Abstract
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.
Highlights
Most coastal countries are affected by out of control proliferation of microalgae—Harmful algal blooms (HABs) [1,2]
Toxic episodes caused by the paralytic toxins are less frequent in Portuguese coastal waters compared to the other types of toxins [6], these compounds are of particular concern due to the severe neurological symptoms they impart to humans
Carbamoylases can use as substrates any carbamate and N-sulfocarbamoyl toxins, though with varying specificity, which is dependent on the origin of the enzyme
Summary
Most coastal countries are affected by out of control proliferation of microalgae—Harmful algal blooms (HABs) [1,2]. Due to the unpredictability of the occurrence of HABs, routine surveillance programs of toxins in commercial bivalves and toxic phytoplankton species in seawater near bivalve harvesting areas were established in EU countries, including Portugal. Three groups of toxins, classified according the symptoms they cause in humans, are included in the monitoring program: diarrheic shellfish toxins (DSTs), paralytic shellfish toxins (PSTs) and amnesic shellfish toxins (ASTs), besides some other lipophilic toxins [4]. Toxic episodes caused by the paralytic toxins are less frequent in Portuguese coastal waters compared to the other types of toxins [6], these compounds are of particular concern due to the severe neurological symptoms they impart to humans. Paralytic shellfish poisoning can be fatal, with overall mortality ranging between 2–14% [5,7]
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