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.

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.

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.

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.

Combined effect of temperature and nutritional regime on the elimination of the lipophilic toxin okadaic acid in the naturally contaminated wedge shell Donax trunculus.

The influence of nutritional regime and water temperature on depuration rates of OA-group toxins in the wedge shell Donax trunculus was examined by exposing naturally contaminated specimens to three nutritional regimes (microalgae, commercial paste of microalgae, and starvation) for 14 days at 16 °C and 20 °C. Total OA was quantified in the whole soft tissues of the individuals collected in days 2, 4, 6, 8, 10, 12 and 14. Mortality, dry weight, condition index, gross biochemical composition and gametogenic stages were surveyed. Low variation of glycogen and carbohydrates during the experiments suggest that wedge shells were under non-dramatic stress conditions. Wedge shells fed with non-toxic diets showed similar depuration rates being 15 and 38% higher than in starvation, at 16 and 20 °C, respectively. Depuration rates under non-toxic diets at 20 °C were 71% higher than at 16 °C. These results highlight the influence of water temperature on the depuration rate of total OA accumulated by D. trunculus, even when the increase is of only 4 °C, as commonly observed in week time scales in the southern Portuguese coastal waters. These results open the possibility of a faster release of OA in harvested wedge shells translocated to depuration systems when under a slight increase of water temperature.

Changes of paralytic shellfish toxins in gills and digestive glands of the cockle Cerastoderma edule under post-bloom natural conditions.

Concentrations of the paralytic shellfish toxins C1+2, C3+4, GTX5, GTX6, dcGTX2+3, dcSTX, dcNEO, GTX2+3, GTX1+4, STX and NEO were determined by LC-FLD in composite samples of digestive glands and gills of Cerastoderma edule cockle. The specimens were sampled in Aveiro lagoon, Portugal, under natural depuration conditions (days 0, 8, 12, 14, 19, 21 and 25) after exposure to a bloom of Gymnodinium catenatum. Individual paralytic shellfish toxins indicated different pathways of elimination and biotransformation in digestive gland and gills. Toxin concentrations in gills were lower than in digestive gland. Most of the quantified toxins in digestive gland decreased during the 25 days of observation according to negative exponential curves, and only GTX5, GTX6 and NEO showed slight irregularities with time. Concentrations of C1+2, C3+4 and dcGTX2+3 in gills decreased progressively, however GTX5, GTX6 and dcSTX showed pronounced increases. Higher concentrations of those toxins in days 8 and 12 in comparison to the initial value (day 0) indicate conversion of other toxins into GTX5, GTX6 and dcSTX during those periods. It appears that inter-conversion of toxins occurs as G. catenatum cells are retained in gills before being transferred to other compartments.