Toxic Alexandrium minutum and Blue Swimmer Crabs: Toxin Dynamics, Impacts and Implications for Public Health, Monitoring and Management Strategies

Cherono S Kwambai

Alexandrium spp. algal blooms and paralytic shellfish poisoning (PSP) events can lead to severe socioeconomic losses on coastal communities and the aquaculture industry. Therefore, establishing effective monitoring and management is critical to ensuring public safety and environmental health. This is particularly important given the rising demand for seafood and that 75% of recreational fishing for Blue Swimmer crab (Portunus armatus) in Western Australia occurs between summer and autumn, coinciding with the Alexandrium bloom season, a relatively recent phenomenon in the state. To enhance management responses to toxic algal blooms impacting Blue Swimmer crab fisheries, four objectives were investigated: (1) using poly-AlCl3 (PAC) modified kaolinite (KPAC) to remove Alexandrium minutum from the environment; (2) adapting an analytical method for toxin analysis (LC-MS/MS) and validating its use in crabs; (3) examining storage and preparation techniques (i.e., freezing and boiling) on paralytic shellfish toxin (PST) load and distribution in P. armatus crabs; and (4) using molecular tools to genetically characterise and assess the toxic potential of an Alexandrium spp. strain from Western Australia. In an effort to manage and mitigate potential risks and socioeconomic impacts associated with Alexandrium bloom events; this study investigated direct environmental management strategies for addressing these blooms. The research specifically examined treatment options for open waters to eliminate these blooms and reduce their toxicity, particularly through clay flocculation, which has shown considerable promise, especially when combined with additives like PAC, Iron salts. PAC was selected for its cost-effectiveness and minimal environmental impact. Thus, the study evaluated the effectiveness of kaolinite clay with and without PAC against Alexandrium blooms. The experiment was conducted in two phases at different cell densities (1.0 x107 and 2.0 x 107 cells L-1), using a factorial design that considered four factors: A. minutum strain (CS-1178; CS-324/16), pH (7 and 8), clay concentration (0.1, 0.25, and 0.3 g L-1), and PAC usage. Results showed that removing A. minutum was possible irrespective of pH, clay concentration, or cell density. KPAC significantly improved removal efficiency and reduced the time required, achieving 100% removal within two hours at very low KPAC concentrations (0.1 gL- 1). Zeta potential analysis revealed that adding PAC changed the surface charge from negative to positive, enhancing electrostatic interactions with negatively charged A. minutum cells and improving flocculation. Low KPAC application rates will minimise potential negative impacts on the benthic environment, warranting larger-scale in-situ trials for verification. Effective monitoring is crucial for managing Alexandrium blooms and their toxins. Toxin detection and molecular methods provide vital insights into these harmful algal species' abundance, presence, and toxicity, essential for developing targeted mitigation strategies. This need for comprehensive monitoring requires the development of tools to characterise blooms. One such tool is a toxin analysis method that detects, identifies, and quantifies PSTs produced by toxic Alexandrium in crabs. This method can aid in assessing potential public health risks associated with ingesting PST-contaminated crabs. Concurrently, molecular methods can also be developed to enhance chemical toxin analysis (PST), creating a more robust framework for monitoring harmful blooms ensuring food safety. A PST toxin analysis method was adopted based on a published protocol that utilised liquid chromatography-tandem mass spectrometry (LC-MS/MS) and served as the foundation for this study’s PST analysis method. Several modifications were made to optimise its effectiveness on a Shimadzu LC-MS-8045 High-Performance Liquid Chromatograph Triple Quadrupole Mass Spectrometer equipped with a Waters HILIC BEH Amide Acuity 1.7 μm column (2.1 x 50 mm). These modifications included extended column conditioning times, adjusted analysis gradients, changes in buffer concentration and recalculated loop times. These changes allowed for the analysis of up to 12 PST analogues in a single sample run. The method achieved limits of detection (LOD) ranging from 0.7 to 6.6 nmol L⁻1, and limits of quantification (LOQ) for all tested analogues were below <200 μg STX di-HCL eq/kg. The adopted method proved instrumental in accurately determining the presence of PSTs crab flesh and was further adapted for detection of PSTs in broth. In order to inform public health management on crab meat safety using the PST analysis method adopted, this research investigated how food storage and preparation techniques (i.e., freezing and boiling) affect the profile, location, and concentration of PSTs in P. armatus crabs. A key aim of this work was to clarify how cooking methods can influence crab meat safety and inform best practices for fishers and consumers. Crabs were fed PST-laden pellets containing approximately 1966 μg saxitoxin di-HCl equivalent per kg twice daily for eleven days. They were then harvested for analysis at the conclusion of the experiment, which employed a complete factorial design with two factors: freezing (fresh or frozen) and cooking (raw or boiled). Crabs were randomly assigned to treatment groups to assess whether PSTs could biotransform or contaminate their edible tissues. Results indicated that crabs accumulated PSTs only in the hepatopancreas when exposed to toxic levels of approximately 1897 μg saxitoxin di-HCl eq/kg. Freezing and thawing significantly reduced total PST concentration. Cooking did not affect toxin concentration, profile, or the interaction between freezing and cooking, nor did it vary by size or sex of the crabs. No PSTs were detected in the cooking broth. This work had significant implications for recreational fishers and public health, providing critical insights into risks associated with consuming crabs during Alexandrium bloom events. Moreover, detecting PSTs enhanced local analysis capabilities, facilitating rapid monitoring and timely warnings for fishers when toxic blooms occur, thereby safeguarding public health and the livelihoods reliant on fishing. The findings provided empirical data supporting the Western Australian Government's recommendation that crabs exposed to toxic A. minutum be eviscerated (head, gut, and gills removed) before cooking to mitigate PST exposure risk. These data are expected to contribute to future policies regulating recreational and commercial crab collection during Alexandrium bloom events. Additionally, freezing whole crabs before cooking can further reduce the risk of PST contamination in crab flesh, although the definitive mechanism behind this outcome requires further investigation. Finally, molecular methods such as qPCR and DNA sequencing can complement toxin analysis techniques to monitor harmful algal blooms (HABs). These tools facilitate early detection and identification of genetic markers associated with toxic HAB species, including those linked to paralytic shellfish toxin (PST) biosynthesis, such as the sxtA gene. This study utilised molecular methods to confirm the presence of a potentially non-toxic strain of A. minutum, which lacks the sxtA4 domain of the sxtA gene, indicating its non-toxicity. This study also reports the genetic identification of HAB species, such as A. minutum, Alexandrium spp., Karlodinium spp., Heterosigma akashiwo, Heterocapsa spp., Akashiwo sanguinea and Prorocentrum triestinum, within the Swan Canning Estuary. The identification of HABs, particularly novel species, enables rapid and accurate detection of toxic taxa, facilitating timely warnings and informed decision-making on mitigation measures such as precautionary closures and targeted bloom treatments. This can help establish guidelines to limit the potential spread of these species, reducing their impacts. This combination of studies now provides a sound scientific basis for the development of reliable Alexandrium bloom management plans by both the crab fishing industry and government departments tasked with ensuring public health in Western Australia. The newly developed tools for toxin analysis and genetic characterisation of toxic Alexandrium significantly enhanced the capacity of local authorities and industry to manage harmful algal blooms, enabling faster diagnostics and improved outcomes for both recreational and commercial sectors.

Toxic Alexandrium minutum and Blue Swimmer Crabs: Toxin Dynamics, Impacts and Implications for Public Health, Monitoring and Management Strategies

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