Alanine to serine substitutions drive thermal adaptation in a psychrophilic diatom cytochrome c6.

In this study, we investigate the thermodynamic mechanisms by which electron transfer proteins adapt to environmental temperature by directly comparing the redox properties and folding stability of a psychrophilic cytochrome c and a mesophilic homolog. Our model system consists of two cytochrome c6 proteins from diatoms: one adapted specifically to polar environments, the other adapted generally to surface ocean environments. Direct electrochemistry shows that the midpoint potential for the mesophilic homolog is slightly higher at all temperatures measured. Cytochrome c6 from the psychrophilic diatom unfolds with a melting temperature 10.4 °C lower than the homologous mesophilic cytochrome c6. Changes in free energy upon unfolding are identical, within error, for the psychrophilic and mesophilic protein; however, the chemical unfolding transition of the psychrophilic cytochrome c6 is more cooperative than for the mesophilic cytochrome c6. Substituting alanine residues found in the mesophile with serine found in corresponding positions of the psychrophile demonstrates that burial of the polar serine both decreases the thermal stability and decreases the midpoint potential. The mutagenesis data, combined with differences in the m-value of chemical denaturation, suggest that differences in solvent accessibility of the hydrophobic core underlie the adaptation of cytochrome c6 to differing environmental temperature.

The relationship between sulfur metabolism and tolerance of hexavalent chromium in Scenedesmus acutus (Spheropleales): Role of ATP sulfurylase.

Sulfur availability and the end products of its metabolism, cysteine, glutathione and phytochelatins, play an important role in heavy metal tolerance, chromium included. Sulfate and chromate not only compete for the transporters but also for assimilation enzymes and chromium tolerance in various organisms has been associated to differences in this pathway. We investigated the mechanisms of Cr(VI)-tolerance increase induced by S-starvation focusing on the role of ATP sulfurylase (ATS) in two strains of Scenedesmus acutus with different chromium sensitivity. S-starvation enhances the defence potential by increasing sulfate uptake/assimilation and decreasing chromium uptake, thus suggesting a change in the transport system. We isolated two isoforms of the enzyme, SaATS1 and SaATS2, with different sensitivity to sulfur availability, and analysed them in S-sufficient and S-replete condition both in standard and in chromium supplemented medium. SaATS2 expression is different in the two strains and presumably marks a different sulfur perception/exploitation in the Cr-tolerant. Its induction and silencing are compatible with a role in the transient tolerance increase induced by S-starvation. This enzyme can however hardly be responsible for the large cysteine production of the Cr-tolerant strain after starvation, suggesting that cytosolic rather than chloroplastic cysteine production is differently regulated in the two strains.

Scenedesnus rotundus isolated from the petroleum effluent employs alternate mechanisms of tolerance to elevated levels of Cadmium and Zinc.

Scenedesmus rotundus was isolated from metal contaminated petroleum industry effluent and its tolerance to Cadmium and Zinc was tested using different concentrations of CdCl2 and ZnCl2 ranging from 0.001 mM to 1.0 mM of Cd and 0.03 mM to 1.21 mM of Zn amended in Bolds Basal medium. The changes in cell count recorded at regular intervals upto a period of 24 days revealed a concentration dependent inhibition in growth. Concentration of the metal, at which 50% of the cells are live and metabolically active referred to as EC50 was calculated as 0.04 mM for Cd and 0.2 mM for Zn. Further, the effect of EC50 of the metals on the protein content, uptake of metals at varying pH, oxidative stress markers including lipid peroxidation, protein oxidation andnd oxygen uptake, levels of enzymatic antioxidants such as catalase and superoxide dismutase and non-enzymatic antioxidants namely, GSH and PC4 were determined. Though a direct correlation could not be drawn between pH and metal uptake, the compartmentalization of the metal during the lag phase and exponential phase was evident, most of the metal was present in extracellular fractions in the former, while in the later it was internalized. Our study shows a clear correlation between toxicity of Cd and the ability of the algae to synthesize PC4 from GSH and chelate it leading to detoxification, while Zn treatment led to an increase in the activity of catalase and superoxide dismutase and replete GSH pools. Further the changes in the cell wall structure at EC50 of Cd and Zn were studied. This is the first report on effect of heavy metals on the structural modifications of the cell wall of Scenedesmus in general and Scenedesmus rotundus in particular, indicating appearance of granules on the entire cell surface in both Cd and Zn treatments, with the degree of granulation increasing in the order of pH 12 > 10 > 8 in Cd treatment. Further structures of higher order resembling minute wheels are observed in Cd treated cells are also reported.

Toxicity evaluation of 4,4'-di-CDPS and 4,4'-di-CDE on green algae Scenedesmus obliquus: growth inhibition, change in pigment content, and oxidative stress.

Polychlorinated diphenyl sulfides and polychlorinated diphenyl ethers are two types of dioxin-like pollutants, which are prevalent in aquatic environments. However, to date, limited information is available regarding their toxicity to green algae. In this study, growth inhibition, effect on pigment content, and oxidative stress potentials of 4,4'-di-CDPS and 4,4'-di-CDE on green algae Scenedesmus obliquus were investigated. The results indicate that the EC50 values of 4,4'-di-CDPS after 24, 48, 72, and 96 h of exposure were 1.736, 1.172, 0.994, and 0.820 mg/L, while the corresponding values for 4,4'-di-CDE were 0.697, 1.087, 0.833, and 0.327 mg/L. As compared to the control group, most of the measured pigment content in algal cells significantly decreased after 96-h exposure to these two chemicals, suggesting their suppressive capability on the photosynthesis process in algal cells. Additionally, oxidative stress occurred as demonstrated by the significantly inhibited activities of the antioxidant enzymes (total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GPx)), and high increases in malondialdehyde (MDA) content in all 4,4'-di-CDE-treated groups and some moderate-dose and high-dose treatments with 4,4'-di-CDPS. Acute toxicity tests and biochemical analysis showed that 4,4'-di-CDE was more toxic than 4,4'-di-CDPS on S. obliquus.

Comparative toxicity of the plasticizer dibutyl phthalate to two freshwater algae.

Phthalate esters (PAEs), a family of emerging environmental contaminants, have been frequently detected in soils and water. However, intensive studies on the toxicity of PAEs have focused on growth response of terrestrial and aquatic animals, while only limited attention has been paid to aquatic plants, especially phytoplankton, the primary producer in aquatic ecosystems. Therefore, the acute toxic effects and underlying mechanisms of dibutyl phthalate (DBP) at different concentrations (0-20mgL-1) on two typical freshwater algae (Scenedesmus obliquus and Chlorella pyrenoidosa) were investigated. The growth of S. obliquus and C. pyrenoidosa was conspicuously inhibited by DBP exposure at 2-20mgL-1. The 96-h median effective concentration values (96h-EC50) were 15.3mgL-1 and 3.14mgL-1 for S. obliquus and C. pyrenoidosa, respectively, implying that the spherical C. pyrenoidosa is more sensitive to DBP than the spindle-shaped S. obliquus. As expected from the damage done to cell organelles (i.e. cell membranes, chloroplasts, and protein rings), cell densities and chlorophyll content conspicuously decreased under DBP treatments. Moreover, the algal growth inhibition was closely linked to the increased production of intracellular reactive oxygen species and malondialdehyde content, indicating oxidative stress and lipid peroxidation in both algae. This was proved by the increased activity of antioxidant enzymes such as superoxide dismutase and catalase. Our findings will contribute to the understanding of toxic mechanisms in PAEs and the evaluation of environmental risks for primary producers in aquatic ecosystems.

Impacts of dissolved organic matter on aqueous behavior of nano/micron-titanium nitride and their induced enzymatic/non-enzymatic antioxidant activities in Scenedesmus obliquus.

Freshwater dispersion stability and ecotoxicological effects of titanium nitride (TiN) with particle size of 20 nm, 50 nm, and 2-10 µm in the presence of dissolved organic matter (DOM) at various concentrations were studied. The TiN particles that had a more negative zeta potential and smaller hydrodynamic size showed more stable dispersion in an aqueous medium when DOM was present than when DOM was absent. Biochemical assays indicated that relative to the control, the TiN particles in the presence of DOM alleviated to some extent the antioxidative stress enzyme activity in Scenedesmus obliquus. In addition, it was found that the TiN with a primary size of 50 nm at a high concentration presented a significant impact on non-enzymatic antioxidant defense in algal cells.

Microalgae respond differently to nitrogen availability during culturing.

Variations in the exogenous nitrogen level are known to significantly affect the physiological status and metabolism of microalgae. However, responses of red, green and yellow-green algae to nitrogen (N) availability have not been compared yet. Porphyridium cruentum, Scenedesmus incrassatulus and Trachydiscus minutus were cultured in the absence of N in the medium and subsequent resupply of N to the starved cells. Culture growth and in-gel changes in isoenzyme pattern and activity of glutamate synthase, glutamate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase were studied. The results demonstrated that the algae responded to the fully N-depleted and N-replete culture conditions by species-specific metabolic enzyme changes, suggesting differential regulation of both enzyme activity and cellular metabolism. Substantial differences in the activities of the antioxidant enzymes between N-depleted and N-replete cells of each species as well as between the species were also found. In the present work, besides the more general responses, such as adjustment of growth and pigmentation, we report on the involvement of specific metabolic and antioxidant enzymes and their isoforms in the mechanisms operating during N starvation and recovery in P. cruentum, T. minutus and S. incrassatulus.

CO2 biofixation and carbonic anhydrase activity in Scenedesmus obliquus SA1 cultivated in large scale open system.

The present study deals with the large scale open system cultivation of the novel microalga: Scenedesmus obliquus SA1 (KC733762) previously isolated in our laboratory. SA1 strain was cultivated in open system at varying CO2 levels ranging from 0.03% to 35% (v/v) and subsequently the carbonic anhydrase activity (CA) and the biochemical properties were monitored. Maximum biomass concentration (1.39 ± 0.023 g L(-1)), CO2 fixation rate (97.65 ± 1.03 mg L(-1)d(-1)) and total CA activity (166.86 ± 3.30 E.U./mg chla) were obtained at 35% CO2. CA inhibitors: acetazolamide and ethoxyzolamide inhibited the external and internal enzyme activity in SA1. High CO2 levels were favorable for the accumulation of lipids and chlorophyll. The present results suggested that SA1 possessed high CO2 tolerance and high carbohydrate, lipid and chlorophyll content when cultivated in open system thus being suitable for CO2 mitigation in outdoor ponds and subsequent generation of value added products.

Acute and chronic toxic effects of chloramphenicol on Scenedesmus obliquus and Chlorella pyrenoidosa.

The acute and chronic toxicological effects of Chloramphenicol (CAP) on Scenedesmus obliquus and Chlorella pyrenoidosa are not well understood. The indoor experiments were carried to observe and analyze the CAP induced changes. Results of the observations have showed that CAP exposure could significantly inhibit the growth of Scenedesmus obliquus in almost all the treated groups, while Chlorella pyrenoidosa exhibited less sensitivity. Chlorophyll-a syntheses of Scenedesmus obliquus were all inhibited by CAP exposure, while Chlorella pyrenoidosa displayed obvious stimulation effect. Catalase (CAT) and Superoxide dismutase (SOD) activities of both algae were promoted in all the treatments. The experimental results indicated that the growth and Chlorophyll-a syntheses of Scenedesmus obliquus were more sensitive in response to CAP exposure than that of Chlorella pyrenoidosa. While for CAT and SOD activities, Chlorella pyrenoidosa showed more susceptible. This research provides a basic understanding of CAP toxicity to aquatic organisms.

Toxic responses and antioxidative enzymes activity of Scenedesmus obliquus exposed to fenhexamid and atrazine, alone and in mixture.

Laboratory studies were conducted to determine the effects of different concentrations of fenhexamid and atrazine (25, 50 and 100 µg L(-1)) on growth and oxidative stress on Scenedesmus obliquus (microalgae) after exposure for 24, 48, and 96 h. In addition, residues of fenhexamid and atrazine were determined in the culture medium after 96 h; 52%, 44% and 43% of fenhexamid remained in the medium for the lowest, middle and highest concentrations, respectively. Atrazine concentration decreased significantly in the medium with time. The reduction was faster with the lowest concentration (-53%), than in the highest concentration (-46%), while it was intermediate with 50 µg L(-1) (-47%). The antioxidative enzyme activities were used as biomarkers to evaluate the toxic effects of fenhexamid and atrazine on the microalgae. Enzymatic activities were measured in the presence of each compound alone after 24, 48 and 96 h and also in mixture after 24h exposure. The results showed that fenhexamid and atrazine induced antioxidative enzyme activities (GST, CAT and GR) at different concentrations. Catalase activities (CAT) in both pesticides treated-algae were significantly increased. Additionally, an increase in gulathione-S-transferase (GST) was observed in algae after 24, 48 and 96 h of exposure to both fenhexamid and atrazine. Antioxidative enzymes in fenhexamid and atrazine mixture treatment showed an antagonistic interaction after 24h of exposure in algae.

Glutathione peroxidase activity in the selenium-treated alga Scenedesmus quadricauda.

The function of selenium in an organism is mediated mostly by selenoproteins including glutathione peroxidase. Glutathione peroxidase is a potent anti-oxidative enzyme, scavenging a variety of peroxides. The green alga Scenedesmus quadricauda was used to investigate the relationship between the toxicity of selenium and the glutathione peroxidase activity. Selenium resistant strains SeIV and SeVI were synchronized and grown in high concentrations of Se (selenite or selenate). As a measure of selenium toxicity the EC(50) values were determined. During growth of the untreated wild type, glutathione peroxidase activity increased slightly and then declined gradually until the end of the cell cycle. A similar pattern was observed in untreated resistant strains and when resistant strains were grown in the presence of selenium in the oxidation state to which they were resistant. In the wild type cultivated with 50 mg Se L(-1) (selenite or selenate), activity increased to a high level and slowly declined until the end of the cell cycle. Similarly, activity increased in strains SeIV and SeVI when grown in the presence of selenium in the oxidation state to which they were not resistant. We followed the effect of selenium on the ultrastructure of S. quadricauda. After exposure to selenite, the chloroplast membranes of wild type were reorganized into thick bundles of thylakoids and the stroma became granulose. When selenate was added, the chloroplast of wild type had a fingerprint-like appearance, the stroma became less dense and starch production increased. In selenium resistant strains, when treated with the selenium form to which they were resistant, the chloroplast was affected, but not to such an extent as in the wild type. The activity of glutathione peroxidase in Scenedesmus was affected by selenium in an oxidation state-dependent manner. The most apparent effects of selenium on the ultrastructure involved impairment of the chloroplast and the overproduction of starch.

Effects of norfloxacin and butylated hydroxyanisole on the freshwater microalga Scenedesmus obliquus.

The toxic effects of norfloxacin (NOR) and butylated hydroxyanisole (BHA) on the microalga Scenedesmus obliquus were assessed in terms of growth rate, concentration of chlorophyll a, activities of 7-ethoxyresorufin-o-dealkylases (EROD), glutathione s-transferase (GST), catalase (CAT) and total malondialdehyde content (MDA). The 96 h EC(50) was 38.49 and 11.12 mg/l for NOR and BHA, respectively. Growth of S. obliquus was affected slightly under low concentrations of BHA (<4 mg/l) and NOR (<15 mg/l) over the 96 h exposure period. With the increasing concentrations of these two compounds, growth of S. oblique decreased significantly. Growth inhibition was 82.4% at 60.0 mg/l for NOR and 60.6% at 16.0 mg/l for BHA after 96 h. A similar trend was also observed for chlorophyll alpha. NOR and BHA affected Phase I and Phase II enzyme activities differently. Upon exposure to NOR, EROD was induced at concentration <15.0 mg/l and depressed at concentrations >30 mg/l significantly. CAT and GST exhibited similar trends during the exposure period. Compared to controls, MDA content only showed high induction at high concentrations of NOR (>30 mg/l). However, EROD activity did not display any change compared to control responses during BHA exposure, whereas GST showed significant induction for all concentrations over the exposure period. CAT activity showed induction at low concentration and depression at high concentration. MDA content increased with the rise of BHA during the exposure period. These types of assays, revealing toxic effects of NOR and BHA to phototrophs, could be employed to assess the potential risks of these xenobiotics to aquatic ecological systems.

[Individual and joint stress of lead and mercury on growth, glutathione and glutathione-related enzymes of Scenedesmus quadricauda].

To understand the toxicity mechanisms of mixed heavy metals on aquatic plant, indicators of algea growth rate,content of reduced glutathione (GSH), activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx) of green algae, Scenedesmus quadricauda were measured to analyze the individual and joint toxic effects of lead and mercury. The results show that the 96h EC50 of algae growth inhibition by lead [Pb(NO3)2] and mercury (HgCl2) are 0.6789 mg/L and 0.1401 mg/L respectively. After 12 h individual and joint lead and mercury exposure, the content of GSH in alga cells is decreased to about 70% of the level of the control, and keeps a steady level with the increase of the exposure concentration. The GST activities are increased to a peak in lower concentration groups and then decrease with the increase of the exposure concentration. Indeed,the higher concentration of lead and mercury combined-poisoning can inhibit the activities of GST significantly, with 13.04% inhibitory rate. The activity of GPx is almost suppressed continuously with the increase of the exposure concentration, and the lowest activity is only 38.77% of the control. The toxic action of the mixture of Pb and Hg on growth inhibition,GSH content,activities of GST and activities of GPx for Scenedesmus quadricauda are addition.

Effect of pulse herbicidal exposure on scenedesmus vacuolatus: a comparison of two photosystem II inhibitors.

Herbicide concentrations fluctuate in rivers following crop application and can reach high levels after rain events, yet the duration of these pulses is short. In the present study, we assessed the effect of atrazine and isoproturon pulse exposure on Scenedesmus vacuolatus (Chlorophyceae; strain 211-8b, Kessler) as well as the recovery in the postexposure period. We further explored whether the time-dependent toxicity is similar for herbicides inhibiting the photosystem II (PSII). The growth rate was assessed for different exposure durations, and in addition the inhibition of the effective quantum yield of PSII was measured to monitor the response at the target site. Atrazine and isoproturon did not have similar time-dependent effects on growth rate, despite their same primary mode of action on PSII. Atrazine was less toxic than isoproturon after 10 h of exposure, but the toxicity of both herbicides was similar after 48 h of exposure. However, both compounds inhibited the PSII effective quantum yield within 1 h following exposure. Similarly, the effective quantum yield recovered completely within 4 h after removal of the toxicants, leading to rapid recovery of algal growth. The rapid onset of effects of isoproturon on the growth of the alga during exposure suggests that a single pulse to this herbicide is likely to induce greater effects than an atrazine pulse at the same concentration, even if these effects are reversible. The information gained in the present study should support the effect assessment of sequential exposures as well as the risk evaluation of fluctuating herbicidal exposure.

Changes in the LHCII-mediated energy utilization and dissipation adjust the methanol-induced biomass increase.

Considerably low methanol concentrations of 0.5% (v/v), induce an immense increase in biomass production in cultures of the unicellular green alga Scenedesmus obliquus compared to controls without additional methanol. The effect is light-regulated and it mimics high-CO2 induced changes of the molecular structure and function of the photosynthetic apparatus. There is evidence that methanol enhances under high light conditions by molecular changes in the LHCII--a decrease of the functional antenna-size per active reaction center--the photochemical effectiveness of the absorbed energy. This means that the non-photochemical quenching (NPQ) is minimized and thereby the overall dissipation energy. Experiments with mutants of Scenedesmus Wt produced evidence that the LHCII is the locus of the mechanism which regulates the methanol effect. The employed mutants were Wt-LHC, lacking a functioning LHCII, the light-dependent greening mutant C-2A', and the double mutant C-2A'-LHC, combining both mutations.

Salt stress impact on the molecular structure and function of the photosynthetic apparatus--the protective role of polyamines.

In the present study the green alga Scenedesmus obliquus was used to assess the effects of high salinity (high NaCl-concentration) on the structure and function of the photosynthetic apparatus and the possibility for alleviation by exogenous putrescine (Put). Chlorophyll fluorescence data revealed the range of the changes induced in the photosynthetic apparatus by different NaCl concentrations, which altogether pointed towards an increased excitation pressure. At the same time, changes in the levels of endogenous polyamine concentrations, both in cell and in isolated thylakoid preparations were also evidenced. Certain polyamine changes (Put reduction) were correlated with changes in the structure and function of the photosynthetic apparatus, such as the increase in the functional size of the antenna and the reduction in the density of active photosystem II reaction centers. Thus, exogenously added Put was used to compensate for this stress condition and to adjust the above mentioned changes, so that to confer some kind of tolerance to the photosynthetic apparatus against enhanced NaCl-salinity and permit cell growth even in NaCl concentrations that under natural conditions would be toxic.

A polyamine- and LHCII protease activity-based mechanism regulates the plasticity and adaptation status of the photosynthetic apparatus.

In the present study we aim to dissect the basis of the polyamine mode of action in the structure and function of the photosynthetic apparatus. Although the modulating effects of polyamines in photosynthesis have been reported since long [K. Kotzabasis, A role for chloroplast-associated polyamines? Bot. Acta 109 (1996) 5-7], the underlying mechanisms remained until today largely unknown. The diamine putrescine was employed in this study, by being externally added to Scenedesmus obliquus cultures acclimated to either low or high light conditions. The results revealed the high efficiency by which putrescine can alter the levels of the major photosynthetic complexes in a concerted manner inducing an overall structure and function of the photosynthetic apparatus similar to that under higher light conditions. The revealed mechanism for this phenomenon involves alterations in the level of the polyamines putrescine and spermine which are bound to the photosynthetic complexes, mainly to the LHCII oligomeric and monomeric forms. In vitro studies point out to a direct impact of the polyamines on the autoproteolytic degradation of LHCII. Concomitantly to the reduction of the LHCII size, exogenously supplied putrescine, induces the reaction centers' density and thus the photosynthetic apparatus is adjusted as if it was adapted to higher light conditions. Thus polyamines, through LHCII, play a crucial role in the regulation of the photosynthetic apparatus' photoadaptation. The protective role of polyamines on the photosynthetic apparatus under various environmental stresses is also discussed in correlation to this phenomenon.

Determination of short-term copper toxicity in a multispecies microalgal population using flow cytometry.

This study was conducted to determine the role of algal-algal interactions in a multispecies microalgal population on their sensitivities to copper based on an enzyme inhibition assay using flow cytometric measures. Autofluorescence (chlorophyll a and phycocyanin) was used to identify species and count algal signals. The effect of multispecies population on copper toxicity of Microcystis aeruginousa was detected (1) at the same initial cell density, (2) at the same surface area, and (3) in the presence and absence of Chlorella pyrenoidosa and Scenedesmus obliquus. As copper concentrations increased, esterase activity of M. aeruginosa changed in a concentration-dependent manner. The 24 h EC(50) value of M. aeruginosa in the multispecies population was significantly (P < 0.05) higher than those in the single-species population. Compared with S. obliquus, the effect of C. pyrenoidosa on M. aeruginosa was more marked (the 24 h EC(50) value of copper on fluorescin diacetate fluorescence of M. aeruginosa was 11 microg/L). At 48 h copper exposure (6 microg/L) analysis of intracellular reactive oxygen species levels also showed similar algal-algal interactions in multispecies microalgal populations. The pigment assay suggested that these algal-algal interactions occurred only at low concentrations (< 13 microg/L, 24 and 48 h copper exposure). This study demonstrates the importance of using multispecies populations to estimate metal toxicity in natural waters.

Individual and combined effect of anthracene, cadmium, and chloridazone on growth and activity of SOD izoformes in three Scenedesmus species.

Short-term (12-48 h) experiments were carried out to examine the effect of anthracene (three-ring aromatic hydrocarbon), cadmium (CdCl(2)), and chloridazone (triazine herbicide), individually and in combination, on growth and SOD activity of three green algae Scenedesmus: S. subspicatus, S. obliquus, and S. microspina, grown in a batch-culture system. The relative toxicity of chemicals to algae was anthracene > chloridazone >> cadmium. The species revealed similar growth sensitivity to individual chemicals after 24 h of exposure but there were differences between them when exposed to their combinations. Two methods were used to determine the modes of interaction effects of the chemical combinations; both led to the same results, with two exceptions of all 36 variants examined. In general, mixtures of two and three chemicals behaved toward algal growth mainly in an antagonistic manner (20 cases), whereas additive and synergistic interaction occurred 13 and 3 times, respectively. Antagonism was the most frequently observed to growth of S. obliquus; antagonistic interaction and additive effect was noted in relation to S. subspicatus, while response of S. microspina depended markedly on applied combination. There is a relationship between SOD activity and growth response to stress. The markedly higher level of SOD isoforms activities was noticed in cells (especially S. microspina and S. obliquus) exposed to 12 h to combined chemicals, as compared to individually treated and control cells. SOD activities in cells of three Scenedesmus grown 24 h were similar in all experimental variants and after 48 h of exposure significantly decreased in almost all cases (especially in S. subspicatus). There were no differences observed between SOD profiles obtained for all variants examined. Chloroplasts seems to be the main target site of interaction effects of dissimilarly acting chemicals.

Determination of photosynthetic and enzymatic biomarkers sensitivity used to evaluate toxic effects of copper and fludioxonil in alga Scenedesmus obliquus.

Modulated PAM fluorometry and Plant Efficiency Analyser methods were used to investigate photosynthetic fluorescence parameters of alga Scenedesmus obliquus exposed to inhibitory effect of fungicides copper sulphate and fludioxonil (N-(4-nitrophenyl)-N'-propyl-uree). The change of those parameters were studied when alga S. obliquus have been exposed during 48 h to different concentrations of fungicides (1, 2 and 3 mgl(-1)). Under the same condition, enzymatic activities of catalase, ascorbate peroxidase, glutathione reductase and glutathione S-transferase were investigated to evaluate antioxidative response to fungicides effects. The change of sensitivity of those parameters was dependent to the mode of fungicide action, their concentration and time of exposure. For copper effects, the most indicative photosynthetic biomarkers were parameters Q(N) as non-photochemical fluorescence quenching, Q(Emax) as the proton induced fluorescence quenching and ABS/RC as the antenna size per photosystem II reaction center. Copper induced oxidative stress was indicated by increased activity of catalase serving as the most sensitive and valuable enzymatic biomarker. On the other hand, fludioxonil effect on photosynthetic parameters was very negligible and consequently not very useful as biomarkers. However, fludioxonil induced strong antioxidative activities associated with cytosol enzymes, as we found for catalase, ascorbate peroxidase and glutathione S-transferase activities. By obtained results, we may suggest for the activation of those enzymes to be sensitive and valuable biomarkers of oxidative stress induced by fludioxonil. Determination of biomarkers sensitivity may offer advantages in providing real criteria to use them for ecotoxicological diagnostic studies.