that this marked decrease could result from an impairment of bile acid metabolism by Cd and energy restriction but also from the recruitment of several genes involved in epigenetic modifications of histones and DNA that lead to gene silencing.

Genotoxic damages in zebrafish submitted to a polymetallic gradient displayed by the Lot River (France)

Genotoxic effects of a polymetallic pollution gradient displayed by the Lot River and one of its tributary have been assessed on zebrafish Danio rerio. Three methods were compared: RAPD-PCR, the comet assay, and 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) formation. The fishes were exposed for 14 days to waters collected from three stations: Joanis, a site polluted by cadmium (Cd) and zinc (Zn) (mean concentrations: 15 μg Cd/L and 550 μg Zn/L), Bouillac (mean concentrations: 0.55 μg Cd/L and 80 μg Zn/L), and Boisse-Penchot, a reference station (mean concentrations:<0.05 μg Cd/L and 7 μg Zn/L). The quantitative RAPD-PCR methodology proved to be sensitive enough to unmask metal genotoxicity after 3 and 7 days of exposure to Joanis water and after 14 days to Bouillac water, whereas the comet assay only detected DNA damages at the most contaminated station (Joanis). The 8-oxodG quantification was not sensitive enough to be used in zebrafish under these environmental conditions.

Cadmium and zinc bioaccumulation and metallothionein response in two freshwater bivalves ( Corbicula fluminea and Dreissena polymorpha) transplanted along a polymetallic gradient

This study was designed to compare the metallothionein (MT) response capacity of two freshwater bivalves, Corbicula fluminea and Dreissena polymorpha, along an environmental gradient of polymetallic pollution. The bivalves were transplanted into 4 stations in southwestern France, with a cadmium and zinc pollution gradient. MT and metal concentrations were measured in the soft bodies of the clams and mussels over 2.5 months. In the zebra mussels, variations in MT concentrations were strictly correlated to progressive Cd and Zn bioaccumulation. In contrast, the faster response in the clams appeared positively correlated to Cd accumulation only, with the activation of efficient detoxification mechanisms which limited Cd bioaccumulation and reduced Zn concentrations over time. Nevertheless, despite stronger metal accumulation factors in D. polymorpha, C. fluminea revealed higher sensitivity of MT response along the pollution gradient.

Sub-cellular partitioning of cadmium, copper, nickel and zinc in indigenous yellow perch ( Perca flavescens) sampled along a polymetallic gradient

Sub-cellular metal distributions were studied in indigenous yellow perch ( Perca flavescens) collected from eight lakes located along a cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) concentration gradient. Ambient dissolved metal concentrations were measured to evaluate exposure and total hepatic metal concentrations were determined as a measure of metal bioaccumulation. Metal partitioning among potentially metal-sensitive fractions (cytosolic enzymes, organelles) and detoxified metal fractions (metallothionein) was determined after differential centrifugation of fish liver homogenates. Major proportions of hepatic Cd and Cu were found in the heat-stable cytosolic peptides and proteins fraction (HSP), a fraction including metallothioneins, whereas the potentially metal-sensitive heat-denaturable proteins fraction (HDP) was the largest contributor to the total Ni and Zn burdens. The concentrations of Cd, Cu and Ni (but not Zn) in each sub-cellular fraction increased along the metal contamination gradient, but the relative contributions of each fraction to the total burden of each of these metals remained generally constant. For these chronically exposed fish there was no threshold exposure concentration below which binding of Cd or Ni to the heat-denaturable protein fraction did not occur. The presence of Cd and Ni in the HDP fraction, even for low chronic exposure concentrations, suggests that metal detoxification was imperfect, i.e. that P. flavescens was subject to some metal-related stress even under these conditions.

Sub-cellular partitioning of metals (Cd, Cu, Zn) in the gills of a freshwater bivalve, Pyganodon grandis: role of calcium concretions in metal sequestration

Indigenous unionid molluscs, Pyganodon grandis, were collected from nine lakes in the Rouyn–Noranda area (Québec, Canada) along a polymetallic concentration gradient (Cd, Cu, Zn). After excision, the gills were gently homogenised and the cellular compartments were separated by a differential centrifugation procedure that yielded the following particulate fractions: “nuclei + cellular debris”, “mitochondria”, “lysosomes + microsomes” and “granules”. The supernatant remaining after the final ultracentrifugation step, i.e., the operationally-defined cytosol, was separated into a “heat-denaturable proteins” (HDP) fraction and a “heat-stable proteins” (HSP) fraction containing metallothionein (MT). The Cd, Cu and Zn content of each particulate and cytosolic fraction was determined and gill metallothionein was quantified independently by a mercury saturation assay. Cytosolic Cd concentrations were significantly related to the dissolved Cd concentrations at each site, but cytosolic Cu and Zn (essential metals) were not related to their respective ambient dissolved metal concentrations. Metallothionein concentrations increased along the metal contamination gradient and were related to cytosolic Cd (and Zn) in a concentration-dependent manner. However mass balance calculations showed that binding to metallothionein could only account for a small proportion of total gill metal (∼10% Cd; ∼3% Cu; ∼1% Zn). Under these chronic exposure conditions, the three metals (Cd, Cu and Zn) were mainly located in calcium concretions present in the gills (respectively 58 ± 13% of the total gill Cd, 64 ± 6% of the total gill Cu and 73 ± 6% of the total gill Zn). The overall contribution of granules to the total gill dry weight remained relatively constant among the different lakes, suggesting that lake-to-lake variations in granule synthesis were independent of the metal contamination gradient, i.e., these constituent elements of unionid gills act as non-inducible metal sinks at the cellular level. Metal concentrations increased proportionally in both the granules and the MT pool along the polymetallic gradient, suggesting a constant partitioning between these two compartments. Overall, despite an increase in Cd in the “mitochondria” fraction, metal sequestration mechanisms seem to be reasonably effective in detoxifying cadmium: in the cytosol, Cd concentrations in the potentially metal-sensitive HDP fraction remained relatively low and constant, even in specimens collected from the most contaminated lakes.

Study of periphytic diatoms communities exposed to metallic contamination through complementary field and laboratory experiments

Periphytic diatom communities are important primary producers in streams where their characteristics are influenced by many environmental factors. To study and cut off real effects of metal pollution (Cd, Zn) on these communities, complementary experiments were performed in order to precise short term effects of pollution during a four weeks colonization process. Early stage of diatom communities exposed to pollution was studied first in outdoor artificial streams connected to a subsystem of the Lot river (SW France), characterized by a polymetallic gradient. Colonization of glass substrates was followed each week in three sites contaminated between 0 to 10 μg Cd.L -1 . In a second step, an experiment was conducted into indoor artificial streams inseminated with homogenized periphyton suspension of the reference stream in situ. Initial development of communities was followed on glass substrates too and collected after two and four weeks of Cadmium exposure to three different metallic concentrations (control in laboratory, 10 μg Cd.L -1 and 100 μg Cd.L -1 ). Structure of diatom communities was assessed by studying density and taxonomic composition. Results of the different approaches are compared and point out marked effects of metallic pollution with clear decrease of diatom global density and perturbation of species composition.

Steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient

The present study was designed to assess the environmental effects of metals in a field setting. We explored exposure→bioaccumulation→effects relationships in freshwater molluscs exposed to metals in their natural habitat. Indigenous floater mussels ( Pyganodon grandis) were collected from ten limnologically similar lakes located along a Cd, Cu and Zn gradient. Ambient free-metal ion concentrations were estimated as a measure of metal exposure. Metallothionein (MT) was measured in mussel gills and metal partitioning among the various cytosolic protein pools was determined by size exclusion chromatography. Various biomarkers were also measured, including malondialdehyde (MDA) concentrations in the gills and in the digestive gland, glutathione-peroxidase and glutathione-reductase activities in the digestive gland, and lipid concentrations in the gonad. Cadmium and MT concentrations in the gill cytosol increased along the contamination gradient, but Cu and Zn levels were independent of the ambient free-metal ion concentrations. The distribution of Cd among the various cytosolic complexes remained quite constant: 80% in the MT-like pool, 7% in the low molecular weight pool (LMW<1.8 kDa) and 13% in the high molecular weight pool (HMW>18 kDa). For these chronically exposed molluscs there was thus no threshold exposure concentration above which spillover of Cd occurred from the MT pool to other cytosolic ligands. However, the presence of Cd in the LMW and HMW fractions suggests that metal detoxification was imperfect, i.e. that P. grandis was subject to some Cd-related stress at low chronic exposure concentrations. Consistent with this suggestion, MDA concentrations, an indicator of oxidative stress, increased with gill cytosolic Cd. In the digestive gland, MDA concentrations were unrelated to any of the measured metals, but glutathione-peroxidase and glutathione-reductase activities increased with gill cytosolic copper. We speculate that cytosolic Cu catalyses the production of reactive oxygen species, to which the organism reacts by increasing activities of the two enzymes, thus preventing the accumulation of reactive oxygen species. Lipid concentrations in the gonad did not decrease with any of the measured toxicological parameters, suggesting that energy reserves for reproduction were not compromised in the metal-contaminated mussels. The results of the present study, where chronically exposed bivalves were collected from their natural habitat along a metal contamination gradient, contrast markedly with what would have been predicted on the basis of experimental metal exposures, and clearly demonstrate the need to study metal exposure→bioaccumulation→effects relationships in natural populations.

Field transplantation of the freshwater bivalve Corbicula fluminea along a polymetallic contamination gradient (river Lot, France): I. Geochemical characteristics of the sampling sites and cadmium and zinc bioaccumulation kinetics

AbstractSpecimens of the Asiatic clam Corbicula fluminea were transplanted from a clean lacustrine site to four stations along a polymetallic gradient in the river Lot (France), downstream from an old Zn ore treatment facility. The bivalves were held in benthic cages for a 5‐month exposure period, April to September 1996; mollusk growth and metal bioaccumulation kinetics (Cd, Zn) were followed by subsampling the cages at t = 0, 21, 49, 85, 120, and 150 d. Rates of Cd bioaccumulation in the whole soft bodies and in individual organs were greater at the upstream stations located close to the pollution source, but there was no direct proportionality between Cd in the bivalves and in the unfiltered or filtered river water samples. Unlike the case for Cd, rates of Zn bioaccumulation did not reflect the contamination gradient. Marked growth differences were measured among the four stations, reflecting both nutritional differences and changes in the degree of metal contamination; these growth differences produced markedly different trends when metal bioaccumulation was expressed in terms of burdens rather than concentrations.