Prevalence and persistence of microcystin in shoreline lake sediments and porewater, and associated potential for human health risk.

Midlatitude waterbodies are experiencing increased cyanobacteria blooms that necessitate health advisories to protect waterbody users. Although surface waters may contain cyanotoxins such as microcystin (MC), at concentrations that pose potential public health risks, little is known about MC contamination of shoreline sediments. Based on growing evidence that lake and reservoir sediments can accumulate MCs, we hypothesized that shoreline sediments (i.e., recreational beaches) may accumulate MCs and thereby pose a potential health risk to recreational users even if people stay out of contaminated water. We sampled nearshore surface water, shoreline sediment, and porewater from seven Washington State, USA, lakes/reservoirs recreational beaches to determine MC presence/absence during or immediately following cyanobacteria blooms. We found MCs in shoreline sediments at all waterbodies using ELISA and LC-MS/MS. MC concentrations in shoreline sediments and porewaters persisted for 20 days following dissipation of cyanobacteria blooms when MC concentrations were near analytical reporting limits in corresponding surface waters. A human health risk assessment based on potential MC exposure through incidental ingestion of porewaters and sediments found, even when very high MC concentrations occur in surface waters (i.e., >11,000 µg/L), estimated ingestion doses are below MC World Health Organization tolerable daily intake and U.S. Environmental Protection Agency's risk reference dose. While our findings suggest MCs in Washington State recreational beaches in 2018 did not present a significant human health risk, future blooms with higher MC concentrations could pose human health risks via the shoreline sediment/porewater exposure pathway.

Status of state cyanoHAB outreach and monitoring efforts, United States.

A widespread effort is underway to improve awareness of cyanobacteria harmful algal blooms (cyanoHABs) across the United States using a variety of monitoring programs and public health outreach measures to protect people, pets, and livestock. To determine the status of cyanoHAB outreach and monitoring efforts, 2 questionnaires were distributed to health/environmental departments in 50 states and the District of Columbia (DC). One questionnaire focused on cyanoHAB exposure to humans from drinking water and the second targeted exposure through recreational activities. All states plus DC responded to the recreational survey; 46 states plus DC responded to the drinking water survey. All states except Alaska answered that microcystins were the cyanotoxins of greatest concern for recreational exposure; microcystins were also of greatest concern for drinking water with the exception of Utah (anatoxin-a in reservoirs was greatest concern) and Rhode Island (microcystins and anatoxin-a in reservoirs/ponds were greatest concern). Regional comparisons disclosed a lack of cyanoHAB programs in southern states relative to northern states that may be related to the higher percentage of water surface area in northern states. Interestingly, recreational outreach is more extensive than drinking water outreach (only 16 states reported having some type of drinking water outreach program, compared with 35 states with recreational outreach), and preferred outreach methods are websites and press releases. Additionally, respondents reported very limited funding for outreach and monitoring programs. Our results establish baseline information to help determine what future direction cyanoHAB outreach and monitoring programs can take at local, regional, and national levels.

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

BACKGROUND: Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture. RESULTS: The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90. CONCLUSION: Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.

Education and notification approaches for harmful algal blooms (HABs), Washington State, USA.

While numerous strategies have been used to educate and notify the public about potential hazards from exposure to Harmful Algal Blooms (HABs), at present there are no national guidelines or suggested outreach approaches. To raise public awareness and determine effective HAB outreach methods, two Washington State agencies and three counties in the Puget Sound region implemented several education and notification strategies. These approaches were rated for effectiveness by state and county public health and water quality professionals. At the state level, the most effective action was a three-tiered advisory posting protocol for notifying external users that was introduced to local health jurisdictions at workshops around the state. Supplemental permanent signage is recommended for lakes with blooms to overcome the time lag between HAB onset and testing/posting. The state also implements effective notification of toxicity test results through a web-based HAB database and listserv. Lake residents were best notified through electronic alerts including email and social media while mailers to lake residents were useful during initial HAB events and to gain subscribers to electronic alerts. Press releases were most valuable when used sparingly for severe blooms or for blooms in large lakes. Initial analyses of lake recreational use indicates these strategies encourage behavior change in lake users. Based on these findings, a general framework for HAB outreach and a specific notification strategy is proposed to assist other regions or agencies that are developing HAB education and notification programs.

Cyanotoxin bioaccumulation in freshwater fish, Washington State, USA.

Until recently, exposure pathways of concern for cyanotoxins have focused on recreational exposure, drinking water, and dermal contact. Exposure to cyanotoxins through fish consumption is a relatively new area of investigation. To address this concern, microcystins and other cyanotoxins were analyzed in fish collected from nine Washington lakes with recurrent toxic blooms using two types of enzyme-linked immunosorbent assays (ELISAs) and liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS). Microcystins or microcystin-like compounds were elevated in fish liver relative to muscle and other tissues (liver>gut>muscle). Microcystin concentrations in fish fillet samples using anti-Adda ELISA (range 6.3-11 µg/kg wet weight) were consistently higher in all fish species than concentrations using anti-microcystin (MC)-leucine-arginine (LR) ELISA (range 0.25-2.4 µg/kg wet weight). MC-leucine-alanine (LA) was the only variant detected in fish (2.5-12 µg/kg in four livers) among the nine variants analyzed by LC-MS/MS. Fish fillets showed no accumulation of the MCs targeted by LC-MS/MS. Other cyanotoxins analyzed (anatoxin-a, saxitoxin, domoic acid, and okadaic acid) were not detected in fish. Based on this and evidence from other studies, we believe that people can safely consume two 8-oz fish fillet meals per week from lakes with blooms producing MCs (clean the fish and discard viscera).

Transfer of microcystin from freshwater lakes to Puget Sound, WA and toxin accumulation in marine mussels (Mytilus trossulus).

Many eutrophic inland freshwater lakes in the Puget Sound Washington region produce toxic cyanobacteria blooms annually. While such blooms in lakes tend to be viewed as a localized phenomenon, there is significant potential for downstream export of toxins to freshwater streams, and marine and brackish water environments. However, monitoring for cyanotoxins typically associated with freshwaters, such as the hepatotoxin, microcystin (MC) in marine receiving waters is rare. In 2013 we studied four eutrophic Puget Sound area lakes to assess both toxin transport to marine waters and its potential accumulation in marine shellfish, specifically mussels. Shellfish beds are extensive throughout Puget Sound, and recreational harvest occurs downstream of our study lakes, so a study goal was to also assess if shellfish consumption poses a human health risk for MC exposure. We confirm, for the first time, freshwater to marine transfer of MCs in Puget Sound with subsequent bioaccumulation of MC by mussels. ELISA analysis estimated maximum MC concentrations in source lakes of 2700 µg/L, up to 0.34 µg/L in marine waters and 6.5 µg/kg in mussels. Confirmatory analyses by LC-MS/MS on water and mussel samples identified MC-LA as the major toxin. Although we found relatively low MC levels in mussels, our study implies that potential concern for human food safety is justified and warrants further investigation.

Integrative monitoring of marine and freshwater harmful algae in Washington State for public health protection.

The more frequent occurrence of both marine and freshwater toxic algal blooms and recent problems with new toxic events have increased the risk for illness and negatively impacted sustainable public access to safe shellfish and recreational waters in Washington State. Marine toxins that affect safe shellfish harvest in the state are the saxitoxins that cause paralytic shellfish poisoning (PSP), domoic acid that causes amnesic shellfish poisoning (ASP) and the first ever US closure in 2011 due to diarrhetic shellfish toxins that cause diarrhetic shellfish poisoning (DSP). Likewise, the freshwater toxins microcystins, anatoxin-a, cylindrospermopsins, and saxitoxins have been measured in state lakes, although cylindrospermopsins have not yet been measured above state regulatory guidance levels. This increased incidence of harmful algal blooms (HABs) has necessitated the partnering of state regulatory programs with citizen and user-fee sponsored monitoring efforts such as SoundToxins, the Olympic Region Harmful Algal Bloom (ORHAB) partnership and the state's freshwater harmful algal bloom passive (opportunistic) surveillance program that allow citizens to share their observations with scientists. Through such integrated programs that provide an effective interface between formalized state and federal programs and observations by the general public, county staff and trained citizen volunteers, the best possible early warning systems can be instituted for surveillance of known HABs, as well as for the reporting and diagnosis of unusual events that may impact the future health of oceans, lakes, wildlife, and humans.

Identifying best methods for routine ELISA detection of microcystin in seafood.

Ingestion of water contaminated with the cyanotoxin, microcystin (MC), can pose serious health risks to humans. MC is also known to accumulate in seafood; however, this exposure pathway is much less understood. A fundamental element of this uncertainty is related to analytical difficulties. Commercially available enzyme-linked immunosorbent assays (ELISAs) offer one of the best options for routine MC detection, but methods of detecting MC in tissue are far from standardized. We spiked freshwater finfish and marine mussel tissues with MC, then compared recovery rates using four different preparation protocols and two ELISA types (polyclonal anti-MC-ADDA/direct monoclonal (DM)). Preparation protocol, type of ELISA, and seafood tissue variety significantly affected MC detection. This is the first known study to use DM ELISA for tissue analyses, and our findings demonstrate that DM ELISA combined with a short solvent extraction results in fewer false positives than other commonly used methods. This method can be used for rapid and reliable MC detection in seafood.