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Atwood, Karen E.
Brevetoxin body burdens in seabirds of Southwest Florida
h [electronic resource] /
by Karen E. Atwood.
[Tampa, Fla.] :
b University of South Florida,
ABSTRACT: Harmful algal blooms (HABs, or "red tides") of the brevetoxin-producing dinoflagellate Karenia brevis occur periodically along Florida's Gulf coast. Mass mortalities of marine birds have long been associated with these blooms, yet there are few data documenting the accumulation of brevetoxins (PbTx) in the tissues of birds. Post-mortem evaluations were performed on 185 birds representing 22 species collected from October 2001 through May 2006 during red tide and non-red tide events to quantify their body burdens of brevetoxins. A variety of tissues and organs were selected for brevetoxin analysis including blood, brain, heart, fat, stomach or gut contents, intestinal contents or digestive tract, muscle, lung, liver or viscera, kidney, gonads, gallbladder and spleen. Brevetoxin levels in avian tissues ranged from < LD (below level of detection) to 9989 ng/g PbTx, with the highest levels generally found in liver, gall bladder, stomach and intestinal contents of affected birds. These results indicate that marine birds are exposed to a range of levels of brevetoxin in their diet during blooms of K. brevis which may amass in various tissues of the body. As a consequence, the birds may exhibit acute brevetoxicosis during red tide events or show chronic accumulation effects during non-red tide events.
Thesis (M.S.)--University of South Florida, 2008.
Includes bibliographical references.
Text (Electronic thesis) in PDF format.
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Co-adviser: Gabriel Vargo, Ph.D.
Co-adviser: Pamela Hallock Muller, Ph.D.
Harmful algal blooms.
Neurotoxic shellfish poisoning.
x Marine Science
t USF Electronic Theses and Dissertations.
Brevetoxin Body Burdens in Seabirds of Southwest Florida by Karen E. Atwood A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science College of Marine Science University of South Florida Co-Major Professor: Gabriel Vargo, Ph.D. Co-Major Professor: Pamela Hallock Muller, Ph.D. Jerome Naar, Ph.D. Date of Approval: March 28, 2008 Keywords: Karenia brevis, harmful algal blooms, HABs, red tide, neurotoxic shellfish poisoning Copyright 2008, Karen E. Atwood
DEDICATION This study is dedicated to Skipper, a liv ely, sweet double crested cormorant who stole my heart but later died from brev etoxicosis at the Save Our Seabirds rehabilitation center in 2001
ACKNOWLEDGEMENTS I would like to thank my family for their support and Michelle van Deventer for all of her hard work in the laboratory. I w ould also like to thank my committee and my fellow employees at the Florida Fish and Wildlife Research Institute for all of their encouragement through this arduous pr ocess. I would especially like to thank Lee Fox, Executive Director of Save Our Seabirds, who showed me that one person can make a difference and to Leanne Flewelling who encouraged me every step of the way. I would also like to thank all of the groups who provided bird collections including SEANET Beached Bird Survey of Shell Key, the St. Petersburg Audubon/Eckerd College Least Tern Nesting Study, Peace River Wildlife Center, The Pelican Man Sanctuary, Save Our Seabirds (SOS), T he Center for Rehabilitation of Wildlife (CROW), The Wildlife Center of Venice (WCV) and Suncoast Seabird Sanctuary (SSS). Profuse gratitude goes to funding institutions including the Tampa Bay Parrot Heads, the Florida Department of Environmental Protection (Award # DO157160), and the Florida Department fo r Health and Centers for Disease Control.
i TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES vi ABSTRACT vii INTRODUCTION 1 METHODS 12 Necropsies 12 Toxin Analysis 12 Extraction Methods for Blood Samples 12 Extraction Methods for Tissue Samples From 2001-2005 13 Extraction Methods for Tissues Samples From 2006-Present 13 ELISA Brevetoxin Methods 14 Karenia brevis cell counts 14 RESULTS 15 Cormor ants (Phalacrocotacidae) 19 Gulls (Laridae) 22
ii Herons and Egrets (Ardeida) 23 Loons and Gannets (Gavii dae and Sulidae) 24 Terns (Sternidae) 25 Pelicans (Pelecanidae) 26 Shorebirds (Laridae, Scol opacidae and Rallidae) 27 Other (Pandionidae, Gr uidae and Ciconiidae) 28 Collection Dates, Locations and Brevetoxin Cell Counts 28 DISCUSSION 36 CONCLUSIONS 47 MANAGEMENT IMPLICATIONS AND AREAS OF FUTURE RESEARCH 49 REFERENCES CITED 51 APPENDICES 56 Appendix 1. 471 years of documented red tide events off of Floridas west coast as shown on the FWRI website. 57 Appendix 2. Brevetoxin levels found in the blood serum samples from 57 double crested cormorants released from the Suncoast Seabird Sanctuary in April of 2006 after treatment for brevetoxicosis symptoms. 58 Appendix 3. Specific results of samples taken for each of the 185 birds used in the study referenced by identification number. 60
iii Appendix 4. Identification num bers for each of the 185 birds used in the study with common name, collection date, region collected, history and miscella neous comments listed. 65 Appendix 5. Date, location and brief summary of Karenia brevis cell count data collected by FWRI. 87
iv LIST OF TABLES Table 1 Illnesses associated with HABs in humans. 2 Table 2 The number of cormorants admitted per year at the Suncoast Seabird Sanctuary. 8 Table 3 List of all species of birds used for toxin assays by group and family and the total number of each species tested. 16 Table 4 Species of birds tested showing general habitat and diet type. 17 Table 5 The fraction of sample types from cormorants that were positive for brevetoxin and the range of toxin levels found in each sample type. 20 Table 6 The fraction of sample types from multiple gull species that were positive for br evetoxin and the range of toxin levels found in each sample type. 22 Table 7 The fraction of sample types from multiple heron and egret species that were positive for brevetoxin and the range of toxin levels found in each sample type. 23
v Table 8 The fraction of sample types from loons and gannets that were positive for brev etoxin and the range of toxin levels found in each sample type. 24 Table 9 The fraction of sample types from multiple tern species that were positive for br evetoxin and the range of toxin levels found in each sample type. 25 Table 10 The fraction of sample types from multiple pelican species that were positive for brevetoxin and the range of toxin levels found in each sample type. 26 Table 11 The fraction of sample types from multiple shorebird species that were positive for brevetoxin and the range of toxin levels found in each sample type. 27 Table 12 The fraction of sample types from other bird species that were positive for brevetoxin and the range of toxin levels found in each sample type. 28 Table 13 Toxin content and bloom distribution for 2001 thru 2004. 29
vi LIST OF FIGURES Figure 1 Karenia br evis SEM photograph. 5 Figure 2 Annual total of double crested cormorants admitted to the Suncoast Seabird Sanctuary plotted per year from 1982-2005 and Karenia brevis bloom duration. 9 Figure 3 The number of birds which tested positive or negative for brevetoxin content. 17 Figure 4 The types of samples which tested positive or negative for brevetoxin content. 19 Figure 5 Brevetoxin levels of blood serum taken from 57 double crested cormorants on their release date from a rehabilitation center in April of 2006. 21 Figure 6 Red tide counts taken by The Florida Wildlife Research Institute from October 29 through November 1, 2001 as represented on the FWRI website. 30 Figure 7 A map of Florida showing the counties. 31
vii Figure 8 Red tide counts taken by The Florida Wildlife Research Institute from February 11 through 15, 2002 as represented on the FWRI website. 32 Figure 9 The number of birds collected for analysis by month and year compared to the average level of bloom presence detected through cell counts by FWRI. 34 Figure 10 Highest brevetoxin concentrations found by tissue sample type and species in logarithmic scale. 35 Figure 11 The highest concentration of brevetoxin found in each type of tissue tested in each group of birds. 39 Figure 12 The average concentration of brevetoxin found in each type of tissue tested in each group of birds. 40 Figure 13 Birds by group compared to the highest concentration of brevet oxin (ng/g) found in a sample from that group. 44 Figure 14 Birds by group compared to the average concentration of brevet oxin (ng/g) found in a sample from that group. 45
viii Brevetoxin Body Burdens in Seabirds of Southwest Florida Karen E. Atwood ABSTRACT Harmful algal blooms (HABs, or red tides) of the brevetoxin-producing dinoflagellate Karenia brevis occur periodically along Floridas Gulf coast. Mass mortalities of marine birds have long been associated with these blooms, yet there are few data documenting the accumu lation of brevetoxins (PbTx) in the tissues of birds. Post-mortem evaluations were per formed on 185 birds representing 22 species collected from October 2001 through May 2006 during red tide and nonred tide events to quantify their body burdens of brevetoxins. A variety of tissues and organs were selected for brevetoxin analysis including blood, brain, heart, fat, stomach or gut contents, intestinal contents or digestive tract, muscle, lung, liver or viscera, kidney, gonads, gallbladder and spleen. Brevetoxin levels in avian tissues ranged from
ix may amass in various tissues of the body. As a consequence, the birds may exhibit acute brevetoxicosis during red tide events or show chronic accumulation effects during non-red tide events.
1 INTRODUCTION Microalgae or phytoplankton are singl e-celled photosynthetic organisms that make up the lowest trophic level of aquatic ecosystems. Of the thousands of species of marine algae found in marine bodies throughout the world, a small number are known to produce chemicals that are toxic to other organisms including fish, shellfish, birds, mari ne mammals and humans (Creekmore, 2001). When these toxic microscopic algae in seawat er proliferate to higher than normal concentrations, they are called Red Tide or Harmful Algae Blooms (HABs) due to the water discoloration they often cause, in cluding colors of red, brown, green or yellow (Anderson, 1994). There are many types of HABs f ound throughout the world in various locations, which can range from recurrent in some areas to episodic or persistent in others (Shumway et al., 2003). The most common toxins involved in these events include domoic acid, saxitoxin, brevetoxin, okadaic acid and ciguatoxins (Table 1).
2Table 1. Illnesses associated with HABs in humans. (Morris, 1999; Anderson et al., 2001; Shumway et al., 2003) Illness Causative organism Associated toxin Clinical symp toms Paralytic shellfish Alexandrium spp., Gymnodinium Saxitoxin & derivatives Neurological manifestations, poisoning (PSP) catentatum, Pyrodinium bahamese respiratory distress, muscular paralysis & death Neurotoxic shellfish Karenia (Gymnodinium) brevis Brevetoxins Gastrointestinal & neurological poisoning (NSP) symptoms, respiratory & eye irritation Diarrhetic shellfish Dinophysis spp., Prorocentrum spp. Okadaic acid & Acute gastroenteritis poisoning (DSP) dinophysis toxins Amnesic shellfish Pseudonitzschia spp. Domoic acid & isomers Gastroenteritis, neurological poisoning (ASP) symptoms leading to severe amnesia & permanent short-term memory loss, coma & death Ciguatera fish Gambierdiscus toxicus Ciguatoxins Gastrointestinal & neurological poisoning symptoms Paralytic Shellfish Poisoning (PSP) is caused by the consumption of shellfish that have been cont aminated with the saxitoxi ns which are produced by several species of dinoflagellate organism s. The effects are neurological and are very fast acting, and can include numbness, loss of coordination, difficulty in breathing, nausea, vomiting, dizziness, loss of sight and headaches. PSP has been found in shellfish on the west and eas t coasts of North America and most reports refer to incidents involving human illness and sometimes human death.
3 During the first recorded outbreak of a to xic dinoflagellate in Massachusetts in 1972, one dead and several sick gulls were noted and a kill of about 100 birds including black ducks and gulls were reported (Shumway et al., 2003). Autopsies showed hemorrhaging as has been seen in other episodes of PSP poisoning, the causative organisms were identified as Gonyaulax tamarensis and toxic shellfish was found in some gut contents of dead birds (Bicknell & Collins, 1972). In November 1987, fourteen dead humpback whales (Megaptera novaeangliae) washed ashore in Massachusetts along Cape Cod Bay and Nantucket Sound. The whales had eaten atlantic mackerel (Scomber scombrus) which tested positively for the PSP toxin (Geraci et al., 1989; Anderson & White, 1992). Amnesic Shellfish Poisoning (ASP) is caused by ingestion of clams, mussels or crabs contaminated with Do moic acid, a neurotoxic produced by several species of pinnate diatoms. E ffects include memory loss, brain damage and even death, as in the case of over 400 sea lions off of the Central Californian coast in 1998. Birds have also been affected as can be seen from the deaths of large numbers of brown pelicans ( Pelecanus occidentalis ) and double crested cormorants ( Phalocrocorax aurelius) in Monterey Bay, CA, in 1991 (Wright & Quilliam, 1995). Diarrhetic Shellfish Poisoning (DSP) is caused by eating shellfish that has been contaminated with Okadaic acid or ot her related toxins. Effects include gastrointestinal distress including di arrhea, nausea, vomiting, chills and abdominal cramps. No human deaths have been reportedly caused by DSP and most symptoms pass within three days. Large and unexplained die-offs of loons
4 in Long Island waters, as well as bird deaths in Europe in the summer of 2002, have been attributed to DSP (Shumway et al, 2003). Although there are over 40 toxic species of algae which live in the Gulf of Mexico, the most common in the Tam pa Bay Florida region is the unarmored toxic dinoflagellate Karenia brevis (Figure 1). Karenia brevis produce brevetoxins, which can cause fish k ills and other marine animal mortalities including birds, manatees and dolphins. Karenia brevis can also cause filterfeeding animals such as oysters or clams to become toxic to humans (i.e., NSP) and cause an air-borne toxin (Steidinger et al., 1998). In addition to the Tampa Bay area, K.brevis has been found throughout the Gulf of Mexico, including along the coasts of Mexico, Texas and Louisiana, along the east coast of Florida and as far north as North Carolina (Steidinger et al., 1998). Karenia brevis is found year round throughout the Gulf of Mexico at concentrations of about 1,000 cells per liter or less and usually blooms in the late summer or early fall. Kim and Martin (1974) found that Karenia brevis thrives in salinity ranges of 30-34 PSU, but can tolerate a wide salinity range (22-39 PSU). Karenia brevis also survives most water temperatures common in t he Gulf of Mexico (Kamykowski, 1981). The primary means of reproduction of t hese organisms is by simple asexual fission, which can increase these blooms to very high concentrations (Anderson, 1994).
5 Figure 1. Karenia brevis SEM photograph. K. brevis cells usually average 20 microns in width. Courtesy of the HAB lab at FWRI. Florida red tide events have been known to cause marine animal deaths since the 1500s (Appendix 1), including episodes of high fish and bird mortality as well as human respiratory illnesses. High mortality rates have also been seen in bottlenose dolphins, turtles and manatees (Fairey et al., 2001). In humans, the effects of NSP include gastrointestinal and neurological symptoms comprising of dizziness and seizures as well as headaches, diarrhea and muscle or joint pain. Symptoms can include difficulty in breathi ng, altered perceptions of hot and cold and double vision. When the toxin becomes airborne in sea spray, asthma-like symptoms in humans have also been documented. Aerosol effects have also been recorded in marine wildlife, including manatees (Bossart et al., 1998) and double crested cormorants (Kreuder, et al., 2002).
6 Brevetoxins act by binding to a spec ific site near voltage-gated sodium channels and then allow an unchecked flow of Na+ ions into and out of the cells. This disruption of ion flow is responsib le for the neurological effects that have been associated with NSP. There seem to be several pathways in which brevetoxins can accumulate in marine wildlife: 1. Aquatic organisms can become contami nated through the direct ingestion of cells, such as the case for filter feeders like sponges, mollusks and crustaceans. In fact, shellfish are often used as an indicator of HAB occurrence in an area and are studied for toxin absorption and retention (Shumway, 1990). 2. Marine life can also be exposed to waterborne toxins after cell lysis caused from wave action. This can happen to birds such as double crested cormorants ( Phalocrocorax auritus) common loons ( Gavia immer ) and red breasted mergansers ( Mergus merganser ) that swim underwater to catch their prey. 3. Marine animals may also be contaminated through aerosolized toxins that can cause respiratory irritation in mammals (e .g., manatees and birds) (Bossart et al., 1998; Kreuder et al, 2002). This can happen when an organism comes up for air in the middle of a bloom or when birds fly over a bloom. Kreuder et al. (2002) reported that between 1995 and 1999, 360 birds showing signs of toxin contamination were admitted to a rehabilitat ion center off of the southwest coast of Florida at the same time that high levels of K. brevis were reported in the area. Brevetoxins were found in the spleens and lungs of all four double crested cormorants tested, which could indicate inhalation as an exposure route.
7 4. Marine life can also be exposed to to xins through the ingestion of other organisms in which the toxin has bioaccumulated (e.g., bivalves such as crustaceans and gastropods) or through bioaccumulation in the organisms own body. The majority of sea birds reportedly sick from brevetoxins are admitted during times in which large HAB blooms have been reported in the area. Save Our Seabirds, Inc. treats about 350-400 bi rds annually and sees anywhere from 12-25 birds exhibiting brevetoxicosis. T he criteria used to diagnose patients with brevetoxicosis at local avian rehabilitat ion centers include states of seizures, shaking, inability to stand, weakness, slumping of the head, nasal discharge, dehydration, reduced body mass or atrophi ed musculature in comparison to a healthy individual (personal communication, Lee Fox). The birds diagnosed with brevetoxin poisoning are treated using a protocol which has a reported 90% success rate (pers. Comm., Lee Fox). Suncoast Seabird Sanctuary (SSS), a large sea bird rehabilitation center in the Redington Beach area of Tampa Bay, treats about 10,000 birds annually. SSS provided data on double crested cormorants admitted to the facility since 1982 (Table 2). Peaks in the numbers of admitted birds tend to occur in years when major red tides were documented (Appendix 1, Figure 2 and Table 2).
8Table 2. The number of cormorants admitted per year at the Suncoast Seabird Sanctuary. Data received from Barbara Suto, head Wildlife Biologist of the sanctuary. See Figure 2 for a plotted graph of same data. Compare to Appendix 1 which shows a pattern similar to the interannual differences in red tide events. Year Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total 1982 19 36 22 31 21 24 8 18 10 17 24 32 262 1983 14 17 27 8 7 3 5 4 8 10 53 27 183 1984 23 19 68 18 19 14 14 7 8 4 6 13 213 1985 7 13 10 15 7 10 20 7 6 11 37 12 155 1986 9 15 22 12 7 1 7 4 5 35 33 8 158 1987 13 19 25 7 13 41 33 16 12 13 16 10 218 1988 7 2 10 17 12 17 20 19 15 19 28 15 181 1989 58 23 114 68 14 18 13 5 8 9 11 1 342 1990 7 45 89 30 17 8 18 14 11 4 18 5 266 1991 15 8 25 32 12 9 7 18 39 53 28 18 264 1992 7 16 26 20 16 8 14 11 10 17 11 3 159 1993 23 6 7 15 7 6 6 18 16 13 18 2 137 1994 3 7 9 9 13 12 14 15 11 41 75 20 229 1995 8 2 23 14 15 52 18 27 12 35 12 15 233 1996 53 76 129 114 15 6 5 6 7 15 13 4 443 1997 8 19 58 21 7 10 10 7 7 14 9 5 175 1998 19 13 10 12 5 6 8 8 7 11 24 14 137 1999 6 8 15 12 11 12 7 6 8 21 25 19 150 2000 10 2 31 31 9 15 21 10 23 29 34 7 222 2001 1 19 37 28 18 17 15 11 13 55 73 116 403
9 For example, in January through June, as well as August and October through December, 1982, 17 or more double crested cormorants per month were admitted at Suncoast Seabird Sanct uary, which corresponded with red tide events reported in the area in January through April and July through October; red tide events were suspected but not c onfirmed in May and June of 1982. This trend is seen again in the following per iods: November and December 1983, January through May 1984, November 1985, October and November 1986, January through July 1987. Sporadic coinci ding months are seen in 1988, 1989, 1990 and 1991, almost all of 1995, the first half of 1996 and early 1997, as well as towards the end of 2001. High year ly averages of affected double crested cormorants also coincide with years of high blooms (Figure 2). 0 50 100 150 200 250 300 350 400 450 5001982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005yearnumber of birds collected (bars)0 2 4 6 8 10 12number of months blooms present (line) Figure 2. Annual total of double crested cormorants admitte d to the Suncoast Seabird Sanctuary plotted per year from 1982-2005 and Karenia brevis bloom duration. Double crested cormor ant data received from Barbara Suto, head Wildlife Biologist of the sanctuary. Bloom duration courtesy of FWRI.
10 Reports are not available for t he total numbers of double crested cormorants admitted during red tide events from The Pelican Man Sanctuary in Sarasota, FL, a rehabilitation center fo r wild birds which treats about 6-7000 birds annually. However, workers at the facility have given anecdotal reports that many birds, mostly double crested cormorant s, have been seen with breveitoxicosis symptoms during red tide events in the area. Brevetoxins have also been implicated in high mortality rates among many other species of marine life. Mortalitie s of frigate birds, terns, gulls, ducks and vultures from Tampa Bay to Key West were reported by Glazier (1882), Moore (1882) and Walker (1884). Dead double crested cormorants ( Phalocrocorax auritus) ducks, frigate birds (Frigata magnificens ), gulls, terns and vultures due to K. brevis were reported in 1973 off of the coast of Florida (Steidenger et al., 1973). Large number of lesser scaup ( 12,000-20,000) and some double crested cormorants and red breasted mergansers di ed during red tides in the Tampa, Florida area in 1975 (Quick & Henderson, 1975). Even though HABs have historica lly been natural, the frequency of occurrence and intensity of some blooms throughout the world seems to have increased in recent decades (Shumway, 1990; Smayda, 1990; Hallagraeff, 1993; Burkholder, 1998; Shumway et al., 2003). Natural events like hurricanes can dilute or terminate toxic algae blooms. Algae can also be transported in ship ballast waters. Hallagraef (1993) postula ted that agricultural runoff into the oceans and other pollutants dispersed into the environment by human activities (including human sewage) has result ed in increased nutrient loading of
11 phosphorus and nitrogen, which can provide conditions favorable to the growth of HABs. Clearly, HABs are adversely affecti ng marine life in many areas around the world and in particular, the Tampa Bay ar ea of Florida. Research in this field of study is direly needed and plainly an opport unity presents itself to study birds coming into the rehabilitation centers in the Tampa Bay area. It is surprising, considering the impacts these toxins have on seabirds, that these types of studies have not been undertaken previously, since sea birds are among the most valuable indicators of environmental problems due to their sensitivity to environmental pollutants (Swennen, 1997; Boersma 1978, 1986). The objectives of my study were: 1. Determine the levels of brevetox ins present in the blood and tissues of various species of sea birds during bloom periods. 2. Compare the capture location and brev etoxin levels in birds with the timing and location of K. brevis blooms to assess any potential inter-relationships.
12 METHODS Necropsies Bird carcasses and blood samples are obtained from local rehabilitation centers, including Suncoast Seabird Sanctuary in Indian Rocks Beach, The Center for Rehabilitation of Wildlife in S anibel, The Wildlife Center of Venice and Save Our Seabirds in Wimauma. Birds ar e stored at -20 C until necropsied. Necropsies are performed following procedur es outlined in The Avian Necropsy Manual by Work (2000). Subsamples of tissues are taken after homogenization and then refrozen until extraction. Toxin Analysis Extraction M ethods for Blood Samples. Blood samples are taken by the staff at the rehabilitati on centers and are placed in either heparanized or nonheparinized glass tubes. Heparinized tubes are kept at room temperature and processed as soon as possible (withi n a few hours). Non-heparinzed samples are refrigerated until processed. The sa mples are centrifuged at 3000 rpm for 15 minutes. The resulting serum is then collected and analyzed directly using the ELISA method developed by Naar et al. (2002).
13 Extraction Methods for Tissue Samples From 2001 to 2005. Three grams of each tissue type were weighed and added to 10 ml of acetone in a 50 ml Falcon tube, then placed on a plate shaker for 30 minutes. The sample was centrifuged at 3000 rpm for 15 minutes and the acetone phase was separated from the tissue. This process was r epeated in the same condition and acetone phase were combined in a separate Falcon tube. The combined acetone extract was the dried and resuspended in 8 ml of MeOH plus 1.5 ml of nanopure water. The aqueous methanol extract was defated using 5 ml of hexane. After hexane addition, the falcon tube was shaken and vented 3 times, then allowed to settle. The resulting separated hexane was removed and the sample was dried. After drying, 3 ml of MeOH was added and the sample was refrigerated until analysis using the ELISA method. Extraction Methods for Tissue Samples From 2006 to Present. In 2006, a new extraction method was developed based on modifications suggested by Paul McNabb (personal comm unication). In this method, tissue samples are homogenized and a sub-sample of 2 grams is weighed out. Nine ml of 80% methanol is added and the sample is then heated at 60 C for 20 minutes. It is then centrifuged at 3000 rpm for 10 minutes and the resulting liquid poured into a new tube. The MeOH extraction is then repeated. Finally, 5ml of hexane is added to the sample, which is shaken and centrifuged at 3000 rpm for 10 minutes. The resulting bottom layer is removed and analyzed in the ELISA method.
14 ELISA Brevetoxin Methods. ELISAs (enzyme-linked immunosorbant assays) are widely used in both clinical and research fields to help rapid, simple, accurate and specific quantitation of m any biological small molecules. For brevetoxins, we are using a brevetoxin competitive ELISA developed by Naar et al. (2002). The original protocol was shor tened for use in the FWRI laboratory by Naar (personal communication). For this assay, samples and controls compete with plate-bound brevetoxin for goat antibrevetoxin antibodies. The antibodies bound to the plate are then visua lized using an HRP-conjugated secondary antibody (rabbit anti-goat antibodies), and the HRP substrate TMB (3,3 ,5,5 Tetramethylbenzidine). Absorbance of t he wells is read at 450 nm. The color intensity is inversely proportional to t he concentration of brevetoxins in the sample. This assay recognizes all congeners and metabolites of brevetoxin that have a PbTx-2-type backbone. Results are reported as nanograms brevetoxin per gram sample (ng/g PbTX) for tissues and nanograms brevetoxin per microliter (ng/ml PbTX) for blood serum samples. Karenia brevis Cell Counts Karenia brevis cell count data used in this study was obtained from the FWRI Harmful Algae Bloom Historical Dat abase, which is updated twice weekly with data collected as part of FWRI's R outine Red Tide Monitoring Program. Samples collected as part of the Routine Red Tide Monitoring Program are processed following the method reported in Naar et al., 2007. Identifications of Karenia brevis were made using Haywood et al., 2004 and Steidinger et al., 2008.
15 RESULTS A total of 185 birds representing 22 species were tested for the presence of brevetoxin (Table 3). To simplify anal ysis, the 22 species were split into 8 groups (Table 3). The groups were chosen on the animals feeding habitat and diet (Table 4). Data for each bird, incl uding identification number, collection date, collection location, tissues tested, specific test results, and history, are provided in Appendices 3 and 4. A variety of tissues and organs were selected for brevetoxin analysis and included blood, brain, heart, fat, stomach, or gut contents, intestinal contents or digestive tract, muscle, lung, liver or viscera, kidney, gonads, gallbladder and spleen. Not all of these tissues were tested for each bird and some types of tissues were tested more often than others, depending on availability. Of the 185 birds tested, 144 tested positive for at least one sample type (Figure 3). A total of 820 tissue and organ samples were analyzed. Of these, 391 were negative and 429 were positive. Gallbladders ( 20 out of 21) showed the highest percentage of positive results at 95% while lungs (19 of 72) showed the lowest percentage of positive results at 26% (F igure 4). Since there was generally considerable variation of brevetoxin content in tissues and organs, I have
16 examined each broad group (Table 3) to det ermine if certain types of birds contain more toxin than others or show a wider distribution in their tissues and organs. Table 3: List of all species of birds used for toxi n assays by group and family and the total number of each species tested. Group Family Common name Species name Number tested Cormorants Phalacrocoracidae double crested corm orant Phalacrocorax auritus 101 Gulls Laridae herring gull Larus arentatus 1 laughing gull Larus atricilla 8 Herons & Egrets Ardeida great blue heron Ardea herodias 6 great white heron Ardea herodias (white morph) 3 green heron Butorides virescens 6 yellow crowned-night heron Nycticorax mauritianus 3 great egret Ardea alba 2 Loons & Gannets Gaviidae common loon Gavia immer 4 Sulidae northern gannet Morus bassanus 5 Terns Sternidae least tern Sternula antillarum 11 royal tern Thalasseus maximus 6 sandwich tern Thala sseus sandvicensis 1 Pelicans Pelicanidae brown pelican Pe lecanus occidentalis 12 white pelican Elecanus erythrorhynchos 1 Shorebirds Laridae black skimmer Rynchops niger 1 Scolopacidae ruddy turnstone Arenaria interpres 1 sanderling Calidris alba 8 Rallidae sora rail Porzana carolina 1 Other Pandionidae osprey P andion haliaetus 2 Gruidae whooping crane Grus americana 1 Ciconiidae wood stork Mycteria americana 1 TOTAL 185
17 0 10 20 30 40 50 60 70 80 90C o rmora n t s Gul ls H e rons & E g re t s Loons & Ga n ne t s Te r ns Pelicans Sho r eb ir ds Othe rbird groupingnumber tested negative positive Figure 3. The number of birds which tested positive or negative for brevetoxin content. Table 4 Species of birds tested showing general habitat and diet type. (adapted from Kaufman, 1996) Species Feeding habitat Diet Black skimmer Ocean beaches, inlets, tidewaters & es tuaries along the coast Mostly small fish & crustaceans Brown pelican Coastal marine & estuari ne environments Mostly fish, small marine invertebrates Common loon Coastal marine near shore areas & lar ge freshwater lakes & ponds Mostly small fish, aquatic vertebrates & invertebrates Double crested cormorant Ponds, lakes, rivers, lagoons estuaries & open coastline Fish, other aquatic animals, insects & amphibians Egret Wetlands, marshlands, swamps, streams, rivers, ponds, lakes, Fish, invertebrates, tidal flats, canals & flooded fields reptiles, birds & small mammals Northern gannet Offshore islands & marine coastlines, often well offshore Mainly fish & some squid
18 Table 4 (Continued) Great blue & white heron Calm, shallow freshwat er & seacoasts Fish, invertebrates, amphibians, reptiles, birds & small mammals Green heron Swamps, creeks, streams, marshes, ponds lake edges, salt Mostly small fish, marshes, ponds & pastures, winters in c oastal areas & mangrove invertebrates, frogs swamps & other small animals Herring gull Along beaches, mudflats & dumps Fish, marine invertebrates, insects, birds, eggs, carrion, garbage Laughing gull Along oceans, on rivers, at landfills & urban parks Aquatic & terrestrial invertebrates, fish, squid, garbage, flying insects & berries Least tern Seacoasts, beaches, bay s, estuaries, lagoons, lakes & rivers Small fish & some invertebrates Osprey Large bodies of water c ontaining fish including boreal fore st ponds, Almost entirely fish desert salt-flat lagoons, temperate lakes & tropical coasts Royal tern Along marine coastlines, sandy beaches & salt bays Fish & shrimp Ruddy turnstone Along rocky shores, s and beaches & mudflats Aquatic invertebrates & insects, carrion, garbage & birds eggs Sanderling Sandy beaches Aquatic & terrestrial invertebrates Sandwich tern Seacoasts, bays, estuaries, mud fl ats & occasionally ocean far Small fish & some from land invertebrates Sora Shallow wetlands Seeds & aquatic invertebrates White pelican Offshore large bodies of water often far from land Fish Whooping crane Freshwater marshes & prairies, shallow lakes, lagoons & saltwater Wide variety of marshes insects, fish, frogs & plant & animal matter, including mollusks, crustaceans, waste grain Wood stork Shallow wetlands Fish, amphibians, reptiles
19 Table 4 (Continued) Yellow crowned night heron Exposed tidal fl ats Crustaceans, water beetles, leeches, mussels, frogs & small fish 0 10 20 30 40 50 60 70blo o d brain heart fat stomach c o n t e nt/ g ut content intest in al content/ d ige s tive tract muscl e lun g liv e r/v is cer a ki d ne y gonad s gal lb ladder splee ntype of samplenumber tested negative positive Figure 4. The types of samples which tested positive or negative for brevetoxin content. Cormorants (Phalacrocoracidae) Of the 101 double crested cormorants test ed, 86 were positive for at least one sample type (Table 5). The fraction testing positive ranged from 54% in lung tissue to 100% of all gallbladders tested (n=12). Brevetoxin concentrations ranged from 0-9,989 ng/g with the highest value reported in a sample of stomach contents. It is interesting to note that the highest level found in any tissue sample in all of the groups tested was this spec ific stomach content. The four sample
20 types with the highest positive brevetoxin levels were gallbladder, stomach contents, intestinal contents and liver/v iscera, whereas the lowest positive brevetoxin levels were found in blood, brain, lung and muscle samples. Table 5. The fraction of sample types from cormorants th at were positive for brevetoxin and the range of toxin levels found in each sample type. Levels have been r ounded to whole numbers. Brevetoxin concentrations are given in ng/g or ng/ml. Sample type # positive over total % posit ive Range PbTXAverage PbTX Median PbTX total 86/101 85 0-9,989 98 10 blood 67/87 77 0-12 3 2 brain 16/25 64 0-65 14 12 heart 18/25 72 0-102 33 35 stomach contents 23/30 77 0-9,989 615 40 intestinal contents 20/24 83 0-2,645 172 60 muscle 18/26 69 0-97 40 45 lung 13/24 54 0-196 19 12 liver/viscera 19/27 70 0-198 63 62 kidney 16/25 64 0-105 31 35 gonads 12/14 86 0-87 48 58 gallbladder 12/12 100 20-949 410 446 spleen 6/11 55 0-84 29 12 In April of 2006, 57 double crested cormorants which had been captured and rehabilitated at the Suncoast Seabird Sanctuary for brevetoxicosis were released. Although original blood samples were not taken for these birds upon admission to the rehabilitation center, most blood samples taken upon release showed detectable levels of brev etoxin (Figure 5 and Appendix 2).
21 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5Jan 9 2005 Sep 28 2005 Oct 16 2005 Oct 23 2005 Nov 7 2005 Nov 7 2005 Nov 8 2005 Nov 15 2005 Nov 16 2005 Nov 20 2005 Nov 21 2005 Nov 22 2005 Nov 26 2005 Nov 27 2005 Nov 28 2005 Nov 29 2005 Dec 2 2005 Dec 16 2005 Dec 18 2005 Dec 19 2005 Jan 1 2006 Jan 2 2006 Jan 2 2006 Jan 2 2006 Jan 3 2006 Jan 5 2006 Jan 6 2006 Jan 7 2006 Jan 27 2006 Jan 10 2006 Jan 10 2006 Jan 10 2006 Jan 11 2006 Jan 11 2006 Jan 12 2006 Jan 12 2006 Jan 13 2006 Jan 14 2006 Jan 15 2006 Jan 16 2006 Jan 17 2006 Jan 19 2006 Jan 21 2006 Jan 21 2006 Jan 22 2006 Jan 24 2006 Jan 24 2006 Jan 24 2006 Jan 29 2006 Jan 29 2006 Jan 31 2006 Feb 7 2006 Feb 10 2006 Feb 21 2006 Feb 21 2006 Feb 28 2006 Feb 28 2006date admitted to facilitylevel of brevetoxin concentration (ng/ml) Figure 5. Brevetoxin levels of blood serum taken from 57 double crested cormorants on their release date from a rehabilitation center in April of 2006. The dates shown represent the date the animals were origin ally brought to the facility for treatment.
22 Gulls (Laridae) Of the 9 gulls from two different species tested for this project (Table 3), 7 were positive for at least one sample type (Table 6). There were no positive brevetoxin levels in either fat or brai n tissue samples. However, like double crested cormorants, all the gallbladders tested were positive (n=2). No blood samples were tested for this group. Br evetoxin concentrations ranged from 02,801 ng/g with the highest value reported in an intestinal content sample. The four sample types with the highest positiv e brevetoxin levels were gallbladder, intestinal contents, stomach contents and liver/viscera whereas the lowest positive brevetoxin levels were found in gonads, heart, lung and spleen. Table 6. The fraction of sample types from multiple gu ll species that were positive for brevetoxin and the range of toxin levels found in each sample type. Levels have been rounded to whole numbers. An asterisk denotes insufficient samples available to calculate. Brevetoxin concentrations are given in ng/g. Sample type # positive over total % posit iv e Range PbTXAverage PbTX Median PbTX total 7/9 78 0-2,801 264 8 brain 0/5 0 0 0 0 heart 1/7 14 0-32 5 0 fat 0/2 0 0 0 stomach contents 8/11 73 0-2,216 418 34 intestinal contents 4/9 44 0-2,801 574 0 muscle 3/9 33 0-177 37 0 lung 3/9 33 0-46 12 0 liver/viscera 6/9 67 0-1,355 290 14 kidney 5/8 63 0-398 91 16 gonads 1/5 20 0-10 2 0 gallbladder 2/2 100 897-2,099 1498 spleen 1/1 100 35 35
23 Herons and Egrets (Ardeida) Twenty birds from 5 species were tested for this group (Table 3), 12 of which were positive for brevetoxin (Table 7). Similar to double crested cormorants, no brevetoxin was detected in the single fat tissue sample. Like cormorants and gulls, 100% of the gallbladder tissue samples tested positive for brevetoxin (n=2), although, the highest level was seen in a stomach content sample (811 ng/g). The four sample types with the highest positive brevetoxin levels were gallbladder, stomach content, intestinal content and liver/viscera, whereas the lowest positive brevetoxin le vels were found in blood, brain, lung and muscle. Table 7. The fraction of sample types from multiple heron and egret species that were positive for brevetoxin and the range of toxin levels found in each sample type. Levels have been rounded to whole numbers. An asterisk denotes insufficient samples available to calculate. Brev etoxin concentrations are given in ng/g or ng/ml. Sample type # positive over total % posit ive Range PbTXAverage PbTX Median PbTX total 12/20 60 0-811 53 0 blood 1/3 33 0-2 1 0 brain 1/10 10 0-15 2 0 heart 6/13 46 0-36 10 0 fat 0/1 0 0 0 stomach contents 10/18 56 0-811 154 20 intestinal contents 7/13 54 0-511 117 40 muscle 5/15 33 0-32 7 0 lung 2/10 20 0-9 2 0 liver/viscera 9/17 53 0-296 71 0 kidney 4/11 36 0-108 17 0 gonads 1/3 33 0-39 13 0 gallbladder 2/2 100 8-315 162 spleen 2/4 50 0-35 15 13
24 Loons and Gannets (Gaviidae and Sulidae) Of the 4 loons and 5 gannets tested, a total of 8 were positive for at least one sample type (Table 8). There were no positive brevetoxin levels found in brain, stomach content and lung tissue samples. The fractions which did test positive ranged from 14% of the muscle tissue samples to 100% of the gallbladders (n=1) and gonads (n=1). Brev etoxin concentrations ranged from 060 ng/g with the highest value reported in an intestinal content sample. The four sample types with the highest positive brevetoxin levels were gallbladder, intestinal content, liver/viscera and gonads, whereas the four sample types with the lowest positive brevetoxin levels were muscle, blood, heart and spleen. Table 8. The fraction of sample types from loons and gannets that were positive for brevetoxin and the range of toxin levels found in each sample type. Levels have been rounded to whole numbers. An asterisk denotes insufficient samples available to calculate. Brevet oxin concentrations are given in ng/g or ng/ml. Sample type # positive over total % posit ive Range PbTXAverage PbTX Median PbTX total 8/9 89 0-60 6 0 blood 2/3 67 0-4 3 3 brain 0/4 0 0 0 0 heart 1/3 33 0-10 3 0 stomach contents 0/7 0 0 0 0 intestinal contents 1/5 20 0-60 12 0 muscle 1/7 14 0-6 1 0 lung 0/5 0 0 0 0 liver/viscera 6/8 75 0-25 17 17 kidney 2/7 29 0-19 4 0 gonads 1/1 100 14 14 gallbladder 1/1 100 58 58 spleen 1/2 50 0-8 4
25 Terns (Sternidae) Of the 18 terns from 3 species tested (Table 3), 11 were positive for at least one sample type (Table 9). There were no positive brevetoxin levels found in gonads and spleen tissue samples. Like all of the previous groups, 100% of the gallbladder samples were positive (n=1 ). Brevetoxin concentrations ranged from 0.0-4,400 ng/g with the highest value r eported in a stomach content sample. No blood samples were tested in this group. The sample types with the highest positive brevetoxin levels other than stomach content were intestinal content, gallbladder and liver/viscera, whereas the sample types with the lowest positive brevetoxin levels were found in brain, muscle, heart and lung tissue samples. Table 9. The fraction of sample types from multiple tern species that were positive for brevetoxin and the range of toxin levels found in each sample type. Levels have been rounded to whole numbers. An asterisk denotes insufficient samples available to calculate. Brevetoxin concentrations are given in ng/g. Sample type # positive over total % positiv e Range PbTX Average PbTX Median PbTX total 11/18 61 0-4,400 83 0 brain 1/7 14 0-8 1 0 heart 2/9 11 0-31 6 0 stomach content 9/20 45 0-4,400 302 0 intestinal content 3/4 75 0-465 131 30 muscle 3/12 25 0-14 3 0 lung 1/4 25 0-33 8 0 liver/viscera 7/20 35 0-141 19 0 kidney 2/8 25 0-90 13 0 gonads 0/3 0 0 0 0 gallbladder 1/1 100 83 83 spleen 0/1 0 0 0
26 Pelicans (Pelecanidae) Of the 13 pelicans tested, 8 birds tested positive for at least one sample type (Table 10). There were no positive brevetoxin levels found in muscle, lung and heart tissue samples and of the fractions that did test positive, 100% of the gallbladders were positive (n=2), much like all the previous groups. Brevetoxin concentrations ranged from 0-2,595 ng/g with the highest level reported in a stomach content sample. The sample ty pes with the highest positive brevetoxin levels other than stomach contents and ga llbladder were intestinal content and liver/viscera, whereas the sample types wit h the lowest positive brevetoxin levels were brain, blood, gonads and kidney. Table 10. The fraction of sample types from multiple pe lican species that were positive for brevetoxin and the range of toxin levels found in each sample type. Levels have been rounded to whole numbers. An asterisk denotes insufficient samples available to calculate. Brev etoxin concentrations are given in ng/g or ng/ml. Sample type # positive over total % positiv e Range PbTX Average PbTX Median PbTX total 8/13 62 0-2,595 54 0 blood 4/7 57 0-12 3 2 brain 2/9 22 0-10 2 0 heart 0/5 0 0 0 0 fat 1/2 50 0-12 6 stomach content 9/14 64 0-2,595 240 17 intestinal content 3/4 75 0-143 47 21 muscle 0/9 0 0 0 0 lung 0/9 0 0 0 0 liver/viscera 4/9 44 0-31 8 0 kidney 2/9 22 0-42 5 0 gonads 1/2 50 0-10 5 gallbladder 2/2 100 17-93 55 spleen 2/4 50 0-16 7 7
27 Shorebirds (Laridae, Scolopacidae and Rallidae) Of the 11 shorebirds tested from 4 species (Table 3), all were positive for at least one sample type (Table 11). There were no positive brevetoxin levels found in gonad or gallbladder tissue samples, contrary to all of the previous groups. No blood samples were tested for th is group. The fractions that did test positive ranged from 14% in lung tissue samples to 100% in liver/viscera (n=11), fat (n=1) and brain tissue samples (n=1). Brevetoxin concentrations ranged from 0-574 ng/g with the highest level reported in a stomach content sample. The tissues with the highest positive brevetoxin levels were liver/viscera, stomach content, intestinal content and kidney, whereas the tissues with the lowest positive brevetoxin levels were lung, muscle, brain and heart. Table 11. The fraction of sample types from multiple s horebird species that were positive for brevetoxin and the range of toxin levels found in each sample type. Levels have been rounded to whole numbers. An asterisk denotes insufficient samples available to calculate. Brevetoxin concentrations are given in ng/g. Sample type # positive over total % positiv e Range PbTX Average PbTX Median PbTX total 11/11 100 0-575 60 16 brain 1/1 100 6 6 heart 3/7 43 0-21 7 0 fat 1/1 100 8 8 stomach contents 8/9 89 0-575 94 40 intestinal contents 9/11 81 0-372 90 30 muscle 3/8 38 0-20 5 0 lung 1/7 14 0-25 4 0 liver/viscera 11/11 100 8-286 152 196 kidney 2/5 40 0-103 27 0 gonads 0/1 0 0 0 gallbladder 0/1 0 0 0 spleen 1/1 100 16 16
28 Other (Pandionidae, Gruidae and Ciconiidae) Of the 4 other birds tested from 3 species (Table 3), only 1 bird was positive, which, surprisingly, was t he wood stork (Table 12). There were no positive brevetoxin levels found in the brain, muscle, lung or kidney of that specific bird and although there were no gallbladders or blood samples tested, the tissue with the highest positive brevetoxin level was the liver/viscera (169 ng/g) whereas the lowest positive brev etoxin level was found in the stomach content. Table 12. The fraction of sample types from other bird sp ecies that were positive for brevetoxin and the range of toxin levels found in each sample type. Levels have been rounded to whole numbers. An asterisk denotes insufficient samples available to calculate. Brevetoxin concentrations are given in ng/g. Sample type # positive over total % positiv e Range PbTX Average PbTX Median PbTX total 1/4 25 0-169 12 0 brain 0/2 0 0 0 stomach content 1/3 33 0-17 16 9 muscle 0/2 0 0 0 lung 0/4 0 0 0 0 liver/viscera 1/4 25 0-169 42 0 kidney 0/2 0 0 0 Collection Dates, Locations and Brevetoxin Cell Counts The first bird was collected on 10-29-01. Karenia brevis cell counts ranged from not present to medium concent rations from off shore St. Petersburg south to Ft. Meyers for that same time frame (Table 13). One bird, a great white heron, which was collected south of the bloom in the Florida Keys, was negative for brevetoxin (Figure 6). Karenia brevis was not present in the Florida Keys
29 during this time frame although low to medium concentrations were detected through the end of December from St. Petersburg south to Ft. Meyers. No birds were collected in that area and time frame for the study. Table 13. Toxin content and bloom distribution for 2001 through 2004. Collection date Species Collection location Bloom geographic range PbTX (+) or (-) 10/29/2001 Great white heron Monroe county St. Petersburg to Ft. Meyers negative 1/10/2002 Common loon Charlotte county St. Petersburg to Naples negative 1/10/2002 Double crested cormorantCharlotte county St. Petersburg to Naples negative 2/5/2002 Brown pelican Monroe county S t. Petersburg to Naples negative 2/13/2002 Brown pelican Monroe county S t. Petersburg to Naples negative 10/17/2002 Great egret Monroe county None negative 3/10/2003 Brown pelican Monroe county Sarasota to Naples negative 7/30/2003 Great white heron Monroe county Tarpon Springs to Naples negative 7/30/2003 Great white heron Monroe county Tarpon Springs to Naples negative 1/30/2004 Laughing gull Sarasota Tarpon Springs to Sarasota negative 1/30/2004 Sanderling Sarasota Tarpon Springs to Sarasota positive 3/29/2004 Double crested cormorantPine llas county Tarpon Springs positive 5/12/2004 Brown pelican Pinellas county None negative 5/27/2004 Laughing gull Pinellas county None positive 6/3/2004 Brown pelican Pinellas county None positive 6/13/2004 Least tern Pinellas county None positive 6/13/2004 Least tern Pinellas county None negative 6/20/2004 Least tern Pinellas county None negative 7/9/2004 Least tern Pinellas county None positive 7/9/2004 Least tern Pinellas county None negative 7/11/2004 Royal tern Pinellas county None positive 7/20/2004 Laughing gull Pinellas county None negative 8/22/2004 Royal tern Pinellas county None positive 8/22/2004 Royal tern Pinellas county None positive 10/3/2004 Laughing gull Pinellas county Sarasota to Naples positive 11/14/2004 Double crested cormorantLee count y Sarasota to Naples positive
30 Figure 6. Red tide counts taken by The Florida Wildlif e Research Institute from October 29 through November 1, 2001 as represented on the FWRI website. In 2002 only five birds were collected for use in this study. Two birds were collected in early January from the Peac e River Wildlife Sanctuary, which is located in Charlotte County (Figure 7). Both birds, a double crested cormorant and a common loon, were negative for brevetoxin presence and cell counts taken by FWRI from the area showed low K. brevis concentrations with ranges of not present to high in various areas from the St. Petersburg coast south to the Naples area (Table 13).
31 Figure 7. A map of Florida showing the counties. Two brown pelicans collected in Februar y from the Florida Keys area were also negative for brevetoxin presence. Du ring the time frame the two birds were collected, K. brevis cell counts had increased in range from not present to high levels detected from offshore St. Petersburg down to Naples, but were not detected in the Florida Keys (Figure 8).
32 Figure 8. Red tide counts taken by The Florida Wild life Research Institute from February 11 through 15, 2002 as represented on the FWRI website. An egret collected in October 2002 in the Florida Keys was also negative for brevetoxin presence and cell counts ta ken by FWRI showed no presence of Karenia brevis in any of the collected areas along the south west coast of Florida. Three birds were collected in 2003 from various locations in the Florida Keys. All three were negative for brevetoxin presence even though very low to medium K. brevis counts were reported up and down the south west coast of Florida throughout the year with a few high patch counts detected in January offshore from the Naples and Ft. Meyers area. Twenty birds were collected during 2004 and very low to medium K. brevis counts seen at the end of 2003 continued along the south west Florida coast from January through April. May to September of that year showed no presence of the red tide organisms but very low to medium counts were reported again
33 through the end of the year offshore from Sarasota south to the Naples area. Although one of the birds collected from the Sarasota area in January was negative for brevetoxin presence, a s anderling collected in the St. Petersburg area in the same month was slightly positive for brevetoxin presence in a liver and intestinal sample. A double crested cormorant collected in St. Petersburg in March was positive for brevetoxin pr esence in its liver, heart and stomach content, however, a brown pelican collected dead from Shell Key in May was negative for brevetoxin presence. A l aughing gull collected on the same island a few weeks later was positive for brevetox in presence in its stomach contents, although negative for all of its other tissue samples. This type of hit and miss positive or negative resultants continued for several months until November of 2004, when patches of medium to high c ounts were seen offshore of the Naples and Ft. Meyers areas (Figure 9). No more birds were collected until March 2005, when 3 double crested cormorants were brought into CROW, a rehabilitation center located in Sanibel (near Ft. Meyers), all 3 of which were positive for brevetoxin presence in all of the tissues collected. In 2005, 101 birds were collected that were used in the study and FWRI records show a clear cut event for the entire year with high cell count concentrations detected in the areas of Tarpon Springs south to Sarasota with some patches even further south to Ft. Meyers, Naples and in the Florida Keys. The event appears to have continued into January and February of 2006 with fina lly no counts being detected in March 2006 along the entire south west coast of Florida. A total of 55 birds were collected in 2006.
34 0 5 10 15 20 25 30 35 40 45Oct-01 Dec-01 Feb-02 Apr-02 Jun-02 Aug-02 Oct-02 Dec-02 Feb-03 Apr-03 Jun-03 Aug-03 Oct-03 Dec-03 Feb-04 Apr-04 Jun-04 Aug-04 Oct-04 Dec-04 Feb-05 Apr-05 Jun-05 Aug-05 Oct-05 Dec-05 Feb-06 Apr-06Month and yearNumber of birds collected for analysis0 1 2 3 4Average level of bloom presence Figure 9. The number of birds collected for analysis by month and year compared to the average level of bloom presence detected through cell counts by FWRI. 0 denotes no presence, 1 is very low counts (<1,000 cells), 2 is low counts (<10,000 cells), 3 is medium counts (<1, 000,000 cells) and 4 is high counts (>1,000,000 cells). Brevetoxin presence in the various tissues tested throughout all 22 species ranged from 0 to 9,989 ng/g. T he highest level was found in a stomach content sample from a double crest ed cormorant that had been collected on August 19, 2005 from Vina del Mar, an area of St. Petersburg Beach in Pinellas County. The bird died en route to the r ehabilitation center and all of the tissues tested from the bird were positive. Karenia brevis cell counts showed low to high patches in the area where the bird had been collected for several months previously and the bloom was ongoing. In fact, 7 of the 12 highest levels found were from double crested cormorants, with the remaining 5 found in laughing gulls (Figure 10).
35 1 10 100 1000 10000blo o d (cormor a nt) brain ( c ormorant) heart (cor m orant) stomach c o n t e nt ( c o rmorant) intest in al content ( gul l) muscl e ( gul l) lun g ( corm o ra n t ) liv e r/v is cer a ( gu ll) ki d ne y (g u ll) gonad s (cormor a nt) gal lb ladder ( g ull) splee n ( c o rmorant)tissue typeng/g or ng/mL brevetoxin level Figure 10. Highest brevetoxin concentrations found by tissue sample type and species in logarithmic scale.
36 DISCUSSION The birds involved in the study were collected between 2001 through May 2006, during red-tide event periods and non-r ed tide event periods. Variability in the collection dates and collection areas ar e a consequence of an assortment of reasons: firstly, we depended on the staff of the rescue centers to contact us when they had birds that died that could be included in the research study. The rescue centers did not always have time for this when personnel were too busy, especially during event periods. Secondly, we were not in contact with every bird rescue center in Southwest Florida. The centers and groups we used were the SEANET Beached Bird Survey of Shell Key located in Pinellas County, The St. Petersburg Audubon/Eckerd College Least Tern Nesting Study located in Pinellas County, Peace River Wildlife Cent er located in Charlotte County, The Pelican Man Sanctuary of Sarasota, Save Our Seabirds (SOS) of Tierra Verde located in Pinellas County, The Center for Rehabilitation of Wildlife (CROW) located in Lee County, The Wildlife Center of Venice (WCV) located in Sarasota County, The Suncoast Seabird Sanctuary (SSS) located in Pinellas County, and birds which came from all over Flori da into the Fish and Wildlife Research Institute (FWRI) located in St. Petersburg. Thirdly, not all birds that die or get
37 sick in a red tide event come ashore and many that do come ashore may not be found. Therefore, the sample size is mi nimal and I have no way of estimating the true impact of brevetoxin concentrations in the tissues and organs of birds. The Karenia brevis cell count data used in this study were provided by the Fish and Wildlife Research Institute (FWRI) located in St. Petersburg, Florida, which is a division of the Florida Fish and Wildlife Commission. Most sampling performed by FWRI is response based, i.e., samples are taken after a bloom had begun and reports of dead fish, discolored wa ter, or respiratory irritation had been made. An independent contractor was hired to perform statistical analysis on cell count data collected from 1954 thru 2002 (comprising over 56,000 samples) by FWRI and that contract or determined that data collected from response-oriented monitoring is incomplete and limited, because it is too late to study the initiation and growth phases of the bloom and because it is logistically difficult to mobilize resources quickly enough to document the event adequately. Therefore, FWRI data, which were used to compare dates and locations of bloom detections, as well as the dates and locations of birds collected for this study, are inconsistent and precluded statistical analysis. Comparisons are therefore descriptive and only semi-quantitative. There were several possible reasons that I observed such high concentrations of brevetoxins in double cr ested cormorants and species of gulls. More numbers of double crested cormorants were collected than any of the other species, although gull species actually rank towards the lower end of the spectrum for numbers collected (Figur e 3). However, both double crested
38 cormorants and gulls inhabit and feed in a wide range of habitats (Table 4), including estuaries and open ocean coastli nes, and therefore may be exposed more frequently to areas where red tide is present. Both species also feed on various planktivorous fish in which br evetoxin has been shown to accumulate (Naar, et al., 2007), including baitfish such as threadfin herring and sardine species common to the Tampa Bay area. Gulls also have a tendency to feed on dead organisms that have washed ashore, gr eatly increasing their chances of being exposed to brevetoxins during events that may cause fish kills (van Deventer, 2007). In addition to finding that double crested cormorants and gulls had the highest values for brevetoxin presence, the tissues that had the highest and the lowest levels for brevetoxins were also consistent for both groups (Figure 11). This was true not only when looking at t he highest concentrations present but at average concentrations of brevetoxin (Figure 12). The highest concentrations were consistently found in stomach contents, intestinal contents, liver/viscera and gallbladder samples not only in double crested cormorants and gulls, but also in herons and egrets, terns and pelicans. Loons and gannets also showed the highest values for intestinal contents, liver/viscera and gallbladder, but differed in that high concentrations were found in gonad samples instead of stomach contents. Concentrations in organs of s horebirds were similar except that high concentrations were found in the kidneys instead of stomach contents or gonads. In the other group, the highest values were found in liver/viscera samples.
39 The lowest concentrations of brevet oxins for tissue samples were most commonly found in the blood, brain, lung and muscle. Such was the case for double crested cormorants and herons and egr ets. Gulls showed the lowest values in gonads, heart, lung and spleen tissues. Loons and gannets showed the lowest values in the muscle, blood, heart and spleen samples and terns showed the lowest values in the brain, muscle, heart and lung samples. Pelicans were similar with the lowest values of br evetoxins shown for brain, blood, gonads and kidney samples, while shorebirds showed the lowest values in lung, muscle, brain and heart samples. Figure 11. The highest concentration of brevetoxin found in each type of tissue tested in each group of birds. 0 500 1000 1500 2000 2500 3000 3500 4000 4500b lo od b r a in h ea r t stomach contents in testinal c o ntents muscle lu n g liver/viscera k id n e y g on a d s ga llb lad d e r spleentissue typebrevetoxin level (ng/g or ng/ml) cormorants gulls herons & egrets loons & gannets terns pelicans shorebirds other
40 0 200 400 600 800 1000 1200 1400 1600blo o d br a in he a rt s t o ma c h c o nt e n t s intest in al c o n t e nts musc le lun g liv e r/v is c e r a k id ne y go n ad s ga llbla dd e r s p lee ntissue typebrevetoxin level (ng/g or ng/ml) cormorants gulls herons & egrets loons & gannets terns pelicans shorebirds other Figure 12. The average concentration of brevetoxin found in each type of tissue tested in each group of birds. There are currently no other published studies of birds associated with K. brevis events with which to compare these values. There is, however, published data concerning manatees and dolphins. During a spring 2002 event, 34 endangered Florida manatees ( Trichechus manatus latirostris ) died in southwest Florida. In the spring of 2004 107 bottlenose dolphins ( Tursiops truncatus) died in waters off the Florida panhandle (Flewe lling, et al., 2005). Of the 63 animals tested (27 manatees, 36 dolphins) all were found to have high concentrations of brevetoxin in their tissues, specifically in the stomach contents (Flewelling et al.,
41 2005), which is comparable to the results f ound in my study. However, that study excluded the possibility of poisoning through aerosol exposure for these particular animals, consistent with t he findings from my study of low concentrations of brevetoxins in the lung tissues. Conversely, in a previous event in 1996 in which 149 manatees died, lung pathology indicated that brevetoxins had been inhaled (Bossart et al., 1998). Brevetoxin concentrations were generally low in the few specimens placed in the other species and the group was unus ual in that they showed the lowest positive values in stomach content sa mples and no positive values in brain, muscle, lung or kidney tissue samples. This observation may result from the small number of individuals (4) available for this group. However, the three species of birds included in this group, the whooping crane, osprey and wood stork, have different feeding habits from birds such as double crested cormorants or gulls. Their habits are more defi ned and limited to certain areas and certain types of prey. For example, wood st orks do eat fish, but are more commonly seen on ponds or ditches where brevetoxin most likely will not be present. However, the wood stork available to my study was the only animal found to have positive brevetoxin levels in tissues fr om this group. Ospreys also eat fish, and can hunt on ponds and in lakes wher e brevetoxins will not be found, in addition to tropical coastlines where bl ooms may occur. Possibly when blooms and fish kills are present, ospreys are may avoid the areas. While working for a rehabilitation center, I also noticed that the resident osprey did not eat fish whole, but picked at its food and usually avoided organs such as the liver, stomach,
42 intestines and kidney where brevetoxins accumulate (Naar et al., 2007), and ate mostly muscle where brevetoxins do not typically concentrate. Whooping cranes, on the other hand, eat a large variety of prey, not only small fish in salt marshes, but insects, frogs and sometimes plant matter, well away from the marine environments where K. brevis blooms. The fact that brevetoxin concentrations in different tissues varies among species seems to be dependant on the animals physiology. A study by Poli et al. (1990) showed that the liver was the major organ of metabolism and that excretion from bile was an important r oute of elimination. They showed that within 30 minutes of intravenous administrat ion of brevetoxin to rats, 69.5% of radiolabelled toxin went to the skeletal muscle, 18% to the liver and 8% to the intestinal tract. They deduced that skeletal muscle does not appear to be a site of metabolism but of storage from which toxin is slowly released prior to clearance by the liver with elimination occurring via feces and urine. Another study by Cattet and Geraci ( 1993) using ingested brevetoxin, also in rats, showed that although brevetoxin wa s distributed widely in the body, that it was concentrated in the liver. That study showed that after 6 hours, mean concentrations in organs were highest in the liver with the stomach next, followed by the intestine, heart, kidney, spleen, lung, fat, muscle, plasma, testes and finally brain. The results of this study were similar to those found by Poli et al. (1990) in that the highest concentrations of brevetoxin was found in stomach contents with intestinal contents next, t hen gallbladder, liver/viscera, kidney, lung, muscle, heart, gonads, spleen, brain and finally blood. Slight differences in the
43 order of highest to lowest can be account ed for in the actual methods of each study. The study by Cattet and Geraci (1993) was controlled in a laboratory setting, whereas the samples obtained for my study were animals from the wild that were all exposed to brevetoxin at di fferent times, at different concentrations and probably by different sources. They were also most likely continuously exposed to brevetoxins due to continued feeding in the environment as opposed to the one time feeding exposure the rats underwent. My study also tested lung tissues of some birds since brevetoxin s can become airborne. Gallbladders, an organ which was absent from the other studies, were also tested and consistently high levels were found in positively exposed birds. I had the lowest brevetoxin concentrations in blood serum samples in contrast to the study by Cattet and Geraci (1993), which found the lowest brevetoxin concentrations in brain samples. This is most likely due to the fact that the majority of blood samples used in this study were from 57 double crested cormorants that were released from the Suncoast Seabird Sanctuary in April of 2006 (Appendix 1), most of which had been at the sanctuary and recovering from brevetoxin exposure for several months Therefore, the average blood sample levels may be lower than they would have been when the animals were first exposed. Original blood samples were not taken for these birds upon admission to the rehabilitation center because most birds that are exposed to brevetoxins are extremely sick and are usually very dehydrated, making it difficult to get enough of a blood sample to be used for brevet oxin testing. On the other hand, it is also interesting to note that the blood samples of these birds had any levels at
44 all considering they had been in rehabilitat ion and not exposed to brevetoxin for up to 3 or 4 months or longer. One animal was at the center for over a year and still showed detectable levels of brevetoxin van Deventer (2007) found generally high levels of brevetoxins in the fish food supplies of at least one rehabilitation center, which could account for this oddity. Differences can also be seen in the highest concentrations and average concentrations of brevetoxin among the different avian groups (Figure 13 and Figure 14). For example, the highest le vel seen in the loon and gannet group was only 60 ng/g, compared to the highes t double crested cormorant value of 9,989 ng/g and the highest gull value of 2,801 ng/g. The highest values for terns and pelicans were 4,400 ng/g and 2,595 ng/g respectively. The highest values seen in the heron and egret group was 810 ng/g and in shorebirds 574 ng/g, with the last group, other, showing a high value of approximately 168 ng/g. 0 2000 4000 6000 8000 10000c or mor ants gulls herons & e g rets l oon s & gannets ter ns pel i cans sho r ebirds othe rgroupbrevetoxin level (ng/g) Figure 13. Birds by group compared to the highest con centration of brevetoxin (ng/g) found in a sample from that group.
45 0 50 100 150 200 250 300corm o ra n t s gul ls herons & e gr e t s loo n s & gan n ets ter n s pel ic a n s shorebirds othe rgroupbrevetoxin level (ng/g) Figure 14. Birds by group compared to the average con centration of brevetoxin (ng/g) found in a sample from that group. Again, these differences are possibly due to habitat and feeding preferences, although feeding habits do not ex plain all of the results reported in my study. Double crested cormorants, gu lls, terns and pelicans eat marine fish which can accumulate brevetoxins in their tissues (Naar et al., 2007) and the levels of brevetoxins seen in the tissues of those birds support those findings. Herons and egrets have a wide variety of feeding habitats that can include organisms in freshwater areas or from land as well as marine areas. Thus, the brevetoxin levels found in my study t end to be lower than in those species found in strictly marine habitats. Common loons often eat in freshwater areas in the northern parts of the US and Canada, alt hough, in Florida, they seem to be strictly estuarine, so it is surprising to see such low levels of brevetoxins in their tissues. Northern gannets often eat well offshore where brevetoxin blooms may
46 not be as concentrated as they are ins hore, although, the individuals occasionally are seen inshore. The shorebird species available for my study perhaps show the most surprising results. It could be argued that these particular species have feeding habits that may keep them from being exposed to areas and foods with high levels of toxins, therefore explaining the low levels seen in the tissues. For example, soras usually eat seeds and aquatic insects and ruddy turnstones, in addition to marine fish, also eat insects, carrion and garbage. Sanderlings eat aquatic and terrestrial invertebrates and sanderlings, ruddy turnstones and black skimmers all feed on crustaceans that have been shown to be highly toxic (Matter, 1994). van Deventer (2007) has show n that these shorebird species also scavenge fish on the beach that showed consis tently high levels of brevetoxin in their tissues, therefore raising the questi on as to why the shorebirds used in my study do not show higher concentrations, similar to double crested cormorants or gulls.
47 CONCLUSIONS Through testing of tissues from 185 birds representing 22 species, collected from October 2001 through May 2006, it has been shown that marine birds accumulate brevetoxins in va rious organs and tissues. Relationships between internal toxin levels and K. brevis cell counts within blooms suggest a high coincidence with avian brevetoxicosis. My observations show that levels of brevetoxin in tissues of birds rose si gnificantly during an ongoing red tide event and even rose slightly over several brief red tide event periods (Appendix 5). Brevetoxins were detected in essentially all internal organs and tissues as well as blood serum. Brevetoxins were det ected in 52% of all tissues tested, with 95% of gallbladders positive, 78% of blood serum positive, 69% of intestinal content/digestive tract samples positive, 60% of stomach/gut content samples positive and 58% of liver/viscera samples pos itive. Values of brevetoxin ranged from 0 to 9,989 ng/g, with the highest leve l found in a stomach content sample of a double crested cormorant. The highest concentrations of brevetoxins occurred in either double crested cormorants or gulls. Although I have no way of estimating the duration of exposure, the fact that toxin was found throughout the
48 body at high levels suggests that multiple toxic prey items were ingested rather than a single acute exposure. Results reveal a clear, acute threat to marine birds and therefore to other marine animals, including numerous species of fish as well as mammals such as dolphins and manatees during red tide event s. Moreover, little is known about chronic, low level exposure effects to ma rine animal health. This fact supports the need for further study in this field, not only during obvious red tide events, but during non-event periods as well. Based on the range of brevetoxin leve ls I found in the various tissues and organs and, due to the expense and time required to test a wide variety of tissues and organs, I would recommend only using muscle, liver, stomach and/or intestinal contents, and lung tissue for ELI SA based tests. Also, given my results for blood serum analyses, a more focused study is in order. Blood should be drawn from all birds upon arrival and release. This will give a data set which will allow for comparisons of blood serum phycotoxin levels in sick animals and allow an assessment of various treatment options.
49 MANAGEMENT IMPLICATIONS AND AREAS OF FUTURE RESEARCH Some previous authors note that inshor e or coastal birds appear to have developed conditioned aversions to algal toxins. Shumway (1990) suggests, based on a 1983 study by Nisbet, that terns developed an aversion response to toxic fish. Nisbet reported many piles of vomit containing the birds major prey, the sandlance, and estimated that they had been regurgitated within 20-30 minutes after ingestion. He suggested t hat more birds vomited than were killed and that only birds feeding on fish during the initial bloom period were impacted. Other studies have shown that birds kill ed in mortality events were nave (e.g., Fritz et al. 1999, Work et al. 1993, S humway et al. 2003, and Kreuder et al. 2002). This research points to a possible field of study on the subject of toxic prey aversion in marine birds. Much research has shown that brevetoxins move through the food web and this bioaccumulation of toxins may affect both ecological communities and individual species, such as sea birds. Seabirds may have the potential to change prey base or feeding location to avoid toxins. Because seabirds may have this ability to move between ecological communi ties, another area of future research may include studies to see if sea bird mortality events have significant affects on
50 seabird populations, based on their slow ra tes of reproduction. Coulson et al. (1968) estimated that 80% of t he breeding population of shags in Northumberland died during an outbreak of PSP. During the summer of 19891990, 150 adult yellow-eyed penguins (out of a population of 240 breeding pairs) were reported dead in New Zealand, apparently due to red tide poisoning (Gill and Darby, 1993). Coulson and Stowger (1999) reported the deaths of over 13,000 black legged kittiwakes in the northeast UK in just two years due to red tide. Many have also noted that the full impact of HABs on marine birds is likely underestimated as many die at sea and never wash ashore, so the need for research obvious (Shumway et al., 2003; Coulson et al., 1968). Further, a protocol has been establis hed at one bird rehabilitation center that reportedly provides a 90% success ra te (pers. Comm., Lee Fox). This claim could be studied and if proven, the prot ocol could be distributed to other rehabilitation centers located in other areas where brevetoxin events occur. The combined answers to these questions could bring us a step closer to managing wildlife in a consistent and prac tical manner and may lead to further research into HABs and its effects on other charismatic marine species, such as manatees, whales and dolphins.
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55 van Deventer, M., 2007. Brevetoxins in marine birds: evidence of trophic transfer and the role of prey fish as toxin vector. Masters Thesis, University of South Florida. Walker, S.T., 1884. Fish mortality in the Gulf of Mexico. Proc. US Natl. Museum 6:105-1090. Work, T.M., Barr, B., Beale, A.M., Frit z, L., Quillam, M.A. and J.L.C. Wright, 1993. Epidemiology of domoic acid poisoning in brown pelicans ( Pelecanus occidentalis ) and Brandts cormorants ( Phalocrocorax penicillatus ) in California. J. Zoo. Wildlife Med. 24:54-62. Work, T. 2000. Avian Necropsy Manual for Biologists in remote refuges. USGS National Wildlife Health center, Hawaii Field Station. 30pp. Wright, J.L.C. & Quilliam, M.A. 1995. 7. Methods for Domoic Acid, the Amnesic Shellfish Poisons. In Hallegraeff, G.M. et al. eds. Manual on Harmful Marine Microalgae IOC Manuals and Guides No. 33. UNESCO. pp.113133.
57 Appendix 1. 471 years of documented red ti de events off of Floridas west coast as shown on the FWRI website.
58 Appendix 2. Brevetoxin levels found in the blood serum samples from 57 double crested cormorants released from the S uncoast Seabird Sanctuary in April of 2006 after treatment for brevetoxicosis symptoms. Identification number Date Toxin level (ng/mL) HABB060417-031 Jan 9 2005 1.53 HABB060417-039 Sep 28 2005 2.44 HABB060417-014 Oct 16 2005 0 HABB060417-041 Oct 23 2005 4.43 HABB060417-006 Nov 7 2005 0 HABB060417-025 Nov 7 2005 3.82 HABB060417-053 Nov 8 2005 1.82 HABB060417-021 Nov 15 2005 4.48 HABB060417-044 Nov 16 2005 4.39 HABB060417-037 Nov 20 2005 3.27 HABB060417-059 Nov 21 2005 1.28 HABB060417-030 Nov 22 2005 1.68 HABB060417-024 Nov 26 2005 0 HABB060417-008 Nov 27 2005 1.28 HABB060417-011 Nov 28 2005 0 HABB060417-035 Nov 29 2005 2.95 HABB060417-034 Dec 2 2005 2.74 HABB060417-047 Dec 16 2005 3.14 HABB060417-023 Dec 18 2005 3.82 HABB060417-015 Dec 19 2005 2.12 HABB060417-026 Jan 1 2006 2.19 HABB060417-046 Jan 2 2006 3.61 HABB060417-051 Jan 2 2006 0 HABB060417-058 Jan 2 2006 3.53 HABB060417-018 Jan 3 2006 3.41 HABB060417-050 Jan 5 2006 1.58 HABB060417-048 Jan 6 2006 2.94 HABB060417-028 Jan 7 2006 3.31 HABB060417-055 Jan 27 2006 1.96 HABB060417-007 Jan 10 2006 0 HABB060417-056 Jan 10 2006 3.38 HABB060418-002 Jan 10 2006 4.31 HABB060417-013 Jan 11 2006 1.91 HABB060418-001 Jan 11 2006 0
59 A ppendix 2 (Continued) HABB060417-038 Jan 12 2006 1.83 HABB060417-054 Jan 12 2006 2.58 HABB060417-029 Jan 13 2006 1.65 HABB060417-032 Jan 14 2006 2.21 HABB060417-022 Jan 15 2006 2.03 HABB060417-017 Jan 16 2006 1.11 HABB060417-010 Jan 17 2006 0 HABB060417-043 Jan 19 2006 2.9 HABB060417-033 Jan 21 2006 2.72 HABB060417-040 Jan 21 2006 3.67 HABB060417-060 Jan 22 2006 0 HABB060417-012 Jan 24 2006 2.27 HABB060417-049 Jan 24 2006 4.28 HABB060417-052 Jan 24 2006 3.18 HABB060417-027 Jan 29 2006 1.45 HABB060417-045 Jan 29 2006 1.77 HABB060417-057 Jan 31 2006 1.43 HABB060417-016 Feb 7 2006 0 HABB060417-042 Feb 10 2006 4.51 HABB060417-009 Feb 21 2006 1.45 HABB060417-036 Feb 21 2006 3.25 HABB060417-019 Feb 28 2006 2.49 HABB060417-020 Feb 28 2006 2.48
60 Appendix 3. Specific results of samples taken for each of the 185 birds used in the study referenced by identification number. Values are in ng/g of brevetoxin concentration. Identification number Blood Brain Digestive tract Fat Gallbl adderGi contentsGonadsGut contentsHeartIntestinal contentsKidney Liver Lung MuscleSi contentsSpleenStomach contents Viscera 02010402 0 02010602 0 HABB031106-002 0 HABB031106-003 0 HABB031106-004 0 HABB031106-005 0 HABB031106-011 0 HABB031106-014 0 0 0 HABB031106-015 0 0 0 HABB040205-002 0 0 0 0 0 0 0 HABB040205-003 0 22.81 0 19.4 0 0 HABB040403-003 33.8 55.7 42.93 HABB040706-001 0 0 0 0 0 0 0 HABB040709-003 0 0 0 0 HABB040709-004 0 0 22.44 HABB040709-005 0 0 0 0 HABB040709-006 0 0 0 0 0 0 0 0 0 33.83 HABB040714-004 0 0 26.08 0 0 33.1 14.06 32.56 HABB040719-001 0 0 0 0 0 0 0 0 13.47 0 HABB040722-009 0 0 0 0 0 0 0 0 0 0 HABB040925-001 0 0 0 0 0 0 HABB040925-002 0 0 7.66 HABB040925-003 0 0 0 HABB040925-004 12.51 26.3 0 13.97 HABB040925-005 40.5 0 HABB041014-007 0 0 0 16.21 13.6 0 0 0 HABB041119-005 34.7 50.614.15 57.03 96.7 32.4 53.37 65.88 60.18
61 Appendix 3 (Continued) HABB041119-006 20.57 46.634. 65 35.24 52.3 18.5 54.11 19.49 HABB050324-007 10.47 0 10.37 0 11.08 0 11.4 0 0 0 19.15 HABB050329-007 0 HABB050526-003 17.67 240.52 51.27 20. 768.16 38.99 61.8 60.9 55.59 182.59 HABB050526-004 0 32.38 0 0 0 0 0 0 0 0 HABB050526-005 26.19 510.69 87.13 51. 7194.85 69.26 137 20.1 90.95 163.82 HABB050526-006 0 10.38 0 8.04 7.53 0 0 34.97 0 HABB050601-003 15.63 424.48 61.11 65. 345.17 45.5 85.9 10.7 77.99 39.32 HABB050601-004 20.98 948.92 56.35 51. 272.7 64.21 98.6 12.5 96.86 61.37 HABB050603-001 13.18 418.13 59.32 39.967.14 39.26 122 18 56.27 442.91 HABB050603-002 28.17 75.65 81.6171 .39 76.05 82.4 22.9 61.13 63.8 99.49 HABB050608-001 18.61 467.76 76.71 35. 7116.57 51.12 108 14.9 45.53 161.53 HABB050608-002 12.19 584.46 82.59 68. 3218.09 41.32 73.4 14.9 56.35 42.96 HABB050609-012 12.09 33.54 55.48 .94 18.32 58.1 0 45.03 12.86 61.9 HABB050609-013 0 10.3 0 88.32 0 0 0 0 0 0 HABB050614-015 7.58 13.6 0 HABB050614-016 0 0 11.31 0 HABB050614-017 23 0 HABB050614-018 0 0 0 0 HABB050614-019 0 0 0 0 HABB050614-020 0 0 0 0 HABB050614-023 0 18.64 23.4 0 HABB050614-024 0 0 0 0 0 HABB050614-025 0 0 11.7 0 HABB050615-001 0 0 0 0 0 257.83 / 48.13 / 1760.01 HABB050615-002 0 0 0 0 0 0 HABB050615-003 0 0 0 0 0 HABB050615-004 0 0 0 0 0 31.68
62 Appendix 3 (Continued) HABB050615-005 0 0 0 0 0 0 HABB050630-010 243.83 15.3 138 HABB050630-011 0 0 0 0 0 0 0 0 0 39.02 HABB050630-012 15.01 122.83 22.3 117 16.99 449.58 HABB050630-013 0 0 0 0 0 0 0 0 0 0 HABB050630-014 0 0 0 68.73 15.68 40.1 0 0 13.16 HABB050630-015 228.58 182 810.51 HABB050630-016 0 21 109.05 34.28 102 0 24.27 25.09 52.96 / 269.92 HABB050701-001 0 0 0 0 0 0 0 0 0 HABB050701-002 0 0 0 0 11.7 0 0 0 0 HABB050701-003 0 0 0 16.02 29.4 0 0 0 15.07 / 0 HABB050705-001 0 11.9 0 31.65 41.88 30.8 0 0 76.47 / 15.15 / 1095.98 HABB050705-002 0 0 0 0 25.5 0 0 0 HABB050705-003 270.76 36.4 227 31.99 727.22 HABB050705-004 0 0 0 0 0 0 0 0 HABB050705-005 0 0 0 0 0 0 0 0 0 0 0 HABB050705-006 0 0 0 42.6 0 HABB050705-007 0 0 21.65 0 0 0 0 0 0 HABB050706-003 0 0 63 0 0 0 0 0 12.94 HABB051020-030 0 0 0 HABB051020-031 0 0 17.19 HABB051020-032 93.36 12.15 0 0 0 13.2 0 0 0 HABB051028-014 2099.21 932.09 2021.28 291.5 1044 18.9 142.92 653.74 HABB051028-015 897.11 2215.8 2800.73 398.2 1355 42.9 176.55 345.17 HABB051220-001 0 / 3.69 HABB051220-002 0 / 4.09 HABB051220-003 0 HABB051220-004 1.82 / 3.88
63 Appendix 3 (Continued) HABB051220-005 3.92 / 0 HABB051220-006 0 HABB051220-007 0 / 2.05 HABB051220-008 0 HABB060112-001 1.74 HABB060112-002 0 / 1.67 HABB060112-003 4.15 HABB060112-004 11.68 HABB060112-005 6.98 HABB060112-006 9.37 HABB060112-007 7.05 / 0 HABB060220-023 5.21 HABB060303-001 12.31 0 20.02 0 0 0 0 0 0 18.36 HABB060303-006 34.45 0 17.25 0 0 0 18.5 8.61 HABB060307-001 23.5 121 718.43 1022645.35 104. 6 198 195 84.07 83.67 2310.32 / 4095.08 / 9988.62 HABB060322-013 64.82 519.13 55.74 35. 5114.74 64.91 147 12.9 39 82.04 525.23 HABB060322-014 8.02 4.93 6.07 11.6 0 8.17 12.25 33.07 HABB060322-015 0 HABB060322-016 1.95 HABB060322-017 1.17 HABB060327-001 371.86 270 19.73 574.67 HABB060327-002 0 0 15.13 0 0 0 0 11.89 HABB060409-001 29.48 48.8 0 28.81 HABB060409-002 0 0 0 0 27.6 0 0 15.54 15.78 HABB060414-006 3.15 0 0 13.4 0 0 HABB060414-007 4.23 0 0 0 0 HABB060414-009 0 13.52 48.9 30.35 HABB060414-010 93.6 263 0 47.92
64 Appendix 3 (Continued) HABB060414-011 12.07 196 0 39.78 HABB060414-016 16.13 23.73 35.4 106 84.45 56.63 39.8 HABB060414-019 23.4 96.9 0 4399.50 / 153.69 HABB060424-006 143.47 19.7 0 170.41 HABB060428-005 0 83.25 0 33.07 0 29.4 0 0 31.79 HABB060428-006 15.896.84 286 25.1 10.84 42.26 HABB060505-020 7.49 0 187.04 202 0 66.4 HABB060515-001 31.7272.89 147 9.98 387.43 HABB060515-002 314.93 21.24 0.27 42.15 100 8.26 21.77 34.5 315.67 HABB060522-004 11.1510.77 107.8 296 13.69 47.72 HABB060530-012 58.21 14.42 9. 9259.58 8.3 29.4 0 5.78 7.67 0 HABB060530-013 0 0 31.4465.1 89.69 141 9.02 733.6 HABB060530-014 5.51 21.4165.88 103.1 303 0 10.92 HABB060530-016 0 0 0 0 0 9.14 0 0 0 HABB060530-017 0 0 0 0 0 HABB060530-018 2.43 0 8.82 0 0 0 0 0 0 0 HABB060613-002 0 0 0 7.86 0
65 Appendix 4. Identification numbers for each of the 185 birds used in the study with common name, collection date, region collected, history and miscellaneous comments listed. Identification number Species Date County Comments 02010402 Common loon1/10/02 Charlotte pooled tissues, intestine, stomach, liver, kidney and spleen 02010602 Cormorant 1/10/02 Charlotte pooled tissues, intestines, stomach, liver, kidney HABB031106-002 Brown pelican 2/5/02 Monroe HABB031106-003 Brown pelican 2/13/02 Monroe HABB031106-004 Brown pelican 3/10/03 Monroe HABB031106-005 Great white Heron 10/29/01Monroe HABB031106-011 Great white Heron 7/30/03 Monroe HABB031106-014 Egret 10/17/02Monroe pooled tissues HABB031106-015 Great white Heron 7/30/03 Monroe pooled tissues
66 Appendix 4 (Continued) HABB040205-002 Laughing gull 1/30/04 Sarasota HABB040205-003 Sanderling 1/30/04 Sarasota HABB040403-003 Cormorant 3/29/04 Pinellas HABB040706-001 Brown pelican 5/12/04 Pinellas found dead on Shell Key, trachea cut; stomach filled with parasites HABB040709-003 Least tern 7/9/04 Pinellas found on ground of parking lot HABB040709-004 Least tern 7/9/04 Pinellas found on ground of parking lot HABB040709-005 Least tern 7/9/04 Pinellas found on ground of parking lot HABB040709-006 Laughing gull 5/27/04 Pinellas no visual problems on outside of body; found dead on Shell Key HABB040714-004 Royal tern 7/11/04 Pinellas found dead on Shell Key
67 Appendix 4 (Continued) HABB040719-001 Brown pelican 6/3/04 Pinellas hit by car on the Skyway Bridge, seemed disoriented before impact, DOA HABB040722-009 Laughing gull 7/20/04 Pinellas found on shell key, disoriented, could not fly, drooping/dragging wings, died on way to rehab center, juvenile; found at 11:19 am, died 11:42 am HABB040925-001 Least tern 6/20/04 Pinellas least tern found dead at Autoway Pontiac in Clearwater, male, most likely died from fall from nest HABB040925-002 Least tern 6/13/04 Pinellas least tern left leg missing, covered in parasites, prob pushed from nest by sibling HABB040925-003 Least tern 6/13/04 Pinellas found on ground of parking lot
68 Appendix 4 (Continued) HABB040925-004 Royal tern 8/22/04 Pinellas royal tern #1 found on Shell Key transect. Found beak of another bird lodged in back. Very emaciated/no broken bones HABB040925-005 Royal tern 8/22/04 Pinellas royal tern #2 found on Shell Key transect/extremely emaciated/no broken bones HABB041014-007 Laughing gull 10/3/04 Pinellas laughing gull found on Shell Key, male, no apparent injuries HABB041119-005 Cormorant 11/14/04Lee par asites in stomach, thin HABB041119-006 Cormorant 11/14/04Lee broken left leg; heart enlarged, aspergillus looking spots in mouth, very red inside of mouth HABB050324-007 White pelican3/19/05 Lee male/probable head impact injury/blood in nares, mouth and eyes, right side of head swollen.
69 Appendix 4 (Continued) HABB050329-007 Whooping crane 3/27/05 Citrus HABB050526-003 Cormorant 3/8/05 Lee found on Sanibel Island with brevetoxicosis symptoms/1.290 kg weight upon admit HABB050526-004 Cormorant 3/9/05 Lee head trauma, bloody eyes and mouth HABB050526-005 Cormorant 3/9/05 Lee 1.195kg upon admit, found on Sanibel Island with brevetoxicosis symptoms HABB050526-006 Herring gull 3/7/05 Pinellas found dead at the Siesta Beach Access #5, white lesions under liver & on the stomach, lung & intestines HABB050601-003 Cormorant 3/7/05 Lee found alive on Sanibel Island, euthanized because of severe hypopoteinemia, showed marked ataxia, moderate head trauma & dull mentation
70 Appendix 4 (Continued) HABB050601-004 Cormorant 3/7/05 Lee found alive on Sanibel Island, 1.190 kg weight on admit, showed moderate ataxia, head tremor, slow blink, respiratory effort, bloodwork showed pcv 3%, TP 1.8g/dl HABB050603-001 Cormorant 3/9/05 Lee found alive on Sanibel Island, 1.195 kg weight upon admit, showed mild ataxia, tarry feces. bloodwork pvc 39%, TP 2.0 g/dl HABB050603-002 Cormorant 3/4/05 Lee found alive on Sanibel Island, 1.345 kg weight upon admit, showed moderate ataxia, head tremor, slow blink, bloodwork pcv 50%, TP 3g/dl HABB050608-001 Cormorant 3/4/05 Lee found alive on Sanibel Island, 1.330 kg weight upon admit, showed moderate ataxia & slow blink, bloodwork pcv 30%, TP 1.4 g/dl
71 Appendix 4 (Continued) HABB050608-002 Cormorant 11/12/04Lee euthanized HABB050609-012 Cormorant 3/10/05 Lee found alive on Sanibel Island, euthanized, showed moderate ataxia, slight head tremor, tarry feces, pcv 33%, TP 2.4g/dl HABB050609-013 Cormorant 5/24/05 Lee DOA HABB050614-015 Least tern 5/30/05 Pinellas found on ground of parking lot HABB050614-016 Least tern 5/30/05 Pinellas found on ground of parking lot HABB050614-017 Least tern 5/30/05 Pinellas found on ground of parking lot HABB050614-018 Least tern 5/30/05 Pinellas found on ground of parking lot HABB050614-019 Least tern 5/30/05 Pinellas found on ground of parking lot
72 Appendix 4 (Continued) HABB050614-020 Cormorant 5/5/05 Sarasota found on Casey Key in Nokomis with propeller wounds on back, not using right leg, immature, indeterminate sex, 640 grams, GI loaded with parasites and lungs congested. HABB050614-023 Northern gannet 6/3/05 Sarasota adult male, 1448.9 grams, blood clot/hemmorhage at left femorotibial junction, bile in peritoneal cavity HABB050614-024 Brown pelican 3/28/05 Pinellas adult female, 1931.8 grams, tan clot/growth on subclavian, worms in gizzard HABB050614-025 Northern gannet 5/20/05 Pinellas adult male, 1590.9 g, low parasite load, slightly emaciated. HABB050615-001 Brown pelican 6/11/05 Pinellas male, second year adult, 2613.6 grams, emaciated.
73 Appendix 4 (Continued) HABB050615-002 Osprey 6/10/05 Pinellas immature female; 852.3 grams, found in backyard of home in Palm Harbor alive, emaciated, black liquid and twist tie in stomach. HABB050615-003 Osprey 6/6/05 Pasco mature female;1306.8 grams; found alive in backyard of home in Holiday zip code 34690, black oily substance in gizzard HABB050615-004 Brown pelican 6/11/05 Pinellas second year adult male, 2045.4 g, fishing line obstructing blood flow to leg, emaciated HABB050615-005 Great blue heron 6/11/05 Pinellas female; 2386.4 g, found alive on roadside HABB050630-010 Green heron 6/1/05 Pinellas male 63.8 grams, emaciated, some large intestine black HABB050630-011 Great blue heron 6/1/05 Pinellas 1051.1 g upon admit, hemoabdomen, hemothorax possible trauma from impact
74 Appendix 4 (Continued) HABB050630-012 Green heron 6/1/05 Pinellas male 85.2 g, yellow uric acid crystals throughout abdominal cavity and covering parts of outside body; liver infarct HABB050630-013 Royal tern 6/1/05 Pinellas mature male, 454.5 g, abscess in cheek, septic, heart hypoxic, one testicle necrotic HABB050630-014 Laughing gull 6/18/05 Pinellas mature female, 198.9 g, found in backyard of home in St. Petersburg alive, evidence of impact, jugular hematoma, hemmorhage in pecs, fractured humerus, necrotic oviduct, very thin HABB050630-015 Green heron 6/1/05 Pinellas male, 28.4 g upon admit, liver infarct, hypoxic heart HABB050630-016 Great blue heron 6/27/05 Pinellas female; 2686.4 g,found in backyard of private home in Largo, FL alive, systemic infection, strongest in lungs, also signs of impact/trauma with multiple hemmorhages, very thin
75 Appendix 4 (Continued) HABB050701-001 Great blue heron 6/20/05 Pinellas second year male, 1988.6 g, found roadside in St. Petersburg alive, hemmorhaging from pectoral muscle, abdomen, kidney, lungs, left femur, liver, possible impact trauma HABB050701-002 Northern gannet 6/16/05 Pinellas immature male, 1647.7 g upon admit, a few worms in GI, very thin HABB050701-003 Cormorant 6/21/05 Pinellas immature female, 965.9 g, very thin, overloaded with worms inside and on top of GI tract, mesentery scattered with hard yellow nodules HABB050705-001 Brown pelican 6/25/05 Pinellas male, 2329.5 g, found floating in water at St. Petersburg beach, multiple fractures to wing, several organs necrotic/hemmorhaging HABB050705-002 Common loon6/22/05 Pinellas mature female, 2329.5 g, found at Caladesi Beach, growths in abdominal cavity
76 Appendix 4 (Continued) HABB050705-003 Green heron 6/25/05 Pinellas 113.6 g, euthanized within 2 hours of admit, emaciated HABB050705-004 Yellow crowned night heron 5/21/05 Pinellas male, 511.4 g, euthanized within 3 hours of arrival, left leg inflamed/broken. HABB050705-005 Cormorant 6/26/05 Pinellas immature male, 1193.2 g, found alive at Indian Rocks Beach, growths under keel in peritoneum, many organs hyperemic. HABB050705-006 Green heron 6/19/05 Pinellas male, 113.6 g, found alive in yard of home in Madiera beach, liver and heart partly necrotic; brown lungs, fractured leg HABB050705-007 Cormorant 6/18/05 Pinellas immature female, 937.5 g, found alive roadside in Dunedin, parasite load high, septic abdomen HABB050706-003 Cormorant 6/5/05 Pinellas juvenile, multiple fractures, systematic infections
77 Appendix 4 (Continued) HABB051020-030 Great egret 8/27/05 Pinellas mature, 850 g, trauma, presumptive, resulting in coelomic hemorrhage and fracture, right ischium, subacute, moderate., ectoparasites and mites present., hemosiderosis, severe, liver, spleen, and kidney HABB051020-031 Wood stork 8/31/05 Pasco shaking and eyes rolling in head, septicemia, subacute, multifocal, severe resulting in necrotizing spleenitis, pneumonia, and cardiomyopathy, emaciation, severetracheitis, mild HABB051020-032 Brown pelican 9/27/05 Pinellas exhibited ascending paralysis HABB051028-014 Laughing gull 8/28/05 Pinellas juvenile HABB051028-015 Laughing gull 8/28/05 Pinellas juvenile HABB051220-001 Cormorant 12/9/05 Pinellas live sample HABB051220-002 Cormorant 12/9/05 Pinellas live sample
78 Appendix 4 (Continued) HABB051220-003 Cormorant 12/9/05 Pinellas live sample HABB051220-004 Cormorant 12/9/05 Pinellas live sample HABB051220-005 Cormorant 12/9/05 Pinellas live sample, had superficial hook puncture in chest HABB051220-006 Cormorant 12/9/05 Pinellas live sample HABB051220-007 Cormorant 12/9/05 Pinellas live sample but bird died later HABB051220-008 Cormorant 12/9/05 Pinellas live sample HABB060112-001 Cormorant 1/11/06 Pinellas mature adult HABB060112-002 Cormorant 1/11/06 Pinellas Immature juvenile HABB060112-003 Cormorant 1/11/06 Pinellas Immature juvenile HABB060112-004 Cormorant 1/11/06 Pinellas Immature juvenile HABB060112-005 Cormorant 1/11/06 Pinellas Immature juvenile HABB060112-006 Cormorant 1/11/06 Pinellas Immature juvenile HABB060112-007 Cormorant 1/11/06 Pinellas Immature juvenile
79 Appendix 4 (Continued) HABB060220-023 Cormorant 1/13/06 Pinellas HABB060303-001 Cormorant 2/27/06 Pinellas HABB060303-006 Cormorant 1/8/05 Lee euthanized, red tide symptoms, emaciated, GI filled with roundworm HABB060307-001 Cormorant 8/19/05 Pinellas juvenile female, 1560 g, red tide symptoms, convulsions, stomach full of roundworm and thread herring HABB060322-013 Cormorant 8/17/05 Pinellas juvenile male 1450g., malnourished, frayed feathers, roundworn HABB060322-014 Cormorant 6/23/05 Pinellas red tide symptoms, very thin, female juvenile 1136g upon admit
80 Appendix 4 (Continued) HABB060322-015 Northern gannet 3/10/06 Pinellas juvenile, generalized fungus infection HABB060322-016 Brown pelican 3/21/06 Pinellas HABB060322-017 Cormorant 3/21/06 Pinellas car impact, unbalanced, head weaving and hyperactive HABB060327-001 Sanderling 2/20/05 Pinellas found dead HABB060327-002 Cormorant 6/18/05 Pinellas female adult 1648g, parasites in GI and respiratory tract HABB060409-001 sanderling 8/26/05 Pinellas adult HABB060409-002 Sora 3/18/05 Pinellas HABB060414-006 Common loon3/21/06 Monroe adult HABB060414-007 Common loon3/21/06 Monroe adult HABB060414-009 sanderling 10/4/05 Pinellas HABB060414-010 Sanderling 10/4/05 Pinellas HABB060414-011 Sanderling 10/4/05 Pinellas
81 Appendix 4 (Continued) HABB060414-016 Cormorant 2/23/06 Pinellas HABB060414-019 Royal tern 8/25/05 Pinellas found dead, evidence of lightning strike HABB060417-006 Cormorant 11/7/05 Pinellas live sample HABB060417-007 Cormorant 1/10/06 Pinellas live sample HABB060417-008 Cormorant 11/27/05Pinellas live sample HABB060417-009 Cormorant 2/21/06 Pinellas live sample HABB060417-010 Cormorant 1/17/06 Pinellas live sample HABB060417-011 Cormorant 11/28/05Pinellas live sample HABB060417-012 Cormorant 1/24/06 Pinellas live sample HABB060417-013 Cormorant 1/11/06 Pinellas live sample HABB060417-014 Cormorant 10/16/05Pinellas live sample HABB060417-015 Cormorant 12/19/05Pinellas live sample HABB060417-016 Cormorant 2/7/06 Pinellas live sample
82 Appendix 4 (Continued) HABB060417-017 Cormorant 1/16/06 Pinellas live sample HABB060417-018 Cormorant 1/3/06 Pinellas live sample HABB060417-019 Cormorant 2/28/06 Pinellas live sample HABB060417-020 Cormorant 2/28/06 Pinellas live sample HABB060417-021 Cormorant 11/15/05Pinellas live sample HABB060417-022 Cormorant 1/15/06 Pinellas live sample HABB060417-023 Cormorant 12/18/05Pinellas live sample HABB060417-024 Cormorant 11/26/05Pinellas live sample HABB060417-025 Cormorant 11/7/05 Pinellas live sample HABB060417-026 Cormorant 1/1/06 Pinellas live sample HABB060417-027 Cormorant 1/29/06 Pinellas live sample HABB060417-028 Cormorant 1/7/06 Pinellas live sample HABB060417-029 Cormorant 1/13/06 Pinellas live sample HABB060417-030 Cormorant 11/22/05Pinellas live sample
83 Appendix 4 (Continued) HABB060417-031 Cormorant 1/9/05 Pinellas live sample HABB060417-032 Cormorant 1/14/06 Pinellas live sample HABB060417-033 Cormorant 1/21/06 Pinellas live sample HABB060417-034 Cormorant 12/2/05 Pinellas live sample HABB060417-035 Cormorant 11/29/05Pinellas live sample HABB060417-036 Cormorant 2/21/06 Pinellas live sample HABB060417-037 Cormorant 11/20/05Pinellas live sample HABB060417-038 Cormorant 1/12/06 Pinellas live sample HABB060417-039 Cormorant 9/28/05 Pinellas live sample HABB060417-040 Cormorant 1/21/06 Pinellas live sample HABB060417-041 Cormorant 10/23/05Pinellas live sample HABB060417-042 Cormorant 2/10/06 Pinellas live sample HABB060417-043 Cormorant 1/19/06 Pinellas live sample HABB060417-044 Cormorant 11/16/05Pinellas live sample
84 Appendix 4 (Continued) HABB060417-045 Cormorant 1/29/06 Pinellas live sample HABB060417-046 Cormorant 1/2/06 Pinellas live sample HABB060417-047 Cormorant 12/16/05Pinellas live sample HABB060417-048 Cormorant 1/6/06 Pinellas live sample HABB060417-049 Cormorant 1/24/06 Pinellas live sample HABB060417-050 Cormorant 1/5/06 Pinellas live sample HABB060417-051 Cormorant 1/2/06 Pinellas live sample HABB060417-052 Cormorant 1/24/06 Pinellas live sample HABB060417-053 Cormorant 11/8/05 Pinellas live sample HABB060417-054 Cormorant 1/12/06 Pinellas live sample HABB060417-055 Cormorant 1/7/06 Pinellas live sample HABB060417-056 Cormorant 1/10/06 Pinellas live sample HABB060417-057 Cormorant 1/31/06 Pinellas live sample HABB060417-058 Cormorant 1/2/06 Pinellas live sample
85 Appendix 4 (Continued) HABB060417-059 Cormorant 11/21/05Pinellas live sample HABB060417-060 Cormorant 1/22/06 Pinellas live sample HABB060418-001 Cormorant 1/11/06 Pinellas live sample HABB060418-002 Cormorant 1/10/06 Pinellas live sample HABB060424-006 Brown pelican 7/7/05 Pinellas live sample HABB060428-005 Sandwich tern 7/28/05 Lee live sample HABB060428-006 Ruddy turnstone 10/1/05 Lee adult, 74g HABB060505-020 Sanderling 8/12/05 Pinellas adult male 58g HABB060515-001 Laughing gull 8/25/05 Pinellas juvenile, 356g HABB060515-002 Great blue heron 8/10/05 Pinellas male, adult, 2125g HABB060522-004 Green heron 7/5/05 Pinellas female juvenile, 142g
86 Appendix 4 (Continued) HABB060530-012 Northern gannet 6/17/05 Lee male juvenile, euthanized HABB060530-013 Royal tern 8/25/05 Pinellas HABB060530-014 Sanderling 9/24/05 Pinellas HABB060530-016 Yellow crowned night heron 5/1/06 Pinellas HABB060530-017 Yellow crowned night heron 5/1/06 Pinellas HABB060530-018 Great blue heron 5/2/06 Pinellas HABB060613-002 Black skimmer 7/7/05 Pinellas euthanized, female, 227g
87 Appendix 5. Date, location and brief summary of Karenia brevis cell count data collected by FWRI. Date Description of detection and location of Karenia brevis cell counts Oct-01 very low to low counts Sarasota south to Ft. Meyers Nov-01 very low to medium counts St. Petersburg south to Ft. Meyers Dec-01 very low to medium counts St. Petersburg south to Ft. Meyers Jan-02 one patch of high counts in Tam pa Bay during early part of the month with very low to medium counts south to Ft. Meyers Feb-02 very low to medium counts along the south west Florida coast with high counts detected between Sarasota and Ft. Meyers towards the end of the month Mar-02 not present St. Petersburg south to Sarasota, very low to medium counts Sarasota south to Ft. Meyers, not present all over by end of the month Apr-02 low counts detected along Sarasota and in the Florida Keys several times during the month May-02 very low counts detected between Sarasota and Ft. Meyers a few times during the month, not pres ent all over by end of the month Jun-02 very low counts off of St. Petersburg and Sarasota by the end of the month Jul-02 very low counts off of St. Petersburg and Sarasota Aug-02 very low to medium counts o ff of St. Petersburg and Sarasota Sep-02 very low to high counts off of St. Petersburg and Sarasota, no presence everywhere by end of the month Oct-02 very low counts between Sarasota and Ft. Meyers with medium counts off of St. Petersburg by the end of the month Nov-02 very low to low counts Tarpon Springs south to Naples Dec-02 very low to medium counts off of Sarasota and Naples
88 Appendix 5 (Continued) Jan-03 very low to low counts from Naples south to Key West with high counts off of Naples and Ft. Meyers by the end of the month Feb-03 very low to medium counts off of Ft. Meyers, Naples and in the Florida Keys Mar-03 very low to medium counts between Sarasota and Naples with some high count patches between Ft. Meyers and Naples Apr-03 very low to medium counts off of Sarasota south ot Ft. Meyers May-03 very low counts detected from Tarpon Springs south to Sarasota and Ft. Meyers Jun-03 very low to medium patches det ected from Tarpon Springs south to Naples all month Jul-03 very low to medium patches detected from tarpon Springs south to Naples all month Aug-03 very low to medium patches detected from tarpon Springs south to Naples all month Sep-03 very low to medium counts detected from St. Petersburg south to Sarasota Oct-03 low counts detected from Sarasota south to Naples Nov-03 very low counts detected from Sarasota south to Naples Dec-03 very low counts off of Ft. Meyers Jan-04 very low to medium counts from Tarpon Springs south to Sarasota Feb-04 high to medium patched off of Sarasota and very low counts detected from Tarpon Springs to St. Petersburg Mar-04 very low counts off of Tarpon Springs at beginning of month, no presence everywhere by end of month Apr-04 very low count patchiness off of Sarasota at beginning of month, no presence anywhere by end of month May-04 no presence
89 Appendix 5 (Continued) Jun-04 no presence Jul-04 no presence Aug-04 no presence Sep-04 no presence Oct-04 very low counts detected off of Sarasota with very low to medium counts between Ft. Meyers and Naples Nov-04 medium counts of off Ft. Meyers with medium to high count patchiness south of Naples, no presence anywhere by end of the month Dec-04 very low to medium counts south of Naples with very low counts between Sarasota and Ft. Meyers, no presence anywhere by end of the month Jan-05 very low to medium counts between St. Petersburg and Sarasota and off of Key West Feb-05 low counts off of St. Petersburg, very low counts in the Florida Keys and high counts of off Sarasota Mar-05 low to medium counts St. Petersburg south to Ft. Meyers with high count patches off of Sarasota Apr-05 high count patches detected between St. Petersburg and Sarasota, very low to not present detected everywhere by the end of the month May-05 medium to high counts detected St. Petersburg south to Sarasota Jun-05 medium to high counts detected St. Petersburg south to Sarasota Jul-05 low to medium counts from Tarpon Springs to St. Petersburg and high counts off of Sarasota Aug-05 low to high counts detected from Ta rpon Springs south to Sarasota all month Sep-05 low to high count patches detect ed from Tarpon Springs south to Ft. Meyers all month
90 Appendix 5 (Continued) Oct-05 high counts detected off of Sarasota with low to medium counts from Tarpon Springs south to Naples Nov-05 very low counts off of Tarpon Spring s, low to medium counts from St. Petersburg to Sarasota with a high count patch detected between Sarasota and Ft. Meyers Dec-05 very low to medium counts from Tarpon Springs to Sarasota with a high patch off of Ft. Meyers and very low to medium counts all over the Florida Keys, no presence anywhere by the end of the month Jan-06 medium counts off of St. Petersburg at beginning of month, very low counts off of Sarasota and Naples with no presence anywhere by end of month Feb-06 very low counts of off St Pete rsburg and in the Florida Keys with no presence anywhere by the end of the month Mar-06 no presence