Detection of the hepatotoxic microcystins in 36 kinds of cyanobacteria Spirulina food products in China

Gel filtration chromatography, ultra-filtration, and solid-phase extraction silica gel clean-up were evaluated for their ability to remove microcystins selectively from extracts of cyanobacteria Spirulina samples after using the reversed-phase octadecylsilyl ODS cartridge for subsequent analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The reversed-phase ODS cartridge/silica gel combination were effective and the optimal wash and elution conditions were: H2O (wash), 20% methanol in water (wash), and 90% methanol in water (elution) for the reversed-phase ODS cartridge, followed by 80% methanol in water elution in the silica gel cartridge. The presence of microcystins in 36 kinds of cyanobacteria Spirulina health food samples obtained from various retail outlets in China were detected by LC-MS/MS, and 34 samples (94%) contained microcystins ranging from 2 to 163 ng g(-1) (mean=1427 ng g(-1)), which were significantly lower than microcystins present in blue green alga products previously reported. MC-RR-which contains two molecules of arginine (R)-(in 94.4% samples) was the predominant microcystin, followed by MC-LR-where L is leucine-(30.6%) and MC-YR-where Y is tyrose-(27.8%). The possible potential health risks from chronic exposure to microcystins from contaminated cyanobacteria Spirulina health food should not be ignored, even if the toxin concentrations were low. The method presented herein is proposed to detect microcystins present in commercial cyanobacteria Spirulina samples.

Distribution of toxins in various tissues of crucian carp intraperitoneally injected with hepatotoxic microcystins

An acute toxicity experiment was conducted to examine the distribution and depuration of microcystins (MCS) in crucian carp (Carassius aurutus) tissues. Fish were injected intraperitoneally with extracted MCs at a dose of 200 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight. Microcystin concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, and 48 h postinjection using liquid chromatography coupled with mass spectrometry. Microcystins were detected mainly in blood (3.99% of injected dose at 1 h), liver (1.60% at I h), gonad (1.49% at 3 h), and kidney (0.14% at 48 h). Other tissues, such as the heart, gill, gallbladder, intestine, spleen, brain, and muscle, contained less than 0.1% of the injected MCs. The highest concentration of MCs was found in blood (526-3,753 ng/g dry wt), followed by liver (103-1,656 ng/g dry wt) and kidney (279-1,592 ng/g dry wt). No MC-LR was detectable in intestine, spleen, kidney, brain, and muscle, whereas MC-RR was found in all examined fish tissues, which might result from organ specificity of different MCs. Clearance of MC-RR in brain tissue was slow. In kidney, the MC-RR content was negatively correlated with that in blood, suggesting that blood was important in the transportation of MC-RR to kidney for excretion.

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.)

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 mu g L-1) for 2, 4, 8, 16 and 24 h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC50 of microcystins in carp hepatocytes was 169.2 mu g L-1. The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 mu g L-1) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 mu g L-1) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 mu g L-1), they died in the form of necrosis. (C) 2006 Elsevier Inc. All rights reserved.

Distribution and bioaccumulation of microcystins in water columns: A systematic investigation into the environmental fate and the risks associated with microcystins in Meiliang Bay, Lake Taihu

For the purpose of understanding the environmental fate of microcystins (MCs) and the potential health risks caused by toxic cyanobacterial blooms in Lake Taihu, a systematic investigation was carried out from February 2005 to January 2006. The distribution of MCs in the water column, and toxin bioaccumulations in aquatic organisms were surveyed. The results suggested that Lake Taihu is heavily polluted during summer months by toxic cyanobacterial blooms (with a maximum biovolume of 6.7 x 10(8) cells/L) and MCs. The maximum concentration of cell-bound toxins was 1.81 mg/g (DW) and the dissolved MCs reached a maximum level of 6.69 mu g/L. Dissolved MCs were always found in the entire water column at all sampling sites throughout the year. Our results emphasized the need for tracking MCs not only in the entire water column but also at the interface between water and sediment. Seasonal changes of MC concentrations in four species of hydrophytes (Eichhornic crassipes, Potamogeton maackianus, Alternanthera philoxeroides and Myriophyllum spicatum) ranged from 129 to 1317, 147 to 1534, 169 to 3945 and 124 to 956 ng/g (DW), respectively. Toxin accumulations in four aquatic species (Carassius auratus auratu, Macrobrachium nipponensis, Bellamya aeruginosa and Cristaria plicata) were also analyzed. Maximum toxin concentrations in the edible organs and non-edible visceral organs ranged from 378 to 730 and 754 to 3629 ng/g (DW), respectively. Based on field studies in Lake Taihu, risk assessments were carried out, taking into account the WHO guidelines and the tolerable daily intake (TDI) for MCs. Our findings suggest that the third largest lake in China poses serious health threats when serving as a source of drinking water and for recreational use. In addition, it is likely to be unsafe to consume aquatic species harvested in Lake Taihu due to the high-concentrations of accumulated MCs. (C) 2007 Elsevier Ltd. All rights reserved.

Biochemical and ultrastructural changes of the liver and kidney of the phytoplanktivorous silver carp feeding naturally on toxic Microcystis blooms in Taihu Lake, China

Many experimental studies have documented the impact of microcystins (MC) on fish based on either intraperitoneal injection, or oral gavaging via the diet, but few experiments were conducted by MC exposure through natural food uptake in lakes. In this study, the phytoplanktivorous silver carp were stocked in a large pen set in Meiliang Bay of Taihu Lake where toxic Microcystis blooms occurred in the warm seasons. Fish samples were collected monthly and MC concentrations in liver and kidney of the fish were determined by LC-MS. The maximum MC concentrations in liver and kidney were present in July when damages in ultrastructures of the liver and kidney were revealed by electron microscope. In comparison with previous studies on common carp, silver carp showed less damage and presence of lysosome proliferation in liver and kidney. Silver carp might eliminate or lessen cell damage caused by MC through lysosome activation. Recovery in the ultrastructures of liver and kidney after Microcystis blooms was companied with a significant decrease or even disappearance of MC. Catalase and glutathione S-transferase in liver and kidney of silver carp during Microcystis blooms were significantly higher than before and after Microcystis blooms. The high glutathione pool in liver and kidney of silver carp suggests their high resistance to MC exposure. The efficient antioxidant defence may be an important mechanism of phytoplanktivorous fish like silver carp to counteract toxic Microcystis blooms. (C) 2007 Published by Elsevier Ltd.

In situ study on the control of toxic Microcystis blooms using phytoplanktivorous fish in the subtropical Lake Taihu of China: A large fish pen experiment

Three large fish pens (0.36 km(2) of each) stocked with silver and bighead carp were set up in Meiliang Bay for controlling toxic Microcystis blooms. The responses of plankton communities and food consumption of silver and bighead carp were studied. Crustacean zooplankton were significantly suppressed in the fish pens. Total phytoplankton biomass, Microcystis biomass and microcystin concentration were lower in the fish pens than in the surrounding lake water, but the difference was not statistically significant. The present stocking density of silver plus bighead carp (about 40 g/m(3) in July) was likely too low to achieve an adequate control of Microcystis. Silver carp fed mainly on phytoplankton but bighead carp mainly on zooplankton: mean zooplankton contribution in the gut was 31.5% for silver carp and 64.7% for bighead carp. Compared with previous studies, both carp species preyed upon more zooplankton because of the abundant food resource. Daily rations of silver and bighead carp were estimated by Egger's model in the main growing season. Filtration rate was calculated from the daily ration and the density of plankton in the lake. During May-October, filtration rates of silver and bighead carp for phytoplankton were 0.22-1.53 L g(-1) h(-1) and 0.02-0.68 L g(-1) h(-1), respectively, and filtration rates for zooplankton were 0.24-0.44 L g(-1) h(-1) and 0.08-1.41 L g(-1) h(-1), respectively. Silver carp had a stronger ability of eliminating phytoplankton than bighead carp. To achieve a successful bioniampulation with a minimum effect of ichthyoeutrophication, the stocking proportion of bighead carp should be controlled in the future practice. (c) 2007 Elsevier B.V All rights reserved.

In situ studies on the distribution patterns and dynamics of microcystins in a biomanipulation fish - bighead carp (Aristichthys nobilis)

The distribution and dynamics of microcystins in various organs of the phytoplanktivorous bighead carp were studied monthly in Lake Taihu, which is dominated by toxic cyanobacteria. There was a good agreement between LC-MS and HPLC-UV determinations. Average recoveries of spiked fish samples were 63% for MC-RR and 71% for MC-LR. The highest MC contents in intestine, liver, kidney and spleen were 85.67, 2.83, 1.70 and 1.57 mu g g(-1) DW, respectively. MCs were much higher in mid-gut walls (1.22 mu g g(-1) DW) than in hind- and fore-gut walls (0.31 and 0.18 mu g g(-1) DW, respectively), suggesting the importance of mid-gut wall as major site for MC absorption. A cysteine conjugate of MC-LR was detected frequently in kidney. Among the muscle samples analyzed, 25% were above the provisional tolerable daily intake level by WHO. Bighead is strongly resistant to microcystins and can be used as biomanipulation fish to counteract cyanotoxin contamination in eutrophic waters. (c) 2006 Elsevier Ltd. All rights reserved.

Genetic diversity: Geographical distribution and toxin profiles of Microcystis strains (Cyanobacteria) in China

Twenty strains of Microcystis Kutz were isolated from different freshwater bodies in China to analyze the diversity, geographical distribution and toxin profiles. Based on whole-cell polymerase chain reaction of cpcBA-IGS nucleotide sequence, the derived neighbor-joining (NJ) and maximum parsimony (MP) trees indicate that these strains of Microcystis can be divided into four clusters. The strains from south, middle and north region of China formed distinct lineages, suggesting high diversity and a geographical distribution from south to north locations. Moreover, the results being indicating high variable genotypes of the strains of the Microcystis strains from the same lake show that there is high diversity of Microcystis within a water bloom population. Comparing the results of the present study with those reported for compared with 43 strains of Microcystis from other locations, also reveals Chinese strains have high similarity with those from regions in the North Hemispherical. This suggests that the Microcystis strains in the world might have a geographical distribution. Analysis of 30 strains using the primers MCF/TER and TOX2P/TOX2M showed that there was no correlation between the gene of cpcBA-IGS and the presence of mcy. Toxic strains were founded to be predominant in different water bodies throughout China.

Bioaccumulation of the hepatotoxic microcystins in various organs of a freshwater snail from a subtropical Chinese lake, Taihu lake, with dense toxic microcystis blooms

In this paper, we describe the seasonal dynamics of three common microcystins (MCs MC-RR, MC-YR, and MC-LR) in the whole body, hepatopancreas, intestine, gonad, foot, remaining tissue, and offspring of a freshwater snail, Bellamya aeruginosa, from Gonghu Bay of Lake Taihu, China, where dense toxic Microcystis blooms occur in the warm seasons. Microcystins were determined by liquid chromatography electrospray ionization mass spectrum. Microcystin (MC-RR + MC-YR + MC-LR) content of the offspring and gonad showed high positive correlation, indicating that microcystins could transfer from adult females to their young with physiological connection. This study is the first to report the presence of microcystins in the offspring of the adult snail. The majority of the toxins were present in the intestine (53.6%) and hepatopancreas (29.9%), whereas other tissues contained only 16.5%. If intestines are excluded, up to 64.3% of the toxin burden was allocated in the hepatopancreas. The microcystin content in the intestine, hepatopancreas, and gonad were correlated with the biomass of Microcystis and intracellular and extracellular toxins. Of the analyzed foot samples, 18.2% were above the tolerable daily microcystin intake recommended by the World Health Organization (WHO) for human consumption. This result indicates that public health warnings regarding human ingestion of snails from Taihu Lake are warranted. In addition, further studies are needed to evaluate the occurrence by Microcystis in relation to spatial and temporal changes in water quality.

Relationships between microcystins and environmental parameters in 30 subtropical shallow lakes along the Yangtze River, China

1. A survey of 30 subtropical shallow lakes in the middle and lower reaches of the Yangtze River area in China was conducted during July-September in 2003-2004 to study how environmental and biological variables were associated with the concentration of the cyanobacterial toxin microcystin (MC). 2. Mean MC concentration in seasonally river-connected lakes (SL) was nearly 33 times that in permanently river-connected lakes (RL), and more than six times that in city lakes (NC) and non-urban lakes (NE) which were not connected to the Yangtze River. The highest MC (8.574 mu g L-1) was detected in Dianshan Lake. 3. MC-RR and MC-LR were the primary toxin variants in our data. MC-RR, MC-YR and MC-LR were significantly correlated with Ch1 a, biomass of cyanobacteria, Microcystis and Anabaena, indicating that microcystins were mainly produced by Microcystis and Anabaena sp. in these lakes. 4. Nonlinear interval maxima regression indicated that the relationships of Secchi depth, total nitrogen (TN) : total phosphorus UP) and NH4+ with MC were characterised by negative exponential curves. The relationships between MC and TN, TP, NO3- + NO2- were fitted well with a unimodal curve. 5. Multivariate analyses by principal component and classifying analysis indicated that MC was mainly affected by Microcystis among the biological factors, and was closely related with temperature among physicochernical factors.

In situ studies on the bioaccumulation of microcystins in the phytoplanktivorous silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms

The phytoplanktivorous silver carp is an important biomanipulation fish to control cyanobacterial blooms and is also a food fish with the greatest production in China. The accumulation of the hepatotoxic microcystins (MCs) determined by LC-MS in various organs of silver carp was studied monthly in Lake Taihu dominated by toxic Microcystis aeruginosa. Average recoveries of spiked fish samples were 78% for MC-RR and 81% for MC-LR. The highest content of MCs was found in the intestine (97.48 mu g g(-1) DW), followed by liver (6.84 mu g g(-1) DW), kidney (4.8 8 mu g g(-1) DW) and blood (1.54 mu g g(-1) DW), and the annual mean MC content was in the order of intestine > liver > kidney > blood > muscle > spleen > gallbladder > gill. Silver carp could effectively ingest toxic Microcystis cells (up to 84.4% of total phytoplankton in gut contents), but showed fast growth (from 141 g to 1759 g in I year in mean weight). Silver carp accumulated less microcystins in liver than other animals in the same site or other fish from different water bodies at similar level of toxin ingestion. There was possible inhibition of the transportation of the most toxic MC-LR across the gutwall. Muscle of silver carp in Lake Taihu should not be consumed during period of dense Microcystis blooms while viscera were risky for consumption in more months. (c) 2006 Elsevier B.V. All rights reserved.

Sorption, degradation and mobility of microcystins in Chinese agriculture soils: Risk assessment for groundwater protection

In the present paper, sorption, persistence, and leaching behavior of three microcystin variants in Chinese agriculture soils were examined. Based on this study, the values of capacity factor and slope for three MCs variants in three soils ranged from 0.69 to 6.00, and 1.01 to 1.54, respectively. The adsorption of MCs in the soils decreased in the following order: RR > Dha(7) LR > LR. Furthermore, for each MC variant in the three soils, the adsorption rate in the soils decreased in the following order: soil A > soil C > soil B. The calculated half-time ranged between 7.9 and 17.8 days for MC-RR, 6.0-17.1 days for MC-LR, and 7.1-10.2 days for MC-Dha(7) LR. Results from leaching experiments demonstrated that recoveries of toxins in leachates ranged from 0-16.7% for RR, 73.2-88.9% for LR, and 8.9-73.1% for Dha 7 LR. The GUS value ranged from 1.48 to 2.06 for RR, 1.82-2.88 for LR, and 1.76-2.09 for Dha(7) LR. Results demonstrated the use of cyanobacterial collections as plant fertilizer is likely to be unsafe in soils. (c) 2006 Elsevier Ltd. All rights reserved.

Development of tolerance against toxic Microcystis aeruginosa in three cladocerans and the ecological implications

This is the first experimental study to compare difference in the development of tolerance against toxic Microcystis among multi-species of cladocerans (Daphnia, Moina and Ceriodaphnia) pre-exposed to two M. aeruginosa PCC7820 strains (MC-containing and MC-free). Zooplankton were divided into S population (fed Scenedesmus), M-F population (fed Scenedesmus + MC-free Microcystis), and M-C population (fed Scenedesmus + MC-containing Microcystis). M-F and M-C populations were pre-exposed to Microcystis strains for 4 weeks, and their newborns were collected for experiments. A pre-exposure to MC-containing or MC-free Microcystis increased tolerance against toxic Microcystis. The marked increases in survival rate and median lethal time (LT50, 100-194% increase) in the M-C population of Ceriodaphnia suggest that small-sized cladocerans may develop stronger tolerance against Microcystis than large-sized ones when both groups are exposed to toxic Microcystis. This may explain why dominant Daphnia is usually replaced by small-sized cladocerans when cyanobacteria bloomed in summer in eutrophic lakes. (c) 2005 Elsevier Ltd. All rights reserved.

Optimization of an effective extraction procedure for the analysis of microcystins in soils and lake sediments

Microcystin analysis in sediments and soils is considered very difficult due to low recovery for extraction. This is the primary limiting factor for understanding the fate of toxins in the interface between water and sediment in both the aquatic ecosystem as well as in soils. In the present study, a wide range of extraction solvents were evaluated over a wide range of pH, extraction approaches and equilibration time to optimize an effective extraction procedure for the analysis of microcystins in soils and lake sediments. The number of extractions required and acids in extraction solutions were also studied. In this procedure, EDTA-sodium pyrophosphate solution was selected as an extraction solvent based on the adsorption mechanism study. The optimized procedure proved to be highly efficient and achieved over 90% recovery. Finally, the developed procedure was applied to field soil and sediment sample collected from Chinese lakes during bloom seasons and microcystins were determined in six of ten samples. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of the phytoplanktivorous silver carp (Hypophthalmichthys molitrixon) on plankton and the hepatotoxic microcystins in an enclosure experiment in a eutrophic lake, Lake Shichahal in Beijing

The responses of nutrients, water transparency, zooplankton, phytoplankton and microcystins to a gradient of silver carp biomass (0, 18, 55, 110 g/m(3)) were assessed using enclosures in Lake Shichahai (Beijing). Picophytoplankton biomass increased with increasing fish stocking density (r=0.64, p=0.09). Silver carp significantly depressed zooplankton biomass, and thus, zooplankton grazing was too low to control phytoplankton. Intracellular microcystin (MC) content in the enclosures was correlated only to Microcystis biomass in the present study. Microcystis spp. biomass and intracellular microcystins content were much higher in lake water than those of enclosures with and without stocking fish. Stocking of silver carp could be an appropriate in highly productive Lake Shichahai, which naturally lacks of large cladoceran zooplankton. A fish stocking density of 55 g/m(3) was most efficient at controlling Microcystis blooms and increasing water clarity. Mean extracellular MC concentration in the lake water was almost the same with that of the enclosures with fish. (c) 2006 Elsevier B.V. All rights reserved.