Monitoring And Early Warning Of Shellfish Toxins Pollution In Oyster

China is the dominant country of shellfish farming and products, and the total output of oysters and the farming area are more than 3.8 million tons and 12 million hectares, respectively. The total output of oysters has accounted for 80.37% of the world’sin 2014. Ostrea gigas, Ostrea rivularis, Ostrea talien whanensis, Ostrea plicatula are major oyster species. In recent years, with the rapid development of industries, lot of industrial pollutants were discharged into the marine ecosystem, which leading to serious water eutrophication, frequent occurrence of harmful red tides. The shellfish toxins that produced by multiplication of toxic and harmful algae was accumulated in shellfish body, transfer to human body through the food chain. It is not only seriously bringing harm to the health of consumers, but also bring serious damage to aquaculture and processing industries. In China, shellfish toxin pollution problems have become the bottleneck of shellfish product quality and safety. At present, there are some limitations in shellfish toxin detecting technology by biological methods. In addition, effective shellfish toxins pollution monitoring and early warning technologies system were failed to establish.The detecting methods of five kinds of diarrhetic shellfish toxins(OA、DTX-1、DTX-2、GYM、PTX-2)and six kinds of paralytic shellfish toxins(STX、dc STX、GTX-1 、 GTX-2 、 GTX-3 、 GTX-4) were established by HPLC-MS/MS(High Performance Liquid Chromatography-Tandem Mass Spectrometry), respectively. The methods of accumulating diarrheal and paralytic shellfish toxin in seawater were established by using HP20 resins and SP700 resins based on the principle of SPATT(Solid Phase Adsorbent and Toxin Tracking. Based on established methods to detect and enrich toxin, oysters and seawater samples were collected and detected the toxins level in aquaculture zone. The relationship of toxins contents between oysters and sea water were researched and variational disciplines of the oyster contents and toxin contents in the seawater were studied The main progresses and conclusions are as follows:1. The methods of detecting DSPs in oysters by LC-MS/MS were optimized and the methods of accumulating diarrheal and paralytic shellfish toxinsfrom seawater were established. The sample being extracted by methanol and water(80% v/v), the extract was cleaned-up by solid phase extraction(SPE) of Max column. The components of the five toxins were performed on a GM II C18 column. The mode of mass spectrometry was selective reaction monitoring mode. OA, DTX-1, DTX-2 were in negative ion mode and GYM, PTX-2 were in positive ion mode. The result showed that the calibration curve were linear(R≥0.99). The average recoveriesof toxins were between 87.5%~102% in different level addition with the relative standard deviations(RSD) of 3.8~9.2%.The limit of quantitation of OA, DTX-1, DTX-2, GYM, PTX-2 were 1.5μg/kg, 0.7μg/kg, 1.2μg/kg, 2.1μg/kg, 0.6μg/kg, respectively. The accumulate of diarrheic shellfish toxins was using HP20 resins. One week after, the resins bags were took out of sea and washed out by methyl, the extract was cleaned by methylene dichloride. The content was detected by LC-MS/MS. The result showed that HP-20 resin accumulated showed a good adsorption ratio of these five DSP toxins the rate was between 62.8 ~ 101.3% and the limit of quantitation of OA, DTX-1、DTX-2、GYM、PTX-2 were 5.2μg/20gresin*week, 6.1μg/20gresin*week, 4.5μg/20gresin*week, 8.3μg/20gresin*week, 7.4μg/20gresin*week in resins, respectively.2. The oysters and seawater samples were collected and detected diarrheal toxin contents, synchronously then explored its variation characteristics and study variation disciplines between the oysters and seawater samples. The results showed that four kinds of diarrheal shellfish toxins except DTX-2 are detected in seawater and oysters. The changes of the toxin contents with temperature in seawater were more obvious than in oyster samples. The toxins contents increased in seawater with the increased by temperature rise, if no weather conditions changed such as typhoons, the peak appear in August and then declined. Affected by the typhoon, the diarrhea shellfish toxin levels increased in seawater. In the spatial distribution, toxin contents in surface seawater is greater than the middle content, and greater than the lower, especially in the year of August and September, the phenomenon was more significant. The four kinds of diarrheic shellfish toxin content in Oyster were low. Its variation with seawater toxin levels were positively correlated, when the toxins content increased in seawater, the toxin levels in the oysters also increased, oyster toxin levels peaked delayed seawater peaked for one or two sampling periods(a sampling period of 7 days), with the decline in the marine environment the toxins content in the oyster have begun to decline, but the decline compared seawater slower.3. The methods of detecting PSPs in oysters by LC-MS/MS and accumulating PSPs in seawater were established. The sample being extracted by water and formic acid(1‰ v/v), the extract was cleaned-up by solid phase extraction(SPE) of HLB column. The components of toxins were performed on a Amide-80 column. The mode of mass spectrometry was positive selective reaction monitoring mode. The result showed that the calibration curve were linear(R≥0.99) The limit of quantitation of STX, dc STX, GTX-1、4, GTX-2, GTX-3 were 7.6μg/kg, 17.3μg/kg, 35.5μg/kg, 25.8μg/kg, 21.5μg/kg, respectively. The average recoveries for the toxins were between 63.7%~79.1% in different level addition with the relative standard deviations(RSD) of 7.2~12.6%.The accumulated of PSPs was using SP700 resins. The resins bags were took out of sea and washed out by water and formic acid(1‰ v/v), the extract was cleaned by HLB-column. The content was detected by LC-MS/MS. The result showed that SP700 resin showed a good adsorption ratio of these PSPs, the rate was between 60.9%~81.5% and the limit of quantitation of STX, dc STX, GTX-1、4, GTX-2, GTX-3 were 10μg/20gresin*week, 20μg/20gresin*week, 45μg/20gresin*week, 30μg/20gresin*week, 25μg/20gresin*week in resins, respectively.4. The oysters and seawater samples werecollected and detected PSPs contents synchronously then explored its variation characteristics and study variation disciplines between the two samples. The results showed that only STX、dc STX were detected in seawater. The changes of the toxin contents with temperature in seawater were more obvious. The toxins contents variation just like DSPs in seawater and the peak appear in August and then declined. Only STX was detected in Oyster. Its variation with seawater toxin levels were positively correlated, oyster toxin levels peaked delayed seawater peaked for two sampling periods(a sampling period of 7 days), with the decline in the marine environment the toxins content in the oyster have begun to decline, but the decline compared seawater slower.