多种产毒藻混合暴露制备麻痹性贝类毒素基体标准物质原料技术

Technological Advances in Preparing Raw Materials of Paralytic Shellfish Toxin Matrix Standards by Mixed Exposure to Multiple Toxic Dinoflagellates

麻痹性贝类毒素(paralytic shellfish toxins,PSTs)在我国乃至全球引发了严峻的生态和食品安全风险,加强该类毒素的安全监控以确保消费者安全成为全球共识。现有检测技术中,高效液相色谱-串联质谱技术因其高通量、高灵敏度的优势成为国际优先发展技术,但符合国际限量目标的参考物质获取成为发展中国家开展风险监测的核心难题。基于此,本研究通过比较6株产毒藻的单细胞产毒量、藻密度和毒素组分,筛选出的4株产毒藻所产毒素可以覆盖我国海域的主要PSTs,平均藻细胞密度达4.0×10^(6)~4.0×10^(7) cells/L。通过4株产毒藻单一暴露实验发现,贻贝中的PSTs含量能够满足国际限量目标,最高蓄积转化率可达83.1%,且藻与贝中毒素组成基本一致。此外,其中的链状裸甲藻可在贻贝体内代谢转化形成dcSTX,并在5 d达到29.6%的占比。根据上述规律,确定混合暴露实验时链状亚历山大藻(Alexandrium catenella)、链状裸甲藻(Gymnodinium catenatum)、塔玛亚历山大藻(A.tamarense)和微小亚历山大藻(A.minutum)投喂比例低密度组为1∶1∶1∶4,高密度组为1∶1.6∶2.4∶8,其中,高密度组的dcNEO、dcSTX、NEO等3种毒素的占比高于低密度组。4株产毒藻混合暴露获得含NEO、dcSTX、dcNEO、GTX1、GTX4、GTX2、GTX3、GTX5、dcGTX2、dcGTX3、C1和C2共12种PSTs,总毒性分别为(535.0±5.6)μg STXeq/kg和(2636.0±12.4)μg STXeq/kg的基体标准物质原料。本研究表明,产毒藻混合暴露可以获得稳定可控的12种PSTs毒素组分,为制备可用于产业监管及行业服务的基体标物提供技术支撑。

Paralytic shellfish toxins(PSTs)pose significant ecological and food safety hazards in China and the world.Consequently,there is a global consensus to intensify safety monitoring of these toxins to safeguard consumers.In recent years,the occurrence of harmful algal blooms(known as“red tides”)has increased due to human activities and climate change.The contamination risk posed by PSTs is a pressing concern.This problem poses significant ecological and food safety risks in China and the world,leading to a global consensus on strengthening the safety monitoring of PSTs to ensure consumer safety.Several countries and regions,including the European Union,the United States,and Canada,have established strict regulatory limits(800μg STXeq/kg)for PSTs and implemented monitoring programs.Currently,the main methods for detecting and monitoring PSTs include enzyme-linked immunosorbent assay,mouse bioassay,high-performance liquid chromatography,and liquid chromatography-tandem mass spectrometry(LC-MS/MS).Among them,LC-MS/MS has gained international attention due to its high throughput and sensitivity,allowing detailed analysis of toxin profiles in contaminated shellfish.However,the complexity of aquatic food matrices often introduces interference during detection,affecting the accuracy and precision of the results.Using matrix reference materials as quality controls during the analysis can effectively mitigate the errors caused by matrix effects.In recent years,research has been conducted in China on the preparation techniques of matrix reference materials.However,existing techniques have limitations,such as unstable raw materials,limited toxin types,and lack of commercial production.There is still a lack of PST standard materials in China that possess traceability,certification,and physical specimens.To meet the quality control and validation needs for PST detection and analysis,and to enhance the regulation of shellfish quality and safety,the preparation of various PST matrix reference materials has become a top priority.In this study,six PST-producing algae species from four major Chinese coastal areas were compared as the raw material for toxin production:Alexandrium catenella(GY-H25),Gymnodinium catenatum(GY-H65),Alexandrium tamarense(GY-H31 and AT5-3),Alexandrium pacificum(TI0989),and Alexandrium minutum(GY-H46).Through three-step extended culture,all strains achieved mean cell densities ranging from 4.0×10^(6) to 4.0×10^(7) cells/L.Single cell toxin producing capacity was ordered from highest to lowest as GY-H46,GY-H31,AT5-3,GY-H25,GY-H65,and TI0989.After comparing the toxin production per cell,algal cell density,and toxin composition of the six strains,GY-H46,GY-H31,GY-H25,and GY-H65 were selected for the exposure experiments.These four species of toxic algae(up to 1.0×10^(5) cells)were fed to mussels(Mytilus galloprovincialis)for 7 days under controlled conditions.The results demonstrated that the toxin composition of contaminated mussels clustered with those of the causative algae and may separate from one another significantly during the different accumulation phases.GY-H46,GY-H31,and GY-H25 showed consistent toxin profiles with those found in mussels,but varied in the proportions of individual toxin components.GY-H65 exhibited significant differences in toxin composition and proportions in mussels.We also detected a new conversion component(dcSTX)with a maximum proportion of 29.6%.The proportion of dcNEO increased from 1.9%to 13.2%in GY-H65 and mussels.In addition,a high proportion of conversion from C2 to C1 was observed in mussels fed GY-H25 and GY-H65.The mean accumulation rates of toxins from the four toxic algae species in mussels ranged from 39.7%to 83.1%,with the highest toxicity reaching(1,151.0±7.6)μg STXeq/kg in mussels.Individual exposure experiments with the four toxic algae species demonstrated that mussels are capable of accumulating multiple components and high levels of PSTs.Based on the above observations,the feeding ratios of the low-and high-density groups in the mixed exposure experiment were determined as 1∶1∶1∶4 and 1∶1.6∶2.4∶8 for GY-H25,GY-H65,GY-H31,and GY-H46,respectively.The total algae feeding amount per mussel per day was 3.5×10^(4) cells in the low-density group and 2.6×10^(5) cells in the high-density group.When examining the PST content in mussels from the two mixed exposure groups,it was observed that the mean accumulation conversion rates were comparable at 41.4%and 44.6%,which were lower than the theoretical rates of 48.7%and 47.5%,respectively.The overall toxicity of the low-and high-density groups was(535.0±5.6)and(2,636.0±12.4)μg STXeq/kg,respectively.After mixed exposure,both experimental groups of mussels accumulated a total of 12 PST components(NEO,dcSTX,dcNEO,GTX1,GTX4,GTX2,GTX3,GTX5,dcGTX2,dcGTX3,C1,and C2).This study demonstrates that mixed exposure of mussels to four toxin-producing algae species,namely A.catenella,A.tamarense,G.catenatum,and A.minutum,can result in the stable and manageable presence of 12 PST components.This provides technical support for the preparation of matrix reference materials that can be used in industrial regulation and industry services.The development of a preparation technology for matrix reference materials with multiple PST components,which do not exceed international limits,holds promise for water quality monitoring and the assessment of marine pollution risks,and provides reference materials for theoretical studies on PSTs risk formation mechanisms.