Investigation on intake, accumulation and toxicity of microcystins to silver carp

Daily intake and accumulation of microcystins (MCYSTs, MCs) in silver carp (Hypophthalmichthys molitrix) were investigated under lab conditions by feeding the fish exclusively with fresh toxic Microcystis bloom at a density of 6 x 10(9) algal cells L-1. The medial lethal dose (LD50) of microcystin-LR to silver carp was estimated to be 270 mu g kg(-1) body-weight, underlining its strong resistance to toxic Microcystis bloom. It can survive after being ingested with high doses of microcystins (about 10 mg kg(-1)) during the 28-days feeding experiment. Enzyme-linked immuno-sorbent assay results show that microcystin concentrations in muscle and liver are 1.57 +/- 0.31 mu g kg(-1) and 4.28 +/- 1.64 mg kg(-1) fresh weight. The former is much lower than the World Health Organization limit recommended for human consumption. These results suggest that silver carps can be widely used in cyanobacterial bloom control, and consumption of fish muscles is safe for human beings.

The optimization of high performance liquid chromatographic method for analysis of trace microcystins in natural water bodies

The ratio of methanol., water and trifluoroacetic acid ( TFA) was regulated to change the polarity and the pH of the rinse solution and the eluent, so as to improve the high performance liquid chromatography HPLC) detection method for trace microcystines (MCs) in natural water bodies. The results showed that 40 % similar to 45 % methanol-water solution containing 0. 1 % TFA could get good effects on the rinse of impurity, and 70% methanol-water solution containing 0. 1% TFA could elute all the MCs in solid phase extraction ( SPE) cartridge ( C-18), In this way. it is suggested that, in analysis of environmental samples with high concentration of impurity, impurity should be washed with 40% similar to 45% methanol-water solution containing 0. 1% TFA, and MCs should be eluted with 70% similar to 100% methanol-water solution containing 0. 1% TFA.

Seasonal dynamics of the hepatotoxic microcystins in various organs of four freshwater bivalves from the large eutrophic Lake Taihu of subtropical China and the risk to human consumption

So far, little is known on the distribution of hepatotoxic microcystin (MC) in various organs of bivalves, and there is no study on MC accumulation in bivalves from Chinese waters. Distribution pattern and seasonal dynamics of MC-LR, -YR and -RR in various organs (hepatopancreas, intestine, visceral mass, gill, foot, and rest) of four edible freshwater mussels (Anodonta woodiana, Hyriopsis cumingii, Cristaria plicata, and Lamprotula leai) were studied monthly during Oct. 2003-Sep. 2004 in Lake Taihu with toxic cyanobacterial blooms in the summer. Qualitative and quantitative determinations of MCs in the organs were done by LC-MS and HPLC. The major toxins were present in the hepatopancreas (45.5-55.4%), followed by visceral mass with substantial amount of gonad (27.6-35.5%), whereas gill and foot were the least (1.8-5.1%). The maximum MC contents in the hepatopancreas, intestine, visceral mass, gill, foot, and rest were 38.48, 20.65, 1.70, 0.64, 0.58, and 0.61 mu g/g DW, respectively. There were rather good positive correlation in MC contents between intestines and hepatopancreas of the four bivalves (r = 0.75-0.97, p < 0.05). There appeared to be positive correlations between the maximum MC content in the hepatopancreas and the delta(13)C (r = 0.919) or delta(15)N (r = 0.878) of the foot, indicating that the different MC content in the hepatopancreas might be due to different food ingestion. A glutathione (GSH) conjugate of MC-LR was also detected in the foot sample of C. plicata. Among the foot samples analyzed, 54% were above the provisional WHO tolerable daily intake (TDI) level, and the mean daily intakes from the four bivalves were 8-23.5 times the TDI value when the bivalves are eaten as a whole, suggesting the high risk of consuming bivalves in Lake Taihu. (C) 2005 Wiley Periodicals, Inc.

Kinetics of the oxidation of MCRR by potassium permanganate

The occurrence of the microcystins in the water bodies, especially in drinking water resources, has received considerable attentions. In situ chemical oxidation is a promising cost-effective treatment method to remove MC from water body. This research investigated the reaction kinetics of the oxidation of MCRR by permanganate. Experimental results indicate that the reaction is second order overall and first order with respect to both permanganate and MCRR, and has an activation energy of 18.9 kJ/mol. The second-order rate constant ranges from 0.154 to 0.225 l/mg/min at temperature from 15 to 30 degrees C. The MCRR degradation rates can be accelerated through increasing reaction temperature and oxidant concentration. The reaction under acid conditions was slightly faster than under alkaline conditions. The half-life of the reaction was less than 1 min, and more than 99.5% of MCRR was degraded within 10 min. (c) 2005 Elsevier Ltd. All rights reserved.

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in two freshwater shrimps, Palaemon modestus and Macrobrachium nipponensis, from a large shallow, eutrophic lake of the subtropical China

So far no information is available on microcystin (MC) contents in shrimps, prawns or crayfish from natural freshwaters. Tissue distributions and seasonal dynamics of the hepatotoxic MC-LR and -RR in two freshwater shrimps, Palaemon modestus and Macrobrachium nipponensis were studied monthly (during June-November, 2003) in a Chinese lake containing toxic cyanobacterial blooms. The shrimps P. modestus and M. nipponensis accumulated high MCs not only in the hepatopancreas (mean 4.29 and 0.53 mu g g(-1) DW, respectively) but also in the gonad (mean 1.17 and 0.48 mu g g-1 DW, respectively), and the crayfish Procambarus clarkii accumulated as much as 0.93 mu g g(-1) DW in the gonad. This indicates that gonads of these invertebrates are the second important target organ of MCs. P. modestus apparently accumulated more MCs in their organs than M. nipponensis, which might be a reflection of their difference in trophic niche. Eggs of the shrimps accumulated 8.4% (M. nipponensis, 0.27 mu g g(-1) DW) and 29.0% (P. modestus, 2.34 mu g g(-1) DW) of total toxin burden, indicating that MCs had been transferred into offspring from their adults. Among the shrimp muscle samples analyzed, 31% were above the provisional WHO TDI level, suggesting the risk of consuming shrimps in Lake Chaohu. (c) 2005 Elsevier Ltd. All rights reserved.

Selective control of toxic Microcystis water blooms using lysine and malonic acid: An enclosure experiment

Three enclosures (10 x 10 x 1.5-1.3 m in depth) were set beside Dianch Lake, Kunming, People's Republic of China, for the period from July 28 to August 26, 2002. The enclosures were filled with cyanobacterial (Microcystis aeruginosa) water bloom-containing lake water. Lake sediment that contained macrophytes and water chestnut seeds was spread over the entire bottom of each enclosure. Initially, 10 g/m(2) of lysine was sprayed in Enclosure B, and 10 g/m(2) each of lysine and malonic acid were sprayed together in Enclosure C. Enclosure A remained untreated and was used as a control. The concentrations of lysine, malonic acid, chlorophyll a, and microcystin as well as the cell numbers of phytoplankton such as cyanobacteria, diatom, and euglena were monitored. On day 1 of the treatment, formation of cyanobacterial blooms almost ceased in Enclosures B and C, although Microcystis cells in the control still formed blooms. On day 7 Microcystis cells in Enclosure B that had been treated with lysine started growing again, whereas growth was not observed in Microcystis cells in Enclosure C, which had been treated with lysine and malonic acid. On day 28 the surface of Enclosure B was covered with water chestnut (Trapa spp.) and the Microcystis blooms again increased. In contrast, growth of macrophytes (Myriophllum spicatum and Potamogeton crispus) was observed in Enclosure C; however, no cyanobacterial blooms were observed. Lysine and malonic acid had completely decomposed. The microcystin concentration on day 28 decreased to 25% of the initial value, and the pH shifted from the initial value of 9.2 to 7.8. We concluded that combined treatment with lysine and malonic acid selectively controlled toxic Microcystis water blooms and induced the growth of macrophytes. (c) 2005 Wiley Periodicals, Inc.

Effects of microcystins on the growth and the activity of superoxide dismutase and peroxidase of rape (Brassica napus L.) and rice (Oryza sativa L.)

Microcystins are naturally occurring hepatotoxic cyclic heptapeptides produced by some toxic freshwater cyanobacterial species. In this study, crude extract of toxic cyanobacterial blooms from Dianchi Lake in southwestern China was used to determine the effects of microcystins on rape (Brassica napus L.) and rice (Oryza sativa L.). Experiments were carried out on a range of doses of the extract (equivalent to 0, 0.024, 0.12, 0.6 and 3 mug MC-LR/ml). Investigations showed that exposure to microcystins inhibited the growth and development of both rice and rape seedlings, however, microcystins had more powerful inhibition effect on rape than rice in germination percentage of seeds and seedling height. Microcystins significantly inhibited the elongation of primary roots of rape and rice seedlings. Determination of the activities of peroxidase and superoxide dismutase demonstrated that microcystin stress was manifested as an oxidative stress. Using ELISA, microcystins were examined from the extract of exposed rape and rice seedlings, indicating that consumption of edible plants exposed to microcystins via irrigation route may have health risks. Significantly different levels of recovered microcystins between exposed rice and rape seedlings Suggested that there might be different tolerant mechanisms toward microcystins. (C) 2004 Elsevier Ltd. All rights reserved.

Dynamics of microcystins-LR and -RR in the phytoplanktivorous silver carp in a sub-chronic toxicity experiment

A sub-chronic toxicity experiment was conducted to examine tissue distribution and depuration of two microcystins (microcystin-LR and microcystin -RR) in the phytoplanktivorous filter-feeding silver carp during a course of 80 days. Two large tanks (A, B) were used, and in Tank A, the fish were fed naturally with fresh Microcystis viridis cells (collected from a eutrophic pond) throughout the experiment, while in Tank B, the food of the fish were M. viridis cells for the first 40 days and then changed to artificial carp feed. High Performance Liquid Chromatography (HPLC) was used to measure MC-LR and MC-RR in the M. viridis cells, the seston, and the intestine, blood, liver and muscle tissue of silver carp at an interval of 20 days. MC-RR and MC-LR in the collected Microcystis cells varied between 268-580 and 110-292 mug g(-1) DW, respectively. In Tank A, MC-RR and MC-LR varied between 41.5-99.5 and 6.9-15.8 mug g(-1) DW in the seston, respectively. The maximum MC-RR in the blood, liver and muscle of the fish was 49.7, 17.8 and 1.77 mug g(-1) DW, respectively. No MC-LR was detectable in the muscle and blood samples of the silver carp in spite of the abundant presence of this toxin in the intestines (for the liver, there was only one case when a relatively minor quantity was detected). These findings contrast with previous experimental results on rainbow trout. Perhaps silver carp has a mechanism to degrade MC-LR actively and to inhibit MC-LR transportation across the intestines. The depuration of MC-RR concentrations occurred slowly than uptakes in blood, liver and muscle, and the depuration rate was in the order of blood > liver > muscle. The grazing ability of silver carp on toxic cyanobacteria suggests an applicability of using phytoplanktivorous fish to counteract cyanotoxin contamination in eutrophic waters. (C) 2003 Elsevier Ltd. All rights reserved.

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.

Determination of trace level microcystins in water using solid-phase extraction and high performance liquid chromatography

This paper presents a method for trace level analysis of microcystins in water using solid-phase extraction and high performance liquid chromatography. The optimized condition enabled the determination of common microcystins at levels as low as 0.02 similar to 0.05 mug/L, and the liner range is from 0.1 mug/L to 50 mug/L. The method has been applied to the analysis of field sample from Dianchi lake.

Comparative study on in vitro inhibition of grass carp (Ctenopharyngodon idellus) and mouse protein phosphatases by microcystins

Microcystins isolated from toxic cyanobacteria are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The inhibitory effects of three structural variants of microcystins (microcystin-LR, -YR, and -RR) on protein phosphatases isolated and purified from the liver and kidney of grass carp (Ctenopharyngodon idellus) were investigated using the P-32 radiometric assay. The relationships between percentage inhibition of protein phosphatase activity and microcystin levels followed a typical dose-dependent sigmoid curve. These results were compared to those obtained from mouse PP2A. The degree and pattern of inhibition of both fish and mouse protein phosphatases by microcystins were similar. Protein phosphatases in crude fish tissue homogenates showed similar inhibition patterns as purified fish PP2A toward microcystins. (C) 2000 by John Wiley & Sons, Inc.

CYCLIC PEPTIDE HEPATOTOXINS FROM FRESH-WATER CYANOBACTERIAL (BLUE-GREEN-ALGAE) WATERBLOOMS COLLECTED IN CENTRAL CHINA

Toxic cyanobacteria (blue-green algae) waterblooms have been found in several Chinese water bodies since studies began there in 1984. Waterbloom samples for this study contained Anabaena circinalis, Microcystis aeruginosa and Oscillatoria sp. Only those waterblooms dominated by Microcystis aeruginosa were toxic by the intraperitoneal (i.p.) mouse bioassay. Signs of poisoning were the same as with known hepatotoxic cyclic peptide microcystins. One toxic fraction was isolated from each Microcystis aeruginosa sample. Two hepatotoxic peptides were purified from each of the fractions by high-performance liquid chromatography and identified by amino acid analysis followed by low and high resolution fast-atom bombardment mass spectrometry (FAB-MS). LD50 i.p. mouse values for the two toxins were 245-mu-g/kg (Toxin A) and 53-mu-g/g (Toxin B). Toxin content in the cells was 0.03 to 3.95 mg/g (Toxin A) and 0.18 to 3.33 mg/kg (Toxin B). The amino acid composition of Toxin A was alanine [1], arginine [2], glutamic acid [1] and beta-methylaspartic acid [1]; for Toxin B it was the same, except one of the arginines was replaced with a leucine. Low- and high-resolution FAB-MS showed that the molecular weights were 1,037 m/z (Toxin A) and 994 m/z (Toxin B), with formulas of C49H76O12N13 (Toxin A) and C49H75O12N10 (Toxin B). It was concluded that Toxin A is microcystin-RR and Toxin B is microcystin-LR, both known cyclic heptapeptide hepatotoxins isolated from cyanobacteria in other parts of the world. Sodium borohydride reduction of microcystin-RR yielded dihydro-microcystin-RR (m/z = 1,039), an important intermediate in the preparation of tritium-labeled toxin for metabolism and fate studies.

Initial Studies on the Occurrence of Cyanobacteria and Microcystins in Irish Lakes

We report the results of a synoptic survey at 14 sites across the north of Ireland undertaken to determine the occurrence of cyanobacteria and their constituent microcystin cyanotoxins. Seven microcystin toxins were tested for, and five of which were found, with MC-LR, MC-RR, and MC-YR being the most prevalent. Gomphosphaeria spp and Microcystis aeruginosa were the most dominant cyanobacterial species encountered. Together with Aphanizomenon flos-aquae, these were the cyanobacteria associated with the highest microcystin concentrations. The occurrence of several microcystin toxins indicates that there may potentially be more than one cyanobacteria species producing microcystins at many sites. Total microcystin concentrations varied over three orders of magnitude dividing the sites into two groups of high (>1000 ngMC/μgChla, six sites) or low toxicity (<200 ngMC/μgChla, eight sites). © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78ngmL and the CCß to be 1ngmL. Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1ngmL, and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1ngmL. This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1µgL. This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4min injection assay to detect total microcystins in water samples below the WHO recommended limit (1µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5ng/mL for extracellular toxins and 0.05ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.