Abstract
Microcystins (MCs) are the most common class of freshwater cyanobacterial toxins and exposure has led to the death of aquatic and terrestrial animals, including humans. The current assumption states that only free MCs (i.e., microcystins that have not undergone a covalent-linkage within the organism) are bioavailable through the food web and should be used to estimate risk to higher trophic levels and humans. Inconsistencies in the literature, however, prevent accurate estimations of risk, as the extraction of free MCs is largely dependent upon the methods and matrices utilized. To standardize extraction efficiency, I developed an internal standard (thiol-LR), a synthetic MC with a unique mass (m/z 1087), that interacts with fish liver tissue in a manner similar to both polar (MC-RR, MC-LR) and non-polar MC variants (MC-LA). Thiol-LR was also a good surrogate for polar variants in fish muscle tissue; however, it was less representative of the matrix's interaction with non-polar variants. The internal standard can be added directly to field samples and quantified along with endogenous MCs using liquid chromatography - mass spectrometry (LC-MS).
In contrast to the current dogma, I propose that covalently-bound MCs should be considered when making predictions regarding risk through food web exposure. Three endopeptidases, pepsin, chymotrypsin, and trypsin, were unable to digest cyclic MC-LR and MC-LY, but were very active against the linear peptide angiotensin II. Therefore, proteases in gut of the consumer will likely digest protein phosphatases that are covalently bound to the toxin in their food, leaving a small peptide attached to the toxin. To test if the resulting MC-peptides are then toxic in the consumer, (1) four peptides were synthesized to replicate sequences that would result from the proteolytic digestion of protein phosphatase 1 (PP1), (2) the four peptides were conjugated to MC-LR, and (3) then tested for toxicity using a protein phosphatase inhibition assay (PPIA). All MC-peptides were approximately half (58%) as toxic as the parent toxin according to PPIA. These results provide a first look into the potential bioavailability of covalently-bound MCs as they transfer up the food web and suggest that covalently-bound MCs should be included in calculations of risk to higher trophic levels (e.g., piscivorous fish) and humans.
MCs were investigated as an impediment to the recolonization of Hexagenia, the burrowing mayfly. Laboratory experiments were conducted to determine if MC-LR produced negative effects on Hexagenia at three points within its life cycle: egg, hatchling nymph, and pre-emergent nymph. Eggs showed a delay to hatching and an altered distribution of hatching over the study period when submerged in 0.1 µg/ml MC-LR (e.g., bloom-scum concentration). The 72-h (1.1 µg/ml) and 96-h (0.049 µg/ml) LC50 values for hatchling nymphs exceeded typical bloom concentrations of North American lakes (0.01 µg/ml). Large nymphs were more tolerant of the toxin, as indicated by 100% survival over seven days exposure to 10 µg/ml, suggesting older larvae can withstand brief encounters with high MC levels for at least short periods of time. The sensitivity of younger nymphs and eggs to MC-LR may have negative implications for the recruitment of the genus in water bodies with persistent summer cyanobacterial blooms.
As a result of my dissertation research, the risk of microcystin exposure to aquatic and terrestrial animals can be better evaluated. I used an interdisciplinary approach to determine toxicological and ecological implications of microcystins for multiple levels of the aquatic food web.