The increasing occurrence of saxitoxins in freshwaters is becoming a concern for water treatment facilities owing to its structural properties which make it resistant to oxidation at pH < 8. Hence, it is crucial to be able to monitor these toxins in surface and drinking water to protect public health. This review aims to outline the current state of knowledge related to the occurrence of saxitoxins in freshwaters and its removal strategies and provide a critical assessment of the detection methods to provide a basis for further development. Temperature and nutrient content are some of the factors that influence the production of saxitoxins in surface waters. A high dose of sodium hypochlorite with sufficient contact time or activated carbon has been shown to efficiently remove extracellular saxitoxins to meet the drinking water guidelines. While HILIC-MS has proven to be a powerful technology for more sensitive and reliable detection of saxitoxin and variants after solid phase extraction, ELISA is cost-effective and easy to use and is used by Ohio EPA for surveillance with a limit of detection of 0.015 μg/L. However, there is a need for the development of cost-effective and sensitive techniques that can quantify the variants of saxitoxin.

Saxitoxin (STX), an exceptionally potent marine toxin for which no antidote is currently available, is produced by methanogens and cyanobacteria. This poses a significant threat to both shellfish aquaculture and human health. Consequently, the development of a rapid, highly sensitive STX detection method is of great significance. The objective of this research is to create a novel approach for identifying STX. Therefore, amplified luminescent proximity homogeneous assay (AlphaLISA) was established using a direct competition method based on the principles of fluorescence resonance energy transfer and antigen-antibody specific binding. This method is sensitive, rapid, performed without washing, easy to operate, and can detect 8-128 ng/mL of STX in only 10 min. The limit of detection achieved by this method is as low as 4.29 ng/mL with coefficients of variation for the intra-batch and inter-batch analyses ranging from 2.61% to 3.63% and from 7.67% to 8.30%, respectively. In conclusion, our study successfully establishes a simple yet sensitive, rapid, and accurate AlphaLISA method for the detection of STX which holds great potential in advancing research on marine biotoxins.

Cyanobacterial biodiversity and potential toxicity in coastal lagoons have barely been studied despite these transitional water systems being very important in conservation and for the preservation of economic resources. Most of these transitional systems have been affected by eutrophication, and climate change will severely affect them by promoting cyanobacteria growth, especially in Mediterranean areas. This study aims to characterize the diversity of epipelic and epiphytic cyanobacteria species in a Mediterranean coastal lagoon and their potential for toxins production (microcystins and saxitoxins). Strains were isolated and genetically identified. Toxins were extracted and quantified by LC/MS-MS. All the taxa belong to the former Oscillatoriales. The presence of Nodosilinea and Toxifilum is reported for the first time for Spanish waters, but Pseudanabaena, Phormidium, Geitlerinema and Synechococcus also formed part of benthic mats. All the strains contained Microcystin-YR (MC-YR), but saxitoxin (STX) was present only in the extracts of Nodosilinea and Pseudanabena. MC-LY, MC-LW and [D-Asp3] MC-LR were detected in the extracts of Synechococcus and MC-LF in Toxifilum, but at concentrations that did not permit quantification. Toxins production by epipelic and epiphytic strains in coastal lagoons may represent a hazard, but also an opportunity to obtain potentially interesting compounds that should be further studied.

Investigating organic carriers\' utilization efficiency and bioactivity within organic-inorganic hybrid nanoflowers is critical to constructing sensitive immunosensors. Nevertheless, the sensitivity of immunosensors is interactively regulated by different classes of biomolecules such as antibodies and enzymes. In this work, we introduced a new alkaline phosphatase-antibody-CaHPO4 hybrid nanoflowers (AAHNFs) microreactor based colorimetric immunoprobe. This system integrates a biometric unit (antibody) with a signal amplification element (enzyme) through the biomineralization process. Specifically, the critical factors affecting antibody recognition activity in the formation mechanism of AAHNFs are investigated. The designed AAHNFs retain antibody recognition ability with enhanced protection for encapsulated proteins against high temperature, organic solvents, and long-term storage, facilitating the selective construction of lock structures against antigens. Additionally, a colorimetric immunosensor based on AAHNFs was developed. After ascorbic acid 2-phosphate hydrolysis by alkaline phosphatase (ALP), the generated ascorbic acid decomposes I2 to I-, inducing the localized surface plasmon resonance in the silver nanoplate, which is effectively tuned through shape conversion to develop the sensor. Further, a 3D-printed portable device is fabricated, integrated with a smartphone sensing platform, and applied to the data of collection and analysis. Notably, the immunosensor exhibits improved analytical performance with a 0.1-6.25 ng·mL-1 detection range and a 0.06 ng·mL-1 detection limit for quantitative saxitoxin (STX) analysis. The average recoveries of STX in real samples ranged from 85.9% to 105.9%. This study presents a more in-depth investigation of the recognition element performance, providing insights for improved antibody performance in practical applications.

Pufferfish is one of the most poisonous marine organisms, responsible for numerous poisoning incidents and some human fatalities due to its capability to accumulate potent neurotoxins such as tetrodotoxins (TTXs) and paralytic shellfish toxins (PSTs). In this study, tissue extracts (muscle, skin, liver, intestinal tract and gonads) obtained from sixteen pufferfish specimens of the Lagocephalus lagocephalus and Sphoeroides pachygaster species, collected along the Spanish Mediterranean coast, were analysed for the presence of voltage-gated sodium channel (also known as Nav channel) blockers using cell-based assay (CBA) and automated patch clamp (APC). No toxicity was observed in any of the S. pachygaster specimens, but toxicity was detected in the liver of most L. lagocephalus specimens. Instrumental analysis of these specimens, as well as in one Lagocephalus sceleratus specimen, by high-performance liquid chromatography coupled to fluorescence detection (HPLC-FLD) was performed, which confirmed the presence of PSTs only in L. lagocephalus specimens. This analysis reported the presence of saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) in all positive samples, being dcSTX the major analogue. These results demonstrate the ability of this species to accumulate PSTs, being the first report of the presence of PSTs in Mediterranean L.lagocephalus specimens. Furthermore, the presence of high PSTs contents in all five tested tissues of one L. lagocephalus specimen pointed the risk that the presence of this toxic fish in the Mediterranean Sea may represent for seafood safety and human health in case of accidental consumption.

Dinoflagellate species that form some of the most frequent toxic blooms are also bioluminescent, yet the two traits are rarely linked when studying bloom development and persistence. P. bahamense is a toxic, bioluminescent dinoflagellate that previously bloomed in Florida with no known record of saxitoxin (STX) production. Over the past 20 years, STX was identified in P. bahamense populations. The goal of this study was to examine toxin dynamics and associated molecular mechanisms in spatially and temporally distinct P. bahamense populations from the Indian River Lagoon, FL. SxtA4 is a key gene required for toxin biosynthesis. SxtA4 genotype analysis was performed on individual cells from multiple sites. Cell abundance, toxin quota cell-1, and sxtA4 and RubisCo (rbcL) transcript abundance were also measured. There was a significant negative correlation between cell abundance and toxin quota cell-1. While the sxtA4+ genotype was dominant at all sites, its frequency varied, but it occurred at 90-100% in many samples. The underlying mechanism for toxin decrease with increased cell abundance remains unknown. However, a strong, statistically significant negative correlation was found between stxA4 transcripts and the sxtA4/rbcL ratio, suggesting cells make fewer sxtA4 transcripts as a bloom progresses. However, the influence of sxtA4- cells must also be considered. Future plans include bioluminescence measurements, normalized to a per cell basis, at sites when toxicity is measured along with concomitant quantification of sxtA4 gene and transcript copy numbers as a means to elucidate whether changes in bloom toxicity are driven more at the genetic (emergence of sxtA4- cells) or transcriptional (repression of sxtA4 in sxtA4+ cells) level. Based on the results of this study, a model is proposed that links the combined traits of toxicity and bioluminescence in P. bahamense bloom development.

Saxitoxin (STX) is a cyanotoxin with high toxicity, and therefore, there is an urgent need to develop a facile detection method for STX. In this study, an ordered nanopillar array-based electrochemical aptasensor was fabricated for the high-performance detection of STX. The anti-STX aptamer with methylene blue (MB) incorporated at the 3\'-end (MB-Apt) was immobilized at the surface of an Au@PAN nanopillar array electrode and used as the recognition element. The proposed aptasensor demonstrated highly sensitive and selective STX detection because of synergistic catalysis effects of MB and ordered nanopillar arrays along with the selection of MB-Apt. The nanopillar array-based electrochemical aptasensor exhibited high sensitivity over a wide linear concentration range of 1 pM-3 nM with a linear regression equation of ΔI (μA) = 28.0 + 6.9 × log[STX] (R2 = 0.98079) and 3-100 nM with a linear regression equation of ΔI (μA) = 10.7 + 43.4 × log[STX] (R2 = 0.98772), where R is the correlation coefficient. In addition, the limit of detection (LOD) was as low as 1 pM. Furthermore, the designed aptasensor demonstrated excellent selectivity toward STX, preventing interference from neo-STX, okadaic acid, and common metal ions. The presented orderly nanopillar array-based strategy to develop an electrochemical aptasensor for STX detection offers a promising method for developing high-performance electrochemical sensors, and the presented aptasensor should find useful application in the detection of shellfish poison.

Contributing to the assessment of potential physiological changes in microalgae subjected to different concentrations and types of cyanotoxins, this study investigated the inhibitory effects of cyanotoxins on the growth, density, biomass, and ecotoxicity of Chlorella vulgaris. Chlorella vulgaris was exposed to crude extracts of cyanobacteria producing microcystin-LR (MC-LR), saxitoxin (SXT), anatoxin-a (ATX-A), and cylindrospermopsin (CYN) with initial concentrations of 5.0, 2.05, 0.61, and 1.42 μg.L-1, respectively. The experiments were conducted under controlled conditions, and monitoring of growth and cell inhibition occurred at 24h, 48h, 72h, and 96h. Chlorophyll-a content and ecotoxicity assessment were conducted with samples collected after 96h of exposure to cyanotoxins. The growth assays of Chlorella vulgaris, with results expressed in terms of average growth rates (doublings/day), indicated the following order for cyanotoxins: SXT (2.03) > CYN (1.66) > MC-LR (1.56) > ATX-A (0.18). This assay revealed the prominent inhibitory potential of ATX-A on Chlorella vulgaris growth compared to the other toxins evaluated. Regarding the inhibition of the photosynthetic process, expressed in terms of the percentage inhibition of Chlorophyll-a, the following order for cyanotoxins was obtained: ATX-A (82%) > MC-LR (76%) > STX (46%) > CYN (16%). These results also indicated that among the cyanotoxins, ATX-A was the most detrimental to the photosynthetic process. However, contrary to the observations in the growth study, SXT proved to be more harmful than CYN in terms of Chlorophyll-a inhibition. Finally, the results of the toxicity assay revealed that only ATX-A and MC-LR exerted a chronic influence on Chlorella vulgaris under the investigated conditions.

In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical printed (DEP) microarray composed of eight individually addressable carbon electrodes. The electrodeposition of gold nanoparticles on the carbon surface offers high conductivity and enlarges the electroactive area. The immobilization of thiolated aptamers on the AuNP-decorated carbon electrodes provides a stable, well-orientated and organized binary self-assembled monolayer for sensitive and accurate detection. A simple electrochemical multiplexed aptasensor based on AuNPs was designed to synchronously detect multiple cyanotoxins, namely, microcystin-LR (MC-LR), Cylindrospermopsin (CYL), anatoxin-α, saxitoxin and okadaic acid (OA). The choice of the five toxins was based on their widespread presence and toxicity to aquatic ecosystems and humans. Taking advantage of the conformational change of the aptamers upon target binding, cyanotoxin detection was achieved by monitoring the resulting electron transfer increase by square-wave voltammetry. Under the optimal conditions, the linear range of the proposed aptasensor was estimated to be from 0.018 nM to 200 nM for all the toxins, except for MC-LR where detection was possible within the range of 0.073 to 150 nM. Excellent sensitivity was achieved with the limits of detection of 0.0033, 0.0045, 0.0034, 0.0053 and 0.0048 nM for MC-LR, CYL, anatoxin-α, saxitoxin and OA, respectively. Selectivity studies were performed to show the absence of cross-reactivity between the five analytes. Finally, the application of the multiplexed aptasensor to tap water samples revealed very good agreement with the calibration curves obtained in buffer. This simple and accurate multiplexed platform could open the window for the simultaneous detection of multiple pollutants in different matrices.

The dinoflagellate Gymnodinium catenatum is considered the primary cause of recurrent paralytic shellfish toxins (PSTs) in shellfish on the Moroccan Mediterranean coasts. The impacts of key environmental factors on the growth, cell yield, cell size and PST content of G. catenatum were determined. Results indicated that increasing salinity from 32 to 39 and nitrate concentrations from 441 μM to 1764 μM did not significantly (ANOVA, P-value >0.63) modify the growth rate of the studied species. Gymnodinium catenatum exhibited the highest growth rate at 24 °C. Cells arrested their division at 15 °C and at ammonium concentration above 441 μM, suggesting that this nitrogen form is toxic for G. catenatum. Furthermore, G. catenatum was unable to assimilate urea as a nitrogen source. In G. catenatum cells, eight analogues of saxitoxin were detected, belonging to the N-sulfocarbamoyl (C1-4, B1 and B2) and decarbamoyl (dc-GTX2/3) toxins. C-toxins contributed 92 % to 98 % of the molar composition of the PSTs. During the exponential growth, C2 tended to dominate, while C3 prevailed during the stationary phase. Toxin content per cell (ranging from 5.5 pg STXeq.cell-1 to 22.4 pg STXeq.cell-1) increased during the stationary growth phase. Cell toxin content increased with the concentrations of nitrate, ranging from 12.1 pg STXeq.cell-1 at 441 μM to 22.4 pg STXeq.cell-1 at 1764 μM during the stationary growth phase. The toxin content of G. catenatum showed the highest values measured at the highest tested temperatures, especially during the stationary phase, where toxicity reached 17.8 pg STXeq.cell-1 and 16.4 pg STXeq.cell-1 at 24 °C and 29 °C, respectively. The results can help understand the fluctuations in the growth and PST content of G. catenatum in its habitat in response to changing environmental variables in the Mediterranean Sea when exposed to increases in warming pressure and eutrophication.