| Since diverse cyanobacteria could produce neurotoxin BMAA (β-N-methylamino-L-alanine, BMAA) and several large lakes (Lake Dianchi, Lake Tai, Lake Chao) have been contaminated by algae-bloom in China recently years, it is urgent for us to investigate BMAA in aquatic environment. In view of Gonghu Bay which is one of the algae-bloom contamined in Lake Tai, firstly, we improved the method for detecting BMAA in environmental samples, secondly, from the period of bloom-emergence to bloom-decline (Apr.～ov.), two forms of BMAA in Microcystis, mollusk, crustaceans and fishes were detected, respectively. Transfer and biomagnification of BMAA in some fodd chains in Gonghu Bay were also investigated. Finally, the fates of BMAA in environments and the interactions of BMAA with seveal proteins (antioxidant enzymes, acetylcholin esterase and bovine serum albumin) were studied. The main results are listed as follows:1. SPE coupled with HPLC-MS/MS was used to detect BMAA in water contaminated by cyanobacteria. Compared with other SPE cartridges, Oasis MCX (waters) cartridge can extract and concentrate BMAA effectively,2%formic acid solution and methanol were used as the washing solutions,5%amonium methanol used as the eluent. The results indicated that this method can effectively concentrate BMAA with the average recovery being above75%. Using0.1Mtrichloroacetic acid together with ultrasonication can successfully extract free BMAA in biological samples (cyanobacteria, benthos, fishes), subsequently, the protein-BMAA can be collected by hydrochloric acid hydrolysis. All derivatized samples were analyzed using HPLC/MS/MS spectrometry system with electrospray ionization (ESI) under positive-ion ionization conditions, and used multiple reaction monitoring (MRM) scan type. HPLC separation was performed on an Eclipse XDB-C18column. The mobile phase was28%acetonitrile in H2O containing0.1%NH4COOH. The average recovery of this method could be above75%.2. During the entire sampling period (Apr.-Nov.), total average BMAA contents in cyanobacteria ranged from2.03μg/dg to7.14μg/dg. BMAA concentration of cyanobacteria reached the peak value in bloom-outbreak period (Jun.～Sep.), however, the BMAA concentrations of cyanobacteria were relatively lower in bloom-emergence (Apr.～May) and bloom-decline (Oct.～Nov.). The results suggested that Micrvcystis.spp from Gonghu Bay have the ability to biosynthesize BMAA. Within the phase of bloom-emergence, the total average BMAA contents in microcystis, mollusks, crustaceans and fish species were2.03、2.0、1.04、1.61μg/dg, respectively, with no significant difference among these different animals (p>0.05). When bloom outbreak, the mean BMAA contents in microcystis, mollusks, crustaceans and fish species were7.14,2.11、5.12、9.12μg/dg, respectively. Higher BMAA concentrations of crustaceans and fish were associated with higher cyanobacteria BMAA concentration. Maximum BMAA concentration was detected in Erythroculter ilishaeformis (35.91ug BMAA/dg). Occurrence of BMAA in different trophic levels of aquatic ecosystems suggests that this toxin may be accumulated within cyanobacteria-based food webs. Within the phase of bloom-decline, the total average BMAA contents in microcystis, mollusks, crustaceans and fish species were3.18、5.66、5.12、7.65μg/dg, respectively. Over the entire sampling period, the highest BMAA concentration of benthos occurred in bloom-decline phase (5.39μg/dg), as well as omnivorous fishes, including Carassius auratus, Cyprinus carpio (the average total BMAA concentration is11.65μg/dg). In summary, the data presented here demonstrate that Microsystis.spp from Gonghu Bay is able to biosynthesize neurotoxin BMAA, and it could be bioaccumulated in different tropic levels. These results indicated that biomagnification of BMAA have been occurred in Gonghu Bay freshwater ecosystem. Based on these studies and BMAA toxicity data, NOAEL value was assumed as0.5g/kg/d. According to the guideline of water drinking which enacted by the WHO, the estimated TDI value of BMAA was calculated as0.5mg/kg/d. The EDI (Estimated Daily Intake) values of BMAA of mollusk, crustacean and fish were5.35、6.27、10.08(μg/kg/d), the average EDI value of these aquatic animals (7.23μg/kg/d) was significantly lower than estimated TDI value (0.5mg/kg/d), suggesting that the health risk of these contaminated aquatic products in Gonghu Bay was relatively low, however, the long-term potential safe problem need further investigation.3. The adsorption of BMAA by unpasteurized and steriled sediment indicated that the adsorption rates both can above95%at15℃,25℃and35℃after7days, which were significantly higher than activated carbon. Different adsorption abilities can be due to different particle morphology. There was no significant difference between unpasteurized and steriled sediment at the same temperature, suggesting that the microorganisms had no apparent effect on the adsorption of BMAA. Additionally, the sediments adsorption capacity rose with increasing temperature.4. Photodegradation rates of BMAA standard solution under different light sources were as followed:UVC> UVA> sunlight with photodegradation rate being38.4%under UVC irradiation after12h continuous irradiation. However, the rates of photodegradation of BMAA in cyanobacteria extracted solution under different light sources as followed:sunlight> UVC> UVA, the photodegradation rates of BMAA under corresponding irradiation was84.8%,79.9%and61.7%after12h continuous irradiation, respectively. These results indicated that the degradation rate of BMAA in cyanobacteria extracted solution containing pigment or photosensitizer was significantly higher than that of in BMAA standard water solution. The effects of illumination intensity, pH and temperature on photodegradation of BMAA were investigated under UVC irradiation as the only light source, the results indicated that temperature was the important influence factor. At the same illumination intensity and pH, temperature had significant influence on the photodegradation rate of BMAA in cyanobacteria extracted solution (p<0.05), with the increase of temperature, the photodegradation rate of BMAA increased gradually. Illumination intensity also strongly influenced the the photodegradation rate of BMAA in cyanobacteria extracted solution at the same temperature. Under15℃and25℃(at the same pH), illumination intensity had’significant influence on the photodegradation rate of BMAA, the photodegradation rate was positively correlated with increase in illumination intensity. Under the same temperature and illumination intensity, alkaline condition is advantageous to the photodegradation of BMAA.The photodegradation rates of BMAA in cyanobacteria extracted solution which were prepared with four solvents, as followed:0.05g/L humic acid> lake water> river water> ultrapure water, the photodegradation rates rose with increasing illumination intensity in above four solvents. At the maximum illumination intensity (4.1mW/m2), the photodegradation rates of BMAA in humic acid, lake water and river water were all above95%within6hours, humic acid can significantly promoted photodegradation of BMAA.5. Five bacterial strains (Pseudomonas, Comamonas, Acinetbactor, Stenotropho-monas, Brevundimonas) were isolated from sediments of Lake Tai, free BMAA and protein-BMAA could be both detected in lag phase, log phase and stationary phase of each bacterial culture. These five bacterial strains can rapidly uptake BMAA in medium, and the order of absorptive ability as follows:Stenotrophomonas> Brevundimonas> Pseudomonas> Comamonas> Acinetbactor, the concentrations of free BMAA in cells were significantly higher than that of protein-BMAA, however, the ability of degradation of BMAA was weak for these bacterium within a short time.6. Results from the study of BMAA interacted with some protein indicated that concentrations of BMAA from10μg/mL to1mg/mL could not affect the enzymatic activity of catalase, superoxide dismutase, glutathione peroxidase and acetyl cholinesterase. The binding ability of BMAA with these antioxidant enzymes was not strong, however, non enzymatic combination could occur between BMAA and acetyl cholinesterase at30℃, in addition, BMAA could effectively bind to bovine serum albumin at30℃, pH7.5, speculated that the distribution of free BMAA may be affected in the body. |
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