Michel Warnau

Distribution of heavy metals in the echinoid Paracentrotus lividus from the Mediterranean Posidonia oceanica ecosystem: seasonal and geographical variations

The concentrations of Zn, Pb, Cd, Fe, Cr, Cu, and Ti were measured in specimens of the echinoid Paracentrotus lividus from three Posidonia oceanica meadows in the NW Mediterranean (Marseille, France; Ischia, Italy; Calvi, Corsica). Investigated metals were selectively distributed among the echinoid body compartments (i.e. body wall, Aristotles lantern, digestive wall, and gonads); their concentrations were influenced by season. The present work emphasises the need to consider both body compartment and sampling period as important factors in studies intending to use P. lividus as a biomonitor species. In particular, digestive wall and, secondarily, body wall and gonads are recommended for use in biomonitoring programmes, though with some restrictions for gonads. Concentrations of most metals differed significantly in the echinoid body compartments according to the seagrass meadow considered. However, these differences were generally of low amplitude, except for Fe and Ti (contaminations by these two metals showed significant departures from background concentrations in echinoids from Ischia). Results suggest that the meadows of Calvi and Marseille, although subject to different pollution status (the meadow of Calvi is considered as a reference meadow while the Marseille meadow is exposed to intense domestic and industrial discharges), showed similar patterns of metal contamination.

Validation of the asteroid Asterias rubens (Echinodermata) as a bioindicator of spatial and temporal trends of Pb, Cd, and Zn contamination in the field

The aim of the present study was to assess the value of Asterias rubens as a bioindicator of metal contamination in the field. Spatial gradients of Pb, Cd, and Zn contamination were identified along Sorfjord, and metal concentrations were consistently higher (1.7–8.3 times) in the body compartments of large asteroids sampled close to the smelters than in those sampled further away. Cd and Pb concentrations in the pyloric caeca varied along the fjord according to a decreasing sigmoidal relationship, while Cd and Pb concentrations in the body wall and the skeleton varied according to a decreasing exponential relationship. This suggests that the bioavailability of these metals has decreased over the last few years at the head of the fjord, which agrees generally with water chemistry data available from the State Pollution Monitoring Programme (Norway). The decrease in metal bioavailability could be a direct consequence of the 1992 remedial action taken in the fjord to reduce heavy metal release from the contaminated sediments. Lead concentrations in the skeletons of large asteroids (12–14cm; 154 ± 63 μg Pb g−1 dry wt) were eight times higher than those in the skeletons of juvenile individuals (0.5–4 cm; 19 ± 8 μg Pb g−1 dry wt) in the population located at the head of the fjord. Asteroids of 12–14 cm length were likely to be 4–7 y old (i.e. born before the remedial action) while small asteroids were born after the remedial action. According to comparisons with allometric relationships in an uncontaminated population and experimental exposures, results indicate that the half-life of Pb in the skeleton is long (i.e. several years) while the half-life of Cd and Zn is much shorter (i.e. several weeks for Cd, and short but not precisely known for Zn). A smaller size and unusual reduction of the skeleton of adult individuals at the head of the fjord were observed and may result from the effects of heavy metal pollution. A comparison between possible sources of contamination in the fjord (metals in water, food, and sediments) indicates that sediment-associated metals are likely to be an important source of contamination in this environment due to the microphagic activity of asteroids.

Allometry of heavy metal bioconcentration in the echinoid Paracentrotus lividus

The relationships between concentration and size in Paracentrotus lividus were studied for seven metals (Zn, Pb, Cd, Fe, Cr, Cu, and Ti). In the calcified body compartment, Zn concentrations showed inverse power relationships, while concentrations of Pb, Cd, and Cr increased as power functions of echinoid size; Fe, Cu, and Ti concentrations did not vary significantly with echinoid size. In the non-calcified body compartment of the echinoid, the concentrations of Zn, Pb, Fe, Cu, and Ti showed inverse power relationships, Cd concentrations increased linearly, and Cr concentrations did not show any significant relationship with echinoid body size. Most relationships were rather weak, except in the cases of Pb and Fe in the non-calcified compartment and Cd in the calcified compartment. The allometric relationships were affected by seasonal factor in the non-calcified compartment only. The results indicate that biomonitoring programs using P. lividus as an indicator of metal contamination must take into account both echinoid size and season as source of metal concentration variations.

Factors influencing the concentrations of heavy metals in the asteroid Asterias rubens L. (Echinodermata)

Abstract Seasonal, geographical, anatomical and sex-related variations in heavy metal concentrations were investigated in the asteroid Asterias rubens collected in four representative biotopes of the NE Atlantic at four periods of an annual gametogenic cycle. Zn, Pb, Cd, Fe, Cr, Cu, Ti and Hg concentrations were measured in the gonads, the central digestive tract, the pyloric caeca, the body wall and the skeleton. According to multi-way analyses of variance, the factors considered in the present study accounted for a large proportion (59–93%) of total variability in concentrations of all metals except Hg (26%). The body compartment appeared as the most important factor (9.7 – 88% of total variability). Interactions between the body compartment and the sampling location factors accounted for up to 29% of total variability indicating that bioaccumulation differed from site to site. Weak interactions between the body compartment and the sampling period factors indicated that bioaccumulation was synchronous within the asteroid body. Concentrations of groups Ib and IIb metals and Fe were significantly higher in the pyloric caeca. The other elements were preferentially accumulated in the skeleton. Most bioconcentration factors (BCF) towards prey were lower than 1 in asteroids feeding on filter-feeders (ascidians and mussels). On the contrary, most BCF in asteroids feeding on grazers (gastropods) were higher than 1. Sex-related differences in the gonads were significant for Zn, Cd, Fe and Cr concentrations (variation ratio: 1.7 – 3.3).

Uptake and Fate of Lead in the Common Asteroid Asterias Rubens (Echinodermata)

Lead uptake kinetics in the body compartments of Asterias rubens individuals exposed through sea water to different concentrations (5, 20, 50 µg Pb l-1) were studied during 30 days. Uptake kinetics were always best described by linear regressions. In every compartment, uptake rate increased significantly with the contaminating concentration. At a given concentration, the uptake rate of the central digestive tract was always significantly higher than that of the other compartments. After short-term exposures (50 µg Pb l-1, 8 days), fate of accumulated Pb was followed in asteroids kept in non-contaminated water during 20 days. The central digestive tract and the pyloric caeca lost Pb according to an inverse exponential trend and concentrations returned to initial values after 4 to 8 days. Pb concentrations in the body wall decreased linearly. Pb concentrations in the skeleton did not vary significantly. In the gonads, Pb concentrations kept on rising while exogenous Pb was no more supplied. Pb loss from the body wall and the digestive compartments could account for such endogenous source to the gonads. It is suggested that Pb accumulated in the gonads is expulsed later through spawning. According to the parameters of uptake and loss kinetics, the central digestive tract and the skeleton are proposed as complementary temporal bioindicators of Pb contamination.

Quantification of metallothioneins in the common asteroid Asterias rubens (Echinodermata) exposed experimentally or naturally to cadmium

Abstract Uptake and intracellular fate of Cd was assessed in the pyloric caeca of the common asteroid Asterias rubens exposed experimentally or naturally to Cd. Cd partitioning among intracellular metal-binding pools was studied by graphite furnace atomic absorption spectrometry of heat-treated cytosolic fractions which had been separated by gel filtration chromatography. Low molecular mass proteins bound > 80% of cytosolic Cd and appeared to become saturated with the metal after experimental exposure to 20 μg Cd 1 −1 for 10–20 days. These proteins presented several features of metallothioneins (MTs), namely molecular mass (13–22 kDa), heat-stability (90 °C, 10 min), high Cd content, and high thiolic content as determined by differential pulse polarography. Analysis of Chromatographie fractions indicated that the majority of thiolic groups (66–73%) present in heat-treated cytosol was associated with the MT pool. Subsequent determinations of MT concentrations were made using whole cytosol extracts after correcting for interference from thiolic groups present in residual, heat-stable, high molecular mass proteins. Asteroids collected from unpolluted sites in SW England, SW Netherlands and SW Norway contained basal levels of 2.5 – 4.5 mg MT g −1 dw. Asteroids experimentally exposed to Cd (20 μg Cd 1 −1 ) responded by a 2-fold increase in the production of MT after 30 days. Turnover of MTs was rapid in A. rubens and probably accounted for the low net increase in protein and characteristic rapid loss of Cd from the pyloric caeca. Alkaline phosphatase (AP) activity was measured as a marker of the functional state of the pyloric caeca. It was not significantly affected during Cd exposure in the laboratory. Asteroids collected in the heavy metal polluted Sorfjord (SW Norway) contained significantly higher MT concentrations (5 – 5.6 mg MT g −1 dw) and significantly lower AP activity in the most heavily contaminated region, though these two parameters were not correlated.

Iron Oxidation and Deposition in the Biofilm Covering Montacuta ferruginosa (Mollusca, Bivalvia)

The shell of the bivalve Montacuta ferruginosa is covered with a rust-colored biofilm. This biofilm includes filamentous bacteria and protozoa encrusted with a mineral, rich in ferric ion and phosphate. The aim of this research was to study two possible microbial iron precipitation pathways in the biofilm, namely, microbial iron oxidation and microbial degradation of organic Fe(III) complexes. The iron-oxidizing activity was assayed spectrophotometrically by monitoring the formation of the dye Wurster blue in biofilm extracts. Iron-oxidizing activity was effectively detected in extracts obtained by oxalic acid treatment of biofilm fragments. Extracts obtained without oxalic acid treatment, heated extracts, or extracts supplemented with HgCl 2 did not show any activity. This suggests that an iron-oxidizing factor (IOF), possibly an enzyme, coprecipitated with the mineral. Additional information gathered by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel-filtration chromatography, and UV spectrophotometry indicate that the IOF would be a small peptide or glycopeptide (1,350 Da). Microbial degradation of organic Fe(III) complexes was assayed with biofilm fragments incubated in a medium containing ferric citrate. Analysis of the supernatants after various intervals revealed that the complex was degraded by living microorganisms much faster than in the heat-killed negative controls. We conclude that ferric iron precipitation in the biofilm may proceed by way of microbial Fe(II) oxidation as well as microbial degradation of organic Fe(III) complexes.The shell of the bivalve Montacuta ferruginosa is covered with a rust-colored biofilm. This biofilm includes filamentous bacteria and protozoa encrusted with a mineral, rich in ferric ion and phosphate. The aim of this research was to study two possible microbial iron precipitation pathways in the biofilm, namely, microbial iron oxidation and microbial degradation of organic Fe(III) complexes. The iron-oxidizing activity was assayed spectrophotometrically by monitoring the formation of the dye Wurster blue in biofilm extracts. Iron-oxidizing activity was effectively detected in extracts obtained by oxalic acid treatment of biofilm fragments. Extracts obtained without oxalic acid treatment, heated extracts, or extracts supplemented with H(g)Cl2 did not show any activity. This suggests that an iron-oxidizing factor (IOF), possibly an enzyme, coprecipitated with the mineral. Additional information gathered by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel-filtration chromatography, and UV spectrophotometry indicate that the IOF would be a small peptide or glycopeptide (1,350 Da). Microbial degradation of organic Fe(III) complexes was assayed with biofilm fragments incubated in a medium containing ferric citrate. Analysis of the supernatants after various intervals revealed that the complex was degraded by living microorganisms much faster than in the heat-killed negative controls. We conclude that ferric iron precipitation in the biofilm may proceed by way of microbial Fe(II) oxidation as well as microbial degradation of organic Fe(III) complexes.

High sensitivity of skeletogenesis to Pb in the asteroid Asterias rubens (Echinodermata)

Abstract The effects of lead bioaccumulation on skeleton morphogenesis was ascertained in the common North Atlantic asteroid Asterias rubens. Regeneration of adambulacral spines was used as an experimental model. Spine regeneration was initiated in asteroids exposed experimentally to 1, 5, 20, or 50 μg Pb l−1 for 15 days. Pb bioaccumulation in the analysed body compartments was dose-dependent. Measures by atomic absorption spectrometry and proton induced X-ray emission microscopy showed that lead effectively reached the skeleton. Scanning electron microscopy showed that skeleton morphogenesis was affected: the trabeculae were shorter and more stocky, secondary bridges were not formed after 15 days, trabeculae outline was irregular and mineral deposition was highly reduced. The size of the regenerates was significantly smaller in the individuals exposed to concentrations ≥5 μg Pb l−1 (up to 70% reduction). Alkaline phosphatase activity was measured as a marker of the functional state of the body wall: it significantly decreased (30% reduction) only in individuals exposed to 50 μg Pb l−1. These results indicate that, in comparison with other phyla, skeletogenesis in asteroids is particularly sensitive to Pb and that Pb toxic effects could arise directly from the incorporation of the metal in the skeleton.