New insights in copper handling strategies in the green alga Chlamydomonas reinhardtii under low-iron condition.

Copper (Cu) is a redox-active transition element critical to various metabolic processes. These functions are accomplished in tandem with Cu-binding ligands, mainly proteins. The main goal of this work was to understand the mechanisms that govern the intracellular fate of Cu in the freshwater green alga, Chlamydomonas reinhardtii, and more specifically to understand the mechanisms underlying Cu detoxification by algal cells in low-Fe conditions. We show that Cu accumulation was up to 51-fold greater for algae exposed to Cu in low-Fe medium as compared to the replete-Fe growth medium. Using the stable isotope 65Cu as a tracer, we studied the subcellular distribution of Cu within the various cell compartments of C. reinhardtii. These data were coupled with metallomic and proteomic approaches to identify potential Cu-binding ligands in the heat-stable proteins and peptides fraction of the cytosol. Cu was mostly found in the organelles (78%), and in the heat-stable proteins and peptides (21%) fractions. The organelle fraction appeared to also be the main target compartment of Cu accumulation in Fe-depleted cells. As Fe levels in the medium were shown to influence Cu homeostasis, we found that C. reinhardtii can cope with this additional stress by utilizing different Cu-binding ligands. Indeed, in addition to expected Cu-binding ligands such as glutathione and phytochelatins, 25 proteins were detected that may also play a role in the Cu-detoxification processes in C. reinhardtii. Our results shed new light on the coping mechanisms of C. reinhardtii when exposed to environmental conditions that induce high rates of Cu accumulation.

Effects and bioaccumulation of Cr(III), Cr(VI) and their mixture in the freshwater mussel Corbicula fluminea.

Chromium has two main oxidation states, Cr(III) and Cr(VI), that can occur simultaneously in natural waters. Current consensus holds that Cr(VI) is of high ecotoxicological concern, but regards Cr(III) as poorly bioavailable and relatively non-toxic. In this work, the effects and bioaccumulation of Cr(III), Cr(VI) and their mixture were studied using the freshwater clam Corbicula fluminea as a model organism. Mixture exposures were carried out using solutions isotopically enriched in 50Cr(III) or 53Cr(VI), allowing to quantify the contribution of each redox form to total Cr accumulation in the clams. Following exposure to individual redox forms, Cr(III) accumulated preferentially in the digestive glands and Cr(VI) in the gills of C. fluminea. In mixture exposures, both redox forms accumulated mainly in the gills; the concentration of Cr(III) in the digestive glands being much lowered compared with individual exposures. Both oxidation states affected the expression of biomarkers related to energy reserves, cellular damage and mitochondrial functioning, as well as the expression of mRNA for detoxification genes. The observed effects differed between gills and digestive glands. The present study suggests that Cr(III) is a bioavailable and biologically active elemental species deserving more consideration by the ecotoxicological community.

Lanthanum and Cerium Toxicity to the Freshwater Green Alga Chlorella fusca: Applicability of the Biotic Ligand Model.

The environmental risk assessment of rare earth elements (REEs) requires data on their potential toxicity. In the present study, the toxicity of lanthanum (La) and cerium (Ce) was studied in relation to metal speciation in solution. For both La and Ce, the use of organic ligands demonstrated that the calculated free ion concentration was a good indicator of toxicity. Whether in the absence or presence of organic ligands, when based on free ion concentrations, the obtained half-maximal effective concentrations were similar. When all generated data were pooled, Ce and La showed identical toxicity thresholds after 120 h of exposure with free ion concentration-based median effective concentration values (95% confidence intervals) of 0.48 (0.38-0.60) µM and 0.47 (0.36-0.61) µM for La3+ and Ce3+ , respectively. The inhibition of algal growth was also correlated with the intracellular lanthanide concentrations, regardless of the ligand used. Finally, increasing the ambient calcium concentration protected the test algae by reducing the amount of lanthanide internalized into the cells. These results suggest that, at constant pH (5.5), REE accumulation and toxicity are linked to the free ion concentration and ambient calcium concentration, as predicted by the biotic ligand model. Environ Toxicol Chem 2020;39:996-1005. © 2020 SETAC.

Chromium hazard and risk assessment: New insights from a detailed speciation study in a standard test medium.

Despite the consensus about the importance of chemical speciation in controlling the bioavailability and ecotoxicity of trace elements, detailed speciation studies during laboratory ecotoxicity testing remain scarce, contributing to uncertainty when extrapolating laboratory findings to real field situations in risk assessment. We characterized the speciation and ecotoxicological effects of chromium (CrIII and CrVI ) in the International Organization for Standardization (ISO) medium for algal ecotoxicity testing. Total and dissolved (< 0.22 µm) Cr concentrations showed little variability in media spiked with CrVI , whereas dissolved Cr concentration decreased by as much as 80% over a 72-h time period in medium amended with CrIII . Analyses by ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS) highlighted the absence of redox interconversion between CrIII or CrVI both in the presence and absence of algal cells (Raphidocelis subcapitata). Furthermore, the concentration of ionic CrIII dropped below detection limits in less than 2 h with the corresponding formation of carbonate complexes and Cr hydroxides. Precipitation of CrIII in the form of colloidal particles of variable diameters was confirmed by nanoparticle (NP) tracking analysis, single particle ICP-MS, and single particle counting. In terms of time-weighted dissolved (< 0.22 µm) Cr concentration, CrIII was 4 to 10 times more toxic than CrVI . However, CrIII ecotoxicity could arise from interactions between free ionic CrIII and algae at the beginning of the test, from the presence of Cr-bearing NPs, or from a combination of the 2. Future ecotoxicological studies must pay more attention to Cr speciation to reliably compare the ecotoxicity of CrIII and CrVI . Environ Toxicol Chem 2018;37:983-992. © 2017 SETAC.

Bioaccumulation and subcellular partitioning of Cr(III) and Cr(VI) in the freshwater green alga Chlamydomonas reinhardtii.

Chromium occurs in aquatic environments under two main redox forms, namely Cr(III) and Cr(VI), with different geochemical and biochemical properties. Cr(VI) readily crosses biological membranes of living organisms and once inside the cells it undergoes a rapid reduction to Cr(III). The route of entry for the latter form is, however, poorly known. Using the radioactive tracer 51Cr we compared the accumulation (absorption and adsorption) of the two Cr forms by the green unicellular alga Chlamydomonas reinhardii after 1h and 72h of exposure to 100nM of either Cr(III) or Cr(VI) at pH 7. Both Cr forms had similar accumulation, with a major part in the extracellular (adsorbed) fraction after 1h and a major part of total accumulated Cr in the intracellular (absorbed) fraction after 72h. We also investigated the intracellular partitioning of Cr using an operational fractionation scheme and found that both Cr forms had similar distributions among fractions: Cr was mostly associated with organelles (23±12% after 1h and 37±7% after 72h) and cytosolic heat-stable proteins and peptides (39±18% after 1h and 35±3% after 72h) fractions. Further investigations using a metallomic approach (SEC-ICP-MS) were performed with the heat-stable proteins and peptides fraction to compare the distribution of the two Cr forms among various biomolecules of this fraction. One Cr-binding biomolecule (∼28kDa) appeared after 1h of exposure for both Cr species. After 72h another biomolecule of lower molecular weight (∼0.7kDa) was involved in binding Cr and higher signal intensities were observed for Cr(VI) than for Cr(III). We show, for the first time, that both Cr(III) and Cr(VI) have similar fate within algal cells, supporting the tenet that a unique redox form occurs within cells.