Harmful Algae 28 (2013) 1–9

Dietary exposure to harmful algal bloom (HAB) toxins inthe endangered manatee (Trichechus manatus latirostris)and green sea turtle (Chelonia mydas) in Florida, USA

Angela Capper a,b,*, Leanne J. Flewelling c, Karen Arthur b

a School of Marine and Tropical Biology and Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook University, Townsville, QLD 4811, Australiab Smithsonian Marine Station, Seaway Drive, Fort Pierce, FL 34949, USAc Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 8th Avenue SE, St. Petersburg, FL 33701, USA

A R T I C L E I N F O

Article history:

Received 8 February 2013

Received in revised form 26 April 2013

Accepted 27 April 2013

Keywords:

Brevetoxin

Okadaic acid

Saxitoxin

Lyngbyatoxin-A

Cyanobacteria

Dinoflagellate

A B S T R A C T

Florida is a hotspot for cyano- and microalgal harmful algal blooms (HABs) with annual red-tide events

off-shore and blooms of Lyngbya spp. commonly observed in both marine and freshwater environments.

This region also provides extensive foraging habitat for large populations of herbivorous green turtles

(Chelonia mydas) and manatees (Trichechus manatus latirostris). The exposure of aquatic organisms to

HAB toxins is not well known and whilst acute exposures are better understood, the vulnerability of

aquatic animals to chronic exposure from multiple HAB toxins over prolonged periods has rarely been

addressed. This study aimed to identify the presence of toxic compounds produced by HAB species

commonly found in Florida (brevetoxins, okadaic acid, saxitoxins and Lyngbya toxins) in tissues and gut

samples from manatee and green sea turtles that stranded in Florida, USA. Muscle, liver and alimentary

tract samples were opportunistically collected from 14 manatees and 13 green turtles that stranded on

the Florida shoreline between December 2003 and February 2006. Samples from each animal were

assessed for the presence of brevetoxin, okadaic acid, lyngbyatoxin-A and saxitoxin. Nine (64%)

manatees and 11 (85%) turtles were found to have been exposed to one or more of the HAB toxins.

Okadaic acid and saxitoxin were only found in alimentary tract samples, whereas brevetoxin was more

widely distributed. No lyngbyatoxin-A was observed in any tissue samples. The majority of turtles (13)

stranded on the Atlantic coast between St. Johns and Monroe counties, with one on the Gulf coast at Bay

County, whereas nine manatees were stranded on the Gulf coast between Levy and Lee counties, with the

remaining five between Volusia and Brevard counties on the Atlantic coast. This HAB toxin screen has

identified that a large proportion of a random sample of turtles and manatees that stranded in Florida in

2003–2006 were exposed to HAB toxins. Most of the concentrations measured were low, and the toxins

were directly linked to the death of only three of these animals. However, the presence of these

compounds, and in some cases the presence of multiple HAB toxins in individual animals, indicates that

turtles and manatees in Florida are exposed to deleterious compounds at sub-lethal levels in their

environment, which could ultimately compromise their health.

� 2013 Elsevier B.V. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Harmful Algae

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ha l

1. Introduction

Harmful algal blooms (HABs) are apparently increasing in bothfrequency and range in aquatic habitats throughout the world(Hallegraeff, 1993, 2010). However, one area of the USA thatappears to be a particular hot-spot for HABs is Florida. The

* Corresponding author at: School of Marine and Tropical Biology and Centre for

Sustainable Tropical Fisheries and Aquaculture, James Cook University, Townsville,

QLD 4811, Australia. Tel.: +61 7 4781 4439; fax: +61 7 4725 1570.

E-mail addresses: angela.capper@jcu.edu.au, capper.a@gmail.com (A. Capper),

Leanne.Flewelling@myfwc.com (L.J. Flewelling), karen.arthur@anu.edu.au

(K. Arthur).

1568-9883/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.hal.2013.04.009

ecological consequences of toxic and bloom-forming species arenumerous with significant impacts noted for the Floridianecosystem, human and aquatic animal health (Landsberg, 2002;Van Dolah et al., 2003; Kirkpatrick et al., 2004; Flewelling et al.,2005; Walsh et al., 2006; Pierce and Henry, 2008; Landsberg et al.,2009; Fleming et al., 2011; Fire and Van Dolah, 2012), and withimplications for fish populations and subsequent recruitment(Flaherty and Landsberg, 2011). The reason why Florida may be soprone to such blooms has been cause of speculation for more than130 years and is related to numerous physico-chemical andbiological drivers exacerbated by long-term global climatic shiftsin conjunction with anthropogenic stressors (Anderson et al.,2008; Paerl and Huisman, 2009; Vargo, 2009).

A. Capper et al. / Harmful Algae 28 (2013) 1–92

More than 70 potentially harmful algal species have beenidentified from Florida estuarine and coastal waters (Abbott et al.,2009a) which include single species or a consortia of cyanobacteria(cyanoHABs) or microalgae (dinoflagellates, diatoms, raphido-phytes) (Badylak and Phlips, 2004; Phlips et al., 2004; Badylaket al., 2007; Livingston, 2007; Badylak and Phlips, 2008; Phlipset al., 2011). Many of these produce toxins and other biologicallyactive secondary metabolites ostensibly as defence againstpredation (Paul et al., 2007). Our understanding of the fate ofcompounds produced by cyanoHABs and toxic microalgal bloomsin their natural environment and potential accumulation inaquatic food webs is still in its infancy. Exposure to, andconsumption of, HAB toxins can impact all trophic levels withsome compounds accumulating through the food web (Landsberget al., 2006; Paul et al., 2007; Fire et al., 2008; Landsberg et al.,2009). The fitness of some aquatic organisms exposed to FloridaHABs may be compromised (Landsberg, 1995, 2002; Prince et al.,2006; Nam et al., 2010). Exposure can be via direct ingestion orinhalation (Bossart et al., 1998; Flewelling et al., 2005; Naar et al.,2007), or via trophic transfer through the food chain (Flewellinget al., 2005; Naar et al., 2007; Deeds et al., 2008; Fire et al., 2008;Landsberg et al., 2009; Sotka et al., 2009). Both direct and indirectexposure can be acute resulting in serious health problems ordeath, or may be chronic with the potential for sub-lethal impacts.

Two endangered species which are closely monitored in Floridaare the manatee (Trichechus manatus latirostris) and the greenturtle (Chelonia mydas). As euryhaline species, manatees living inFlorida ecosystems forage in both freshwater and marineenvironments consuming a range of aquatic vegetation andassociated epiphytes (Hartman, 1979). The large resident feedingpopulations of green turtles in Florida (Ehrhart, 1995; Ehrhart andRedfoot, 1995; Ehrhart et al., 2007) have a high site fidelity withinsmall feeding grounds (Musick and Limpus, 1996; Bresette et al.,1998) consuming a wide range of seagrass and algae (Mendonca,1983). Both species are likely to be affected by HABs throughcontamination of food sources, toxins in the water column andaerosolised toxins released due to wave action.

The health of manatees is notably adversely affected each yearby exposure to the frequent and periodic red-tide bloomsproducing brevetoxins (PbTX) in Florida (Landsberg et al., 2009).There has also been a long-standing association with aquaticorganism red-tide deaths in Florida including: fish kills since 1844(Ingersoll, 1882); dolphins since 1946–1947 (Gunter et al., 1948);and manatees since 1963 (Layne, 1965). Dietary exposure to PbTXduring a Karenia brevis bloom has been demonstrated byFlewelling et al. (2005) who found a high concentration of PbTXin stranded manatee stomach contents associated with largequantities of seagrass (Thalassia testidunum). Subsequent analysisof seagrass collected from the area of strandings showed highconcentrations of PbTX, not only in epiphytes on the seagrass, butalso in the seagrass blades and rhizomes. This indicated thatseagrass was the primary brevetoxins vector. Manatees may alsobe exposed to PbTX via inhalation of aerosolised toxins leading torespiratory problems and lung damage (Bossart et al., 1998; VanDolah et al., 2003). PbTX has also been detected in brain and livertissues as well as lymph nodes, urine and milk from lactatingmanatees (FWC, 2007, 2008; Flewelling, 2008).

The health of turtles is also adversely affected by exposure toKarenia spp. During prolonged Karenia brevis blooms in 2005 and2006, PbTXs were detected in blood and tissues of multiple speciesof both live and dead stranded sea turtles, including green turtles.In the majority of these animals brevetoxin exposure wasdetermined to have caused or contributed to the strandings(Fauquier et al., 2013).

Another dietary route of HAB toxin exposure are benthicdinoflagellates potentially ingested with seagrass. Prorocentrum

lima, Prorocentrum concavum and Prorocentrum mexicana havebeen observed at numerous locations in Florida (Bomber et al.,1988) with Prorocentrum micans in the Indian River Lagoon (Phlipset al., 2010) and Prorocentrum rhathymum in Florida Bay (An et al.,2010) Many of these species can produce okadaic acid (OA) (seereview by Landsberg, 2002), which can lead to diarrheic shellfishpoisoning (DSP) in humans (Yasumoto et al., 1978), acts as atumour promoter in mice (Suganuma et al., 1988; Fujiki andSuganuma, 2009) and has been implicated as an environmental co-factor in the aetiology of the debilitating neoplastic diseasefibropapillomatosis (FP) in turtles (Landsberg et al., 1999; Arthuret al., 2008). FP is most prevalent in inshore areas of high humanimpact (Herbst, 1994) with the highest recorded incidences inHawaii (92%, Balazs, 1991; Chaloupka et al., 2009), Florida (33–61%, Ehrhart, 1991; Ehrhart et al., 2007) and Moreton Bay, Australia(40–70%, Aguirre et al., 1999).

The potent tumour promoter, lyngbyatoxin-A (LTA), has alsobeen implicated as a co-factor in FP aetiology in the turtles in thePacific (Arthur et al., 2008). LTA is produced by Lyngbya majuscula

(Cardellina et al., 1979) and has been detected in high concentra-tions in a number of locations worldwide (Capper et al., 2005;Arthur et al., 2008). This highly adaptive benthic cyanobacteria hasbeen increasingly observed in Florida freshwater, estuarine andmarine ecosystems in recent years (Landsberg et al., 2003; Burns,2008; Pinowska et al., 2009), proliferating in the reef-associatedcommunities of south Florida (Paul et al., 2005); with ephemeralblooms in the Indian River Lagoon (Capper and Paul, 2008) andSanibel, west Florida (Paerl et al., 2008). Lyngbya spp. often blanketseagrass beds hindering access to important food sources for turtleand manatee populations (Arthur et al., 2006; EPA, 2010;Yasumoto, 2000) leading to potential exposure through directingestion and dietary assimilation. Consumption of L. majuscula byChelonia mydas has been observed in field and laboratory trials inAustralia and in field sampling in Hawaii (McMaster et al., 2003;Arthur and Balazs, 2008; Arthur et al., 2008). Preferential grazing ofLyngbya spp. by manatees is unlikely; many of the compounds itproduces are unpalatable to generalist herbivore meso-grazerssuch as sea urchins and crabs (Nagle et al., 1996; Pennings et al.,1996; Nagle and Paul, 1998; Capper et al., 2006a; Capper and Paul,2008), and macro-grazers such as fish (Thacker et al., 1997; Nagleand Paul, 1998; Capper et al., 2006b). Nonetheless, incidentalgrazing whilst feeding on preferred dietary items may occur.

Whilst a diverse array of compounds (displaying immunosup-pressant, antimitotic and chemotrypsin inhibitory activity) havebeen isolated from three Lyngbya chemotypes in the reefs of southeastern Florida (Sharp et al., 2009), LTA has not been detected inany marine Lyngbya spp. sampled (n = 38) from sites along theeastern coast ranging from Cape Canaveral to the Florida Keys fromJuly 2004 to June 2006 (pers. obs.). LTA has however, been detectedin freshwater Lyngbya wollei (Burns, 2008) occurring in Florida’sfreshwater springs and riverine systems (Cowell and Botts, 1994;Pinowska et al., 2009). L. wollei also produces saxitoxins (STX) andtheir analogues (Carmichael et al., 1997; Onodera et al., 1997;Thacker and Camacho, 2008; Foss et al., 2012). These potentcompounds are neurotoxic and cause paralytic shellfish poisoning(PSP) in humans (Etheridge, 2004; Pearson et al., 2010). STXs havebeen implicated as the aetiological agent in the deaths of dolphins,whales and seals following ingestion of organisms found with highSTX concentrations (Van Dolah et al., 2003; Lowenstein, 2004; Fireand Van Dolah, 2012).

The diversity of HAB organisms in Florida coastal waterssuggests the possibility of simultaneous environmental HAB toxinexposure, particularly when many of these species have the abilityto co-occur (Kubanek et al., 2005). A recent example of this wasobserved in the coastal embayments of Sanibel and Captiva Islandsin 2006 with a co-occuring bloom of Karenia spp. and L. majuscula

A. Capper et al. / Harmful Algae 28 (2013) 1–9 3

(Paerl et al., 2008). Although many studies have been carried out onindividual aquatic organisms and specific HAB toxins, very fewstudies have focussed on the detection of a range of HAB toxins inthe same organism. A recent study by Fire et al. (2011) reported theco-occurrence of multiple HAB toxins (okadaic acid, domoic acid,and brevetoxins) in bottlenose dolphins that stranded along theTexas coast. While the toxins were not conclusively linked to thecause of the strandings, the findings highlight the need to betterunderstand the impacts of HABs on wildlife health. Additionalstudies by Twiner et al. (2011), Twiner et al. (2012) also noted thatdolphins on the southwest coast of Florida were commonlyexposed to both brevetoxins and domoic acid over a ten-yearperiod (1999–2009).

The extent to which aquatic organisms are exposed to multipleHAB toxins is not well known. Chronic and acute poisoningepisodes have the potential to compromise animal fitness. Thesynergistic effect of multiple stressors over prolonged periods hasnot been assessed. This study aimed to identify the presence ofmultiple HAB toxins (brevetoxins, saxitoxins, okadaic acid andlyngbyatoxin-A) in the tissues of manatees and green sea turtlesstranded along the coast of Florida in order to identify regionswhere animals may be at risk of exposure to HAB toxins, whichmay provide important information for the conservation of theseendangered species.

2. Material and methods

2.1. Tissue sample collection

The Florida Fish and Wildlife Conservation Commission (FWC)and the Sea Turtle Stranding and Salvage Network strive to collectall manatees and sea turtles respectively that strand along theFlorida coast. Stranded carcasses provide an excellent opportunityfor tissue sampling to screen for exposure to HAB toxins and assesswhether any exposure may have been acute, sub-lethal or played arole in animal death, which may have implications for generalpopulation health.

Several green turtles (n = 13) and manatees (n = 14) thatstranded between May 2004 and February 2006 were sampledduring necropsies conducted at the FWC Marine MammalPathobiology Laboratory in St. Petersburg, FL. Wild carcasses weresourced opportunistically from predominantly the east and westcoast of Florida (Table 1; Fig. 1). For green turtles tissue samplesconsisted of muscle (n = 13), liver (n = 8), and fibropapillomas(n = 2). Stomach contents (where available) and small intestinecontents combined herein referred to as upper GI (n = 12), colon/faecal matter, herein referred to as lower GI. Manatee samplesincluded muscle, liver, mouth contents, stomach contents andfaecal matter in the lower GI. Samples were placed into sealed glasscontainers and transported on ice to the Smithsonian MarineStation, Fort Pierce (SMSFP), FL. Wet weights were recorded andsamples stored at �20 8C prior to analysis.

2.2. Brevetoxin analyses

Turtle lower GI (n = 6), liver (n = 7) and fibropapilloma (n = 2)tissues were tested for the presence of PbTX and their congeners.Freeze-dried (1 g dwt) tissue samples were mascerated andextracted for PbTX as described in Flewelling (2008). A competitiveELISA was performed according to Naar et al. (2002) whichrecognises all congeners and metabolites of brevetoxin that have aPbTX-2-type backbone. Toxin concentrations are calculated usinga PbTX-3 standard curve and results are reported in PbTX-3 eq g�1.Samples from the manatees included in this study were previouslytested for PbTX by FWC using the same method as described above

and are provided in Table 1 for comparison (NOAA, FWC, pers.comm.).

Whilst this ELISA does not distinguish among individual PbTXcongeners, it does show sensitivity and specificity for the family oftoxins at low concentrations and is a reliable, high-throughputassay. As performed in this study, the limit of quantification was 5–10 ng PbTX-3 eq g�1 dry wt. (1–1.5 ng PbTX-3 eq g�1 wet wt.).

2.3. Okadaic acid analyses

Turtle lower GI (n = 6), liver (n = 7) and fibropapilloma (n = 2)and manatee lower GI/faecal (n = 11) and liver (n = 13) were testedfor the presence of OA using a colourimetric protein phosphataseinhabitation assay (PPIA). To extract the toxins, tissue sampleswere prepared as described in Section 2.2 for brevetoxin analysis.

The PPIA was performed using a method modified from Tubaroet al. (1996). The PPIA detects compounds that inhibit proteinphosphatase, including the marine algal toxins okadaic acid anddinophysistoxins as well as the predominantly freshwater micro-cystins. To differentiate and confirm the identity of the toxins,samples that were positive by PPIA were analysed for microcystinsusing the Microcystins-DM ELISA kit (Abraxis, Warminster, PA,USA) performed as specified in the product insert and for okadaicacid and dinophysistoxin-1 using ultrahigh-performance liquidchromatography with tandem mass spectrometry (UPLC–MS/MS).UPLC–MS/MS analyses were performed using an Acquity UPLCsystem coupled to a Quattro microTM API triple quadrupole massspectrometer (Waters, Milford, MA, US) according to Deeds et al.(2010). Quantification was performed using a 6-point calibrationof OA reference solution purchased from NRC Canada.

2.4. Saxitoxin analyses

The RIDASCREEN1FAST Saxitoxin Assay (R-Biopharm AG,Damstadt, Germany) ELISA was used to screen for the potentialpresence of STX and analogues in manatee stomach (n = 10) andlower GI (n = 1) samples. Turtle upper GI (n = 8) and lower gut(n = 1) samples were also tested. For each tissue sample, 1 g offreeze-dried material was analysed. Samples were extracted in10 mL 0.1 M acetic acid, boiled for 5 mins, then centrifuged at3500 rpm for 10 min. The supernatant (100 mL) was removed andadded to 980 mL buffer solution.

For toxin characterisation, potential positive samples (manateestomach, n = 5; turtle upper GI sample (n = 4); turtle lower GI/faecalsample (n = 1)) were subjected to high performance liquidchromatography with fluorescence detection (HPLC-Fl). Prior toHPLC analysis, sample extracts were cleaned by passing through aSupelclean LC-18 cartridge (500 mg/3 mL; Supelco, Bellefonte, PA,USA) After preconditioning the cartridge with 6 mL of methanol andthen 6 mL deionized water, 1 mL of extract was passed through thecolumn and collected in graduated centrifuge tube. The column wasrinsed with 2 mL of deionized water, which was also collected in thecentrifuge tube. The sample was then adjusted to pH 6–7 andbrought to a final volume of 4 mL. Extracts were analysed for PSPtoxins using HPLC-Fl according to Lawrence et al. (2005).

2.5. Lyngbyatoxin analyses

To test for the presence of LTA, gut contents and tissues werefreeze-dried. Turtle upper GI (n = 11) and lower GI/faecal (n = 12)samples and manatee stomach (n = 13) and lower GI/faecalsamples (n = 12) were freeze-dried and extracted three times in1:1 ethyl acetate: methanol during a 24 h period to obtain a non-polar extract. The sample was further extracted three times in 1:1ethanol: distilled H2O for an additional 24 h to obtain a polarextract. Samples were filtered and dried by rotary evaporation.

Table 1Concentrations of lyngbyatoxin-A (LTA), saxitoxins (STX), okadaic acid (OA) and brevetoxins (PbTX) detected in manatees and green turtles opportunistically sampled

between December 2003 and February 2006. Results are reported in ng per gram wet wt or ng per ml fluid.

Animal ID Date Location

(County)

Probable cause

of deatha

LTA STX OA PbTXg

Manatees

(1)MNW0548 09 September 05 Pinellas Red tide nd Potential pos

(stomach)b

nd Stomach 579.1,

Liver 71.4

Lung 15.9, brain 8.2

Urine 9.3, milk 54.4

(2)SWFTm0520b 15 September 05 Volusia Watercraft collision nd – nd nd

(3)LPZ102106 16 September 05 Hillsborough Watercraft collision nd nd nd Urine 1.4

(4)MNW0552 17 September 05 Manatee Watercraft collision nd Potential pos

(stomach)b

nd Stomach 61.7

Liver 16.8

(5)MNW0555 28 September 05 Pasco Red tide nd nd nd Stomach 330.7

Liver 105

Kidney 17.1, urine 7.2

(6)MSW5136 26 October 05 Lee Red tide nd Potential pos

(stomach)b

Lower

GIe 16.1

Stomach 1221.8

Liver 79.6

Kidney 23.3, urine 12.5

(7)MNW0566 13 December 05 Citrus Watercraft collision nd Stomach 148

(dcGTX-2/3

+ dcSTX)c

nd nd

(8)NMW0567 15 December 05 Pinellas Watercraft collision nd nd nd Stomach 9.8

(9)LPZ102127 22 December 05 Levy Watercraft collision nd nd nd nd

(10)MEC0615 14 February 06 Brevard Watercraft collision nd – nd –

(11)MEC0616 14 February 06 Brevard Cold stress nd nd nd nd

(12)MSW0630 19 February 06 Lee Cold stress nd nd nd nd

(13)MEC0620 19 February 06 Brevard Natural nd nd nd Urine 1.8

(14)MEC0621 20 February 06 Brevard Natural nd Potential pos

(stomach)b

nd nd

Turtles

(1)SKD 031221-03 21 December 03 Volusia Unknown nd Potential pos

(stomach)b

– –

(2)EPD 040520-01 20 May 04 Unknown Unknown nd Potential pos

(stomach)b

– Lower GI 4.1

(3)NME 040523-01 23 May 04 Bay Watercraft collision nd nd – Liver 40.9

Lower GI 6.3

(4)CXC 040528-01 28 May 04 Brevard Propeller wound nd – – Lower GI 4.4

(5)DBH 040704-01 4 July 04 Indian River Unknown (emaciated) nd Potential pos

(stomach)b

– Lower GI 7.8

(6)NMY 041007-01 07 October 04 Brevard Watercraft collision nd – nd –

(7)CM 0413041019-01 19 October 04 Palm Beach Fishing line on flippers.

(Fibropapillomas)

nd nd nd Lower GI 7.8

(8)GWW 041123-01 23 October 04 Broward Watercraft collision nd – nd Liver 25.2

(9)MXW 04110101 02 November 04 Broward Unknown nd nd Lower

GIf 8.7

Liver 11.4

(10)KES 050203-01 03 February 05 Martin Unknown nd Potential pos

(stomach)b

nd Liver 16.2

Lower GI 16.3

(11)CAD 050315-01 15 March 05 Palm Beach Watercraft collision nd – nd nd

(12)EDS 050508-01 8 May 05 St. Johns Vehicle collision nd – nd Liver 5.6

Lower GI 5.1

(13)MLW 050621-01 21 Jun 05 Palm Beach Watercraft collision

(Fibropapillomas)

nd Potential pos

(stomachb, lower GId)

nd Liver 20.9

–, not tested; nd, toxin not detected.a Cause of death determined by FWC Marine Mammal Pathology Laboratory (manatees) and Sea Turtle Stranding and Salvage Network (turtles).b Positive result using RIDASCREEN1 assay but below level of detection for HPLC confirmation.c Positive result with ELISA, confirmed by HPLC.d Sample too viscous to pass through C18 column for clean-up.e Protein Phosphatase Inhibition Assays followed by MY ELISA to test for microcystins (negative) and OA UPLC–MS/MS for okadaic acid (positive).f Sample size small and unable to carry out confirmatory analysis using MY ELISA and UPLC–MS/MS for OA.g Manatee PbTX analyses conducted by FWC previously during the 2005–2006 Unusual Marine Mammal Mortality Event (NOAA, FWC in preparation).

A. Capper et al. / Harmful Algae 28 (2013) 1–94

For LTA turtle tissue extractions, 10 g of freeze-dried materialwas used (where available) for muscle (n = 13) and fibropapillomatissue (n = 2), whilst 6 g was used for liver (n = 7) extractions (livertissue samples obtained from mass necropsies were smaller thanthose of muscle). For manatee muscle (n = 12) and liver tissue(n = 3), 20 g of freeze-dried tissue was extracted where available.Samples were extracted in methanol and sonicated twice over a24 h period with a final extraction in methanol:acetone (1:1).Solvents were filtered and loaded onto a pre-rinsed (acetone,

methanol and distilled water) HP20 column (C18 Bond Elut LRC,Analytichem, Harbor City, CA). The filtered extract was passedthrough the column twice and rinsed through with distilled water.A solvent scheme with a gradient of distilled water and acetonewas used. A final rinse of 100% methanol was used for each tissuesample. Compounds eluted in the 20% H2O: 80% acetone fraction.

Thin layer chromatography (TLC) was used as an initialassessment to determine the presence of LTA in turtle andmanatee gut contents and tissue extracts using an LTA standard

Fig. 1. Map to show stranding locations and toxins present in tissues of necropsied

(A) manatee (Trichechus manatus latirostris) and (B) green sea turtle (Chelonia

mydas), in Florida, USA.

A. Capper et al. / Harmful Algae 28 (2013) 1–9 5

which was purified in-house at SMSFP from Lyngbya sp. andconfirmed by nuclear magnetic resonance (NMR).

2.5.1. Gut examination for Lyngbya filaments

Where available, the crop/stomach contents of turtles (n = 9;ranging 61.4–164.74 g wwt) and mouth contents (n = 2; 4.2 and7.3 g wwt) of manatees were visually assessed for Lyngbya

filaments using a compound microscope (40� magnification).Twenty random 500 ml sub-samples were taken from each gutsample and the whole microscope slide was scanned using a grid toconduct transects horizontally across the slide. If filaments wereobserved, filament width (including sheath) and cell length, wasrecorded to help identify Lyngbya spp.

3. Results

3.1. Brevetoxin analyses

Ten of the 11 turtles tested for PbTX were positive by ELISA,with low PbTX concentrations detected in seven lower GI samples

and six liver samples (Table 1; Fig. 1). At the time of testing,confirmatory analysis by LC–MS/MS at FWC was not available.Seven of the 14 manatees also tested positive in analyses carriedout by the FWC in response to a red-tide event (Table 1).

3.2. Okadaic acid analyses

OA was detected in faecal samples taken from the lower GI inone manatee (16.1 ng g�1) and one turtle (8.7 ng g�1) usingcolourimetric PPIA (Fig. 1; Table 1). OA was confirmed in themanatee sample by UPLC–MS/MS analysis however, insufficientturtle faecal sample remained for confirmatory analysis.

3.3. Saxitoxin analyses

Five of the 11 manatees initially tested positive by ELISA for STXin stomach samples with RIDASCREEN1 however, four of thesewere below the detection limit for HPLC confirmation (Fig. 1;Table 1). In the fifth sample dcGTX-2/3 and dcSTX were confirmed(Table 1) by HPLC-Fl. The total concentration (148 ng g�1) waspredominantly dcGTX-2/3 (70%) with less dcSTX (30%). Five turtlestomach samples also tested positive with RIDASCREEN1, butcould not be confirmed (Fig. 1, Table 1).

3.4. Lyngbyatoxin-A analyses

No LTA was detected in any of the manatee and turtle tissuetested. As no tissue samples tested positive with TLC, no furtheranalysis was carried out.

3.4.1. Gut examination

Visual examination of crop and stomach contents of greenturtle (Chelonia mydas) revealed Lyngbya filaments in three turtlecrop/stomach contents consistent with Lyngbya confervoides

(20.82 mm � 0.95 SE filament width, 1.99 mm � 0.09 SE cell width)(turtle #5, n = 7; turtle #2, n = 18; and turtle #7, n = 1, Fig. 1; Table 1)and one turtle sample (#2, n = 1, Fig. 1; Table 1) yielded a filamentconsistent with Lyngbya majuscula (39.6 mm filament width, 2.05 mmcell width).

4. Discussion

In this study nine out of 14 stranded manatees (64%) and 11 outof 13 stranded green sea turtles (85%) opportunistically sampledfrom around the coast of Florida during 2003–2006 were found tohave been exposed to one or more HAB toxins. Several manateesand turtles revealed evidence of exposure to two or more HABtoxins, with one manatee potentially positive for three of the HABtoxins tested. OA and STX were predominantly found in the GI tractcontents rather than tissues of both manatees and turtles, whereasPbTXs were also found in the liver. Previous testing by FWC of themanatees used in this study found PbTX in stomach, liver, urineand multiple other tissues, with three of these deaths attributed tored tide events. The stranded manatees in this study were from thewest coast of Florida between Pasco and Lee counties in the Gulf ofMexico where red tides are prevalent and most strandings occur.The majority of stranded turtles were from along the Florida eastcoast in the Atlantic Ocean ranging from St. Johns to Miami-DadeCounty.

On the west coast of Florida, mass animal mortalities arecommonly associated with Karenia spp. bloom (O‘Shea et al., 1991;Bossart et al., 1998; Flewelling et al., 2005, 2010; Landsberg et al.,2009). Animals are not only exposed to lethal doses of brevetoxinsduring bloom periods but also, due to toxin circulating throughfood webs, for weeks or months after a bloom has dissipated,prolonging the risk to marine animals (Flewelling et al., 2005;

A. Capper et al. / Harmful Algae 28 (2013) 1–96

Landsberg et al., 2009). High concentrations of PbTX in seagrassprovide a vector source of toxin transfer in manatees long afterblooms have dissipated (Flewelling et al., 2005). This may haveoccurred with the manatee that stranded on October 26, 2005 inBoca Grande, Gasparilla Sound, Lee County. A bloom beganSeptember 6–8 (�10,000 cells l�1), peaking between September26–30 (100,000 to <1,000,000 cells l�1), returning to low levels byOctober 3–6 (�10,000 cells l�1), dissipating by October 9 (ww-myfwc). Yet this manatee had high levels of PbTX in its stomach(1221.8 ng g�1). This provides additional evidence that even whenHAB species and toxins are present at only background levels, deathsmay still occur and chronic sub-lethal exposure is taking place.

During the sampling period of December 2003 and February2006, Florida red tides were observed during the whole periodexcept March to September 2004 (FWC, 2013a). The 2005 red tidelasted the whole year and while the intensity varied throughout theyear, the bloom affected all areas of the Florida Gulf coast at somepoint. Large numbers of manatees stranded and died during thisperiod, six of which are included in our study. PbTX was detected inmultiple tissues ranging from stomach, liver, brain, lymph nodes,urine and milk. However, only three of these deaths were attributedto natural causes by red tide whilst the other three had a probablecause of death by watercraft collision (FWC, 2013b). These had muchlower levels of PbTX in urine (1.4 ng mL�1), stomach (9.8 ng g�1) andliver (16.8 ng g�1) compared to levels as high as 1221.8 ng g�1 instomach and 105.0 ng g�1 in liver in manatees whose deaths wereattributed to red tides. It is possible that sub-lethal exposure to PbTXmay result in stressed animals, and prolonged exposure along withother co-factors such as cold-stress could reduce fitness even further(Walsh et al., 2005). Stressed animals may also become moresusceptible to boating accidents or accidental death. Of specialconcern is the finding of brevetoxins in milk from a lactatingmanatee (MNW0548). Placental transport of brevetoxin and/or itsmetabolites following maternal acute exposure and repeated low-dose exposure has been shown in mice (Benson et al., 2006), andbrevetoxins have been detected in the tissues of foetal shark pups(Flewelling et al., 2010). The consequences of exposure in earlydevelopmental stages have not been considered in manatee andturtle populations.

Brevetoxins were present in lower gut samples (64%) and liver(55%) in turtles tested. One turtle stranded in May 2004 in BayCounty in northwest Florida. Dolphin mortalities (107) due tobrevetoxicosis also occurred in this area in March of the same year(NOAA, 2004). Interestingly, two turtles both suffering from FP alsohad low concentrations of PbTX in their tissues but were from PalmBeach County on the east coast where no red tides had beenreported that year. Several other brevetoxin-positive turtles alsostranded on the east coast in 2004. Many had been severelydamaged in accidents (fishing line on flippers, shell cut andcracked, propeller wounds). Whilst it is possible that exposure tosub-lethal levels of toxins may make animals more susceptible toaccidental death, reduce their ability to evade boats, fishing linesetc. or may facilitate morbidity during cold weather snaps, the datafrom this paper cannot confirm this. Immune system function mayalso be impaired by exposure to brevetoxins in loggerhead turtles(Walsh et al., 2010).

No red tide bloom was observed on Florida’s east coast in 2004;however, in a separate study several sharks representing multiplespecies collected from Florida’s east coast during the summer of2004 contained surprisingly high concentrations of brevetoxins intheir tissues, including gills (Flewelling et al., 2010). Because redtide monitoring on the east coast is not routine and largely event-driven, blooms that remain offshore, below the surface, or atmodest cell concentrations may have no noticeable effects andmay remain undetected. As turtles have high site fidelity withregard to feeding grounds (Bresette et al., 1998) it is likely these

were exposed to PbTX where they feed. Studies such as this,especially with regard to turtles which show site fidelity, couldprovide a starting point for background level monitoring toascertain potential sub-lethal exposure levels to turtles.

Unlike many other harmful species, blooms of benthic dino-flagellates such as Prorocentrum spp. are often inconspicuous anddemonstrate a great plasticity in colonising a vast array of algalspecies (Landsberg et al., 1999). In Florida OA producing Prorocen-

trum lima has been found in low abundance on the east coast in theIndian River Lagoon (IRL), (Badylak and Phlips, 2004) andProrocentrum rhathymum in the northwest coast in Florida Bay(An et al., 2010). Being associated with seagrass and macroalgalspecies means that Prorocentrum spp. may incidentally be consumedby turtles and manatees whilst feeding. OA has been found onseagrass associated with dugong and turtle feeding grounds inAustralia, however, OA was not detected in tissues (Takahashi et al.,2008). In Hawai’i, OA producing Prorocentrum spp. were also foundon numerous species of algae preferentially consumed by turtles andpresumptive OA was detected in turtle kidney tissue (Landsberget al., 1999). A correlation between high OA concentration andsevere FP affliction in turtles led to suggestions that OA may play arole in aetiology of FP in Hawaiian turtles (Landsberg et al., 1999). Inthis study OA was detected in a turtle that stranded in BrowardCounty but it did not exhibit signs of FP. One manatee also testedpositive for OA, which stranded in Boca Grande, Lee County. This isthe first report of OA in manatees. This particular manatee alsotested positive for PbTX and STX. The concentrations of OA in thetissues of both these animals were at very low levels. Whilst this maynot be a major threat to animal health, the combined effects ofmultiple HAB toxins may lead to more harmful effects. Fire et al.(2011) documented the first report of OA in a marine mammal inTexas following an unusual mortality event of 100 bottlenosedolphins during a Prorocentrum spp. and Dinophysis spp. bloom. BothOA and domoic acid (DA) were detected at low levels in tissuessamples however, it is not known if OA and or DA were the cause ofstranding. Whilst OA does not appear to be causing a problem atpresent in Florida waters, this study should heed as a warning to thepotential impacts of such a bloom.

As a benthic cyanobacterium, Lyngbya can smother seagrassand macroalgae in sites where turtles have high fidelity and, assuch, it can be incidentally ingested during bloom periods (Arthuret al., 2006). Turtles held in captivity have been observed ingestingLyngbya majuscula (McMaster et al., 2003). In Australia, L.

majuscula often produces high quantities LTA (Capper et al.,2006b) which has been linked to the debilitating FP disease ingreen sea turtles (Arthur et al., 2008). L. cf. majuscula blooms wereobserved in the IRL in Florida in both 2004 and 2005 (pers. obs.). Asthe IRL has large numbers of resident green sea turtles which sufferfrom FP (Ehrhart, 1991), this prompted a study to determine if LTAwas present and whether this might play a potential role in theaetiology of FP in IRL turtles. Whilst no LTA was detected in thisstudy, three turtles were found with a sparse number of Lyngbya

filaments in their crop/stomach contents which demonstrates thatturtles are consuming small quantities of Lyngbya in Florida.However, this is in such small quantities it is likely to be incidentalconsumption. Both turtles tested with extensive FP (stranded inPalm Beach County) had not been exposed to LTA or OA tumourpromoters but had been exposed to brevetoxins and saxitoxins.Whilst neither brevetoxins nor saxitoxins are likely to play a role intumour promotion, vector-mediated sub-lethal exposure to thesecompounds can compromise animal fitness (Samson et al., 2008;Walsh et al., 2005).

Manatees are exposed to Lyngbya blanketing their favouredfood sources of turtle grass and eelgrass (EPA, 2010) in both marine(L. c.f. majuscula, L. confervoides, L. cf. polychroa) and freshwater(Lyngbya wollei) environments. No LTA was observed in manatee

A. Capper et al. / Harmful Algae 28 (2013) 1–9 7

tissues in this study or filaments observed in manatee upper gutsamples. However, external tumours have been documented inmanatees residing in the Crystal River and Homosassa Springsareas (Bennett, 2002; Bossart et al., 2002; Woodruff et al., 2003). Astudy by Harr et al. (2008) observed Lyngbya mats growing onmanatees on the east coast of Florida, further increasing theirchances of exposure to a wide range of Lyngbya toxins.

The Crystal River, located in Citrus County, is one of the world’slargest spring water fed ecosystems and provides a warm waterrefuge for manatees during winter periods (Evans et al., 2007). Thisarea is also subjected to large smothering blooms of Lyngbya wollei

(Evans et al., 2007), which not only produces toxins such as LTA(Burns, 2008) but also saxitoxins including STX, dcGTX-2/3, anddcSTX (Carmichael et al., 1997; Onodera et al., 1997; Foss et al.,2012). Two of these compounds (dcGTX-2/3 and dcSTX) weredetected in the stomach of a manatee that stranded in this area.This is the first finding of STX in manatees. Saxitoxins were alsodetected in four other manatees (although at levels too low to beconfirmed by HPLC) that stranded on both the west and east coastof Florida. These may have been from a dinoflagellate rather thancyanobacterial origin. One of the manatees with STX detected instomach samples stranded in the upper section of the IRL. This areahas been subjected to Pyrodinium bahamense var. bahamense

blooms on numerous occasions (Badylak and Phlips, 2004; Badylaket al., 2004; Phlips et al., 2004, 2010) and was also implicated as theputative organism in an STX poisoning incident from consumptionof toxic puffer fish between 2002 and 2004 in the north and centralIRL (Landsberg et al., 2006). STX was routinely detected in pufferfish over a four-year period (Abbott et al., 2009b), which alsoencompassed the time period when three turtles that had STX instomach and gut contents stranded south of the IRL. Saxitoxins andtheir derivatives are also found in a number of marine dinoflagel-late species and freshwater cyanobacterial species (Deeds et al.,2008). Unless bloom proportions of an organism are noted,surreptitious smaller blooms may mean both turtles and manateesare being chronically exposed to low-levels of STXs.

5. Conclusions

Both manatees and turtles are being exposed to multiple HABtoxins (okadaic acid, brevetoxins, saxitoxins, and likely others) inFlorida. Most of the compounds observed in this study were foundat low levels suggesting sub-lethal low-level exposure, but little isknown about the accumulation and metabolism of HAB toxins inhigher vertebrates, and low tissue concentrations may notaccurately represent exposure level in all cases. Sub-lethalexposure and the potential synergistic effects of multiple HABtoxins leading to reduced animal fitness cannot be ruled out.Further studies are required to determine the effects of thesetoxins at sub-lethal levels.

Acknowledgements

This research was funded by a Smithsonian Marine StationFellowship to A.C., Grant Number NA05 NOS 4781194 from the USDept. of Commerce (DoC, NOAA, ECOHAB programme) throughUNC-CH, and the Florida Fish and Wildlife Conservation Commis-sion. The findings, opinions and recommendations expressed inthis publication are those of the authors and not necessarily thoseof the DoC. The authors thank veterinarians and staff at the MarineMammal Pathobiology Laboratory and Karrie Minch and staff atthe Sea Turtle Stranding and Salvage Network for their assistancewith tissue acquisition and stranding data. Tissue collection andanalysis was carried out in accordance with Permit No. MA773494-8 for manatees and Marine Turtle Permit No. 105 for green sea

turtles. This is contribution 911 of the Smithsonian Marine Station,Fort Pierce, Florida.[SS]

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.hal.2013.04.009.

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