Analysis Of Paralytic Shellfish Toxins Produced By Dinoflagellate Gymnodinium Catenatum And Their Biokinetics In Bivalves

Paralytic shellfish toxins(PSTs)are neurotoxic alkaloids mainly produced by marine dinoflagellates.Bivalves may accumulate PSTs through feeding on toxic dinoflagellates,thus leading to human intoxication and,as a result,posing severe threat to human health.On one hand,the toxin composition and concentration of toxin-producing dinoflagellates strongly affect the toxin load and toxicity of bivalves.On the other hand,the biokinetic processes in bivalves including accumulation,distribution,biotransformation,and elimination may determine the toxin burden as well.Gymnodinium catenatum is an important PST producer that have caused multiple intoxication incidents around the world.Human poisoning events caused by G.catenatum also occurred in the costal areas of the East China Sea.Most PSTs are hydrophilic toxins,yet a novel group of lipophilic PSTs were revealed in G.catenatum recently and named GC toxins by their source.Little is known on the GC toxin production feature of G.catenatum from the East China Sea as well as the biokinetics of GC toxins in bivalves.What are the toxin production features of G.catenatum in the costal waters of China?How do environmental factors affect toxin production?What are the biokinetic processes of GC toxins in bivalves?What are the mechanisms behind such metabolic processes?Anwsering these questions helps assessing the risks of GC toxins produced by G.catenatum.The current dissertation estabilished an HPLC-MS method that is able to analyze PSTs including GC toxins,and examined the toxin production features of G.catenatum isolated from the East China Sea along with effects of environmental factors on toxin production feature.The biokinetic processes including accumulation,distribution,biotransformation,and elimination and the mechanisms behind these processes in blood clam(Scarpharca subcrenata)and bay scallop(Argopecten irradians)were revealed through simulation experiments.In this study,an HPLC-MS method has been established,which could analyze most PST analogues including GC toxins.The limits of detection of PSTs range between 1.05–9.63 ng/g with the limits of quantitation between 3.15–28.90 ng/g.The recovery of most hydrophilic PSTs are between 80%–120%,which matchs the analytical requirements.The matrix effects of some PSTs in shellfish samples could exceed 60%,and the matrix-mixed standards should be used to reduce the strong matrix effect.The HPLC-MS method could be used to perform qualitative and semi-quantitative detection of GC toxins.The toxin production features of G.catenatum(strain MEL11)isolated from the East China Sea were examined using the established HPLC-MS method,and the effects of temperature and nutrients on toxin production of G.catenatum was examined G.catenatum strain MEL11 mainly produce N-sulfocarbamoyl toxins and GC toxins that counts up to 50%of total PSTs in the algal cell.The maximum cell densities of G.catenatum(strain MEL11)cultivated in 20–26℃exceeded 0.70×10~4cells/m L,and the cellular toxin concentration ranged between 189–219 fmol/cell,indicationg the growth and toxin production of were rather constant under 20-26℃.When cultivated under nitrogen or phosphorus limited condition,the growth of G.catenatum were suppressed,maximum cell densities declined to 0.3×10~4 cells/m L.Under nitrogen or phosphorus limiting condition,the proportion of GC toxins dropped from over 50%to around 30%when the culture reached stationary growth phase and decline phase.Cellular toxin concentration raised up to 1100±150fmol/cell when replacing nitrate with ammonium at a concentration of 88.3μmol/L.The results revealed the toxin production feature of G.catenatum in costal waters of China and the effects of temperature and nutrients.The biokinetics including accumulation,distribution,biotransformation,and elimination of PSTs produced by G.catenatum in blood clam and bay scallop were examined by indoor simulation experiments focusing on the biokinetics of GC toxins.Toxin accumulation rate of blood clam was 18.5%,lower than of the bay scallop(90%).The detoxificaiton rate of blood clam was only 0.021 per day.The GC toxins were converted to decarbamoyl toxins through the hydrolysis of hydroxybenzoate group.The proportion of GC toxins remained under 7.7%in bay scallops,much lower than 30%in blood clams,suggesting that biotransformation of GC toxins were stronger in bay scallops.Most toxins accumulated in the viscera of bay scallop,yet the toxicity of adductor muscle in scallops exposed to G.catenatum reached1780±720μg STXeq/kg,with the total toxin burden of adductor muscle counts up to30%of the whole scallop.The amphiphilicity of GC toxins probably accelerated its transfer between scallop tissues,which may lead to the high toxin concentration of adductor muscle.This discovery revealed that GC toxins may increase the risk of paralytic shellfish poisoning by prompting toxin distribution in scallops.The mechanisms,however,remain to be further investigated.Based on the results of the simulating experiments,toxin biotransformation processes of GC toxins and its mechanism were further examined by in vitro incubation experiments.It was found that decarbamoyl toxins in the tissue homogenates of blood clam and bay scallop mainly derived from the hydrolysis of GC toxins,not N-sulfocarbamoyl toxins.The hydrolysis of GC toxins requires enzyme catalysis,the enzymatic activity governing the hydrolysis of GC toxins were higher in viscera of bay scallop than other tissues.The molecular weight of“GC toxin hydrolase”is likely larger than 50 k Da,with optimum temperature at 15–30℃and optimum p H around 7.0.Two para-nitrobenzyl esterase-like proteins probably catalyse the hydrolysis of GC toxins.Besides,the reduction of hydroxyl on R1 of GC toxins were detected in blood clam,not bay scallops.Due to the limited knowledge on toxicity of GC toxins,the effects of hydrolysis and reduction of GC toxins on the toxicity of bivalves still remain to be further confirmed.In general,this dissertation established an HPLC-MS method focusing on the analysis of GC toxins produced by G.catenatum.With the method,the toxin production features of a G.catenatum isolate from the East China Sea was revealed.The strain produces a high proportion of GC toxins,and its toxin profile are strongly affected by nutrient concentration and species of nitrogen.It was discovered,for the first time,that toxin content of the adductor muscle in bay scallop was high when scallops are exposed to G.catenatum.The results suggest that GC toxins might alter the distribution of PSTs and increase the risks of paralytic shellfish poisoning.Transformation of GC toxins,either through reduction of hydroxyl on R1 or enzymatic hydrolysis of hydroxybenzoate group on R4,were confirmed in bivalves.The potential enzymes involved in the hydrolysis of GC toxins were tentatively identified,and their activity in different species and tissues of bivalves were identified.The current study improved the understanding of toxin production of G.catenatum in the Chinese costal waters,gained further in-depth knowledge of GC toxin biokinetics in bivalves and its mechanism,and provided scientific basis of preventing the risks of PSTs caused by G.catenatum.