Profiling And Photodegradation Kinetics Of Extracellular Toxins Associated With Diarrhetic Shellfish Poison From Marine Microalgae

In recent years,biotoxins released by marine harmful algal blooms pose a serious threat to marine ecosystems,mariculture and human health,with the global climate change and the intensification of coastal environmental pollution.Diarrhetic shellfish poison?DSP?are secondary metabolites mostly produced by marine algae,which can be accumulated or transformed in bivalves,and could cause severe diarrhetic poisoning after human consumption of shellfish contaminated by DSP.To date,research reports on DSP at home and abroad mainly focused on the detection of DSP toxins in seafood,food safety risk assessment,the discovery and characterization of DSP toxins in toxin-producing algae cells,and the effect of environmental factors on the accumulation of DSP toxins in toxin-producing algae.However,there are few studies on the characterization of DSP toxins released into the extracellular environment of toxigenic algae and its degradation in the natural environment.In this study,an effective method for the profiling,complete characterization,and quantification of extracellular DSP compounds in the culture medium of toxigenic algae was developed and applied to the analysis of real sample.Besides,two typical DSP toxins,Okadaic Acid?OA?and Dinophysistoxin 1?DTX1?,were used as model compounds to investigate the degradation of OA and DTX1 under natural conditions and the effects of various environmental factors on photodegradation systematically.The details are as follows:1.In this study,a new method for profiling and characterization of extracellular DSP compounds in the culture medium of marine toxigenic algae was developed by high performance liquid chromatography?HPLC?-high resolution mass spectrometry?HR-MS?/tandem mass spectrometry?MS/MS?.The proposed method was also applied for the identification and quantitation of the extracellular DSP compounds in the culture medium of Prorocentrum lima.The results showed that solid phase extraction can effectively enrich and clean the DSP compounds in the culture medium of P.lima,and satisfactory recoveries?94.80-100.58%?and repeatability?relative standard deviation?RSD??9.27%?of the method were achieved.Commercial software associated with the accurate mass information of known DSP toxins and their derivatives were used to screen and identify DSP compounds.Nine extracellular DSP compounds were identified,of which seven toxins?including OA-D7b,OA-D9b,OA-D10a/b,etc?were found in the culture medium of P.lima for the first time.The results of quantitative analysis showed that the content of extracellular DSP compounds in the culture medium of P.lima were relatively high,and the types and contents of intracellular and extracellular toxins have obviously varied in different growth stages of P.lima.The concentrations of okadaic acid?OA?and dinophysistoxin 1?DTX1?in the culture medium of P.lima are in the range of19.9-34.0?g/L and 15.2-27.9?g/L,respectively.The total concentration of the DSP compounds is in the range of 57.70-79.63?g/L,indicating that the concentration of extracellular toxins should not be ignored.The results showed that the proposed method is an effective tool for profiling of the extracellular DSP compounds in the culture medium of marine toxigenic algae,which provides technical support for obtaining comprehensive information on the species and content of intracellular and extracellular toxins of toxin-producing algae and the study of the toxicity mechanism and environmental chemical behavior of extracellular toxins.2.The photodegradation kinetics and degradation products of two typical DSP toxins in seawater were preliminary studied with indoor and outdoor simulated degradation experiments.Firstly,the degradation of OA and DTX1 in different months and under different concentrations was investigated under the natural sunlight outdoor.No obvious loss of OA and DTX1 occurred in dark controls,indicating that this was primarily a photo-mediated process.The removal rate of high concentrations of OA and DTX1 in seawater was higher than that of the low-concentration group,and the removal rate was greater than 90%within 30 days.The significant fluctuation of the toxin content during natural photodegradation indicates that the photodegradation of OA and DTX1 is a complex process involving both reversible and irreversible reactions.Secondly,in the case of mercury lamp as the light source,the effects of various factors,including the pH of seawater,trace metals,dissolved oxygen and organic matter,on the photodegradation of OA and DTX1 have been investigated system atically through indoor simulation experiments.The first-order reaction kinetics equation of OA was observed under the irradiation of mercury lamp.The higher the humic acid content and the pH,the slower the photodegradation rate of OA and DTX1.Trace metal and dissolved oxygen content can promote the photodegradation of OA and DTX1.In addition,a preliminary identification of the conversion products of OA and DTX1 was performed using high performance liquid chromatography-high resolution mass spectrometry.Both OA and DTX1 produce three major degradation products under light conditions:C₂₅H₃₈O₉,C₄₄H₇₀O₁₄,C₄₃H₆₆O₁₁1 and C₂₅H₃₈O₉,C₄₅H₇₂O₁₄,C₄₄H₆₈O₁₁,respectively.Relevant studies on the identification of OA and DTX1 degradation products and toxicity analysis need to be further explored.In summary,a new method for the profiling of extracellular toxin metabolites of DSP-producing algal based on high performance liquid chromatography-high resolution mass spectrometry/tandem mass spectrometry was established in this paper.The proposed method realizes the screening and identification of extracellular DSP toxin in the culture medium of P.lima and elucidated the release characteristics of extracellular DSP toxins from P.lima in different growth stages initially.With OA and DTX1 as research subjects,the period of removal of OA and DTX1 in seawater under natural light conditions and the effects of different environmental factors on the photodegradation kinetics of OA and DTX1 were preliminary clarified,and the light-transformation products were preliminary identified.The results in this paper can provide technical support for the profiling of metabolites of extracellular DSP toxins produced by marine toxigenic algae,and scientific evidence for elucidating the fate of DSP toxins in natural environments and environmental risks,which have important implications for the objective evaluation of the ecological risk caused by DSP toxins released harmful algae.