Comparative determination of paralytic shellfish toxins (PSTs) using five different toxin detection methods in shellfish species collected in the Aleutian Islands, Alaska

Paralytic shellfish poisoning (PSP), a human illness caused by the ingestion of shellfish contaminated with paralytic shellfish toxins (PSTs), has been reported in Alaska for decades. These poisoning incidents have resulted in losses to local economies due to shellfish harvest closures. Thus the development of an effective biotoxin monitoring program designed specifically for the remote regions of Alaska would provide protection for public health and allow for a viable shellfish industry. The present study provides data useful for the development of an effective toxin screening protocol by comparing PST levels quantified in shellfish by many of the currently available PST detection techniques. Seven bivalve species were collected along beaches of the Aleutian Islands from June 2006 to September 2007. The concentration of PSTs was quantified and compared using five different analytical methods: the mouse bioassay, high performance liquid chromatography (HPLC), receptor-binding assay, the commercially available Jellett Rapid PSP Test strips, and an enzyme linked immunosorbent assay technique. The Association of Official Analytical Chemists (AOAC)-approved HPLC method proved to be valuable for characterizing the suite of individual PSTs in each species for research purposes, but was not considered practical for rapid toxin screening in remote Alaskan regions due to its time-consuming nature and requirement of expensive equipment and considerable expertise. In the present study, Jellett test strips were shown to be an effective tool for rapid screening, however due to the high percentage of false positives, subsequent validation via AOAC-approved methods would be required to prevent unnecessary closures.     

Pre- versus post-column oxidation liquid chromatography fluorescence detection of paralytic shellfish toxins

Both pre- and post-column oxidation liquid chromatography methods with fluorescence detection are available for detecting paralytic shellfish toxins. Each method has been evaluated in multiple laboratories and validated as a potential alternative to the mouse bioassay. This communication compares the advantages and limitations of both methods. For a given laboratory, the selection of either method may be based primarily on practicality and less on any deficiencies in scientific merit.     

Accumulation and depuration of paralytic shellfish poisoning toxins by laboratory cultured purple clam Hiatula diphos Linnaeus.

Purple clams (Hiatula diphos Linnaeus) accumulate paralytic shellfish poisoning (PSP) toxins produced by a toxic strain of the dinoflagellate Alexandrium minutum Halim in a laboratory study. The maximal toxicity of PSP toxins attained 31.3m MU/g after 20 days exposure. The toxin profile of H. diphos was similar to that reported for A. minutum at the end of the exposure period; and GTX1 was dominant. GTX congeners were found in muscle on day 16 and day 20, these substances could be detected during the depuration period as well. GTX1 was detected in the siphon only on day 32. The results show that H. diphos accumulates PSP toxins according to the amount and toxin profile of ingested A. minutum.     

Determination of paralytic shellfish poisoning toxins in Norwegian shellfish by liquid chromatography with fluorescence and tandem mass spectrometry detection.

A novel extraction and clean-up method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish samples. Raw shellfish material was extracted with an acidic acetonitrile/water (80:20, v/v) solution, whilst being homogenised. During the homogenisation the sample extraction solution was cooled with ice water. Subsequently, the extract was frozen at -20 degrees C for at least 4h. During freezing, two layers were formed, only the lower predominantly aqueous layer was used for the determination. The final extract solution was cleaned-up using a combination of Oasis HLB and Carbograph activated carbon SPE columns. The developed extraction and clean-up methods combined with gradient elution liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS) has resulted in a method which can determine the analogs GTX 1-5, C1-2, DcGTX 2-3, DcSTX, Neo, STX in a single analysis with an overall detection limit of 313mug STXdiHCL-eq./kg shellfish meat. The use of the developed extraction method with post-column high performance liquid chromatography (HPLC) with fluorescence detection (FLD) method provided an overall limit of detection of 89mug STXdiHCL-eq./kg shellfish meat for the same toxins. Both post-column HPLC-FLD and LC-MS/MS was used to investigate the Norwegian PSP toxin profile. It was found that the PSP toxins could be detected in shellfish samples from the Norwegian coastline for 10 months of the year, from March till December. The toxin profile consisted mainly of the carbamate toxins, GTX 1-4, Neo and STX, in terms of both concentrations and contribution to the overall toxicity. In addition, several of the n-sulfo-carbamoyl toxins were either detected in the samples at relatively low concentrations or their presence in the samples were indicated but could not be confirmed by the post-column HPLC-FLD and LC-MS/MS analyses.     

Comparative analysis of pre- and post-column oxidation methods for detection of paralytic shellfish toxins

Paralytic shellfish poisoning (PSP) toxins are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. Shellfish contaminated with these toxins create significant public health threat and economic losses to the shellfish industry. For this reason, several methods of high performance liquid chromatography (HPLC) with fluorescence detection have been developed in order to gain better knowledge of toxins profiles in shellfish and dinoflagellates samples. These methods have been subjected to continuous modifications to improve and shorten the run time of analysis in the routine monitoring control. In this paper, different samples are analyzed by pre- and post- column HPLC methods to compare toxin profiles. All PSP toxins were individually identified and quantified within the post-column oxidation method. However, although the pre-column oxidation method is significantly more sensitive and detects lower toxin levels, it provides a total amount of toxins that co-elute together, as GTX2 and 3, GTX1 and 4 and dcGTX2 and dcGTX3. The results obtained by both HPLC methods showed similar toxin concentration (expressed in μg/mL) in mussel samples, however when dinoflagellates samples were analyzed the toxin profile and concentration were different. In summary, the post-column oxidation method is accurate to determine the amount of each individual PSP toxin and to know the real toxic profile of non-transformed samples. In addition, this method is easy and faster to screen a large number of samples. The pre-column HPLC method is useful when mussel samples are analyzed even though the time required to prepare the samples is longer.     

Solid core column technology applied to HPLC-FD of paralytic shellfish toxins

Pre-column oxidation liquid chromatography with fluorescence detection is a chemical method for analyzing paralytic shellfish toxins. In order to improve the sample throughput and efficiency of AOAC Method 2005.06, solid core particle column technology was evaluated. We demonstrate that supplanting the original fully porous particle column with a solid core particle column reduces sample analysis time from 15 to 5 min per sample and improves resolution.     

Interactions of paralytic shellfish toxins with xenobiotic-metabolizing and antioxidant enzymes in rodents.

Paralytic shellfish toxins (PSTs) are neurotoxins known to block voltage-gated sodium channels in intoxicated animals and humans. Their metabolism in mammalian systems and their effects on other receptors are not as well understood. In this study, we investigated the in vitro metabolism of two classes of PSTs, gonyautoxin 2/3 (GTX2/3) and C1/2 toxins (C1/2), using rat and mouse liver enzyme preparations. We also analyzed the effects of these toxins on several antioxidant and xenobiotic-metabolizing enzymes in mice. These toxins were selected for their prevalence in the coastal waters of Southern China. When the toxins were incubated with liver preparations containing Phase I and Phase II xenobiotic metabolizing enzymes and appropriate co-factors, no transformation of the toxins was detectable. When mice were given sub-lethal doses of GTX2/3, a loss of activity was observed in hepatic ethoxyresorufin-O-deethylase, penthoxyresorufin-O-deethylase, glutathione peroxidase and superoxide dismutase, but not in glutathione S-transferase, catalase and glutathione reductase. Exposure to the same mouse units of C1/2 caused only a slight reduction in the activity of penthoxyresorufin-O-deethylase and glutathione peroxidase. Our results indicated that these toxins may not be metabolized readily in mammals and that they may cause adverse effects other than sodium channel blocking.     

麻痹性贝类毒素在大鼠体内的分布与累积

目的检测低剂量摄入麻痹性贝类毒素(PSP)后,多种毒素在大鼠内脏器官中的分布和累积情况,为进一步完善贝类水产品中麻痹性贝毒的安全标准提供依据。方法按美国公职分析化学家协会(AOAC)推荐的麻痹性贝毒小鼠测定法从自然毒化的海湾扇贝和华贵栉孔扇贝中提取PSP粗毒素,分别给大鼠灌胃51.2,25.6和10.2小鼠单位.kg-1,每天1次,连续28d。同时设溶剂(未毒化贝组织的盐酸提取液)对照组和空白对照组。停毒后的d1,d3和d14心脏取血,处死大鼠,收集肝、肾、脾和脑等组织,采用高效液相色谱法检测各组织中PSP的种类和含量。结果在所检测的4个内脏组织中,3个剂量的肝和肾脏组织中均检测出PSP,并且在停毒d14时仍有毒素存留,在脑和脾脏组织未检测出PSP。肝和肾组织中累积的PSP种类不同,所检测的各类PSP中,膝沟藻毒素4最易在大鼠肝和肾脏中累积。结论低剂量经口染毒的PSP可分布于大鼠的肝和肾脏,且在肝和肾脏中累积。     

A rapid detection method for paralytic shellfish poisoning toxins by cell bioassay.

We report here a rapid detection method for paralytic shellfish poisoning (PSP) toxins using a cultured neuroblastoma cell line, modified from the bioassay system previously established by Manger et al. [Manger, R.L., Leja, L.S., Lee, S.Y., Hungerford, J.M., Kirkpatrick, M.A., Yasumoto, T., Wekell, M.M., 2003. Detection of paralytic shellfish poison by rapid cell bioassay: antagonism of voltage-gated sodium channel active toxins in vitro. J. AOAC Int. 86 (3), 540-543]. In the present study, we made two major modifications to the previous method. The first is the use of maitotoxin, a marine toxin of ciguatera fish poisoning, which enables the incubation period to be reduced to 6 h when applied to the microplate 15 min prior to the end of the incubation. The second is the use of WST-8, a dehydrogenase detecting water-soluble tetrazolium salt for determining the target cell viability, which permits the omission of a washing step and simplifies the counting process. In addition, we attempted to reduce the required materials as much as possible. Thus, our modified method should be useful for screening the PSP-toxins from shellfish.     

Differential accumulation of paralytic shellfish toxins from Alexandrium minutum in the pearl oyster, Pinctada imbricata

To investigate the potential for differential accumulation of paralytic shellfish toxins (PSTs) in various tissues of the akoya pearl oyster, Pinctada imbricata, two feeding trials were carried out using the PST-producing dinoflagellate, Alexandrium minutum. When fed with A. minutum at concentrations between 100 and 1300 cells ml⁻¹, the maximum clearance by P. imbricata was shown to occur at a density of 300 cells ml⁻¹. When fed twice daily at this rate for up 12 days, P. imbricata accumulated analogues of gonyautoxins (GTXs): GTXs 1,4 and 2,3. The levels of GTXs in the viscera increased progressively on days 4, 8 and 12 to peak at 17.9 ± 4.47 μg STX-equivalent 100 g⁻¹ biomass. Following 12 days of depuration, in the absence of A. minutum, GTX levels fell by approximately 65% to 6.0 ± 2.20 μg STX-equivalent 100 g⁻¹ biomass. No GTX was found in the oysters at the start of the trial or in untreated controls. The accumulation of GTX was found to be tissue specific. No GTX was detected in the muscle tissue of P. imbricata during the feeding trial.     

The effect of temperature on growth and production of paralytic shellfish poisoning toxins by the cyanobacterium Cylindrospermopsis raciborskii C10.

Cylindrospermopsis raciborskii is a cyanobacterium which produces either cylindrospermopsine or paralytic shellfish poisoning (PSP) toxins. We studied the effect of temperature on growth and production of PSP toxins by C. raciborskii C10, isolated from a freshwater reservoir in Brazil. We analyzed the extracellular and intracellular content of PSP toxins at two different temperatures: 19 and 25 degrees C. C. raciborskii C10 produces STX, GTX2, and GTX3 at both temperatures. dcSTX was also detected at 25 degrees C in the intracellular extracts obtained at the end of the stationary phase. The growth achieved at 25 degrees C and estimated by optical density at 700 nm was three times greater than at 19 degrees C. However, no significant differences were observed in the content of PSP toxins in either the cells or the extracellular media. The kinetics of accumulation of PSP toxins within the cells rather than in the media suggests an active PSP toxins-export process that is not related to cell lysis. The extracellular accumulation of PSP toxins at 19 degrees C suggested a biotransformation of STX to the epimers GTX2 and GTX3. The stability of the PSP toxins produced by C. raciborskii C10 was high enough for them to remain active in the media after 30 days (at 25 degrees C) or after 50 days (at 19 degrees C).     

Investigations into matrix components affecting the performance of the official bioassay reference method for quantitation of paralytic shellfish poisoning toxins in oysters

Significant differences previously observed in the determination of paralytic shellfish poisoning toxins (PSTs) in oysters using official method AOAC 2005.06 and 959.08 were investigated in detail with regard to possible matrix effects. Method AOAC 2005.06 gave results 2-3 times higher than the mouse bioassay method, 959.08, differences thought to be due to underestimation of PSTs by the mouse bioassay. In order to prove the cause of these large differences, work was conducted here to examine the presence and effects of matrix components on the performance of each of the two assays. A range of oyster, cockle and mussel samples were extracted using the AOAC 959.08 hydrochloric acid (HCl) extraction method and analysed for PSP by both MBA and LC-FLD. In addition, extracts were analysed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for metals as well as being subjected to a range of nutritional testing methods. Whilst there was no evidence for effect of nutritional components on either assay, ICP-MS analysis revealed a relationship between samples exhibiting the largest differences in relative method performance, specifically those with the largest LC-FLD/MBA toxicity ratio, and samples containing the highest concentrations of zinc and manganese. In order to prove the potential effect of the metals on either the LC-FLD and/or MBA assays, HCl extracts of a range of shellfish were subjected to a number of matrix modifications. Firstly, a number of PSP-positive oyster samples were processed to reduce the concentrations of metals within the extracts, without significantly reducing the concentrations of PSTs. Secondly, a range of mussel and cockle extracts, plus a standard solution of saxitoxin di-hydrochloride were spiked at variable concentrations of zinc. All treated and non-treated extracts, plus a number of controls were subjected to ICP-MS, LC-FLD and MBA testing. Results proved the absence of any effect of metals on the performance of the LC-FLD, whilst showing a large suppressive effect of the metals on the MBA. As such, the results show the performance of the official MBA is potentially unsafe for application to the routine monitoring of PSP toxicity in oysters or in any other shellfish found to contain high concentrations of metal ions.     

Paralytic shellfish poisoning toxins in green mussels (Perna viridis) from the Gulf of Paria, Trinidad.

Paralytic shellfish poisoning (PSP) toxins were determined in green mussels (Perna viridis) collected from one collection site in the Gulf of Paria in Trinidad in 1999 and 2000. Aqueous extracts of PSP were purified by passage through C-18 SPE cartridges, oxidized with peroxide and periodate, respectively, then analyzed by HPLC with fluorescence detection. This procedure provided rapid and highly sensitive screening of samples for PSP toxins. Further purification of PSP-containing extracts using COOH SPE cartridges resulted in the separation and identification of individual PSP toxins. The method of analysis was validated by spike and recovery experiments, with 85-103% recoveries of mixed toxins. PSP toxins determined in our samples in both years were GTX2,3, dcGTX2,3, STX, and dcSTX, while GTX1,4 and NeoSTX were only identified in 1999 and 2000, respectively. In 1999, GTX1,4, GTX2,3 and dcGTX2,3 predominated, as compared to NeoSTX, GTX2,3 and dcGTX2,3 in 2000. However, mussel samples in 2000 contained higher total concentrations of detected PSP toxins than those of 1999. These results represent the first identification of specific PSP toxins in local shellfish and provide a basis for effective monitoring and control of these toxins in Trinidad.     

Occurrence of paralytic shellfish toxins in Cambodian Mekong pufferfish Tetraodon turgidus: selective toxin accumulation in the skin.

The toxicity of two species of wild Cambodian freshwater pufferfish of the genus Tetraodon, T. turgidus and Tetraodon sp., was investigated. Tetraodon sp. was non-toxic. The toxicity of T. turgidus was localized mainly in the skin and ovary. Paralytic shellfish toxins (PSTs), comprising saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX), account for approximately 85% of the total toxicity. Artificially reared specimens of the same species were non-toxic. When PST (dcSTX, 50 MU/individual) was administered intramuscularly into cultured specimens, toxins were transferred via the blood from the muscle into other body tissues, especially the skin. The majority (92.8%) of the toxin remaining in the body accumulated in the skin within 48h. When the same dosage of tetrodotoxin (TTX) was similarly administered, all specimens died within 3-4h, suggesting that this species is not resistant to TTX. Toxin analysis in the dead specimens revealed that more than half of the administered TTX remained in the muscle and a small amount was transferred into the skin. The presence of both toxic and non-toxic wild specimens in the same species indicates that PSTs of T. turgidus are derived from an exogenous origin, and are selectively transferred via the blood into the skin, where the toxins accumulate.     

Transformation of paralytic shellfish poisoning toxins in Crassostrea gigas and Pecten maximus reference materials

Matrix reference materials are an important requirement for the assessment of method performance characteristics and for routine quality control. In the field of marine toxin testing where biological assays have been used and where modern analytical testing methods are now becoming available, this requirement has become an urgent one. Various approaches are utilised for preparation of such materials in the absence of available naturally occurring toxic shellfish samples. Toxin-free shellfish may be artificially fortified through the addition of cultured toxic phytoplankton or shellfish may be incurred through natural feeding on toxic algae in a laboratory environment. Both of these approaches may be potentially affected by issues relating to the degradation or transformation of toxin analytes, so studies were conducted to assess these effects within our laboratory. A range of PSP-toxic shellfish tissues were prepared using the two approaches, in both Pacific oyster (Crassostrea gigas) and king scallops (Pecten maximus). Additionally, sub-samples of incurred Pacific oyster tissue were further treated, through addition of artificial chemical stabilisers and gamma irradiation. Two separate month-long stability trials were conducted at +4 °C on each material. Results highlighted clear evidence for improved stability of materials following shellfish feeding experiments in comparison with the tissues which had been spiked with plankton. In addition, there were clear differences in stability of toxins between the two shellfish species studied. There was evidence for good stability of C1&2 toxins in both the incurred tissues and improved stability of some toxins in tissues which had been subjected to either gamma irradiation or treatment with chemical additives. The results therefore highlighted the benefits of conducting shellfish feeding if suitable stable reference materials are to be prepared containing a full range of PSP toxin analytes. The study also highlighted the benefits of post-production treatment to prolong the stability of the materials. Work is ongoing to assess the full characteristics of candidate reference materials prepared with these approaches with the aim of producing a homogenous and stable PSP reference material in Pacific oysters.     

Accumulation and depuration rates of paralytic shellfish poisoning toxins in the shore crab Telmessus acutidens by feeding toxic mussels under laboratory controlled conditions.

Accumulation and depuration rates of paralytic shellfish poisoning toxins (PSP) in the crab Telmessus acutidens were investigated by feeding toxic and non-toxic mussels under laboratory controlled conditions. The crab accumulated toxins in the hepatopancreas in proportion to the amount of toxic mussels they ingested, and the toxicity in the crab hepatopancreas became 3.2 fold of that in the prey mussels after 20 days of feeding. During depuration, a fast reduction of the total toxicity was observed in the crab, and the retention rate of the toxicity after 5 days depuration with feeding of non-toxic mussels was 45.8+/-18.7%. The reduction of the toxicity was moderated in the later period of depuration, and the retention rates of the total toxicity after 10 and 20 days were 54.1+/-29.8% and 14.5+/-9.0%, respectively. The toxin profiles in the crab and mussel were investigated by high performance liquid chromatography, and reductive conversions of the toxins were observed when the toxins were transferred from the mussel to the crab. Consequently, high concentrations of GTX2 and GTX3, and STX that were not detected in the prey mussels, were found in the crab.     

Adopting alternatives for the regulatory monitoring of shellfish for paralytic shellfish poisoning in Canada: Interface between federal regulators, science and ethics

To ensure the safety of Canada’s shellfish, the Canadian Shellfish Sanitation Program (CSSP) relies on the mouse bioassay to detect toxins known to cause paralytic shellfish poisoning (PSP). This assay uses a large number of mice and requires death as an endpoint. Canadian research has led to the development of a pre-column High Performance Liquid Chromatography (HPLC) method that is more sensitive and more reliable than the mouse bioassay. However, it is not being used by Canadian regulators despite its acceptance by the AOAC and adoption by the United Kingdom. An ethnography study of stakeholders in the CSSP was conducted to determine the opportunities and obstacles to adopting analytical testing methods.The results of the study indicate that the major obstacles are a lack of certified reference materials (CRMs) and the direction of resources towards the development of new instrument-based methods rather than towards the refinement of the existing pre-column HPLC method for regulatory use. To move away from the mouse bioassay, Canada should invest in: basic research to develop a complete set of CRMs for PSP toxins; method refinement to increase sample throughput; and exercises to gain international acceptance of the pre-column HPLC method.     

Assessment of fitness for purpose of an insect bioassay using the desert locust (Schistocerca gregaria L.) for the detection of paralytic shellfish toxins in shellfish flesh.

We have developed a bioassay using 5th instar desert locusts (Schistocerca gregaria L.) for the detection of saxitoxin-the paralytic shellfish poison in shellfish flesh. The bioassay procedure is to inject 10 locusts with a shellfish extract, and assess their conditions at time points up to 2h post injection, looking for an endpoint of paralysis. From the proportion responding, the equivalent dose of pure saxitoxin could be estimated. Performance characteristics of the bioassay were assessed using shellfish samples spiked with saxitoxin, and we found the bioassay could detect and quantify toxin levels in the range of regulatory relevance. Relative toxicities of selected saxitoxin analogues differed from those reported in mammalian systems. Variation for repeatability conditions was acceptable but variation was higher under reproducibility conditions. This was related to (a) batches of insects from different suppliers, (b) different operators, and (c) different observers assessing the endpoint. We also noted adverse reactions with some shellfish species. These problems may be resolved by further refinement of the method and operator training, before formal validation. Nevertheless, we suggest the method potentially offers a simple, ethically acceptable, broad-specificity functional bioassay, which is desirable in any toxin-monitoring programme.     

Paralytic shellfish poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords.

In July 5, 2002 fishermen working in harvesting sea urchin (Loxechinus albus) in the Patagonia Chilean fjords were intoxicated by consumption of filter-feeder bivalve Aulacomya ater. After the ingestion of 7-9 ribbed mussel, two fishermen died 3-4 h after shellfish consumption. The forensic examination in both victims did not show pathological abnormalities with the exception of the lungs conditions, crackling to the touch, pulmonary congestion and edema. The toxic mussel sample showed a toxicity measured by mouse bioassay of 8575 microg of STX (saxitoxin) equivalent by 100 g of shellfish meat. Using post-column derivatization HPLC method with fluorescent on line detection was possible to measure mass amount of each paralytic shellfish poisoning (PSP) toxin yielding individual toxin concentrations. These PSP toxins were identified in the gastric content, body fluids (urine, bile and cerebrospinal fluid) and tissue samples (liver, kidney, lung, stomach, spleen, heart, brain, adrenal glands, pancreas and thyroids glands). The toxin profiles of each body fluid and tissue samples and the amount of each PSP toxin detected are reported. The PSP toxins found in the gastric content, were STX and the gonyautoxins (GTX4, GTX1, GTX5, GTX3 and GTX2) which showed to be the major amount of PSP toxins found in the victims biological samples. The PSP toxin composition in urine and bile showed as major PSP toxins neoSaxitoxin (neoSTX) and GTX4/GTX1 epimers, both STX analogues with an hydroxyl group (-OH) in the N(1) of the tetrahydropurine nucleus. The neoSTX was not present in the gastric content sample, indicating that the oxidation of N(1) in the STX tetrahydropurine nucleus resulted neoSTX, in a similar way that GTX3/GTX2 epimers were transformed in GTX4/GTX1 epimers. Beside this metabolic transformation, also the hydrolysis of carbamoyl group from STX to form its decarbomoyl analogue decarbamoylsaxitoxin was detected in liver, kidney and lung. These two findings show that PSP toxins went under metabolic transformation during the 3-4 h of human intoxication period, in which PSP toxins showed enzymatic oxidation of N(1) in the tetrahydropurine nucleus, producing neoSTX and GTX4/GTX1 epimers starting from STX and GTX3/GTX2 epimers, respectively. This study conclude, that PSP toxins are metabolically transformed by humans and that they are removed from the body by excretion in the urine and feces like any other xenobiotic compound.