Carbamoylase-based impedimetric electronic tongue for rapid detection of paralytic shellfish toxins.

Phytotoxins produced by marine microalgae, such as paralytic shellfish toxins (PSTs), can accumulate in bivalve molluscs, representing a human health concern due to the life-threatening symptoms they cause. To avoid the commercialization of contaminated bivalves, monitoring programs were established in the EU. The purpose of this work is the implementation of a PST transforming enzyme-carbamoylase-in an impedimetric test for rapid simultaneous detection of several carbamate and N-sulfocarbamoyl PSTs. Carbamoylase hydrolyses carbamate and sulfocarbamoyl toxins, which may account for up to 90% of bivalve toxicity related to PSTs. Conformational changes of carbamoylase accompanying enzymatic reactions were probed by Fourier transform mid-infrared spectroscopy (FT-MIR) and electrochemical impedance spectroscopy (EIS). Furthermore, a combination of EIS with a metal electrode and a carbamoylase-based assay was employed to harness changes in the enzyme conformation and adsorption on the electrode surface during the enzymatic reaction as an analytical signal. After optimization of the working conditions, the developed impedimetric e-tongue could quantify N-sulfocarbamoyl toxins with a detection limit of 0.1 µM. The developed e-tongue allows the detection of these toxins at concentration levels observed in bivalves with PST toxicity close to the regulatory limit. The quantification of a sum of N-sulfocarbamoyl PSTs in naturally contaminated mussel extracts using the developed impedimetric e-tongue has been demonstrated.

A review on aquatic toxins - Do we really know it all regarding the environmental risk posed by phytoplankton neurotoxins?

Aquatic toxins are potent natural toxins produced by certain cyanobacteria and marine algae species during harmful cyanobacterial and algal blooms (CyanoHABs and HABs, respectively). These harmful bloom events and the toxins produced during these events are a human and environmental health concern worldwide, with occurrence, frequency and severity of CyanoHABs and HABs being predicted to keep increasing due to ongoing climate change scenarios. These contexts, as well as human health consequences of some toxins produced during bloom events have been thoroughly reviewed before. Conversely, the wider picture that includes the non-human biota in the assessment of noxious effects of toxins is much less covered in the literature and barely covered by review works. Despite direct human exposure to aquatic toxins and related deleterious effects being responsible for the majority of the public attention to the blooms' problematic, it constitutes a very limited fraction of the real environmental risk posed by these toxins. The disruption of ecological and trophic interactions caused by these toxins in the aquatic biota building on deleterious effects they may induce in different species is paramount as a modulator of the overall magnitude of the environmental risk potentially involved, thus necessarily constraining the quality and efficiency of the management strategies that should be placed. In this way, this review aims at updating and consolidating current knowledge regarding the adverse effects of aquatic toxins, attempting to going beyond their main toxicity pathways in human and related models' health, i.e., also focusing on ecologically relevant model organisms. For conciseness and considering the severity in terms of documented human health risks as a reference, we restricted the detailed revision work to neurotoxic cyanotoxins and marine toxins. This comprehensive revision of the systemic effects of aquatic neurotoxins provides a broad overview of the exposure and the hazard that these compounds pose to human and environmental health. Regulatory approaches they are given worldwide, as well as (eco)toxicity data available were hence thoroughly reviewed. Critical research gaps were identified particularly regarding (i) the toxic effects other than those typical of the recognized disease/disorder each toxin causes following acute exposure in humans and also in other biota; and (ii) alternative detection tools capable of being early-warning signals for aquatic toxins occurrence and therefore provide better human and environmental safety insurance. Future directions on aquatic toxins research are discussed in face of the existent knowledge, with particular emphasis on the much-needed development and implementation of effective alternative (eco)toxicological biomarkers for these toxins. The wide-spanning approach followed herein will hopefully stimulate future research more broadly addressing the environmental hazardous potential of aquatic toxins.

Alzheimer's Disease and Toxins Produced by Marine Dinoflagellates: An Issue to Explore.

This paper examined the toxins naturally produced by marine dinoflagellates and their effects on increases in ß-amyloid plaques along with tau protein hyperphosphorylation, both major drivers of Alzheimer's disease (AD). This approach is in line with the demand for certain natural compounds, namely those produced by marine invertebrates that have the potential to be used in the treatment of AD. Current advances in AD treatment are discussed as well as the main factors that potentially affect the puzzling global AD pattern. This study focused on yessotoxins (YTXs), gymnodimine (GYM), spirolides (SPXs), and gambierol, all toxins that have been shown to reduce ß-amyloid plaques and tau hyperphosphorylation, thus preventing the neuronal or synaptic dysfunction that ultimately causes the cell death associated with AD (or other neurodegenerative diseases). Another group of toxins described, okadaic acid (OA) and its derivatives, inhibit protein phosphatase activity, which facilitates the presence of phosphorylated tau proteins. A few studies have used OA to trigger AD in zebrafish, providing an opportunity to test in vivo the effectiveness of new drugs in treating or attenuating AD. Constraints on the production of marine toxins for use in these tests have been considered. Different lines of research are anticipated regarding the action of the two groups of toxins.

Major characteristics of microplastics in mussels from the Portuguese coast.

The present study reports the quantity, shape, colour and chemical properties of microplastics (MP) and MP-like in whole soft tissues of the mussel Mytilus spp. collected in January and February 2019 from four natural banks in the Portuguese coast. Three sites are located in estuarine areas influenced by anthropogenic pressures and freshwater discharges, and one in the coast far from urbanised areas. An alkaline digestion (KOH) of biological tissues was used and a polymeric identification of 20% of the visually sorted particles was achieved using the Fourier-transform mid-infrared spectroscopy (FT-MIR). MP and MP-like concentrations ranged from 0.54 to 3.0 items g-1 without significant differences among the sites. Particle size varied from 36 to 4439 µm, being fibers the most abundant shape (50%) followed by films (22%) and spherules (18%). FT-MIR revealed that 69% of the analysed particles were plastic, being identified six polymers and two polymeric blends, and 32% were cellulose-based materials. Fibers identified in mussel tissues were mainly composed of cotton and viscose (77%). This study emphasizes the importance of the polymer's spectroscopic identification after microscopic observation to recognise MP.

Biochemical performance of mussels, cockles and razor shells contaminated by paralytic shellfish toxins.

Marine toxins in bivalves pose an important risk to human health, and regulatory authorities throughout the world impose maximum toxicity values. In general, bivalve toxicities due to paralytic shellfish toxins (PSTs) above the regulatory limit occur during short periods, but in some cases, it may be extended from weeks to months. The present study examines whether cockles (Cerastoderme edule), mussels (Mytilus galloprovincialis) and razor shells (Solen marginatus) naturally exposed to a bloom of Gymnodinium catenatum activated or suppressed biochemical responses as result of the presence of PSTs in their soft tissues. Toxins (C1+2, C3+4, GTX5, GTX6, dcSTX, dcGTX2+3 and dcNEO) and a set of biomarkers (ETS, electron transport system activity; GLY, glycogen; PROT, protein; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferases; LPO, lipid peroxidation; reduced (GSH) and oxidized (GSSG) glutathione contents and AChE, acetylcholinesterase activity) were determined in the three bivalve species. Specimens were harvested weekly in Aveiro lagoon, Portugal, along thirteen weeks. This period included three weeks in which bivalve toxicity exceeded largely the regulatory limit and the subsequence recovery period of ten weeks. Biochemical performance of the surveyed species clearly indicated that PSTs induce oxidative stress and neurotoxicity, with higher impact on mussels and razor shells than in cockles. The antioxidant enzymes CAT and GPx seemed to be the biomarkers better associated with toxin effects.

A Carbamoylase-Based Bioassay for the Detection of Paralytic Shellfish Poisoning Toxins.

Out of control proliferation of toxic phytoplankton, called harmful algal blooms (HABs), have a significant economic impact on bivalve aquaculture and harvesting in coastal waters. Some phytotoxins, such as paralytic shellfish toxins (PSTs), are of concern due to the life-threatening symptoms they can cause. Development of rapid and low-cost screening tools would be a welcome addition to the laboratory methodologies employed in routine monitoring programs. However, most of the assays and biosensors for the screening of PSTs, are restricted to a single target, saxitoxin (STX), which is the most potent PST. The present study aimed at developing an assay for the detection of N-sulfocarbamoyl PST-GTX5, which is one of the most abundant toxins in bivalves during G. catenatum blooms as found on the Portuguese coast. Enzymatic assay employing PSTs' transforming enzyme-carbamoylase-was proposed. Carbamoylase was extracted and purified from the surf clam S. solida. Carbamoylase displayed similar specificity to both carbamate (STX) and N-sulfocarbamate toxins (GTX5 and C1+2) converting them into decarbamoyl saxitoxin (dcSTX) and decarbamoyl gonyautoxins 2+3 (dcGTX2+3), respectively. The enzymatic assay involved hydrolysis of GTX5 by carbamoylase and quantification of the product of enzymatic reaction, dcSTX, using a potentiometric chemical sensor. A potentiometric sensor with plasticized PVC membrane that displayed sensitivity to dcSTX and selectivity in the presence of GTX5 was employed. Enzymatic assay allowed determination of GTX5 in the concentration range from 0.43 to 3.30 µmolL-1, which encompasses levels of GTX5 in contaminated bivalve extracts with toxicities above PSTs regulatory limits. The feasibility of the carbamoylase-based potentiometric assay for detection of GTX5 was demonstrated.

Tracking the Spatial Fate of PCDD/F Emissions from a Cement Plant by Using Lichens as Environmental Biomonitors.

In an area with multiple sources of air pollution, it is difficult to evaluate the spatial impact of a minor source. Here, we describe the use of lichens to track minor sources of air pollution. The method was tested by transplanting lichens from a background area to the vicinity of a cement manufacturing plant that uses alternative fuel and is located in a Natural Park in an area surrounded by other important sources of pollution. After 7 months of exposure, the lichens were collected and analyzed for 17 PCDD/F congeners. The PCDD/F profiles of the exposed lichens were dominated by TCDF (50%) and OCDD (38%), which matched the profile of the emissions from the cement plant. The similarity in the profiles was greatest for lichens located northeast of the plant (i.e., in the direction of the prevailing winds during the study period), allowing us to evaluate the spatial impact of this source. The best match was found for sites located on the tops of mountains whose slopes faced the cement plant. Some of the sites with highest influence of the cement plant were the ones with the highest concentrations, whereas others were not. Thus, our newly developed lichen-based method provides a tool for tracking the spatial fate of industrially emitted PCDD/Fs regardless of their concentrations. The results showed that the method can be used to validate deposition models for PCDD/F industrial emissions in sites with several sources and characterized by complex orography.

Partitioning of paralytic shellfish toxins in sub-cellular fractions of the digestive gland of the cockle Cerastoderma edule: Changes under post-bloom natural conditions.

Concentrations of paralytic shellfish toxins (C1+2, B1, dcGTX2+3, dcSTX, GTX2+3 and STX) were determined by LC-FLD in composite samples of digestive glands of the cockle Cerastoderma edule and in each sub-cellular particulate fractions obtained after differential centrifugation (nuclei+debris, mitochondria, lysosomes and microsomes). The specimens were sampled during the exposure to a bloom of Gymnodinium catenatum (day 0) and in the subsequent 8, 12, 14, 19, 21 and 25 days under natural depuration conditions. Toxin profiles of digestive glands were dominated by C1+2 followed by B1 and dcGTX2+3, although the proportion between C1+2 and B1 contents decreased with the time, indicating a slower elimination of B1. All toxins, except GTX2+3 and STX, were quantified in the four sub-cellular fractions. The content of the quantified toxins decreased most markedly in nuclei+debris and microsomal fractions, during the first eight and 12 days, respectively. Conversely, different patterns were observed among toxins in mitochondrial and lysosomal fractions. The less accentuated decreases of dcGTX2+3 and dcSTX contents in the mitochondrial fraction may have resulted from the conversion of other toxins, like C1+2 and B1, associated with enzymatic activities in that fraction. The largest discrepancy was registered in lysosomal fraction for B1, since its content increased after eight days of post-bloom conditions. Input of B1 may come from the conversion of other toxins, like the abundant B2 and C1+2. These transformations are associated to the major role of lysosomes in the intra-cellular digestive process of materials acquired through vesicular transport.

Can exposure to Gymnodinium catenatum toxic blooms influence the impacts induced by Neodymium in Mytilus galloprovincialis mussels? What doesn't kill can make them stronger?

The presence in marine shellfish of toxins and pollutants like rare earth elements (REEs) poses a major threat to human well-being, coastal ecosystems, and marine life. Among the REEs, neodymium (Nd) stands out as a widely utilized element and is projected to be among the top five critical elements by 2025. Gymnodinum catenatum is a phytoplankton species commonly associated with the contamination of bivalves with paralytic shellfish toxins. This study evaluated the biological effects of Nd on the mussel species Mytilus galloprovincialis when exposed to G. catenatum cells for fourteen days, followed by a recovery period in uncontaminated seawater for another fourteen days. After co-exposure, mussels showed similar toxin accumulation in the Nd and G. catenatum treatment in comparison with the G. catenatum treatment alone. Increased metabolism and enzymatic defenses were observed in organisms exposed to G. catenatum cells, while Nd inhibited enzyme activity and caused cellular damage. Overall, this study revealed that the combined presence of G. catenatum cells and Nd, produced positive synergistic effects on M. galloprovincialis biochemical responses compared to G. catenatum alone, indicating that organisms' performance may be significantly modulated by the presence of multiple co-occurring stressors, such those related to chemical pollution and harmful algal blooms. ENVIRONMENTAL IMPLICATIONS: Neodymium (Nd) is widely used in green technologies like wind turbines, and this element's potential threats to aquatic environments are almost unknown, especially when co-occurring with other environmental factors such as blooms of toxic algae. This study revealed the cellular impacts induced by Nd in the bioindicator species Mytilus galloprovincialis but further demonstrated that the combination of both stressors can generate a positive defense response in mussels. The present findings also demonstrated that the impacts caused by Nd lasted even after a recovery period while a previous exposure to the toxins generated a faster biochemical improvement by the mussels.

A short-term exposure to saxitoxin triggers a multitude of deleterious effects in Daphnia magna at levels deemed safe for human health.

Harmful algal blooms and the toxins produced during these events are a human and environmental health concern worldwide. Saxitoxin and its derivatives are potent natural aquatic neurotoxins produced by certain freshwater cyanobacteria and marine algae species during these bloom events. Saxitoxins effects on human health are well studied, however its effects on aquatic biota are still largely unexplored. This work aims at evaluating the effects of a pulse acute exposure (24 h) of the model cladoceran Daphnia magna to 30 µg saxitoxin L-1, which corresponds to the safety guideline established by the World Health Organization (WHO) for these toxins in recreational freshwaters. Saxitoxin effects were assessed through a comprehensive array of biochemical (antioxidant enzymes activity and lipid peroxidation), genotoxicity (alkaline comet assay), neurotoxicity (total cholinesterases activity), behavioral (swimming patterns), physiological (feeding rate and heart rate), and epigenetic (total 5-mC DNA methylation) biomarkers. Exposure resulted in decreased feeding rate, heart rate, total cholinesterases activity and catalase activity. Contrarily, other antioxidant enzymes, namely glutathione-S-transferases and selenium-dependent Glutathione peroxidase had their activity increased, together with lipid peroxidation levels. The enhancement of the antioxidant enzymes was not sufficient to prevent oxidative damage, as underpinned by lipid peroxidation enhancement. Accordingly, average DNA damage level was significantly increased in STX-exposed daphnids. Total DNA 5-mC level was significantly decreased in exposed organisms. Results showed that even a short-term exposure to saxitoxin causes significant effects on critical molecular and cellular pathways and modulates swimming patterns in D. magna individuals. This study highlights sub-lethal effects caused by saxitoxin in D. magna, suggesting that these toxins may represent a marked challenge to their thriving even at a concentration deemed safe for humans by the WHO.

Snapshot of resistome, virulome and mobilome in aquaculture.

Aquaculture environments can be hotspots for resistance genes through the surrounding environment. Our objective was to study the resistome, virulome and mobilome of Gram-negative bacteria isolated in seabream and bivalve molluscs, using a WGS approach. Sixty-six Gram-negative strains (Aeromonadaceae, Enterobacteriaceae, Hafniaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Vibrionaceae, and Yersiniaceae families) were selected for genomic characterization. The species and MLST were determined, and antibiotic/disinfectants/heavy metals resistance genes, virulence determinants, MGE, and pathogenicity to humans were investigated. Our study revealed new sequence-types (e.g. Aeromonas spp. ST879, ST880, ST881, ST882, ST883, ST887, ST888; Shewanella spp. ST40, ST57, ST58, ST60, ST61, ST62; Vibrio spp. ST206, ST205). >140 different genes were identified in the resistome of seabream and bivalve molluscs, encompassing genes associated with ß-lactams, tetracyclines, aminoglycosides, quinolones, sulfonamides, trimethoprim, phenicols, macrolides and fosfomycin resistance. Disinfectant resistance genes qacE-type, sitABCD-type and formA-type were found. Heavy metals resistance genes mdt, acr and sil stood out as the most frequent. Most resistance genes were associated with antibiotics/disinfectants/heavy metals commonly used in aquaculture settings. We also identified 25 different genes related with increased virulence, namely associated with adherence, colonization, toxins production, red blood cell lysis, iron metabolism, escape from the immune system of the host. Furthermore, 74.2 % of the strains analysed were considered pathogenic to humans. We investigated the genetic environment of several antibiotic resistance genes, including blaTEM-1B, blaFOX-18, aph(3″)-Ib, dfrA-type, aadA1, catA1-type, tet(A)/(E), qnrB19 and sul1/2. Our analysis also focused on identifying MGE in proximity to these genes (e.g. IntI1, plasmids and TnAs), which could potentially facilitate the spread of resistance among bacteria across different environments. This study provides a comprehensive examination of the diversity of resistance genes that can be transferred to both humans and the environment, with the recognition that aquaculture and the broader environment play crucial roles as intermediaries within this complex transmission network.

How Biological Activity in Sea Cucumbers Changes as a Function of Species and Tissue.

Biological activity and bioactive compound content in sea cucumbers was assessed, considering Parastichopus regalis, Holothuria mammata, Holothuria forskali, and Holothuria arguinensis as species and intestine, muscle band, respiratory tree, body wall, and gonads as tissues. P. regalis had the lowest content in phenolic compounds and antioxidant activity in contrast to Holothuria species. In the respiratory tree, the highest phenolic concentration was recorded in H. arguinensis, 76.4 ± 1.2 mg GAE/100 g dw vs. 21.0-49.0 mg GAE/100 g dw in the other species. H. arguinensis had the highest DPPH and FRAP results in the gonads, 13.6 ± 0.7 mg AAE/100 g dw vs. 2.6-3.5 mg AAE/100 g dw and 27.1 ± 0.3 µmol Fe2+/g dw vs. 8.0-15.9 µmol Fe2+/g dw, respectively. Overall, P. regalis biomass presented the highest anti-inflammatory activity levels and H. arguinensis the lowest anti-inflammatory levels. The respiratory tree was the most anti-inflammatory (measured by the inhibition of cyclooxygenase-2, COX-2) tissue in H. mammata and H. forskali (also the muscle band in this case), 76.3 ± 6.3% and 59.5 ± 3.6% COX-2 inhibition in 1 mg/mL aqueous extracts, respectively. The results demonstrated a variable bioactive potential and advantage in targeting antioxidant properties in the muscle band and anti-inflammatory activity in the respiratory tree, which may constitute a starting point for a biorefinery approach envisaging multiple applications.

Assessing the Bacterial Community Composition of Bivalve Mollusks Collected in Aquaculture Farms and Respective Susceptibility to Antibiotics.

Aquaculture is a growing sector, providing several products for human consumption, and it is therefore important to guarantee its quality and safety. This study aimed to contribute to the knowledge of bacterial composition of Crassostrea gigas, Mytilus spp. and Ruditapes decussatus, and the antibiotic resistances/resistance genes present in aquaculture environments. Two hundred and twenty-two bacterial strains were recovered from all bivalve mollusks samples belonging to the Aeromonadaceae, Bacillaceae, Comamonadaceae, Enterobacteriaceae, Enterococcaceae, Micrococcaceae, Moraxellaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Staphylococcaceae, Streptococcaceae, Vibrionaceae, and Yersiniaceae families. Decreased susceptibility to oxytetracycline prevails in all bivalve species, aquaculture farms and seasons. Decreased susceptibilities to amoxicillin, amoxicillin/clavulanic acid, cefotaxime, cefoxitin, ceftazidime, chloramphenicol, florfenicol, colistin, ciprofloxacin, flumequine, nalidixic acid and trimethoprim/sulfamethoxazole were also found. This study detected six qnrA genes among Shewanella algae, ten qnrB genes among Citrobacter spp. and Escherichia coli, three oqxAB genes from Raoultella ornithinolytica and blaTEM-1 in eight E. coli strains harboring a qnrB19 gene. Our results suggest that the bacteria and antibiotic resistances/resistance genes present in bivalve mollusks depend on several factors, such as host species and respective life stage, bacterial family, farm's location and season, and that is important to study each aquaculture farm individually to implement the most suitable measures to prevent outbreaks.

Selective incorporation of rare earth elements by seaweeds from Cape Mondego, western Portuguese coast.

This study examined the mechanism of incorporation of the rare earth elements (REEs), La, Ce, Nd, Eu, Gd, Tb, Yb, into green (Codium tomentosum, Ulva rigida), red (Gracilaria gracilis, Osmundea pinnatifida, Porphyra sp), and brown seaweeds (Saccorhiza polyschides, Undaria pinnatifida) collected from a single site near the coastline of the Cape Mondego, western Portugal. The concentrations of REEs, Mg, Ca, Al, Fe, Zn, and Cu in the biomasses were determined by inductively-coupled plasma mass spectrometry (ICP-MS). The species showed differences in their incorporation and fractionation of REEs from the same environment: the sum of REEs was higher in U. rigida, C. tomentosum, G. gracilis, and O. pinnatifida (0.7-1.7 µg g-1) than in Porphyra sp., S. polyschides, and U. pinnatifida (0.1-0.2 µg g-1). Ratios of Ce/Yb ranged from 13 (in S. polyschides) to 103 (in U. rigida), indicating different proportions of light and heavy REEs among species. Good correlations were found between Al and Fe (R2 = 0.98), and between these elements and La, Ce, Nd, Gd (R2 = 0.88-0.97) and Yb (R2 = 0.66-0.71) for all species except C. tomentosum and G. gracilis. Profiles of REE values normalised to average upper-continental crust composition indicated positive anomalies of Eu and Tb that reinforced the singularity of these elements in the REE group. Correlations between the REEs and Al or Fe suggest that detrital terrigenous particles, adhered to seaweed walls, may be an important mechanism for the incorporation of REEs by seaweeds. Different patterns for C. tomentosum and G. gracilis may also be indicative of the higher influence of cell wall composition on REE incorporation.

Depuration kinetics of paralytic shellfish toxins in Mytilus galloprovincialis exposed to Gymnodinium catenatum: laboratory and field experiments.

The kinetics of paralytic shellfish toxins in Mytilus galloprovincialis, previously exposed to Gymnodinium catenatum, was studied under depuration laboratory conditions and over a declining bloom of the dinoflagellate in the field. The variation of the levels observed throughout the laboratory experiment was characterized by a fast depuration of B1, C1 + 2, dcSTX and dcGTX2 + 3, possibly due to the gut evacuation of unassimilated toxins or microalgae cells, or loss during digestive mechanisms. Subsequent enhancements were observed for all compounds with emphasis to dcSTX and dcGTX2 + 3, pointing to biotransformation of the assimilated toxins. Then levels decreased gradually. A first-order depuration kinetic model fitted well to the decrease of B1, C1 + 2 and dcGTX2 + 3 concentrations, but not for dcSTX. Mussels exposed to a declining bloom of Gymnodinium catenatum exhibited a loss of toxins following the same pattern. Despite the low abundance of this dinoflagellate, a similar kinetic model was applied to the field data. The depuration rate of dcGTX2 + 3 in the field experiment (0.153 ± 0.03 day(-1)) significantly exceeded the value calculated in the laboratory (0.053 ± 0.01 day(-1)), while smaller differences were obtained for B1 (0.071 ± 0.02 and 0.048 ± 0.01 day(-1)) and similar values for C1 + 2 (0.082 ± 0.03 and 0.080 ± 0.03 day(-1)). The slower depuration rate of dcGTX2 + 3 in the heavily contaminated mussels at the laboratory may be related to a more effective contribution of C1 + 2 biotransformation.

Isolation and Characterization of Cylindrospermopsis raciborskii Strains from Finished Drinking Water.

In the summer of 2015, an intense cyanobacterial bloom producing geosmin/2-methylisoborneol (MIB) occurred in the Roxo freshwater reservoir in Alentejo, Portugal. The drinking water supplied from the Roxo water treatment plant (WTP) exhibited an unpleasant odor/taste and a significant cyanobacteria density was detected in the finished water at the exit of the WTP. Cyanobacteria were not evaluated downstream of the WTP, namely, at the city reservoir. The aim of this work was to isolate and characterize viable cyanobacteria present in finished water (exit of the WTP and city reservoir) that withstand conventional water treatment. Treated water samples collected at both sites were inoculated in Z8 culture medium to provide the conditions for putative cyanobacterial growth. After 30 days, filamentous cyanobacteria were observed in cultures inoculated with samples from the exit point of the WTP. Viable trichomes were isolated and identified as Cylindrospermopsis raciborskii by morphometric and molecular analysis. None of the isolates were cylindrospermopsin/microcystin producers, as confirmed by ELISA and amplification of corresponding genes (PS/PKS and mcyA-cd/mcyAB/mcyB). ELISA results were positive for saxitoxin, but saxitoxin and derivatives were not detected by liquid chromatography with fluorescence detection (LC-FLD), nor were their related genes (sxtA/sxtA4/sxtB/sxtM/sxtPer/sxtI). To our knowledge, this is the first report on the establishment of cultures of C. raciborskii that resisted water treatment processes.

Paralytic shellfish toxin profiles in mussel, cockle and razor shell under post-bloom natural conditions: Evidence of higher biotransformation in razor shells and cockles.

Concentrations of the paralytic shellfish toxins GTX6, C1+2, GTX5, C3+4, dcSTX, dcNEO and dcGTX2+3 were determined by LC-FLD in composite samples of whole soft tissues of mussels (Mytilus galloprovincialis), cockles (Cerastoderma edule) and razor shells (Solen marginatus) after exposure to a Gymnodinium catenatum bloom. Specimens were harvested weekly during three months under natural depuration conditions in the Mira branch of Aveiro lagoon, Portugal. Under the decline of G. catenatum cell densities, elimination or transformation of the uptake toxins associated with the ingestion of toxic cells differed among the surveyed species. Ratio between the toxins dcSTX plus dcGTX2+3 plus dcNEO and toxins GTX6 plus GTX5 plus C1+2 plus C3+4 was used to illustrate the biotransformation occurring in the bivalves. Enhancement of the ratios was observed for razor shells and cockles seven weeks after the peak of the algal bloom. Most likely it reflects more intense biotransformation in razor shells and cockles than in mussels. Conversion into toxins of higher toxicity may prolong the bivalve toxicity. These results show the complexity of toxin elimination in bivalves under post-bloom conditions and emphasize the pertinence of monitoring programs of bivalve toxicity in order to protect human health.

Paralytic Shellfish Toxins (PST)-Transforming Enzymes: A Review.

Paralytic shellfish toxins (PSTs) are a group of toxins that cause paralytic shellfish poisoning through blockage of voltage-gated sodium channels. PSTs are produced by prokaryotic freshwater cyanobacteria and eukaryotic marine dinoflagellates. Proliferation of toxic algae species can lead to harmful algal blooms, during which seafood accumulate high levels of PSTs, posing a health threat to consumers. The existence of PST-transforming enzymes was first remarked due to the divergence of PST profiles and concentrations between contaminated bivalves and toxigenic organisms. Later, several enzymes involved in PST transformation, synthesis and elimination have been identified. The knowledge of PST-transforming enzymes is necessary for understanding the processes of toxin accumulation and depuration in mollusk bivalves. Furthermore, PST-transforming enzymes facilitate the obtainment of pure analogues of toxins as in natural sources they are present in a mixture. Pure compounds are of interest for the development of drug candidates and as analytical reference materials. PST-transforming enzymes can also be employed for the development of analytical tools for toxin detection. This review summarizes the PST-transforming enzymes identified so far in living organisms from bacteria to humans, with special emphasis on bivalves, cyanobacteria and dinoflagellates, and discusses enzymes' biological functions and potential practical applications.

Sorption of okadaic acid lipophilic toxin onto plastics in seawater.

The present study tested under laboratorial conditions the sorption of okadaic acid (OA), a lipophilic toxin produced by marine phytoplankton, onto 6-mm plastic circular fragments. Fragments (4 g L-1) of polyethylene terephthalate (PET), polypropylene (PP), expanded polystyrene (EPS) and non-expanded polystyrene (PS) were exposed for 96 h to seawater spiked with 10 ng mL-1 of OA. Results of the experiments showed a broad percentage of OA removed from the water by plastics after 48 h of exposure: 30 ± 5.1% (PET), 37 ± 9.5% (PP), 62 ± 7.1% (EPS) and 83 ± 1.9% (PS). Sorption appears to be highly influenced by polymer's characteristics, such as polarity and degree of crystallinity. Further studies are needed to clarify the effect of the contact area on sorption by expanded plastics. These results point to the plausible interaction of OA and plastics in coastal waters.

Seasonal and multi-annual trends of bivalve toxicity by PSTs in Portuguese marine waters.

Temporal and spatial trends of paralytic shellfish toxins (PSTs) in bivalves from Portuguese estuarine and coastal waters, and connectivity of bivalve toxicity among the harvest areas, were examined using long-term data from the national biotoxin monitoring programme. Data from 1994 to 2017 were chosen for commercial bivalve species sensitive to PSTs, and for production areas exhibiting recurrent episodes of bivalve toxicity. Mussels (Mytilus spp.) and cockles (Cerastoderma edule) from the Ria de Aveiro, Mondego estuary, Óbidos lagoon and Ria Formosa, and wedge clams (Donax trunculus) and surf clams (Spisula solida) from the coastal areas Aguda and Olhão were selected. Bivalve toxicity data point to higher incidents of PST episodes in autumn and winter, although in 2008 the toxicity of mussels and cockles in the three estuarine areas was registered in summer. Most likely, favourable oceanographic conditions triggered the bloom formation of Gymnodinium catenatum, which is the species responsible for paralytic shellfish poisoning in Portuguese waters. Episodes in the southern coast of Portugal were less recurrent, although values above the PST regulatory limit displayed also a seasonal signal with a peak between autumn and early winter. On the basis of the number of weeks per month that bivalves showed elevated toxicity values, a connectivity index was defined for the surveyed areas. High connectivity was obtained among Aveiro, Mondego and Óbidos, which are 180 km apart, suggesting that G. catenatum cells are imported from blooms formed or reaching the coastal waters adjacent to these systems. During episodes of elevated toxicity, toxin profiles in contaminated mussels and cockles were dominated by N-sulfocarbamoyl compounds, which are the major toxins produced by the toxic dinoflagellate G. catenatum. The identification of coupled systems relatively to bivalve toxicity has an impact on monitoring programmes and allows improved decision-making on closures of bivalve harvest areas affected by toxic algae.