Water and Marine Toxins

Microcystins detection The quality of water intended for human consumption is one of the main concerns globally. Health criteria are increasingly strict and include new analytical parameters for quality determination, such as the detection of microcystins. Microcystins are toxins generated by the uncontrolled growth of cyanobacteria present in water under specific conditions of temperature, light or with the presence of nutrients or metals; over 80 variants with toxic capacity have been classified. Several studies show the relationship between these toxins and different allergic and stomach pathologies in humans and animals, which are considered by the WHO as promoters of liver or colon cancer in the medium and long term. For these reasons, there is a new Royal Decree with stricter technical and health criteria for the supply and quality control of drinking water to increase public protection against any adverse effects. This transposes the Directive (EU) 2020/2184 on the quality of water intended for human consumption and replaces RD 140/2003; it includes a new series of analytical parameters to determine drinking water quality. METHODOLOGY At Zeulab, we have developed a microcystin detection kit based on the use of biological targets, specifically phosphatase inhibition, to determine the potential toxicity of the sample. It detects all variants regardless of their chemical structure and will reflect the potential toxicity associated with the water sample. It can be used to analyse both natural water and water in the process of being purified, and can identify both free and intracellular toxins. This methodology reproduces the mechanism of action of microcystins in humans and is based on the ability of phosphatase to hydrolyse a substrate, forming a product detectable at 405 nm. The enzymatic activity of phosphatase is inhibited by microcystins proportionally to their amount in the sample. In addition to determining the potential toxicity of the sample, Microcytest is notable for its simplicity of use. The test is easily implementable in laboratories and provides very quick results in less than 1 hour. Assay procedure: The quality of water intended for human consumption is one of the main concerns globally. Health criteria are increasingly strict and include new analytical parameters for quality determination, such as the detection of microcystins. Microcystins are toxins generated by the uncontrolled growth of cyanobacteria present in water under specific conditions of temperature, light or with the presence of nutrients or metals; over 80 variants with toxic capacity have been classified. Several studies show the relationship between these toxins and different allergic and stomach pathologies in humans and animals, which are considered by the WHO as promoters of liver or colon cancer in the medium and long term. For these reasons, there is a new Royal Decree with stricter technical and health criteria for the supply and quality control of drinking water to increase public protection against any adverse effects. This transposes the Directive (EU) 2020/2184 on the quality of water intended for human consumption and replaces RD 140/2003; it includes a new series of analytical parameters to determine drinking water quality. Leer más Enzimáticos Microcystest Leer más [button target="_self" hover_type="default" font_weight="400" text_align="center" text="Discover Zeulab's complete line for the detection of water/marine toxins." link="https://www.zeulab.iatic.es/en/marine-water-toxins/"][button target="_self" hover_type="default" font_weight="400" text_align="center" text="Leer este artículo en español" link="https://www.zeulab.iatic.es/centro-de-conocimiento/detectar-microcistinas-en-una-hora/"]

Algal blooms in freshwater lakes Cyanobacteria (blue-green algae) are unicellular prokaryotes that thrive mainly in freshwater lakes. Being autotroph organisms they accomplish oxygenic photosynthesis playing a significant role in the food chain of several ecosystems. However, under certain circumstances (eutrophication, climatic change, etc.), they can grow and lead to uncontrolled blooms. Cianobacteria bloom in Saint Claire lake (USA). Cyanobacteria blooms are observable even from space. http://earthobservatory.nasa.gov/NaturalHazards/view.php?id=86327&src=twitter-nh&linkId=16012048 Harmful effect of toxin release The risk of these uncontrolled blooms is related to the capacity that some particular genera have in producing pathogenic toxins. The most common and dangerous toxin is called microcystin and is produced by Microcystis, Anabaena, Planktothrix, Nostoc and Anabaenopsis. It is generally accepted that half of the blooms are toxic. Microcystins are secondary metabolites that are normally located inside cyanobacteria cells. However, when toxins are released, normally due to cellular lysis, the freshwater becomes contaminated and its consumption turns hazardous not only just for humans but for animals too. It is important to address that toxin analysis does not indicate the presence of the microorganism. This issue may become a problem in drinking water treatment plants as toxin may release after cellular lysis. Therefore, it is advisable to perform an intracellular microcystin analysis prior to drinking water treatment. During the last years the concern regarding cyanotoxin toxicity has not been limited to drinking and recreational water but to the intake of food that has been exposed to toxins (i.e. vegetables, fish and mollusks, which specially have a great bio-concentrating capacity). Until recent years, microcystins were linked exclusively to freshwater environments. Nevertheless, these toxins are very stable and persistent and may appear in marine water as well (due to downstream toxin transport). In fact, several studies have verified microcystin accumulation in mollusk bivalbes ready for human consumption. Microcystins Microcystins are mainly hepatotoxic and its long term exposition leads to a direct carcinogenic effect in the liver. They specifically inhibit serin/threonine phosphatases type 1 and 2A from mammals, which have tumor suppressor functions. Once the toxin is accumulated in the liver, they promote cytoskeleton disorganization, lipid peroxidation, loose of plasmatic membrane integrity, DNA fragmentation, apoptosis and finally necrosis. Microcystins are cyclic heptapeptides composed by natural and no natural amino acids. They are very stable and thermoresistent and more than 100 variants have been described. Four of them (LR, RR, LA and YR) are especially frequent. WHO recommends consumption of drinking water with microcystin LR concentration bellow 1 µg/L. Molecular structure of the main microcystins. Microcystins are cyclic heptapeptides formed by natural and no natural amino acids. Amino acids variations in X and Y positions lead to microcystin diversity (more than 100 known variants). Factors promoting toxin release Cyanobacteria grow is associated to high concentration of nutrients being fosforous the element that controls cell proliferation. Nitrogen availability is also important especially in those species that cannot fix atmospheric nitrogen. Temperature rise, luminosity rise or low water turbulence are factors that also trigger cyanobacteria grow. Potentially cyanotoxin producing blooms can thrive without toxin synthesis and release. One of the reasons arises from the factor that toxin and no toxin producing cyanobacteria normally coexist in a given bloom. The ratio between toxic and no toxic strains can vary significantly in space and time; factors involved are not completely understood at present. Besides, the presence of a toxic strain does not imply that the toxin is being synthesized. A number of studies have tried to find a correlation between toxin synthesis and physic-chemical parameters (lake depth, temperature, nutrients, etc.) and biological parameters (number of genes related with toxin biosynthesis, amount of chlorophyll, biomass, etc.). However, no one of them has been successful and direct analysis of the toxin is the only reliable method so far. Toxin detection Several methodologies have been described concerning microcystin analysis in water. The choice of a specific method is related to the use of the analytical results derived. The following table summarizes the main features among the current methodologies. METODO PROPIEDADES BIOASSAY Real toxicity measurement No specific No respect animal welfare PHOSPHATASE INHIBITION Fastest Specific and very sensitive Real toxicity measurement  (target: PP2A) HPLC Specific but less sensitive Need of standards for every molecule Does not measure real toxicity ELISA Fast and sensitive Irregular cross reactivity against other microcystins/ False positives Does not measure real toxicity The methods can be divided in screening and confirmatory methods. Mouse bioassay, ELISA and PP2A inhibition assays are among the first ones. Screening methods have to be sensitive enough to avoid false negatives, fast, simple and allow the treatment of a high number of samples. On the other hand, the confirmatory methods are the only methods that allow unequivocal identification of the molecule, being HPLC and LC-MS the representative ones. The disadvantage of these instrumental methods is that they require standards for every molecule that want to be identified. Sometimes, in the case of microcystins, this is not possible as more than 100 variants exist. The methods can also be classified as structural or functional methods. The first ones are based on its physic-chemical properties (HPLC, immunochemical methods) while the second ones use the biological target of the toxins (PP2A inhibition assay and mouse bioassay). It is clear that both strategies are perfectly complementary. It is advisable to use a screening method first, and if possible based on the toxigenic potential, followed by a confirmatory method. Sometimes, a positive sample could never be confirmed as there are no standard for all the discovered microcystins. An important factor to take into account in microcystin analysis is that toxins can be confined inside the cells and have to be released first for the correct analysis. There are several documented methods concerning lysis and toxin extraction. However, not every method has been tested in terms of recovery and may give rise to toxin underestimation. More importantly, de extraction method has to be compatible with the analytical technique. Microcystest MicroCystest is a simple and rapid enzymatic test for the detection of microcystins and nodularins in drinnking and recreational water. The toxicity of microcystins is associated with the inhibition of

  Laboratorios de España, Croacia, Holanda o Namibia se reunieron el pasado 14 y 15 de Junio en el Marine Institute (Ireland) en un taller acerca de “Análisis de Biotoxinas Marinas”. Expertos en la materia como Dr. Arjen Gerssen, del laboratorio de referencia holandés, o Dra. Begoña Ben-Gigirey, del EURLMB (Laboratorio de Referencia Europeo para Toxinas Marinas) hablaron sobre análisis de PSP y toxinas lipofílicas. Dra. Elena Domínguez de ZEULAB realizó una demostración del ensayo de inhibición de la fosfatasa OkaTest para la cuantificación de toxinas del grupo del ácido okadaico en moluscos bivalvos. Los participantes tuvieron la oportunidad de ver la sencillez de un ensayo que puede ser utilizado para el análisis oficial, complementado con LC-MS o HPLC, o análisis de producto final, de acuerdo la legislación vigente EC. No 25/2011. Otras sesiones prácticas de laboratorio sobre PSP y toxinas lipofílicas completaron la demostración de OkaTest es este taller. Presentaciones orales y demostraciones prácticas permitieron una participación activa de todos los asistentes. Los participantes compartieron experiencias y procedimientos de trabajo que les permitió obtener diferentes puntos de vista y poder implementar y mejorar sus metodologías actuales.