Search

Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid) moiety. Variations include 9-O-desmethylAdda (DMAdda) and 9-O-acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda5]MCs and [DMAdda5]MCs, which were analyzed using multiple approaches. The cross-reactivities of the [DMAdda5]- and [ADMAdda5]MC standards were similar to that of MC-LR when analyzed with a protein phosphatase 2A (PP2A) inhibition assay, but were, Graphical abstract Image 1, Highlights • Toxic ADMAdda/DMAdda microcystins (MCs) exhibit low cross-reactivity with Adda ELISA. • A method for oxidative cleavage was expanded to test for ADMAdda/DMAdda MCs. • Two sites of oxidative cleavage of the Adda/DMAdda/ADMAdda were observed. • Oxidation products were identified and analytically tested to quantitate ‘total MCs’.

In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL −1 of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener ( m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu 1 ]MC-LR using NMR spectroscopy, LC-HRMS and LC–MS 2 , which slowly converted to [D-Leu 1 ,Glu(OMe) 6 ]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC–MS 2 in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard ( Anas platyrhynchos ) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC–MS 2 were used to measure free MCs, while ‘total’ MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63–112 ng g −1 ) accounted for 33–41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu 1 ]MC-LR was quantitated in tissues at 25–67 ng g −1 (LC–MS 2 ). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present.

In 2016, the Pennsylvania Department of Environmental Protection conducted a limited survey of streams in the Susquehanna River basin in Pennsylvania, USA, to screen for microcystins/nodularins, anatoxin-a (ATX) and homoanatoxin-a (HTX). Testing revealed the presence of HTX in samples collected from the Pine Creek basin, with ATX present at lower levels. Microcystins/nodularins (MCs/NODs) were also tested and found to be concomitant, with NOD-R confirmed present by LC-MS/MS.

In 2013 and 2015, the Pennsylvania Department of Environmental Protection conducted a survey of lotic habitats within the Susquehanna, Delaware, and Ohio River basins in Pennsylvania, USA, to screen for microcystins/nodularins (MCs/NODs) in algae communities and smallmouth bass (Micropterus dolomieu). Periphyton (68 from 41 sites), juvenile whole fish (153 from 19 sites) and adult fish liver (115 from 16 sites) samples were collected and screened using an Adda enzyme-linked immunosorbent assay (ELISA). Samples that were positive for MCs/NODs were further analyzed using LC-MS/MS, including 14 variants of microcystin and NOD-R and the MMPB technique. The ELISA was positive for 47% of the periphyton collections, with NOD-R confirmed (0.7–82.2 ng g−1 d.w.) in 20 samples. NOD-R was confirmed in 10 of 15 positive juvenile whole fish samples (0.8–16.7 ng g−1 w.w.) and in 2 of 8 liver samples (1.7 & 2.8 ng g−1 w.w.). The MMPB method resulted in total MCs/NODs measured in periphyton (2.2–1269 ng g−1 d.w.), juvenile whole fish (5.0–210 ng g−1 d.w.) and adult livers (8.5–29.5 ng g−1 d.w.). This work illustrates that NOD-R is present in freshwater benthic algae in the USA, which has broader implications for monitoring and trophic transfer.

In the summer of 2018, six dogs exposed to a harmful algal bloom (HAB) of Microcystis in Martin County Florida (USA) developed clinicopathological signs of microcystin (MC) intoxication (i.e., acute vomiting, diarrhea, severe thrombocytopenia, elevated alanine aminotransferase, hemorrhage). Successful supportive veterinary care was provided and led to survival of all but one patient. Confirmation of MC intoxication was made through interpretation of clinicopathological abnormalities, pathological examination of tissues, microscopy (vomitus), and analytical MC testing of antemortem/postmortem samples (vomitus, blood, urine, bile, liver, kidney, hair). Gross and microscopic examination of the deceased patient confirmed massive hepatic necrosis, mild multifocal renal tubular necrosis, and hemorrhage within multiple organ systems. Microscopy of a vomitus sample confirmed the presence of Microcystis. Three analytical MC testing approaches were used, including the MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) technique, targeted congener analysis (e.g., liquid chromatography tandem-mass spectrometry of MC-LR), and enzyme-linked immunosorbent assay (ELISA). Total Adda MCs (as MMPB) were confirmed in the liver, bile, kidney, urine, and blood of the deceased dog. Urinalysis (MMPB) of one surviving dog showed a high level of MCs (32,000 ng mL&minus, 1) 1-day post exposure, with MCs detectable &gt, 2 months post exposure. Furthermore, hair from a surviving dog was positive for MMPB, illustrating another testable route of MC elimination in canines. The described cases represent the first use of urine as an antemortem, non-invasive specimen to diagnose microcystin toxicosis. Antemortem diagnostic testing to confirm MC intoxication cases, whether acute or chronic, is crucial for providing optimal supportive care and mitigating MC exposure.

Potentially harmful freshwater algal blooms of cyanobacteria (CHAB) are increasingly reported among US surface waters. Clear Lake in Lake County, CA is regularly impacted by CHABs during the spring...

β-Methylamino-L-alanine (BMAA) has been identified as the potential cause of the amyotrophic lateral sclerosis/parkinsonism–dementia complex (ALS/PDC) observed in the Chamorro people of Guam. The principal hypothesis for BMAA exposure and intoxication relies on the biomagnification of BMAA in flying fox specimens ingested by the Chamorro people. Although high levels of BMAA were quantitated in flying fox specimens utilizing liquid chromatography-fluorescence (LC-FL), there have not been any confirmatory analyses conducted to date. Therefore, a method for the tissue homogenization, extraction and direct analysis of BMAA (including BAMA, 2,4-DAB and AEG) was utilized. The approach was applied to mammalian dried skin and hair from various rodent species (negative controls) and archived flying fox (Pteropus mariannus mariannus) specimens. A positive control sample of homogenized mussel (Mytelius edulis) with native BMAA was used to verify the method. It was determined that the direct analysis using HILIC MS/MS required additional quality control in order to allow for the confident identification of BMAA due to the near co-elution of BAMA. BMAA was not present above 0.2 μg g−1 (free fraction) or 2.8 μg g−1 (total fraction) in the flying fox specimens. While analysis did not result in BMAA detection in flying fox or negative control samples, the positive control sample and spiked samples were successfully detected.

Microcystins have been detected in raw and finished drinking water using a variety of techniques, including assays (immunoassay, phosphatase inhibition) and HPLC (UV, MS/(MS)). The principal challenge to microcystin analysis is accounting for the over 150 variants that have been described. A confirmatory individual variant HPLC analysis is prone to under-reporting total microcystins due to method specificity. One method that allows for total microcystin quantitation is the MMPB technique. In this study, water samples with native microcystins were oxidized to cleave the Adda moiety, common to all microcystin variants. LC-MS/MS analysis was conducted on the subsequent MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) molecule and calibrated using a certified reference standard (microcystin-LR) and 4-phenylbutyric acid. Total microcystin concentrations from MMPB were compared to Adda ELISA and individual variant analyses (LC-UV, LC-MS/(MS)). Variants of microcystin, including [DAsp(3)]MC-RR, [Dha(7)]MC-RR, MC-RR, MC-YR, MC-LR, [DAsp(3)]MC-LR, [Dha(7)]MC-LR, MC-WR, MC-LA, and MC-LY were detected and quantified in samples. The individual variant analyses did not account for total microcystins present in samples, as indicated by ELISA and MMPB data. Results demonstrated the MMPB technique is a simple and valuable approach to confirm ELISA data when analyzing microcystins, with method detection limits of 0.05 μg L(-1) for total microcystins.