2008 NORTHEASTERN NATURALIST 15(3):403–416 Cyanotoxins in Tidal Waters of Chesapeake Bay Peter J. Tango1,2,* and Walt Butler3 Abstract - Cyanobacteria blooms have long been described for Chesapeake Bay nontidal and tidal waters, but measurable toxin has only been recently recorded. During September 2000, the earliest tidal-water records of cyanotoxins in the Bay identified microcystin from a Microcystis-dominated bloom on the Sassafras River. Between 2000 and 2006, opportunistic samples collected from cyanobacteria blooms were analyzed for toxin concentration to better inform natural resource, agriculture, and human-health management agencies on potential bloom-related health risks. The hepatotoxin microcystin was detected most frequently and over a range of concentrations from 2.9 x 10-2 to 6.58 x 102 μg L-1. Microcystin levels exceeded literature-based chronic drinking-water guidance values of 1 μg L-1 and recreational safety guidance for children of 10 μg L-1 in 71% and 31% of samples, respectively. Samples from tidal fresh and oligohaline habitats showed a log-normal distribution of toxin concentrations, and microcystin had positive log-linear relationship with Microcystis aeruginosa cell counts (r2 = 0.42). A subset of the samples positive for microcystin was also tested for neurotoxins and showed anatoxin-a as the next-most common toxin encountered (46% of samples tested) at concentrations from 3 x 10-3 to 3 μg L-1. Saxitoxin (PSP-toxin) was present in trace amounts (3 x 10-3 μg L-1) in one sample. Cylindrospermopsis raciborskii has occasionally been found in abundance, but all tests for cylindrospermopsin were negative. Microcystin and anatoxin-a have been identified in association with fish kills, bird kills, and human-health events. Virginia and Maryland state management agencies conducted beach closures during 2000, 2003, and 2004 and provided waterway health advisories in 2005 and 2006 in response to the findings. Introduction The awareness for the toxic nature of freshwater cyanobacteria has existed worldwide for at least 150 years (Codd et al. 2005a). There are earlier indications that there was traditional knowledge for toxicity associated with cyanobacterial blooms among native North Americans, Africans, and Australians (Codd et al. 2005b). The earliest documented investigation into the potentially poisonous nature of cyanobacteria in the United States was Arthur 1883 (cited in Carmichael and Stukenberg 2005). The first description of a cyanotoxic event in the US is considered 1925, when a farmer lost 127 hogs and 4 cows after they drank bloom-affected water from Big Stone Lake, SD (Carmichael and Stukenberg 2005). Further, Carmichael and Stukenberg (2005) indicate the first documented cases of human illness in North America attributed to unclassified cyanobacterial toxins were !Maryland Department of Natural Resources, 580 Taylor Avenue D-2, Annapolis, MD 21401. 2Current address - 410 Severn Avenue, Suite 109, Annapolis, MD 21403. 3Maryland Department of Natural Resources Field Office, 1919 Lincoln Drive, Annapolis, MD 21401. *Corresponding author - ptango@chesapeakebay.net. 404 Northeastern Naturalist Vol. 15, No. 3 from the Potomac (Chesapeake Bay watershed) and Ohio River drainages associated with massive Microcystis blooms in 1930–31 (Tisdale 1931a,b; Veldee 1931) when 5000 to 8000 people were sickened. In the 1970s, pyrogenic effects were noted in 49 dialysis patients in Washington, DC, retrospectively considered a result of cyanotoxin exposure delivered from cyanobacteria-bloom waters within the Potomac River basin (Hindman et al. 1975, WHO 2003). Between the 1930s and 1970s, noxious cyanobacteria blooms increased on the tidal Potomac River and other northern Bay waters as nutrient loading increased and submerged aquatic vegetation populations declined (Lear and Smith 1976, Stevenson et al. 1979). Cyanobacteria blooms were further evident on the Potomac River in the 1980s (Jones et al. 1992). The first acknowledgment for probable toxicity of such blooms on the estuarine tidal waters occurred when Maryland Department of Agriculture reported two dogs were sickened after drinking bloom-affected waters of the Elk River, northern Chesapeake Bay in 1998. In spite of the recognition for toxigenic cyanobacteria taxa among the phytoplankton community (Marshall et al. 2005a), episodic evidence of likely toxicity, and decades-long records of cyanobacteria blooms in the region, direct detection of any of the cyanotoxins (hepatotoxins and neurotoxins) has only recently been documented. (Driscoll et al. 2002, Marshall et al. 2008, Tango et al. 2008). Some of the most significant impacts associated with exposure to cyanobacteria- derived hepato- and neurotoxins have been impaired health or death of livestock, wildlife, pets, and humans (Chorus and Bartram 1999, Codd et al. 2005a). Less-obvious effects of cyanotoxins on living resources include allelopathic interactions with microbial, zooplankton, nektonic, benthic, and aquatic macrophyte taxa (Christoffersen 1996, Pflugmacher 2002, Sivonen and Jones 1999, Smayda 1997). Additional evidence indicates their toxins can biomagnify in the food web (Christoffersen 1996, Driscoll et al. 2002, Prepas et al. 1997, Simoni et al. 2004), posing a further mechanism for possible impacts on food-web structure. Cylindrospermopsin shows evidence in the laboratory for inducing chromosome breakage and loss in vitro (Humpage et al. 2000). Cyanobacteria are also used in food supplements, providing direct exposure risk for human consumption (Backer 2002), with additional risk a function of oral intake and aerosol inhalation during recreational activities. For many countries, drinking-water safety guidance for microcystin has tended to focus on World Health Organization (WHO) recommendations (Codd et al. 2005a). Safety guidance for recreational water use related to cyanotoxin levels continues to evolve and be refined (e.g., Chorus and Bartram 1999, NHMRC 2005, Stone and Bress 2007). While there are presently no US federal guidance values for drinking water or recreational safety dealing with cyanotoxins, states such as Vermont and Oregon are moving forward and adopting their own criteria (Stone and Bress 2007). The purpose 2008 P.J. Tango and W. Butler 405 of this paper is to 1) use toxin-testing results from cyanobacteria bloom investigations between 2000–2006 to describe toxins detected for Chesapeake Bay tidal waters, 2) illustrate the distribution of cyanotoxin findings, 3) relate microcystin levels to the cell counts of common toxigenic cyanobacteria Microcystis aeruginosa (Kutzing) Lemmermann, and 4) describe toxin findings as they relate to literature-derived guidance on human-health risk thresholds. Study Area With a watershed area of 172,000 km2, the Chesapeake Bay watershed is the largest estuary in the United States. The watershed drains portions of New York, Pennsylvania, West Virginia, Virginia, Maryland, and Delaware, and all of Washington, DC. The Bay extends 300 km from the mouth of the Susquehanna River to the Atlantic Ocean. Habitat conditions include a salinity gradient from freshwater (<0.5 ppt salinity) and oligohaline (0.5–5 ppt) conditions in tidal end members to polyhaline (>18 ppt) conditions at its ocean boundary. Phytoplankton taxonomic diversity in the tidal Chesapeake Bay has expanded coincident with long-term water-quality monitoring in the system and improved monitoring techniques. During the 1990s, 708 species of phytoplankton were recognized (Marshall 1994), while over 1450 species are recognized today (Marshall et al. 2005). The list of toxigenic phytoplankton species for the Bay has also increased from 12 (Marshall 1996) to 34, including 15 for cyanobacteria (Marshall et al. 2005). Excessive nutrients in Chesapeake Bay and its tidal tributaries promote undesirable water-quality conditions that include low dissolved oxygen, reduced water clarity, and excessive algal growth (Kemp et al. 2005, Koroncai et al. 2003). A 2000-year history of sedimentation, eutrophication, anoxia, and diatom community structure was reconstructed from strategraphic records preserved in the mesohaline sediments showing eutrophication and related symptoms increasing since the time of European settlement in the watershed (Cooper 1995). Before the 17th century, the landscape was largely influenced by climate and the settlement and agriculture of Native Americans. During the 1800s, it is estimated that the population first exceeded one million residents (Cooper 1995). The watershed population has increased exponentially in the last century to approximately 16 million (Kemp et al. 2005), continually affecting land-use patterns and subsequent nutrient delivery and availability in the estuary. Eutrophic symptoms continue Bay-wide, and watershed States remain committed to water-quality protection and ecosystem restoration (Koroncai et al. 2003). Methods Cyanobacteria bloom samples (1–4 L) were collected opportunistically as surface grabs. State management activities require responses to citizen 406 Northeastern Naturalist Vol. 15, No. 3 reports from an aquatic-health hotline that initiate potential harmful algal bloom (HAB) event investigations (e.g., fish kill, bird kill, water-color complaints, or human-health concerns). If bloom conditions were identified, then bloom tracking continued until risk situations declined. Plankton monitoring, from the Chesapeake Bay Program long-term water-quality program stations, periodically encountered blooms, which also prompted additional focus sampling to evaluate duration, magnitude, and distribution of events. Cyanobacteria were most often enumerated under light and epi-fluorescent microscopy. The most-frequent assessments were done using a Zeiss Axiovert 200. The live sample was mixed for 45 seconds, then one milliliter was pipetted into a Sedgewick-Rafter counting chamber, covered, inverted, and allowed to settle for 25 minutes. After the settling time, the sample is placed on the microscope, where one strip is counted across the counting chamber. The 640X magnification used is obtained with a 40X objective, a 10X eyepiece, and a 1.6X optovar. Cyanobacteria-dominated samples typically contained an array of toxigenic species. The presence and abundance of Microcystis spp., Anabaena spp., Aphanizomenon flos-aquae J. Ralfs ex Bornet and Flah., and Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju in some samples prompted testing for microcystin and, due to limited financial resources, a subset of samples for anatoxin-a, PSP-toxins, and cylindrospermopsin. Samples for toxin analysis were shipped as 1-L whole-water samples or a select volume was filtered and the filter frozen and shipped to outside analytical laboratories. Cyanotoxin detections involved the use of biological and biochemical methods (Carmichael 2001, Carmichael et al. 2001). Microcystins were analyzed using enzyme-linked immunosorbent assay (ELISA; An and Carmichael 1994, Carmichael and An 1999). Anatoxin-a was the form of anatoxin analyzed for by either high-performance liquid chromatography with fluorescence detection (HPLC-FD) (James and Sherlock 1998) or liquid chromatography/mass spectrometry (LC/MS) (G. Boyer, State University of New York College of Enviromental Science and Forestry, Syracuse, NY, pers. comm.). PSP-toxins were analyzed by HPLC after fluorescent derivitization (Oshima 1995). Cylindrospermopsin was run by HPLC with a photoiodide array detector (G. Boyer, pers. comm.). Detection limits varied according to sample volume, toxin, and analytical technique. Results Toxin detections: timeline of first reports During September 2000, 4 samples collected from a Microcystisdominated bloom on the Sassafras River were positive for microcystins, representing the first direct measurement of cyanotoxins in the tidal estuary of Chesapeake Bay (Tango et al. 2005). Microcystin levels were 5.91 x 102, 9.38 x 102, 9.66 x 102, and 1.041 x 103 μg g-1 dry-weight 2008 P.J. Tango and W. Butler 407 concentrations and considered potentially lethal levels (Carmichael report to MD DNR 2000). The Sassafras River located in northern Chesapeake Bay is characterized by tidal freshwater and oligohaline habitats. In contrast, two samples collected from mesohaline waters of the western shore of Chesapeake Bay on June 19, 2006, showed low levels of microcystin (2.9 x 10-2 and 3.9 x 10-2 μg L-1) associated with sample concentrations of 9.02 x 104 and 1.92 x 105 cells ml-1 Cyanobium sp., respectively. Cyanobium sp., previously synonymous with Synechococcus sp., has been identified as a microcystin toxin producer and has been associated with waterfowl kills (Carmichael and Li 2006, Hallegraeff et al. 2003). Water temperatures and salinity associated with the shoreline and mid-channel sampling locations were 27.9 and 22.7 oC and 14.7 and 14.9 ppt, respectively. Microcystin detections in the open-Bay mesohaline habitat represented were unique compared with previous toxin derivation from freshwater and oligohaline cyanobacteria communities. During 2001, waterbird deaths on Kent Island, MD were considered a function of cyanotoxin burden (Driscoll et al. 2002). A composite of pondwater samples from the area showed microcystin present. Liver tissues of Ardea herodias Linnaeus (Great Blue Heron) had 1.1 x 102 and 4.5 x 102 ng g-1 microcystin (MMPB or 2-methyl-3-methoxy-4-phenylbutyric acid test results). Presently, this is the only record identifying food-web transfer of cyanotoxins in Chesapeake Bay. Another bird kill investigated in 2002 occurred around an active dredge-spoil island pond located on the Bay (Poplar Island). Anatoxin-a was identified at low levels (9 x 10-3 μg L-1) from a cyanobacteria bloom sample collected from the pond water. No direct linkage was made between the toxin and the kills in this event; however, the anatoxin-a results represented the first confirmation of this neurotoxin in the tidewater region. During a cyanobacteria bloom on the Sassafras River in 2003 dominated by Aphanizomenon flos-aquae, saxitoxin was detected (3 x 10-3 μg L-1). PSP-toxins are better known for their association with marine dinoflagellates, but can be cyanobacteria-derived from species such as Aphanizomenon flos-aquae, Aphanizomenon gracile, Anabaena circinalis Rabenhorst, C. raciborskii, and Lyngbya wollei Farlow ex Gomont comb. nov. (Cronberg and Annadotter 2006). PSP-toxin testing has been very limited in Chesapeake Bay, and presently, this 2003 detection is the only positive record. It is notable that saxitoxin illness was recently detected in two human cases in Virginia (Bodager 2002, Quilliam et al. 2004). The source of the illness was consumption of Sphoeroides nephelus Goode & Bean (Southern Puffer Fish) taken from the Indian River Lagoon, fl, and not Chesapeake Bay. Toxin concentrations during bloom surveys: microcystin, anatoxin-a, and saxitoxin Microcystin concentrations across all habitats has ranged from 7 x 10-3 μg L-1 (Poplar Island dredge pool) to 6.58 x 102 μg L (Potomac River) (n = 408 Northeastern Naturalist Vol. 15, No. 3 70, period 2002–2006). Concentrations approximate a log-normal distribution (Fig. 1). Compared with WHO recommendations for chronic exposure in drinking-water safety of 1 μg L-1, 71% of cyanobacteria bloom samples tested exceeded this threshold. Considering the recreational water safety recommendation of NHMRC (2005) with exposure to less than 10 μg L-1 microcystins protective of children, 31% of bloom-related samples exceeded this recommended guidance value. On an annual basis, between 25% and 60% of cyanobacteria bloom samples tested exceeded 10 μg L-1. A positive log-linear relationship was evident between Microcystis cell counts and microcystin toxin concentrations (Fig. 2) (Log10microcystin = 0.53*log10Microcystis cells ml-1 - 1.84, r2 = 0.42, n = 40). The relationship is characterized by a consistent 2–3 order of magnitude range of toxin values relative to any given Microcystis cell count across the density gradient. The data show toxin assays are better than cell counts for risk assessment, but cell counts provide a tool for making an initial assessment of the risk situation. Used together, the cell counts and toxin assays are valuable for management assessments. Spatially, tributaries with recurrent cyanobacteria blooms in tidal fresh and oligohaline habitats demonstrated toxin production, with levels exceeding the 10-μg L-1 threshold (Fig. 3). While all samples tested positive for microcystin, a subset was further tested for neurotoxins. Anatoxin-a was found in 19 of 41 (46%) such samples, and concentrations ranged from 5 x 10-4 to 3 μg L-1 (Fig. 1). Lowest levels were associated with Bay island pond samples, while highest concentrations Figure 1. Frequency distribution for concentrations of microcystin (n = 70) and anatoxin-a (n = 20), Chesapeake Bay 2000–2006. 2008 P.J. Tango and W. Butler 409 were found in tributary samples. There is no clear safety guidance available for anatoxin-a at this time. Samples exceeding 1 μg L-1 are considered of concern for elevated human health risk. There are important management considerations regarding possible interactive effects with exposure to multiple toxins. Investigation of health effects due to simultaneous exposure to more than one cyanotoxin remains in its infancy. Cyanobacteria bloom impacts Recognition of cyanobacteria bloom events with associated toxin measurements since 2000 has resulted in public beach closures on the Sassafras River (Tango et al. 2008) and the Potomac River (cited in Marshall et al. 2008). Health advisories for recreational activities were again issued in Maryland for a toxic bloom on the Transquaking River (2005) and the Potomac River (2006). Investigations of blooms have taken place in response to human-health reports of skin rashes, nausea, fever, and vomiting in citizens recreating on bloom-affected waters. Bird kills were notably investigated in 2001 in the Kent Island, MD area and were linked with cyanotoxin exposure and accumulations (Driscoll et al. 2002). A fish kill Figure 2. A positive log-linear relationship was evident between Microcystis cell counts and microcystin toxin concentrations for Maryland-based data (Log10microcystin = 0.53*log10Microcystis cells ml-1 - 1.84, r2 = 0.42, n = 40). Curved lines above and bleow the regression line are 95% confidence intervals 410 Northeastern Naturalist Vol. 15, No. 3 of approximately 1800 fish on the Bush River, in July 2003, largely Dorosoma cepedianum Lesueur (Gizzard Shad) was investigated coincident with a toxic cyanobacteria bloom dominated by Microcystis aeruginosa (1.6 x 107 cells ml-1) and subdominated by Anabaena spp. (2.6 x 105 cells ml-1). Unfortunately, the fish were advancing in their state of decomposition and not suitable for further testing. Extensive blooms such as those observed on the Sassafras River in 2003 (http://mddnr.chesapeakebay.net/ hab/news_7_29_03.cfm) and Potomac River in 2004 (http://mddnr.chesapeakebay. net/hab/news_072204.cfm) represent obvious reduction in light resources to submerged aquatic vegetation. The events have been associated with Microcystis cell densities exceeding 1 million cells ml-1 and the production of thick surface scums. Discussion Toxigenic cyanobacteria blooms presently represent the most-significant plankton-related, annually recurrent risk to human health in Chesapeake Bay. Such toxic blooms represent one of a suite of important plankton-based symptoms affecting overall water quality and living resources across the estuary. HABs in general represent a significant and expanding threat to Figure 3. Spatial distribution of cyanobacteria toxin concentrations found in Chesapeake Bay. 2000–2006. “Star” denotes location of findings from Marshall et al. (2008); “Cross” denotes findings associated with Driscoll et al. (2002). “Triangle” represents the first reports for microcystins in 2000 (n = 4) on the Sassafras River that were provided in μg g-1 dry weight. 2008 P.J. Tango and W. Butler 411 aquatic life, human health, and regional economies (Ramsdell et al. 2005). As long as eutrophic symptoms continue in the Chesapeake Bay, the persistence of cyanobacteria blooms will remain a signature indicator of impaired Bay health, helping target restoration activities. The relationship between microcystin and Microcystis in this study expresses a continuous trend complimenting the underlying categorical gradient approach frequently recommended for risk management (e.g., World Health Organization [WHO] in Chorus and Bartram 1999 and NHMRC 2005). Real time, in situ toxin monitors, or inexpensive field tests with high precision are not yet widely available for most phycotoxins. Graham et al. (2006) found clear associations between particulate microcystin, cyanobacteria, and the environment. Chorus and Bartram (1999) also indicate work in Germany that showed relatively stable toxin quota within species that would support such a relationship. Graham et al. (2006) further noted that in field experiments, microcystin never responded independently of net chlorophyll, but that light and nutrients influenced microcystin indirectly by influencing cyanobacteria biomass rather than intracellular toxin production. It is acknowledged that some studies do not find consistent relationships between toxin levels and cyanobacteria measures. Toxin levels have been shown to vary with season, environmental conditions, population growth phases, and genetics surrounding proportions of toxic and non-toxic strains present in the population (Sivonen and Jones 1999). Toxin assays are better than cell counts for risk assessments. For management, the option of estimating toxin content from measures of dominant cyanobacteria can still be helpful (Chorus and Bartram 1999). Consistent results between cell counts and microcystin here provide one valuable basis in support of management needs and risk evaluations in Chesapeake Bay. Both approaches should be used together for the best risk assessments. These approaches are further improving our understanding of the Chesapeake Bay ecosystem. There is a need for the scientific community to identify a commonly acceptable threshold for recreational safety for microcystin and the methods to make that assessment. While the NHMRC (2005) has proposed a 10-μg L-1 threshold for microcystins for waterway closures by management agencies, Stone and Bress (2007) show that US State agencies are imposing even more conservative measures. Vermont has recently selected 6 μg L-1 to represent significant risk level to human and animals, and Oregon uses cyanobacterial cell counts that are estimated to correlate with 8 μg L-1 for guidance to post health advisories on recreational waters (Stone and Bress 2007). Nebraska conducts weekly monitoring of lakes in the summer season and closes lakes to swimming based on a 20-μg L-1 total microcystin threshold (J. Lund, Nebraska Department of Environmental Qaulity, Lincoln, NE, pers. comm.). Maryland provides immediate citizen advisories based on cell counts, although advisories may be extended when results of toxin analyses indicate 412 Northeastern Naturalist Vol. 15, No. 3 microcystin concentrations in excess of 10 μg L-1. Standardization of toxin thresholds can improve management decisions. Additionally, interactions among toxins may lead to synergistic effects (Fitzgeorge et al. 1994) that would require consideration for more conservative thresholds when multispecies co-dominance in blooms is evident. There is an increasing appreciation for the growing list of both toxigenic prokaryotic and eukaryotic plankton species across Chesapeake Bay habitats that include not only cyanobacteria but also diatoms, dinoflagellates, and raphidophytes (Marshall et al. 2005a). Further, there is an expanding understanding about the variety of their now-recognized toxic activities, helping to explain a range of past and present ecosystem events (e.g., Deeds et al. 2002, Goshorn et al. 2004, this paper). Characterizing plankton-related impacts and risks associated with cyanobacteria blooms with continuing eutrophic conditions around Chesapeake Bay remains an important step in understanding how to target restoration activities. These results provide a new context for previously reported incidental cyanotoxin findings and illustrate a broad geographic distribution of significant toxic activity. Results show links with a diversity of environmental impacts. Demonstration of toxin diversity and concentrations significant to suggested public safety guidance thresholds further establishes a foundation for additional research and management while improving our understanding of the Chesapeake Bay ecosystem. Acknowledgments The authors acknowledge the field monitoring, lab, and office personnel involved in conducting long-term water quality and HAB-specific response monitoring, data, and grant management from Maryland Department of Natural Resources (MD DNR), Maryland Department of the Environment, Maryland Department of Health and Mental Hygiene, Virginia Department of Environmental Quality, University of Maryland Biotechnology Institute Center of Marine Biotechnology, Johns Hopkins University, and Morgan State University Estuarine Research Center. We thank Wayne Carmichael of Wright State University and Greg Boyer of SUNY College of Environmental Science and Forestry and their respective laboratories for toxin analyses. Funding for water-quality monitoring, and living resource and toxin analyses has been provided by the State of Maryland, US Environmental Protection Agency, US Centers Disease Control, and National Oceanic and Atmospheric Administration (NOAA) CSCOR Program. 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