DETECTION OF CYANOBACTERIA AND THEIR TOXINS IN WATER
The major objective for this task is to develop analytical methods to detect problematic cyanotoxins in water. A preconcentration/extraction procedure will be initially developed to remove interfering substances with the detection of cyanotoxins in various water matrices. These methods will be used to determine the occurrence/prevalence of cyanotoxins in water and in the cyanobacterial cell culture collection in-house, and compare with the effectiveness of innovative detection methods developed for biological toxins.
Blooms of cyanobacteria, also known as blue-green algae, have recently become more prevalent worldwide as a result of human activities. The long-term chronic human health hazard attributable to toxic cyanotoxins in drinking water has caused considerable concern in humans. Continuous sublethal or low-level exposures to cyanotoxins can potentially lead to the development of gastrointestinal, neurological, and liver disorders.
The frequency in which blooms occur and the amounts of toxic cyanotoxins in bodies of waters during blooms are largely unknown. Cyanobacteria and their toxins are currently on the Agency's Contaminant Candidate List (CCL) and require additional research to assess the magnitude and extent of any risk to humans. Very few studies have been done to determine the occurrence and prevalence of cyanobacteria and their toxins in surface and finished water, yet such knowledge is important to future regulatory decisions about whethr and how to limit consumption or exposure to these toxins. The goal of this study is to develop a method to simultaneously extract and detect cyanotoxins of interest to the Agency. Developed methods will be used to determine the occurrence of problematic cyanobacteria and their toxins in water. Initially, studies will be focused on microcystins and cyanobacteria-producing microcystins in surface water. Eventually, methods will be developed to detect other cyanobacteria and the toxins they produce such as cylindrospermopsin, anatoxins, and saxitoxins in water. Such toxins have also been reported to have significant adverse health effects in humans and are potential biological toxins that terrorists can use to poison our drinking water systems.
Projected Completion Date:09/01/2006
Keywords:CYANOBACTERIA, BLUE-GREEN ALGAE, CYANOTOXINS, MICROCYSTINS,
The anti-microcystin monoclonal antibody (mAb) was successfully coupled with Dynal immunomagnetic beads using two different approaches. Control beads showed no nonspecific binding to microcystin-LR (MC-LR). Unbound MC-LR was monitored by HPLC with PDA detection. An alternate approach is being pursued due to the unreliability of the mAb supply. A protein phosphatase peptide binding site of MC has been synthesized and polyclonal antibody against this peptide is currently being produced. This polyclonal antibody and/or weakly binding MC variant will be coupled with quantum dots (QD). An initial attempt to directly couple the mAb with QD via amine coupling showed polymerization of QD resulting in the reduction of signal. An attempt will be made to use linkers/spacers or try alternate coupling reactions to solve this problem. The CRADA partner is currently optimizing the microfluidic portion of the sensor. They have also successfully shown displacement of their target analyte.
The stability of six hepatotoxins at different temperatures, turbidities, pHs, and ionic strengths are currently being analyzed. Preliminary data showed that in sterile environment, microcystins showed very little degradation over a year at temperatures -20oC, 4oC, and 18oC. Water soluble microcystins appear to be stable for > 3 months at pH 5, 6,7 and 8. More hydrophobic microcystin variants degraded at lower pHs. Varying turbidities (1-1000 NTU) had minimal effect on the stability and recovery of microcystins at higher NTUs only.
The hazards of cyanotoxins to human health provide grounds for the development of monitoring and control programs of cyanobacteria and their toxins in aquaculture, recreational and drinking water supplies. Cyanobacteria and their toxins are currently on the CCL II that require further research. The methods developed in this study could be used in a future Unregulated Contaminant Monitoring Rule round of occurrence data collection. This information could then be used by the USEPA Office of Ground Water and Drinking Water in making the final decision on whether or not cyanobacteria or their toxins should be regulated. The innovative field-portable continuously monitoring sensor to detect and monitor cyanotoxins in water will allow immediate assessment of water quality so remedial measures can be put in place.
NRMRL, OGWDW, Homeland Security