Multiple regression analysis to assess the role of plankton on the distribution and speciation of mercury in water of a contaminated lagoon.

Spatial and seasonal variation of mercury species aqueous concentrations and distributions was carried out during six sampling campaigns at four locations within Laranjo Bay, the most mercury-contaminated area of the Aveiro Lagoon (Portugal). Inorganic mercury (IHg(II)) and methylmercury (MeHg) were determined in filter-retained (IHgPART, MeHgPART) and filtered (<0.45µm) fractions (IHg(II)DISS, MeHgDISS). The concentrations of IHgPART depended on site and on dilution with downstream particles. Similar processes were evidenced for MeHgPART, however, its concentrations increased for particles rich in phaeophytin (Pha). The concentrations of MeHgDISS, and especially those of IHg(II)DISS, increased with Pha concentrations in the water. Multiple regression models are able to depict MeHgPART, IHg(II)DISS and MeHgDISS concentrations with salinity and Pha concentrations exhibiting additive statistical effects and allowing separation of possible addition and removal processes. A link between phytoplankton/algae and consumers' grazing pressure in the contaminated area can be involved to increase concentrations of IHg(II)DISS and MeHgPART. These processes could lead to suspended particles enriched with MeHg and to the enhancement of IHg(II) and MeHg availability in surface waters and higher transfer to the food web.

Electrochemical oxidation of imazapyr with BDD electrode in titanium substrate.

In this work we have studied the treatment of imazapyr by electrochemical oxidation with boron-doped diamond anode. Electrochemical degradation experiments were performed in a one-compartment cell containing 0.45 L of commercial formulations of herbicide in the pH range 3.0-10.0 by applying a density current between 10 and 150 mA cm(-2) and in the temperature range 25-45 °C. The maximum current efficiencies were obtained at lower current densities since the electrochemical system is under mass transfer control. The mineralization rate increased in acid medium and at higher temperatures. The treatment was able to completely degrade imazapyr in the range 4.6-100.0 mg L(-1), although the current charge required rises along with the increasing initial concentration of the herbicide. Toxicity analysis with the bioluminescent bacterium Vibrio fischeri showed that at higher pollutant concentrations the toxicity was reduced after the electrochemical treatment. To clarify the reaction pathway for imazapyr mineralization by OH radicals, LC-MS/MS analyses we performed together with a theoretical study. Ions analysis showed the formation of high levels of ammonium in the cathode. The main final products of the electrochemical oxidation of imazapyr with diamond thin film electrodes are formic, acetic and butyric acids.

Pentachlorophenol toxicity to a mixture of Microcystis aeruginosa and Chlorella vulgaris cultures.

Pentachlorophenol (PCP) is a priority pollutant due to its persistence and high toxicity. For the first time, PCP effects were investigated at laboratory scale on co-cultures of two ubiquitous freshwater phytoplankton species: the cyanobacterium Microcystis aeruginosa and the microalgae Chlorella vulgaris. The cells were exposed to environmental levels of PCP for 10 days in Fraquil culture medium, at nominal concentrations from 0.1 to 10,000 µg L(-1). Growth was assessed by area under growth curve (cell count vs. time). The phytoplankton community structure can be changed as a consequence of a PCP contamination. Low µg L(-1) levels of PCP are advantageous to M. aeruginosa. This is the first report of the promoting effect of PCP on the growth of aquatic cyanobacteria, using mixtures with microalgae. As a result of the direct toxic effects of high PCP concentrations on M. aeruginosa, C. vulgaris cell count increased given that in biological controls M. aeruginosa inhibited the C. vulgaris growth. At 16.7 mg L(-1), PCP already had direct toxic effects also on the microalga. The pH of culture medium tended to decrease with increasing PCP concentrations, which was mostly related to the growth inhibition of cyanobacterium caused by PCP. The PCP concentration was stable in the co-cultures, which differed from what has been observed in monocultures of the same two species. Short-term laboratory assays with two phytoplankton species gives important information on the species interactions, namely possible direct and indirect effects of a toxicant, and must be considered in ecotoxicity studies regarding environmental extrapolations.

Effects of depuration on oxidative biomarkers in tilapia (Oreochromis niloticus) after subchronic exposure to cyanobacterium producing cylindrospermopsin.

Cylindrospermopsin (CYN) is a cytotoxic polyketide-derived alkaloid produced by several freshwater cyanobacterial species. It is now considered the second most studied cyanotoxin worldwide. Among the toxic mechanisms suggested for CYN pathogenicity are inhibition of protein and glutathione synthesis, genotoxicity by DNA fragmentation, and oxidative stress. The study of depuration of cyanobacterial toxins by aquatic organisms, particularly by fish, is important for fish economy and public health, but in the case of CYN is practically nonexistent. In this work, we investigated the efficiency of two distinct depuration periods, 3 or 7d, in a clean environment, as a mean of restoring the levels of several oxidative stress biomarkers in tilapia (Oreochromis niloticus) subchronically exposed to CYN by immersion in an Aphanizomenon ovalisporum culture (by adding 10 µg CYN/L every two days during 14 d). Lipid peroxidation (LPO) and DNA oxidation returned to normal values after 7d of depuration, whereas the time needed for restoring of the oxidatively damaged proteins was longer. Superoxide dismutase (SOD) and gamma-glutamyl-cysteine-synthetase (γ-GCS) activities recovered after just 3d of depuration, while catalase (CAT) activity needed up to 7d to return to control values. Ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) returned to control levels after 7d of depuration in both organs. These results validate the depuration process as a very effective practice for detoxification in fish contaminated with these toxins.

Cyanobacterium Microcystis aeruginosa response to pentachlorophenol and comparison with that of the microalga Chlorella vulgaris.

Pentachlorophenol (PCP) effects on a strain of the cyanobacterium Microcystis aeruginosa were investigated at laboratory scale. This is the first systematic ecotoxicity study of the effects of PCP on an aquatic cyanobacterium. The microalga Chlorella vulgaris was studied in the same conditions as the cyanobacterium, in order to compare the PCP toxicity and its removal by the species. The cells were exposed to environmental levels of PCP during 10 days, in Fraquil culture medium, at nominal concentrations from 0.01 to 1000 µg L(-1), to the cyanobacterium, and 0.01 to 5000 µg L(-1), to the microalga. Growth was assessed by area under growth curve (AUC, optical density vs time) and chlorophyll a content (chla). The toxicity profiles of the two species were very different. The calculated effective concentrations EC20 and EC50 were much lower to M. aeruginosa, and its growth inhibition expressed by chla was concentration-dependent while by AUC was not concentration-dependent. The cells might continue to divide even with lower levels of chla. The number of C. vulgaris cells decreased with the PCP concentration without major impact on the chla. The effect of PCP on M. aeruginosa is hormetic: every concentration studied was toxic except 1 µg L(-1), which promoted its growth. The legal limit of PCP set by the European Union for surface waters (1 µg L(-1)) should be reconsidered since a toxic cyanobacteria bloom might occur. The study of the removal of PCP from the culture medium by the two species is an additional novelty of this work. M. aeruginosa could remove part of the PCP from the medium, at concentrations where toxic effects were observed, while C. vulgaris stabilized it.

Cyanobacterium producing cylindrospermopsin cause oxidative stress at environmentally relevant concentrations in sub-chronically exposed tilapia (Oreochromis niloticus).

Cylindrospermopsin (CYN) is a potent cyanobacterial cytotoxin produced by certain freshwater cyanobacteria. Structurally, it is an alkaloid with a tricyclic guanidine moiety combined with hydroxymethyluracil. It has proved to be a potent inhibitor of protein synthesis, and to deplete hepatic glutathione. Recently, some studies have shown that CYN produces changes in some oxidative stress biomarkers in fish acutely exposed to pure CYN by oral and intraperitoneal (i.p.) routes. In the present study tilapia (Oreochromis niloticus) were exposed by immersion to lyophilized Aphanizomenon ovalisporum cells added to the aquaria using two concentration levels, 10 or 100 µg CYN L(-1), during two different exposure times: 7 and 14 d. Fish were sacrificed and liver and kidney were extracted. The oxidative status of fish was evaluated by analyzing in both organs the following biomarkers: lipid peroxidation (LPO), protein oxidation, DNA oxidation, reduced-oxidized glutathione ratio (GSH/GSSG), and changes in the activity of Glutathione-S-transferase (GST), Glutathione Peroxidase (GPx), Superoxide dismutase (SOD), Catalase (CAT), and γ-Glutamyl-cysteine synthetase (GCS). In general, major changes were observed in tilapia treated with 100 µg CYN L(-1) after 14 d of exposure. However, some endpoints were altered at the lowest concentration assayed only after 7d of exposure, such as DNA oxidation and γ-GCS in kidney, and CAT and GSH/GSSG decrease in the liver and kidney. The kidney was the most affected organ. These findings confirm that the oxidative stress play a role in the pathogenicity induced by CYN in this fish species, and the results obtained could be useful for future ecotoxicological risks assessment studies, for the protection of fish and aquatic ecosystems. To our knowledge this is the first study dealing with the oxidative stress changes induced by cyanobacterial cells containing CYN and its derivative deoxy-CYN on fish exposed sub-chronically under laboratory conditions.

Reversed-phase HPLC/FD method for the quantitative analysis of the neurotoxin BMAA (ß-N-methylamino-L-alanine) in cyanobacteria.

A method has been developed and optimized in order to detect and quantify the non-protein amino acid ß-N-methylamino-L-alanine(BMAA) in cyanobacteria. The novelty of the method is that we have used methanol instead of acetonitrile as the eluent. The method includes extraction with 0.1 M trichloroacetic acid (free BMAA) or protein hydrolysis with 6 M hydrochloric acid (total BMAA), derivatization with AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) and reversed-phase high-performance liquid chromatography analysis with fluorescence detection (HPLC/FD). Detection limits ranged from 0.35 to 0.75 pg injected, while quantification limits ranged from 1.10 to 2.55 pg injected for total and free BMAA hydrolysis, respectively. The linear response range was up to 850 pmol in both methods, embracing three orders of magnitude. The method was successfully applied to a lyophilized estuarine species of Nostoc (LEGE 06077). All previous published methods for BMAA quantification, using HPLC/FD, have reported the usage of acetonitrile. This is the first report using methanol as the mobile phase. Although the elution strength differs with both solvents, the final method proved efficient for the quantification of BMAA in this complex sample. The method resulted effective, low-priced, and simple, being suitable for routine monitoring of BMAA in cyanobacteria.

Identification and quantification of microcystins from a Nostoc muscorum bloom occurring in Oukaïmeden River (High-Atlas mountains of Marrakech, Morocco).

Health risks generated by cyanobacterial toxins in drinking and recreational waters are clearly recognised. During the monitoring programme on the distribution of toxic freshwater cyanobacteria in various water bodies including reservoirs, ponds and rivers of Morocco, many toxigenic cyanobacteria bloom-forming species have been identified. Particular attention was given to the investigation of the toxicology of a benthic Nostoc species-Nostoc muscorum Ag. (cyanobacteria, Nostocales, Nostocaceae)-that was found dominant in Oukaïmeden river located at 2,600 m of altitude in High-Atlas mountains of Marrakech. The massive growth of the mat-forming N. muscorum occurred yearly during the period of March-October, when the water temperature was above 10 degrees C. During 1997-1999, samples were collected from either floating or benthic mats. Hepatotoxicity associated to gastrointestinal (diarrhoea) intoxication symptoms was confirmed by intraperitoneal (i.p.) injection in mice of N. muscorum thallus extract. The survival time was estimated to be from 2-5 h, and the calculated i.p. LD(50) in mice ranged from 15 to 125 mg kg(-1) body weight. The application of the high performance liquid chromatography with photodiode array detection confirmed the occurrence of microcystin-LR (MC-LR) and three others microcystin variants from the methanolic Nostoc extract. The MC-LR represented a proportion of 39% of the total microcystin content however, the total concentration equivalents-eq-of MC-LR was estimated to be 139 microg MC-LR eq per gram dry weight. The existence of a benthic microcystin-producing N. muscorum strain under the particular environmental conditions of Oukaïmeden region may be a potential human health hazard and the ecological harmful effects of these cyanobacterial toxins need to be assessed. This paper constitutes the first report of the occurrence of a toxic benthic Nostoc in Morocco. So, the benthic species should be considered during monitoring of toxic Cyanobacteria particularly for river used for source of drinking water.

Detection of microcystin contamination by the measurement of the variability of the in vivo chlorophyll fluorescence in aquatic plant Lemna gibba.

In recent years, chlorophyll fluorescence analysis has become one of the most powerful and widely used techniques available to plant ecophysiologists. In this work, the chlorophyll fluorescence is used in order to evaluate the biotic stress induced by exposure to cyanobacterial toxins (microcystins). Experiments were carried on the aquatic plant Lemna gibba exposed to various concentrations of a microcystins (0.01, 0.03, 0.05, 0.07, 0.15, 0.22 and 0.3mug equivalent MC-LR.mL(-1)) during 5h. The reversibility of the stress changes was also studied following 24h of treatment. The efficiency and the utility of this biophysical technique were compared to biochemical analysis priory used to evaluate the plant stress induced by such contamination. The results showed that there is a concentration-dependent effect on the measured in vivo chlorophyll fluorescence with significant differences between the control and all concentrations except for 0.01mug equivalent MC-LR.mL(-1). The reversibility tested showed also that after avoiding the contact with the microcystins, the chlorophyll fluorescence measurements were not significantly different from the control. The results showed that if the contact with the microcystins is short and not repeated plants may not suffer from a significant stress. We concluded that this simple and rapid technique based on the variable fluorescence, could be recommended and applied to test the plant stress caused by cyanobacterial toxins.

Antimicrobial and cytotoxic assessment of marine cyanobacteria - Synechocystis and Synechococcus.

Aqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.

Dynamics of glutathione-S-transferases in Mytilus galloprovincialis exposed to toxic Microcystis aeruginosa cells, extracts and pure toxins.

Molluscs and especially bivalves are able to accumulate dinoflagelates, diatoms and cyanobacteria toxins, and, being vectors for these toxins, transfer them along food chains. The data obtained from laboratory experiments showed that bivalve molluscs are resistant to cyanobacteria toxins. In this work, we wanted to test if Mytilus galloprovincialis organs react to microcystins and other cyanobacteria compounds by inducing or decreasing its GST activity. Acclimated mussels M. galloprovincialis were exposed to the toxic Microcystis aeruginosa M13 strain. Exposure of mussels to toxins was done in three ways: living Microcystis cells, crude Microcystis extracts and pure toxins. The measurement of soluble and microsomal GST activity in the different mussel organs was done by using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dichloro-1-nitrobenzene (DCNB). Analysis of the GST activity of the control mussels using CDNB as a substrate showed that cytosolic activity is much more significant than microsomal. Intact M. aeruginosa cells did not induce any significant response from the mussels, showing that these animals are quite resistant to the cyanobacteria if they are intact. On the other hand, cell extracts caused an important effect in the gut, in the gills and in the labial palps, although in different ways. There was an increase in GST activity in the gut and gills of mussels exposed to Microcystis extracts, showing a response of this detoxication pathway, but in the labial palps a severe reduction in GST activity occurred. Pure MC LR+YR induced an increase in GST activity in all organs but the labial palps. The results showed that other substances apart from microcystins may cause stress to mussels and affect detoxication enzymes such as GST.

Phototactic behavior in Daphnia magna Straus as an indicator of toxicants in the aquatic environment.

In this study, the phototactic behavior of Daphnia magna was investigated as a possible bioindicator for the following 11 chemicals commonly found in the aquatic environment: benzo(b)fluoranthene, mercury (II) chloride, dimethoate, lindane, linuron, MCPA, TBTO, carbon tetrachloride, thiram, 2,4,6-trichlorophenol and arsenic trioxide. Phototactic response was monitored as the movement of 7 to 8 day-old D. magna individuals. The analysis was carried out using glass test tubes divided radially into two zones, with increasing distance from a light source. For each of the compounds, different concentrations and exposure times were analyzed, and the behavior of the D. magna in each of the treatments compared to the controls in which the chemicals were not added. Using the experimental model described here, all of the 11 chemicals could be detected following exposure times of between 15 min and 48 h. The lowest concentrations detected using this technique were between 2 and 43 times lower than the LC(50) and EC(50) values reported for D. magna. The results of this study show that the analysis of phototactism is a useful method for detecting the presence of a wide range of potentially toxic chemicals found in the aquatic environment.

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river.

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.

Detection of microcystin synthetase genes in health food supplements containing the freshwater cyanobacterium Aphanizomenon flos-aquae.

In this study we investigated the presence of toxin-producing cyanobacterial contaminants in food supplements manufactured from blooms of the non-toxic freshwater cyanobacterium Aphanizomenon flos-aquae. Previous reports investigating the contamination of health food supplements with toxin-producing cyanobacteria have used chemical and or biochemical methods such as HPLC, ELISA and protein phosphatase assays. Whilst these studies have drawn attention to the presence of hepatotoxic microcystins in some commercially available food supplements, the methods used do not provide any information on the source of the contaminant. Such information would be useful for the quality control of food supplements produced for human consumption. In this study we applied a molecular technique, involving the amplification of the 16s rRNA gene, the phycocyanin operon, and two genes of the microcystin synthetase gene cluster to show that all 12 food supplement samples, sourced from various internet distributors and containing non-toxic A. flos-aquae, also contained toxigenic cyanobacteria. Sequencing of the microcystin synthetase genes detected in all of the food supplements showed that M. aeruginosa was the organism responsible for the production of microcystins in the samples. The presence of microcystins in the food supplements was confirmed by ELISA, with concentrations within the range of 0.1--4.72 microgg(-1) (microcystin-LR equivalents). Given that the molecular methods applied here are highly sensitive, and show good agreement with the results obtained from ELISA, we believe that they could potentially be used as a quality control technique for food products that contain cyanobacteria.

Cyanobacteria diversity and toxicity in a wastewater treatment plant (Portugal).

Cyanobacteria are common in eutrophic natural waters. Being favoured by warm, stable and nutrient-enriched waters they may constitute an important part of the phytoplankton community in Wastewater Treatment Plants (WWTP). The phytoplankton communities of two ponds (facultative and maturation) of the WWTP of Esmoriz (North Portugal) were studied, with particular importance given to cyanobacteria. Mouse bioassays were performed with cyanobacteria samples during some of the blooms and ELISA assays specific for hepatotoxic microcystins were carried out. During the study period (January-July 1999) cyanobacteria were frequently dominant in the ponds ranging from 15.2 to 99.8% of the total phytoplankton density. The main species were Planktothrix mougeotii, Microcystis aeruginosa and Pseudanabaena mucicola. Mouse bioassays were performed during Oscillatoria bloom period but the results were negative, in spite of the high cyanobacteria biomass. ELISA assays were performed for both ponds but only in the maturation pond positive values were found. Microcystin concentrations (as MCYST-LR equivalents) varied from 2.3 to 56.0 micrograms/l on the margin of the pond and between 1.7 and 4.6 micrograms/l in the outflow of this pond. These values indicate that WWTP may be a source of contamination of water bodies with cyanobacteria toxins.

Cyanobacterial toxins in Portugal: effects on aquatic animals and risk for human health.

Toxic cyanobacteria are common in Portuguese freshwaters and the most common toxins are microcystins. The occurrence of microcystin-LR (MCYST-LR) has been reported since 1990 and a significant number of water reservoirs that are used for drinking water attain high levels of this toxin. Aquatic animals that live in eutrophic freshwater ecosystems may be killed by microcystins but in many cases the toxicity is sublethal and so the animals can survive long enough to accumulate the toxins and transfer them along the food chain. Among these, edible mollusks, fish and crayfish are especially important because they are harvested and sold for human consumption. Mussels that live in estuarine waters and rivers where toxic blooms occur may accumulate toxins without many significant acute toxic effects. In this study data are presented in order to understand the dynamics of the accumulation and depuration of MCYST-LR in mussels. The toxin is readily accumulated and persists in the shellfish for several days after contact. In the crayfish the toxin is accumulated mainly in the gut but is also cleared very slowly. In carps, although the levels of the toxins found in naturally caught specimens were not very high, some toxin was found in the muscle and not only in the viscera. This raises the problem of the toxin accumulation by fish and possible transfer through the food chain. The data gathered from these experiments and from naturally caught specimens are analyzed in terms of risk for human consumption. The occurrence of microcystins in tap water and the incidence of toxic cyanobacteria in fresh water beaches in Portugal are reported. The Portuguese National Monitoring Program of cyanobacteria is mentioned and its implications are discussed.