Combined effects of alpha particles and depleted uranium on Zebrafish (Danio rerio) embryos.

The combined effects of low-dose or high-dose alpha particles and depleted uranium (DU) in Zebrafish (Danio rerio) embryos were studied. Three schemes were examined-(i) [ILUL]: 0.44 mGy alpha-particle dose + 10 µg/l DU exposure, (ii) [IHUH]: 4.4 mGy alpha-particle dose + 100 µg/l DU exposure and (iii) [IHUL]: 4.4 mGy alpha-particle dose + 10 µg/l DU exposure-in which Zebrafish embryos were irradiated with alpha particles at 5 h post fertilization (hpf) and/or exposed to uranium at 5-6 hpf. The results were also compared with our previous work, which studied the effects of [ILUH]: 0.44 mGy alpha-particle dose + 100 µg/l DU exposure. When the Zebrafish embryos developed to 24 hpf, the apoptotic signals in the entire embryos, used as the biological endpoint for this study, were quantified. Our results showed that [ILUL] and [IHUL] led to antagonistic effects, whereas [IHUH] led to an additive effect. The effect found for the previously studied case of [ILUH] was difficult to define because it was synergistic with reference to the 100 µg/l DU exposure, but it was antagonistic with reference to the 0.44 mGy alpha-particle dose. All the findings regarding the four different schemes showed that the combined effects critically depended on the dose response to each individual stressor. We also qualitatively explained these findings in terms of promotion of early death of cells predisposed to spontaneous transformation by alpha particles, interacting with the delay in cell death resulting from various concentrations of DU exposure.

Uranium accumulation and toxicity in the green alga Chlamydomonas reinhardtii is modulated by pH.

The effects of pH on metal uptake and toxicity in aquatic organisms are currently poorly understood and remain an evolving topic in studies about the biotic ligand model (BLM). In the present study, the authors investigated how pH may influence long-term (4 d) uranium (U) accumulation and chronic toxicity in batch cultures of the freshwater green alga Chlamydomonas reinhardtii. The toxicity expressed as a function of the free uranyl ion was much greater at pH 7 (effective concentration, 50% [EC50] = 1.8 × 10(-9) M UO2 (2+) ) than at pH 5 (EC50 = 1.2 × 10(-7) M UO2 (2+) ). The net accumulation rate of U in algal cells was much higher at pH 7 than at pH 5 for the same free [UO2 (2+) ], but the cells exposed at pH 5 were also more sensitive to intracellular U than the cells at pH 7 with EC50s of 4.0 × 10(-15) and 7.1 × 10(-13) mol of internalized U cell(-1) , respectively. The higher cellular sensitivity to U at pH 5 than at pH 7 could be explained partly by the increase in cytosolic U binding to algal soluble proteins or enzymes at pH 5 as observed by subcellular fractionation. To predict U accumulation and toxicity in algae accurately, the important modulating effects of pH on U accumulation and U cellular sensitivity should be considered in the BLM.

Genotoxicity of uranium contamination in embryonic zebrafish cells.

Uranium is a metal used in the nuclear industry and for military applications. Studies on mammals have shown that uranium is genotoxic. However the molecular and cellular mechanisms responsible for the genotoxicity of uranium are poorly known for other types of vertebrates such as fish. Since unrepaired DNA double-strand breaks (DSBs) are considered to be key lesions in cell lethality, the activity of one of the major DSB-repair pathways, i.e. non-homologous end-joining (NHEJ), has been evaluated in embryonic zebrafish cells (ZF4) exposed to uranium. Genotoxicity of uranium in ZF4 cells was further assessed by comet and micronucleus assays. Exposure to uranium results in the production of DSBs a few hours after incubation. These breaks trigger the phosphorylation of H2AX proteins. We showed that the DNA-PK kinase activity, essential for NHEJ, is altered by the presence of uranium. The presence of uranium in cells disturbs but does not inhibit the repair rate of DSBs. Such a result suggests an impact of uranium upon the reparability of DSBs and the potential activation of alternative DSBs repair pathway leading to the propagation of possible misrepaired DSBs. In parallel, we performed a transmission electron microscopy analysis of cells exposed to uranium and were able to localize internalized uranium using an Energy Dispersive X-ray microanalyser. We observed the formation of precipitates in lysosome-like vesicles for 250 µM of uranium in the medium. The appearance of these precipitates is concomitant with the decrease of the number of DSBs per cell. This process might be a part of a defence system whose role in counteracting cytotoxicity calls for further dedicated research.

Internal distribution of uranium and associated genotoxic damages in the chronically exposed bivalve Corbicula fluminea.

Uranium (U) internal distribution and involved effects in the bivalve Corbicula fluminea have been studied after direct chronic exposure (90 d, 10 µg.L-1). U distribution was assessed at the subcellular level (Metal Rich Granules -MRG-, pellets and cytosol fractions) in two main organs of the bivalve (gills and visceral mass). Micro-localisation was investigated by TEM-EDX analysis in the gills epithelium. DNA damage in gill and hemolymph samples was measured by the Comet assay. The 90-d exposure period led to a significant increase of U concentration in gills over time (× 5) and a large U quantity in subcellular granules in gills. Finally, a significant increase (× 2) in DNA damage was noted in exposed gills and haemocytes. This study shows that the accumulation levels and consequently the potential toxicity cannot be successfully predicted only on the basis of concentration in water or in tissues and subcellular fractions after chronic exposure.

Mitochondrial energetic metabolism perturbations in skeletal muscles and brain of zebrafish (Danio rerio) exposed to low concentrations of waterborne uranium.

Anthropogenic release of uranium (U), originating from the nuclear fuel cycle or military activities, may considerably increase U concentrations in terrestrial and aquatic ecosystems above the naturally occurring background levels found throughout the environment. With a projected increase in the world-wide use of nuclear power, it is important to improve our understanding of the possible effects of this metal on the aquatic fauna at concentrations commensurate with the provisional drinking water guideline value of the World Health Organization (15 µg U/L). The present study has examined the mitochondrial function in brain and skeletal muscles of the zebrafish, Danio rerio, exposed to 30 and 100 µg/L of waterborne U for 10 and 28 days. At the lower concentration, the basal mitochondrial respiration rate was increased in brain at day 10 and in muscles at day 28. This is due to an increase of the inner mitochondrial membrane permeability, resulting in a decrease of the respiratory control ratio. In addition, levels of cytochrome c oxidase subunit IV (COX-IV) increased in brain at day 10, and those of COX-I increased in muscles at day 28. Histological analyses performed by transmission electron microscopy revealed an alteration of myofibrils and a dilatation of endomysium in muscle cells. These effects were largest at the lowest concentration, following 28 days of exposure.

Effects of chronic uranium exposure on life history and physiology of Daphnia magna over three successive generations.

Daphnia magna was exposed to waterborne uranium (U) at concentrations ranging from 10 to 75 microgL(-1) over three successive generations (F0, F1 and F2). Progeny was either exposed to the same concentration as mothers to test whether susceptibility to this radioelement might vary across generations or returned to a clean medium to examine their capacity to recover after parental exposure. Maximum body burdens of 17, 32 and 54 ng U daphnid(-1) were measured in the different exposure conditions and converted to corresponding internal alpha dose rates. Low values of 5, 12 and 20 microGy h(-1) suggested that radiotoxicity was negligible compared to chemotoxicity. An increasing sensitivity to toxicity was shown across exposed generations with significant effects observed on life history traits and physiology as low as 10 microgL(-1) and a capacity to recover partially in a clean medium after parental exposure to

Sublethal effects of waterborne uranium exposures on the zebrafish brain: transcriptional responses and alterations of the olfactory bulb ultrastructure.

The toxic action modes of uranium (U) in fish are still scarcely known. U is known to modify the acetylcholinesterase activity in the fish brain. To gain further insight into U neurotoxicity in fish, we examined transcriptional responses in the brain of the zebrafish, Danio rerio, exposed to 15 microg L(-1) and 100 microg L(-1) of waterborne U for 3 and 10 days. In parallel, an ultrastructure analysis of the neuropil of the olfactory bulb, an area in the brain of fish sensitive to metal contamination, was performed after 10 days of U exposure. This combined transcriptomic and histological study is the first report performed in the brain and specifically the olfactory bulb of fish exposed to U. We found that 56 transcripts responded to the metal exposure, and the anatomical structure of the olfactory bulb was damaged. The greatest gene response occurred at the lower U concentration and the numbers of responding genes common to any two U exposures were much smaller than those unique to each exposure. These data showed that the intensity of gene response may not correlate positively with toxicant concentrations according to our experimental design. Instead, different patterns of gene expression are expected for each exposure. Gene responses were categorized into eight functional classes, and the transcriptional responses of genes involved in the olfactory system were significantly affected. Collectively, the data suggest that genes in the olfactory region may be ecologically relevant and sensitive transcriptional biomarkers of U waterborne exposure.

A probabilistic assessment of the chemical and radiological risks of chronic exposure to uranium in freshwater ecosystems.

Uranium (U) presents a unique challenge for ecological risk assessments (ERA) because it induces both chemical and radiological toxicity, and the relative importance of these two toxicities differs among the various U source terms (i.e., natural, enriched, depleted). We present a method for the conversion between chemical concentrations microg L(-1)) and radiological dose rates (microGy h(-1)) for a defined set of reference organisms, and apply this conversion method to previously derived chemical and radiological benchmarks to determine the extent to which these benchmarks ensure radiological and chemical protection, respectively, for U in freshwater ecosystems. Results show that the percentage of species radiologically protected by the chemical benchmark decreases with increasing degrees of U enrichment and with increasing periods of radioactive decay. In contrast, the freshwater ecosystem is almost never chemically protected by the radiological benchmark, regardless of the source term or decay period considered, confirming that the risks to the environment from uranium's chemical toxicity generally outweigh those of its radiological toxicity. These results are relevant to developing water quality criteria that protect freshwater ecosystems from the various risks associated with the nuclear applications of U exploitation, and highlight the need for (1) further research on the speciation, bioavailability, and toxicity of U-series radionuclides under different environmental conditions, and (2) the adoption of both chemical and radiological benchmarks for coherent ERAs to be conducted in U-contaminated freshwater ecosystems.

Comparative analysis of gene expression in brain, liver, skeletal muscles, and gills of zebrafish (Danio rerio) exposed to environmentally relevant waterborne uranium concentrations.

The effects of waterborne uranium (U) exposure on gene expression were examined in four organs (brain, liver, skeletal muscles, and gills) of the zebrafish (Danio rerio). Adult male fish were exposed to three treatments: No added uranium (control), 23 +/- 6 microg U/L, and 130 +/- 34 microg U/L. After 3, 10, 21, and 28 d of exposure and an 8-d depuration period, gene expression and uranium bioaccumulation were analyzed. Bioaccumulation decreased significantly in liver during the depuration phase, and genes involved in detoxification, apoptotic mechanism, and immune response were strongly induced. Among these genes, abcb311, which belongs to the adenosine triphosphate (ATP)-binding cassette transporter family, was induced 4- and 24-fold in organisms previously exposed to 23 +/- 6 and 130 +/- 34 microg U/L, respectively. These results highlight the role of liver in detoxification mechanisms. In gills, at the highest uranium concentration, gpx1a, cat1, sod1, and sod2 genes were up-regulated at day 21, indicating the onset of an oxidative stress. Mitochondrial metabolism and DNA integrity also were affected, because coxI, atp5f1, and rad51 genes were up-regulated at day 21 and during the depuration phase. In skeletal muscles, coxI, atp5f1, and cat were induced at day 3, suggesting an impact on the mitochondrial metabolism and production of reactive oxygen species. In brain, glsI also was induced at day 3, suggesting a need in the glutamate synthesis involved with neuron transmission. No changes in gene expression were observed in brain and skeletal muscles at days 21 and 28, although bioaccumulation increased. During the depuration phase, uranium excretion was inefficient in brain and skeletal muscles, and expression of most of the tissue-specific genes was repressed or unchanged.

Impact of hypoxia on hemolymph contamination by uranium in an aquatic animal, the freshwater clam Corbicula fluminea.

Multi-stress situations are a major question and low-oxygenated waters (hypoxia) are a growing problem. Importantly, hypoxia stimulates the ventilatory flow rate in aquatic animals and this increases gill exposure to contaminants. Surprisingly, in the freshwater clam Corbicula fluminea, this is associated with increased bioaccumulation of uranium in gills but not in deep tissues. We searched for an explanation by analyzing hemolymph U-transport in Corbicula exposed to 0.36 microM dissolved uranium at various O2-levels for 10 days. In hypoxia, one observed an increased U concentration in the arterial hemolymph flowing from gills to tissues but this was not associated with an increased U concentration in the venous hemolymph nor in the other tissues. We conclude that the cardiac flow rate must have decreased to explain this absence of over-accumulation. In addition to its already known deleterious effects, uranium can thus deeply impair cardiac flow rate in exposed aquatic animals during multi-stress exposures.

Effects of waterborne uranium on survival, growth, reproduction and physiological processes of the freshwater cladoceran Daphnia magna.

Acute uranium toxicity (48 h immobilisation test) for Daphnia magna was determined in two different exposure media, differing in pH and alkalinity. LC(50) varied strongly between media, from 390+/-40 microgL(-1)U at pH 7 to 7.8+/-3.2 mgL(-1)U at pH 8. According to the free ion activity model uranium toxicity varies as a function of free uranyl concentration. This assumption was examined by calculating uranium speciation in our water conditions and in those reported in the literature. Predicted changes in free uranyl concentration could not solely explain observed differences in toxicity, which might be due to a competition or a non-competitive inhibition of H(+) for uranium transport and/or the involvement of other bioavailable chemical species of uranium. Chronic effects of uranium at pH 7 on mortality, ingestion and respiration, fecundity and dry mass of females, eggs and neonates were investigated during 21-day exposure experiments. A mortality of 10% was observed at 100 microgL(-1)U and EC(10) for reproduction was 14+/-7 microgL(-1)U. Scope for growth was affected through a reduction in feeding activity and an increase in oxygen consumption at 25 microgL(-1)U after 7 days of exposure. This had strong consequences for somatic growth and reproduction, which decreased, respectively, by 50% and 65% at 50 microgL(-1)U after 7 days and at 25 microgL(-1)U after 21 days. Uranium bioaccumulation was quantified and associated internal alpha dose rates from 2.1 to 13 microGyh(-1) were estimated. Compared to the toxicity of other alpha-emitting radionuclides and stable trace metals, our results confirmed the general assumption that uranium chemical toxicity predominates over its radiotoxicity.

Metal-phytoplankton interactions: modeling the effect of competing ions (H+, Ca2+, and Mg2+) on uranium uptake.

The influence of pH and hardness cation concentrations on uranium uptake by a green alga, Chlamydomonas reinhardtii, was investigated through short-term exposure experiments. Uranium uptake at pH 5 and at pH 7 was measured over a large concentration range (0.020-2.0 microM 233U), and the effects of hardness cations were studied over environmentally pertinent concentration ranges (approximately 0.05-2 mM) at a constant uranium concentration (0.25 microM). Calcium and magnesium inhibited uranyl uptake, but the influence of pH was more complex than anticipated. The equilibrium biotic ligand paradigm of metal bioavailability predicts that two distinct phenomena of antipathetic effect will influence uranium availability as pH is varied. Increasing pH reduces the concentration of protons, thus reducing competition for the physiologically active sites, whereas the concomitant complexation by carbonates and hydroxides reduces the free uranyl activity. Maximum uranium uptake rates observed at pH 7, however, were far greater than those observed at pH 5, suggesting a noncompetitive inhibition of metal transport by protons. Modeling on the basis of our results strongly suggests that cells grown and exposed at pH 7 have either a greater internalization rate of uranyl or a higher number of transport sites compared with cells grown and exposed at pH 5. We thus conclude that the simple proton-metal competition described by the biotic ligand model cannot successfully depict uranium-algae interactions. The development of an appropriate model incorporating the influence of protons to predict metal uptake and toxicity will be more challenging than anticipated.

Effect of selenate on growth and photosynthesis of Chlamydomonas reinhardtii.

Algal communities play a crucial role in aquatic food webs by facilitating the transfer of dissolved inorganic selenium (both an essential trace element and a toxic compound for a wide variety of organisms) to higher trophic levels. The dominant inorganic chemical species of selenium in freshwaters are selenite (SeO(3)(2-)) and selenate (SeO(4)(2-)). At environmental concentrations, selenite is not likely to have direct toxic effects on phytoplankton growth [Morlon, H., Fortin, C., Floriani, M., Adam, C., Garnier-Laplace, J., Boudou, A., 2005a. Toxicity of selenite in the unicellular green alga Chlamydomonas reinharditii: comparison between effects at the population and sub-cellular level. Aquat. Toxicol. 73(1), 65-78]. The effects of selenate, on the other hand, are poorly documented. We studied the effects of selenate on Chlamydomonas reinhardtii growth (a common parameter in phytotoxicity tests). Growth inhibition (96-h IC(50)) was observed at 4.5+/-0.2 microM selenate (p<0.001), an effective concentration which is low compared to environmental concentrations. Growth inhibition at high selenium concentrations may result from impaired photosynthesis. This is why we also studied the effects of selenate on the photosynthetic process (not previously assessed in this species to our knowledge) as well as selenate's effects on cell ultrastructure. The observed ultrastructural damage (chloroplast alterations, loss of appressed domains) confirmed that chloroplasts are important targets in the mechanism of selenium toxicity. Furthermore, the inhibition of photosynthetic electron transport evaluated by chlorophyll fluorescence induction confirmed this hypothesis and demonstrated that selenate disrupts the photosynthetic electron chain. Compared to the classical 'growth inhibition' parameter used in phytotoxicity tests, cell diameter and operational photosynthetic yield were more sensitive and may be convenient tools for selenate toxicity assessment in non-target plants.

Selenium bioaccumulation in Chlamydomonas reinhardtii and subsequent transfer to Corbicula fluminea: role of selenium speciation and bivalve ventilation.

The uptake of Se by the freshwater alga Chlamydomonas reinhardtii and the subsequent transfer to the Asiatic clam Corbicula fluminea was investigated. The objective was to investigate the bioavailability of algal-bound Se for C. fluminea while taking into account Se speciation and bivalve ventilation. First, uptake rates of waterborne Se (selenite, selenate, and selenomethionine) in the algae during a 1-h exposure period were determined for a range of concentrations up to 2,000 microg/L. Fluxes for selenite uptake were constant in the range of concentrations tested, whereas fluxes for selenate and selenomethionine uptake decreased with increasing concentrations, suggesting a saturated transport system at high concentrations (approximately 1,000 microg/L for selenate and 100 microg/L for selenomethionine). These data were used to set the algal contamination for the study of trophic transfer to the clam. Three parameters were studied: The Se form, the algal density, and the Se burden in the algae. The results show that for a fixed algal density, an Se-contaminated algal diet does not modify ventilation. In this case, the driving factor for ventilation is the algal density, with ventilation being enhanced for low algal densities. On the basis of ventilatory flow rate measurements and Se burdens in algae, it was found that bioaccumulation of Se in C. fluminea was proportional to the total quantity of Se passing through the whole organism, but with a lesser extraction coefficient for selenomethionine than for the inorganic forms. These results underline the importance of both physiological factors and speciation in understanding the trophic transfer of Se.

Selenite transport and its inhibition in the unicellular green alga Chlamydomonas reinhardtii.

The influence of time, ambient concentration, and medium composition on selenite (Se(IV)) uptake by the unicellular green alga Chlamydomonas reinhardtii has been investigated. The aims of the performed experiments were to describe the kinetics of accumulation, to characterize transport capacities, to identify key nutrients influencing absorption, and to establish links between speciation and bioavailability. Our results suggested that the adsorbed fraction was negligible compared to the absorbed one. Over the short time scale considered, the absorption was linear with time, with an estimated conductance of approximately 0.2 nmol/m2/h/nM. Uptake was proportional to ambient levels in a broad range of intermediate concentrations (from nM to microM). However, conductances were higher at low concentrations (< nM) and then decreased with increasing concentrations (> microM). These results suggested that a specific but rapidly saturated transport system was involved at low concentrations, coupled with a nonspecific one that was only saturated at high ambient concentrations (approximately mM). The latter could involve transporters used by anionic macronutrients, which is supported by the fact that increasing sulfate and nitrate concentrations induced significant inhibition of Se(IV) uptake. Finally, Se(IV) speciation changes caused by varying pH did not significantly affect bioavailability.

Modulation of uranium bioaccumulation by hypoxia in the freshwater clam Corbicula fluminea: induction of multixenobiotic resistance protein and heat shock protein 60 in gill tissues.

The influence of hypoxia on the bioaccumulation of uranium in the clam Corbicula fuminea was investigated in ecologically relevant conditions. The cellular impact at the gill-tissue level was assessed by analyzing the induction of multixenobiotic resistance protein (MXR) and heat shock protein 60. Analyses were performed at three biological levels. First, at the organism level, uranium induced a significant decrease in the valve open duration under normoxia, but not under hypoxia, in which oxygen drive imposed an increase of the valve open duration. Second, at the tissue level, the uranium bioaccumulation rate in the gills was higher under hypoxia than under normoxia. Third, at the cellular level, MXR was induced by uranium but not by hypoxia. The threshold of tissular uranium concentration triggering MXR induction was between 3 and 5 nmol/g. On the contrary, Hsp60 was induced by hypoxia but not by uranium.

Laboratory and field assessment of uranium trophic transfer efficiency in the crayfish Orconectes limosus fed the bivalve C. fluminea.

At present, ecotoxicological information regarding the impact of natural uranium (U) on freshwater ecosystems via the trophic contamination route is scarce. We generated an experimental trophic food chain involving the prey species, Corbicula fluminea, and a predator, Orconectes limosus, for a 10-day and a 30-day feeding periods (food ration: one whole soft body/day/crayfish). We studied the efficiency of U trophic transfer and the distribution of U in the predator. During the test, we varied the quantity of dietary U (from beforehand contaminated bivalves at concentrations ranging from 0.9+/-0.1 to 20.2+/-9 microg/g fw provided to each crayfish over the 10 days) applying a daily feeding rate equal to 3.9+/-0.8% fw. The efficiency of U trophic transfer from clams to crayfish varied between 1 and 13% depending on the prey exposure modalities. Accumulation of U was observed in the digestive gland but also in gills, in the muscle, and in the molt of the crayfish after trophic exposure treatments. Under high-level exposure conditions, the digestive gland was the main target-organ, however a significant accumulation was also observed in the stomach. With regard to low levels of trophic exposure, accumulation of U in gills, in the stomach, and in the digestive gland was of the same order of magnitude. Longer exposure period which incorporated a crayfish molt, resulted in a decrease of trophic transfer ratio and a modified U tissue distribution.

Valve closure response to uranium exposure for a freshwater bivalve (Corbicula fluminea): quantification of the influence of pH.

Laboratory experiments were carried out to analyze the first valve closure response of a freshwater bivalve (Corbicula fluminea) exposed to uranium during a 5-h period. Experiments were performed in a well-defined artificial water at two pH values, 5.5 and 6.5, with a noninvasive method of valve recording. Sensitivity thresholds, based on percentage of bivalve that close their valves in a given time, were determined. Response thresholds depended on the total uranium concentration, integration time of response (fast responses could only be observed for the highest concentrations), and pH. The bivalve is much more sensitive to total uranium concentration at pH 5.5 than pH 6.5. The minimal sensitivity threshold determined, expressed as the uranium concentration inducing the valve closure of 50% of the bivalves, was 0.05 micromol/L at pH 5.5 after 5 h of exposure. Moreover, higher concentrations of the free ion UO2(2-) are required at pH 5.5 than at pH 6.5 to illicit the same response. Two hypotheses can be proposed, that UO2(2-) is not the only detected species or that competition exists between H- and UO2(2-) for binding sites.

Kinetic analysis of uranium accumulation in the bivalve Corbicula fluminea: effect of pH and direct exposure levels.

The bioaccumulation of natural uranium in the freshwater bivalve Corbicula fluminea was investigated subsequent to the bivalve's experimental waterborne exposures. A first experiment determined the accumulation rate (transfer efficiency, tissular distribution) and subcellular distribution of uranium in organs after over 42 days of uranium exposure (100 microg l(-1); pH 7) and later following 60 days of depuration. Results showed that there was direct transfer of uranium to the bivalve organs ([U]organism/[U]water = 0.16, fresh weight, fw). The highest accumulation levels occurred in the visceral mass and remained constant throughout the exposure duration, although a linear increase in the U concentration in the gills was observed (2.98 +/- 1.3-10.9 +/- 3.7 microg g(-1) between days 2 and 42). A second set of experiments were performed in order to test the influence of the exposure levels (100; 500; 1500 microg l(-1)) and pH (7 and 8.1) on the bioaccumulation capacities. A marked difference of U distribution is observed as a function of exposure levels (gills were favoured in the case of high exposure levels-relative burden: 49.1 +/- 3% (1500 microg l(-1)), whereas the visceral mass presented higher accumulation levels at environmentally relevant U concentrations). Uranium concentration in the insoluble fraction (80%) in the whole body does not depend upon exposure levels in the water column or upon duration. These experiments did not allow any link to be established between the free-metal ion concentration and the bioaccumulation efficiency. Results showed a significant pH effect and indicated a link between the exposure conditions and the distribution of uranium in the bivalve organs.

Uranium complexation and uptake by a green alga in relation to chemical speciation: the importance of the free uranyl ion.

The bioavailability and toxicity of dissolved metals are closely linked to the metals' chemical speciation in solution. Normally the complexation of a metal by a ligand would be expected to decrease its bioavailability. The aqueous speciation of uranium (U) undergoes tremendous changes in the presence of ligands commonly found in natural waters (carbonate, phosphate, hydroxide, and natural organic matter). In the present project, links between speciation, medium composition, and bioavailability of uranium toward Chlamydomonas reinhardtii, a unicellular green alga, were investigated. Short-term metal uptake rates were determined in simple inorganic media at constant low pH (5.0) and hardness with particular emphasis on the differentiation between adsorbed and intracellular metal. While intracellular uptake was fairly linear over 1 h, partly reversible adsorption reached steady-state within minutes. Both adsorption and absorption were saturable processes (with a half-saturation constant Km of 0.51 microM). Addition of phosphate, citrate, or ethylenediaminetetraacetic acid (EDTA) as ligands decreased uranium bioavailability. No evidence indicating the transport of intact uranyl complexes was found (i.e., facilitated diffusion of metal bound to an assimilable ligand such as uranium-phosphate complexes). Within these experimental conditions, uranium uptake was correlated with the free uranyl ion concentration as predicted by the free-ion activity model (FIAM) and biotic ligand model (BLM).