Potential cellular targets of platinum in the freshwater microalgae Chlamydomonas reinhardtii and Nitzschia palea revealed by transcriptomics.

Platinum group element levels have increased in natural aquatic environments in the last few decades, in particular as a consequence of the use of automobile catalytic converters on a global scale. Concentrations of Pt over tens of µg L-1 have been observed in rivers and effluents. This raises questions regarding its possible impacts on aquatic ecosystems, as Pt natural background concentrations are extremely low to undetectable. Primary producers, such as microalgae, are of great ecological importance, as they are at the base of the food web. The purpose of this work was to better understand the impact of Pt on a cellular level for freshwater unicellular algae. Two species with different characteristics, a green alga C. reinhardtii and a diatom N. palea, were studied. The bioaccumulation of Pt as well as its effect on growth were quantified. Moreover, the induction or repression factors of 16 specific genes were determined and allowed for the determination of possible intracellular effects and pathways of Pt. Both species seemed to be experiencing copper deficiency as suggested by inductions of genes linked to copper transporters. This is an indication that Pt might be internalized through the Cu(I) metabolic pathway. Moreover, Pt could possibly be excreted using an efflux pump. Other highlights include a concentration-dependent negative impact of Pt on mitochondrial metabolism for C. reinhardtii which is not observed for N. palea. These findings allowed for a better understanding of some of the possible impacts of Pt on freshwater primary producers, and also lay the foundations for the investigation of pathways for Pt entry at the base of the aquatic food web.

Genetic and physiological responses of three freshwater diatoms to realistic diuron exposures.

This study examined the effects of diuron on strains of three major freshwater diatom species, Eolimna minima, Nitzschia palea and Planothidium lanceolatum. These species are frequently recorded in the Morcille River, where diuron runs off during phytosanitary treatments of the vineyards around. Here, there were three diatom exposure groups for each species: 0, 1 and 10 µg/L diuron during a 14-day laboratory assessment. Diuron water concentration, cell number, photosynthetic activity and gene expression were assessed at 6 h and 2, 7 and 14 days after contamination. Diuron exposure altered photosynthetic activity in that the optimal quantum yield of photosystem II (PSII) decreased between 40 and 50% and, for P. lanceolatum at 10 µg/L, there was complete inhibition. Genetic responses indicated diuron effects on both photosystem II and mitochondrial metabolism in all three species at both diuron exposure levels. Thus, analysis of the expression of psaA, d1, cox1, nad5 and 12s could be an early biomarker to detect pesticide pollution. Overall, this study revealed differences in diuron sensitivity among the three species: E. minima and N. palea appeared to be more tolerant than P. lanceolatum. These results suggest that the development of molecular tools, and more precisely of biomarkers, will aid in early assessment of contamination and water quality.

Detoxification and recovery capacities of Corbicula fluminea after an industrial metal contamination (Cd and Zn): a one-year depuration experiment.

This study aimed to assess the recovery capacity of the freshwater bivalve Corbicula fluminea subjected to industrial metal discharges (Cd, Zn). After a 24-day exposure in a metal-contaminated river, bivalves were transferred and maintained in the laboratory for one year under metal-free conditions. Metal accumulation, metallothionein production and genetic expressions of genes involved in metal stress were studied. Results demonstrated the high persistence of Cd in tissues (only 73% eliminated after 365 days) whereas Zn was rapidly depurated. The Cd half-life was estimated around 240 days. Metallothioneins were strongly induced within the 28 first days of decontamination, then decreased by 45% after 365 days. The metal exposure of bivalves led to a significant gene induction. After 28 days, most of the genes were no longer overexpressed, suggesting that the bivalves may withstand small amounts of non-essential metals in their tissues without showing signs of detrimental effects on the tested genes.

What are the outcomes of an industrial remediation on a metal-impacted hydrosystem? A 2-year field biomonitoring of the filter-feeding bivalve Corbicula fluminea.

The Riou-Mort watershed (southwest France) exhibits high metal contaminations (Cd and Zn) related to an important mining past. In this context, a remediation process has been implemented in 2007 to reduce the watershed contamination. The aim of this study was to assess the early effectiveness of the remediation process on the hydrosystem contamination state. A biomonitoring was realized over two years (2008-2010) with the filter-feeding bivalve Corbicula fluminea, exposed along a contamination gradient. Several biological parameters were monitored: (1) Cd and Zn bioaccumulation, (2) Metallothionein (MT) production as detoxification mechanism, (3) differential gene expression (cat, sod, gst, 12S, cox1, mt). The physicochemical data highlighted strong metal contamination persistence in the river water and failed to demonstrate a significant decrease of metal contamination during the 2-year monitoring. The bioaccumulation results confirmed the persistence of a water contamination despite remediation works, with maximum values measured downstream from the industrial site (Joany). The bioaccumulation increased in 2010, reaching 69.3±5.3 µg Cd g(-1) DW at Joany in July 2010, whereas it did not exceed 1.4±0.2 µg Cd g(-1) DW at the reference site throughout the biomonitoring. MT concentrations were closely related to the contamination gradient, especially at Joany, demonstrating their strong involvement in the detoxification processes. The mt gene induction was strongly correlated to the MT and metal concentrations. The gene inductions of cat, sod, gst and 12S were correlated to both the metal concentrations and the seasonal variations, especially temperatures. This suggests that environmental factors require serious consideration for the interpretation of bioaccumulation kinetics and thus for the assessment of the remediation effectiveness. Consequently, the whole results did not yet highlight strong beneficial effects of remediation work on the hydrosystem contamination state. First benefits of that process should be progressively felt, once the remediation achieved, and should grandly accelerate the decontamination process of the contaminated area.

Chemical and structural status of copper associated with oxygenic and anoxygenic phototrophs and heterotrophs: possible evolutionary consequences.

Copper adsorption on the surface and intracellular uptake inside the cells of four representative taxons of soil and aquatic micro-organisms: aerobic rhizospheric heterotrophs (Pseudomonas aureofaciens), anoxygenic (Rhodovulum steppense) and oxygenic (cyanobacteria Gloeocapsa sp. and freshwater diatoms Navicula minima) phototrophs were studied in a wide range of pH, copper concentration, and time of exposure. Chemical status of adsorbed and assimilated Cu was investigated using in situ X-ray absorption spectroscopy. In case of adsorbed copper, XANES spectra demonstrated significant fractions of Cu(I) likely in the form of tri-coordinate complexes with O/N and/or S ligands. Upon short-term reversible adsorption at all four studied micro-organisms' cell surface, Cu(II) is coordinated by 4.0 ± 0.5 planar oxygens at an average distance of 1.97 ± 0.02 Å, which is tentatively assigned to the carboxylate groups. The atomic environment of copper incorporated into diatoms and cyanobacteria during long-term growth is similar to that of the adsorbed metal with slightly shorter distances to the first O/N neighbor (1.95 Å). In contrast to the common view of Cu status in phototrophic micro-organisms, XAFS failed to detect sulfur in the nearest atomic environment of Cu assimilated by freshwater plankton (cyanobacteria) and periphyton (diatoms). The appearance of S in Cu 1st coordination shell at 2.27-2.32 Å was revealed only after long-term interaction of Cu with anoxygenic phototrophs (and Cu uptake by soil heterotrophs), suggesting Cu scavenging in the form of sulfhydryl, histidine/carboxyl or a mixture of carboxylate and sulfhydryl complexes. These new structural constraints suggest that adsorbed Cu(II) is partially reduced to Cu(I) already at the cell surface, where as intracellular Cu uptake and storage occur in the form of both Cu(I)-S linked proteins and Cu(II) carboxylates. Obtained results allow to better understand how, in the course of biological evolution, micro-organisms elaborated various mechanisms of Cu uptake and storage, from passive adsorption and uptake to active, protein-controlled surface reduction, and intracellular storage.

Impacts of Cd and Zn on the development of periphytic diatom communities in artificial streams located along a river pollution gradient.

Many field and experimental studies have been conducted to reveal modifications of periphytic diatom communities under metal pollution, but most of the approaches presented drawbacks: variability of environmental factors for field studies and lack of representativity for experimental ones. An original approach that allows growth of periphytic communities under conditions more controlled than in the field and more realistic than experimental investigations was developed to try conclusions being drawn on the real impact of metallic pollution. Artificial streams were placed on the river bank at three sites along the pollution gradient (Cd/Zn) of the River Lot (SW France): reference (upstream pollution discharge), slightly polluted (around 1.5 microg Cd x L(-1) and 50 microg Zn x L(-1)), and polluted (around 6 microg Cd x L(-1) and 400 microg Zn x L(-1)) sites. Structural characteristics of diatom communities growing in these artificial streams, on glass substrates, and under homogenized current velocity and water depth were compared after four durations (1, 2, 3, and 4 weeks) to study effects of metal on first steps of community development. Marked perturbations of diatom community development were observed overall in the polluted site: after 1 week, early biofilms differed already by taxonomic composition; after 4 weeks, cell density was significantly lower than in the reference site and taxonomic composition was very different. These results, generated by a methodology intermediate between field and experimental approaches, compare well with literature findings based on more traditional approaches, strongly attesting that metallic pollution affects diatom community architecture and induce rising of abnormal cells.

Role of temperature on isoproturon bioaccumulation and effects on two freshwater rooted macrophytes: Elodea densa and Ludwigia natans.

The effects of temperature on the bioaccumulation of the phenylurea herbicide isoproturon (IPU) and on the growth inhibition induced on two freshwater rooted macrophytes--Elodea densa and Ludwigia natans--were investigated using indoor microcosms. The experimental protocol was based on five temperatures (12, 16, 20, 24, and 28 degrees C) and two contamination levels of the water column (30 and 60 micrograms IPU.liter-1), plus a control condition. Variations in temperature had little effect on the decrease in [IPU] in the water column during the 21-day experiment. The IPU concentration in the two macrophyte species was not significantly modified by the large range of temperatures when results were expressed using the concentration criterion. IPU burdens in the E. densa cuttings, on the other hand, increased significantly when the temperature rose from 12 to 28 degrees C; small differences were observed between the two exposure conditions, in relation to the antagonistic effects of IPU and temperature on the growth of the cuttings. Bioconcentration factors in the plants (stems + leaves) were close to 10 and 13 after exposure to 30 and 60 micrograms IPU.liter-1, respectively.

Effects of the phenylurea herbicide isoproturon on periphytic diatom communities in freshwater indoor microcosms.

The toxic effects of the phenylurea herbicide Isoproturon -IPU: (3-(4-isopropylphenyl)-1, 1-dimethylurea)-were studied on the colonization of periphytic diatom communities, within indoor microcosms consisting of a mixed biotope (water column and natural sediment) and two biological species-rooted macrophyte cuttings (Elodea densa) and benthic bivalve molluscs (Corbicula fluminea). The periphyton, essentially composed of diatoms, was collected on artificial substrata (glass slides) in the upper layers of the water column, after two periods of exposure (34 and 71 days). IPU was initially added in the water or in the sediment compartment, at two nominal concentrations (L1 and L2 levels) for each contamination source-5 and 20 microg litre(-1) and 100 and 400 microg kg(-1) in sediment (w/w) respectively. The effects of IPU on the density and community structure of periphytic diatoms are described. A marked reduction in the diatom density was observed after 34 days exposure to the lower concentration of IPU in the water (5 microg litre(-1)). For the L2 levels, the very small number of live cells present did not permit quantification of the diatom density. After 71 days, recovery in community parameters occurred for the two contamination levels of the sediment and water column sources. Samples collected in the experimental units contaminated with the L2 levels were dominated by heterotrophic and smaller diatom species, such as Sellaphora seminulum. Data treatment based on factorial discriminant analysis enabled us to distinguish the different contamination conditions, with only 11 species from the 130 taxa identified.

Herbicide isoproturon-specific binding in the freshwater macrophyte Elodea densa--a single-cell fluorescence study.

The experimental approach to the effects of the phenylurea herbicide Isoproturon (IPU), on the photosynthesis activity of leaf cells of the freshwater macrophyte Elodea densa was based on the fast induction kinetics of the PSII chlorophyll a in vivo fluorescence. FI/FP ratios determined on the induction curves exhibited a noticeable effect at 5 micrograms IPU.liter-1. They appeared to be the most reliable parameter for the quantitative evaluation of the dissociation constant of the complex "IPU/D1 protein" (Kd = 1.2 x 10(-7) M) and of the concentration of free IPU either at the thylakoid level or in the surrounding medium of the leaf epithelial cells. Total IPU bioaccumulation at the whole plant level as a function of [IPU] in the medium indicated that the D1 protein represented the main binding site for IPU in this aquatic plant species.