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.

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail (Bellamya aeruginosa) from a large shallow, eutrophic lake of the subtropical China

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail (Bellamya aeruginosa) were studied monthly in a large shallow, eutrophic lake of the subtropical China during June-November, 2003. Microcystins (MCs) were quantitatively determined by High-Performance Liquid Chromatography (HPLC) with a qualitative analysis by a Finnigan LC-MS system. On the average of the study period, hepatopancreas was the highest in MC contents (mean 4.14 and range 1.06-7.42 mug g(-1) DW), followed by digestive tracts (mean 1.69 and range 0.8-4.54 mug g(-1) DW) and gonad (mean 0.715 and range 0-2.62 mug g(-1) DW), whereas foot was the least (mean 0.01 and range 0-0.06 mug g(-1) DW). There was a positive correlation in MC contents between digestive tracts and hepatopancreas. A constantly higher MC content in hepatopancreas than in digestive tracts indicates a substantial bioaccumulation of MCs in the hepatopancreas of the snail. The average ratio of MC-LR/MC-RR showed a steady increase from digestive tracts (0.44) to hepatopancreas (0.63) and to gonad (0.96), suggesting that MC-LR was more resistant to degradation in the snail. Since most MCs were present in the hepatopancreas, digestive tracts and gonad with only a very small amount in the edible foot, the risk to human health may not be significant if these toxic parts are removed prior to snail consumption. However, the possible transference of toxins along food chains should not be a negligible concern. (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.

Determinations of MC-LR and [Dha(7)] MC-LR Concentrations and Physicochemical Properties by Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography electrospray mass spectrometry (LC/ESI/MS) method working in multiple reactions monitoring mode for the determination of trace amounts of microcystin variants (MC-LR and [Dha(7)] MC-LR) in waters was developed. The limit of quantification was 0.05 mu g/L and the limit of detection was 0.015 mu g/L for MC-LR and [Dha(7)] MC-LR, respectively. Recoveries for MCs were in the range of 68%-81%. MC-LR and [Dha(7)] MC-LR were chemically stable with similar physiochemical behavior.

Determination of microcystin-LR and its metabolites in snail (Bellamya aeruginosa), shrimp (Macrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) from Lake Taihu, China

This paper describes seasonal changes of microcystin-LR (MC-LR) and its glutathione (MC-LR-GSH) and cysteine conjugates (MC-LR-Cys) in three aquatic animals - snail (Bellamya aeruginosa), shrimp (fMacrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) collected from Lake Taihu, China. MC-LR, MC-LR-GSH, and MC-LR-Cys were determined by liquid chromatography electrospray ionization mass spectrum (LC-ESI-MS). The mean MC-LR concentrations in the hepatopancreas of snail and shrimp and liver of silver carp were 6.61, 0.24, and 0.027 mu g g(-1) dry weight (DW), respectively: while the average MC-LR-Cys concentrations were 0.50, 0.97, and 5.72 mu g g(-1) DW, respectively. MC-LR-GSH was usually not detectable in these samples. The above results suggest that: (1) in aquatic animals, especially fish, the main excretion form of MC-LR could be MC-LR-Cys, but not MC-LR-GSH, whereas MC-LR-Cys might play an important role in detoxication of MC-LR and (2) that efficiency of MC-LR-Cys formation differs among species. The main detoxication pathway of MC-LR in aquatic animals is suggested as follows: when MC-LR enters into liver/hepatopancreas, it firstly conjugates with polypeptide or protein (including GSH, PP-1 and 2A) containing Cys residues, perhaps also some free cysteine; subsequently, MC-LR-Cys is degraded from these polypeptide or protein; and finally is excreted from animals by the compound of MC-LR-Cys. (C) 2009 Elsevier Ltd. All rights reserved.

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA-Na-2-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 mu g g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 mu g g(-1) DW, with a limit of quantification (LOQ) of 0.02 mu g g(-1) DW. The limit of detection (LOD) of the method was 0.007 mu g g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR. (c) 2007 Elsevier B.V. All rights reserved.

Photodegradation dynamics of pure microcystin variants with illumination of fixed wavelength UV-lights

Photolysis of microcystins by UV irradiation and the effects of different environmental factors on efficiency of UV degradation were studied. The results indicated that the rates of the photolytical degradation reactions of microcystin-LR and RR-follow pseudo-first-order kinetic process. The results also showed that the concentrations of two microcystin variants decreased significantly by UV-C Irradiation; the wavelength and intensitiy of UV irradiation are two very important factors affecting the rate of degradation; temperature and pH value could also affect the half life of degradation rates. When irradiated by weaker UV-Iight, isomerization could be detected in the course of photolytical degradation. The concentrations of two isomers transformed from microcystin-LR reached its maximum at the third minute and decreased with the time afterwards. To simulate photolysis of microcystins in the field water body, microcystins with low concentration were used. It was found that UV-C illumination was capable of decomposing over 95% of microcystins within 40 min. In the presence of humic substances the photodecomposition slowed down to a certain extent. These results are valuable in using UV irradiation for elimination microcystins from raw water.

Hepatic Histopathological Characteristics and Antioxidant Response of Phytoplanktivorous Silver Carp Intraperitoneally Injected with Extracted Microcystins

Objective To investigate the hispathological characteristics and antioxidant responses in liver of silver carp after intraperitoneal administration of microcystins (MCs) for further understanding hepatic intoxication and antioxidation mechanism in fish. Methods Phytoplanktivorous silver carp was injected intraperitoneally (i.p.) with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq./kg body weight, and liver histopathological changes and antioxidant responses were studied at 1, 3, 12, 24, and 48 h, respectively, after injection. Results The damage to liver structure and the activities of hepatic antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxide (GPX) were increased in a time-dependent manner. Conclusion In terms of clinical and histological signs of intoxication and LD50 (i.p.) dose of MC-LR, silver carp appears rather resistant to MCs exposure than other fishes. Also, the significantly increased SOD activity in the liver of silver carp suggests a higher degree of response to MCs exposure than CAT and GPX.

In vivo studies on the toxic effects of microcystins on mitochondrial electron transport chain and ion regulation in liver and heart of rabbit

This study examined the toxic effects of microcystins on mitochondria of liver and heart of rabbit in vivo. Rabbits were injected i.p. with extracted microcystins (mainly MC-RR and -LR) at two doses, 12.5 and 50 MCLReq. mu g/kg bw, and the changes in mitochondria of liver and heart were studied at 1, 3,12, 24 and 48 h after injection. MCs induced damage of mitochondrial morphology and lipid peroxidation in both liver and heart. MCs influenced respiratory activity through inhibiting NADH dehydrogenase and enhancing succinate dehydrogenase (SDH). MCs altered Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities of mitochondria and consequently disrupted ionic homeostasis, which might be partly responsible for the loss of mitochondrial membrane potential (MMP). MCs were highly toxic to mitochondria with more serious damage in liver than in heart. Damage of mitochondria showed reduction at 48 h in the low dose group, suggesting that the low dose of MCs might have stimulated a compensatory response in the rabbits. (C) 2008 Elsevier Inc. All rights reserved.

Accumulation of hepatotoxic microcystins in freshwater mussels, aquatic insect larvae and oligochaetes in a large, shallow eutrophic lake (Lake Chaohu) of subtropical China

This paper studied the seasonal changes of two common microcystins (MCs), MC-RR and -LR, in the commercially important mussel Corbicula fluminea in Lake Chaohu, where there occurred dense cyanobacteria. Occasional measurements were also made for MC in the mussel Arconaia lanceolat, the oligochaete Limnodilus hoffineisteri and the insect larva Chironomus sp. Mean MC of C. fluminea was much higher in hepatopancreas than in intestine and foot. Our study is the first to report accumulation of MCs in oligochaetes and aquatic insect larvae. The hi-h contents of MCs in the insect larvae suggest a great possibility for the transfer of MCs to benthos-feeding omnivores like common carp. According to the provisional standard by the WHO, 28.6% of the collected C. fluminea were harmful for human consumption, assuming a daily consumption of 300 by a person. It is recommended that edible mussels should not be collected for human consumption during toxic cyanobacterial blooms in Lake Chaohu.

Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix

Up to now, in vivo studies on the toxic effects of microcystins (MCs) on the ultrastructures of fish liver have been very limited. The phytoplanktivorous silver carp was injected i.p. with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq. kg(-1) body weight, showing a time-dependent ultrastructural change in liver as well as significant increases in enzyme activity of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). We observed for the first time the occurrence of a large amount of activated secondary lysosomes, which might be an adaptive mechanism to eliminate or lessen cell damage caused by MCs through lysosome activation. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS2, respectively. MCs concentration in the liver reached the maximum (114.20 mu g g(-1) dry weight) after 3 h post-injection, and then rapidly dropped to 7.57 mu g g(-1) dry weight at 48 h, indicating a deputation of 99% accumulated MC-LReq. On the other hand, a decrease trend in glutathione (GSH) concentration was observed in the liver of silver carp while the activity of glutathione S-transferase (GST) increased significantly after injection. The high tolerance of silver carp to MCs might be due to the high basic GSH level in their liver, and/or an increased GSH synthesis. (C) 2007 Elsevier Inc. 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.

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.

In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocytes of the phytoplanktivorous bighead carp i.p.-injected with extracted microcystins

Phytoplanktivorous bighead carp were injected i.p. with extracted microcystins (mainly MC-RR and -LR) at two doses, 200 and 500 MC-LReq. mu g kg(-1) bw, and the changes in extractable MCs in liver and in the ultrastructure of hepatocytes were studied at 1, 3, 12, 24 and 48 h after injection. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS, respectively. MC concentration in the liver reached the maxima at 12 It (2.89 mu g MCs g(-1) dry weight at the lower dose) or at 3 h (5.43 mu g MCs g(-1) dry weight at the higher dose) post-injection, followed by sharp declines afterwards, whereas the ultrastructural changes of hepatocytes in both dose groups suggest progressive increases in severity toward the directions of apoptosis and necrosis from I to 24 h, respectively. There were two new findings in fish: widening of intercellular spaces was among the early ultrastructural changes induced by MCs and ultrastructural recovery of hepatocytes was evident at 48 h post-injection in both dose groups. Both the present and previous studies suggest that with in vivo or in vitro exposure to microcystins, hepatocyte damage in fish tends to proceed toward the direction of apoptosis at lower MC concentrations but toward the direction of necrosis at high MC concentrations. The temporal dynamics of MCs in the liver suggest that bighead carp may have a mechanism to degrade or bind MC-LR actively after it enters the blood system. (c) 2005 Elsevier Ltd. All rights reserved.

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.