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Article

Mitigation of Karenia brevis Cells and Brevetoxins Using Curcumin, a Natural Supplement

by
Emily R. Hall
*,
Cynthia A. Heil
,
Jessica D. Frankle
,
Sarah Klass
,
Victoria Devillier
,
Vincent Lovko
,
Jennifer H. Toyoda
and
Richard Pierce
Mote Marine Laboratory and Aquarium, Sarasota, FL 34236, USA
*
Author to whom correspondence should be addressed.
Water 2024, 16(10), 1458; https://doi.org/10.3390/w16101458
Submission received: 26 April 2024 / Revised: 6 May 2024 / Accepted: 13 May 2024 / Published: 20 May 2024
(This article belongs to the Special Issue Eutrophication and Harmful Algae in Aquatic Ecosystems)
Browse Figures
Versions Notes

Abstract

:
Curcumin, a natural plant product, was investigated as a mitigation tool against Karenia brevis, the toxic dinoflagellate responsible for Florida red tides. A series of laboratory bench-top studies were conducted with additions of 0.1, 1, 2, 3, 5, 10, 20, 30, and 40 mg/L curcumin to K. brevis at an average of 1.0 × 106 cells/L to determine the efficacy of curcumin against K. brevis cells and brevetoxins and to optimize treatment dosage. Treatment with 5 mg/L of curcumin reduced K. brevis cell abundance by 89% and total brevetoxins by 60% within 24 h. Lower concentrations of curcumin (0.1–3 mg/L) exhibited between a 2 and 45% reduction in K. brevis and a reduction in brevetoxins of between 2 and 44% within 24 h. At the highest curcumin doses, 30 and 40 mg/L, a 100% reduction in cell abundance was observed by 6 h, with reduction in total brevetoxins by at least 64% in 48 h. These results suggest that curcumin, used alone or potentially in combination with other technologies, is a promising K. brevis bloom mitigation option.

1. Introduction

Harmful algal blooms (HABs) of both fresh and marine origin have wreaked havoc on the state of Florida dating back hundreds of years, with impacts documented as early as the 1500s [1,2]. These HABs often have significant ecological, economical, and human health impacts, which have been increasing in severity alongside increasing population growth, rapid development, eutrophication, and climate change occurring in this region [1]. Among Florida HABs, blooms of the toxic marine dinoflagellate Karenia brevis are perhaps the most severe and formidable, due to the frequency and geographic spread of these blooms, and the complex natural and anthropogenic drivers that make these blooms challenging to mitigate. First officially identified and described in 1948 [3], K. brevis blooms occur in the eastern Gulf of Mexico almost annually [4,5,6] and produce brevetoxins that cause respiratory irritation [7,8,9], neurotoxic shellfish poisoning [10,11], and extensive marine and coastal organism mortalities [12,13]. Karenia brevis blooms can also cause significant socio-economic problems for the state of Florida, due to the impact on fisheries and the degradation of shorelines and coastal waters [14,15].
Given the severity of these blooms, there has been recent renewed attention on potential mitigation strategies and technologies, and efforts are being made at multiple levels to manage and mitigate K. brevis blooms. Public education, monitoring and reporting programs, and the implementation of policies to reduce sources of nutrient pollution serve to reduce the economic and health impacts of blooms, and attempt to reduce the severity of blooms before they occur [16,17]. In addition to these management tools, and given the challenges of comprehensive nutrient reduction strategies, it is desirable to develop methods of reducing K. brevis cells and toxins during bloom development. Currently, no method has achieved widespread accepted use in the US. Past attempts to control blooms chemically, along with other mitigation strategies, have so far been unsuccessful, due to issues including high costs, poor scalability, elevation of other pollutants, and detrimental impacts on non-targeted species (e.g., copper sulfate is lethal to marine organisms [18,19,20]. The severe impacts of the long lasting 2017–2019 K. brevis bloom on the west coast of Florida reinvigorated public and academic interest in the development of suitable mitigation methods in the state of Florida and led to the creation of the Red Tide Mitigation & Technology Development Initiative (RTMTDI), a research program within Mote Marine Laboratory funded by the state in the 2019 bill, S.B. 1552 [21]. To date, this program has investigated a variety of mitigation techniques in benchtop experiments, large-scale mesocosm experiments, and in situ field tests, with the goal of identifying compounds and technologies that efficiently remove K. brevis cells and toxins while prioritizing environmental safety.
Natural pesticide products are active substances derived from plants used for pest management and are often touted as safe alternatives to synthetic chemicals [22]. A number of natural products have been proposed as effective and environmentally benign methods to mitigate HABs [23,24,25,26,27,28,29], though none have been tested on K. brevis. One of the promising natural products, based on availability, cost, effectiveness on the mitigation of other harmful algae species and toxins [30], and benefit to marine fish [31,32], is the phytochemical curcumin, (1E,6E)-1,7-Bis(4(hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), the primary bioactive compound found in turmeric (Curcuma longa), a member of the ginger family (Zingiberaceae). Curcumin has been found to be effective in controlling animal and agricultural pests [22,33], and is an increasingly popular health supplement in human diets due to its anti-inflammatory, anti-angiogenic, antioxidant and anticancer effects [29,34,35].
To test the efficacy of curcumin in mitigating K. brevis cells and associated toxins, we conducted multiple laboratory studies with a commercially available food-grade curcumin (a mixture of all curcuminoids found in turmeric) to determine the efficacy of curcumin as a viable product for the mitigation of K. brevis blooms. These studies included initial tests on K. brevis cell and toxin reduction, experiments to identify ideal treatment dosage and the method of preparation, and potential impacts of treatment on measures of water quality, including nutrients, dissolved oxygen, turbidity, and color.

2. Materials and Methods

2.1. Experimental Setup

A total of 13 total laboratory experiments assessing the effects of curcumin on K. brevis were completed from 2019 to 2024. Studies were initially conducted in 1.5 L beakers for 48 h to determine the efficacy of curcumin on the reduction of cultured K. brevis cells and brevetoxins, then in 10 L aquaria for a minimum of 48 h, and up to 336 h to continue examination of the effects of curcumin on K. brevis and any residual effects on water quality. Beaker experiments were conducted on the benchtop in the laboratory under ambient fluorescent light, while aquaria experiments were conducted under a 12 h light–dark cycle at 213 ± 12 μmol/m2/s (mean ± standard error [SE]). The majority of experiments were conducted using Turmeric Extract Powder, an orange curcumin powder, supplied from Bulk Supplements. One study was conducted with white curcumin product (Zedoary Root (Curcuma zedoaria) and CuroWhiteTM (hydrogenated form of Curcuma longa) [36] to attempt to address issues of orange water color seen in previous experiments. In every experiment, each treatment group was replicated in duplicate at minimum. Each experiment was conducted with a control group (K. brevis + seawater) to account for container-related effects.
As curcumin is not 100% soluble in seawater, curcumin stock solutions were made by multiple methods: 1. adding curcumin (>95% curcuminoid, Bulk Supplements) to deionized water and vigorously mixing, 2. mixing curcumin with ozonated and filtered (20 μm) seawater and vigorously mixing, 3. mixing curcumin with ozonated and filtered seawater and heating (40 °C for 1 h), 4. mixing curcumin with ozonated and filtered seawater with 0.001% ethanol additions, 5. mixing curcumin with ozonated and filtered seawater with 0.004% ethanol, and 6. mixing curcumin with 99.5–100% ethanol [28,37,38]. All curcumin stock solutions were stored in glass bottles held in the dark to prevent photodegradation.
Karenia brevis (Mote New Pass Clone/CCMP 2228) was cultured in modified L1 media (omission of SiO4 additions) at 24 °C, salinity 32–34 ppt, and a 12 h L:D cycle at 50–60 μmol/m2/s. Cultures of 20.0–30.0 × 106 cells/L were diluted with filtered (Whatman GFF) seawater collected from Mote’s New Pass dock (salinity, 33 ppt) immediately prior to the start of each experiment to a final target cell abundance of ~1.0 × 106 cells/L. For each experiment, diluted cultures of K. brevis were added to triplicate cleaned (10% HCl followed by repeated Milli-Q rinses) glass containers (size depending on experiment, see Table S1), covered lightly with parafilm and allowed to acclimate at room temperature for 1–2 h. Curcumin stock solution was added to each treatment container (0.05, 0.1, 1.5, or 10 L depending on the experiment) by gently pouring over the surface of each treatment container. Samples of K. brevis live cell counts were collected at T0 (before curcumin addition), T2 (2 h post curcumin addition), T4 or T6 (4–6 h post curcumin addition), T24 (24 h post curcumin addition), then every 24 h after. In experiments where brevetoxins were analyzed, water samples were typically collected at T0, T24, and T48. Water quality (temperature, salinity, dissolved oxygen, pH) were measured in each treatment at each time point, and when nutrients were analyzed, samples were collected at each time point. Nutrients (dissolved ammonium, nitrate-nitrite, and phosphate) were monitored in experiments in 10 L tanks only (due to water volume restriction).

2.2. Analyses

Prior to sampling, each treatment was stirred gently with a glass pipette. Water quality parameters (temperature, salinity, pH, and dissolved oxygen) were measured at each sampling time point with a calibrated multimeter (YSI ProDSS, Xylem, Yellow Springs, OH, USA) in all experiments. Approximately 60 mL of water was collected and apportioned into a clean scintillation vial (10 mL) for determination of cell abundance and into clean glass bottles (50 mL) for brevetoxin analysis. Once collected, samples were counted live after staining with neutral red [39] and/or preserved (~1% Utermohl’s solution) in glass scintillation vials and stored in the dark until analysis. Cells of K. brevis were counted using a Sedgwick-Rafter counting chamber and a Zeiss PrimoStart Microscope. Brevetoxins were extracted from 50 mL aliquots of water using Strata C-18 cartridges (Phenomenex, Torrance, CA, USA) in a PromoChrom SPE-O3 automated sample preparation system (PromoChrom, Cincinnati, OH, USA). Intracellular toxins (BTX-1, BTX-2) and two major degradation products (BTX-3, BTX-B5) were quantified by HPLC-MS/MS analysis using a Vanquish HPLC system coupled to a TSQ Quantis triple quadrupole (TQ) mass spectrometer equipped with an electrospray interface (LC/ESI/MS/MS) (Thermo Fisher Scientific Inc., Waltham, MA, USA) (Modified from Pierce et al., 2011 [8]). Chromatographic separation was performed on a Hypersil Gold Vanquish Aq UHPLC C18 reversed phase polar end-capped column at 30 °C (1.9 µm particle size, 100 mm × 2.1 mm ID; Thermo Fisher Scientific Inc., Waltham, MA, USA). The mobile phase consisted of water fortified with 0.1% formic acid (solvent A) and acetonitrile fortified with 0.1% formic acid (solvent B). Gradient elution consisted of a solution of 50:50 (v/v) (A:B) for 1 min, followed by a linear increase in solvent B to 95% over 9 min. After achieving this composition, solvent B was reduced back to initial conditions over a period of 1 min, and this was held for the remaining 4 min of the method. The injection volume was 5 µL and the flow-rate was 200 µL/min throughout the entire 15 min gradient. Ionization in the ESI source was achieved using nitrogen as a nebulizer and drying gas. ESI source spray voltage was 4200 V positive mode only, the ion transfer capillary temperature was 350 °C, the vaporization temperature was 75 °C, and the sheath gas, auxiliary and sweep gas pressure were 30, 5 and 0, respectively, in arbitrary units used by Thermo Scientific. A multiple reaction monitoring method was used with collision-induced dissociation (CID) using argon as the collision gas at a fixed pressure of 1.5 mTorr and a cone voltage that was optimized for each individual compound (Table S2). Commercially available reference standards (BTX-1, BTX-2, BTX-3 and BTX-B5; MARBIONC University of North Carolina, Wilmington, NC, USA) were used for instrument calibration and extraction and analyses quality. Samples for dissolved ammonium (DNH4-N), nitrite-nitrate (DNO2,3-N), and phosphate (DPO4-P) were filtered through Pall Supor 450 0.45 µm membrane filters immediately after sample collection and analyzed within 24 h of collection. Analyses for dissolved nutrients were conducted on an Auto Analyzer (Bran + Luebbe/Seal) following methods by Dixon et al. [40].

2.3. Data Reduction and Statistical Analyses

In order to compare results between experiments, given different starting K. brevis cell abundances and other differences between experiments, cell and toxin measurements were converted to percent reduction data for statistical analyses. The percent reduction (%R) in cell and toxin concentrations with additions of different concentrations of curcumin were calculated at 2, 4, 6, 24, 48, 72, and 96 h (if applicable) relative to controls following methods by Liu et al. [28]. Data distributions were tested for normality and homoscedasticity according to the Shapiro–Wilk and Levene tests, respectively. Given the strong left skew of these distributions and the presence of negative values, due to the necessary use of percentages, Kruskal–Wallis and Dunn’s non-parametric tests were used to examine statistical differences between experimental treatments and other parameters. Generalized Linear Models were used to visualize the data and provide estimates of confidence intervals. Data analyses were performed using R.

3. Results

3.1. Cell Reduction

The abundance of K. brevis throughout each experiment is presented in Table S3. A target initial cell abundance of approximately 1,000,000 cells/L of K. brevis culture (mean ± SE; 1,124,179 ± 58,225 cells/L; Table S3) was used to both approximate a medium bloom abundance range [5] and to allow for direct comparisons between experiments. Overall, the percent reduction (%R) in K. brevis cell abundance, relative to the control, was significantly different over time and between curcumin concentrations (Figure 1 and Figure 2, Kruskal–Wallis p < 0.001).
Within the lower concentration tests (0.1–5 mg/L curcumin), no significant differences were found in %R between 0.1, 1, or 2 mg/L curcumin treatments at any time point (Figure 1, Tables S4–S10, Dunn’s tests p > 0.05). Treatments of 3 mg/L began to show significantly greater reduction over 0.1 mg/L starting at 72 h (Table S9, Dunn’s test p < 0.05), and greater reduction over 1 mg/L at 96 h (Table S10, Dunn’s test p < 0.05). Treatments of 5 mg/L began to show significantly greater reduction over all lower concentrations beginning at 24 h (Table S7, Dunn’s test p < 0.05). At 24 h, the mean %R with 5 mg/L was 89.26% ± 3.57 SE, compared to 3 mg/L at 44.62% ± 10.12 SE (Table 1).
Within the higher concentration tests (10–40 mg/L curcumin), post hoc analyses found no significant differences between 5, 10, and 20 mg/L curcumin treatments at any time point (Figure 2, Tables S3–S9, Dunn’s tests p > 0.05). The two highest concentrations (30 and 40 mg/L) were not significantly different from each other at any time point (Dunn’s tests, p > 0.05), with mean %R > 90% beginning at 4 h (Table 1), and had significantly higher reduction rates than lower concentrations (0.1–5.0 mg/L curcumin) beginning at 2 h (Dunn’s tests, p < 0.05).
One experiment was conducted with a control group containing K. brevis and ethanol without curcumin to examine the potential contribution of the solvent to reduction rates. %R of the ethanol control, relative to 0 h, did not significantly differ from the K. brevis control (Kruskal–Wallis p = 0.77), and did not significantly differ over time (p = 0.08), indicating little impact of the solvent on cell abundance.
White curcumin (CuroWhite and Zedoary Root) failed to achieve > 50% reduction at any time point at any concentration investigated (5–50 mg/L, Table 2). The performance of white curcumin was therefore determined to be unsatisfactory, and these products were not explored further.

3.2. Reduction of Brevetoxins

Statistical analyses for total K. brevis toxins were conducted on the sums of the four brevetoxin analogs measured: BTX-1, BTX-2, BTX-3, and BTX-B5. For 1, 2, 3, and 5 mg/L curcumin treatments, measurements of toxins were taken between 0 and 96 h. For 10, 20, 30, and 40 mg/L, measurements were taken at 24 and 48 h only. Overall, the percent reduction (%R) in total toxins, relative to control, were significantly different over time (Figure 3 and Figure 4, Kruskal–Wallis p = 0.05) and between curcumin concentrations (Kruskal–Wallis p < 0.0001).
Within the lower concentration tests (1–5 mg/L curcumin), no significant differences in %R between 1 or 2 mg/L curcumin concentrations were found at any time point (Figure 3, Tables S11–S16, Dunn’s tests p > 0.05). Concentrations of 3 and 5 mg/L began to show significantly greater reduction over 1 and 2 mg/L beginning at 24 h (Table S13, Dunn’s test p < 0.05), but were not significantly different from each other at any time point. At 72 h, the mean %R with 2 mg/L was 41.94% ± 1.71 SE, compared to 5 mg/L at 79.28% ± 2.33 SE (Table 3).
Considerable overlap in toxin reduction performance was seen between low and high curcumin dosages, indicating a weaker relationship compared to cell reduction. Within the higher concentration tests (10–40 mg/L curcumin), post hoc analyses found no significant differences in %R between 3, 5, 20, 30, and 40 mg/L curcumin treatments at either 24 or 48 h (Figure 4, Tables S12–S13, Dunn’s tests p > 0.05). Brevetoxin concentrations after curcumin dosage of 10 mg/L did not differ from 1, 2, or 3 mg/L at either time point, and performed significantly lower than 40 mg/L at both time points. Within the highest concentration, 40 mg/L, the mean %R was 64.35% ± 1.43 SE at 48 h. However, the highest %R overall was seen with 5 mg/L at 72 h, at 79.28% ± 2.33 SE.

3.3. Water Quality and Nutrients

Water quality parameters (pH, salinity, temperature, dissolved oxygen) occurred within narrow ranges and showed little variation throughout each study, with the exception of dissolved oxygen (DO) in the ethanol test. All water quality data are presented in Table S17. At T48, DO dropped from 6–7 mg/L to 1.55 mg/L and was likely due to a bacterial bloom. Nutrients (dissolved ammonium (DNH4-N), nitrate-nitrite (DNO2,3-N) and phosphate (DPO4-P)) were only measured throughout laboratory tests that were performed in containers greater than 10 L (3 and 5 mg/L curcumin) due to the volume required for analyses. There were no significant differences (ANOVA on ranks; DNO2,3-N, p = 0.957; DNH4-N, p = 0.095; DPO4-P, p = 0.95) between nutrient concentrations throughout or between experiments (Table S18).

4. Discussion

Investigation of compounds and technologies for the mitigation of K. brevis blooms are currently under investigation and scrutiny with the understanding that the mitigation product should be effective against K. brevis cells and brevetoxins, and not be more harmful to the environment than an untreated bloom. Curcumin is commercially available, has been widely used in aquaculture, food, cosmetics and pharmaceuticals [41,42] and has been tested as an anti-parasitic, anti-algal compound on other HABs and pests [28,29,33,43]. To the authors’ knowledge, no other mitigation studies have been conducted on K. brevis mitigation using curcumin. In our study, curcumin displayed the ability to eliminate cells of K. brevis, reduce total brevetoxins, and have little to no impact on water quality in laboratory mitigation testing. A focus on laboratory mitigation studies prior to in situ deployment within a bloom allows for evaluation of a range of concentrations that might be efficient at reducing the intended organism. Liu et al. [28] found that 10 mg/L curcumin inhibits the growth of Chatonella marina with inhibitory rates of approximately 80% after 72 h. The minimum concentration of curcumin for a reduction in K. brevis in our study was 5 mg/L (89–90% at T24); however, the best mitigation results occurred with higher curcumin concentrations of 20–40 mg/L (60–100% at T2–T6).
The mechanism of K. brevis destruction by curcumin was not evaluated in this study, yet it will be important to determine long-term efficacy as a mitigation product. Curcumin, is a photocatalyst and is a known scavenger of reactive oxygen species (ROS) [44] that mainly accumulate in chloroplast and mitochondrial electron transport chains of phytoplankton, yet may also increase ROS [45]. When ROS is elevated, it serves as an intracellular signal to activate caspase-like activity in phytoplankton cells, leading to reduced chlorophyll a and cell death [46,47]. Other studies have found that potential mitigation products such as dibutyl phthalate inhibit cell growth, decrease cell abundance, reduce chlorophyll a, and cause oxidative stress to K. brevis by causing overproduction of ROS [48]. Photocatalytic methods are based on the interaction between molecular oxygen and a photocatalyst agent (such as curcumin) that produces ROS that can kill target organisms by oxidizing them [49].
A reduction in and/or the destruction of HAB cells alone is insufficient to mitigate a HAB bloom. Cell death can also release additional toxins, so eliminating toxins is also important when determining the efficacy of a mitigation compound or technology. Yuan et al. [30] found that 80 mmol/L curcumin mixture inhibits accumulation of diarrheal shellfish toxins from an epibenthic dinoflagellate (Prorocentrum lima) by inhibiting activity of uptake genes. In our study, total brevetoxins (in seawater) were significantly reduced by 48 h post treatment in curcumin concentrations greater than 3 mg/L and by 24 h in concentrations from 5 to 40 mg/L. The suite of brevetoxins in seawater during and after a bloom consists of intra-cellular (BTX-1, BTX-2) and extra-cellular (BTX-3, BTX-B5) toxins. BTX-1 and BTX-2 are made of similar, yet distinct polycyclic ether backbones, while the rest of the brevetoxins are thought to be metabolic derivatives, or oxidation products, of these two parent compounds [8,50,51,52,53]. Interestingly, parent toxins were not typically converted to analog forms over time with curcumin treatment (Figure 5 and Figure 6). This is unlike other mitigation strategies such as modified PAC clay where parent toxins were modified, indicative of dying K. brevis cells [54]. It is possible that due to its scavenging properties on oxygen, curcumin prevented the conversion into metabolites [44].
Like any mitigation product, there are possible negative effects from application that need to be investigated. Curcumin is a naturally orange-yellow organic molecule that readily turns seawater a light yellow or dark orange color depending on concentration. Liu et al. [28] chose not to investigate curcumin further beyond cell abundance reduction due to the color change. Karenia brevis blooms can also turn seawater a dark red or brown color [2]. If the concentration of curcumin used for mitigation does not exceed the already impacted color from a K. brevis bloom (or if color is diluted with the application of the product), then it is potentially viable as a mitigation tool. There are currently no restrictions on color according to the EPA Clean Water Act for Class II water bodies (which includes estuarine environments such as Sarasota Bay). Therefore, more investigation needs to occur on color changes and timeliness of dilution of color impacts.
Solubility and shelf life of curcumin powder are also limiting factors for the use of curcumin as a mitigation product. Curcumin is only slightly soluble in water [34,38,44]. During our study, curcumin was mixed with deionized water, filtered seawater, a small percent of ethanol (0.001–0.004%), and 99.9% ethanol. Liu et al. [28] dissolved curcumin in absolute ethanol, while Yuan et al. [30] dissolved very small amounts in NaOH. Other studies have used DMSO as a solvent [55]. Low concentrations (e.g., 0.004%) of ethanol did not negatively impact K. brevis growth; however, further studies are needed to determine potential impacts of a carrier solvent when application is scaled up. The shelf life of store-bought curcumin is limited to a few years, even when stored in sealed containers in low light [56]; however, the cost of 99.5% powdered curcumin is approximately USD 100/kg, so if stored properly, it is a relatively economical solution.
Methods of distribution of curcumin as a mitigation product on K. brevis blooms are also important to consider, though they were not investigated in these laboratory studies. Questions to be answered include: solubility, optimum dose, dispersal method, spatial distribution, and the number of dispersals per bloom event. Strategies for application of other HAB mitigation compounds include surface spraying (e.g., clay; [57]), pellet distribution, distribution via air, and in-water distribution. Tidal cycles are also important to evaluate. Typical tidal cycles in and around estuaries where K. brevis is often found (e.g., Sarasota Bay, Florida) depends on navigation channels, the number of inlets, and bathymetry [58]. Karenia brevis blooms are not typically associated with tidal movement; however, mitigation product deployment may be, depending on water turnover time, solubility, density, etc.
In conclusion, curcumin is an increasingly common and popular health supplement that could be a cost-effective way to treat K. brevis blooms. In these laboratory studies, lower dose concentrations of curcumin (0. 1–5 mg/L) were less effective against K. brevis compared to higher concentrations (10–40 mg/L). There were also no significant impacts on water quality in these controlled studies. Further studies need to be conducted to evaluate the performance and dynamics of curcumin on K. brevis blooms in natural marine and coastal waters.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w16101458/s1, Table S1: Laboratory experimental design. Water quality parameters were measured in all experiments except for the first 10 and 20 mg/L study; Table S2: Toxin compound cone voltage optimization; Table S3: Abundance of K. brevis throughout the experiments up to 96 h. S.E.—standard error, N.S.—not sampled; Table S4: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control 2 h after application; Table S5: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control 4 h after application; Table S6: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control 6 h after application; Table S7: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control 24 h after application; Table S8: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control 48 h after application, Table S9: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control 72 h after application; Table S10: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control 96 h after application; Table S11: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis toxins (sum of analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L) relative to control 4 h after application; Table S12: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis toxins (sum of analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L) relative to control 6 h after application; Table S13: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis toxins (sum of analogs BTxX-1, BTX-2, BTX-3, and BTX-B5 in ng/L) relative to control 24 h after application; Table S14: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis toxins (sum of analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L) relative to control 48 h after application; Table S15: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis toxins (sum of analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L) relative to control 72 h after application; Table S16: Dunn’s pairwise nonparametric tests of percent reduction (%R) of curcumin treatments on K. brevis toxins (sum of analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L) relative to control 96 h after application; Table S17: Average (SE) water quality conditions in each test; Table S18: Average (SE) nutrient conditions in the 3 and 5 mg/L curcumin tests.

Author Contributions

The idea for the studies originated with E.R.H., C.A.H., V.L. and R.P. The studies were initiated by C.A.H., E.R.H., V.L. and R.P. Studies were planned and conducted by C.A.H., E.R.H., J.D.F., S.K. and V.D. Sample collection and instrument analyses were performed by J.D.F., S.K., V.D. and J.H.T. Data analyses were performed by E.R.H. and V.D. E.R.H., C.A.H. and J.D.F. wrote the first draft of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Florida Red Tide Mitigation and Technology Development Initiative State of Florida, Florida Fish and Wildlife Conservation Commission grant to Mote Marine Laboratory (initiative agreement #19153).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors would like to acknowledge Amanda Muni-Morgan and Erin Cuyler who assisted with the initial planning of this work. The authors would also like to recognize a wide team of contributors associated with Mote Marine Laboratory who participated in sample collection and analysis including: Ari Nissanka, Susan Launay, Camia Charniga, David Gordon, Jess Hankins, Kelsey Thompson, Jaci Martinez, Reese Kober, Ryan McGoldrick, Stephanie Stanton, Caroline Matute, Kathleen Sway, Kelley Breeden, Chloe Manley, Claire Neal, Tristyn Bercel, Sara Turner, James Javaruski, Bill Geisbert, Patricia Blum, Sam Harlow, Jessica Donald, Megan Arp, Aileen Maldonado, Devin Burris, William Scott, Val Palubok, Briana Mays, and Giandria Green. We also thank all student interns who participated including: Olivia Shaffer, Emmanuel Perry, Lucas Patt, and Victoria Vossler.

Conflicts of Interest

The authors declare no conflicts of interest. The authors declare that the research was conducted in absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Water 16 01458 g001
Figure 1. Percent reduction (%R) of low-dose (0.1–5 mg/L) curcumin treatments on K. brevis cell abundance (cells/L) relative to control. Lines represent mean ± 95% CI from additive GLM of time and treatment as explanatory variables. N varies by treatment and time point, N = 3 at minimum.
Figure 1. Percent reduction (%R) of low-dose (0.1–5 mg/L) curcumin treatments on K. brevis cell abundance (cells/L) relative to control. Lines represent mean ± 95% CI from additive GLM of time and treatment as explanatory variables. N varies by treatment and time point, N = 3 at minimum.
Water 16 01458 g001
Water 16 01458 g002
Figure 2. Percent reduction (%R) of high-dose (10–40 mg/L) curcumin treatments on K. brevis cell abundance (cells/L) relative to control. Lines represent mean ± 95% CI from additive GLM of time and treatment as explanatory variables. N varies by treatment and time point, N = 3 at minimum.
Figure 2. Percent reduction (%R) of high-dose (10–40 mg/L) curcumin treatments on K. brevis cell abundance (cells/L) relative to control. Lines represent mean ± 95% CI from additive GLM of time and treatment as explanatory variables. N varies by treatment and time point, N = 3 at minimum.
Water 16 01458 g002
Water 16 01458 g003
Figure 3. Percent reduction (%R) of low-dose (1–5 mg/L) curcumin treatments on K. brevis total toxins relative to control. Lines represent mean ± 95% CI from additive GLM of time and treatment as explanatory variables. Total toxin measurements are sums of brevetoxin analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L. N varies by treatment and time point, N = 3 at minimum.
Figure 3. Percent reduction (%R) of low-dose (1–5 mg/L) curcumin treatments on K. brevis total toxins relative to control. Lines represent mean ± 95% CI from additive GLM of time and treatment as explanatory variables. Total toxin measurements are sums of brevetoxin analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L. N varies by treatment and time point, N = 3 at minimum.
Water 16 01458 g003
Water 16 01458 g004
Figure 4. Percent reduction (%R) of high-dose (10–40 mg/L) curcumin treatments on K. brevis total toxins relative to control. Lines represent mean ± 95% CI from additive GLM of time and treatment as explanatory variables. Total toxin measurements are sums of brevetoxin analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L. N varies by treatment and time point, N = 2 at minimum.
Figure 4. Percent reduction (%R) of high-dose (10–40 mg/L) curcumin treatments on K. brevis total toxins relative to control. Lines represent mean ± 95% CI from additive GLM of time and treatment as explanatory variables. Total toxin measurements are sums of brevetoxin analogs BTX-1, BTX-2, BTX-3, and BTX-B5 in ng/L. N varies by treatment and time point, N = 2 at minimum.
Water 16 01458 g004
Water 16 01458 g005
Figure 5. Total brevetoxin concentrations (ng/L) in low (1–5 mg/L)-dose treatments over time divided by congener: BTX-1, BTX-2, BTX-3, and BTX-B5. In the x-axis, C indicates control treatment (no curcumin addition), while T indicates curcumin treatment, with the number of hours post addition indicated after the letter. N varies by treatment and time point, N = 3 at minimum.
Figure 5. Total brevetoxin concentrations (ng/L) in low (1–5 mg/L)-dose treatments over time divided by congener: BTX-1, BTX-2, BTX-3, and BTX-B5. In the x-axis, C indicates control treatment (no curcumin addition), while T indicates curcumin treatment, with the number of hours post addition indicated after the letter. N varies by treatment and time point, N = 3 at minimum.
Water 16 01458 g005
Water 16 01458 g006
Figure 6. Total brevetoxin concentrations (ng/L) in high (10–40 mg/L)-dose treatments over time divided by congener: BTX-1, BTX-2, BTX-3, and BTX-B5. In the x-axis, C indicates control treatment (no curcumin addition), while T indicates curcumin treatment, with the number of hours post addition indicated after the letter. N varies by treatment and time point, N = 2 at minimum.
Figure 6. Total brevetoxin concentrations (ng/L) in high (10–40 mg/L)-dose treatments over time divided by congener: BTX-1, BTX-2, BTX-3, and BTX-B5. In the x-axis, C indicates control treatment (no curcumin addition), while T indicates curcumin treatment, with the number of hours post addition indicated after the letter. N varies by treatment and time point, N = 2 at minimum.
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Table 1. Mean and standard error of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control by sample event.
Table 1. Mean and standard error of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control by sample event.
Experiment TreatmentTime (h)NMeanSE
0.1 mg/L23−4.0925313.73799
0.1 mg/L43−12.698428.23137
0.1 mg/L2462.04517710.18821
0.1 mg/L486−14.32949.434375
0.1 mg/L726−3.7694211.52276
1 mg/L23−34.045138.45247
1 mg/L43−10.58213.60313
1 mg/L63−1.960782.995148
1 mg/L24917.142175.885734
1 mg/L48911.1202814.83291
1 mg/L72931.970178.466663
1 mg/L96312.6712317.26313
2 mg/L6323.856213.682819
2 mg/L24335.517246.792316
2 mg/L48326.470596.878833
2 mg/L72343.243245.479966
2 mg/L96342.465756.840748
3 mg/L238.6956529.584471
3 mg/L43−2.8089921.91011
3 mg/L666.1949425.37714
3 mg/L24944.6213310.11696
3 mg/L48927.898277.832955
3 mg/L72654.696433.472442
3 mg/L96664.726034.073759
5 mg/L21434.3623214.05441
5 mg/L41116.1716816.11522
5 mg/L6522.533427.324052
5 mg/L242089.26213.573754
5 mg/L48966.936365.136131
5 mg/L721287.412924.515164
10 mg/L222.4866791.598579
10 mg/L24238.106829.36893
10 mg/L48263.144332.319588
20 mg/L2863.2839315.23073
20 mg/L24883.632287.094545
20 mg/L48598.144331.147993
30 mg/L21675.386789.131826
30 mg/L41391.743823.06602
30 mg/L61099.427120.324572
30 mg/L241699.598070.232054
30 mg/L48111000
40 mg/L2399.264710.735294
40 mg/L2431000
40 mg/L4831000
Ethanol + KB Control23−5.327874.420407
Ethanol + KB Control4322.8007219.76823
Ethanol + KB Control2435.76687110.12772
Ethanol + KB Control483−19.05661.799885
Table 2. Mean and standard error of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control by sample event for white curcumin only.
Table 2. Mean and standard error of percent reduction (%R) of curcumin treatments on K. brevis cell abundance relative to control by sample event for white curcumin only.
Experiment TreatmentTime (h)NMeanSE
5 mg/L2621.0242611.18452
5 mg/L24644.034329.778153
5 mg/L48640.9265714.96852
10 mg/L237.4123992.139422
10 mg/L2431.2769353.959779
10 mg/L48310.265287.934202
25 mg/L2331.578952.905701
25 mg/L243−2.592592.592593
25 mg/L483−0.218343.647007
50 mg/L2329.401090.654365
50 mg/L243−11.55563.449817
50 mg/L48315.938863.86284
Table 3. Mean and standard error of percent reduction (%R) of curcumin treatments on K. brevis toxins (sum of analogs BTx-1, BTx-2, BTx-3, and BTx-B5 in ng/L) relative to control by sample event.
Table 3. Mean and standard error of percent reduction (%R) of curcumin treatments on K. brevis toxins (sum of analogs BTx-1, BTx-2, BTx-3, and BTx-B5 in ng/L) relative to control by sample event.
Experiment TreatmentTime(h)NMeanSE
1 mg/L6338.665093.967214
1 mg/L2432.67006616.2927
1 mg/L483−34.276517.4238
1 mg/L723−9.927914.43228
1 mg/L96334.275886.965364
2 mg/L6343.861694.237249
2 mg/L243−4.7313919.13836
2 mg/L4836.41247914.36292
2 mg/L72341.945641.712205
2 mg/L96346.854261.304109
3 mg/L4319.3694620.81235
3 mg/L6352.505453.286115
3 mg/L24643.530528.099593
3 mg/L48353.822851.938302
3 mg/L72673.788732.462797
3 mg/L96379.096340.540372
5 mg/L4338.2957323.27433
5 mg/L24360.2302215.44914
5 mg/L72379.284452.337412
10 mg/L24221.312840.902916
10 mg/L48222.506168.595048
20 mg/L24533.998843.749756
20 mg/L48551.376549.761752
30 mg/L24328.413012.527997
30 mg/L48369.530115.037381
40 mg/L24348.4688410.62812
40 mg/L48364.353531.435036
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Hall, E.R.; Heil, C.A.; Frankle, J.D.; Klass, S.; Devillier, V.; Lovko, V.; Toyoda, J.H.; Pierce, R. Mitigation of Karenia brevis Cells and Brevetoxins Using Curcumin, a Natural Supplement. Water 2024, 16, 1458. https://doi.org/10.3390/w16101458

AMA Style

Hall ER, Heil CA, Frankle JD, Klass S, Devillier V, Lovko V, Toyoda JH, Pierce R. Mitigation of Karenia brevis Cells and Brevetoxins Using Curcumin, a Natural Supplement. Water. 2024; 16(10):1458. https://doi.org/10.3390/w16101458

Chicago/Turabian Style

Hall, Emily R., Cynthia A. Heil, Jessica D. Frankle, Sarah Klass, Victoria Devillier, Vincent Lovko, Jennifer H. Toyoda, and Richard Pierce. 2024. "Mitigation of Karenia brevis Cells and Brevetoxins Using Curcumin, a Natural Supplement" Water 16, no. 10: 1458. https://doi.org/10.3390/w16101458

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