Development of methods for measuring total microcystins in Fish Tissue using the 2-methoxy-3-methyl-4-phenylbutyric acid (MMPB) procedure (Region 8 webinar)
Citation:
Sanan, T., Jim Lazorchak, D. Sundaravadivelu, J. Kickish, J. Jones, AND R. Venkatapathy. Development of methods for measuring total microcystins in Fish Tissue using the 2-methoxy-3-methyl-4-phenylbutyric acid (MMPB) procedure (Region 8 webinar). Region 8, Webinar, Webinar, January 16, 2020.
Impact/Purpose:
This will inform Region 8 and there states and tribes on the Cyanotoxin research ORD is doing.
Description:
There are limited methods for the analyses of multiple algal toxins in aquatic food webs, phytoplankton, zooplankton, periphyton, macroinvertebrates, forage fish, bottom feeders and top carnivore fish. Algal toxins in freshwater systems do not necessarily occur as single contaminants; mixtures of toxins may be produced from Cyanobacteria, Prymnesium parvum , and Euglena sanguinea, including microcystins, saxitoxins, cylindrospermopsin, anatoxin-a, prymnesins and euglenophycin. The objective of the first phase of this research was to spike existing fillet and whole fish homogenates with 3 congeners of microcystins (LR, LA and RR) individually and as mixtures, and to develop a method for their recovery and measurement using the MMPB derivatization method. The second phase of the project is to field-test this method on fish collected from water bodies experiencing algal blooms and compare results with individual congener measurements. Extraction methods and analytical methods being developed for this research will be a starting point for developing extraction procedures for plankton, periphyton, and macroinvertebrates. Fish homogenates weighing 10 and 100 mg from fish containing 1, 4 and 14% lipids were spiked with 4 and 40 ng of each of the microcystin congeners, LR, LA and RR. Various extraction techniques and conditions were tested to optimize recovery and simplify the procedure. Overall toxin recoveries were found to range from 30 to 50%. The lipid content was found to not interfere with generation of MMPB; however, it did impact the workup/extraction procedure in ways which were quantifiable through the use of a surrogate standard and standard addition procedures. The MMPB technique can be reliably employed for microcystin quantification in fish tissue. During the second phase of the project fathead minnows that were deployed in experimental streams that were dosed with different nitrogen and phosphorus concentrations in 2017 and 2018 were analyzed as well as periphyton slurries collected from the streams in 2018. In addition, fish collected in 2018 from lakes with historic algal blooms in Utah, Florida, Connecticut and New Jersey were analyzed. For toxin quantification in tissues, the measurement of total microcystins via the MMPB method provides improvements over extraction of individual toxin congeners, particularly with very polar or hydrophobic MCs. Results from phases 1 and 2 will be presented.