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Harmful algal and cyanobacterial toxins in foraging green turtles (Chelonia mydas) in Florida's Big Bend

Numerous toxin-producing harmful algal (HAB) species occur in Florida's coastal waters. Exposure to these toxins has been shown to have sublethal effects in sea turtles. The objective of this study was to establish concentrations of 10 HAB toxins in plasma samples from green turtles (Chelonia mydas) foraging in Florida's Big Bend. Domoic acid, lyngbyatoxin-A, microcystins, nodularin, and okadaic acid were detected, demonstrating exposure to these HAB toxins, which are also a public health concern.

Harmful algal and/or cyanobacterial species have a global distribution, are found in all aquatic environments (e.g., marine, brackish, freshwater) (Abbott et al., 2009), and can pose significant threats to wildlife species (Landsberg, 2002). When conditions are favorable, these species proliferate and present at high concentrations (Abbott et al., 2009) in phenomena known as harmful algal blooms, or HABs, which appear to be increasing in frequency, duration, and range due to agriculture runoff, overfishing, and climate change (Glibert et al., 2005;Hallegraeff, 2010;Anderson et al., 2012). Toxins released from the single-celled organisms that cause these blooms can negatively impact wildlife health and survival (Landsberg, 2002;Fire and Van Dolah, 2012). Exposure to these toxins can result in strandings (dead or alive) of endangered species such as sea turtles, in addition to sublethal impairments including alterations in blood biochemistry, immunity, and neurologic function, thereby impacting their health (Arthur et al., 2006;Walsh et al., 2010Walsh et al., , 2019Perrault et al., 2014Perrault et al., , 2016Cocilova and Milton, 2016;Foley et al., 2019). This is of particular concern in Florida's coastal waters, where >70 freshwater, estuarine, and marine harmful algal species have been documented, and five species of threatened or endangered sea turtles (leatherbacks, Dermochelys coriacea; loggerheads, Caretta caretta; green turtles, Chelonia mydas; Kemp's ridleys, Lepidochelys kempii; hawksbills, Eretmochelys imbricata) inhabit coastal waters year-round (Foley et al., 2019). As many of Florida's turtle species inhabit both marine and estuarine environments (Ehrhart, 1983), there is the potential for exposure to numerous algal toxins. (saxitoxins) (Steidinger, 1993;Holloway-Adkins, 2001;Landsberg, 2002;Jacobson et al., 2006, Geselbracht, 2007Paul et al., 2008;Phlips et al., 2015;WHOI, 2016;Foley et al., 2019, Table 1). Sea turtles and other marine fauna become exposed to HAB toxins primarily through ingestion of contaminated prey (Flewelling et al., 2005;Manire et al., 2017). Immature green turtles are omnivorous and thus can secondarily ingest these toxins through the consumption of (1) primary consumers that have accumulated toxins, and/or (2) benthic algae and seagrasses and their associated epibiota (Bjorndal, 1997;Landsberg et al., 1999;Holloway-Adkins, 2001;Williams et al., 2014). Toxin exposure has been documented long after a bloom has dissipated, indicating persistence in both the environment and within organismal tissues (Flewelling et al., 2005;Capper et al., 2013;Fauquier et al., 2013;Perrault et al., 2014Perrault et al., , 2016. With the exception of brevetoxins, studies investigating the prevalence and impacts of HAB toxins on sea turtles are few; without directed monitoring of toxin concentrations, our understanding of exposure dynamics will remain limited. To address this knowledge gap, the objective of this study was to establish concentrations of HAB toxins in plasma samples collected from green turtles captured in Florida's Big Bend during a non-bloom period. The sampling site for this study was located in and adjacent to the shallow seagrass (e.g., Halodule wrightii, Siringodium filidorme, Thalassia testudinum, etc.) habitat of the St. Martins Marsh Aquatic Preserve. This relatively pristine area serves as an important foraging habitat for five sea turtle species in addition to containing the second largest expanse of seagrass habitat in the eastern Gulf of Mexico (Hale et al., 2004). In the Big Bend region, land coverage is primarily natural, much of the coastline and inland areas are under conservation protection, and human population densities are among the lowest in the state (Main and Allen, 2007;Seavey et al., 2011). Despite these factors, loss of oyster beds and seagrasses and a high estimated prevalence rate of fibropapillomatosis (FP; a tumor disease linked to chelonid herpesvirus 5 infection and degraded habitats; Lackovich et al., 1999;Van Houtan et al., 2010) in green turtles still occur in this area (Hale et al., 2004;Seavey et al., 2011). Even though this region has a perceived "pristine" ecosystem status, HABs are still considered a "very high-rated threat" to Big Bend coastal waters (Geselbracht, 2007). Juvenile green turtles were captured using dip nets and sampled in Big Bend (28.8324 � N, À 82.7596 � W) from 18 to 23 June 2015. Upon capture, blood (up to 10 mL, depending on the turtles' size) was sampled from the external jugular vein using a lithium heparin Vacutainer® (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) blood collection system fitted with a 22-gauge, 1-inch needle. The venipuncture site was swabbed with alternating applications of betadine and 70% isopropyl alcohol before and after blood collection. The blood samples were immediately chilled on ice until return to land, whereupon whole blood was centrifuged at 1318 g (3400 rpm) for 8 min for plasma separation and harvest. Plasma was stored frozen at À 80 � C until analysis.
Turtles were weighed and minimum straight carapace length (SCL min ) was measured. Additionally, we visually assessed and recorded the presence, number, and size of FP tumors to assign a tumor severity score of 0-3 . After sampling, turtles were tagged with flipper (metal Inconel; National Band and Tag Co., Newport, Kentucky, USA) and passive integrated transponder (PIT; Biomark®, Inc., Boise, Idaho, USA) tags for identification. Turtles were released into the water at their capture site.
Plasma samples were shipped overnight on dry ice to the Center for Environmental Sciences and Engineering at the University of Connecticut, a lab certified by the Connecticut Department of Public Health for the analysis of organic compounds in biological tissues. Plasma samples (approximately 1 g) were homogenized and split into 2 aliquots of an equal mass to separately prepare for lipophilic and hydrophilic toxin analyses. The lipophilic fraction was suspended in 1 mL methanol (0.1% formic acid), sonicated in the dark for 30 min, and vortexed for an additional 30 min. The resulting extract was centrifuged at 5443 g for Table 1 Summary of harmful algal toxins, their causative organisms with a Florida distribution, habitats, mechanisms of action, hydrophilic or lipophilic characteristics, and associated poisoning names. Data below are summarized from Landsberg (2002) and Abbott et al. (2009 , 7, 11, 14, 16-18, 20, 22, 25 References: 1, Yasumoto, 1998 and internal standard was added. The hydrophilic fraction was extracted in 1 mL of methanol:water (50:50), vortexed for 30 min, followed by centrifugation (5443 g � 10 min), and 190 μL of the supernatant transferred to a liquid chromatography vial. The hydrophilic and the lipophilic extracts were characterized for HAB toxins separately using a Waters, Inc. (Milford, Massachusetts, USA) Acquity ultra-performance liquid chromatograph/tandem mass spectrometer (UPLC-MS/MS) (Yang et al., 2017;Rodríguez et al., 2018). The UPLC was equipped with an HSS T3 column (2.1 mm � 100 mm, particle size 1.8 μm; Waters, Inc.), with water (0.1% formic acid) and acetonitrile (0.1% formic acid) used as mobile phases for separation of the HAB toxins (Provatas et al., 2014). Standard quality assurance procedures were employed including the analysis of surrogate, laboratory controls, method blanks, duplicates, matrix spikes, and matrix spike duplicate samples. All quality control results were within the acceptance criteria. Duplicate and matrix spike duplicate recoveries were within 20% relative percent difference from each other. Laboratory control and matrix spike recoveries were within �25% of the anticipated value. Instrument response was evaluated initially and after every 10 samples using calibration verification standards and a blank. All calibration verifications were within the acceptance criterion of 85-115% recovery, and all method blank values were below the reporting limit.
To the authors' knowledge, this is the first report of toxin concentrations in sea turtles residing in Florida's Big Bend, and represents the most comprehensive toxin analysis of sea turtles to date with regards to the number of toxins/toxin classes analyzed (Table 1). Overall, 13/21 (61.9%) green turtles tested positive for at least one toxin, with 4/21 (19.1%) testing positive for two toxins (Table 2). 8/21 (38.1%) turtles had plasma with all toxins below the detection limits. The most frequently detected toxin was domoic acid (6/21 turtles, 28.6%), followed by lyngbyatoxin-A, microcystin-LR, microcystin-RR, nodularin, and okadaic acid (2/21 turtles, 9.5% each), and microcystin-LA detected in 1/21 turtles (4.8%). Anatoxin-A, brevetoxin-B, brevetoxin-3, cylindrospermopsin, microcystin-YR, neosaxitoxin, and saxitoxin were not detected in any samples (Table 2). Due to low sample size, in-depth statistical tests could not be conducted; however, using SPSS (v. 25, SPSS, Inc. Chicago, Illinois, USA), a Fisher's exact test revealed that turtles with external evidence of FP were not more likely (P ¼ 0.617) to test positive for at least one plasma toxin (11/17 turtles, 64.7%) than turtles that did not have external FP (2/4 turtles, 50%).
The positive results seen here are concerning, especially in the absence of a temporally related, detected HAB, as several toxins (e.g., anatoxins, lyngbyatoxins, microcystins, nodularins, okadaic acid) have been implicated as tumor promoters in laboratory cell lines and mammalian models and/or are of public health concern (Landsberg, 2002;Humpage, 2008). Toxin exposure has been hypothesized to be a co-factor in FP tumor development in green turtles, since harmful algal and cyanobacterial species tend to occur in regions where FP prevalence rates are high (Landsberg et al., 1999;Anderson et al., 2001;Arthur et al., 2008a;Perrault et al., 2017). Here, we did not find evidence that turtles with FP tumors were more likely to test positive for toxin exposure. Identifying causal relationships between toxin exposure and FP development is problematic due to various unknown factors such as virus exposure timing and duration, and the possibility that biological factors or synergistic exposure to multiple toxins may potentiate or mask any observable effects .
Numerous harmful algal species regularly occur in Florida's coastal waters; therefore, co-occurrence of these HAB organisms and subsequent exposure to their associated toxins is possible, and in some areas is likely, for sea turtles that reside there (Kubanek et al., 2005;Paerl et al., 2008;Fire et al., 2011;Twiner et al., 2011;Capper et al., 2013). Out of the published studies to date that have documented toxin exposure in sea turtles (Table 1), only five (20.8%) examined more than one toxin/class of toxins (e.g., brevetoxins) with regards to cause of death or stranding (Jacobson et al., 2006;Capper et al., 2013;Manire et al., 2015Manire et al., , 2016Reckendorf et al., 2016). We can expect HAB toxin exposure and subsequent sea turtle strandings in Florida to increase in future years as HAB-related impacts to public health, tourism, fisheries, organismal health, and ecosystem functioning have increased in the last several decades and are expected to continue rising (Anderson et al., 2012). Therefore, it is imperative to gain a better understanding of toxin concentrations in sea turtles from Florida's Big Bend, a region vulnerable to the effects of anthropogenic disturbances including nutrient enrichment, increased agricultural use, and surface drainage (Frazer et al., 2001;Seavey et al., 2011;USFWS, 2013). Ongoing and future research by the authors of this study will explore relationships between toxin exposure and specific health biomarkers in sea turtles, which may point to toxin-related disruption of key physiological systems and elucidate any effects that exist between toxin exposure and disease risk or severity (Capper et al., 2013). Effective conservation measures must take species health into consideration, because a population cannot be sustainable if it consists primarily of unhealthy individuals (Karesh and Cook, 1995). Because sea turtles are sentinels of ecosystem health (Aguirre and Lutz, 2004), more research is needed to determine the prevalence of HAB species, HAB-associated toxins, and their impacts on sea turtle health.

Ethics statement
All sampling was conducted under Florida Fish and Wildlife Conservation Commission Marine Turtle Permit #125 and National Marine Fisheries Service Permit #16598.

Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.