Interactive effects of cyanobacterial metabolites aeruginosin-98B, anabaenopeptin-B and cylindrospermopsin on physiological parameters and novel in vivo fluorescent indicators in Chironomus aprilinus larvae.

We aimed to determine the effects of single cyanobacterial metabolites aeruginosin-B (AER-B), anabaenopeptin-B (ANA-B), cylindrospermopsin (CYL), their binary and ternary mixtures on biomarkers of Chironomus aprilinus larvae: oxygen consumption, fat body structure and two novel fluorescent indicators: imaging of nuclei in cells of body integument, and the catecholamine level. The obtained results showed that oxygen consumption was inhibited by single tested cyanobacterial metabolites except for ANA-B at the lowest concentration (250 µg/L). Although the mixtures of the metabolites inhibited oxygen consumption with antagonistic interactions between the components stimulation was noted in the group exposed to the lowest concentrations of AER-B + CYL (125 µg/L + 125 µg/L, respectively) and the ternary mixture of AER-B + ANA-B + CYL (83.3 µg/L + 83.3 µg/L + 83.3 µg/L, respectively). In vivo fluorescent staining with Hoechst 34580 showed that single AER-B had lower cytotoxic potential on body integument cells than ANA-B and CYL and most binary mixtures except for AER-B + CYL induced synergistic toxicity. Catecholamine level was decreased in animals exposed to single metabolites, their binary and ternary mixtures; however, the interactions between the components in the ternary mixture were antagonistic. Fat body was found to be disrupted in the larvae exposed to single metabolites and their combinations. Antagonistic toxic interactions between the oligopeptide components were found in most binary and the ternary mixtures; however, synergistic effect was noted in the binary mixture of AER-B + CYL. The results suggest that in natural conditions Chironomus larvae and possibly other benthic invertebrates may be affected by cyanobacterial metabolites, however various components and in mixtures and their concentrations may determine varied physiological effects and diverse interactions.

First report on adverse effects of cyanobacterial anabaenopeptins, aeruginosins, microginin and their mixtures with microcystin and cylindrospermopsin on aquatic plant physiology: An experimental approach.

Cyanobacteria produce a variety of oligopeptides beyond microcystins and other metabolites. Their biological activities are not fully recognized especially to aquatic plants. Acute toxicity tests on Spirodela polyrhiza and Lemna minor exposed to a range of concentrations of cyanobacterial metabolites: anabaenopeptins (ANA-A, ANA-B), aeruginosins 98 (Aer-A, Aer-B), microginin-FR1 (MG-FR1), microcystin-LR (MC-LR) and cylindrospermopsin (Cyl) were carried out to compare their influence on plant physiology. Effects of their binary mixtures were determined by isobole approach and calculation of the combination index (CI) that indicates a type of metabolites' interaction. Cyclic oligopeptides microcystin-LR and anabaenopeptin-A revealed the strongest inhibition of S. polyrhiza growth while other metabolites appeared less toxic. Oxygen evolution was inhibited by Cyl, MC-LR, ANA-A, ANA-B, while both variants of aeruginosins and MG-FR1 did not affect this process. Photosynthetic pigments' contents decreased in S. polyrhiza exposed to ANA-A and Cyl, while MC-LR and Aer-A caused their slight increase. 96 h-EC50 values showed that the growth of L. minor was more sensitive to MC-LR, ANA-A, MG-FR1 and Cyl than the growth of S. polyrhiza. In S. polyrhiza synergistic effects of all the binary mixtures of peptides with MC-LR on oxygen evolution were observed, while antagonistic one on the growth of S. polyrhiza exposed to the mixtures with aeruginosins and ANA-A. The mixtures of MC-LR and MG-FR1 with cylindrospermopsin revealed synergistic effects on the growth but antagonistic one to the O2 evolution. Quadruple mixtures (ANA-A + MC-LR + MG-FR1+Cyl) did not reveal any inhibitive effect on the plant growth and very slight on the oxygen evolution, irrespectively of their total concentrations. Various effects caused by ANA-A and ANA-B suggest the importance of molecule structures of metabolites for toxicity. Composition of the mixtures of cyanobacterial metabolites was essential for the observed effects.

Cyanobacterial metabolites: Aeruginosin 98A, microginin-FR1, anabaenopeptin-A, cylindrospermopsin and their mixtures affect behavioral and physiological responses of Daphnia magna.

We determined the effects influence of cyanobacterial products metabolites: aeruginosin-A (AER-A), microginin-FR1 (MG-FR1), anabaenopeptin-A (ANA-A), cylindrospermopsin (CYL) and their binary and quadruple mixtures on swimming behavior, heart rate, thoracic limb activity, oxygen consumption and in vivo cell health of Daphnia magna. The study showed that CYL induced mortality of daphnids at the highest concentrations, however three oligopeptides had no lethal effect. All the tested Each single metabolites inhibited swimming speed. The mixtures AER+MG-FR1 and AER-A+ANA-A induced antagonistic and the quadruple mixture synergistic effects. Physiological endpoints were depressed by CYL, however they were simulated by the oligopeptides and their binary mixtures. The quadruple mixture inhibited the physiological parameters with antagonistic interactions between the components were antagonistic. Single CYL, MG-FR1 and ANA-A induced cytotoxicity with synergistic interactions and the metabolites in mixtures showed. The study suggests that swimming behavior and physiological parameters may be affected by single cyanobacterial oligopeptides, however their mixtures may induce different total effects.

Searching for Optimal Substitute Habitats for Plants by Biological Experiments-A Case Study of the Endangered Species Aldrovanda vesiculosa L. (Droseraceae).

The selection of appropriate locations for the reintroduction of endangered plant species is an important process, because it usually influences the success of the conservation. The aim of this study was to select the optimal substitute habitats for Aldrovanda vesiculosa, taking into account the influence of physical-chemical factors (light intensity, temperature, pH, concentration of dissolved forms of nitrogen and cyanobacterial toxin microcystin-LR) on the efficiency of plant growth. Water analysis and field observations of the habitats of six lakes in Eastern Poland typified as potential substitute habitats for aldrovanda were carried out. The results of the experiments showed that both the concentration and the form in which nitrogen compounds are present in the environment were the factors limiting the growth rate and condition of plants. The second factor that caused the inhibition of aldrovanda growth was microcystin-LR. It was found that the habitat conditions in Lake Brzeziczno were within the ecological tolerance of the species. Particularly important was the low content of mineral compounds and the available forms of nitrogen and phosphorus in the water. Therefore, the probability of development of toxic cyanobacteria, the metabolites of which may affect the growth of A. vesiculosa, is also minimal.

Behavioral disturbances induced by cyanobacterial oligopeptides microginin-FR1, anabaenopeptin-A and microcystin-LR are associated with neuromotoric and cytotoxic changes in Brachionus calyciflorus.

Aquatic animals are exposed to various cyanobacterial products released concomitantly to the environment by decaying blooms. Although there exist results on the toxicity of cyanobacterial extracts little is known on the influence of pure oligopeptides or their mixtures and elucidated mechanisms of behavioral toxicity in zooplanktonic organisms. Therefore, the aim of the present study was to assess the effects of single and mixed pure cyanobacterial oligopeptides: microginin FR-1 (MG-FR1), anabaenopeptin-A (ANA-A) and microcystin-LR (MC-LR) at various concentrations on the swimming behavior and catecholamine neurotransmitter activity, muscular F-actin structure, DNA nuclear content and cell viability of a model rotifer Brachionus calyciflorus. Swimming behavior was analyzed with the use of video digital analysis. Fluorescent microscopy imaging was used to analyze neuromotoric biomarkers in the whole organisms: neuromediator release (by staining with EC517 probe), muscle F-actin filaments (by staining with blue phalloidin dye). DNA content and cytotoxicity was also determined by Hoechst 34580 and propidium iodide double staining, respectively. The results showed that single oligopeptides inhibited all the tested endpoints. The binary mixtures induced synergistic interaction on swimming speed except for MG-FR1 +MC-LR which was nearly additive. Both binary and ternary mixtures also synergistically degraded F-actin and triggered cytotoxic effects visible in the whole organisms. Antagonistic inhibitory effects of all the binary mixtures were found on catecholamine neurotransmitter activity, however the ternary mixture induced additive toxicity. Antagonistic effects of both binary and ternary mixtures were also noted on nuclear DNA content. The results of the study suggest that both depression of neurotransmission and impairment of muscle F-actin structure in muscles may contribute to mechanisms of Brachionus swimming speed inhibition by the tested single cyanobacterial oligopeptides and their mixtures. The study also showed that natural exposure of rotifers to mixtures of these cyanobacterial metabolites may result in different level of interactive toxicity with antagonistic, additive synergistic effects depending on the variants and concentrations present in the environment.

Synergistic toxicity of some cyanobacterial oligopeptides to physiological activities of Daphnia magna (Crustacea).

Anabaenopeptins and microcystins are oligopeptides produced by bloom-forming cyanobacteria. We determined in vivo effects of anabaenopeptin-B (AN-B) and two variants of microcystins of different hydrophobicity (MC-LR and MC-LF) on the physiology of Daphnia magna. Heart rate, thoracic limb activity and post-abdominal claw activity were determined by digital video analysis and oxygen consumption by Oxygraph + system. EC50 calculation and isobole methodology for interactive effects of AN-B and MC-LR mixture were used. Daphnids' responses to all three oligopeptides were concentration- and time-dependent. MC-LF was the most potent inhibitor of heart rate, thoracic limb activity, post-abdominal claw activity and oxygen consumption. AN-B was more toxic than MC-LR toward oxygen consumption; it inhibited the movements of limbs and post-abdominal claw similarly to MC-LR, but did not inhibit heart rate. The strongest toxic effects were induced by the binary mixture of AN-B with MC-LR at the sum concentration equal to the concentration of the single compounds. First time direct synergistic toxic effects of the cyanopeptides on all the physiological parameters were found. The obtained results explain stronger disturbances in aquatic organisms caused by cyanobacterial cell contents than the individual cyanopeptides present even at higher concentrations. Other metabolites and their interactions need further studies.

Cyanobacterial anabaenopeptin-B, microcystins and their mixture cause toxic effects on the behavior of the freshwater crustacean Daphnia magna (Cladocera).

Comparison of the toxic effects caused by the pure cyanobacterial cyclic hexapeptide anabaenopeptin-B (AN-B), the heptapeptides: microcystin-LR (MC-LR) and MC-LF as well as a binary mixture of AN-B with MC-LR on the swimming speed and hopping frequency - essential activities of Daphnia, was experimentally determined. Till now, no information on behavioral effects of AN-B and its mixture with microcystins, commonly produced by cyanobacteria, was available. Also MC-LF effect on aquatic crustaceans was determined for the first time. The results showed that AN-B exerted considerable inhibition of D. magna swimming speed and hopping frequency similar to MC-LR and MC-LF. The mixture of AN-B and MC-LR caused stronger toxic effects, than the individual oligopeptides used at the same concentration. The much lower 48 h- EC50 value of the AN-B and MC-LR mixture (0.95 ± 0.12 µg/mL) than those of individual oligopeptides AN-B (6.3 ± 0.63 µg/mL), MC-LR (4.0 ± 0.27 µg/mL), MC-LF (3.9 ± 0.20 µg/mL) that caused swimming speed inhibition explains the commonly observed stronger toxicity of complex crude cyanobacterial extracts to daphnids than individual microcystins. The obtained results indicated that AN-B, microcystins and their mixture exerted time- and concentration-dependent motility disturbances of crustaceans and they can be good candidates for evaluation of toxicity in early warning systems. Other cyanobacterial oligopeptides beyond microcystins should be considered as a real threat for aquatic organisms.

The Effects of Cyanobacterial Bloom Extracts on the Biomass, Chl-a, MC and Other Oligopeptides Contents in a Natural Planktothrix agardhii Population.

Blooms of the cyanobacterium Planktothrix agardhii are common in shallow, eutrophic freshwaters. P. agardhii may produce hepatotoxic microcystins (MCs) and many other bioactive secondary metabolites belonging mostly to non-ribosomal oligopeptides. The aim of this work was to study the effects of two extracts (Pa-A and Pa-B) of P. agardhii-predominated bloom samples with different oligopeptide profiles and high concentration of biogenic compounds on another natural P. agardhii population. We hypothesised that the P. agardhii biomass and content of oligopeptides in P. agardhii is shaped in a different manner by diverse mixtures of metabolites of different P. agardhii-dominated cyanobacterial assemblages. For this purpose, the biomass, chlorophyll a and oligopeptides content in the treated P. agardhii were measured. Seven-day microcosm experiments with four concentrations of the extracts Pa-A and Pa-B were carried out. Generally, aeruginosins (AERs), cyanopeptolins (CPs) and anabaenopeptins (APs) were the most numerous peptides; however, only 16% of them were common for both extracts. The addition of the extracts resulted in similar effects on P. agardhii: an increase in biomass, Chl-a and MC content in the exposed P. agardhii as well as changes in its oligopeptide profile were observed. MCs present in the extracts did not inhibit accumulation of P. agardhii biomass, and did not have any negative effect on MC and Chl-a content. No evidence for bioaccumulation of dissolved peptides in the P. agardhii exposed was found. As the two tested extracts differed considerably in oligopeptide composition, but contained similar high concentrations of nutrients, it seems that biogenic compounds, not oligopeptides themselves, positively influenced the mixed natural P. agardhii population.

Early indicators of behavioral and physiological disturbances in Daphnia magna (Cladocera) induced by cyanobacterial neurotoxin anatoxin-a.

The majority of reports on the toxic effect of cyanobacterial metabolites on the freshwater invertebrates is based on determination of two endpoints: mortality or immobilization. However, detection of sub-lethal effects requires more sensitive indicators The aim of the present study was to evaluate the applicability of digital-video analysis for determination of early behavioral and physiological responses in the assessment of effects caused by the cyanobacterial neurotoxin, anatoxin-a (ANTX) at a broad range of its concentration (0.5-50 µg/mL). Swimming speed (SS), heart rate (HR), oxygen consumption (OC), thoracic limb activity (TLA) and abdominal claw movement (ACM) of Daphnia magna were evaluated. Swimming speed and abdominal claw movements were determined by digital analysis of video clips by Tracker® software; OC by Oxygraph Plus System® while HR, TLA and ACM by digital frame-by-frame analysis of video clips of microscopic view with the use of a media player software. The experimental study showed a concentration- and time-dependent decrease of SS, HR, OC, TLA and ACM. SS was inhibited as early as after 10 s of the exposure of Daphnia magna to ANTX, and the other physiological responses after 2 h. Further inhibition of these parameters was also noted after 24 h of the exposure. On the other hand, stimulation of ACM was noted at the lower (0.5 and 2.5 µg/mL) ANTX concentrations after both 2 h and 24 h of exposure. The results indicated that some behavioral and physiological biomarkers measured by video analysis may be a valuable tool for an early determination of toxic effects induced by cyanobacterial metabolites in zooplankters.

Effects of secondary metabolites produced by different cyanobacterial populations on the freshwater zooplankters Brachionus calyciflorus and Daphnia pulex.

Cyanobacterial blooms in eutrophic water bodies are a worldwide problem. Combined effects of mixtures of secondary metabolites produced by different cyanobacterial species on aquatic fauna are still not well recognised. We compared the survivorship of Brachionus calyciflorus Pallas (Rotifera) and Daphnia pulex Leyding (Cladocera) exposed to pure microcystin LR (MC-LR), anatoxin-a (ANTX) and to five extracts obtained from bloom-forming cyanobacteria Microcystis, Planktothrix and Dolichospermum. The obtained results revealed different response of the organisms to high concentrations of pure MC-LR, ANTX and complex cyanobacterial extracts. The extracts' toxicity to invertebrates was higher than that exerted by pure cyanotoxins and was dependent on the composition of cyanobacterial metabolites: Microcystis spp. extract containing anabaenopeptins A and B, aeruginosamide, four variants of cyanopeptolins and five MCs was not toxic to either of the organisms, whereas Planktothrix agardhii extract (I), containing anabaenopeptins A, B, F, 915, oscillamide Y, five different aeruginosins and four variants of MC was more toxic to daphnids than to rotifers. The extracts of another P. agarhdii (II) biomass and two different biomass samples of Dolichospermum spp. also affected survivorship of the rotifer and cladoceran, however, to various extent. It strongly suggests that non-ribosomal oligopeptides, other than MCs, had essential contribution to the observed toxicity to invertebrates and their effects on particular species or populations can vary depending on the secondary metabolite profiles of cyanobacteria.

Effects of experimental addition of nitrogen and phosphorus on microbial and metazoan communities in a peatbog.

Interactions between microbial communities and the classical grazing food chain are essential for understanding the functioning of peatbog ecosystems. However, little is known of how short-term intensification of eutrophication processes may influence microbial and metazoan communities in transitional peatbog. We addressed the hypothesis that an increase in the concentration of nutrients will affect the species richness and abundance of microorganisms and small metazoans and cause changes in the food web structure in a peatbog. The experiments were performed in a transitional peatbog. Four experimental variants were conducted (control and nutrient-enriched: +P, +N and P+N). Increased habitat fertility was found to modify the taxonomic composition and functioning of microbial communities. We observed a strong reduction in the species richness of testate amoebae-top predators, and a substantial increase in the abundance of bacteria, flagellates and ciliates. A better understanding of which parameters regulate microbial populations in peatbogs is critical for more accurate prediction of how peatbogs will respond to global climate change or anthropogenic disturbances.

Mass Development of Diazotrophic Cyanobacteria (Nostocales) and Production of Neurotoxic Anatoxin-a in a Planktothrix (Oscillatoriales) Dominated Temperate Lake.

In spite of extensive studies on multispecies toxigenic cyanobacterial blooms, they are still difficult to eliminate, and factors regulating their succession and toxin production remain still to discover. A 4-year study revealed periodical mass development of diazotrophic Nostocales such as Dolichospermum spp. (previously Anabaena), Aphanizomenon gracile and expansive Cuspidothrix (previously Aphanizomenon) issatschenkoi in a lake affected by perennial blooms of Planktothrix agardhii (Oscillatoriales). Compared to Oscillatoriales, Nostocales reached the highest total biomass (up to 16 mg L-1) and contributed nearly 33-85 % to the total biomass of filamentous cyanobacteria at higher water temperatures (average values 17.5-22.6 °C) and higher ratio (11.8-14.1) of dissolved inorganic nitrogen to dissolved inorganic phosphorus (DIN/DIP). Species structure of Nostocales changed considerably from year to year as indicated by the Jaccard similarity index (0.33-0.78). Concentrations of intracellular anatoxin-a (ANTX) ranged from 0.03 to 2.19 µg L-1 of the lake water, whilst extracellular toxin reached up to 0.55 µg L-1. The highest positive correlations were found between the intracellular ANTX and the biomass of Dolichospermum spp. (R2 = 0.73) and C. issatschenkoi (R2 = 0.43-0.65). Our study suggests that ANTX production by Dolichospermum depended mainly on water temperature, whereas that by C. issatschenkoi was related to water conductivity and DIN/DIP ratio. P-PO4 concentrations also seemed to be important. The relatively short-term mass development of neurotoxic Nostocales is an additional threat to shallow, highly eutrophic water bodies continuously affected by Oscillatoriales blooms and may be controlled mainly by the DIN/DIP ratio. ANTX should be considered as a pollutant of freshwaters.

Adaptation strategies of two closely related Desmodesmus armatus (green alga) strains contained different amounts of cadmium: a study with light-induced synchronized cultures of algae.

During the Desmodesmus armatus cell cycle, 8-celled coenobia of 276-4d strain accumulated a much lower amounts of cadmium than unicells of B1-76 strain. Cadmium reduced growth and photosynthesis in the cells of strain B1-76, but not those of 276-4d strain. Cells of 276-4d strain revealed a higher activity of superoxide dismutase (SOD) isoforms, in particular the activity and protein content of Fe-SOD. Cu/Zn-SOD was earlier and much stronger induced by cadmium in 276-4d than in B1-76 strain, whereas Fe- and Mn-SOD activity and Fe-SOD synthesis were induced only in 276-4d strain. Cadmium did not affect the heat shock protein 70 synthesis in B1-76 strain, but significantly stimulated this process in 276-4d strain. The level of glutathione increased 30-fold during cell development of Cd-exposed 276-4d strain, while in B1-76 it increased about 12 timed. Matured cells of both strains exposed to cadmium produced comparable amounts of phytochelatins and other thiol peptides, but their production in young cells of B1-76 strain was much higher than in 276-4d strain. In conclusion, a complex of internal detoxification mechanisms appeared to be more efficient in cells of 276-4d strain than B1-76 one.

Influence of photoperiods on the growth rate and biomass productivity of green microalgae.

The effect of different photoperiods: 24 h illumination and a 12:12-h light/dark (12L:12D) cycle on the growth rate and biomass productivity was studied in five algal species: Neochloris conjuncta, Neochloris terrestris, Neochloris texensis, Botryococcus braunii and Scenedesmus obliquus. The green microalgae examined differ in the reproduction mode. Continuous illumination stimulated the growth of B. braunii and S. obliquus more effectively than the growth of the microalgal species from the genus Neochloris. However, under shorter duration of light of the same intensity (12L:12D cycle), the growth of all the three species of Neochloris was stimulated. Under continuous illumination, the specific growth rate in the first phase of B. braunii and S. obliquus cultures was higher than the growth rate of Neochloris, whereas under the 12L:12D cycle, the specific growth rate of all the three Neochloris species was generally higher than that in B. braunii and S. obliquus. As a result, the light regime influenced algal biomass productivity differently. The maximum biomass productivity was obtained in B. braunii and S. obliquus cultures carried out at continuous illumination. All the Neochloris species produced biomass more efficiently at the 12L:12D cycle, which was two-threefold higher than that of B. braunii and S. obliquus. The unicellular species of the green microalgae from the genus Neochloris, examined for the first time in this study, are promising prospective objects for algal biotechnology.

Effects of microcystin-containing cyanobacterial extract on hematological and biochemical parameters of common carp (Cyprinus carpio L.).

The aim of the study was to assess the effects of a cyanobacterial extract containing microcystins (MCs) on selected hematological and biochemical parameters in common carp (Cyprinus carpio L.), as well as to determine the accumulation of toxins in fish tissues. The fish were immersed for 5 days in water containing toxins at a final concentration of 12 µg/L of microcystin LR equivalent. Microcystin LR residues were detected in fish liver, reaching 207, 238 and 260 ng/g f.w. of the tissues taken 24 h, 72 h and 5 days after the end of intoxication, respectively. The most substantial changes were found in fish plasma, including increases in creatine kinase, lactate dehydrogenase, ammonia, glucose, aspartate aminotransferase and alanine aminotransferase levels. A decline of about 50% in lysozyme activity was observed by the end of the experimental period. Moreover, a marked increase in ceruloplasmin activity was detected 24 h after the end of intoxication with a subsequent decrease in its activity after 72 h and 5 days. This study concludes that not only consumption of food containing toxins but also MCs dissolved in water may pose a threat to fish health. Additionally, detected changes in lysozyme and ceruloplasmin activity may have distinct effects in fish resistance against pathogens or oxidative stress, which should be taken into account in the future studies.

Toxicity of cyanobacterial bloom in the eutrophic dam reservoir (Southeast Poland).

Cyanobacterial bloom was observed in a highly eutrophic dam reservoir, Zemborzycki, near Lublin (SE Poland) over a warm period in the year 2007. The water bloom consisted of several cyanobacterial taxa: Anabaena circinalis, Anabaena spiroides, Anabaena flos-aquae, Planktothrix agardhii, Aphanizomenon flos-aquae, Aphanizomenon gracile, and Microcystis flos-aquae. Anabaena spp., and Aphanizomenon spp., potential producers of neurotoxic anatoxin-a, quantitatively predominated in the studied bloom. High-performance liquid chromatography (HPLC) analysis of surface scum sampled during Anabaena circinalis domination revealed the presence of anatoxin-a at a high concentration (1,035.59 microg per liter of surface scum). At the same time, neither gas chromatography/mass spectrometry (GC/MS) nor microcystin enzyme-linked immunosorbent assay (ELISA) test showed the presence of other frequently found cyanotoxins, microcystins. Toxicity of cyanobacterial bloom was assessed by the crustacean acute toxicity test Daphtoxkit F pulex using Daphnia pulex, and by the chronic toxicity test Protoxkit F with a ciliate protozoan Tetrahymena thermophila. The crude extract of cyanobacterial scum showed high toxicity for Daphnia pulex, with 24-h median effective concentration (EC50) value of 90.3 microg/L of anatoxin-a, which corresponded to the cyanobacterial density in the scum of 1.01 g dry weight/L. For Tetrahymena thermophila, 24-h EC50 was lower, evaluated to be 60.48 microg/L of anatoxin-a, which corresponded to a cyanobacterial density of 0.68 g dry weight/L of the scum. On the basis of evaluated toxicity units, the cyanobacterial extract was classified at class IV toxicity, which means high toxic hazard.

Response of two terrestrial green microalgae (Chlorophyta, Trebouxiophyceae) isolated from Cu-rich and unpolluted soils to copper stress.

Some algae inhabit Cu-polluted soils. Intracellular Cu-accumulation and production of non-protein thiols in response to copper stress were compared in Stichococcus minor and Geminella terricola isolated from Cu-polluted and unpolluted soils, respectively. Cu-exposed (0.5 microM) S. minor accumulated lower amounts of copper (0.38 mM) than G. terricola (4.20 mM) and maintained 8.5-fold higher level of glutathione (GSH) than G. terricola. The ratio GSH/0.5 GSSG in the Cu-treated S. minor (7.21) was 7-times higher than in G. terricola. Reduced and oxidized forms of phytochelatins were found in both algae. Under copper stress (5 microM) the ratio -SH(total)/Cu(intracellular) in S. minor ranged from 2.3 to 6.2, while it was lower than 1.0 in G. terricola. Low intracellular Cu-accumulation and maintenance of high GSH level concomitant with PCs production seem to be responsible for a higher Cu-resistance of S. minor than G. terricola.

Concentrations of phytochelatins and glutathione found in natural assemblages of seaweeds depend on species and metal concentrations of the habitat.

The occurrence of the metal-complexing thiol peptides, phytochelatins (PC) in natural populations of brown, red and green seaweeds (marine macroalgae) was studied. Concentrations of PCs and their precursor glutathione (GSH) were measured in seaweeds collected from locations in south-west England with different levels of contamination by trace metals, to evaluate their role under natural environmental conditions. The non-protein thiols were identified and quantified in seaweed extracts by HPLC and the molecular structures of PCs were confirmed by LC-ESIMS. The capacity for production of PCs of representative seaweeds under Cd and Zn exposure was also assessed, experimentally. The concentrations of metals/metalloids (As, Cu, Cd, Pb and Zn) accumulated by the seaweeds were determined by ICP-MS. For the first time, PCs are reported in native Phaeophyceae (Fucus spp.), Rhodophyceae (Solieria chordalis) and Chlorophyceae (Rhizoclonium tortuosum) but not in thalli of Ulva spp. and Codium fragile (Chlorophyceae). The concentrations of PCs in brown and red seaweeds correlated with the contamination history of sampling sites and total metal burden of thalli. The highest concentrations of metals (5.6-7.1micromolg(-1) DW), PCs (200-240nmolSHg(-1)DW) and GSH (1,550-3,960nmolSHg(-1)DW), and the longest PC chain lengths (PC(2-4)) were found in Fucus spp. collected from the most contaminated site. A combination of PC-production and maintenance of high concentrations of GSH allows Fucus spp. and R. tortuosum (2,000nmolGSHg(-1)DW) to thrive in highly contaminated environments whereas in Ulva spp. high concentrations of GSH (1,000-1,500nmolSHg(-1)DW) together with thick cells walls and a high polysaccharide content appear to be responsible for metal-resistance. The lack of production of PCs in these green macroalgae suggests lower intracellular metal accumulation rather than an inability for synthesizing PCs. The higher concentrations of Cu (approximately 3.4micromolg(-1)DW) found in thallus of S. chordalis, compared with the Fucus spp. (1.5-2.4micromolg(-1)DW) from the same site, may induce stronger oxidative stress and result in lower concentrations of reduced glutathione (648nmolSHg(-1)DW) and PCs (70nmolSHg(-1)DW). As a consequence S. chordalis at this site may have a lower resistance to metals and a more restricted distribution than the fucoids. Both fucoid species and the red seaweed Gracilaria gracilis, but not Ulva spp. or C. fragile, from low contaminated sites synthesized PCs under laboratory conditions when exposed to very high concentration of Cd. Our results clearly show that natural assemblages of seaweeds, belonging to disparate phylogenetic groups produce PCs when exposed to a mixture of metals in their environment. However, the involvement of thiol peptides in metal homeostasis, detoxification and resistance varies between seaweed species that are growing under the same environmental conditions.

Response to copper stress in aposymbiotically grown lichen mycobiont Cladonia cristatella: uptake, viability, ergosterol and production of non-protein thiols.

The mycobiont of lichens usually determines the morphology of the symbiotic organism and is also dominates in terms of biomass. However, its role for sensitivity or tolerance of lichens to heavy metals is almost unknown. In the present study, the influence of copper (Cu) on the aposymbiotically-grown mycobiont of Cladonia cristatella was assessed. Intracellular Cu uptake was correlated with increasing Cu concentrations over a 24-h exposure time. Viability, measured as the degree of reduction of triphenyltetrazolium chloride to triphenyl formazan, as well as to ergosterol levels, decreased with growing Cu concentrations tested. Reduced glutathione (GSH) was found to be the most abundant low-molecular-weight thiol in the hyphae of C. cristatella and its intracellular content increased at concentrations of 10mum Cu. Higher Cu concentrations caused a significant decrease in GSH, possibly due to heavy metal-induced oxidation of GSH to glutathione disulphide (GSSG). Free cysteine levels were relatively constant. As expected, we did not observe the production of phytochelatins in the mycobiont, contrary to what is found in intact lichens and axenic cultures of their photobionts.

Differential responses to Cr(VI)-induced oxidative stress between Cr-tolerant and wild-type strains of Scenedesmus acutus (Chlorophyceae).

A wild-type and a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus were used to investigate if the difference in tolerance to Cr(VI) could depend on a different response to oxidative stress in terms of free cysteine (Cys) and reduced glutathione (GSH), and in preventing membrane lipid peroxidation. The growth of the two strains in standard medium in the presence of Cr(VI) 9.6, 19.2 or 38.4microM was compared, and the content of thiobarbituric acid reactive substances (TBARS) after a 4-day treatment was determined as marker of lipid peroxidation. The Cys and GSH contents were determined in both strains treated with Cr(VI) for 24h in HEPES buffer both enriched and not with sulphate. The treated algae were also subjected to recovery test in standard medium. The growth of wild type was inhibited at all Cr(VI) concentrations, while that of the Cr-tolerant strain only at the highest one. After exposure to 19.2microM Cr(VI), TBARS levels ranging from 0.091 to 0.263micromol/g d.w. were detected in the wild type, while only a slight increase (0.090+/-0.014micromol/g d.w.) was observed in the Cr-tolerant strain. Following treatment with 38.4microM Cr(VI), TBARS levels rose significantly and were similar in the two strains. The Cys content did not vary significantly in the cells exposed to Cr(VI) in either sulphate-lacking or sulphate-enriched buffer, and the differences between the two strains were insignificant. In the wild-type strain, the GSH content showed a significant downward trend with the increase in Cr(VI) concentration in the sulphate-lacking buffer, while it remained as high as the one of control cells in the sulphate-enriched buffer. In the Cr-tolerant strain, the GSH content did not vary significantly when the treatment took place in the sulphate-lacking buffer, while it showed a significant rise with the increase in Cr(VI) concentration in the sulphate-enriched buffer. The growth of both strains during recovery was significantly faster after treatment in the sulphate-enriched than in the sulphate-lacking buffer, the Cr-tolerant strain showing a much higher recovery capacity than the wild type. It appears that the Cr-tolerant strain, when exposed to Cr(VI) in the presence of a sulphur source, can increase GSH pool to levels not achievable by the wild type, and is thus able to recover better. This first report on the role of thiol compounds in Cr tolerance in algae suggests that tolerance to Cr(VI) in S. acutus could depend on a prompt up-regulation of the pathways leading to GSH synthesis.