Studies Of Phycotoxin Contamination In The Coastal Waters Of China And Preparation Of Toxin Reference Matreials

Some microalgae in the sea can produce phycotoxins,which can be divided into hydrophilic marine toxins and lipophilic algal toxins based on their polarity.Phycotoxins can be accumulated in filter-feeding marine animals,and pose potent threats to seafood consumers.Previous studies have shown that contamination of phycotoxins in shellfish is a serious issue along the coast of China,and many poisoning incidents caused by contaminated seafood were recorded.Therefore,it is urgent to carry out systematic investigation to figure out the contamination status of phycotoxins and to strengthen the monitoring and research efforts on phycotoxins.However,there are no ceritified reference materials of phycotoxins yet in China,which restricts the relevant monitoring and research work on phycotoxins.Therefore,this study aims to reveal the contamination status via analyzing phycotoxins in phytoplankton and shellfish samples collected from coastal waters of China,and promote the preparation and declaration of toxin reference materials.In order to reveal the contamination status of phycotoxins,methods of highperformance liquid chromatography-fluorescence detection(HPLC-FLD)and high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS)were established to analyze paralytic shellfish toxins(PSTs)and lipophilic algal toxins,respectively.Besides,samples of phytoplankton and shellfish were collected during four surveys in representative mariculture zones(including Yantai,Laizhou,Hangu,Qinhuangdao and Huludao)around the Bohai Sea from December 2013 to November 2014,three surveys in the Yellow Sea and the East China from May 2014 to August 2015,and surveys in the Changjiang River estuary and its adjacent waters from February to September 2015.Phycotoxins in theses samples were analyzed with the established methods.Moreover,an experiment of PST accumulation and transformation in scallops Patinopecten yesoensis was conducted in the present study.Based on the analytical results,PSTs were commonly found in phytoplankton samples collected from coastal waters of China.PSTs in most of the phytoplankton samples were dominated by low-potency N-sulfonylcarbamyl toxins(C1&2).But in the phytoplankton samples from Qinhuangdao and Laishan,PSTs were dominated by highly potency carbamate toxins(GTX1-4)and decarbamoly toxins(dcGTX2&3 and dcSTX),respectively.Based on toxin profiles of PSTs,it is deduced that PSTs in the Yellow Sea and the East China Sea are mainly produced by Alexandrium tamarense species complex,while Gymnodinium catenatum and other toxic species may be responsible for PSTs in the Bohai Sea.Toxin content of PSTs in the Changjiang River estuary and its adjacent waters(0-215 nmol m-3)was much higher than that in the Yellow Sea and East China Sea(0-156 nmol m-3),while that in the Bohai Sea is the lowest(0-3.35 nmol m-3).For the shellfish samples collected from the Bohai Sea,PST toxicity in 12% of the samples exceeded the food safety standard(80 ?gSTXeq 100g-1).The composition of PSTs in shellfish was dominated by high potency gonyautoxin 1&4(GTX1&4),gonyautoxin 2&3(GTX2&3),neosaxitoxin(NEO)and saxitoxin(STX),significantly different from toxin profiles of phytoplankton.According to the simulation experiment,scallops exposed to toxic Alexandrium could quickly accumulate PSTs,and the toxicity level exceeded the regulatory limit for PSTs within 5 days.Toxin composition of scallops was different from toxic algae,and the kidney was the major organ for toxin transformation in scallops.Lipophilic algal toxins were commonly found in phytoplankton samples from the coastal waters in China.Pectenotoxin2(PTX2),okadaic acid(OA),dinophysis toxin 1(DTX1),yessotoxin(YTX)and homo-YTX were major toxin components,and 13-desmethyl spirolide C(desMec),gymnodimine(GYM)and azaspiracids(AZAs)were occasionally detected.For some groups of phycotoxins,homologues of toxins could be detected in phytoplankton samples,such as PTX11 and PTX1 of PTX2,and trinor-YTX,45-OH-YTX,45-OH-homo-YTX,COOH-45-OH-YTX of YTX.The highgest content of lipophilic algal toxins in phytoplankton generally occurred during the period from June to September.In shellfish samples collected from the aquculture zones of the Bohai Sea,OA,DTX1,PTX2,YTX,homo-YTX,AZA2,AZA3,GYM and desMec were detected.Among the different species of shellfish,scallops,clams and mussels were more likely to accumulated lipophilic algal toxins.The content of lipophilic algal toxins in shellfish was also the highest from June to September.However,toxin content is much lower than the food safety standards,and the risk of seafood poisoning caused by lipophilic algal toxins is low.Based on the investigations,PST contamination in coastal waters of China is the most serious.Therefore,C1&2,GTX2&3 and GTX1&4 were chosed to prepare reference materials.C1&2,GTX1&4,GTX2&3 were extracted from cultured cells of Alexandrium,and purified with multiple columns.The purified toxins were assessed for homogeneity and stability,and determined for their values and uncertainties.The purified toxins were then declared for reference material to National Administrative Committee for CRM's of China.In conclusion,based on the systematic investigations of phycotoxins,this study figures out the contamination status of phycotoxins in coastal waters of the Bohai Sea,Yellow Sea and East China Sea.Moreover,some PSTs were purified from toxic algae and then examined for homogeneity and stability.The purified toxins were determined for their values and uncertainties,and declared for reference materials.The results of the current study will promote the declaration of toxin reference materials,and lay a sound foundation for monitoring and research of phycotoxins in China.