Effects of the microcystin profile of a cyanobacterial bloom on growth and toxin accumulation in common carp Cyprinus carpio larvae.

A 12 day growth trial was conducted to compare the effect of the variation in microcystins (MC) composition of two bloom samples of Microcystis aeruginosa on the growth performance and microcystin accumulation in common carp Cyprinus carpio larvae. Two M. aeruginosa natural bloom samples with different MC profiles were collected and larvae were exposed to cyanobacterial cells through their diet. Three diets, a basal control diet and two diets prepared from the basal diet plus the same toxins content (60 ng MC g(-1) diet) of each cyanobacterial bloom, were given at the same ration level to three groups of larvae during the experimental period. Larval mass and standard length from day 9 were significantly different between cyanobacterial treatments and in both cases lower than that of the control. The MC accumulation by larvae, inversely correlated with the growth performance, was also significantly different between cyanobacterial treatments (26.96 v. 17.32 ng g(-1) at the end of the experimental period). These results indicate that MC variants profile may have effects on the toxin uptake and toxicity. To date, this is the first laboratory study to show that fish accumulate MC depending on the toxin profile of the cyanobacterial bloom.

Identification and quantification of cyanobacterial toxins (microcystins) in two Moroccan drinking-water reservoirs (Mansour Eddahbi, Almassira).

Mansour Eddahbi (MED) (30 degrees 55'N, 6 degrees 53'W) and Almassira (ALM) (31 degrees 95'N, 6 degrees 72'W) are two Moroccan lake reservoirs located at an arid and semi-arid hydrographic basin, respectively. Both are used for irrigation, recreational activities and drinking-water production. This paper deals with the characterization and quantification of microcystins (MC) from two Microcystis aeruginosa blooms occurring in those reservoirs. The toxicity of the blooms was confirmed and evaluated by both mouse and Artemia bioassays. The calculated LD50 values revealed that the MED bloom had a medium toxicity (LD50=358 mg kg(-1) body weight), whereas the ALM bloom had low toxicity (LD50=829 mg kg(-1) body weight). The 24-h LC50 values were 1.88 and 4.15 mg ml(-1) for the MED and ALM blooms, respectively, using Artemia assay. The identification and quantification of MC variants were carried out by high performance liquid chromatography (HPLC) equipped with a photodiode array detector, and HPLC coupled to mass spectrometry. The MC content, as Microcystin-LR (MC-LR) equivalents, was higher in the MED bloom (64.4 microg g(-1) dry weight) than in the ALM bloom (9.9 microg g(-1) dry weight). Five MC variants were identified in the MED cyanobacteria bloom (MC-RR, MC-YR, MC-LR, MC-FR, and MC-WR) and only one (MC-LR) in the ALM bloom. The results show that the occurrence of toxic cyanobacteria blooms in the studied reservoirs may be regarded as a health hazard; therefore, cyanotoxin monitoring in them is highly recommended.

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.

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 and quantification of microcystins from cyanobacteria strains isolated from reservoirs and ponds in Morocco.

In Morocco, the occurrence of toxic cyanobacteria blooms is confirmed in some water bodies used for recreational and/or as drinking water reservoirs. According to WHO recommendations, the establishment of a monitoring program for microcystins is a necessity. This paper presents toxicological studies of 19 toxic cyanobacteria strains of Microcystis, Synechocystis, Pseudanabaena, and Oscillatoria. These strains were isolated from various water bodies including natural lakes, reservoirs, and ponds located in central regions of Morocco. The isolation, culture, and biomass production of these strains was made on Z8 or BG13 media under laboratory controlled conditions. The hepatotoxicity of cyanobacterial lyophilized material was confirmed by mouse bioassays. The amount of microcystins produced by each strain was determined by the enzyme-linked immunosorbent assay (ELISA). The detection and identification of microcystin variants was performed by high performance liquid chromatography (HPLC) with photodiode array detection. Almost all strains showed medium to high toxicity, the estimated LD50 i.p. mice bioassay ranged between 28 to 350 mg/kg body weight. The concentrations of microcystins varied between 2.16 to 944 micrograms/g and 26.8 to 1884 micrograms/g dry weight determined by ELISA and HPLC, respectively. The screening of bloom-forming and microcystin producer cyanobacteria strains in these fresh water bodies leads us to propose the need for the establishment of a survey of cyanobacteria and a cyanotoxin-monitoring program.

Bioactive compounds from Pseudanabaena species (Cyanobacteria).

Bioactive products of the multicellular filamentous benthic cyanobacterium, Pseudanabaena species, isolated from wastewater stabilization ponds at Marrakech, were tested against some pathogenic micro-organisms. Extracellular and intracellular products released by this blue-green alga in the stationary, growth phase, reduced the survival of Escherichia coli, Salmonella sp., Staphylococcus aureus and Candida albicans, and stimulated the growth of non-O1 V. cholerae, whereas no significant effect was noted on the growth of Candida tropicalis. Intracellular substances (methanolic extract) had a stronger inhibitory effect on these micro-organisms than that produced by extracellular substances. These antibacterial and antifungal substances may have a pharmacological value. They may have an important ecological effect on the composition of the bacterial community in Marrakech stabilization ponds when blooms of Pseudanabaena species occurred in this sewage treatment ecosystem during hot periods.

Isolation, characterization and quantification of microcystins (heptapeptides hepatotoxins) in Microcystis aeruginosa dominated bloom of Lalla Takerkoust lake-reservoir (Morocco).

This paper presents the first data on the identification, characterization and quantification of microcystins isolated from both an extract of a cyanobacteria natural bloom, collected from a eutrophic Moroccan reservoir (Lalla Takerkoust, Marrakesh) and an isolated strain cultivated under laboratory conditions. The isolation and purification of toxins was performed by reverse phase HPLC and then characterized by amino acid analysis and fast atom bombardment mass spectrometry (FAB-MS). Chemical characterization of the toxins from the bloom revealed variants of microcystins such as Mcyst-LR, Mcyst-RR, Mcyst-YR and [D-Asp3]Mcyst-LR. However, the Microcystis aeruginosa strain produced only Mcyst-RR. Using an ELISA assay the total microcystin contents of eight bloom samples collected from 1994 to 1997 ranged from 0.7 to 8.8 microg/mg of lyophilized material. The two isolated Microcystis strains contained higher amounts of microcystins (0.65 microg/ mg of dry weight) than the Pseudanabaena strains (0.021 microg/mg of dry weight). Our results show that the presence of cyanobacteria toxins in water used for drinking in a North African country may be regarded as an health hazard. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, namely in north African countries.