Scenedesmus Vacuolatus Research Articles

Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli

We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along with digestive gland bacteriolytic activity were higher in EG than in SV mussels. Both SV and EG mussels showed increased gill mass upon E. coli challenge, while digestive gland mass was increased by bacterial challenge only in SV mussels. Bacterial challenge produced no effect on humoral reactive oxygen species levels of both groups. Total oxyradical scavenging capacity levels was reduced in challenged SV mussels but remained unaffected in EG ones. In general, EG diet decreased glutathione S-transferase and catalase activities in gill and digestive gland, compared with SV diet; but increased enzyme activity was evident in challenged mussels of both groups. Gill and digestive gland lipid peroxidation levels were higher in EG than in SV mussels but E. coli challenge had stronger effect on SV mussels. Adductor muscle RNA:DNA ratio was higher in EG mussels than in SV ones, and increased upon E. coli challenge in mussels of both groups. E. gracilis can be suggested as a nutritional and protective diet complement suitable for filtering bivalves. However, our results obtained from starved mussels show that starvation periods after supplying this diet should be avoided, since these could revert part of the acquired benefits and/or exacerbate detrimental effects.

Predictive QSAR modelling of algal toxicity of ionic liquids and its interspecies correlation with Daphnia toxicity.

Predictive toxicology using chemometric tools can be very useful in order to fill the data gaps for ionic liquids (ILs) with limited available experimental toxicity information, in view of their growing industrial uses. Though originally promoted as green chemicals, ILs have now been shown to possess considerable toxicity against different ecological endpoints. Against this background, quantitative structure-activity relationship (QSAR) models have been developed here for the toxicity of ILs against the green algae Scenedesmus vacuolatus using computed descriptors with definite physicochemical meaning. The final models emerged from E-state indices, extended topochemical atom (ETA) indices and quantum topological molecular similarity (QTMS) indices. The developed partial least squares models support the established mechanism of toxicity of ionic liquids in terms of a surfactant action of cations and chaotropic action of anions. The models have been developed within the guidelines of the Organization of Economic Co-operation and Development (OECD) for regulatory QSAR models, and they have been validated both internally and externally using multiple strategies and also tested for applicability domain. A preliminary attempt has also been made, for the first time, to develop interspecies quantitative toxicity-toxicity relationship (QTTR) models for the algal toxicity of ILs with Daphnia toxicity, which should be interesting while predicting toxicity of ILs for an endpoint when the data for the other are available.

Toxicity of anthelmintic drugs (fenbendazole and flubendazole) to aquatic organisms.

Flubendazole (FLU) and fenbendazole (FEN) belong to benzimidazoles—pharmaceuticals widely used in veterinary and human medicine for the treatment of intestinal parasites as well as for the treatment of systemic worm infections. In recent years, usage of these drugs increased, which resulted in a larger contamination of the environment and possible negative effects on biota. Hence, in our research, we investigated an aquatic ecotoxicity of these pharmaceuticals towards: marine bacteria (Vibrio fischeri), green algae (Scenedesmus vacuolatus), duckweed (Lemna minor) and crustacean (Daphnia magna). Ecotoxicity tests were combined with chemical analysis in order to investigate the actual exposure concentration of the compounds used in the experiment as well as to stability and adsorption studies. As a result, study evaluating sensitivity of different aquatic organisms to these compounds and new ecotoxicological data is presented. The strongest negative impact of FLU and FEN was observed to D. magna.

Development of sensitive and reliable LC-MS/MS methods for the determination of three fluoroquinolones in water and fish tissue samples and preliminary environmental risk assessment of their presence in two rivers in northern Poland

Antibiotic consumption (e.g. fluoroquinolones (FQs)) and, as a consequence, their presence in the environment, have received a lot of attention in the last several years due to increasing numbers of diseases and infections that are becoming resistant to traditional treatments for both humans and animals. In addition, even though antibiotics are safe for human and veterinary usage, ecosystems may be exposed to these substances. In this study, analytical methods for determining enrofloxacin (ENR), norfloxacin (NOR) and ciprofloxacin (CIP) in water samples and fish tissue based on the LC-MS/MS technique were developed and validated. As there is no data available concerning the risks posed by antibiotics in Poland, the proposed methods were applied for monitoring drug presence in environmental samples collected from two rivers in northern Poland. Evaluations of the ecotoxicity of ENR, NOR and CIP towards four different species of aquatic organisms: marine bacteria (Vibrio fischeri), green algae (Scenedesmus vacuolatus), duckweed (Lemna minor) and crustacean (Daphnia magna), were also carried out. All the investigated compounds were detected at least once in the survey. NOR was found to be the most ubiquitous drug with concentrations of up to 442.8ngL−1. Moreover, it was established that L. minor is the most sensitive species to the investigated drugs (EC50NOR=0.13mgL−1, EC50ENR=0.22mgL−1 and EC50CIP=0.34mgL−1). The calculated risk quotient (RQ) values confirmed that the concentrations of the investigated FQs in the environmental samples were at a level of moderate environmental risk (1<RO≤10), however, for CIP they were found to be closer to a level of high environmental risk (RQCIP=8.1).

Beta-blockers in the environment: Part II. Ecotoxicity study

The increasing consumption of beta-blockers (BB) has caused their presence in the environment to become more noticeable. Even though BB are safe for human and veterinary usage, ecosystems may be exposed to these substances. In this study, three selected BB: propranolol, metoprolol and nadolol were subjected to ecotoxicity study. Ecotoxicity evaluation was based on a flexible ecotoxicological test battery including organisms, representing different trophic levels and complexity: marine bacteria (Vibrio fischeri), soil/sediment bacteria (Arthrobacter globiformis), green algae (Scenedesmus vacuolatus) and duckweed (Lemna minor). All the ecotoxicological studies were supported by instrumental analysis to measure deviation between nominal and real test concentrations. Based on toxicological data from the green algae test (S. vacuolatus) propranolol and metoprolol can be considered to be harmful to aquatic organisms. However, sorption explicitly inhibits the hazardous effects of BB, therefore the risks posed by these compounds for the environment are of minor importance.

The reversible degeneration of heat-treated Scenedesmus vacuolatus under continuous light cultivation conditions

To find out how microalgae cope with heat stress, the small vegetative cells of a synchronous Scenedesmus vacuolatus culture were subjected to heat treatment and then cultured under continuous illumination. The heat-treated cells were found first to enter a degenerative intermediate stage with low cellular activities almost right after the start of the cultivation, which was then followed by a revival. The changes in physiological activities and morphology of the treated cells throughout the whole period of regeneration were explored. The variations in cellular DNA content and protein composition were also investigated. Stressed cells at the end of the degeneration stage were completely bleached and were also characterized by condensed but undegraded chromatin, partially disintegrated chloroplasts but with the thylakoid membrane system retained, partially operating mitochondria, intact plasma membranes, and a dramatically changed profile of cellular proteins. All of our data indicate they were still alive but in a different physiological state than the control cells. Recovery started with regeneration of mitochondrial cristae and redispersion of chromatins. These were followed by regreening and resuscitation of chloroplasts, which often started from one part of a thylakoid membrane system and then spread out. This study provided a unicellular model for studying how plant cells react to a period of stress and recover.

In silico modelling for predicting the cationic hydrophobicity and cytotoxicity of ionic liquids towards the Leukemia rat cell line, Vibrio fischeri and Scenedesmus vacuolatus based on molecular interaction potentials of ions

In this study we present prediction models for estimating in silico the cationic hydrophobicity and the cytotoxicity (log [1/EC50]) of ionic liquids (ILs) towards the Leukemia rat cell line (IPC-81), the marine bacterium Vibrio fischeri and the limnic green algae Scenedesmus vacuolatus using linear free energy relationship (LFER) descriptors computed by COSMO calculations. The LFER descriptors used for the prediction model (i.e. excess molar refraction (E), dipolarity/polarizability (S), hydrogen-bonding acidity (A), hydrogen-bonding basicity (B) and McGowan volume (V)) were calculated using sub-descriptors (sig2, sig3, HBD3, HBA4, MR, and volume) derived from COSMO–RS, COSMO and OBPROP. With the combination of two solute descriptors (B, V) of the cation we were able to predict cationic hydrophobicity values (log ko ) with r 2 = 0.987 and standard error (SE) = 0.139 log units. By using the calculated log k o values, we were able to deduce a linear toxicity prediction model. In the second prediction study for the cytotoxicity of ILs, analysis of descriptor sensitivity helped us to determine that the McGowan volume (V) terms of the cation was the most important predictor of cytotoxicity and to simplify prediction models for cytotoxicity of ILs towards the IPC-81 (r 2 of 0.778, SE of 0.450 log units), Vibrio fischeri (r 2 of 0.762, SE of 0.529 log units) and Scenedesmus vacuolatus (r 2 of 0.776, SE of 0.825 log units). The robustness and predictivity of the two models for IPC-81 and Vibrio fischeri were checked by comparing the calculated SE and r 2 (coefficient of determination) values of the test set.

Characterization of the decline and recovery of heat-treated Scenedesmus vacuolatus.

BackgroundTo find out how algal cells cope with and recover from heat stress, the small vegetative cells of the synchronous Scenedesmus vacuolatus culture were subjected to a heat pretreatment (46.5°C for 1 h) followed by dark recultivation. The changes in physiological activities and morphology of Scenedesmus cells were continuously monitored throughout the course of decline and recovery.ResultsIt was found that the heat treatment, though completely inhibited photosynthesis, did not kill Scenedesmus cells. These cells, during dark recultivation, could make a fast repair and regained the ability of proliferation. We suggest that they entered a ‘stand-by’ state, which was characterized by condensed chromatin, partially functional but morphologically altered chloroplasts, disappeared vacuoles, slightly shrunk protoplast and intact plasma membranes. These stressed cells, on the surface, seemingly were undergoing some kind of disintegration, could readily and quickly return to normal cells upon illumination. Cell death occurred only after a long period of darkness (>48 h).ConclusionsOur results suggest that the recovery of algal cells from stress damage may actually proceed in two steps. The middle “stand-by’ stage normally is gone through too rapidly to be detected unless cells are kept in the dark.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-3) contains supplementary material, which is available to authorized users.

Aquatic toxicity of four veterinary drugs commonly applied in fish farming and animal husbandry

Doramectin (DOR), metronidazole (MET), florfenicol (FLO), and oxytetracycline (OXT) are among the most widely used veterinary drugs in animal husbandry or in aquaculture. Contamination of the environment by these pharmaceuticals has given cause for concern in recent years. Even though their toxicity has been thoroughly analyzed, knowledge of their ecotoxicity is still limited. We investigated their aquatic toxicity using tests with marine bacteria (Vibrio fischeri), green algae (Scenedesmus vacuolatus), duckweed (Lemna minor) and crustaceans (Daphnia magna). All the ecotoxicological tests were supported by chemical analyses to confirm the exposure concentrations of the pharmaceuticals used in the toxicity experiments, since deviations from the nominal concentration can result in underestimation of biological effects. It was found that OXT and FLO have a stronger adverse effect on duckweed (EC50=3.26 and 2.96mgL−1 respectively) and green algae (EC50=40.4 and 18.0mgL−1) than on bacteria (EC50=108 and 29.4mgL−1) and crustaceans (EC50=114 and 337mgL−1), whereas MET did not exhibit any adverse effect in the tested concentration range. For DOR a very low EC50 of 6.37×10−5mgL−1 towards D. magna was determined, which is five orders of magnitude lower than values known for the toxic reference compound K2Cr2O7. Our data show the strong influence of certain veterinary drugs on aquatic organisms and contribute to a sound assessment of the environmental hazards posed by commonly used pharmaceuticals.

Elicitation of the most important structural properties of ionic liquids affecting ecotoxicity in limnic green algae; a QSAR approach

Many ionic liquids are soluble in water and their impact on the aquatic environment has to be evaluated. However, due to the large number of ionic liquids and lack of experimental data, it is necessary to develop estimation procedures in order to reduce the materials and time consumption. In this study using multilayer perceptron neural network (MLP), ant colony optimization (ACO) and multiple linear regression (MLR) strategies, good predictive quantitative structure–activity relationships (QSAR) models were introduced and structural parameters affecting ecotoxicity of ionic liquids in limnic green algae (Scenedesmus vacuolatus) were revealed. Moreover, principal component analysis (PCA) and cluster analysis (CA) approaches were also applied to visualize any possible patterns or relationships among ionic liquids data. It was revealed that selected descriptors of the MLR model are also capable of clustering ionic liquids according to their four level of toxicity.

Flow cytometry as a diagnostic tool for the effects of polyphenolic allelochemicals on phytoplankton

We investigated the impact of the polyphenol tannic acid (TA) on the green algae Desmodesmus armatus and Scenedesmus vacuolatus and the diatom Stephanodiscus minutulus in order to find new diagnostic tools for allelopathic effects on phytoplankton. Esterase activity, membrane integrity and production of reactive oxygen species (ROS) were tested using flow cytometry with specific fluorescent markers. For comparison, growth rate and photosynthesis, two variables known to be affected by TA, were evaluated. Algae were exposed to TA concentrations between 0.6 and 30μmolL−1 for 3, 14 and 24h. A significant inhibition of esterase activity was detected at every time point in all three tested algal species at 30μmolL−1 TA and in most other treatments when TA concentrations exceeded 3μmolL−1. A significant production of ROS could also be detected in all three algal species, but only after a longer exposure period. Changes in membrane rigidity revealed no consistent patterns of enhancement or inhibition when tested with different TA concentrations, algal species and exposure time. Growth rates of all algae were significantly inhibited after 24h, whereas D. armatus was the only species for which the photosynthetic yield did not decline.The effects on esterase activity and ROS production indicate a general influence of polyphenolic allelochemicals on phytoplankton, but also reveal patterns which vary between species, concentrations and exposure times. Changes in esterase activity were the most sensitive variable, and could be detected after short exposure periods and at naturally occurring concentrations. Thus, esterase activity may be a suitable variable for future investigations into the allelopathic effects of submerged macrophytes on phytoplankton.

The use of quantum-chemical descriptors for predicting the photoinduced toxicity of PAHs

The geometries of 19 polyaromatic hydrocarbons (PAHs) were fully optimized and calculated by a density functional method (B3LYP) with the 3-21G basis set. Various quantum chemical descriptors such as the energy of the highest occupied molecular orbital (E (HOMO)), the energy of the lowest unoccupied molecular orbital (E (LUMO)), the difference in energy between those orbitals (E (GAP)), electronegativity (χ), chemical potential (μ), chemical hardness (η), softness index (S), electrophilicity (ω), and polarizability (α) were employed along with physicochemical descriptors to construct useful quantitative structure-activity relationship (QSAR) models for the photoinduced toxicity of PAHs toward two aquatic species (Daphnia magna and Scenedesmus vacuolatus). E (LUMO), E (HOMO), E (GAP), S, χ, the molar refractivity (MR), and the molecular weight provide valuable information and play a significant role in the assessment of PAH phototoxicity. The resulting models are not expected to be useful per se for making genuine predictions for much larger test sets, but the various results do demonstrate the potential benefits of incorporating quantum-chemical descriptors into QSAR models for predicting the phototoxicity of PAHs.

5-Fluorouracil Accumulation in Green Microalgae and its Biogenetic Transfer into Ciliate Protozoan

The study has demonstrated that anticancer drug 5-fluorouracil causes acute toxicity and interferes with the growth of green microalgae, Scenedesmus vacuolatus. It accumulates in microalgae biomass with bioaccumulation factor of 1.84×10(4) and further integrates into the DNA and RNA of microalgae. In addition, the labelled microalgae genome is transferred into protozoan Tetrahymena pyriformis on feeding and is retained in the food vacuoles of predator organisms. This biotransfer of labelled 5-fluorouracil via genomic material was evaluated using radioactivity in Tetrahymena cell pellets though radioactivity did not detect anticancer drug in the genome of the predator organism.

Ecotoxicity evaluation of selected sulfonamides

Sulfonamides (SAs) are a group of antibiotic drugs widely used in veterinary medicine. The contamination of the environment by these pharmaceuticals has raised concern in recent years. However, knowledge of their (eco)toxicity is still very basic and is restricted to just a few of these substances. Even though their toxicological analysis has been thoroughly performed and ecotoxicological data are available in the literature, a systematic analysis of their ecotoxicological potential has yet to be carried out. To fill this gap, 12 different SAs were chosen for detailed analysis with the focus on different bacteria as well as non-target organisms (algae and plants). A flexible (eco)toxicological test battery was used, including enzymes (acetylcholinesterase and glutathione reductase), luminescent marine bacteria ( Vibrio fischeri), soil bacteria ( Arthrobacter globiformis), limnic unicellular green algae ( Scenedesmus vacuolatus) and duckweed ( Lemna minor), in order to take into account both the aquatic and terrestrial compartments of the environment, as well as different trophic levels. It was found that SAs are not only toxic towards green algae (EC 50 = 1.54–32.25 mg L −1) but have even stronger adverse effect on duckweed (EC 50 = 0.02–4.89 mg L −1) than atrazine – herbicide (EC 50 = 2.59 mg L −1).

Application of nd-SPME to determine freely dissolved concentrations in the presence of green algae and algae-water partition coefficients

Biological tests are essential for toxicity assessment of chemicals to aquatic organisms. Since awareness is increasing that freely dissolved concentrations in test media may change during exposure and deviate significantly from nominal concentrations there is a need for concentration monitoring in biotests. Biotests are increasingly miniaturized with effect amounts of toxicants that are close to or below quantification limits of conventional analytical methods. Thus, the suitability of pre-equilibrium non-depletive (nd) solid phase microextraction (SPME) as an analytical method for small test volumes in the presence of green algae Scenedesmus vacuolatus was investigated . The method was validated for several compounds with different physical–chemical properties and effect concentrations. Limits of quantification were at least about 10 times smaller than the EC 50 of the compounds determined in earlier studies in a cell multiplication inhibition tests with Scenedesmus vacuolatus. Fouling of the fibres due to attached algae cells could be excluded and the method was found to be well suited to measure free concentrations in the algae test. The nd-SPME-method was applied to determine partitioning coefficients between algae and the surrounding aqueous phase and can be used to determine real exposure concentrations in a cell multiplication inhibition test with green algae.

Anchoring metabolic changes to phenotypic effects in the chlorophyte Scenedesmus vacuolatus under chemical exposure

In order to derive a causal understanding of toxic effects in organisms, ecotoxicology may benefit from linking molecular changes, evaluated by ‘omics’-techniques, to phenotypic observations. However, an approach to link these observation levels is still lacking. The aim of this study was to relate metabolic changes in the chlorophyte Scenedesmus vacuolatus to established parameters of toxicity. Therefore, synchronized cultures of the alga were exposed for 14 h to the phytotoxicant N-phenyl-2-naphthylamine (PNA) in the range of 0.00089 μmol L −1 (environmental concentrations) up to 1.82 μmol L −1. Cell growth and photosynthesis inhibition were evaluated but revealed no effect of PNA at experimental concentrations below 0.456 μmol L −1. Changes in the biochemical composition of algae were measured by GC–MS in both polar and non-polar phases. PCA uncovered no separation in the multivariate pattern of mass spectral features at exposure concentrations below 0.00356 μmol L −1 of PNA. However, a clear separation was detected at concentrations higher than 0.00713 μmol L −1. A combined visualization of PCA results for metabolic changes and concentration–response relationships for growth and photosynthesis inhibition revealed (I) a two orders of magnitude higher sensitivity of metabolomics to detects changes after PNA exposure compared to the phenotypic parameters measured and (II) two types of metabolic responses: one group of features was reflecting pharmacological effects at low exposure concentrations and the second group corresponded to adverse effects along with conventional observations of toxicity.

Analyzing Cytotoxic Effects of Selected Isothiazol-3-one Biocides Using the Toxic Ratio Concept and Structure−Activity Relationship Considerations

To demonstrate how baseline toxicity can be separated from other more specific modes of toxic action and to address possible pitfals when dealing with hydrophobic substances, the four isothiazol-3-one biocides N-methylisothiazol-3-one (MIT), 5-chloro-N-methylisothiazol-3-one (CIT), N-octylisothiazol-3-one (OIT), and 4,5-dichloro-N-octylisothiazol-3-one (DCOIT) as an example for reactive electrophilic xenobiotics were tested for their cytotoxic effects on the human hepatoblastoma cell line Hep G2, on the marine bacterium Vibrio fischeri, and on the limnic green alga Scenedesmus vacuolatus. In each of the three test systems, toxic effects were observed in a consistent pattern. The two chlorinated compounds and OIT were found to be significantly more toxic than MIT. As compared to baseline toxicants, the small and polar MIT and CIT exhibited pronounced excess toxicity in each of the three test systems that is presumably triggered by their intrinsic reactivity toward cellular thiols. In contrast, OIT and DCOIT showed mainly toxicities that could be explained by their hydrophobicity. Analyzing and comparing these results using the toxic ratio concept and with data that indicate a dramatic depletion of cellular glutathione levels after incubation with DCOIT reveals that for highly hydrophobic substances, baseline level toxicity in an assay for acute toxicity can lead to an oversight of other more specific modes of toxic action that may cause significant effects that might be less reversible than those caused by unreactive baseline toxicants. This possibility should be taken into account in the hazard assessment of chemicals that are both hydrophobic and reactive.

Uptake and toxicity of hexafluoroarsenate in aquatic organisms

The arsenic species hexafluoroarsenate has been described as a contaminant of surface waters of anthropogenic origin. Here, we undertake to identify the most sensitive biological receptor among several sentinel aquatic species used in eco-toxicological assessment and to understand toxicity in terms of internal dose. Therefore, a screening of short-term effects using different aquatic organisms (bacterium Vibrio fischeri, fish Danio rerio, crustacean Daphnia magna and green alga Desmodesmus subspicatus and Scenedesmus vacuolatus) was conducted. For most organisms tested, effects were not detectable even at very high hexafluoroarsenate concentrations (up to 9.6 mM) and thus the ecotoxic potential was found to be low in comparison to other arsenic compounds. The only organisms showing a clear response were the unicellular green alga, e.g. S. vacuolatus with an EC 50 value of 1.12 mM (84 mg L −1 As). A linear relationship between ambient and internal concentration was found for this organism with a slope of 1.63 × 10 −3. Therefore, the internal concentration which shows a significant effect, e.g. 20% of inhibition of reproduction, was found to occur at a relatively low internal dose of 0.98 μM. Moreover, no biotransformation products inside the algae could be detected using arsenic speciation analysis with HPLC–ICP–MS, thus biological effects must be attributed to the untransformed compound. We conclude that the very low uptake observed for hexafluoroarsenate may be interpreted as preventive against toxic effects for the organisms.

The host-range of Paraphysoderma sedebokerensis, a chytrid that infects Haematococcus pluvialis

A parasitic chytrid that attacks the green alga Haematococcus pluvialis was recently isolated in our laboratory and identified as a novel species from the phylum Blastocladiomycota, named herein Paraphysoderma sedebokerensis (nom. prov.). A method for early and precise detection of chytrid infections was developed using the fluorescent dye, Nile red, which stained the chytrids’ sporangia. Using this technique we determined the specificity of Paraphysoderma sedebokerensis for 13 algal species belonging to the Chlorophyta. Algal species tested including: Chlamydomonas nivalis, Chlorella emersonii, Chlorella vulgaris, Chlorococcum sp., Chlorogonium elongatum, Monoraphidium braunii, Muriella zofigiensis, Scenedesmus obliquus, Scenedesmus vacuolatus (two strains), and Scotiellopsis oocystiformis were either resistant to infection, or only experienced slight levels of infection during exponential growth. During exponential growth phase 100% Chlorella zofigiensis Donz cells were infected, but none developed any infection during resting stage. Only in cultures of H. pluvialis did infections develop rapidly (3–4 days) and intensively (100% cells infected) during both the logarithmic and stationary stages of growth. We suggest that the newly isolated chytrid, Paraphysoderma sedebokerensis (nom. prov.), is highly specific for H. pluvialis, but has a limited capacity to infect other green algae.

Dietary copper effects in the estuarine crab, Neohelice ( Chasmagnathus) granulata, maintained at two different salinities

We analyzed the dietary copper effects in the estuarine crab Neohelice ( Chasmagnathus) granulata and its interaction with water salinity. Crabs were maintained at 2‰ and 30‰ salinity for 5 weeks and they were fed with commercial food supplemented with the green alga Scenedesmus vacuolatus previously exposed to copper. No mortalities were observed, but crabs maintained at 2‰ salinity accumulated on average 40% more copper compared to animals maintained at 30‰ salinity. At 2‰ salinity, superoxide dismutase (SOD) activity and reduced glutathione (GSH) levels were increased at the first and second weeks, respectively, while lipid peroxidation and protein oxidation were evident after 4 weeks of copper exposure. At 30‰ salinity, all measured variables increased progressively but were significantly higher only at the end of the assay (5th week), except for protein oxidation that remained unchanged throughout the experiment. The hepatosomatic index (HSI) was significantly decreased in response to copper exposure, but only in crabs acclimated to 2‰. These findings have suggested that dietary copper exposure induces greater metal accumulation and larger oxidative stress responses in crabs maintained at 2‰ salinity.