|Other Abstract||Recently, eutrophication due to the excessive input of nutrients is often accompanied with the occurrence of toxic cyanobacterial blooms in freshwater systems all over the world. Cyanobacterial blooms can produce various toxins that can cause poisonings of animals and human. Among these toxins, microcystins (MCs) are the most common and dangerous group in eutrophic lakes. Potential risks of these toxins (especially microcystins) to aquatic ecosystem and human health are considered to be one of the hot spots of life and environmental sciences and the public. In China, eutrophication of lakes is quite serious. Cyanobacterial surface blooms occur frequently in eutrophic lakes in the warm seasons each year and pose potential risks to both aquatic animals and human health. Hence, great attention should be paid to bioaccumulation, distribution, metabolization and seasonal changes of microcystins in aquatic products and the effects of MCs on safety of aquatic products need to be evaluated. However, so far, studies on the metabolization of microcystins in animals have been limited, and no quantitative study has been conducted to examine the distribution of various metabolites of MCs in animals. Hence, it is very necessary to determinate the metabolites of microcystins quantitatively and discuss the metabolization pathway of microcystins in aquatic animals. The aims of the present study are to 1) examine the bioaccumulation, distribution, transfer and metabolization of microcystins in the food web in a larage eutrophic lake, Taihu Lake, China; 2) to analyze microcystins content in aquatic products from 9 lakes with dense cyanobacterial blooms along the Yangtze River with evaluation on the potential risks of the contaminated aquatic products to human health；3）to determinate the concentration of MC-LR and its two metabolites (MC-LR-GSH and MC-LR-Cys) in aquatic animals with discussion on the metabolization pathway of MC-LR in aquatic animals . The main results and conclusions are as follows:
1. Contents of three common microcystins (microcystin-RR (MC-RR), microcystin-YR (MC-LR), and microcystin-LR (MC-LR)) were examined monthly in hepatopancreas, intestine, gonad, foot, rest, remaining tissue, and offspring of a freshwater snail, Bellamya aeruginosa, from three sites of Gonghu Bay of Lake Taihu during the period between November 2004 and October 2005. Microcystins were present in the offspring and a high positive correlation in microcystin (MCs (RR+YR+LR)) content was found between the offspring and gonad, indicating that microcystins were able to transfer from adult females to their young snail with physiological connection. The majority of the toxins were present in the intestine (53.6%) and hepatopancreas (29.9%), whereas other tissues comprised only 16.5%. If intestines are excluded, up to 64.3% of the toxin burden was allocated in the hepatopancreas. There were great seasonal variations in MCs content in various tissues of the snail. The highest concentrations in the hepatopancreas, intestine, and gonad were all observed in July when Microcystis biomass in the lake water was the highest, and decresed to low level in the spring and winter, while the maxmum MCs contents in muscle and rest tissues were respectively observed in November and December when Microcystis blooms disappeared. Microcystin contents in the intestine, hepatopancreas and gonad were correlated with Microcystis biomass and intracellular and extracellular toxins. 18.2% of the analyzed foot samples were above the tolerable daily microcystin intake recommended by World Health Organization (WHO) for human consumption.
2. Spatial and temporal variations of MC-RR, -YR, -LR in the hepatopancreas of a freshwater snail (Bellamya aeruginosa) were studied monthly at five sites of two bays of Lake Taihu during November 2004 and October 2005. The MCs concentrations in hepatopancreas were higher at Site 1 than at other sites, which was in agreement with the changes of intracellular MCs concentrations in the water column. There was a significant correlation between MCs concentrations in the hepatopancreas and that in the seston, suggesting that spatial variances of MCs concentrations in hepatopancreas among the five sites were due to spatial changes of toxic Microcystis cells in the water column. PCCA indicates that in addition to Microcystis, other factors (e.g., water temperature) also substantially affected the accumulation of MCs in hepatopancreas of the snail.
3. Accumulation and distribution of microcystins (MCs) were examined monthly in six species of fish with different trophic levels in Meiliang Bay, Lake Taihu from June to November 2005. Average recoveries of spiked fish samples were 67.7% for MC-RR, 85.3% for MC-YR, and 88.6% for MC-LR, respectively. MCs (-RR, -YR, -LR) concentration in liver and gut content was highest in phytoplanktivorous fish, followed by omnivorous fish, and was lowest in carnivorous fish; while MCs concentration in muscle was highest in omnivorous fish, followed by phytoplanktivorous fish, and was lowest in carnivorous fish. This is the first study reporting MCs accumulation in the gonad of field fish. The main uptake of MC-YR in fish seems to be through the gills from the dissolved MCs. Only MCs content in common carp muscle exceeded the tolerable daily intake suggested by WHO.
4. Two experiments were carried out to examine the metabolization pathway of MC-LR in aquatic animals. In the first experiment, seasonal changes of MC-LR and its two metabolites (MC-LR-Cys and MC-LR-GSH conjugates) were studied quantitatively in three aquatic animals –snail (Bellamya aeruginosa), shrimp (Macrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) collected from Lake Taihu. The mean MC-LR concentration in hepatopancreas of snail and shrimp and liver of silver carp were 6.61μg/g DW, 0.24 μg/g DW, and 0.027μg/g DW, respectively, while the average MC-LR-Cys were 0.50 μg/g DW, 0.97 μg/g DW, and 5.72 μg/g DW, respectively. MC-LR-GSH was usually not detectable in these samples. The second experiment included both field and laboratory studies. In the laboratory, bighead carp were injected intraperitoneally with pure MC-LR at a dose of 500 µg MC-LR /kg bw, and then MC-LR and its two metabolites (LR-GSH and LR-Cys) concentration in liver were analyzed at 1h, 3h, 6h, and 12h postinjection. In the field, MC-LR and its two metabolites (LR-GSH and LR-Cys) concentration were detected in liver of bighead carps from five lakes of Hunan and Jiangsu Provinces during July and August 2008. In the field, LR-Cys is the main form of MC-LR in the liver of bighead carp (LR-Cys concentration was as 13-fold as that of MC-LR), and very low level of LR-GSH was detected in the samples of the two lakes. In the laboratory experiment, free MC-LR was the main form of MC-LR in the liver of bighead carp within 12h, and the two MC-LR metabolites were detected quantitatively in the liver samples during the whole study period, but MC-LR-Cys concentration was as 6.6-fold as that of LR-GSH. 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; 2) that 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, and subsequently, MC-LR-Cys is degraded from these polypeptide or protein, and finally is excreted from animals by the compounds of MC-LR-Cys or/and MC-LR conjugates with polypeptide; 3) that the MC-LR detoxification pathway varied among species of aquatic animals; and 4) that the MC-LR detoxification pathway differed among different exposure routs.
5. Distributions of microcystins and MC-LR conjugates were examined in liver and muscle of silver carp (Hypophthalmichthys molitrix) from 9 subtropical shallow lakes (Taihu, Chaohu, Huanggaihu, Bajiaohu, Donghu, Dongjiu, Gehu, Dianshanhu, and Wushanhu) with cyanobacterial blooms along the Yangtze River to evaluate the potential risks of microcystins-contaminated fish to human health during July and August. In spite of significant variation of MCs concentrations in seston, MCs (mainly MC-RR) content in muscle of silver carp from these lakes only showed slight variation. High amount of MC-LR and MC-LR-Cys was found in the liver of silver carp from these lakes, while in muscle samples, no MC-LR-Cys was detected and MC-LR contents were below the detectable levels, suggesting that 1) MC-LR was metabolized and transformed primarily in the liver of fish, and little entered into muscle, and that 2) MC-LR in muscle might be excreted via a different pathway from liver. Assuming that a 60 kg person would consume 300g muscle per day, MC contents in muscle materials from all these lakes did not exceed TDI proposed by WHO, indicating that it is still safe to consume muscle of silver carp from these lakes in spite of the presence of cyanobacteria blooms. However, potential risks from long-term exposure to MC-contaminated fish product can not be ignored.|