Exposure of blue crab (Callinectes sapidus) to modified clay treatment of Karenia brevis as a bloom control strategy

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Karenia brevis
Harmful algal blooms (HABs) of the toxic marine dinoflagellate Karenia brevis, a natural phenomenon known as red tide, are an almost annual occurrence on the west coast of Florida, USA (Walsh et al., 2006;Stumpf et al., 2008).K. brevis populations are monitored by the Florida Fish and Wildlife Conservation Commission, and their database shows that HABs of this species most frequently initiate in the summer and fall months and most frequently affect the coast between Hillsborough and Collier counties (Fig. 1, Florida Fish and Wildlife Conservation Commission (2021)).These data also show that K. brevis blooms vary in frequency, distribution, and intensity from year to year, and the variable nature of this phenomenon makes it difficult to predict, mitigate, and control.
K. brevis blooms have far-reaching impacts in the coastal environment and communities in Florida.K. brevis produce neurotoxins (brevetoxins), which can lead to impairment and mortality in many forms of marine life (Pierce et al., 2005;Abraham et al., 2006;Walsh et al., 2006;Pierce and Henry, 2008;Plakas and Dickey, 2010;Pierce et al., 2011).HABs can also potentially lead to anoxic or hypoxic zones, and the combined toxic and hypoxic conditions resulting from K. brevis blooms can inflict mass mortalities in fish and other forms of life, including sharks, dolphins, manatees, sea turtles, and sea birds (Landsberg et al., 2002;Naar et al., 2007;Gannon et al., 2009;Shi et al., 2012;DiLeone and Ainsworth, 2019;Turley et al., 2022).In humans, ingestion of brevetoxins leads to neurotoxic shellfish poisoning (NSP), and inhalation of brevetoxins causes respiratory distress and airway constriction.Toxic aerosol is a widespread problem on coasts afflicted by K. brevis, which can significantly disrupt tourism and recreational activities (Watkins et al., 2008;Fleming et al., 2011).Declines of fish stocks, disruption of tourism and recreation, medical costs, and costs of beach cleanup result in the loss of millions of dollars to the local economy during each K. brevis HAB event (Hoagland et al., 2014;Adams, 2017;Murphy, 2018;Bechard, 2020).

Bloom control
In Florida, USA, K. brevis bloom control strategies are a relatively new avenue of mitigation that are currently being researched and developed.Historically, K. brevis bloom management has focused on the development of environmental policies to reduce sources of nutrients that sustain these blooms (Sengco, 2009;Heil et al., 2014).Other mitigation strategies include programs that monitor K. brevis populations, regulate seafood, remove fish kills, and educate the public (Sengco, 2009).However, natural resource managers are currently lacking tools that directly control or suppress K. brevis blooms as they are occurring.To address this issue, multiple institutions have invested in the research and development of physical or chemical algicides, which are applied to an active bloom to remove cells and/or toxins (hereafter, referred to as bloom control).
A variety of HAB control materials and techniques are in development, but there are still many factors to be considered before these methods can be widely applied to an active bloom in open waters.The effectiveness of any bloom control technique at removing cells and toxins must be balanced against the costs of production and transportation of the control agent, feasibility of application over a large area, and minimization of negative impacts to the environment.In addition, the managers and officials responsible for using bloom control in public waters must consider public opinion (Sengco, 2009).Bloom control is perhaps the least developed area of marine HAB research and management, and is arguably the most controversial, given the myriad stakeholders who use the coastal ecosystem for business and recreation and who want to ensure bloom control will not be detrimental to fisheries, ecosystems, or human health.
To date, there has yet to be a K. brevis bloom control method to gain public and/or regulatory acceptance.However, the intense K. brevis blooms that have afflicted Florida in the past decade have led to increased efforts in research circles to develop bloom controls that prioritize environmental safety.Following the 2018 K. brevis bloom, which impacted over 150 miles of Florida coast and cost millions of dollars in damages (Murphy, 2018;NOAA, 2022), the state of Florida pledged $18 million to the creation of the Florida red tide mitigation & technology development initiative (RTMTDI), a collaborative research program between the Fish and Wildlife Conservation Commission and Mote Marine Laboratory to pursue development of K. brevis bloom control and mitigation strategies (S.B. 1552, 2019).This program funds and investigates projects, including the one presented here, with the goal of finding the most efficient and benign methods for K. brevis bloom control in Florida waters.

Modified clay II as a bloom control
At this time, the most advanced and globally widespread method of marine HAB control is clay flocculation, which has been actively used in China and Korea for over 25 years, often on HABs over 100 km 2 in size.Among the various options for bloom control, clay is appealing because it is inexpensive to source and transport, is easily scalable over large areas, and has low environmental impacts, which has been demonstrated in the extensive laboratory experiments and field applications in China and Korea (Park et al., 2013;Yu et al., 2017;Song et al., 2021), as well as experiments taking place within the Florida RTMTDI program by the authors of this paper (currently unpublished).
In the process of clay flocculation, a clay-seawater solution (typically 5 -20 g m − 2 ) is sprayed over the surface of an affected area.As the clay particles travel through the water column, the particles interact with each other and with the bloom, lysing cells and aggregating (aka "flocculating") with cells and toxins (Pierce et al., 2004;Sengco et al., 2005; Park et al., 2013;Seger et al., 2015;Yu et al., 2017;Liu et al., 2019;Song et al., 2021;Seger et al., 2022).The clay and cells create sinking aggregates (aka "floc"), clearing bloom biomass from the water column and depositing on the bottom sediments to be dispersed by natural currents.A variety of clay compounds have been developed for this purpose, and here we used the formulation known as Modified Clay II (MC II; described in detail in Yu et al., 2017).MC II is a kaolinite (silica-based) clay treated with polyaluminum chloride (PAC), a compound that coats clay particles and carries a positive charge, thereby allowing MC II to electrically attract particles that carry a negative charge, including K. brevis cells.The chemical and electrical properties of PAC-modified clay create microscopic net-like structures that capture algae cells as the clay falls through the water column, providing high cell removal efficiency (Pierce et al., 2004;Park et al., 2013;Yu et al., 2017;Song et al., 2021).
Conceptually, if clay flocculation was used to treat K. brevis blooms, then cells and brevetoxins would be delivered to bottom sediments; therefore, researchers and managers must consider potential impacts to organisms living at or near the bottom of the water column, such as seagrass, shellfish, mollusks, crustaceans, and benthic fish.A body of previous research has been conducted in which a non-target organisms were exposed to a various clay compounds and HAB species (Park et al., 2013;Yu et al., 2017;Zhang et al., 2020;Song et al., 2021Song et al., , 2022)).Collectively, these studies conclude that clay alone is generally not harmful to non-target organisms at the concentrations used to treat blooms (typically 5 -20 g m − 2 ), and that clay treatment may reduce mortality compared to organisms exposed to an untreated bloom.However, there have been few studies on the potential impacts of clay treatment of K. brevis on animals (e.g., Lewis et al., 2003), and to our knowledge, this study is the first that investigates the combination of K. brevis with the MC II formula on animals.Given the numerous concerns and possible outcomes of adding non-endemic compounds to natural systems, it is imperative to investigate how MC II might potentially affect the marine food web, including recreational and commercial species that are important components of Florida's ecological and economic viability.

Research objectives
The goal of this experiment was to observe potential lethal and sublethal (behavioral) effects of Modified Clay II (hereafter referred to as clay) and K. brevis on a bottom-dwelling species with ecological and economic importance in Florida.It should be noted that this study was not conducted to assess the toxicity of K. brevis to blue crabs, the cell or toxin removal efficiency of MC II, or the impact of clay treatment on water quality, since these aspects have been studied separately our research group as a part of the Florida RTMTDI program (currently unpublished).However, we will present cursory results of some of these topics as they relate to our specific questions outlined below.
Blue crabs (Callinectes sapidus) were chosen for this study for their importance in the marine food web and significance as a fishery species.Blue crabs are a vital component of the benthic marine food web and are known as a dominant prey source for marine fish, birds, and mammals, and can also serve as keystone species by consuming herbivores that affect habitat structure (e.g., Littoraria irrorate, which feeds on marsh grass; Boudreau and Worm, 2012).Blue crab is also a valuable fishery species throughout the Gulf of Mexico and Eastern United States, with over a century of history in Florida (Steele and Bert, 1998).In 2020 alone, commercial landings of blue crab in Florida were reported at over 3000 t, with a worth of over 13 million USD (NOAA Fisheries, 2021).News articles have reported blue crabs appearing in fish kills associated with recent K. brevis blooms (Allen, 2018;O'Brien, 2021) and blue crabs were identified in 29 fish kills in the Florida Fish and Wildlife database in 2018 alone (Florida Fish and Wildlife Conservation Commission, 2023a).
The objective of this study, based on Lewis et al. (2003) and other previous studies conducted with various clays and animals, outlined in Park et al. (2013), Yu et al. (2017), andSong et al. (2021), was to determine if clay increased mortality or caused behavioral changes in blue crab.Specifically, we hypothesized that mortality and reflex impairment of blue crabs exposed to clay-treated K. brevis bloom would be less than or equal to crabs exposed to an untreated K. brevis bloom.

Experimental laboratory
This study took place at Mote Marine Laboratory in Sarasota, FL.Blue crabs (Callinectes sapidus) were collected between July and October of 2020 from Sarasota Bay at Bird Key Park using baited crab traps (FL Special Activity License 20-2204-A-SR).A total of 48 crabs were collected, 39 male and 9 female, with a mean carapace length of 14 cm (± 1.5 cm).According to historic records, K. brevis blooms typically initiate offshore in the summer months and achieve bloom density (> 100,000 cells/L) between June and October (Walsh et al., 2006).When this study took place, according to the state database, K. brevis cell concentrations at west coast sampling sites were not above background levels (0 -1000) at any time, and most cell counts were zero (Florida Fish and Wildlife Conservation Commission, 2023c).

Experimental setup
Crabs were randomly assigned to individual 20 L tanks (42 cm length, 27 cm height, and 22 cm width) containing filtered and ozonated seawater supplied by an indoor flow through system.Tanks were arranged in random positions within two rows within a raceway and randomly assigned to treatments.Each tank was treated as an experimental unit in statistics.Additionally, separating individuals prevented potential mortality and stress through interaction.Each tank was aerated with Penn Plax air pumps via two tubes without air stones (20 cm length, 0.5 inner diameter).Before the experiments, during acclimation, water was allowed to overflow the tanks into the raceway; for the duration of the experiments, flow was turned off and tanks were static to prevent loss of cells or clay from the tanks.Crabs were allowed to acclimate to laboratory conditions for a minimum of 24 h before experiments began.Crabs were fed with frozen shrimp provided daily, and uneaten food and solid waste were removed daily using small aquarium dip nets.Due to limited space, only eight tanks could be tested at a time; therefore, this study was conducted over seven experiments that ran between July and October in 2020.

Experimental design
Tanks were randomly assigned to one of four treatments: seawater only, clay only, Karenia only, or clay-treated Karenia (Fig. 2, n = 12 tanks per treatment).Crabs were exposed to their respective treatments for two consecutive periods of four days (96 h each), for a total treatment time of eight days (192 h total).After four days, tanks were given a complete water change and were re-dosed with culture and clay in the same concentrations described below.Because water flow remained off for the duration of the experiments, a water change was necessary to prevent dissolved ammonia waste from building to toxic levels, which can induce stress in crabs (Weihrauch, 2004).
K. brevis culture was added to Karenia only and clay-treated Karenia tanks to achieve treatment concentrations of 1 × 10 6 cells L − 1 , which has been used as a target in previous K. brevis exposure studies (Lewis et al., 2003;Gravinese et al., 2018Gravinese et al., , 2019) ) and is representative of a "high" intensity bloom according to the Florida Fish and Wildlife Conservation Commission (2023b).Mean initial cell concentrations were measured at 1023,333 ± SE 55,683 cells L − 1 on day 1 and 923,333 ± SE 90,265 cells L − 1 on day 5. K. brevis was cultured at Mote in L1 media (enriched f/2 media from Guillard and Ryther, 1962)  16.1%.Clay was applied one hour after culture was applied.For application, clay was mixed with seawater in a squeeze bottle and sprayed on the surface of tanks to achieve a final concentration of 0.5 g L − 1 .It should be noted that this concentration is a relatively high, as high K. brevis cell removal can be achieved at 0.1 g L − 1 of MC II (unpublished data), but a higher concentration was chosen as it better reflects the clay deposition expected from clay treatment of natural waters in actual practice, compared to these shallow aquarium tanks.
Environmental parameters were monitored for stasis to ensure the drivers of stress on the crabs were the K. brevis and clay, and not a confounding effect of water quality.Water quality parameters, including temperature, salinity, pH, and dissolved oxygen, were measured daily using a calibrated multimeter (ProDSS YSI).Ammonia was monitored using Salifert aquarium test kits (0.5 -2.0 ppm limit).To monitor daily cell populations, 10 mL integrated samples were taken from the water column at the center of the tank with a pipette.These samples were preserved with Lugol's iodine solution to conduct cell counts on a Sedgewick rafter slide under a compound light microscope.Toxins were measured in addition to cell populations, since toxins were the mechanism by which the K. brevis would induce stress and mortality in the crabs in this experiment.To measure toxins, integrated 100 mL water samples were taken from the water column at the center of the tank with a pipette at the beginning and end of each dosing period (days 0, 4, 5, and 8).Brevetoxin types and concentrations were analyzed by liquid chromatography-mass spectrometry using a ThermoFinnigan AqA HPLC/MS (methods described in Pierce et al., 2011).For this study, we measured the two parent toxins produced by K. brevis, BTx-1 and BTx-2 (intracellular toxins), and the two major degradation products (extracellular toxins) of BTx-2, which are BTx-3 and BTx-2-CA (extracellular toxins).The parent toxins create a suite of other products that were not included in analysis due to the lack of standards and inconsistent alterations in mass spectrometric detector response.For our instrument, the limits of detection were 54 ng L − 1 for BTx-1, 23 ng L − 1 for BTx-2, 9 ng L − 1 for BTx-3 and 14 ng L − 1 for BTx-2-CA.
Trends in cell concentrations, total toxins, and water quality parameters were examined with multivariate generalized linear models (GLMs) to determine if treatments had an impact on these measurements, and to ensure water quality was in stasis and not having a confounding effect on mortality and reflexes of the crabs apart from experimental treatments.If data did not meet assumptions of normality and variance of a Gaussian distribution, alternative distributions were explored.
To evaluate lethal and sublethal responses to treatments, we monitored mortality and conducted reflex tests daily using a binary data system.In this experiment, we measured righting reflex and eyestalk reflex daily, modeled after the pesticide exposure experiment conducted on blue crabs by Schroeder-Spain et al. (2018).Crabs were handled inside the tanks and were not handled for more than five minutes each day to minimize stress.The righting reflex was measured by using forceps to flip the crab on its dorsal side.Presence of the reflex (value of 1) was noted if the crab righted itself in ≤ 5 s; absence of the reflex (value of 0) was noted if the crab failed to right itself after > 5 s.The eyestalk reflex was measured by touching the forceps to the eyestalks.Presence of the reflex (1) was noted if the crab retracted both eyestalks; absence of the reflex (0) was noted if the crab did not retract one or both eyestalks in response to touch.Active, responsive crabs were considered alive (1).Crabs were considered dead (0) if they were inactive, limp, and unresponsive to both reflex tests.

Statistical analyses
To analyze crab mortality, we used Cox proportional hazard models (Cox, 1972) using the "survival" and "survminer" packages in R (R Core Team, 2020).Cox models are a method of modeling survival as a function of one or more variables, in which the primary assumption is that the risk of mortality between groups is constant and proportional over time.The base formula structure for the hazard function h(t) is: In this formula, t represents survival time, x represents covariates, b represents the effect size of covariates, and h 0 is the value of the hazard when all covariates equal zero.Cox models have been used in previous studies to analyze crab mortality in response to stressors (Gravinese et al., 2018;Tankersley and Wieber, 2000).Sublethal responses of the crabs were examined using multivariate generalized linear models  (GLMs) using logistic regression.Variables considered in these models, apart from time and treatment, included crab sex, crab size, experimental trial, tank placement, and water quality parameters; however, these covariates were not found to have any significant effect on mortality and were not included in the final model presented here.
In our model exploration process, to compare between GLMs and select the distribution and formula structure that best represented each response variable, we used Akaike's Information Criteria (AIC) scores (Akaike, 1973), a measure of model predictive error, in which lower scores indicates a better fit to the data.All statistical analyses were conducted in the statistical program R (R Core Team, 2020).

Changes in experimental environment
Water quality parameters remained within narrow ranges and showed little variation within or between treatments, with pH as the notable exception (Supplementary Tables 1 -2, Supplementary Fig. 1).Temperature ranged between 22 -26 • C, salinity between 34 -37, and dissolved oxygen remained above 6.0 mg/L, falling within observations of the ranges blue crabs occupy in nature (Fisher, 1999).In clay only and clay-treated Karenia tanks, application of clay caused pH to drop significantly (GLM, p < 0.0001).On day 1, mean pH of clay only tanks was 7.25 (0.07 SE) compared to a pH of 7.86 (0.03 SE) in seawater tanks.After this initial drop, pH of clay tanks gradually increased over time and returned to ambient pH of the seawater tanks after 72 h.Lower levels of pH have been shown to affect survival rates of larval blue crab (Tomasetti et al., 2018), but did not appear to have an effect on mortality and reflexes of our adult crabs.Despite the observed effect on pH, water quality parameters did not contribute significant effects to models of trends in cell concentrations, toxins, mortality, or reflexes.
Cell concentrations in clay-treated Karenia tanks decreased by 95% after 24 h and 98% after 48 h.In models, cell concentrations were significantly lower in clay-treated Karenia tanks compared to Karenia only tanks (GLM, p < 0.0001, Supplementary Table 3 -4).The percent removal efficiency of clay was 93% after 24 h (Fig. 3).

Mortality and reflexes
Out of the 48 crabs tested, 2 mortalities occurred in seawater only tanks, 1 in clay only tanks, and 1 in Karenia only tanks.No mortalities occurred in the clay-treated Karenia tanks, and no mortalities occurred in any treatment within the first 48 h of exposure.The survivorship analysis found no significant differences among treatments (Cox model, p > 0.05, Table 1 and Fig. 5).There were also no significant differences observed among treatments for either the eyestalk reflex or the righting reflex (GLM, p > 0.05, Tables 2 and 3).

Effects of clay on experimental subjects
In this study, we investigated the potential impacts of Modified Clay II treatment of K. brevis on blue crab (C.sapidus), an ecologically and commercially important species in Florida.Supporting our initial hypothesis, mortality and reflexes in crabs were not significantly different in clay-treated Karenia tanks compared to Karenia only tanks (Fig. 5, Tables 1 -3).Our study can be most closely compared to Lewis et al. (2003), which to our knowledge is the only other study that examined the impact of modified clay treatment of K. brevis on marine animals, using a formulation of phosphatic clay.In that study, researchers found that mortality of amphipods (Leptocheirus plumulosus, Ampelisca abdita), grass shrimp embryos (Palaemonetes pugio), and sheepshead larvae (Cyprinodon variegatus) in clay-treated K. brevis tanks were not significantly different from mortalities in untreated K. brevis tanks, similar to the findings presented in this paper.Additionally, our results fall in line with other previous research with modified clays, which indicate clay has negligible impacts on animal survival (Park et al., 2013;Yu et al., 2017;Zhang et al., 2020;Song et al., 2021Song et al., , 2022)).We conclude a 0.5 g L − 1 dosage of MC II combined with bloom-level densities of K. brevis had zero to negligible impacts on the lethal and sublethal responses of crabs taken within the time frame of this study.As stated previously, the clay Fig. 3. Percent removal efficiency of Karenia brevis cells over time from tanks treated with clay subtracted from untreated tanks.Plots are separated by measurements taken during the first dosing period on 1 -4 (A) and the second dosing period on days 5 -8 (B).loading concentration used in this study is significantly higher than levels that are known to be effective at removing Karenia cells with modified clay (ex.0.1 -0.2 g/L; Yu et al., 2017).Our results here are thus conservative, based on an initial clay loading that is as much as five times higher than is needed for effective cell removal, but which is representative of a clay loading that might occur in a practical application where multiple doses might be needed.
This study contributes to ongoing research of bloom control technologies as one of the few investigations on the impact of clay treatment    of K. brevis on non-target organisms, and to our knowledge, is the first animal study conducted using MC II in combination with K. brevis.Our findings suggest that MC II treatment of a natural bloom may not be more harmful to adult blue crab populations than untreated bloom conditions.Further, because the use of clay will reduce growth and spread of the bloom, the crab mortalities typically seen in fish kills from prolonged exposure to K. brevis (Florida Fish and Wildlife Conservation Commission, 2023a) could be prevented.Therefore, crab populations could see fewer mortalities and may recover more quickly with treatment than if the bloom were allowed to persist untreated.Any form of bloom control would be most effective at preserving fish and shellfish populations if treatment can be applied before the bloom reaches high intensities, perhaps multiple times throughout the bloom event.

Effects of clay on experimental environment
This study did not focus on the impact of MC II on water quality or the effectiveness of clay on cell and toxin removal since these aspects are being examined in past and future work within our research team.But we do find it pertinent to provide a brief overview of these findings in the context of the goals of this paper.Cell removal capabilities of the clay reported in this study (Fig. 3) are in line with preliminary experiments of MC II conducted by our research team, and extensive literature describing the removal capabilities of various clay types on different algae species (Pierce et al., 2004;Sengco and Anderson, 2004;Park et al., 2013;Yu et al., 2017;Song et al., 2021).Other previous studies reported high removal rates of toxins with various clays and algae species (Pierce et al., 2004 (90% decrease  .Other water quality characteristics, including pH, nutrients, and organic matter, may also have an impact on the ability of clay to remove cells and toxins (Sengco et al., 2005;Seger et al., 2015;Liu et al., 2019).In examining total toxins (sum of the four toxin analogs), we did not observe a significant difference between treatments in this study (Fig. 2).Although we were not attempting to examine the effectiveness of clay on toxins, this finding may be of interest to examine further in future work.We did observe a significant decline in total toxins in both treatments over time, which has several potential explanations.First, conversion between the parent and metabolite toxins is not one-to-one, and parent toxins were likely converted to a suite of other metabolites over time (Abraham et al., 2006;Roth et al., 2007;Pierce et al., 2011) which are not normally measured using our current HPLC procedures.It is also possible that some toxins were degraded (Kieber et al., 2010) or sequestered in the clay floc (Pierce et al., 2004).Other possible routes of toxin loss may be through the organisms, where toxins may accumulate or depurate (Hinton and Ramsdell, 2008), or through aerosolization (Pierce et al., 2005).These potential routes of toxin fate are of interest but were outside the scope of this study and will be considered in future research within our team.
HAB control technologies have the potential to significantly reduce ecological impacts and economic losses in areas affected by K. brevis blooms.It is important to consider that the water quality changes induced by clay are temporary (i.e., turbidity, pH, etc.), and that the alternative to utilizing bloom control technologies is to allow an untreated bloom to run its course, thus allowing continuous damage to the environment over weeks or months.Shumway et al. (2003) and Archambault et al. (2004) expressed caution towards the use of clay for bloom control.In these studies, researchers exposed various invertebrates to yellow loess and phosphatic clay, respectively, without the addition of a toxic HAB species.Shumway et al. (2003) reported reduced clearance rates in their various filter-feeding organisms exposed to relatively high concentrations of continuously suspended clay, and Archambault et al. (2004) reported reduced growth rates in juvenile hard clams (Mercenaria mercenaria) exposed to continuously suspended clay and nontoxic algae.However, managers might consider the findings of Shumway et al. (2003) and Archambault et al. (2004) acceptable outcomes compared to the widespread economic and ecological damage inflicted by an untreated toxic bloom, recognizing also that the amount of clay being used in more recent work is considerably lower than was the case twenty years ago, nor is continuous resuspension a realistic scenario, except in very shallow waters.

Future directions
Further study is needed on the fate of K. brevis cells and toxins and on potential sublethal impacts of clay on the marine food web.As we are currently lacking a holistic understanding of how clay might affect the marine ecosystem as a whole, scientists, managers, officials, and the public are reluctant to apply clay on active blooms.Although the use of clay is prevalent in other parts of the world, further study is needed on its impacts in the United States and in Florida waters to determine if this is a safe and effective technology to use on K. brevis blooms.To that end, our team will continue our research of clay in iterative experiments of increasing physical scale.The next step in this process will be to conduct experiments in 1400 L mesocosm tanks to test Modified Clay II for cell and toxin removal, changes in water quality and nutrients, and impacts on a benthic community representative of Sarasota Bay, which will include hard clam (Mercenaria campechiensis), sea urchin (Lytechinus variegatus), and blue crab (C.sapidus).In these experiments, we intend to further explore potential sublethal impacts of treatment and the fate of toxins by measuring respiration rates in organisms and measuring toxins in organs.Following these land-based experiments, our team plans to conduct experiments with MC II in field tests on natural K. brevis blooms to gain additional information on clay dispersal dynamics and gage practicality of clay application.
The overall goal of our work is advance the development of K. brevis bloom control in Florida by providing a foundation of study on the potential impacts of clay treatment on non-target species so that resource managers can make informed decisions on the use of bloom control technologies in their local waters.Our continued studies on the ecological impacts of clay will include additional important species, such as the macrobenthic and microbial communities that support the marine ecosystem.If clay proves to be a safe and effective bloom control method, this technique may be adapted to treat HABs across the nation.

Fig. 1 .
Fig. 1.Cumulative map of K. brevis cell counts taken from 1990 to 2021 illustrating relative geographic extent and intensity of K. brevis blooms in Florida waters.Data provided by the Florida Fish and Wildlife Conservation Commission (2021).

Fig. 2 .
Fig. 2. Conceptual diagram of the experimental setup.Water was supplied to tanks via spigots from a single header tank.Water was allowed to overflow the tanks during acclimation and drain into the raceway, and flow was turned off during the experimental period.Tanks were randomly assigned to one of four treatments: seawater only (blue), clay only (yellow), Karenia only (orange), or clay-treated Karenia (gray).

Fig. 4 .
Fig. 4. Mean ± SE concentrations of sum of BTx-1, BTx-2, BTx-3, and BTx-2-CA from tanks containing either K. brevis only or K. brevis treated with clay (n = 12 tanks per treatment).Plots are separated by measurements taken during the first dosing period on days 1 and 4 (A) and the second dosing period on days 5 and 8 (B).

Fig. 5 .
Fig. 5. Cumulative survivorship of Callinectes sapidus in tanks containing seawater only, clay only, K. brevis only, or K. brevis treated with clay (CK) over eight days.Lines represent means of the model and shading represents 95% CI (n = 12 tanks per treatment).No significant differences were detected among treatments (p > 0.05).

Table 1
Summary of Cox survivorship model of Callinectes sapidus survivorship as a function of time and treatment.

Table 2
Summary of Generalized Linear Model of Callinectes sapidus eyestalk reflex as a function of day and treatment over eight days.