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Recent studies demonstrate that most cyanobacteria produce the neurotoxin beta-N-methylamino-L-alanine (BMAA) and that it can biomagnify in at least one terrestrial food chain. BMAA has been implicated as a significant environmental risk in the development of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis (ALS). We examined several blooms of cyanobacteria in South Florida, and the BMAA content of resident animals, including species used as human food. A wide range of BMAA concentrations were found, ranging from below assay detection limits to approximately 7000 μg/g, a concentration associated with a potential long-term human health hazard.
There is a general consensus that Harmful Algal Blooms (HABs) are increasing worldwide (Smayda, 1990, Hallegraeff, 1993, Anderson et al., 2002, Glibert et al., 2005). This is primarily the result of an increasing human population generating increasing nutrient runoff from fertilizer, animal waste, sewage, and soil erosion (Nixon, 1995; Richardson and Jorgensen, 1996; Moffat, 1998; Heisler et al., 2008; Anderson et al., 2002, 2008). The increased mobilization of nutrients has led to the eutrophication, first of small water bodies such as ponds, lakes and rivers (Vollenweider, 1992a); then of estuaries such as Chesapeake Bay (Cooper and Brush, 1991; Harding and Perry, 1997); and more recently of large seas such as the Black Sea (Bodeanu, 1992; Mee, 1992; Cociasu et al., 1996), Baltic Sea (Larsson et al., 1985; Nehring, 1992), and Adriatic Sea (Vollenweider et al., 1992b; Justic et al., 1995); and of continental shelf areas such as the Mississippi River delta (Turner and Rabalais, 1991, 1994; Justic et al., 1995).
Blooms of cyanobacteria appear to also be increasing worldwide in response to increased nutrient inputs (Chorus and Bartram, 1999; Paerl, 2008). Cyanobacteria have the ability to produce a wide array of secondary metabolites (Moore, 1996; Welker and von Dohren, 2006; Sivonen and Borner, 2008), many of which are noxious or toxic to animals and/or humans (Carmichael and Falconer, 1993; Chorus and Bartram, 1999; Carmichael, 2001; Carmichael et al., 2001; Ibelings et al., 2008; Falconer, 2008; Pilotto, 2008; Stewart et al., 2008). Many of these toxins are dermatoxins, hepatotoxins, or neurotoxins, but only a few species of cyanobacteria are known to produce each of these toxins. Recently Cox et al. (2005) have produced laboratory results that suggest that virtually all cyanobacteria species produce the neurotoxin beta-N-methylamino-L-alanine (BMAA). The data demonstrate a 500-fold variation in the amount of BMAA produced among the cyanobacteria species examined, but how much of this variation is genetic and how much is environmental-physiological is not known at the present time. Metcalf et al. (2008) and Esterling and Downing (2008) have also presented data suggesting that most cyanobacteria produce BMAA in the environment.
In vitro studies have shown that BMAA is toxic to neurons at concentrations as low as 10-30 nM (Weiss and Choi, 1988; Weiss et al., 1989; Rao et al, 2006; Lobner et al., 2007). BMAA is structurally similar to glutamate and binds to glutamate receptors (Richter and Mena, 1989; Copani et al., 1990; Smith and Meldrum, 1990; Allen et al, 1995; Mash, 2008). Abnormal stimulation of these receptors may play a role in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis (Takahashi et al., 1997). BMAA has also been shown to be neurotoxic in a variety of animal models Spencer et al, 1987; Karamyan and Speth, 2008).
BMAA, as an amino acid, is water soluble, not lipid soluble, and this has a low octanol-water partition coefficient (Yunger and Cramer, 1981). It therefore would not be expected to biomagnify up the food chain (Mackay, 1982; Connolly and Pedersen, 1988; Arnot and Gobar, 2006; Kelly et al., 2007). However, unlike the more moderate bioaccumulation observed with most water soluble cyanobacteria toxins (Xie et al., 2005; Ibelings and Chorus, 2007; Ibelings and Havens, 2008; Funari and Testai, 2008), Cox et al. (2003), Banack and Cox (2003), Murch et al. (2004), and Banack et al. (2006) have demonstrated a 10,000-fold biomagnification of free BMAA and 50-fold biomagnification of total BMAA in a food chain in Guam from symbiotic cyanobacteria to cycads to fruit bats (Pteropids, also known as “flying foxes”). Cox and Sacks (2002) hypothesized that the increased consumption of these fruit bats by one ethnic group of people in Guam, the Chamorros, in the 1940s led to a 100-fold increase in the development of Amyotrophic Lateral Sclerosis (ALS)-Parkinsonism dementia complex in the Chamorros living in Guam. The observation of high concentrations of BMAA in the autopsied brains of Chamorros who died of these neurodegenerative diseases and the absence of BMAA in the age-matched non-neurological control brains of Canadians who died of other causes suggested a possible link between BMAA and neurodegenerative diseases (Murch et al., 2004). Recently Pablo et al. (2009) have demonstrated high concentrations of BMAA in Americans who died of Alzheimer’s disease or ALS, but little or no BMAA in the brains of age-matched non-neurological controls, or in cases of Huntington’s disease, a genetic disorder. These data suggest that the unusual Guam situation is not unique, and that BMAA may biomagnify in other food chains, enter the human diet, and potentially trigger neurodegenerative disease.
Together, these recent studies suggest that BMAA from cyanobacteria could be involved in neurodegenerative diseases. As BMAA is not lipophilic, it would not be expected to biomagnify, yet the indirect evidence suggests that it can biomagnify in at least certain circumstances. To determine if BMAA is present and can biomagnify in aquatic food chains, BMAA concentrations were analyzed in animals collected from water bodies in South Florida (Fig. 1) known to have blooms of cyanobacteria.
Water samples were collected on a roughly monthly basis at numerous stations in Florida Bay (Fig. 2), Biscayne Bay (Fig. 3), and the Caloosahatchee River (Fig. 4). Sample tissue to be analyzed for BMAA content were collected from various frozen animals that had been collected for a variety of other research projects in these water bodies.
Cyanobacteria abundance in water samples was estimated by measuring concentrations of phycocyanin. Water samples were filtered through GF/F glass fiber filters and the filters were frozen at −20°C until extracted. These filters were extracted overnight with a phosphate buffer (0.05M H2KPO4,0.05M HK2PO4; pH 6.5; with 0.01%(v/v) mercaptoethanol added) at 5°C and analyzed for phycocyanin with a SPEX Fluorolog-3 spectrofluorometer, using 580 nm excitation and 640 nm emission. The spectrofluorometer was calibrated using pure phycocyanin.
Water flow and nutrient data were obtained from the hydrometeorological and water quality databases of the South Florida Water Management District and the Lee County Environmental Laboratory.
Quantitation of BMAA was performed on frozen, unfixed tissues by a modification of previously validated methods (Cox et al., 2003; Banack and Cox, 2003). All tissue samples were analyzed and assayed in duplicate. Briefly, frozen-pulverized liquid nitrogen submerged (Model# 36903-10, Cole-Parmer, Quebec, Canada) tissues (1:10 w/v) were hydrolyzed overnight in 6 M HCl (1:10 w/v) at 110°C. Particulate matter was removed in 500 μl aliquots by ultrafiltration (Ultrafree-MC, Millipore) at 15,800 × g. Two hundred microliters of the extract was vacuum centrifuged in a speed-vac (Thermo-Savant SC250DDA Speed Vac Plus with a Savant refrigerator trap RVT 4104). The lyophilized residue was resuspended in 200 μl 0.1 M trichloroacetic acid (TCA) then washed with 100 μl of chloroform for extraction of any residual lipids. The aqueous layer was then transferred to a fresh tube and the chloroform layer was discarded. Samples (5 μl) and standards were derivatized with 6-aminoquinolyl-N-hydrosuccinimidyl carbamate (AQC) using the AccQ-Fluor reagent (Waters Corp, Millford, MA; WAT052880). BMAA was separated from the protein amino acids by reverse-phase elution (Waters Nova-Pak C18 column, 3.9 × 300 mm using 140 mM sodium acetate, 5.6 mM triethylamine, pH 5.7 (mobile phase A), and 58% acetonitrile in water (mobile phase B) at 1 ml per min at 37°C. The elution gradient (30 min) was as follows: time 0 = 75% A; 2 min = 75% A curve 6; 17 min = 63% A curve 7; 18.5 min = 100% B curve 6; 23.5 min = 100% B curve 6; 25 min = 75% A curve 6; 30 min = 75% A. Samples were run in duplicate followed by an AQC blank (containing borate buffer and AQC tag) to ensure that there was no carryover between samples. BMAA was quantified by detection of the AQC fluorescent tag (Waters 2475 Multi λ-Fluorescence Detector) with excitation at 250 nm and emission at 395 nm. Experimental samples were compared to standard spiked similar matrix negative for endogenous BMAA, containing commercial (Sigma B-107; >95% purity) or authentic standards of BMAA (99% purity). The percentage of recovery of BMAA was >94%.
Identification of a BMAA peak detected by reverse phase HPLC was verified by LC/MS/MS using product ion mode in a triple quadrupole system. The frozen (unfixed) sample was hydrolyzed for 18 hours in 6N HCL at 110°C (Fountoulakis et al., 1998) and then dried to remove HCl in a Thermo-Savant SC250DDA Speed Vac Plus (Waltham, MA). The sample was reconstituted in dilute HCl (20 mM) and derivatized with AQC, which increased the molecular weight of the BMAA analyte from 118 to 458. Individual compounds were eluted from the column with a gradient elution of 140 mM sodium acetate buffer, 5.6 mM triethylamine, pH 5.7 (mobile phase A) and 52% acetonitrile (mobile phase B) with a flow rate of 1.0 ml/min. Separation was achieved by liquid chromatography (Thermo model Surveyor LC, San Jose, CA) using a Thermo Hypersil GOLD 100 × 2.1 mm, 3 μm particle column at 0.28 ml/min using the eluents of 0.1% formic acid (v/v) in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B) with the following gradient: time 0 = 100% A, 2 min = 100% A, 16 min = 80% A, 20 min = 2% A, 22 min = 2% A, 24 min = 81% B, 26 min = 81% A, 27 min = 100% A, 31 min = 100% A. Nitrogen gas was supplied to the H-ESI probe (heated electrospray ionization) with a nebulizing pressure of 25 psi and a vaporizing temperature of 300°C. The mass spectrometer was operated in the positive electrospray ionization (ESI) mode under the following conditions: nebulizing pressure of 25 psi, vaporizing temperature of 300°C, capillary temperature set at 280°C, capillary offset of 35, tube lens offset of 94, source collision energy of 5v. The protonated molecular ion of double derivatized BMAA (m/z 459) was used as the precursor ion for SRM (single reaction monitoring) analysis. Three transitions were monitored: 459 to 119 (collision energy of 19v), 459 to 171(collision energy of 32v), 459 to 289 (collision energy of 16v). The ratios of these three product ions were compared to the ratios of the product ions created by injection of AQC-derivatized pure BMAA standard (synthesized and then triple crystallized by Peter Nunn, University of Portsmouth, U.K.) into the triple quadrupole LC/MS/MS under the same conditions.
The effects of BMAA are purported to affect glutamate-associated mechanisms (Rao et al., 2006; Lobner et al, 2007). To validate the methods used for amino acid hydrolysis/extraction, we quantified the area ratio of glutamate (rt = 2.47 min) compared to an internal standard (istd) norleucine (rt = 18.91 min.) using standard curves of the specific matrix taken from various species. Briefly, increasing amounts (0-250 ng/injection)of glutamate along with one concentration (50ng/injection) of istd were spiked into 0.1 mg tissue/injection and analyzed by HPLC as per methods similarly described for BMAA.
Using HPLC we were able to detect significant concentrations of BMAA in various marine samples taken from various locations of the South Florida marine ecosystems (see Fig. 5 for a representative chromatogram). Initial assays using BMAA-free matrix spiked with increasing amounts of L-BMAA were conducted to determine if there were any differences in the lower limits of detection (LOD) and lower limits of quantitation (LOQ) for the extraction/instrumental method on the various matrices assayed. Results from these assays demonstrated an increasing threshold for detection in matrix dependent on origin (buffer<mollusc<teleost fish<<crustacean), and a recovery range of 94-98% (Fig. 6). The average linear dynamic range (1-250 ng) for BMAA spiked standard curves in matrix (Grey snapper, Lutjanus griseus, muscle) gave an R2 value of 0.998 (P < 0.0001). The presence of BMAA from marine samples found to have positive HPLC results were confirmed using triple quadrupole LC/MS/MS (Fig. 7). The application of triple quadrupole LC/MS/MS has four checks to verify the identity of the BMAA peak: (i) a specific single parent mass [molecular weight (MW): 459] is selected in the first quadrupole with all other masses excluded from the second quadrupole; (ii) the column retention time of the peak is unique; (iii) collision-induced dissociation of product ions in the second quadrupole must be all detected in the third quadrupole; (iv) the product ion ratios must match repetitive runs of the standard injected at a similar concentration within 5% variation. All four of these LC/MS/MS checks were verified with ratios of the product ions (MW: 289, 171, 119) of BMAA matching the daughter ion ratios of the derivatized standard (Fig. 7).
HPLC analysis of glutamate was conducted on select marine samples (mollusc (1 sample), crustacean (2 samples), and teleost fish (2 samples)) as an internal control and reference value for comparison to BMAA concentrations. To determine the significance of the BMAA concentrations, averages of glutamate and BMAA concentrations were converted to mole units and the calculated ratios gave a range of 18-45 for the measured glutamate to BMAA concentrations (Table 1). Glutamate measured in these samples were in good agreement with previous estimates (Dunn et al., 1949), providing validation for the amino acid extraction methods described here.
A large bloom of cyanobacteria developed in north central Florida Bay in the 1980s, which has persisted ever since (Brand, 2002). Although there are no quantitative data before 1989, many fishers and other boaters who were frequently in Florida Bay were quoted by DeMaria (1996) as observing algal blooms and water “discoloration” beginning in 1981 and increasing thereafter. They stated “In 1981, the water got dirtier, the blooms grew, and the seagrass started dying.” and “From 1981 to 1986, the decline [of the bay] was gradual. In 1987, the bay started to collapse quickly.”
The bloom is located primarily in northcentral Florida Bay and is primarily cyanobacteria (Fig. 8). It occurs in an area where high phosphorus in the west (Fig. 9) mixes with high nitrogen in the east (Fig. 10). It has been hypothesized that the phosphorus in western Florida Bay is from natural phosphorite deposits tens of meters below being transported by geothermal circulation to the surface (Top et al., 2001; Brand, 2002). Phosphorus is low in eastern Florida Bay because there are no phosphorite deposits in that area and the calcium carbonate that is the primary mineral of the Florida peninsula chemically scavenges phosphate from seawater (DeKanel and Morse, 1978; Kitano et al., 1978). This occurs in both groundwater flowing through limestone as well as shallow surface water in which calcareous sediments are resuspended. The nitrogen in eastern Florida Bay, on the other hand, is from the decomposition of nitrogen-rich organic peat in the northern Everglades as a result of the drainage of the Everglades and conversion into agricultural fields (now called the Everglades Agricultural Area), primarily sugar cane (Brand, 2002). Figure 11 shows the high nitrogen and phosphorus in this water in the north and the gradual decline to the south. Plants scavenge phosphorus from the water as it flows to the south through the Everglades, but they cannot scavenge all of the nitrogen because of the initial high N:P ratio (Brand, 2002). Diversion of this nitrogen-rich water in the 1980s into eastern Florida Bay (Fig. 12) led to the blooms of cyanobacteria developing in northcentral Florida Bay where nitrogen from the east meets phosphorus from the west (Brand, 2002). Because of the highly seasonal nature of the rainfall and runoff in South Florida, cyanobacteria abundance is low during the dry season and high during the wet season (Fig. 13). Florida Bay is a major nursery area for shrimp, and critical to the Florida shrimping industry. Samples of pink shrimp in Florida Bay have high concentrations of BMAA (Table 2). Grey snapper collected south of the bloom have either no or only moderate concentrations of BMAA (Table 1).
South Biscayne Bay-Eastern Florida Bay normally has extremely low concentrations of phosphorus (Fig. 14A) because of calcium carbonate scavenging (Brand, 2002), and low abundance of cyanobacteria (Fig. 14C). In the summer of 2005, large areas of mangroves in this area were destroyed in a road widening project (Fig. 3), and the organic material was buried in the surrounding sediments of South Biscayne Bay-Eastern Florida Bay. The subsequent anaerobic conditions and acidification of the calcium carbonate sediments released large amounts of phosphorus (Figs. 14B, ,15).15). As phosphorus was the limiting nutrient in this area, the additional phosphorus led to large blooms of cyanobacteria (Figs. 14D, ,15)15) that persisted for approximately two years. Samples of fish and crustaceans collected just north of this area in the spring and summer of 2007 (Fig. 3) as the bloom was on the decline show some species with no BMAA and others with very high concentrations (Table 3).
Some of the nutrient rich water from the Everglades Agricultural Area is pumped into Lake Okeechobee and then to the west down the Caloosahatchee River (Fig. 1, Brand, 2002). Because of the phosphorite deposits, waters along the western side of the Florida peninsula tend to be rich in phosphorus and thus nitrogen limited (Brand, 2002). High concentrations of nitrogen flow out of Lake Okeechobee and down the Caloosahatchee River (Fig. 16) and large blooms of cyanobacteria develop (Fig. 17). Moderate amounts of BMAA were found in molluscs and high concentrations were found in fish in the river (Table 4).
Because the animal samples were collected for other research projects unrelated to our research on cyanobacteria blooms and the BMAA hypothesis, the spatial and temporal patterns of animal samples are not optimal for examining the hypothesized link between cyanobacteria blooms and BMAA accumulation in animals. Nevertheless, these initial results indicate that high concentrations of BMAA can accumulate in some aquatic animals in areas of cyanobacteria blooms. Not enough samples have been analyzed to discern any clear patterns, but some possibilities do appear.
In south Biscayne Bay, there was large variation in the BMAA concentrations between individuals of a species in pink shrimp, blue crabs, mojarra, band tail puffers, least puffers, and bluestriped grunts (Table 3). The replicability of our methods suggest that these differences are real and not the result of methodological problems. The cause of this variation is unknown but several possibilities exist. As these animals were collected from an area north of the bloom, depending on their migration patterns, some animals may have spent considerable time in the bloom (or the organisms in their diet did so) while others may have spent little or no time in the bloom. While our research focussed on planktonic cyanobacteria, benthic cyanobacteria and cyanobacteria epiphytic on seagrass and macroalgal blades are also present. In these shallow waters, microalgal abundance per square meter is typically around 20 times higher on the sediment surface than in the water column above (Brand and Suzuki, 1999). Microhabitat differences in the relative abundance of these different groups of cyanobacteria and their BMAA content might account for the differences observed. Genetic differences between individuals in their ability to accumulate BMAA are also a possibility (discussed below), just as there are genetic differences among humans in their susceptibility to develop neurodegenerative diseases. Whatever the cause of this variation, it must be kept in mind when examining data on only a few individuals of a species.
Many crustaceans appear to have high concentrations of BMAA. Pink shrimp in Florida Bay (Table 2) have high concentrations of BMAA, comparable to those found in the fruit bats of Guam (3556 μg/g; Cox et al., 2003). Pink shrimp and blue crabs in south Biscayne Bay have a wide range of concentrations (Table 3). All three species of fish from the Caloosahatchee River (Table 4) have high concentrations of BMAA similar to that of the fruit bats of Guam. The filter feeding molluscs in the Caloosahatchee River and estuary have only moderate amounts of BMAA (Table 4). Grey snapper in Florida Bay south of its cyanobacteria bloom have moderate amounts of BMAA (Table 2), while grey snapper in Biscayne Bay north of its cyanobacteria bloom have none (Table 3).
In south Biscayne Bay, a wide range of BMAA concentrations was observed among the 17 species examined. A perusal of the data does not reveal a classical biomagnification pattern, as top carnivores such as barracuda and grey snapper show no BMAA, while species near the bottom of the food chain, such as pink shrimp, pufferfish, and sea bream have relatively high concentrations. A pattern that does appear, however, is a tendency for higher concentrations of BMAA in species that feed on the benthos, and lower concentrations in species that feed on the plankton. An exception to this pattern is pinfish, which primarily feed on benthic vegetation, but had no BMAA. No BMAA was found in anchovies, silversides, needlefish, and barracuda that feed in the water column, while high concentrations were found in pink shrimp, blue crabs, pufferfish, and cowfish that feed on the bottom. This could be an indication that benthic cyanobacteria produce more BMAA than planktonic species, but we have no direct information on this. A perusal of the data of Cox et al. (2005) does suggest that such a pattern may exist.
At the present time, we do not know why cyanobacteria produce BMAA and thus, what environmental patterns may exist. Similarly, we do not know the mechanism of bioaccumulation of this amino acid. Because BMAA is water soluble, the standard mechanism for biomagnification of lipophilic compounds is not plausible. One possibility is differential uptake and excretion. Because BMAA is structurally similar to glutamate in the presence of bicarbonate as a carbamate (Weiss et al., 1988), neutral acid uptake enzymes in the gastrointestinal tract and elsewhere may take up BMAA from dietary sources, but enzymes involved in the turnover, degradation, and excretion of amino acids may not be able to recognize BMAA and eliminate it. A differential in uptake vs. excretion enzymes may be one possible mechanism for the large differences observed between individuals and species in this study.
Ultimately there are many sources of variation that could account for the results shown here. The laboratory data of Cox et al. (2005) have shown a 500-fold variation in the amount of BMAA produced by cyanobacteria. Much of this is likely the result of genetic differences between species, but some is likely due to physiological differences in response to environmental differences. As a result, one would expect microhabitat variation among benthic and epiphytic cyanobacteria, and differences between planktonic and benthic communities. As BMAA propagates up the food chain, differential uptake and excretion will cause varying amounts of biomagnification. The age of animals could be important, with older animals accumulating more BMAA. The time course of BMAA exposure over the lifetime of an animal could be important, depending on the ability of an animal to slowly or quickly excrete BMAA after short term exposure. While it is clear that most cyanobacteria produce BMAA, we cannot, at this time, be sure that there are no other sources of BMAA such as heterotrophic bacteria.
Based upon this study and other data, it appears that the situation in Guam is not unique, and that high concentrations of BMAA do occur in parts of aquatic food webs. To enhance the chances of finding BMAA in aquatic animals, this study focussed on areas of unnatural blooms of cyanobacteria caused by nutrient enrichment by human activities. Cyanobacteria are of course a significant part of natural aquatic habitats unaffected by human activities. Cyanobacteria are approximately 50% of the phytoplankton community of the open ocean covering over half of the earth (Li et al., 1983; Itturiaga and Mitchell, 1986). It is plausible that humans have been exposed to some level of BMAA throughout their evolutionary history. The increase in cyanobacteria blooms as a result of human activities is probably increasing this exposure, but by how much is not yet known. Our research is now expanding from this initial exploratory analysis to determine the distribution of BMAA in aquatic food webs and seafood in both natural ecosystems and eutrophic ecosystems.
Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis (ALS) are also increasing (Kokmen, et al., 1993; Gauthier, 1997; Chio, 2005). Increased longevity alone may not account for all of this increase. Heritability of these diseases is low, accounting for less that 10% of cases. While certain genetic factors are known to influence susceptibility to these diseases, some unknown environmental factors probably play a major role. BMAA may be a significant environmental factor. Cyanobacteria and the presence of BMAA in portions of aquatic food webs used as human food could be a significant human health hazard.
These data on BMAA concentrations in animals in South Florida waters indicate that the situation in Guam is not unique. These data further suggest that BMAA could be found in high concentrations in aquatic animals in many areas of the world where cyanobacteria blooms occur. This possibility includes not only natural water bodies, but also nutrient rich habitats such as aquaculture ponds and flooded rice fields that have high concentrations of cyanobacteria (Paerl and Tucker, 1995; Massout, 1999; Kankaanpaa et al., 2005; Zimba, 2008). Agricultural soils can also have high concentrations of cyanobacteria (Ramsey and Ball, 1983; Shimmel and Darley, 1985; Whitton, 2000).
As an increasing human population on the earth increases the flux of sewage, agricultural fertilizer, animal wastes, and eroded soil into various aquatic habitats, it is predicted that blooms of cyanobacteria will increase. It is predicted that human exposure to cyanobacteria and BMAA will increase, leading to a possible increased incidence of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis (ALS).
We would like to acknowledge John Cassani, Maria Criales, John Lamkin, and Aswani Volety for some of the animal samples used in this study, and Margaret Basile for help in the laboratory. Nutrient and water flow data were obtained from the South Florida Water Management District and Lee County Environmental Laboratory. We thank Lora Fleming for reviewing an earlier draft of this paper. We would like to acknowledge the Cove Point Foundation, National Science Foundation (#OCE0742285 and #OCE0432368) and National Institute of Environmental Health Sciences (#1 P50 ES12736) for their financial support of this research. Partial support for this study was provided by gifts in kind from the Institute for Ethnomedicine.
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