Harmful algal blooms (HABs) are frequently associated with morbidity and large-scale mortalities of marine mammals, due to exposure to potent neurotoxins produced during such events (Broadwater et al., 2018; NOAA, 2020). Assessing the impacts of HAB toxins on marine mammals is often problematic, and is complicated by complex trophic interactions, time lags between blooms and observable impacts, and logistical constraints inherent in sampling free-ranging animals. Important marine sentinel species such as the common bottlenose dolphin (Tursiops truncatus) are apex predators that are typically distanced from toxic phytoplankton by several trophic links, with the organisms in each link having distinct rates of toxin accumulation and/or depuration (Landsberg, 2002; Doucette et al., 2006; Naar et al., 2007). Should a strong correlative relationship exist between the toxin concentration detected in an animal and the severity of the HAB to which it was exposed, this would serve as a useful tool to predict toxin accumulation in individuals or back-calculate toxic phytoplankton abundances associated with mortality events. Previous work has suggested that the abundance of toxic phytoplankton alone is a poor predictor of toxin accumulation rates and likelihood of exposure in dead-stranded marine mammals (Fire et al., 2007, 2020a; Landsberg et al., 2009). This is primarily due to the fact that apex predator marine mammals do not graze on phytoplankton nor ingest large amounts of seawater, accumulate negligible amounts of HAB toxins transdermally or via inhalation, leaving dietary intake (through large fish and invertebrates) as the only feasible route of biologically significant exposure. However, the relationship between phytoplankton abundance and toxin accumulation in exposed animals has not been specifically demonstrated for live, free-ranging marine mammals.

Bottlenose dolphins endemic to the west coast of Florida are frequently and severely impacted by blooms of the red tide organism Karenia brevis, which occur on a nearly annual basis (Steidinger et al., 1998; Steidinger, 2009) and produce a suite of neurotoxins known as brevetoxins (PbTx). These blooms typically occur over very large spatial areas (∼3,000 km²), can last for several months, and often result in mass mortality events of multiple species of marine megafauna (Zhao et al., 2013; Broadwater et al., 2018). This is of particular concern for populations of non-migratory, resident marine mammals that do not travel long distances and therefore reside in red tide-affected areas for the entire duration of each bloom. Although high PbTx tissue loads and acute PbTx exposure have been assigned as the cause of several large-scale dolphin mortality events in Florida waters, corresponding information for the impacts on living dolphins that survive such events is limited (Twiner et al., 2012; Litz et al., 2014). The present study addresses this lack of knowledge by (1) providing a long-term dataset of PbTx loads in resident dolphins from a region that frequently experiences severe K. brevis blooms, and (2) by investigating the relationship between K. brevis abundance associated with individual dolphins sampled in the field and their degree of PbTx exposure. Here we present PbTx data from live, free-ranging bottlenose dolphins resident to Sarasota Bay, Florida from 2000–2018, and test the utility of K. brevis abundance data as an index of exposure that corresponds to PbTx concentrations detected in various toxicologically important sample matrices.

In total, 253 dolphins were tested for PbTx between 2000 and 2018, yielding 599 samples of urine, feces, gastric, milk or blood. Twenty-eight percent of dolphins (n = 70) were associated with K. brevis abundance values of ≥10,000 cells/L, and were therefore assigned to the Exposed group; the remaining 72% (n = 183) were assigned to the Baseline group. Maximum K. brevis cell abundances associated with each dolphin ranged from background levels (≤1,000 cells/L) to 60,000,000 cells/L (Supplementary Table 1). Overall, 41% (n = 104) of dolphins tested positive for PbTx in at least one sample type (Table 1). Comparing between exposure groups, the proportion of PbTx-positive animals was significantly greater in the Exposed group relative to the Baseline group, with 60% and 34% testing positive, respectively (p < 0.001).

PbTx was detected most frequently in feces (57%, n = 38), followed by gastric (35%, n = 37), urine (32%, n = 55), and blood (7%, n = 17) (Table 1). PbTx concentrations by sample type were highest in feces (2–231 ng/g; mean 46), followed by urine (0.8–90 ng/g; mean 7.2), gastric (0.8–61 ng/g; mean 12), and blood (0.3–5 ng/g; mean 1.3). PbTx was not detected in milk samples, which were therefore excluded from statistical analyses.

PbTx concentrations were significantly higher in the Exposed group relative to the Baseline group for feces (p < 0.001), gastric (p = 0.032), and urine (p = 0.013) samples, but were not significantly different for blood samples (p = 0.133; Figure 1).

Simple regression analyses of K. brevis cell abundance as an index of exposure vs. corresponding PbTx concentration found no statistically significant relationship for feces (p = 0.120), gastric (p = 0.349), urine (p = 0.053), or blood (p = 0.729) samples. Spearman correlation analyses indicated that while correlation was statistically significant for all sample types, correlation coefficients were close to zero for all sample types except feces (rho = 0.715 feces; 0.365 gastric; 0.399 urine; 0.256 blood; Figure 2). Regression analysis of index of exposure vs. presence/absence of PbTx in individual dolphins indicated no statistically significant relationship (p = 0.580; data not shown).

Overall, our findings indicate that the majority of live, free-ranging bottlenose dolphins exposed to K. brevis cell abundances of >10,000 cells/L accumulate detectable levels of PbTx, and do so at approximately twice the frequency of individuals sampled at background K. brevis abundances. Similarly, the concentrations of PbTx detected in three of four sample types were also higher in dolphins sampled during elevated levels of K. brevis vs. those sampled during baseline conditions. This complements earlier work demonstrating large differences in the PbTx concentrations detected and proportion of PbTx-positive dolphin carcasses recovered during red tides vs. those recovered during non-bloom conditions (Fire et al., 2007). However, our results contrast with previous findings demonstrating that live dolphins sampled during non-bloom conditions show no evidence of PbTx exposure (Fire et al., 2008b). These conflicting results are likely due to differences in sampling design and sample size, as the previous preliminary effort included only a handful of non-bloom individuals, all of which were sampled during the same 2-week health assessment. We suggest that the results of the present study are a more accurate representation of degree and frequency of PbTx exposure in live dolphins from this region.

Our results indicate that K. brevis is a poor predictor of detectable PbTx concentrations in gastric, urine and blood samples, and a weak predictor of PbTx concentration in feces samples. While analysis of some sample types showed significant correlation with index of exposure, correlation coefficients approaching zero indicate that these relationships were not biologically meaningful. We found relatively low prevalence of PbTx in blood samples, and narrow ranges of PbTx concentrations in feces, gastric, and urine samples despite high variability in their associated K. brevis abundance values. PbTx concentrations typically ranged over two orders of magnitude between minimum and maximum values and did not scale with corresponding indices of exposure which ranged over three orders of magnitude or more.

These findings indicate that despite the feasibility of collecting feces, urine, gastric, and blood samples from live, free ranging dolphins, and their utility in determining presence/absence of PbTx in exposed individuals, they have limited use in estimating the degree of exposure to K. brevis blooms. This is likely due to the uncertainty with which K. brevis exposure levels were assigned to the animals in the present study, as the 30-day window used to assign indices of exposure to each dolphin was based on limited information on estimated PbTx transfer rates through the food web (Landsberg, 2002; Doucette et al., 2006; Naar et al., 2007). Likewise, the 16-km buffer used to assign K. brevis cell counts to each dolphin sampling event may be insufficiently precise to account for the actual movements of individuals used in this study. In addition, the spatial distribution of K. brevis is highly heterogeneous at scales of a few kilometers, and the discontinuous, patchy nature of these blooms may account for much of the variation observed in the indices of exposure used herein (Tester and Steidinger, 1997; Zhao et al., 2013). The type of sample able to be collected safely from living, wild dolphins may also be a limitation in estimating exposure. Ideally, liver tissue is very useful for addressing PbTx detoxification and depuration rates in experimental dosing models, as well intestinal tissue for determining how much PbTx is assimilated from the gastrointestinal tract, however, these samples were not available from the live animals in this study. While PbTx concentrations in blood are theoretically at equilibrium with the rest of the tissues in an exposed organism, serum PbTx values in bottlenose dolphins are typically at concentrations near the limit of detection for most PbTx analytical methods, and may often go undetected.

Further efforts to assess the relationship between K. brevis exposure and PbTx accumulation in live dolphins may benefit from more physiologically relevant biomarkers of toxin exposure, potentially including plasma cortisol concentration or immune response. Feces and gastric samples are only proxies for uptake and assimilation of toxins, and feces and urine as indicators of detoxification and depuration via renal and biliary routes do not provide information on real-time health impacts. In vitro exposure of dolphin peripheral blood cells to PbTx is known to increase proliferation of multiple immune cell types, and this merits investigation as a complementary data set to those used in the present study (Gebhard et al., 2015). In addition, targeted fine-scale seawater sampling combined with tracking movements of live dolphins immediately prior to health assessments may give more accurate estimates of individual exposure to K. brevis cells and reduce the impact of patchy cell distribution on future analyses.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Mote Marine Laboratory annual Institutional Animal Care and Use Committee.

SF designed and oversaw all aspects of the study. GM provided statistical guidance and analyses, and assisted with development of the manuscript. ES carried out laboratory analyses and assisted with development of the manuscript. RW provided field logistical support, field sampling design, sample collection, and assisted with development of the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.