Search

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.

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.

Microcystin poisoning was diagnosed in a dog exposed to a Microcystis aeruginosa-dominated, freshwater, harmful algal bloom at Milford Lake, Kansas, which occurred during the summer of 2011. Lake water microcystin concentrations were determined at intervals during the summer, using competitive enzyme-linked immunosorbent assays, and indicated extremely high, localized microcystin concentrations of up to 126,000 ng/ml. Multiple extraction and analysis techniques were used in the determination of free and total microcystins in vomitus and liver samples from the poisoned dog. Vomitus and liver contained microcystins, as determined by enzyme-linked immunosorbent assays, and the presence of microcystin-LR was confirmed in vomitus and liver samples using liquid chromatography coupled with tandem mass spectrometry. Major toxic effects in a dog presented for treatment on the day following exposure included fulminant liver failure and coagulopathy. The patient deteriorated rapidly despite aggressive treatment and was euthanized. Postmortem lesions included diffuse, acute, massive hepatic necrosis and hemorrhage, as well as acute necrosis of the renal tubular epithelium. A diagnosis of microcystin poisoning was based on the demonstration of M. aeruginosa and microcystin-LR in the lake water, as well as in vomitus produced early in the course of the poisoning; the presence of microcystin-LR in liver tissue; and a typical clinical course including gastroenteritis and fulminant liver failure.

We conducted a study of recreational exposure to microcystins among 81 children and adults planning recreational activities on either of three California reservoirs, two with significant, ongoing blooms of toxin-producing cyanobacteria, including Microcystis aeruginosa (Bloom Lakes), and one without a toxin-producing algal bloom (Control Lake). We analyzed water samples for algal taxonomy, microcystin concentrations, and potential respiratory viruses (adenoviruses and enteroviruses). We measured microcystins in personal air samples, nasal swabs, and blood samples. We interviewed study participants for demographic and health symptoms information. We found highly variable microcystin concentrations in Bloom Lakes (10 microg/L to500 microg/L); microcystin was not detected in the Control Lake. We did not detect adenoviruses or enteroviruses in any of the lakes. Low microcystin concentrations were found in personal air samples (0.1 ng/m(3) [limit of detection]-2.89 ng/m(3)) and nasal swabs (0.1 ng [limit of detection]-5 ng). Microcystin concentrations in the water-soluble fraction of all plasma samples were below the limit of detection (1.0 microg/L). Our findings indicate that recreational activities in water bodies that experience toxin-producing cyanobacterial blooms can generate aerosolized cyanotoxins, making inhalation a potential route of exposure. Future studies should include collecting nasal swabs to assess upper respiratory tract deposition of toxin-containing aerosols droplets.