Dietary Exposure to Harmful Algal Bloom (HAB) Toxins In

Harmful Algae 28 (2013) 1–9

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Harmful Algae

jo urnal homepage: www.elsevier.com/locate/hal

Dietary exposure to harmful algal bloom (HAB) toxins in

the endangered manatee (Trichechus manatus latirostris)

and green sea turtle (Chelonia mydas) in Florida, USA

a,b, c b

Angela Capper *, Leanne J. Flewelling , Karen Arthur

School of Marine and Tropical Biology and Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook University, Townsville, QLD 4811, Australia

Smithsonian Marine Station, Seaway Drive, Fort Pierce, FL 34949, USA

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 A B S T R A C T

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

Received 8 February 2013

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

Received in revised form 26 April 2013

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

Accepted 27 April 2013

(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

Keywords:

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

Brevetoxin

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

Okadaic acid

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

Saxitoxin

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

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

Dinoﬂagellate 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.

1. Introduction ecological consequences of toxic and bloom-forming species are

numerous with signiﬁcant impacts noted for the Floridian

Harmful algal blooms (HABs) are apparently increasing in both ecosystem, human and aquatic animal health (Landsberg, 2002;

frequency and range in aquatic habitats throughout the world Van Dolah et al., 2003; Kirkpatrick et al., 2004; Flewelling et al.,

(Hallegraeff, 1993, 2010). However, one area of the USA that 2005; Walsh et al., 2006; Pierce and Henry, 2008; Landsberg et al.,

appears to be a particular hot-spot for HABs is Florida. The 2009; Fleming et al., 2011; Fire and Van Dolah, 2012), and with

implications for ﬁsh populations and subsequent recruitment

(Flaherty and Landsberg, 2011). The reason why Florida may be so

* Corresponding author at: School of Marine and Tropical Biology and Centre for prone to such blooms has been cause of speculation for more than

Sustainable Tropical Fisheries and Aquaculture, James Cook University, Townsville, 130 years and is related to numerous physico-chemical and

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

biological drivers exacerbated by long-term global climatic shifts

E-mail addresses: [email protected], [email protected] (A. Capper),

in conjunction with anthropogenic stressors (Anderson et al.,

[email protected] (L.J. Flewelling), [email protected]

(K. Arthur). 2008; Paerl and Huisman, 2009; Vargo, 2009).

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

More than 70 potentially harmful algal species have been lima, Prorocentrum concavum and Prorocentrum mexicana have

identiﬁed from Florida estuarine and coastal waters (Abbott et al., been observed at numerous locations in Florida (Bomber et al.,

2009a) which include single species or a consortia of cyanobacteria 1988) with Prorocentrum micans in the Indian River Lagoon (Phlips

(cyanoHABs) or microalgae (dinoﬂagellates, diatoms, raphido- et al., 2010) and Prorocentrum rhathymum in Florida Bay (An et al.,

phytes) (Badylak and Phlips, 2004; Phlips et al., 2004; Badylak 2010) Many of these species can produce okadaic acid (OA) (see

et al., 2007; Livingston, 2007; Badylak and Phlips, 2008; Phlips review by Landsberg, 2002), which can lead to diarrheic shellﬁsh

et al., 2011). Many of these produce toxins and other biologically poisoning (DSP) in humans (Yasumoto et al., 1978), acts as a

active secondary metabolites ostensibly as defence against tumour promoter in mice (Suganuma et al., 1988; Fujiki and

predation (Paul et al., 2007). Our understanding of the fate of Suganuma, 2009) and has been implicated as an environmental co-

compounds produced by cyanoHABs and toxic microalgal blooms factor in the aetiology of the debilitating neoplastic disease

in their natural environment and potential accumulation in ﬁbropapillomatosis (FP) in turtles (Landsberg et al., 1999; Arthur

aquatic food webs is still in its infancy. Exposure to, and et al., 2008). FP is most prevalent in inshore areas of high human

consumption of, HAB toxins can impact all trophic levels with impact (Herbst, 1994) with the highest recorded incidences in

some compounds accumulating through the food web (Landsberg Hawaii (92%, Balazs, 1991; Chaloupka et al., 2009), Florida (33–

et al., 2006; Paul et al., 2007; Fire et al., 2008; Landsberg et al., 61%, Ehrhart, 1991; Ehrhart et al., 2007) and Moreton Bay, Australia

2009). The ﬁtness of some aquatic organisms exposed to Florida (40–70%, Aguirre et al., 1999).

HABs may be compromised (Landsberg, 1995, 2002; Prince et al., The potent tumour promoter, lyngbyatoxin-A (LTA), has also

2006; Nam et al., 2010). Exposure can be via direct ingestion or been implicated as a co-factor in FP aetiology in the turtles in the

inhalation (Bossart et al., 1998; Flewelling et al., 2005; Naar et al., Paciﬁc (Arthur et al., 2008). LTA is produced by Lyngbya majuscula

2007), or via trophic transfer through the food chain (Flewelling (Cardellina et al., 1979) and has been detected in high concentra-

et al., 2005; Naar et al., 2007; Deeds et al., 2008; Fire et al., 2008; tions in a number of locations worldwide (Capper et al., 2005;

Landsberg et al., 2009; Sotka et al., 2009). Both direct and indirect Arthur et al., 2008). This highly adaptive benthic cyanobacteria has

exposure can be acute resulting in serious health problems or been increasingly observed in Florida freshwater, estuarine and

death, or may be chronic with the potential for sub-lethal impacts. marine ecosystems in recent years (Landsberg et al., 2003; Burns,

Two endangered species which are closely monitored in Florida 2008; Pinowska et al., 2009), proliferating in the reef-associated

are the manatee (Trichechus manatus latirostris) and the green communities of south Florida (Paul et al., 2005); with ephemeral

turtle (Chelonia mydas). As euryhaline species, manatees living in blooms in the Indian River Lagoon (Capper and Paul, 2008) and

Florida ecosystems forage in both freshwater and marine Sanibel, west Florida (Paerl et al., 2008). Lyngbya spp. often blanket

environments consuming a range of aquatic vegetation and seagrass beds hindering access to important food sources for turtle

associated epiphytes (Hartman, 1979). The large resident feeding and manatee populations (Arthur et al., 2006; EPA, 2010;

populations of green turtles in Florida (Ehrhart, 1995; Ehrhart and Yasumoto, 2000) leading to potential exposure through direct

Redfoot, 1995; Ehrhart et al., 2007) have a high site ﬁdelity within ingestion and dietary assimilation. Consumption of L. majuscula by

small feeding grounds (Musick and Limpus, 1996; Bresette et al., Chelonia mydas has been observed in ﬁeld and laboratory trials in

1998) consuming a wide range of seagrass and algae (Mendonc¸a, Australia and in ﬁeld sampling in Hawaii (McMaster et al., 2003;

1983). Both species are likely to be affected by HABs through Arthur and Balazs, 2008; Arthur et al., 2008). Preferential grazing of

contamination of food sources, toxins in the water column and Lyngbya spp. by manatees is unlikely; many of the compounds it

aerosolised toxins released due to wave action. produces are unpalatable to generalist herbivore meso-grazers

The health of manatees is notably adversely affected each year such as sea urchins and crabs (Nagle et al., 1996; Pennings et al.,

by exposure to the frequent and periodic red-tide blooms 1996; Nagle and Paul, 1998; Capper et al., 2006a; Capper and Paul,

producing brevetoxins (PbTX) in Florida (Landsberg et al., 2009). 2008), and macro-grazers such as ﬁsh (Thacker et al., 1997; Nagle

There has also been a long-standing association with aquatic and Paul, 1998; Capper et al., 2006b). Nonetheless, incidental

organism red-tide deaths in Florida including: ﬁsh kills since 1844 grazing whilst feeding on preferred dietary items may occur.

(Ingersoll, 1882); dolphins since 1946–1947 (Gunter et al., 1948); Whilst a diverse array of compounds (displaying immunosup-

and manatees since 1963 (Layne, 1965). Dietary exposure to PbTX pressant, antimitotic and chemotrypsin inhibitory activity) have

during a Karenia brevis bloom has been demonstrated by been isolated from three Lyngbya chemotypes in the reefs of south

Flewelling et al. (2005) who found a high concentration of PbTX eastern Florida (Sharp et al., 2009), LTA has not been detected in

in stranded manatee stomach contents associated with large any marine Lyngbya spp. sampled (n = 38) from sites along the

quantities of seagrass (Thalassia testidunum). Subsequent analysis eastern coast ranging from Cape Canaveral to the Florida Keys from

of seagrass collected from the area of strandings showed high July 2004 to June 2006 (pers. obs.). LTA has however, been detected

concentrations of PbTX, not only in epiphytes on the seagrass, but in freshwater Lyngbya wollei (Burns, 2008) occurring in Florida’s

also in the seagrass blades and rhizomes. This indicated that freshwater springs and riverine systems (Cowell and Botts, 1994;

seagrass was the primary brevetoxins vector. Manatees may also Pinowska et al., 2009). L. wollei also produces saxitoxins (STX) and

be exposed to PbTX via inhalation of aerosolised toxins leading to their analogues (Carmichael et al., 1997; Onodera et al., 1997;

respiratory problems and lung damage (Bossart et al., 1998; Van Thacker and Camacho, 2008; Foss et al., 2012). These potent

Dolah et al., 2003). PbTX has also been detected in brain and liver compounds are neurotoxic and cause paralytic shellﬁsh poisoning

tissues as well as lymph nodes, urine and milk from lactating (PSP) in humans (Etheridge, 2004; Pearson et al., 2010). STXs have

manatees (FWC, 2007, 2008; Flewelling, 2008). been implicated as the aetiological agent in the deaths of dolphins,

The health of turtles is also adversely affected by exposure to whales and seals following ingestion of organisms found with high

Karenia spp. During prolonged Karenia brevis blooms in 2005 and STX concentrations (Van Dolah et al., 2003; Lowenstein, 2004; Fire

2006, PbTXs were detected in blood and tissues of multiple species and Van Dolah, 2012).

of both live and dead stranded sea turtles, including green turtles. The diversity of HAB organisms in Florida coastal waters

In the majority of these animals brevetoxin exposure was suggests the possibility of simultaneous environmental HAB toxin

determined to have caused or contributed to the strandings exposure, particularly when many of these species have the ability

(Fauquier et al., 2013). to co-occur (Kubanek et al., 2005). A recent example of this was

Another dietary route of HAB toxin exposure are benthic observed in the coastal embayments of Sanibel and Captiva Islands

dinoﬂagellates potentially ingested with seagrass. Prorocentrum in 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 on and are provided in Table 1 for comparison (NOAA, FWC, pers.

individual aquatic organisms and speciﬁc HAB toxins, very few comm.).

studies have focussed on the detection of a range of HAB toxins in Whilst this ELISA does not distinguish among individual PbTX

the same organism. A recent study by Fire et al. (2011) reported the congeners, it does show sensitivity and speciﬁcity for the family of

co-occurrence of multiple HAB toxins (okadaic acid, domoic acid, toxins at low concentrations and is a reliable, high-throughput

and brevetoxins) in bottlenose dolphins that stranded along the assay. As performed in this study, the limit of quantiﬁcation was 5–

À1 À1

Texas coast. While the toxins were not conclusively linked to the 10 ng PbTX-3 eq g dry wt. (1–1.5 ng PbTX-3 eq g wet wt.).

cause of the strandings, the ﬁndings highlight the need to better

understand the impacts of HABs on wildlife health. Additional 2.3. Okadaic acid analyses

studies by Twiner et al. (2011), Twiner et al. (2012) also noted that

dolphins on the southwest coast of Florida were commonly Turtle lower GI (n = 6), liver (n = 7) and ﬁbropapilloma (n = 2)

exposed to both brevetoxins and domoic acid over a ten-year and manatee lower GI/faecal (n = 11) and liver (n = 13) were tested

period (1999–2009). for the presence of OA using a colourimetric protein phosphatase

The extent to which aquatic organisms are exposed to multiple inhabitation assay (PPIA). To extract the toxins, tissue samples

HAB toxins is not well known. Chronic and acute poisoning were prepared as described in Section 2.2 for brevetoxin analysis.

episodes have the potential to compromise animal ﬁtness. The The PPIA was performed using a method modiﬁed from Tubaro

synergistic effect of multiple stressors over prolonged periods has et al. (1996). The PPIA detects compounds that inhibit protein

not been assessed. This study aimed to identify the presence of phosphatase, including the marine algal toxins okadaic acid and

multiple HAB toxins (brevetoxins, saxitoxins, okadaic acid and dinophysistoxins as well as the predominantly freshwater micro-

lyngbyatoxin-A) in the tissues of manatees and green sea turtles cystins. To differentiate and conﬁrm the identity of the toxins,

stranded along the coast of Florida in order to identify regions samples that were positive by PPIA were analysed for microcystins

where animals may be at risk of exposure to HAB toxins, which using the Microcystins-DM ELISA kit (Abraxis, Warminster, PA,

may provide important information for the conservation of these USA) performed as speciﬁed in the product insert and for okadaic

endangered species. acid and dinophysistoxin-1 using ultrahigh-performance liquid

chromatography with tandem mass spectrometry (UPLC–MS/MS).

UPLC–MS/MS analyses were performed using an Acquity UPLC

2. Material and methods system coupled to a Quattro micro API triple quadrupole mass

spectrometer (Waters, Milford, MA, US) according to Deeds et al.

2.1. Tissue sample collection (2010). Quantiﬁcation was performed using a 6-point calibration

of OA reference solution purchased from NRC Canada.

The Florida Fish and Wildlife Conservation Commission (FWC)

and the Sea Turtle Stranding and Salvage Network strive to collect 2.4. Saxitoxin analyses

all manatees and sea turtles respectively that strand along the

Florida coast. Stranded carcasses provide an excellent opportunity The RIDASCREEN FAST Saxitoxin Assay (R-Biopharm AG,

for tissue sampling to screen for exposure to HAB toxins and assess Damstadt, Germany) ELISA was used to screen for the potential

whether any exposure may have been acute, sub-lethal or played a presence of STX and analogues in manatee stomach (n = 10) and

role in animal death, which may have implications for general lower GI (n = 1) samples. Turtle upper GI (n = 8) and lower gut

population health. (n = 1) samples were also tested. For each tissue sample, 1 g of

Several green turtles (n = 13) and manatees (n = 14) that freeze-dried material was analysed. Samples were extracted in

stranded between May 2004 and February 2006 were sampled 10 mL 0.1 M acetic acid, boiled for 5 mins, then centrifuged at

during necropsies conducted at the FWC Marine Mammal 3500 rpm for 10 min. The supernatant (100 mL) was removed and

Pathobiology Laboratory in St. Petersburg, FL. Wild carcasses were added to 980 mL buffer solution.

sourced opportunistically from predominantly the east and west For toxin characterisation, potential positive samples (manatee

coast of Florida (Table 1; Fig. 1). For green turtles tissue samples stomach, n = 5; turtle upper GI sample (n = 4); turtle lower GI/faecal

consisted of muscle (n = 13), liver (n = 8), and ﬁbropapillomas sample (n = 1)) were subjected to high performance liquid

(n = 2). Stomach contents (where available) and small intestine chromatography with ﬂuorescence detection (HPLC-Fl). Prior to

contents combined herein referred to as upper GI (n = 12), colon/ HPLC analysis, sample extracts were cleaned by passing through a

faecal matter, herein referred to as lower GI. Manatee samples Supelclean LC-18 cartridge (500 mg/3 mL; Supelco, Bellefonte, PA,

included muscle, liver, mouth contents, stomach contents and USA) After preconditioning the cartridge with 6 mL of methanol and

faecal matter in the lower GI. Samples were placed into sealed glass then 6 mL deionized water, 1 mL of extract was passed through the

containers and transported on ice to the Smithsonian Marine column and collected in graduated centrifuge tube. The column was

Station, Fort Pierce (SMSFP), FL. Wet weights were recorded and rinsed with 2 mL of deionized water, which was also collected in the

samples stored at À20 8C prior to analysis. centrifuge tube. The sample was then adjusted to pH 6–7 and

brought to a ﬁnal volume of 4 mL. Extracts were analysed for PSP

2.2. Brevetoxin analyses toxins using HPLC-Fl according to Lawrence et al. (2005).

Turtle lower GI (n = 6), liver (n = 7) and ﬁbropapilloma (n = 2) 2.5. Lyngbyatoxin analyses

tissues were tested for the presence of PbTX and their congeners.

Freeze-dried (1 g dwt) tissue samples were mascerated and To test for the presence of LTA, gut contents and tissues were

extracted for PbTX as described in Flewelling (2008). A competitive freeze-dried. Turtle upper GI (n = 11) and lower GI/faecal (n = 12)

ELISA was performed according to Naar et al. (2002) which samples and manatee stomach (n = 13) and lower GI/faecal

recognises all congeners and metabolites of brevetoxin that have a samples (n = 12) were freeze-dried and extracted three times in

PbTX-2-type backbone. Toxin concentrations are calculated using 1:1 ethyl acetate: methanol during a 24 h period to obtain a non-

À1

a PbTX-3 standard curve and results are reported in PbTX-3 eq g . polar extract. The sample was further extracted three times in 1:1

Samples from the manatees included in this study were previously ethanol: distilled H2O for an additional 24 h to obtain a polar

tested for PbTX by FWC using the same method as described above extract. Samples were ﬁltered and dried by rotary evaporation.

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

Table 1

Concentrations 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 Probable cause LTA STX OA PbTX a

(County) of death

Manatees

(1)MNW0548 09 September 05 Pinellas Red tide nd Potential pos nd Stomach 579.1, (stomach)b

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 nd Stomach 61.7 (stomach)b

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 Lower Stomach 1221.8 (stomach)b GIe 16.1

Liver 79.6

Kidney 23.3, urine 12.5

(7)MNW0566 13 December 05 Citrus Watercraft collision nd Stomach 148 nd nd (dcGTX-2/3 c

+ dcSTX)

(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 nd nd (stomach)b

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 – Lower GI 4.1

(stomach)b

(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 – Lower GI 7.8

(stomach)b

(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. nd nd nd Lower GI 7.8

(Fibropapillomas)

(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 Liver 11.4

GIf 8.7

(10)KES 050203-01 03 February 05 Martin Unknown nd Potential pos nd Liver 16.2

(stomach)b

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 nd Potential pos nd Liver 20.9

b d

(Fibropapillomas) (stomach , lower GI )

–, not tested; nd, toxin not detected.

Cause of death determined by FWC Marine Mammal Pathology Laboratory (manatees) and Sea Turtle Stranding and Salvage Network (turtles).

b 1

Positive result using RIDASCREEN assay but below level of detection for HPLC conﬁrmation.

Positive result with ELISA, conﬁrmed by HPLC.

Sample too viscous to pass through C18 column for clean-up.

Protein Phosphatase Inhibition Assays followed by MY ELISA to test for microcystins (negative) and OA UPLC–MS/MS for okadaic acid (positive).

Sample size small and unable to carry out conﬁrmatory analysis using MY ELISA and UPLC–MS/MS for OA.

Manatee PbTX analyses conducted by FWC previously during the 2005–2006 Unusual Marine Mammal Mortality Event (NOAA, FWC in preparation).

For LTA turtle tissue extractions, 10 g of freeze-dried material methanol and distilled water) HP20 column (C18 Bond Elut LRC,

was used (where available) for muscle (n = 13) and ﬁbropapilloma Analytichem, Harbor City, CA). The ﬁltered extract was passed

tissue (n = 2), whilst 6 g was used for liver (n = 7) extractions (liver through the column twice and rinsed through with distilled water.

tissue samples obtained from mass necropsies were smaller than A solvent scheme with a gradient of distilled water and acetone

those of muscle). For manatee muscle (n = 12) and liver tissue was used. A ﬁnal rinse of 100% methanol was used for each tissue

(n = 3), 20 g of freeze-dried tissue was extracted where available. sample. Compounds eluted in the 20% H2O: 80% acetone fraction.

Samples were extracted in methanol and sonicated twice over a Thin layer chromatography (TLC) was used as an initial

24 h period with a ﬁnal extraction in methanol:acetone (1:1). assessment to determine the presence of LTA in turtle and

Solvents were ﬁltered and loaded onto a pre-rinsed (acetone, manatee gut contents and tissue extracts using an LTA standard

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

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 carried

out 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 in

À1 À1

one manatee (16.1 ng g ) and one turtle (8.7 ng g ) using

colourimetric PPIA (Fig. 1; Table 1). OA was conﬁrmed in the

manatee sample by UPLC–MS/MS analysis however, insufﬁcient

turtle faecal sample remained for conﬁrmatory analysis.

3.3. Saxitoxin analyses

Five of the 11 manatees initially tested positive by ELISA for STX

in stomach samples with RIDASCREEN however, four of these

were below the detection limit for HPLC conﬁrmation (Fig. 1;

Table 1). In the ﬁfth sample dcGTX-2/3 and dcSTX were conﬁrmed

À1

(Table 1) by HPLC-Fl. The total concentration (148 ng g ) was

predominantly dcGTX-2/3 (70%) with less dcSTX (30%). Five turtle

stomach samples also tested positive with RIDASCREEN , but

could not be conﬁrmed (Fig. 1, Table 1).

3.4. Lyngbyatoxin-A analyses

No LTA was detected in any of the manatee and turtle tissue

tested. As no tissue samples tested positive with TLC, no further

analysis was carried out.

3.4.1. Gut examination

Visual examination of crop and stomach contents of green

turtle (Chelonia mydas) revealed Lyngbya ﬁlaments in three turtle

crop/stomach contents consistent with Lyngbya confervoides

(20.82 mm Æ 0.95 SE ﬁlament width, 1.99 mm Æ 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 ﬁlament

consistent with Lyngbya majuscula (39.6 mm ﬁlament width, 2.05 mm

cell width).

4. Discussion

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 In this study nine out of 14 stranded manatees (64%) and 11 out

mydas), in Florida, USA.

of 13 stranded green sea turtles (85%) opportunistically sampled

from around the coast of Florida during 2003–2006 were found to

have been exposed to one or more HAB toxins. Several manatees

which was puriﬁed in-house at SMSFP from Lyngbya sp. and and turtles revealed evidence of exposure to two or more HAB

conﬁrmed by nuclear magnetic resonance (NMR). toxins, with one manatee potentially positive for three of the HAB

toxins tested. OA and STX were predominantly found in the GI tract

2.5.1. Gut examination for Lyngbya ﬁlaments contents rather than tissues of both manatees and turtles, whereas

Where available, the crop/stomach contents of turtles (n = 9; PbTXs were also found in the liver. Previous testing by FWC of the

ranging 61.4–164.74 g wwt) and mouth contents (n = 2; 4.2 and manatees used in this study found PbTX in stomach, liver, urine

7.3 g wwt) of manatees were visually assessed for Lyngbya and multiple other tissues, with three of these deaths attributed to

ﬁlaments using a compound microscope (40Â magniﬁcation). red tide events. The stranded manatees in this study were from the

Twenty random 500 ml sub-samples were taken from each gut west coast of Florida between Pasco and Lee counties in the Gulf of

sample and the whole microscope slide was scanned using a grid to Mexico where red tides are prevalent and most strandings occur.

conduct transects horizontally across the slide. If ﬁlaments were The majority of stranded turtles were from along the Florida east

observed, ﬁlament width (including sheath) and cell length, was coast in the Atlantic Ocean ranging from St. Johns to Miami-Dade

recorded to help identify Lyngbya spp. County.

On the west coast of Florida, mass animal mortalities are

3. Results commonly associated with Karenia spp. bloom (O‘Shea et al., 1991;

Bossart et al., 1998; Flewelling et al., 2005, 2010; Landsberg et al.,

3.1. Brevetoxin analyses 2009). Animals are not only exposed to lethal doses of brevetoxins

during bloom periods but also, due to toxin circulating through

Ten of the 11 turtles tested for PbTX were positive by ELISA, food webs, for weeks or months after a bloom has dissipated,

with low PbTX concentrations detected in seven lower GI samples prolonging the risk to marine animals (Flewelling et al., 2005;

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

Landsberg et al., 2009). High concentrations of PbTX in seagrass were exposed to PbTX where they feed. Studies such as this,

provide a vector source of toxin transfer in manatees long after especially with regard to turtles which show site ﬁdelity, could

blooms have dissipated (Flewelling et al., 2005). This may have provide a starting point for background level monitoring to

occurred with the manatee that stranded on October 26, 2005 in ascertain potential sub-lethal exposure levels to turtles.

Boca Grande, Gasparilla Sound, Lee County. A bloom began Unlike many other harmful species, blooms of benthic dino-

À1

September 6–8 (10,000 cells l ), peaking between September ﬂagellates such as Prorocentrum spp. are often inconspicuous and

À1

26–30 (100,000 to <1,000,000 cells l ), returning to low levels by demonstrate a great plasticity in colonising a vast array of algal

À1

October 3–6 (10,000 cells l ), dissipating by October 9 (ww- species (Landsberg et al., 1999). In Florida OA producing Prorocen-

myfwc). Yet this manatee had high levels of PbTX in its stomach trum lima has been found in low abundance on the east coast in the

À1

(1221.8 ng g ). This provides additional evidence that even when Indian River Lagoon (IRL), (Badylak and Phlips, 2004) and

HAB species and toxins are present at only background levels, deaths Prorocentrum rhathymum in the northwest coast in Florida Bay

may still occur and chronic sub-lethal exposure is taking place. (An et al., 2010). Being associated with seagrass and macroalgal

During the sampling period of December 2003 and February species means that Prorocentrum spp. may incidentally be consumed

2006, Florida red tides were observed during the whole period by turtles and manatees whilst feeding. OA has been found on

except March to September 2004 (FWC, 2013a). The 2005 red tide seagrass associated with dugong and turtle feeding grounds in

lasted the whole year and while the intensity varied throughout the Australia, however, OA was not detected in tissues (Takahashi et al.,

year, the bloom affected all areas of the Florida Gulf coast at some 2008). In Hawai’i, OA producing Prorocentrum spp. were also found

point. Large numbers of manatees stranded and died during this on numerous species of algae preferentially consumed by turtles and

period, six of which are included in our study. PbTX was detected in presumptive OA was detected in turtle kidney tissue (Landsberg

multiple tissues ranging from stomach, liver, brain, lymph nodes, et al., 1999). A correlation between high OA concentration and

urine and milk. However, only three of these deaths were attributed severe FP afﬂiction in turtles led to suggestions that OA may play a

to natural causes by red tide whilst the other three had a probable role in aetiology of FP in Hawaiian turtles (Landsberg et al., 1999). In

cause of death by watercraft collision (FWC, 2013b). These had much this study OA was detected in a turtle that stranded in Broward

À1 À1

lower levels of PbTX in urine (1.4 ng mL ), stomach (9.8 ng g ) and County but it did not exhibit signs of FP. One manatee also tested

À1 À1

liver (16.8 ng g ) compared to levels as high as 1221.8 ng g in positive for OA, which stranded in Boca Grande, Lee County. This is

À1

stomach and 105.0 ng g in liver in manatees whose deaths were the ﬁrst report of OA in manatees. This particular manatee also

attributed to red tides. It is possible that sub-lethal exposure to PbTX tested positive for PbTX and STX. The concentrations of OA in the

may result in stressed animals, and prolonged exposure along with tissues of both these animals were at very low levels. Whilst this may

other co-factors such as cold-stress could reduce ﬁtness even further not be a major threat to animal health, the combined effects of

(Walsh et al., 2005). Stressed animals may also become more multiple HAB toxins may lead to more harmful effects. Fire et al.

susceptible to boating accidents or accidental death. Of special (2011) documented the ﬁrst report of OA in a marine mammal in

concern is the ﬁnding of brevetoxins in milk from a lactating Texas following an unusual mortality event of 100 bottlenose

manatee (MNW0548). Placental transport of brevetoxin and/or its dolphins during a Prorocentrum spp. and Dinophysis spp. bloom. Both

metabolites following maternal acute exposure and repeated low- OA and domoic acid (DA) were detected at low levels in tissues

dose exposure has been shown in mice (Benson et al., 2006), and samples however, it is not known if OA and or DA were the cause of

brevetoxins have been detected in the tissues of foetal shark pups stranding. Whilst OA does not appear to be causing a problem at

(Flewelling et al., 2010). The consequences of exposure in early present in Florida waters, this study should heed as a warning to the

developmental stages have not been considered in manatee and potential impacts of such a bloom.

turtle populations. As a benthic cyanobacterium, Lyngbya can smother seagrass

Brevetoxins were present in lower gut samples (64%) and liver and macroalgae in sites where turtles have high ﬁdelity and, as

(55%) in turtles tested. One turtle stranded in May 2004 in Bay such, it can be incidentally ingested during bloom periods (Arthur

County in northwest Florida. Dolphin mortalities (107) due to et al., 2006). Turtles held in captivity have been observed ingesting

brevetoxicosis also occurred in this area in March of the same year Lyngbya majuscula (McMaster et al., 2003). In Australia, L.

(NOAA, 2004). Interestingly, two turtles both suffering from FP also majuscula often produces high quantities LTA (Capper et al.,

had low concentrations of PbTX in their tissues but were from Palm 2006b) which has been linked to the debilitating FP disease in

Beach County on the east coast where no red tides had been green sea turtles (Arthur et al., 2008). L. cf. majuscula blooms were

reported that year. Several other brevetoxin-positive turtles also observed in the IRL in Florida in both 2004 and 2005 (pers. obs.). As

stranded on the east coast in 2004. Many had been severely the IRL has large numbers of resident green sea turtles which suffer

damaged in accidents (ﬁshing line on ﬂippers, shell cut and from FP (Ehrhart, 1991), this prompted a study to determine if LTA

cracked, propeller wounds). Whilst it is possible that exposure to was present and whether this might play a potential role in the

sub-lethal levels of toxins may make animals more susceptible to aetiology of FP in IRL turtles. Whilst no LTA was detected in this

accidental death, reduce their ability to evade boats, ﬁshing lines study, three turtles were found with a sparse number of Lyngbya

etc. or may facilitate morbidity during cold weather snaps, the data ﬁlaments in their crop/stomach contents which demonstrates that

from this paper cannot conﬁrm this. Immune system function may turtles are consuming small quantities of Lyngbya in Florida.

also be impaired by exposure to brevetoxins in loggerhead turtles However, this is in such small quantities it is likely to be incidental

(Walsh et al., 2010). consumption. Both turtles tested with extensive FP (stranded in

No red tide bloom was observed on Florida’s east coast in 2004; Palm Beach County) had not been exposed to LTA or OA tumour

however, in a separate study several sharks representing multiple promoters but had been exposed to brevetoxins and saxitoxins.

species collected from Florida’s east coast during the summer of Whilst neither brevetoxins nor saxitoxins are likely to play a role in

2004 contained surprisingly high concentrations of brevetoxins in tumour promotion, vector-mediated sub-lethal exposure to these

their tissues, including gills (Flewelling et al., 2010). Because red compounds can compromise animal ﬁtness (Samson et al., 2008;

tide monitoring on the east coast is not routine and largely event- Walsh et al., 2005).

driven, blooms that remain offshore, below the surface, or at Manatees are exposed to Lyngbya blanketing their favoured

modest cell concentrations may have no noticeable effects and food sources of turtle grass and eelgrass (EPA, 2010) in both marine

may remain undetected. As turtles have high site ﬁdelity with (L. c.f. majuscula, L. confervoides, L. cf. polychroa) and freshwater

regard to feeding grounds (Bresette et al., 1998) it is likely these (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 gut turtles. This is contribution 911 of the Smithsonian Marine Station,

samples. However, external tumours have been documented in Fort Pierce, Florida.[SS]

manatees residing in the Crystal River and Homosassa Springs

areas (Bennett, 2002; Bossart et al., 2002; Woodruff et al., 2003). A Appendix A. Supplementary data

study by Harr et al. (2008) observed Lyngbya mats growing on

manatees on the east coast of Florida, further increasing their

Supplementary data associated with this article can be found, in

chances of exposure to a wide range of Lyngbya toxins.

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

The Crystal River, located in Citrus County, is one of the world’s

largest spring water fed ecosystems and provides a warm water

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