1. Phycotoxin loads in bottlenose dolphins (Tursiops truncatus) within the
Indian River Lagoon Estuary System and surrounding coastal waters,
Florida, 2002-2011
Jeremy Browning – M.S. Candidate Marine Biology
Advisor: Spencer. E. Fire

2. Background
• Marine mammals are sentinel organisms
• Experience large scale ‘die-offs’
• Increase in die off events
• Marine mammal health in general

3. nmfs.noaa.gov
Unusual Mass Mortality Events - UMEs
40.4%
Biotoxin = phycotoxin (phytoplankton produced)

4. Harmful Algal Bloom (HAB)
• Acute proliferation of marine
phytoplankton (80 toxic / >5000 species)
– Origin of phycotoxins
– Perturb ecosystems
– Cause UMEs
– Extreme – ecological regime changes

5. IRL Study Area; Two HAB Species
Karenia brevis
‘red-tide’
organism
Brevetoxin
(PbTx)
Neurotoxic
Shellfish
Poisoning – NSP
𝑳𝑫 𝟓𝟎 800ug/kg
Pyrodinium
bahamense
bioluminescence
Saxitoxin (STX)
Paralytic Shellfish
Poisoning – PSP
𝑳𝑫 𝟓𝟎 6-24ug/kg

6. IRL bottlenose dolphin (Tursiops truncatus)

7. Fire et al. 2015
2007 UME-HAB Event; the IRL
K. brevis associated mortalities
STRANDINGS (PbTx detected)
*Dolphins (92% positive)
*Manatees (69% positive)
• Baseline?
• Severity?
• Comparisons?

8. Objectives
1.) DETERMINE THE BASELINE FOR DOLPHINS IN
THE IRL EXPOSED TO PBTX AND STX IN non-HAB
conditions.
2.) COMPARE OUR TOXIN CONCENTRATION OF
DOLPHINS RECOVERED DURING HAB conditions
AND DURING non-HAB conditions IN OTHER
LOCATIONS OF FLORIDA.

9. Hypotheses
𝐻1: For PbTx & STX higher tissue toxin amounts are in
dolphins RECOVERED during HAB conditions (exposed)
versus dolphins RECOVERED in non-HAB conditions
(non-exposed).
𝐻2: Spatio-temporal toxic phytoplankton cell abundance
correlates positively with increased likelihood of toxin
detection in dolphins.

10. • Dolphin Samples
– Stranded dolphins recovered from 2002-2011
• IRL and immediate Atlantic Ocean Coast (AO)
– dolphin livers for biotoxin analysis (n=119)

11. Extractions
• Saxitoxin (STX); 0.1M HCl acid extraction with
Deproteinization step
– Water soluble
• Brevetoxin(PbTx); solvent extraction with
more mechanical homogenization
– Lipophilic

12. Toxin Analysis
• Enzyme Linked Immunosorbent Assay (ELISA)
– Competitive antibody binding assay
– Colorimetric (blueyellow)
• ‘Toxin-like Activity’ Reported
– 490 nm with 96-well plate reader
– 4-parameter dose response curve
(%B/Bmax)
Step 1.) Add sample and toxin-enzyme conjugate
Step 2.) Sample competes with toxin conjugate for
binding sites on antibody
Step 3.) Antibody bound toxin conjugate produces
color change
?
??
?
?TMB
Toxin-HRP-TMB

13. • HAB Phytoplankton Data
– Florida Fish and Wildlife Conservation Commission
(FWC) Historical database, 2001-2014
• Criteria for a ‘bloom’
*K. brevis ≥100,000 cells/L
*P. bahamense ≥ 15,000 cells/L
Defining Dolphin HAB Exposure

14. Estimating HAB Exposure
• HAB vs. non-HAB exposed dolphins
– Subset of FWC phytoplankton data
– Max abundances near stranding location
• Within a radius of 20 km
– Max abundances in stranding window
• 30 days prior to stranding (exposure)
• ArcMap 10.2
– Buffer Tool
• Mean linear home range of 40km
• Digital circle as limiting feature
– Intersect tool
• FWC phytoplankton data captured

15. Logistic Model for Predictions
1. Probability of positive group affiliation increases with
algal cell abundance (K. brevis & P. bahamense).
2. At what cell abundance produces a 50% chance of
belonging to the positive group ?
P 𝑑𝑒𝑡𝑒𝑐𝑡𝑖𝑜𝑛 = 1
1+𝑒
−𝛽1(
𝑐𝑒𝑙𝑙𝑠
𝐿
)+𝛽0

16. K. brevis abundance & PbTx detected
100
1000
10000
100000
1000000
10000000
100000000
Date
Karenia brevis PbTx positive dolphins PbTx negative dolphins
K.brevis,cells/L(logscale)

17. PbTx Results
• All Dolphins
– 12% of dolphins were positive (14
of 119)
• HAB-exposed dolphins
– 80% of dolphins positive (8 of 10)
– Range: 0.16 – 12.3 ng/g
– avg. 6.3 ± 5.3 ng/g
• non-HAB exposed dolphins
– 6% of dolphins positive (6 of 109)
– Range: 0.17 – 1.2 ng/g (baseline)
– avg. 0.62 ± 0.42 ng · g -1
t = 2.9106, df = 9.002, p = 0.0086, 1-β = 0.99
n=109
n=10
0
1
2
3
4
5
6
7
8
9
non-HAB Tt HAB Tt
PbTxConcentration(ng/g)
non-HAB Dolphins HAB Dolphins
*

18. Probability of detecting PbTx
𝜋(𝑥) =
1
1 + 𝑒−0.00002903𝑥+2.340
At an exposure of ~625,000 cells/L, probability of a dolphin testing positive is 0.5
χ2
=10.657, df=1, P<0.001, pseudo-R²=0.42
𝜋(𝑥) =
1
1 + 𝑒−0.000003697𝑥+2.282

19. P. bahamense abundance & STX detected
100
1000
10000
100000
1000000
10000000
Date
Pyrodinium bahamense STX positive dolphins STX negative dolphins
P.bahamense,cells/L(logscale)

20. STX Results
• All Dolphins
– 13% of dolphins were positive
(16 of 119)
• HAB-exposed dolphins
– 60% were positive (9 of 15)
– Range: 0.97 – 9.5 ng/g
– avg. 3.2 ± 2.9 ng/g
• non-HAB exposed dolphins
– 7% were positive (7 of 104)
– Range: 0.16 – 1.9 ng/g (baseline)
– avg. 0.92 ± 0.73 ng/g
t = 2.4834, df = 14.045, p = 0.0131, 1-β = 0.98
n=104
n=15
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
non-HAB Tt HAB Tt
STXConcentration(ng/g)
*
non-HAB Dolphins HAB Dolphins

21. Probability of detecting STX
𝜋(𝑥) =
1
1 + 𝑒−0.00002903𝑥+2.340
At an exposure of ~82,000 cells/L, probability of a dolphin testing positive is 0.5
χ2
=14.975, df=1, P<<0.01, pseudo-R²=0.32

22. Summary
• Dolphins exposed to K. brevis HABs
– PbTx is 12-fold higher than baseline
• HAB exposed range: 0.70 – 12.3 ng/g
• Baseline: 0.20 to 1.2 ng/g
• Other regions - HAB exposed
• Gulf of Mexico (2005/2006); 278 ng/g
• FL-panhandle (1999/2000); 138 ng/g
• FL-panhandle (2004); 104 ng/g
• Sarasota, FL (2002); 71 ng/g
• Other regions - Baseline
• Sarasota, FL (3 mo previous HAB); 21 ng/g
• Dolphins exposed to P. bahamense HABs
– STX is 5-fold higher than baseline
• HAB exposed range: 0.97 - 9.5 ng/g
• Baseline: 0.16 - 1.9 ng/g
• Suspected STX driven mortalities
• Not confirmed as HAB event
• First STX baseline report locally

23. Bigger Picture
1. Sub-lethal exposures to phycotoxins in
marine mammals lead to deteriorating
immune response
• In-vitro immune response to HAB
toxins (WBC and eosinophils
increase)
• STX exposure increases
morbillivirus infection rate in-
vitro; neutrophil increase
(Bogolmoni et al. 2016)
2. Location of HAB species near dolphin
communities can be important!
3. Food web contamination by STX
4. Prey vector IRL spotted sea trout
(STX in skin 359ng/g; baseline)

24. Acknowledgments
Hubbs Seaworld
Research Institute
• Wendy Durden
• Teresa Jablonski
Florida Atlantic
University
• Marilyn Mazzoil
FWC Staff
• Karen Atwood
• Jim Ivey
• Alina Corcoran
St. Johns River
Water Management
District
• Ali Simpson
• Wendy Tweedale

26. STX found in puffers of IRL Proper
This problem is unique to Florida and the IRL; Brevard County