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Development of a Non-Commercial Microcystin ELISA and Its Impact on Public Health Policy

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Development of a Non-Commercial Microcystin ELISA and Its Impact on Public Health Policy ITREOH Building of Regional Capacity to Monitor Recreational Water: Development of a Non-commercial Microcystin ELISA and Its Impact on Public Health Policy BEATRIZ M. BRENA, PHD, LAURA DÍAZ, DANIEL SIENRA, GRACIELA FERRARI, NATALIA FERRAZ, ULF HELLMAN, PHD, GUALBERTO GONZALEZ-SAPIENZA, PHD, JEROLD A. LAST, PHD In 2001, a University of California, Davis–University of Cyanobacteria species produce hepatotoxins, neuro- the Republic, Montevideo, partnership created a Foga- toxins, cytotoxins, or dermatotoxins, which can pose a rty ITREOH program to exploit the potential of ELISA public health risk to humans and animals that drink to provide a low-cost environmental analysis attractive this water or use it for recreational purposes. The most to economically distressed countries of temperate common of these cyanobacterial toxins are the micro- South America. This paper describes the development cystins, hepatotoxins produced by the genera Micro- and validation of an ELISA method for the determina- cystis, Anabaena, and Plankthotrix, which are structurally tion of Cyanobacteria microcystin toxins in algal blooms, which release hepatotoxic metabolites that can reach related cyclic heptapeptides that contain several D- toxic levels in rivers, lakes, or coastal estuaries used for amino acids, N-methyldehydroalanine, and an unusual recreation or water supplies. The assay made possible 20-carbon amino acid, 3-amino-9-methoxy-2-6,8- the first systematic monitoring of water from the Rio de trimethyl-10-phenyldeca-4,6-dienoic acid (Adda). The la Plata at Montevideo over two summers. The project relevance of cyanobacterial toxins such as the micro- has been integrated into a bi-national effort to monitor cystins to human health is underscored by a tragedy the Rio de la Plata. Key words: ELISA; microcystins; that occurred in a hemodialysis clinic in Brazil in 1996, cyanobacteria; Fogarty International Center; Uruguay, where over 100 patients became ill and developed Rio de la Plata; water pollution; monitoring. acute liver failure, and more than 50 died, as a result of microcystin intoxication from contaminated water.2 INT J OCCUP ENVIRON HEALTH 2006;12:377–385 Cyanobacterial toxins can also cause severe effects on targets other than humans, including acute large-scale yanobacteria, or blue-green algae, can produce fish kills and subtler effects such as decreased food dense cellular growth called blooms in nutrient- sources for fish, for example zooplankton. The World rich fresh water such as eutrophic lakes and Health Organization has proposed a provisional public C health guideline of less than 1 µg/L of microcystin-LR rivers contaminated by anthropogenic sources such as 3 Bioaccumulation of sewage effluent and agricultural runoff.1 Some of the equivalents in drinking water. microcystins in fish has been reported, so exposure to contaminated food is another major public health con- cern.4,5 Thus, sensitive and robust analytical methods Received from Cátedra de Bioquímica, Facultad de Quimíca, are required for detection of microcystins in waters at Universidad de la Republica, Montevideo, Uruguay (BMB, LD, NF); risk of algal blooms. ELISA diagnostic kits for micro- the Laboratory of Environmental Hygiene, Municipality of Montev- cystins are commercially available, and have been used ideo, Uruguay (BMB, DS); the National Direction of Aquatic 6 Resources (DINARA), Uruguay (GF); the Ludwig Institute for for analysis of toxins in water. Cancer Research, Uppsala, Sweden (UH); the Cátedra de Cyanobacterial blooms and scums have been Inmunología, Facultad de Quimíca, Universidad de la Republica, reported to occur commonly in lakes, ponds, and reser- Montevideo, Uruguay (GG-S); and the Department of Pulmonary voirs in Argentina,7,8 Brazil,9,10 and Chile,11,12 where a and Critical Care Medicine, University of California, Davis, Califor- major industry—fish farming—-is threatened, and in nia (JAL). Supported in part by Fogarty International Center grant 13 TW-05718, Fulbright Commission grant ASJ 4-1362, PEDECIBA (Pro- Uruguay, including the Rio de la Plata. Over the last gram for the Development of Basic Sciences, United Nations, few years, blooms of Cyanobacteria have been an Uruguay), and CSIC (Scientific Research Sectorial Commission, Uni- increasing phenomenon in the waters of the Rio de la versidad de la República, Uruguay). Plata, with unforeseen intensity after the summer of Address correspondence and reprint requests to: Jerold Last, 2001.14 To characterize the potential hazards associated Department of Pulmonary and Critical Care Medicine, 6519 GBSF, 451 East Health Sciences Drive, University of California, Davis, CA with the different uses of water, it is important to carry 95616, U.S.A.; e-mail: <[email protected]>. out adequate monitoring programs, including deter- 377 TABLE 1 WHO Guidelines for the Safe Management of Recreational Waters* Guidance Level or Situation Health Risks Relatively low probability of adverse health effects Short-term adverse outcomes, e.g., skin irritations, 20,000 cyanobacterial cells /mL or gastrointestinal illness 10 µg chlorophyll a/L 2–4 mg microcystin/L (up to 10 mg/L for very toxic blooms) Moderate probability of adverse health effects 100,000 cyanobacterial cells/mL or Potential for long-term illness 50 µg/L chlorophyll a Short-term adverse health outcomes 20 µg microcystin /L (up to 50 µg/L for very toxic blooms) High probability of adverse health effects Cyanobobacterial scum formation Potential for acute poisoning Potential for long-term illness Short-term adverse health outcomes Source: Chorus and Bartram.15 minations of chlorophyll a (an indicator of phyto- water rich in nitrogen and phosphorus,17 giving rise to plankton biomass), salinity, and surface current circu- ideal conditions for encouraging algal and Cyanobac- lation, in order to evaluate transport of harmful algae, teria blooms. as well as other factors involved, such as the temporal The coast of the Rio de la Plata, including Montev- and geographic distribution of inorganic nutrients and ideo, the capital of Uruguay and a city with a popula- other phytoplankton growth factors. WHO experts tion of about 1.5 million, has an extensive network of have proposed a classification of the risk associated beaches directly on the river. The recreational use of all with recreational activities15 based on the chlorophyll a of these beaches for swimming, boating, and fishing content, the number of cyanobacterial cells, and the during the summer and warmer months has major eco- different ranges of toxin contents associated with these nomic and social implications for the local population parameters as calculated from the average toxin con- and their government agencies. The health risks asso- tents of the most common species (Table 1). ciated with the use of these beaches for recreational In most South American countries, the lack of purposes have remained unknown, due to the difficul- financial resources for environmental monitoring pro- ties of developing cost-effective methods for monitor- grams restricts the information available to inform gov- ing cyanotoxins. ernmental decision makers to set rational public poli- In 2001, a University of California, Davis–University cies. The Rio de la Plata, which drains the second of the Republic, Montevideo, partnership created a largest watershed in South America after the Amazon new Fogarty ITREOH program to exploit the potential river, is a tidal river in the upper (western) part and an of ELISA to provide a low-cost approach to environ- estuary in the areas near Montevideo.16 The numerous mental analysis that could be attractive to the econom- dams built in the Rio de la Plata basin, excessive use of ically distressed countries of temperate South America. fertilizers, and inadequate treatment of sewage in the We describe the development and first results of the whole region make the river and the nearby ocean application of a locally developed ELISA method for microcystins, as well as our approach and progress thus far towards assisting local agencies to develop a ABBREVIATIONS regional network for monitoring Cyanobacteria BSA bovine serum albumin blooms, especially in the Rio de la Plata, which forms DMSO dimethylsulfoxide the border between Argentina and southern Uruguay. FIC Fogarty International Center HRP Horseradish peroxidase MATERIALS AND METHODS IC50 midpoint of the standard curve of inhibition Materials MALDI-TOF MS matrix-assisted laser desorption/ ionization time-of-flight mass Microcystins were obtained from Alexis Biochemicals spectrometry MC. microcystins (San Diego, CA) as MC-LR (purity > 98.0 %); MC-RR PBS phosphate-buffered saline PBS-T PBS containing 0.05 % v/v Tween 20 (purity > 97 %), and MC-LW (purity > 95.0 %). The TFA trifluoroacetic acid monoclonal antibody AD4G2 was kindly provided by Dr. Michael Weller from the Technical University of 378 • Brena et al. www.ijoeh.com • INT J OCCUP ENVIRON HEALTH Munich (Germany). Goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate was from Pierce Biotech- nology (Rockford, IL), bovine serum albumin (BSA, fraction V, purity 98%) was obtained from Sigma- Aldrich (Saint Louis, MO). Purified water was prepared with a Milli-Q plus 185, (Millipore), C 18 cartridges, 500 mg C18-E (55 µm, 70 A) were from Phenomenex, (Torrance, CA), and HPLC solvents were from J. T. Baker (Phillipsburg, NJ). All other chemicals were reagent or analytical grade. Figure 1—a) Uruguay and South-America; b) Locations of sampling sites along the coast of Montevideo (sites Site Description 1–13). Site 14 is 200 km west of Montevideo. The coast of Montevideo, located in the southern cone of South America towards the northern side of the Rio Extraction and Cleanup de la Plata (Figure 1a), is characterized by an abun- dance of sand beaches. During the summer season, the Water samples for the analysis of microcystins were water-quality monitoring program includes 20 bathing stored at –20° C prior to use. To a 100-mL aliquot of beaches, where water samples are obtained every other this sample, 5% acetic acid (v/v) was added; after three day and analyzed routinely for fecal coliforms.
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