连云港海州湾麻痹性贝类毒素中毒分析

为分析连云港地区麻痹性贝类毒素中毒特征,对连云港海州湾织纹螺进行形态鉴定,收集近年麻痹性贝类中毒资料,应用小白鼠生物测定法检测螺肉中麻痹性贝类毒素。结果连云港海州湾存有4种织纹螺,其中以半褶织纹螺含麻痹性贝类毒素量高(1.6×103μg100g螺肉),并导致连云港地区10起、银川1起麻痹性贝类中毒;1992年还发生一起由泥螺引起的麻痹性贝类中毒。中毒者均表现为下行性神经麻痹症状,重者致死。鉴于海洋污染的严重性,为保障海洋贝类的食用安全,应对贝类进行毒素等安全指标的监测。     

Development and validation of an accurate and sensitive LC-ESI-MS/MS method for the simultaneous determination of paralytic shellfish poisoning toxins in shellfish and tunicate

An analytical method using liquid chromatography-tandem mass spectrometry has been developed for the determination of 5 paralytic shellfish poisoning (PSP) toxins (saxitoxin, gonyautoxin1-4) in shellfish and tunicate. The tested parameters included extraction solvent, clean-up method, mobile phase and mass spectrometric detection parameters including multiple reaction monitoring (MRM) conditions. The PSP toxins were extracted with 0.1 N hydrochloric acid and cleaned up using C18 SPE cartridges. Chromatographic separation of the 5 PSP toxins was carried out on a TSK-gel Amide-80 column with gradient elution. The developed method was validated according to the requirements of SANCO 12571/2013. The average recoveries of the PSP toxins ranged from 82.7% to 110.7%, the intra-day precisions were in the range of 0.3–10.9%, the inter-day precisions were in the range of 0.8–10.0% and the limits of quantification (LOQs) were in the range of 25–100 μg/kg, depending on the analytes and matrices. The validated method was successfully applied for the determination of 5 PSP toxins in real shellfish and tunicate samples, including mussel, oyster, ark shell, sea squirt and styela clava.     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. I: Interlaboratory studies of methods of analysis

This paper describes the first part of a project undertaken to develop mussel reference materials for Paralytic Shellfish Poisoning (PSP) toxins. Two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX) in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the second part of the project: the certification exercise. In the first study, 18 laboratories were asked to measure STX and dc-STX in rehydrated lyophilized mussel material and to identify as many other PSP toxins as possible with a method of their choice. In the second interlaboratory study, 15 laboratories were additionally asked to determine quantitatively STX and dc-STX in rehydrated lyophilized mussel and in a saxitoxin-enriched mussel material. The first study revealed that three out of four postcolumn derivatization methods and one pre-column derivatization method sufficed in principle to determine STX and dc-STX. Most participants (13 of 18) obtained acceptable calibration curves and recoveries. Saxitoxin was hardly detected in the rehydrated lyophilized mussels and results obtained for dc-STX yielded a CV of 58% at a mass fraction of 1.86 mg/kg. Most participants (14 out of 18) identified gonyautoxin-5 (GTX-5) in a hydrolysed extract provided. The first study led to provisional criteria for linearity, recovery and separation. The second study revealed that 6 out of 15 laboratories were able to meet these criteria. Results obtained for dc-STX yielded a CV of 19% at a mass fraction of 3.49mg/kg. Results obtained for STX in the saxitoxin-enriched material yielded a CV of 19% at a mass fraction of 0.34mg/kg. Saxitoxin could not be detected in the PSP-positive material. Hydrolysis was useful to confirm the identity of GTX5 and provided indicative information about C1 and C2 toxins in the PSP-positive material. The methods used in the second interlaboratory study showed sufficiently consistent analysis results to undertake a certification exercise to assign certified values for STX and dc-STX in lyophilized mussel.     

Measurement of paralytic shellfish toxins in molluscan extracts: comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay

This study compared five methods of measuring paralytic shellfish toxins (PSTs) including the long-used mouse lethality bioassay, a commercially available cell culture test (MIST ® Quantification kit), HPLC analysis, and two newly developed radioreceptor assays utilizing mammalian sodium channels and saxiphilin. Methods were challenged with toxic shellfish extracts prepared according to the AOAC official method. The best correlations between predicted toxicity values being 0.9 or better, were those between HPLC analysis when compared with both radioreceptor assays and the mouse lethality bioassay, as well as that between the saxiphilin and the sodium channel radioreceptor assays. In all cases, statistically significant correlations existed between the toxicity measurements of the same extracts. The ratios between some methods were not unitary as measured by the slopes of the regression lines used for correlation analyses. HPLC analysis predicted more toxicity than all of the bioassays. The saxiphilin assay underestimated toxicity relative to the mouse bioassay, the MIST ® kit determinations and the sodium channel assay. The sodium channel assay predicted there to be less toxicity than the mouse bioassay and the MIST ® kit. Of all of the techniques used, the MIST ® kit correlation with the mouse bioassay was nearest to one. Each method possesses different virtues and it may be that a multi-method approach would harness the benefits of each method for various aspects of a shellfish testing regime.     

Determination of rate constants and half-lives for the simultaneous biodegradation of several cyanobacterial metabolites in Australian source waters

The fate of five cyanobacterial metabolites was assessed in water sourced from Lake Burragorang (Warragamba Dam) in New South Wales, Australia. All of the studied metabolites were shown to be biodegradable in this water source. For some metabolites, biodegradation was influenced by factors, including temperature, location (within the water body) and seasonal variations. The biodegradation of the metabolites was shown to follow pseudo-first-order kinetics with rate constants ranging from 8.0 × 10⁻⁴ to 1.3 × 10⁻² h⁻¹. Half-lives of the metabolites were also estimated and ranged from 2.2 to 36.1 d. The order of ease of biodegradability in this water source followed the trend: microcystin-LR ≥ cylindrospermopsin > saxitoxins > geosmin ≥ 2-methylisoborneol. The lack of detection of the mlrA gene during microcystin biodegradation suggests that these toxins may be degraded via a different pathway. While no metabolite-degrading organisms were isolated in this study, the inoculation of previously isolated geosmin- and microcystin-degrading bacteria into Lake Burragorang water resulted in efficient biodegradation of the respective metabolites. For example, microcystin-degrading isolate TT25 was able to degrade three microcystin variants to concentrations below analytical detection within 24 h, suggesting that inoculation of such bacteria has the potential to enhance biodegradation in Lake Burragorang.     

First report of Paralytic Shellfish Poisoning (PSP) in mussels (Mytilus galloprovincialis) from eastern Adriatic Sea (Croatia)

The chromatographic HPLC-FLD method was introduced for the first time to identify and quantitatively determine individual Paralytic Shellfish Poisoning toxins accumulated in aquacultured shellfish from Croatian coastal waters. Populations of Mediterranean mussels (Mytilus galloprovincialis) were contaminated with PSP toxins throughout January to April 2009 leading to the positive test results by Mouse Bioassay (MBA). Until 2009 there was no evidence of PSP toxins in the examined samples. For the first time an instrumental method revealed the PSP toxin profile of samples taken along the eastern Adriatic coast and identified saxitoxin (STX) as the main representative of this toxin group that may cause paralysis and death in consumers of contaminated shellfish. This phenomenon may have serious health and economic consequences. Following these potential consequences, marine biotoxins (PSP, ASP and DSP) are continuously assessed in bivalves from 25 breeding and harvesting areas along the Croatian Adriatic coast. Positive MBA results were confirmed by instrumental method in two out of three recorded samples. Saxitoxin was the dominant PSP toxin extracted from contaminated mussels within the range of 53.17-1298.17 μg g⁻¹, that contributed more than 70% to the total shellfish toxicity, followed by gonyautoxins 2 and 3 (GTX 2,3) which contributed 27% and decarbamoylsaxitoxin (dcSTX) that accounted for less than 2%, considering all stations.