New sensitive tools to characterize meta-metabolome response to short- and long-term cobalt exposure in dynamic river biofilm communities.

Untargeted metabolomics is a non-a priori analysis of biomolecules that characterizes the metabolome variations induced by short- and long-term exposures to stressors. Even if the metabolite annotation remains lacunar due to database gaps, the global metabolomic fingerprint allows for trend analyses of dose-response curves for hundreds of cellular metabolites. Analysis of dose/time-response curve trends (biphasic or monotonic) of untargeted metabolomic features would thus allow the use of all the chemical signals obtained in order to determine stress levels (defense or damage) in organisms. To develop this approach in a context of time-dependent microbial community changes, mature river biofilms were exposed for 1 month to four cobalt (Co) concentrations (from background concentration to 1 × 10-6 M) in an open system of artificial streams. The meta-metabolomic response of biofilms was compared against a multitude of biological parameters (including bioaccumulation, biomass, chlorophyll a content, composition and structure of prokaryotic and eukaryotic communities) monitored at set exposure times (from 1 h to 28 d). Cobalt exposure induced extremely rapid responses of the meta-metabolome, with time range inducing defense responses (TRIDeR) of around 10 s, and time range inducing damage responses (TRIDaR) of several hours. Even in biofilms whose structure had been altered by Co bioaccumulation (reduced biomass, chlorophyll a contents and changes in the composition and diversity of prokaryotic and eukaryotic communities), concentration range inducing defense responses (CRIDeR) with similar initiation thresholds (1.41 ± 0.77 × 10-10 M Co2+ added in the exposure medium) were set up at the meta-metabolome level at every time point. In contrast, the concentration range inducing damage responses (CRIDaR) initiation thresholds increased by 10 times in long-term Co exposed biofilms. The present study demonstrates that defense and damage responses of biofilm meta-metabolome exposed to Co are rapidly and sustainably impacted, even within tolerant and resistant microbial communities.

Meta-metabolomic responses of river biofilms to cobalt exposure and use of dose-response model trends as an indicator of effects.

The response of the meta-metabolome is rarely used to characterize the effects of contaminants on a whole community. Here, the meta-metabolomic fingerprints of biofilms were examined after 1, 3 and 7 days of exposure to five concentrations of cobalt (from background concentration to 1 × 10-5 M) in aquatic microcosms. The untargeted metabolomic data were processed using the DRomics tool to build dose-response models and to calculate benchmark-doses. This approach made it possible to use 100% of the chemical signal instead of being limited to the very few annotated metabolites (7%). These benchmark-doses were further aggregated into an empirical cumulative density function. A trend analysis of the untargeted meta-metabolomic feature dose-response curves after 7 days of exposure suggested the presence of a concentration range inducing defense responses between 1.7 × 10-9 and 2.7 × 10-6 M, and of a concentration range inducing damage responses from 2.7 × 10-6 M and above. This distinction was in good agreement with changes in the other biological parameters studied (biomass and chlorophyll content). This study demonstrated that the molecular defense and damage responses can be related to contaminant concentrations and represents a promising approach for environmental risk assessment of metals.

How do biomarkers dance? Specific moves of defense and damage biomarkers for biological interpretation of dose-response model trends.

Omics studies are currently increasingly used in ecotoxicology to highlight the induction of known or novel biomarkers when organisms are exposed to contaminants. Although it is virtually impossible to identify all biomarkers from all organisms, biomarkers can be grouped as defense or damage biomarkers, exhibiting a limited number of response trends. Our working hypothesis is that defense and damage biomarkers follow different dose-response patterns. A meta-analysis of 156 articles and 2595 observations of dose-response curves of defense and damage biomarkers was carried out in order to characterize the response trends of these biological parameters in a large panel of living organisms (18 phyla) exposed to inorganic or organic contaminants (176 in total). Using multinomial logistic regression models, defense biomarkers were found to describe biphasic responses (bell- and U-shaped) to a greater extent (2.5 times) than damage biomarkers. In contrast, damage biomarkers varied mainly monotonically (decreasing or increasing), representing 85% of the observations. Neither the nature of the contaminant nor the type of organisms belonging to 4 kingdoms, influence these specific responses. This result suggests that cellular defense and damage mechanisms are not specific to stressors and are conserved throughout life. Trend analysis of dose-response models as a biological interpretation of biomarkers could thus be a valuable way to exploit large omics datasets.

Metal localisation in gastropod shells: New insights from mass spectrometry techniques.

Gastropod shells are calcified structures made of several crystal layers. They grow throughout the lifecycle of mollusks by integrating some of the chemical elements present in their environment, including metals. This characteristic means mollusks can be useful bioindicators of metal exposure. The present study aimed to better understand the role of layer composition on metal accumulation. To that end, the gastropods Radix balthica were collected in a French river adjacent to a municipal wastewater treatment plant. Microchemical metal analyses in the different shell layers were performed by Femtosecond-Laser Ablation Inductively Coupled Plasma Mass Spectrometry (Fs-LA-ICP-MS) and analyses of the molecular environment of the metals were performed by Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS). Strontium, Ba and Mn were well distributed within the whole shell and the high concentrations of these elements were found to be related to the aragonite structure of the shell. Copper, Ni, Pb and Zn were mostly present at the outer surfaces of the shell where the organic constituents were more concentrated. The analysis of metal distribution in shell layers could improve our understanding of the relationships between metal exposure and accumulation in mollusks, therefore providing evidences of their use as powerful integrated bioindicator of metal contamination.

Temperature and Photoperiod Affect the Sensitivity of Biofilms to Nickel and its Accumulation.

Whereas metal impacts on fluvial communities have been extensively investigated, effects of abiotic parameters on community responses to contaminants are poorly documented. Variations in photoperiod and temperature commonly occur over the course of a season and could affect aquatic biofilm communities and their responses to contaminants. Our objective was to characterize the influence of environmental conditions (photoperiod and temperature) on nickel (Ni) bioaccumulation and toxicity using a laboratory-grown biofilm. Environmental parameters were chosen to represent variations that can occur over the summer season. Biofilms were exposed for 7 days to six dissolved Ni treatments (ranging from 6 to 115 µM) at two temperatures (14 and 20 °C) using two photoperiods (16:8 and 12:12-h light:dark cycle). Under these different scenarios, structural (dry weight biomass and chlorophyll-a) and functional biomarkers (photosynthetic yield and Ni content) were analyzed at four sampling dates, allowing us to evaluate Ni sensitivity of biofilms over time. The results highlight the effects of temperature on Ni accumulation and tolerance of biofilms. Indeed, biofilms exposed at 20 °C accumulated 1.6-4.2-fold higher concentrations of Ni and were characterized by a lower median effect concentration value using photosynthetic yield compared with those exposed at 14 °C. In terms of photoperiod, significantly greater rates of Ni accumulation were observed at the highest tested Ni concentration for biofilms exposed to a 12:12-h compared with a 16:8-h light:dark cycle. Our study demonstrates the influence of temperature on biofilm metabolism and illustrates that environmental factors may influence Ni accumulation response and thus Ni responses of phototrophic biofilms. Environ Toxicol Chem 2022;41:1649-1662. © 2022 SETAC.

Exploring the role of water chemistry on metal accumulation in biofilms from streams in mining areas.

Biofilms play a key role in aquatic ecosystems. They are ubiquitous, even in the most contaminated ecosystems, and have great potential as biomonitors of exposure to contaminants such as metals. Freshwater biofilms and surface waters were sampled in two active mining areas of Canada: in the northern part of Nunavik (Quebec) and in the Greater Sudbury area (Ontario). Significant linear relationships were found between both total dissolved and free metal ion concentrations with biofilm metal contents for Cu and Ni, but not for Cd. When pH was below 6, biofilms accumulated less metals than at higher pHs. These results confirm that protons have a protective effect, leading to lower internalized metal concentrations. When considering only the sites where pH was above 6, the linear relationships between metal concentrations in water and in biofilms were improved for all three studied metals. The presence of metal ions could also modify the internalization of a given metal. To further study the role of cations as competitors to Cu, Ni and Cd uptake, relationships between the ratio of biofilm metal contents (Cu, Ni and Cd) on the ambient free metal ion concentrations were built as a function of potential cation competitors, such as major cations and metals. Surprisingly, our data suggest that calcium plays a minor role in preventing metal accumulation as compared to magnesium and possibly other metals. At a global scale, metal accumulation remained highly consistent between the two studied regions and over the sampling period, despite differences in ambient physicochemical water characteristics, climate or types of ecosystems. Metal bioaccumulation is thus a promising biomarker to assess metal bioavailability in a mining context. Nevertheless, more data are still required to further highlight the contribution of each competitor in metal accumulation by biofilms and to be able to build a unifying predictive model.

Development of Quantitative Ion Character-Activity Relationship Models to Address the Lack of Toxicological Data for Technology-Critical Elements.

Recent industrial developments have resulted in an increase in the use of so-called technology-critical elements (TCEs), for which the potential impacts on aquatic biota remain to be evaluated. In the present study, quantitative ion character-activity relationships (QICARs) have been developed to relate intrinsic metal properties to their toxicity toward freshwater aquatic organisms. In total, 23 metal properties were tested as predictors of acute median effect concentration (EC50) values for 12 data-rich metals, for algae, daphnids, and fish (with and without species distinction). Simple and multiple linear regressions were developed using the toxicological data expressed as a function of the total dissolved metal concentrations. The best regressions were then tested by comparing the predicted EC50 values for the TCEs (germanium, indium, gold, and rhenium) and platinum group elements (iridium, platinum, palladium, rhodium, and ruthenium) with the few measured values that are available. The 8 "best" QICAR models (adjusted r2 > 0.6) used the covalent index as the predictor. For a given metal ion, this composite parameter is a measure of the importance of covalent interactions relative to ionic interactions. Toxicity was reasonably well predicted for most of the TCEs, with values falling within the 95% prediction intervals for the regressions of the measured versus predicted EC50 values. Exceptions included Au(I) (all test organisms), Au(III) (algae and fish), Pt(II) (algae, daphnids), Ru(III) (daphnids), and Rh(III) (daphnids, fish). We conclude that QICARs show potential as a screening tool to review toxicity data and flag "outliers," which might need further scrutiny, and as an interpolating or extrapolating tool to predict TCE toxicity. Environ Toxicol Chem 2021;40:1139-1148. © 2020 SETAC.

Mercury bioavailability, transformations, and effects on freshwater biofilms.

Mercury (Hg) compounds represent an important risk to aquatic ecosystems because of their persistence, bioaccumulation, and biomagnification potential. In the present review, we critically examine state-of-the-art studies on the interactions of Hg compounds with freshwater biofilms, with an emphasis on Hg accumulation, transformations, and effects. Freshwater biofilms contain both primary producers (e.g., algae) and decomposers (e.g., bacteria and fungi), which contribute to both aquatic food webs and the microbial loop. Hence they play a central role in shallow water and streams, and also contribute to Hg trophic transfer through their consumption. Both inorganic and methylated mercury compounds accumulate in biofilms, which could transform them mainly by methylation, demethylation, and reduction. Accumulated Hg compounds could induce diverse metabolic and physiological perturbations in the microorganisms embedded in the biofilm matrix and affect their community composition. The bioavailability of Hg compounds, their transformations, and their effects depend on their concentrations and speciation, ambient water characteristics, biofilm matrix composition, and microorganism-specific characteristics. The basic processes governing the interactions of Hg compounds with biofilm constituents are understudied. The development of novel conceptual and methodological approaches allowing an understanding of the chemo- and biodynamic aspects is necessary to improve the knowledge on Hg cycling in shallow water as well as to enable improved use of freshwater biofilms as potential indicators of water quality and to support better informed risk assessment. Environ Toxicol Chem 2017;36:3194-3205. © 2017 SETAC.

Transcriptomic approach for assessment of the impact on microalga and macrophyte of in-situ exposure in river sites contaminated by chlor-alkali plant effluents.

Water quality degradation is a worldwide problem, but risk evaluation of chronic pollution in-situ is still a challenge. The present study aimed to evaluate the potential of transcriptomic analyses in representative aquatic primary producers to assess the impact of environmental pollution in-situ: the microalga Chlamydomonas reinhardtii and the macrophyte Elodea nuttallii were exposed 2 h in the Babeni Reservoir of the Olt River impacted by chlor-alkali plant effluent release resulting in increased concentrations of Hg and NaCl in receiving water. The response at the transcriptomic level was strong, resulting in up to 5485, and 8700 dysregulated genes (DG) for the microalga and for the macrophyte exposed in the most contaminated site, respectively. Transcriptomic response was congruent with the concentrations of Hg and NaCl in the water of the impacted reservoir. Genes involved in development, energy metabolism, lipid metabolism, nutrition, and RedOx homeostasis were dysregulated during in-situ exposure of both organisms. In addition, genes involved in the cell motility of C. reinhardtii and development of the cell wall of E. nuttallii were affected. DG were in line with adverse outcome pathways and transcriptomic studies reported after exposure to high concentrations of Hg and NaCl under controlled conditions in the laboratory. Transcriptomic response provided a sensitive measurement of the exposure as well as hints on the tolerance mechanisms of environmental pollution, and is thus promising as an early-warning tool to assess water quality degradation.

Environmental quality assessment of reservoirs impacted by Hg from chlor-alkali technologies: case study of a recovery.

Mercury (Hg) pollution legacy of chlor-alkali plants will be an important issue in the next decades with the planned phase out of Hg-based electrodes by 2025 within the Minamata convention. In such a context, the present study aimed to examine the extent of Hg contamination in the reservoirs surrounding the Oltchim plant and to evaluate the possible improvement of the environmental quality since the closure of its chlor-alkali unit. This plant is the largest chlor-alkali plant in Romania, which partly switched to Hg-free technology in 1999 and definitely stopped the use of Hg electrolysis in May 2012. Total Hg (THg) and methylmercury (CH3Hg) concentrations were found to decrease in the surface waters and sediments of the reservoirs receiving the effluents of the chlor-alkali platform since the closure of Hg units. Hence, calculated risk quotients (RQ) indicated no adverse effect of Hg for aquatic organisms from the ambient water exposure. RQ of Hg in sediments were mostly all higher than 1, showing important risks for benthic organisms. However, ecotoxicity testing of water and sediments suggest possible impact of other contaminants and their mixtures. Hg hotspots were found in soils around the platform with RQ values much higher than 1. Finally, THg and CH3Hg concentrations in fish were below the food safety limit set by the WHO, which contrasts with previous measurements made in 2007 revealing that 92 % of the studied fish were of high risk of consumption. Discontinuing the use of Hg electrodes greatly improved the surrounding environment of chlor-alkali plants within the following years and led to the decrease environmental exposure to Hg through fish consumption. However, sediment and soil still remained highly contaminated and problematic for the river reservoir management. The results of this ecological risk assessment study have important implications for the evaluation of the benefits as well as limits of the Minamata Convention implementation.

Leachates draining from controlled municipal solid waste landfill: Detailed geochemical characterization and toxicity tests.

Management of municipal solid wastes in many countries consists of waste disposal into landfill without treatment or selective collection of solid waste fractions including plastics, paper, glass, metals, electronic waste, and organic fraction leading to the unsolved problem of contamination of numerous ecosystems such as air, soil, surface, and ground water. Knowledge of leachate composition is critical in risk assessment of long-term impact of landfills on human health and the environment as well as for prevention of negative outcomes. The research presented in this paper investigates the seasonal variation of draining leachate composition and resulting toxicity as well as the contamination status of soil/sediment from lagoon basins receiving leachates from landfill in Mpasa, a suburb of Kinshasa in the Democratic Republic of the Congo. Samples were collected during the dry and rainy seasons and analyzed for pH, electrical conductivity, dissolved oxygen, soluble ions, toxic metals, and were then subjected to toxicity tests. Results highlight the significant seasonal difference in leachate physicochemical composition. Affected soil/sediment showed higher values for toxic metals than leachates, indicating the possibility of using lagoon system for the purification of landfill leachates, especially for organic matter and heavy metal sedimentation. However, the ecotoxicity tests demonstrated that leachates are still a significant source of toxicity for terrestrial and benthic organisms. Therefore, landfill leachates should not be discarded into the environment (soil or surface water) without prior treatment. Interest in the use of macrophytes in lagoon system is growing and toxic metal retention in lagoon basin receiving systems needs to be fully investigated in the future. This study presents useful tools for evaluating landfill leachate quality and risk in lagoon systems which can be applied to similar environmental compartments.

Species-specific isotope tracers to study the accumulation and biotransformation of mixtures of inorganic and methyl mercury by the microalga Chlamydomonas reinhardtii.

The present study demonstrates that species-specific isotope tracing is an useful tool to precisely measure Hg accumulation and transformations capabilities of living organisms at concentrations naturally encountered in the environment. To that end, a phytoplanktonic green alga Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) was exposed to mixtures of (199)-isotopically enriched inorganic mercury ((199)IHg) and of (201)-isotopically enriched monomethylmercury ((201)CH3Hg) at a concentration range between less than 1 pM to 4 nM. Additionally, one exposure concentration of both mercury species was also studied separately to evaluate possible interactive effects. No difference in the intracellular contents was observed for algae exposed to (199)IHg and (201)CH3Hg alone or in their mixture, suggesting similar accumulation capacity for both species at the studied concentrations. Demethylation of (201)CH3Hg was observed at the highest exposure concentrations, whereas no methylation was detected.

Towards Mechanistic Understanding of Mercury Availability and Toxicity to Aquatic Primary Producers.

The present article reviews current knowledge and recent progress on the bioavailability and toxicity of mercury to aquatic primary producers. Mercury is a ubiquitous toxic trace element of global concern. At the base of the food web, primary producers are central for mercury incorporation into the food web. Here, the emphasis is on key, but still poorly understood, processes governing the interactions between mercury species and phytoplankton, and macrophytes, two representatives of primary producers. Mass transfer to biota surface, adsorption to cell wall, internalization and release from cells, as well as underlying toxicity mechanisms of both inorganic mercury and methylmercury are discussed critically. In addition, the intracellular distribution and transformation processes, their importance for mercury toxicity, species-sensitivity differences and trophic transfer are presented. The mini-review is illustrated with examples of our own research.

Interactions between mercury and phytoplankton: speciation, bioavailability, and internal handling.

The present review describes and discusses key interactions between mercury (Hg) and phytoplankton to highlight the role of phytoplankton in the biogeochemical cycle of Hg and to understand direct or indirect Hg effects on phytoplankton. Phytoplankton are exposed to various Hg species in surface waters. Through Hg uptake, phytoplankton affect the concentration, speciation, and fate of Hg in aquatic systems. The mechanisms by which phytoplankton take up Hg are still not well known, but several studies have suggested that both facilitated transport and passive diffusion could be involved. Once internalized, Hg will impact several physiological processes, including photosynthesis. To counteract these negative effects, phytoplankton have developed several detoxification strategies, such as the reduction of Hg to elemental Hg or its sequestration by intracellular ligands. Based on the toxicological studies performed so far in the laboratory, Hg is unlikely to be toxic to phytoplankton when they are exposed to environmentally relevant Hg concentrations. However, this statement should be taken with caution because questions remain as to which Hg species control Hg bioavailability and about Hg uptake mechanisms. Finally, phytoplankton are primary producers, and accumulated Hg will be transferred to higher consumers. Phytoplankton are a key component in aquatic systems, and their interactions with Hg need to be further studied to fully comprehend the biogeochemical cycle of Hg and the impact of this ubiquitous metal on ecosystems.

Effects of untreated hospital effluents on the accumulation of toxic metals in sediments of receiving system under tropical conditions: case of South India and Democratic Republic of Congo.

Physicochemical and ecotoxicological analyses have been performed to assess the quality of sediments receiving untreated hospital effluents from Indian and Democratic Republic of Congo (DRC) hospitals. The sediments were collected monthly and characterized for grain size, organic matter, total organic carbon, total carbon, nitrogen, phosphorus, toxic metals and ecotoxicity. The results highlight the high concentration of toxic metals from the Indian hospital effluent receiving systems, especially for Cr, Cu, As, Zn and Hg. On the other hand, the metal concentrations in the sediment receiving system from DRC are low (e.g. maximum Hg and Zn concentration were 0.46 and 48.84 mg kg(-1) respectively). Ostracods exposed to sediment samples H2 (September month sample) and H3 (June and September month samples) were found dead after 6d of exposure whereas the higher mortality rate for Congo sediments was 23% but was accompanied with 33 ± 7% of growth inhibition. The results of this study show the variation of sediment composition on toxic metal levels as well as toxicity related to both, the type of hospitals and the sampling period. Additionally, hospital effluent disposal practices at the study sites can lead to the pollution of water resources and may generate risks for aquatic organisms and human health.

THE INFLUENCE OF pH ON ALGAL CELL MEMBRANE PERMEABILITY AND ITS IMPLICATIONS FOR THE UPTAKE OF LIPOPHILIC METAL COMPLEXES(1).

Uptake of lipophilic metal complexes by freshwater algae has recently been shown to be pH dependent. Here we look at different physiological aspects that could influence the diffusion of the lipophilic Cd complex, Cd(diethyldithiocarbamate)2 (0) (Cd(DDC)2 (0) ), into algal cells at different exposure pH values. Changes in cell membrane permeability were assessed as a function of pH for three species of green algae [Chlamydomonas reinhardtii P. A. Dang., Pseudokirchneriella subcapitata (Korshikov) Hindák, and Chlorella fusca var. vacuolata Shihira et R. W. Kraus] using two neutral, nonionic probes, fluorescein diacetate (FDA) and D-sorbitol. In parallel experiments, we exposed algae to inorganic Cd or to Cd(DDC)2 (0) and monitored Cd intracellular metal distribution, together with phytochelatin synthesis. For the three algal species acclimated at pH 5.5 (w/wo DDC 1 µM) and exposed at this pH, their permeability to FDA and D-sorbitol was consistently lower than for algae growing at pH 7.0 and exposed at this pH (P < 0.001). The ratio of the FDA hydrolysis rate measured at pH 7.0 with respect to the rate measured at pH 5.5 (both in the presence of DDC) correlated with the ratio of the Cd(DDC)2 (0) initial internalization rate constant obtained at pH 7.0 versus that obtained at pH 5.5 (three algae species, n = 9, r = 0.85, P = 0.004). Our results strongly suggest that acidification affects metal availability to algae not only by proton inhibition of facilitated metal uptake but also by affecting membrane permeability.

UPTAKE OF LIPOPHILIC CADMIUM COMPLEXES BY THREE GREEN ALGAE: INFLUENCE OF HUMIC ACID AND ITS pH DEPENDENCE(1).

Cadmium forms neutral, lipophilic CdL2 (0) complexes with diethyldithiocarbamate (L = DDC) and with ethylxanthate (L = XANT). In a synthetic solution and in the absence of natural dissolved organic matter (DOM), for a given total Cd concentration, uptake of these complexes by unicellular algae is much faster than the uptake of the free Cd(2+) cation. The objective of the present study was to determine how this enhanced uptake of the lipophilic CdL2 (0) complexes was affected by the presence of natural DOM (Suwannee River humic acid, SRHA). Experiments were performed with Cd(DDC)2 (0) and Cd(XANT)2 (0) at two pH values (7.0 and 5.5) and with the three chlorophytes [Chlamydomonas reinhardtii P. A. Dang., Pseudokirchneriella subcapitata (Korshikov) Hindák, Chlorella fusca var. vacuolata Shihira et R. W. Krauss]. Short-term uptake (30-40 min) of the CdL2 (0) complexes was followed in the absence and presence of SRHA (6.5 mg C · L(-1) ). Acidification from pH 7.0 to 5.5 decreased CdL2 (0) uptake by the three algae, in the presence or absence of humic acid (HA). The dominant effect of the HA was to decrease Cd uptake, due to its interaction with the CdL2 (0) complexes in solution. However, if uptake of the free CdL2 (0) complexes was compared in the presence and absence of HA, in four of eight cases initial uptake rate constants (ki ) were significantly higher (P < 0.05) in the presence of the HA, suggesting the operation of an interfacial effect of the HA at the algal cell membrane, favoring uptake of CdL2 (0) . Overall, the experimental results suggest that neutral metal complexes will be less bioavailable in natural waters than they are in synthetic laboratory media in the absence of natural DOM.

Cadmium detoxification strategies in two phytoplankton species: metal binding by newly synthesized thiolated peptides and metal sequestration in granules.

The aim of this study was to evaluate whether intracellular detoxification mechanisms could explain, at least partially, the different sensitivity to Cd of two freshwater green algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata. Subcellular Cd distribution and the synthesis of metal-binding thiolated peptides were thus examined in both algae exposed to a range of free [Cd(2+)] from 0.7 to 253 nM. Cadmium partitioning among five subcellular fractions (cellular debris, granules, organelles, heat-denaturable proteins - HDP, and heat-stable proteins - HSP) was determined after differential centrifugation of algal homogenates. Thiolated-peptides, phytochelatins (PC(n)) and precursors, were analyzed by HPLC with pre-column monobromobimane derivatization. Cadmium accumulation per cell was 2-4 times greater for C. reinhardtii than for P. subcapitata, yet C. reinhardtii was more resistant to Cd with an EC(50) of 273 nM Cd(2+) [244-333 nM Cd(2+) CI(95%)]) compared to 127 nM Cd(2+) [111-143 nM Cd(2+) CI(95%)] for P. subcapitata. Although [Cd] generally increased in the organelle fractions when free [Cd(2+)] increased in the experimental media, their relative contributions to the total Cd cellular content decreased, suggesting that partial protection of some metal sensitive sites was achieved by the initiation of cellular detoxification mechanisms. An increase in the proportion of Cd in the granules fraction was observed for C. reinhardtii between 6 and 15 nM Cd(2+) (i.e., at [Cd(2+)]

Thiols in Scenedesmus vacuolatus upon exposure to metals and metalloids.

Phytochelatins are intracellular metal ligands produced by algae when exposed to elevated metal concentrations. In freshwater ecosystems, algae are exposed to a wide range of metals and metalloids. The aim of this study was thus to investigate phytochelatin induction in freshwater algae upon metal and metalloid exposure. To that purpose, the unicellular green alga Scenedesmus vacuolatus, was exposed to Cu, Zn, Ni, Pb and Ag, as well as to As(III), As(V), Sb(III) and Sb(V), and examined for its thiol content (gamma-glutamylcysteine, glutathione and phytochelatins). Glutathione content was found to decrease upon the exposure to Zn and to increase upon the exposure to Pb and Ag. Phytochelatins were only induced by Cu (at [Cu2+] = 8x10(-11) M) and Pb (at [Pb2+] = 8x10(-11) to 8x10(-10) M), where [Cu2+] and [Pb2+] are computed free metal ion concentrations. Glutathione content also decreased upon the exposure to Sb(V) whereas an increase was observed as a result as the exposure to As(III) and As(V). The metalloids As(III), As(V) and Sb(III) in the concentration range from 8x10(-6) to 2x10(-4) M (total concentrations of oxyanions) were inducing phytochelatins. Glutathione and phytochelatin content in S. vacuolatus do thus sensitively respond to exposure to a number of metals and metalloids.

Thiol and metal contents in periphyton exposed to elevated copper and zinc concentrations: a field and microcosm study.

Phytochelatins are metal-binding polypeptides produced by algae under metal exposure. The aim of this study was to investigate the effects of metal concentration variations in natural systems on periphyton at the biochemical level by analyzing its intracellular thiol content, in particular phytochelatins. To that purpose, two field campaigns were conducted in a stream subject to an increase of dissolved metal concentrations (particularly Cu and Zn) during rain events, which results in an increase of their accumulation in periphyton. At background metal concentrations, several thiols were detectable in periphyton, namely, glutathione (GSH), gamma-glutamylcysteine (gammaGluCys), phytochelatins (PC2), and some unidentified thiols, U1 and U2. Glutathione and gammaGluCys contents were found to vary independently of the rain, as well as U1 and U2, whereas the phytochelatin content increased during the rain events. To investigate whether Cu or Zn may be responsible for this increase, microcosm experiments were carried out with natural water enriched with Cu, Zn, and Cd separately, and Cu and Zn in combination. In this study, GSH, PC2, and U1 were also detected, but not gammaGluCys. An increase in accumulated Cu content did not induce any changes in thiol content, whereas an increase of the Zn content induced a decrease in GSH content and an increase in phytochelatin content. Zinc rather than Cu may thus induce a phytochelatin content increase in periphyton in the field studies. Addition of Cu and Zn in combination also induced an increase in phytochelatin content. Cadmium was found to be the most effective inducer, with the production of larger phytochelatins (PC3-4). This study is the first one to report changes in thiol content in periphyton in response to an increase of the metal concentration in natural freshwaters.